Dongyou Liu - Molecular Detection of Human Fungal Pathogens-CRC Press (2011) PDF

MOLECULAR
DETECTION OF
HUMAN FUNGAL
PATHOGENS
© 2011 by Taylor & Francis Group, LLC

MOLECULAR
DETECTION OF
HUMAN FUNGAL
PATHOGENS
EDITED BY
DONGYOU LIU
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an informa business

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CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
Printed in the United States of America on acid-free paper

Version Date: 20110510
International Standard Book Number: 978-1-4398-1240-2 (Hardback)
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and
information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and
publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission
to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any
future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or
retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact
the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides
licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment
has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation
without intent to infringe.
Visit the Taylor & Francis Web site at
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and the CRC Press Web site at
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This volume is dedicated to a group of international mycologists, whose in-depth knowledge and
technical expertise have made an all inclusive coverage of major human fungal pathogens possible.

Contents
Preface......................................................................................................................................................................................... xv
Editor.........................................................................................................................................................................................xvii
Contributors................................................................................................................................................................................xix
Chapter 1 Introductory Remarks.............................................................................................................................................. 1

Dongyou Liu
Part I Ascomycota
Pezizomycotina: Dothideomycetes
Chapter 2 Alternaria............................................................................................................................................................... 27
Giuliana Lo Cascio and Marco Ligozzi
Chapter 3 Aureobasidium....................................................................................................................................................... 37
Miia Pitkäranta and Malcolm D. Richardson
Chapter 4 Bipolaris and Drechslera....................................................................................................................................... 49

Dongyou Liu and Joanna Gray
Chapter 5 Botryomyces........................................................................................................................................................... 57
Dongyou Liu and R.R.M. Paterson
Chapter 6 Botryosphaeria and Lasiodiplodia........................................................................................................................ 61

Dongyou Liu
Chapter 7 Corynespora........................................................................................................................................................... 65
Dongyou Liu and Po-Ren Hsueh
Chapter 8 Curvularia.............................................................................................................................................................. 71
Audrey N. Schuetz
Chapter 9 Exserohilum........................................................................................................................................................... 83
K. Lily Therese and H.N. Madhavan
Chapter 10 Fusicoccum and Scytalidium................................................................................................................................. 93

Marie Machouart and Jean Menotti
Chapter 11 Hortaea.................................................................................................................................................................101
Dongyou Liu and Larry Hanson
vii

viii Contents
Chapter 12 Leptosphaeria...................................................................................................................................................... 105

Dongyou Liu
Chapter 13 Macrophomina..................................................................................................................................................... 109

Artur Alves, Alan J.L. Phillips, and António Correia
Chapter 14 Madurella..............................................................................................................................................................117
Wendy W.J. van de Sande, Ahmed H. Fahal, G. Sybren de Hoog, and Alex van Belkum
Chapter 15 Neodeightonia...................................................................................................................................................... 129

Chapter 16 Phoma and Phomopsis......................................................................................................................................... 135

Dongyou Liu
Chapter 17 Piedraia.................................................................................................................................................................141
Dongyou Liu
Chapter 18 Pyrenochaeta....................................................................................................................................................... 145

Dongyou Liu
Chapter 19 Ramichloridium.................................................................................................................................................... 151

Dongyou Liu
Chapter 20 Ulocladium........................................................................................................................................................... 157

Dongyou Liu
Pezizomycotina: Eurotiomycetes
Chapter 21 Acrophialophora.................................................................................................................................................. 163

Dongyou Liu
Chapter 22 Arthrographis....................................................................................................................................................... 167

Dongyou Liu
Chapter 23 Aspergillus.............................................................................................................................................................171
Maiken Cavling Arendrup, Yanan Zhao, and David S. Perlin
Chapter 24 Blastomyces.......................................................................................................................................................... 189

Mark D. Lindsley
Chapter 25 Chrysosporium..................................................................................................................................................... 203


Contents ix
Chapter 26 Cladophialophora................................................................................................................................................ 209

Rubén Lopez-Martínez and Francisca Hernández-Hernández
Chapter 27 Coccidioides..........................................................................................................................................................217
Rossana de Aguiar Cordeiro, Raimunda Sâmia Nogueira Brilhante, Marcos Fábio Gadelha Rocha, and
José Júlio Costa Sidrim
Chapter 28 Cyphellophora...................................................................................................................................................... 231

Dongyou Liu
Chapter 29 Emmonsia............................................................................................................................................................. 235

Chapter 30 Epidermophyton................................................................................................................................................... 241

Dongyou Liu and Susan Coloe
Chapter 31 Exophiala............................................................................................................................................................. 247

Dongyou Liu and Shoo Peng Siah
Chapter 32 Fonsecaea............................................................................................................................................................. 255

Hideki Miyagi
Chapter 33 Histoplasma......................................................................................................................................................... 263

Rosely Maria Zancopé Oliveira, Allan Jefferson Guimarães, Joshua D. Nosanchuk,
Mauro de Medeiros Muniz, Priscila Costa Albuquerque, and Rodrigo de Almeida Paes
Chapter 34 Lacazia................................................................................................................................................................. 275

Dongyou Liu and Yi-Wei Tang
Chapter 35 Lecythophora....................................................................................................................................................... 281

Dongyou Liu
Chapter 36 Microsporum........................................................................................................................................................ 285

Rahul Sharma and Yvonne Gräser
Chapter 37 Myriodontium....................................................................................................................................................... 299

Dongyou Liu
Chapter 38 Onychocola.......................................................................................................................................................... 303

Dongyou Liu
Chapter 39 Paecilomyces........................................................................................................................................................ 309

Ana Alastruey-Izquierdo, Maria Victoria Castelli, Leticia Bernal-Martinez, and Manuel Cuenca-Estrella

x Contents
Chapter 40 Paracoccidioides...................................................................................................................................................317
Eduardo Bagagli, Sandra de Moraes Gimenes Bosco, Virgínia Bodelão Richini-Pereira,
Raquel Cordeiro Theodoro, and Sílvio Alencar Marques
Chapter 41 Penicillium: Mycoses and Mycotoxinoses........................................................................................................... 329

R.R.M. Paterson and N. Lima
Chapter 42 Phialophora.......................................................................................................................................................... 345

Chapter 43 Rhinocladiella.......................................................................................................................................................351
Dongyou Liu
Chapter 44 Trichophyton........................................................................................................................................................ 357

Sharon C.-A. Chen, David Ellis, Tania C. Sorrell, and Wieland Meyer
Chapter 45 Veronaea.............................................................................................................................................................. 377

Dongyou Liu
Pezizomycotina: Sordariomycetes
Chapter 46 Acremonium......................................................................................................................................................... 385

Dongyou Liu, Xianghong Du, and Song Weining
Chapter 47 Beauveria............................................................................................................................................................. 391

Dongyou Liu
Chapter 48 Chaetomium......................................................................................................................................................... 397

Chapter 49 Colletotrichum..................................................................................................................................................... 401

M.R. Shivaprakash, Abhishek Baghela, and Arunaloke Chakrabarti
Chapter 50 Cylindrocarpon.....................................................................................................................................................411
Dongyou Liu
Chapter 51 Fusarium...............................................................................................................................................................417
Palanisamy Manikandan, László Galgóczy, Kanesan Panneer Selvam,
Coimbatore Subramanian Shobana, Sándor Kocsubé,
Csaba Vágvölgyi, Venkatapathy Narendran, and László Kredics
Chapter 52 Microascus, Including Scopulariopsis................................................................................................................. 435

Jouni Issakainen and Dongyou Liu

Contents xi
Chapter 53 Myceliophthora and Thielavia............................................................................................................................. 445

Dongyou Liu
Chapter 54 Neocosmosporas.................................................................................................................................................. 449

Palanisamy Manikandan, Csaba Vágvölgyi, Venkatapathy Narendran,
Kanesan Panneer Selvam, and László Kredics
Chapter 55 Ochroconis........................................................................................................................................................... 459

Ayako Sano and Kyoko Yarita
Chapter 56 Phaeoacremonium............................................................................................................................................... 469

László Galgóczy, Laura Kovács, Tamás Papp, and Csaba Vágvölgyi
Chapter 57 Phialemonium...................................................................................................................................................... 481

Chapter 58 Pseudallescheria and Scedosporium................................................................................................................... 485

Ana Alastruey-Izquierdo, Maria Victoria Castelli, Leticia Bernal-Martinez, and
Juan Luis Rodríguez Tudela
Chapter 59 Sarcopodium........................................................................................................................................................ 493

Chapter 60 Sporothrix and Sporotrichosis.............................................................................................................................. 497

Conchita Toriello, María del Rocío Reyes-Montes, Armando Pérez-Torres, and Amelia Pérez-Mejía
Chapter 61 Stachybotrys..........................................................................................................................................................511
Chapter 62 Trichoderma..........................................................................................................................................................517
László Kredics, Lóránt Hatvani, László Manczinger, Csaba Vágvölgyi, and Zsuzsanna Antal
Chapter 63 Verticillium........................................................................................................................................................... 535

Malena P. Pantou and Milton A. Typas
Saccharomycotina and Taphrinomycotina
Chapter 64 Candida.................................................................................................................................................................551
P. Lewis White, Michael D. Perry, and Rosemary A. Barnes
Chapter 65 Debaryomyces...................................................................................................................................................... 569

María J. Andrade, Mar Rodríguez, Elena Bermúdez, Félix Núñez, Miguel A. Asensio,
and Juan J. Córdoba

xii Contents
Chapter 66 Geotrichum.......................................................................................................................................................... 581

Silvia D’Arezzo, Paolo Visca, and Corrado Girmenia
Chapter 67 Kluyveromyces..................................................................................................................................................... 591

Dongyou Liu
Chapter 68 Pichia and Kodamaea.......................................................................................................................................... 595

Dongyou Liu
Chapter 69 Pneumocystis........................................................................................................................................................ 603

Steve M. Taylor and Steven R. Meshnick
Chapter 70 Saccharomyces......................................................................................................................................................615
Franca Rossi and Sandra Torriani
Part II Bastidiomycota
Chapter 71 Coprinopsis and Hormographiella...................................................................................................................... 629

Chapter 72 Cryptococcus........................................................................................................................................................ 633

Massimo Cogliati, Anna Maria Tortorano, and Maria Anna Viviani
Chapter 73 Malassezia............................................................................................................................................................ 643

Takashi Sugita, Mami Tajima, Hisae Tsubuku, Mayumi Miyamoto, Enshi Zhang, Ryoji Tsuboi,
Masako Takashima, Yoshio Ishibashi, and Akemi Nishikawa
Chapter 74 Rhodotorula......................................................................................................................................................... 653

Diego Libkind
Chapter 75 Schizophyllum...................................................................................................................................................... 669

Sophie Cassaing, Marie-Denise Linas, and Antoine Berry
Chapter 76 Sporobolomyces................................................................................................................................................... 677

Dongyou Liu
Chapter 77 Trichosporon........................................................................................................................................................ 681

Takashi Sugita, Reiko Ikeda, Akemi Nishikawa, Masako Takashima, Nanthawan Mekha,
Natteewan Poonwan, Ayse Kalkanci, and Semra Kustimur
Chapter 78 Ustilago and Pseudozyma.................................................................................................................................... 687

Dongyou Liu

Contents xiii
Chapter 79 Wallemia............................................................................................................................................................... 693

Dongyou Liu
Part III Entomohpthoromycotina and Mucoromyotina
Chapter 80 Apophysomyces.................................................................................................................................................... 699

Arunaloke Chakrabarti, Shiv Sekhar Chatterjee, and Varghese K. George
Chapter 81 Cokeromyces........................................................................................................................................................ 709

Dongyou Liu
Chapter 82 Cunninghamella................................................................................................................................................... 713

Nancy L. Wengenack and D. Jane Hata
Chapter 83 Entomophthorales................................................................................................................................................ 723

Johannes E. Rothhardt, Volker U. Schwartze, and Kerstin Voigt
Chapter 84 Lichtheimia (Absidia-Like Fungi)....................................................................................................................... 735

Kerstin Hoffmann and Kerstin Voigt
Chapter 85 Mortierella........................................................................................................................................................... 749

Tamás Papp, Kerstin Hoffmann, Ildikó Nyilasi, Tamás Petkovits, Lysett Wagner, Csaba Vágvölgyi, and
Kerstin Voigt
Chapter 86 Mucor................................................................................................................................................................... 759

Peter C. Iwen
Chapter 87 Rhizopus............................................................................................................................................................... 773

Dongyou Liu and Frank W. Austin
Chapter 88 Rhizomucor.......................................................................................................................................................... 783

Tamás Papp, Ildikó Nyilasi, Miklós Takó, László G. Nagy, and Csaba Vágvölgyi
Chapter 89 Saksenaea............................................................................................................................................................. 791

Eric Dannaoui
Chapter 90 Syncephalastrum.................................................................................................................................................. 799

Dongyou Liu
Part IV Microsporidia
Chapter 91 Anncaliia (Brachiola)........................................................................................................................................... 807

Govinda S. Visvesvara and Lihua Xiao

xiv Contents
Chapter 92 Encephalitozoon....................................................................................................................................................817
Dongyou Liu and Elizabeth S. Didier
Chapter 93 Enterocytozoon..................................................................................................................................................... 827

Jaco J. Verweij and Dongyou Liu
Chapter 94 Nosema, Vittaforma, and Microsporidium.......................................................................................................... 837

Dongyou Liu
Chapter 95 Pleistophora and Trachipleistophora................................................................................................................... 843

Dongyou Liu
Part V Oomycota, Chlorophyta, and Mesomycetozoea
Chapter 96 Pythium.............................................................................................................................................................. 851

Theerapong Krajaejun, Boonmee Sathapatayavongs, and Thomas D. Sullivan
Chapter 97 Prototheca.......................................................................................................................................................... 865

Uwe H. Roesler
Chapter 98 Rhinosporidium................................................................................................................................................. 871

S.N. Arseculeratne
Part VI Panfungal and Drug Resistance Detection
Chapter 99 Nucleic Acid-Based Panfungal Detection......................................................................................................... 891

Sandra Preuner and Thomas Lion
Chapter 100 Molecular Characterization of Fungal Drug Resistance................................................................................... 903

Maurizio Sanguinetti, Brunella Posteraro, and Patrizia Posteraro
Index.......................................................................................................................................................................................... 923

Preface
Fungi are a diverse group of eukaryotic organisms that range developments to know which are most appropriate to use for
from yeasts, molds, mushrooms, lichens, rusts, smuts, to streamlined identification and detection of fungal organisms
microsporidia. Forming a kingdom of their own and being of interest.
ubiquitously distributed in all environments, most fungi are With contributions from international scientists in respec-
saprophytes involved in the decomposition and recycling tive fungal pathogen research and diagnosis, this book aims
of organic matters as well as in the formation of symbiotic to provide a reliable and comprehensive source relating the
relationship with plants and animals. However, some fungi molecular detection and identification of major human fun-
have the capacity to cause diseases in plants, animals, and gal pathogens. Each chapter consists of a brief review on the
humans. Often occurring as a result of trauma or underlying classification, epidemiology, clinical features, and diagno-
immunosuppression, human mycoses may manifest as super- sis of one or a group of related fungal species; an outline
ficial, cutaneous, subcutaneous, or systemic diseases. The of clinical sample collection and preparation procedures; a
inability to distinguish human mycoses caused by various selection of representative stepwise molecular protocols;
fungal pathogens on clinical ground necessitates the develop and a discussion on additional research for further improv-
ment and use of laboratory diagnostic procedures in order to ing the diagnosis. This book represents an indispensable tool
facilitate their treatment and prevention. for both upcoming and experienced medical, veterinary, and
Given their complex life cycle and their tendency to pro- industrial laboratory scientists engaged in fungus character-
duce morphologically similar structures, fungi are notori- ization and provides an essential reference for undergraduate
ously difficult to identify on the basis of their macroscopic and graduate students majoring in mycology.
and microscopic features, even for an experienced mycolo- An all-encompassing book such as this clearly demands a
gist. To increase the accuracy, sensitivity, and efficiency of concerted team’s efforts. I am fortunate and extremely hon-
fungal identification, molecular techniques such as PCR ored to have had a large group of international mycologists
and nucleotide sequencing have been increasingly adopted as chapter contributors, whose in-depth knowledge and tech-
and applied in research and clinical laboratories worldwide. nological insights into human fungal pathogen detection have
Consequently, a large number of molecular protocols have significantly enriched this book. Additionally, the profession-
been described in the literature for the identification and alism and dedication of executive editor Barbara Norwitz and
detection of fungal organisms. As the saying goes, one per- senior project coordinator Jill Jurgensen at CRC Press have
son’s medicine could easily turn into another’s poison. There enhanced its presentation. Finally, without the understand-
is certainly no exception here. The overabundance of origi- ing and support of my family, Liling Ma, Brenda, and Cathy,
nal protocols and subsequent modifications has created a the compilation of this comprehensive book would have been
dilemma for anyone who was not directly involved in their unimaginable.
xv

Editor
Dongyou Liu, PhD, undertook his veterinary science edu- dermatophyte fungi (Trichophyton, Microsporum, and
cation at Hunan Agricultural University, Changsha, China. Epidermophyton), and listeriae (Listeria species). He is the
Upon graduation, he received an overseas postgraduate first author of more than 50 original research and review
scholarship from the Chinese Ministry of Education to articles in various international journals and the editor of
pursue further training at the University of Melbourne, the recently released Handbook of Listeria monocytogenes
Melbourne, Victoria, Australia, where he worked toward (2008), Handbook of Nucleic Acid Purification (2009),
improved immunological diagnosis of human hydatid dis- Molecular Detection of Foodborne Pathogens (2009),
ease. During the past two decades, he has crisscrossed Molecular Detection of Human Viral Pathogens (2010),
between research and clinical laboratories in Australia and and Molecular Detection of Human Bacterial Pathogens
the United States, with focuses on molecular characteriza- (2011), as well as the forthcoming Molecular Detection of
tion and virulence determination of microbial pathogens Human Parasitic Pathogens (2012), all of which are pub-
such as ovine footrot bacterium (Dichelobacter nodosus), lished by CRC Press.
xvii

Contributors
Ana Alastruey-Izquierdo Eduardo Bagagli Raimunda Sâmia Nogueira
Centro Nacional de Microbiologia Departamento de Microbiologia e Brilhante
Instituto de Salud Carlos III Imunologia Specialized Medical Mycology Center
Majadahonda, Spain Instituto de Biociências Federal University of Ceará
Universidade Estadual Ceará, Brazil
Priscila Costa Albuquerque Paulista-Botucatu
Laboratório de Micologia Sao Paulo, Brazil Sophie Cassaing
Instituto de Pesquisa Clinica Evandro Department of Parasitology and
Chagas Abhishek Baghela Mycology
Fundação Oswaldo Cruz Division of Mycology Faculty of Medicine Purpan
Rio de Janeiro, Brazil Department of Medical Microbiology Toulouse University Hospitals
Postgraduate Institute of Medical Toulouse University
Artur Alves Education and Research Toulouse, France
Departamento de Biologia Chandigarh, India
Centro de Estudos do Ambiente e do Maria Victoria Castelli
Mar Rosemary A. Barnes Centro Nacional de Microbiologia
Universidade de Aveiro School of Medicine Instituto de Salud Carlos III
Aveiro, Portugal University Hospital of Wales Majadahonda, Spain
Cardiff University
María J. Andrade Cardiff, United Kingdom Arunaloke Chakrabarti
Higiene y Seguridad Alimentaria Division of Mycology
Facultad de Veterinaria Alex van Belkum Department of Medical Microbiology
Universidad de Extremadura Department of Medical Microbiology Postgraduate Institute of Medical
Cáceres, Spain and Infectious Diseases Education and Research
Erasmus Medical Center Chandigarh, India
Rotterdam, the Netherlands
Zsuzsanna Antal
Laboratoire de Génomique Shiv Sekhar Chatterjee
Elena Bermúdez
Fonctionnelle des Champignons Division of Mycology
Higiene y Segurided Alimentaria
Pathogènes des Plantes Department of Medical Microbiology
Facultad de Veterinaria
Université Claude Bernard Lyon 1 Postgraduate Institute of Medical
Universidad de Extremadura
Villeurbanne, France Education and Research
Cáceres, Spain
Chandigarh, India
Maiken Cavling Arendrup Leticia Bernal-Martinez
Unit of Mycology and Parasitology Centro Nacional de Microbiologia Sharon C.-A. Chen
Statens Serum Institute Instituto de Salud Carlos III Centre for Infectious Diseases and
Copenhagen, Denmark Majadahonda, Spain Microbiology
Westmead Hospital
S.N. Arseculeratne Antoine Berry Westmead, New South Wales, Australia
Faculty of Medicine Department of Parasitology and and
University of Peradeniya Mycology Molecular Mycology Research
Peradeniya, Sri Lanka Faculty of Medicine Rangueil Laboratory
Toulouse University Hospitals Sydney Medical School—Western
Miguel A. Asensio Toulouse University University of Sydney at Westmead
Higiene y Seguridad Alimentaria Toulouse, France Hospital
Facultad de Veterinaria Sydney, New South Wales, Australia
Universidad de Extremadura Sandra de Moraes Gimenes Bosco
Cáceres, Spain Departamento de Microbiologia e Massimo Cogliati
Imunologia Laboratory of Medical Mycology
Frank W. Austin Instituto de Biociências Department of Public Health –
College of Veterinary Medicine Universidade Estadual Microbiology – Virology
Mississippi State University Paulista-Botucatu Università degli Studi di Milano
Mississippi State, Mississippi Sao Paulo, Brazil Milan, Italy
xix

xx Contributors
Susan Coloe Xianghong Du Larry Hanson

Microbiology Department College of Agronomy College of Veterinary Medicine
Melbourne Pathology Northwest A&F University Mississippi State University
Collingwood, Australia Shaanxi, China Mississippi State, Mississippi
Rossana de Aguiar Cordeiro David Ellis

Mycology Unit D. Jane Hata
Specialized Medical Mycology Center
South Australia Pathology Division of Clinical Microbiology
Federal University of Ceará
Adelaide, South Australia, Australia Mayo Clinic College of Medicine
Fortaleza, Brazil
Jacksonville, Florida
Ahmed H. Fahal
Juan J. Córdoba Mycetoma Research Centre
Higiene y Seguridad Alimentaria Lóránt Hatvani
University of Khartoum
Facultad de Veterinaria Faculty of Science and Informatics
Khartoum, Sudan
Universidad de Extremadura Department of Microbiology
Cáceres, Spain University of Szeged
László Galgóczy
Faculty of Science and Informatics Szeged, Hungary
António Correia Department of Microbiology
Departamento de Biologia University of Szeged
Centro de Estudos do Ambiente e do Szeged, Hungary Francisca Hernández-Hernández
Mar Faculty of Medicine
Universidade de Aveiro Department of Microbiology and
Varghese K. George
Aveiro, Portugal Parasitology
Division of Mycology
National Autonomous University of
Department of Medical Microbiology
Mexico
Manuel Cuenca-Estrella Postgraduate Institute of Medical
Mexico City, Mexico
Centro Nacional de Microbiologia Education and Research
Instituto de Salud Carlos III Chandigarh, India
Majadahonda, Spain Kerstin Hoffmann
Corrado Girmenia Jena Microbial Resource Collection
Dipartimento di Ematologia, (JMRC)
Eric Dannaoui
Oncologia, Anatomia Patologica e Institute of Microbiology
Unité de Mycologie Moléculaire
Medicina Rigenerativa University of Jena
Centre National de Référence
Azienda Policlinico Umberto I and
Mycologie et Antifongiques
Sapienza Università de Roma Department of Microbiology and
Institut Pasteur
Rome, Italy Molecular Biology
and
Faculté de Médecine Leibniz-Institute for Natural Product
Université Paris Descartes Yvonne Gräser Research and Infection Biology e.V.
Unité de Parasitologie—Mycologie Institute für Mikrobiologie und Hans-Knöll-Institute (HKI)
Assistance Publique-Hôpitaux de Paris Hygiene Neugasse, Jena, Germany
Hôpital Européen Georges Pompidou Charité Universitätsmedizin Berlin
Paris, France Berlin, Germany
G. Sybren de Hoog
Joanna Gray Central Bureau of Fungal Cultures
Silvia D’Arezzo Royal College of Pathologists of Fungal Biodiversity Centre
Unità di Microbiologia Molecolare Australasia Royal Netherlands Academy of Arts
Azienda Policlinico Umberto I BioSecurity Quality Assurance and Sciences
Istituto Nazionale per le Malattie Programs Utrecht, the Netherlands
Infettive “Lazzaro Spallanzani” Surrey Hills, New South Wales,
Sapienza Università de Roma Australia
Rome, Italy
Po-Ren Hsueh
Allan Jefferson Guimarães Departments of Laboratory Medicine
Elizabeth S. Didier Laboratório de Micologia and Internal Medicine
Division of Microbiology Instituto de Pesquisa Clinica Evandro National Taiwan University Hospital
Tulane National Primate Research Chagas National Taiwan University College of
Center Fundação Oswaldo Cruz Medicine
Covington, Louisiana Rio de Janeiro, Brazil Taipei, Taiwan

Contributors xxi
Reiko Ikeda Semra Kustimur Dongyou Liu

Department of Microbiology Department of Microbiology Royal College of Pathologists of
Meiji Pharmaceutical University School of Medicine Australasia
Tokyo, Japan Gazi University BioSecurity Quality Assurance
Ankara, Turkey Programs
Surrey Hills, New South Wales,
Yoshio Ishibashi Australia
Department of Immunobiology Diego Libkind
Meiji Pharmaceutical University Laboratorio de Microbiología Aplicada Giuliana Lo Cascio
Tokyo, Japan y Biotecnología Departmento ad Attività Integrata di
Centro Regional Universitario Patologia e Diagnostica
Bariloche
Jouni Issakainen Azienda Ospedaliera Universitaria
Consejo Nacional de Investigaciones
Department of Biology Integrata
Científicas y Tecnológicas
University of Turku Verona, Italy
Instituto de Investigaciones en
Turku, Finland Biodiversidad y Medio Ambiente
Rubén Lopez-Martínez
Universidad Nacional del Comahue
Faculty of Medicine
Bariloche, Argentina
Peter C. Iwen Department of Microbiology and
Department of Pathology and Parasitology
Microbiology Marco Ligozzi National Autonomous University of
University of Nebraska Medical Center Dipartimento di Patologia Mexico
Omaha, Nebraska Università di Verona Mexico City, Mexico
Verona, Italy
Marie Machouart
Ayse Kalkanci
Faculté de Médecine
Department of Microbiology
N. Lima Centre Hospitalier Universitaire de
School of Medicine
Centre of Biological Engineering Nancy
Gazi University
Institute for Biotechnology and Hôpital Brabois
Ankara, Turkey
Bioengineering Université Henri Poincaré
University of Minho Vandoeuvre-les-Nancy, France
Sándor Kocsubé Braga, Portugal
Faculty of Science and Informatics H.N. Madhavan
Department of Microbiology L&T Microbiology Research Centre
University of Szeged Marie-Denise Linas Vision Research Foundation, Sankara
Szeged, Hungary Department of Parasitology and Nethralaya
Mycology Chennai, India
Faculty of Medicine Purpan
Laura Kovács Toulouse University Hospitals László Manczinger
Faculty of Science and Informatics Toulouse University Faculty of Science and Informatics
Department of Microbiology Toulouse, France Department of Microbiology
University of Szeged University of Szeged
Szeged, Hungary Szeged, Hungary
Mark D. Lindsley
Mycotic Diseases Branch
Theerapong Krajaejun Centers for Disease Control and Palanisamy Manikandan
Faculty of Medicine Prevention Department of Microbiology
Department of Pathology Atlanta, Georgia Aravind Eye Hospital and Postgraduate
Ramathibodi Hospital Institute of Ophthalmology
Mahidol University Coimbatore, India
Bangkok, Thailand Thomas Lion
Division of Molecular Microbiology Sílvio Alencar Marques
and Development of Genetic Faculdade de Medicina
László Kredics Diagnostics Departamento de Dermatolologia e
Faculty of Science and Informatics Children’s Cancer Research Institute Radioterapia
Department of Microbiology and Universidade Estadual
University of Szeged LabDia Labordiagnostik Paulista-Botucatu
Szeged, Hungary Vienna, Austria Sao Paulo, Brazil

xxii Contributors
Nanthawan Mekha Venkatapathy Narendran Tamás Papp

Department of Medical Sciences Department of Microbiology Faculty of Science and Informatics
National Institute of Health Aravind Eye Hospital and Postgraduate Department of Microbiology
Ministry of Public Health Institute of Ophthalmology University of Szeged
Nonthaburi, Thailand Coimbatore, India Szeged, Hungary
Jean Menotti R.R.M. Paterson

Akemi Nishikawa
Hôpital Saint-Louis Centre for Biological Engineering
Department of Immunobiology
Assistance Publique-Hôpitaux de Paris Institute for Biotechnology and
Meiji Pharmaceutical University
Université Paris-Diderot Bioengineering
Tokyo, Japan
Paris, France University of Minho
Braga, Portugal
Steven R. Meshnick Joshua D. Nosanchuk
Department of Epidemiology Division of Infectious Disease Amelia Pérez-Mejía
Gillings School of Global Public Departments of Medicine and Facultad de Medicina
Health Immunology Departamento de Microbiología y
University of North Carolina Albert Einstein College of Medicine Parasitología
Chapel Hill, North Carolina Yeshiva University Universidad Nacional Autónoma de
Bronx, New York México
México City, Mexico
Wieland Meyer
Centre for Infectious Diseases and
Microbiology Félix Núñez Armando Pérez-Torres
Westmead Hospital Higiene y Seguridad Alimentaria Facultad de Medicina
Westmead, New South Wales, Australia Facultad de Veterinaria Departamento de Biología Celular y
and Universidad de Extremadura Tisular
Molecular Mycology Research Cáceres, Spain Universidad Nacional Autónoma de
Laboratory México
Sydney Medical School—Western México City, Mexico
University of Sydney at Westmead Ildikó Nyilasi
Hospital Faculty of Science and Informatics
Department of Microbiology David S. Perlin
Sydney, New South Wales, Australia
University of Szeged Public Health Research Institute
Szeged, Hungary New Jersey Medical School
Hideki Miyagi University of Medicine and Dentistry
Division of Dermatology of New Jersey
University of the Ryukyus Rosely Maria Zancopé Oliveira Newark, New Jersey
Okinawa, Japan Laboratório de Micologia
Instituto de Pesquisa Clinica Evandro Michael D. Perry
Mayumi Miyamoto Chagas Public Health Wales
Department of Dermatology Fundação Oswaldo Cruz Microbiology—Cardiff
Tokyo Medical University Rio de Janeiro, Brazil University Hospital of Wales
Tokyo, Japan Cardiff, United Kingdom
Rodrigo de Almeida Paes

Mauro de Medeiros Muniz Tamás Petkovits
Laboratório de Micologia
Laboratório de Micologia Faculty of Science and Informatics
Instituto de Pesquisa Clinica Evandro
Instituto de Pesquisa Clinica Evandro Department of Microbiology
Chagas
Chagas University of Szeged
Fundação Oswaldo Cruz
Fundação Oswaldo Cruz Szeged, Hungary
Rio de Janeiro, Brazil
Rio de Janeiro, Brazil
Alan J.L. Phillips
László G. Nagy Malena P. Pantou Faculdade de Ciências e Tecnologia
Faculty of Science and Informatics Molecular Immunopathology and Departamento de Ciências da Vida
Department of Microbiology Histocompatibility Laboratory Centro de Recursos Microbiológicos
University of Szeged Onassis Cardiac Surgery Center Universidade Nova de Lisboa
Szeged, Hungary Athens, Greece Caparica, Portugal

Contributors xxiii
Miia Pitkäranta Virgínia Bodelão Richini-Pereira Maurizio Sanguinetti

DNA Sequencing and Genomics Departamento de Microbiologia e Istituto di Microbiologia
Laboratory Imunologia Istituto Di Ricovero e Cura a Carattere
Institute of Biotechnology Instituto de Biociências Scientifico
University of Helsinki Universidade Estadual Istituto Dermopatico dell’Immacolata
Helsinki, Finland Paulista-Botucatu Ospedale San Carlo
Sao Paulo, Brazil Università Cattolica del Sacro Cuore
Natteewan Poonwan Rome, Italy
Department of Medical Sciences
National Institute of Health Marcos Fábio Gadelha Rocha
Faculty of Veterinary Medicine Ayako Sano
Ministry of Public Health
Postgraduate Program in Veterinary Medical Mycology Research Center
Nonthaburi, Thailand
Science Chiba University
State University of Ceará Chuo-ku, Japan
Brunella Posteraro
Istituto di Microbiologia and
Istituto Dermopatico dell’Immacolata Specialized Medical Mycology Center Boonmee Sathapatayavongs
Istituto Di Ricovero e Cura a Carattere Federal University of Ceará Faculty of Medicine
Scientifico Ceará, Brazil Department of Medicine
Ospedale San Carlo Ramathibodi Hospital
Università Cattolica del Sacro Cuore Mahidol University
Mar Rodríguez
Rome, Italy Bangkok, Thailand
Higiene y Seguridad Alimentaria
Facultad de Veterinaria
Patrizia Posteraro Universidad de Extremadura Audrey N. Schuetz
Laboratory of Clinical Pathology and Cáceres, Spain Weill Cornell Medical College
Microbiology NewYork-Presbyterian Hospital
Istituto Dermopatico dell’Immacolata New York, New York
Istituto Di Ricovero e Cura a Carattere Uwe H. Roesler
Scientifico Institute of Animal Hygiene and
Ospedale San Carlo Environmental Health Volker U. Schwartze
Università Cattolica del Sacro Cuore Freie University Berlin Institute of Microbiology
Rome, Italy Berlin, Germany School of Biology and Pharmacy
University of Jena
Sandra Preuner Jena, Germany
Division of Molecular Microbiology Franca Rossi
and Development of Genetic Biotechnology Department Kanesan Panneer Selvam
Diagnostics Children’s Cancer University of Verona Department of Microbiology
Research Institute Verona, Italy Dr. G.R. Damodaran College of
and Science
LabDia Labordiagnostik Johannes E. Rothhardt Coimbatore, India
Vienna, Austria Jena Microbial Resource Collection
(JMRC) Rahul Sharma
María del Rocío Reyes-Montes Institute of Microbiology Plant Science Division
Facultad de Medicina University of Jena Agharkar Research Institute
Departamento de Microbiología y and Pune, India
Parasitología Department of Microbiology and
Universidad Nacional Autónoma de Molecular Biology
México M.R. Shivaprakash
Leibniz-Institute for Natural Product Department of Medical Microbiology
México City, Mexico Research and Infection Biology e.V. Postgraduate Institute of Medical
Hans-Knöll-Institute (HKI) Education and Research
Malcolm D. Richardson Neugasse, Jena, Germany
Regional Mycology Laboratory Chandigarh, India
Education and Research Centre
Wythenshawe Hospital Wendy W.J. van de Sande Coimbatore Subramanian Shobana
University Hospital of South Department of Medical Microbiology Department of Microbiology
Manchester and Infectious Diseases Dr. G.R. Damodaran College of
University of Manchester Erasmus Medical Center Science
Manchester, United Kingdom Rotterdam, the Netherlands Coimbatore, India

xxiv Contributors
Shoo Peng Siah Steve M. Taylor Juan Luis Rodríguez Tudela

Human Genetic Signatures Department of Epidemiology Centro Nacional de Microbiologia
North Ryde, New South Wales, Gillings School of Global Public Instituto de Salud Carlos III
Australia Health Majadahonda, Spain
University of North Carolina
José Júlio Costa Sidrim Chapel Hill, North Carolina
Specialized Medical Mycology Center and Milton A. Typas
Federal University of Ceará Division of Infectious Diseases and Faculty of Biology
Ceará, Brazil International Health Department of Genetics and
Duke University Medical Center Biotechnology
Tania C. Sorrell Durham, North Carolina University of Athens
Centre for Infectious Diseases and Athens, Greece
Microbiology Raquel Cordeiro Theodoro
Westmead Hospital Departamento de Microbiologia e
Westmead, New South Wales, Australia Imunologia Csaba Vágvölgyi
and Instituto de Biociências Faculty of Science and Informatics
Sydney Medical School Universidade Estadual Department of Microbiology
Western University of Sydney at Paulista-Botucatu University of Szeged
Westmead Hospital Sao Paulo, Brazil Szeged, Hungary
Sydney, New South Wales, Australia
K. Lily Therese
Takashi Sugita L&T Microbiology Research Centre Jaco J. Verweij
Department of Microbiology Vision Research Foundation, Sankara Department of Parasitology
Meiji Pharmaceutical University Nethralaya Leiden University Medical Center
Tokyo, Japan Chennai, India Leiden, the Netherlands
Conchita Toriello
Thomas D. Sullivan
Facultad de Medicina Paolo Visca
Department of Pediatrics
Departamento de Microbiología y Dipartimento di Biologia
School of Medicine and Public Health
University of Wisconsin Parasitología Università Roma Tre
Universidad Nacional Autónoma de and
Madison, Wisconsin
México Azienda Policlinico Umberto I
México City, Mexico Sapienza Università de Roma
Mami Tajima
and
Department of Dermatology Sandra Torriani Unità di Microbiologia Molecolare
Tokyo Medical University Biotechnology Department Istituto Nazionale per le Malattie
Tokyo, Japan University of Verona Infettive “Lazzaro Spallanzani”
Verona, Italy Rome, Italy
Masako Takashima
Japan Collection of Microorganisms, Anna Maria Tortorano
RIKEN BioResource Center, Laboratory of Medical Mycology Govinda S. Visvesvara
Saitama, Japan Department of Public Health – Department of Health and Human
Microbiology – Virology Services
Miklós Takó Università degli Studi di Milano Public Health Service
Faculty of Science and Informatics Milan, Italy Centers for Disease Control and
Department of Microbiology Prevention
University of Szeged Ryoji Tsuboi Atlanta, Georgia
Szeged, Hungary Department of Dermatology
Tokyo Medical University
Yi-Wei Tang Tokyo, Japan Maria Anna Viviani
Departments of Pathology and Laboratory of Medical Mycology
Medicine Hisae Tsubuku Department of Public Health –
Vanderbilt University School of Department of Dermatology Microbiology – Virology
Medicine Tokyo Medical University Università degli Studi di Milano
Nashville, Tennessee Tokyo, Japan Milan, Italy

Contributors xxv
Kerstin Voigt Nancy L. Wengenack Enshi Zhang

Jena Microbial Resource Collection Division of Clinical Microbiology Department of Dermatology
(JMRC) Mayo Clinic College of Medicine Tokyo Medical University
Institute of Microbiology Rochester, Minnesota Tokyo, Japan
University of Jena
and P. Lewis White Yanan Zhao
Department of Microbiology and Public Health Wales Public Health Research Institute
Molecular Biology Microbiology—Cardiff New Jersey Medical School of New
Leibniz-Institute for Natural Product University Hospital of Wales Jersey
Research and Infection Biology e.V. Cardiff, United Kingdom University of Medicine and Dentistry
Hans-Knöll-Institute (HKI) Newark, New Jersey
Neugasse, Jena, Germany Lihua Xiao
Department of Health and Human
Lysett Wagner Services
Institute of Microbiology Public Health Service
School of Biology and Pharmacy Centers for Disease Control and
University of Jena Prevention
Jena, Germany Atlanta, Georgia
Song Weining Kyoko Yarita

College of Agronomy Medical Mycology Research Center
Northwest A&F University Chiba University
Shaanxi, China Chuo-ku, Japan

1 Introductory Remarks
Dongyou Liu
Contents
1.1 Preamble............................................................................................................................................................................... 1
1.2 Classification, Biology, Genetics, and Clinical Presentation................................................................................................ 2
1.2.1 Classification............................................................................................................................................................. 2
1.2.2 Biology...................................................................................................................................................................... 4
1.2.3 Genetics.................................................................................................................................................................... 4
1.2.4 Clinical Presentation................................................................................................................................................ 5
1.3 Phenotypic Characterization................................................................................................................................................ 5
1.3.1 Sample Collection and Processing........................................................................................................................... 5
1.3.1.1 General Guidelines for Specimen Handling.............................................................................................. 5
1.3.1.2 Sputum, Bronchial Washings, and Throat Swabs...................................................................................... 6
1.3.1.3 Blood, Bone Marrow, and Body Fluids..................................................................................................... 6
1.3.1.4 Pus, Exudate, and Drainage....................................................................................................................... 6
1.3.1.5 Vaginal Swabs............................................................................................................................................ 6
1.3.1.6 Urine.......................................................................................................................................................... 6
1.3.1.7 Cerebrospinal Fluid................................................................................................................................... 6
1.3.1.8 Tissue Biopsies from Visceral Organs....................................................................................................... 6
1.3.1.9 Nail, Hair, Skin Scraping, and Swabs........................................................................................................ 6
1.3.2 Microscopic Examination......................................................................................................................................... 7
1.3.3 In Vitro Cultivation................................................................................................................................................... 7
1.3.4 Biochemical and Antifungal Testing...................................................................................................................... 15
1.4 Genotypic Characterization................................................................................................................................................ 15
1.4.1 Nucleic Acid Purification........................................................................................................................................ 15
1.4.2 Target Genes........................................................................................................................................................... 15
1.4.3 Template Amplification.......................................................................................................................................... 16
1.4.4 Product Detection................................................................................................................................................... 19
1.5 Result Interpretation, Standardization, Quality Control, and Assurance........................................................................... 20
1.5.1 Key Performance Characteristics........................................................................................................................... 20
1.5.2 Result Interpretation............................................................................................................................................... 20
1.5.3 Standardization and Validation.............................................................................................................................. 21
1.5.4 Quality Control and Assurance.............................................................................................................................. 21
1.5.4.1 Quality Control........................................................................................................................................ 21
1.5.4.2 Quality Assurance................................................................................................................................... 21
1.6 Conclusions......................................................................................................................................................................... 22
References.................................................................................................................................................................................... 22
1.1 Preamble (exons), possess membrane-bound cytoplasmic organelles

(e.g., mitochondria), sterol-containing membranes, and 80S
Fungi (singular fungus, meaning “mushroom” in Latin) are a ribosomes and produce a variety of soluble carbohydrates
diverse group of eukaryotic organisms (ranging from yeasts, and storage compounds, including sugar alcohols, disac-
molds, mushrooms, lichens, rusts, smuts to microsporidia) charides, and polysaccharides. Furthermore, Fungi resemble
that constitute one of the five kingdoms (i.e., Prokaryotae, Protista and Animalia by the lack of chloroplasts and thus
Fungi, Protista, Plantae, and Animalia) in the current classi- the requirement of preformed organic compounds as energy
fication system for living organisms. Similar to other eukary- sources. Although both Fungi and Plantae possess a cell wall
otic kingdoms (Protista, Plantae, and Animalia), fungi harbor and vacuoles, reproduce by sexual as well as asexual means,
membrane-bound nuclei with chromosomal DNA, which generate spores (as in ferns and mosses), and have haploid
consists of noncoding regions (introns) and coding regions nuclei (as in mosses and algae), Fungi differ from Plantae

2 Molecular Detection of Human Fungal Pathogens
by the presence of chitin (which also exists in the exoskel- Fungi consists of one subkingdom (Dikarya including phyla
eton of arthropods), instead of cellulose in the cell walls, and Ascomycota and Basidiomycota), seven phyla (all with the
the absence of chloroplasts. On the other hand, Fungi dif- suffix -mycota except Microsporidia; i.e., Ascomycota,
fer from Prokaryotae by having nuclear membrane, plasma Basidiomycota, Chytridiomycota, Glomeromycota, Blasto
membrane, and cell wall. cladiomycota, Neocallimastigomycota, and Microsporidia,
In this introductory chapter, a brief overview is presented in addition to Fungi incertae sedis, which encompasses fungi
on the classification, biology, and genetics of fungal organ- with indeterminate taxonomical status) (Table 1.1), 10 sub-
isms, and clinical manifestations in human hosts resulting phyla (with the suffix -mycotina), 35 classes (with the suffix
from their infections. This is followed by a summary of labo- -mycetes), 12 subclasses (with the suffix -mycetidae), and 129
ratory approaches that are useful for phenotypic character- orders (with the suffix -ales) [10–13]. A most notable feature
ization of fungi, including sample collection and processing, of this taxonomical scheme is the reorganization of the for-
microscopic examination, in vitro cultivation, biochemi- mer phylum Zygomycota into the phylum Glomeromycota
cal and anti-fungal testing. The subsequent section focuses and four separate subphyla (Mucoromycotina,
on key attributes relating to molecular characterization of Entomophthoromycotina, Zoopagomycotina, and Kick
fungi, such as nucleic acid extraction, target gene selection, xellomycotina), which may form independent phyla upon fur-
template amplification, and amplicon detection. Finally, the ther confirmation (Table 1.1). However, this scheme does not
importance of rational result interpretation, standardization, take into account of organisms such as oomycetes and slime
and quality control and assurance in the molecular fungal molds that were formerly included in the kingdom Fungi
testing is emphasized. [12]. Also, the genera Caulochytrium, Olpidium, Rozella
(formerly of Chytridiomycota), and Basidiobolus (formerly
of Entomophthorales, Zygomycota) are not included in any
1.2 C
lassification, Biology, Genetics, higher taxa in this scheme, pending further taxonomical
and Clinical Presentation resolutions [12]. In addition, a clade (i.e., Symbiomycota)
sharing similarity between Glomeromycota and Dikarya is
1.2.1 Classification
not included in the current scheme as Symbiomycota may
Fungi are an extremely diverse and abundant group of possibly constitute a rank between kingdom and subking-
eukaryotic organisms, whose sizes range from single-celled dom [12].
aquatic chytrids to large mushrooms and whose number has Most human pathogenic fungi are found in the phyla
been estimated to be between 700,000 and 1.5 million spe- Ascomycota, Basidiomycota, and Microsporidia as well
cies, with nearly 100,000 species being described to date as Fungi incertae sedis (principally Mucoromycotina
[1–8]. However, fewer than 500 of the recognized fungal spe- and Entomophthoromycotina of the former phylum
cies (including about 200 yeast species) have been shown to Zygomycota). From a medical mycologist’s perspec-
cause human infections. tive, human pathogenic fungi are conveniently separated
Based on morphological criteria, fungi are often divided into eight subgroups: (i) dermatophytes (represented by
into two major categories: filamentous fungi (true fungi) Epidermophyton, Microsporum, and Trichophyton); (ii)
and yeasts. Accounting for the bulk of fungal species, fila- yeasts (represented by Blastoschizomyces, Candida,
mentous fungi produce tubular, elongated, and thread-like Cryptococcus, Lacazia, Malassezia, Rhodotorula,
(filamentous) cellular structures (known as hyphae), which Saccharomyces, and Trichosporon); (iii) dimorphic fungi
contain multiple nuclei and extend at their tips. With about (represented by Blastomyces, Coccidioides, Histoplasma,
700 known species, yeasts are single-celled organisms that and Paracoccidioides); (iv) hyaline hyphomycetes (hyaline
reproduce by budding or binary fission. In addition, a few molds) (represented by Acremonium, Aspergillus, Beauveria,
fungal species are able to switch between a yeast phase Chrysosporium, Cylindrocarpon, Fusarium, Geotrichum,
and a hyphal phase in response to environmental condi- Gliocladium, Graphium, Madurella, Malbranchea,
tions and are referred to as dimorphic fungi. Despite their Onychocola, Paecilomyces, Penicillium, Scedosporium,
relatively insignificant proportion in relation to filamen- Scopulariopsis, Sepedonium, Trichoderma, Trichothecium,
tous fungi, about 200 of the 700 recognized yeast species and Verticillium); (v) dematiaceous hyphomycetes (dematia-
are responsible for a majority of clinical cases of human ceous molds) (represented by Acrophialophora, Alternaria,
mycoses. Aureobasidium, Bipolaris, Cladophialophora, Cladosporium,
Using a combination of morphological characteristics Curvularia, Drechslera, Exophiala, Exserohilum, Fonsecaea,
and mechanisms of reproduction, fungi have been tradi- Hortaea, Lecythophora, Ochroconis, Phaeoacremonium,
tionally separated into five phyla: Ascomycota (sac fungi), Phialophora, Ramichloridium, Rhinocladiella, Scedosporium,
Basidiomycota (club fungi), Mycophycophyta (lichens Sporothrix, Ulocladium, and Veronaea); (vi) coelomycetes
fungi), Zygomycota (conjugation fungi), and Deuteromycota (represented by Colletotrichum, Lasiodiplodia, Nattrassia, and
(imperfect fungi, or mitosporic fungi, which are fungi with Phoma); (vii) zygomycetes (represented by Apophysomyces,
no known sexual cycle) [9]. Basidiobolus, Conidiobolus, Cunninghamella, Mortierella,
Recent phylogenetic analyses of 18S rRNA, 28S rRNA, 5.8S Mucor, Absidia, Rhizomucor, Rhizopus, Saksenaea, and
rRNA, rpb1, rpb2, and tef1 genes indicate that the kingdom Syncephalestrum); and (viii) basidiomycetes [7].

Introductory Remarks 3
TABLE 1.1
Classification of the Kingdom Fungi
Subphylum (Former
Phylum (Subkingdom) Classification) Brief Description
Ascomycota (Dikarya) Pezizomycotina Ascomycota (commonly known as sac fungi) represents the largest phylum of Fungi, with over
64,000 species grouped under three subphyla (Taphrinomycotina, Saccharomycotina, and
Pezizomycotina). Ascomycota produce ascus (from Greek askos, meaning “sac” or “wineskin”), in
which nonmotile spores (a sexual structure also known as ascospores) are formed. However, some
Ascomycota (formerly belonging to Deuteromycota) are asexual, do not have a sexual cycle, and
thus do not form asci (or ascospores).
Forming part of Ascomycota, Pezizomycotina consist of Orbiliomycetes, Pezizomycetes,
Dothideomycetes, Arthoniomycetes, Eurotiomycetes, Laboulbeniomycetes, Lichinomycetes,
Lecanoromycetes, Leotiomycetes, and Sordariomycetes, as well as three unassigned orders
(Lahmiales, Medeolarials, and Triblidiales). Pezizomycotina cover all ascomycetes that produce
ascocarps (fruiting bodies), except for Neolecta in Taphrinomycotina.
Saccharomycotina Forming part of Ascomycota, Saccharomycotina consist of the “true” yeast class Saccharomycetes
Taphrinomycotina Forming part of Ascomycota, Taphrinomycotina consist of four classes: Neolectomycetes (hyphal
fungi), Pneumocystidomycetes (mammalian lung pathogen Pneumocystis), Schizosaccharomycetes
(fission yeasts), and Taphrinomycetes (hyphal fungi).
Basidiomycota Pucciniomycotina Basidiomycota (commonly known as club fungi) is the second largest phylum of Fungi, with 31,515
(Dikarya) (Urediniomycetes) species grouped under three subphyla (Pucciniomycotina, Ustilaginomycotina, and
Agaricomycotina, in addition to two separate classes Wallemiomycetes and Entorrhizomycetes).
Forming part of Basidiomycota, Pucciniomycotina consist of eight classes of rust fungi (i.e.,
Classiculomycetes, Cryptomycocolacomycetes, Mixiomycetes, Atractiellomycetes,
Agaricostilbomycetes, Cystobasidiomycetes, Pucciniomycetes, and Microbotryomycetes).
Ustilaginomycotina Forming part of Basidiomycota, Ustilaginomycotina consist of two smut fungus classes
(Ustilaginomycetes) Exobasidiomycetes and Ustilaginomycetes, as well as a separate smut fungus order Malasseziales.
Agaricomycotina Forming part of Basidiomycota, Agaricomycotina consist of three classes: Agaricomycetes (hymenia-
(Basidiomycetes) forming fungi), Dacrymycetes (hymenia-lacking fungi), and Tremellomycetes (jelly fungi).
Chytridiomycota Consisting of two classes Chytridiomycetes (with three orders: Chytridiales, Spizellomycetales, and
Rhizophydiales) and Monoblepharidomycetes (with one order Monoblephariales), Chytridiomycota
include more than 1000 known species. Chytridion (meaning “little pot”) describes the structure
containing unreleased spores. Chytrids are mostly primitive, aquatic, saprobic fungi involved in the
degradation of chitin and keratin, have coenocytic thalli, and usually form rhizoids (instead of true
mycelium).
Neocallimastigomycota (Chytridiomycota) Consisting of Neocallimastigales, a traditional member of Chytridiomycota, Neocallimastigomycota
include a small group of anaerobic fungi that inhabit the digestive system of larger herbivorous
mammals and possibly other terrestrial and aquatic environments. Although lacking mitochondria,
Neocallimastigomycota possess hydrogenosomes of mitochondrial origin. Similar to chrytrids,
neocallimastigomycetes form zoospores with posteriorly uniflagellate or polyflagellate. However,
neocallimastigomycetes are distinct from other chytrids on the basis of both morphology and
molecular phylogeny.
Blastocladiomycota (Chytridiomycota) Consisting of Blastocladiales, also a traditional member of Chytridiomycota, Blastocladiomycota are
saprotrophs, and also parasites of all eukaryotic groups. Blastocladiomycota undergo sporic
meiosis in contrast to chytrids, which mostly exhibit zygotic meiosis.
Glomeromycota (Zygomycota) Forming part (commonly known as “sugar” and “pin” molds) of former Zygomycota, and consisting
of one class Glomeromycetes (with four orders: Glomerales, Diversisporales, Paraglomerales, and
Archaeosporales) with about 200 described species (all of which reproduce asexually),
Glomeromycota produce arbuscular mycorrhizas with roots or thalli, and are obligate biotrophs,
dependent on symbiosis with land plants for carbon and energy.
Fungi incertae sedis Mucoromycotina Fungi that were placed in Zygomycota are now being reassigned to Glomeromycota, and Fungi
(Zygomycota) incertae sedis (including four subphyla Mucoromycotina, Entomophthoromycotina,
Zoopagomycotina, and Kickxellomycotina).
Consisting part of Fungi incertae sedis, Mucoromycotina cover three orders: Mucorales,
Endogonales, and Mortierellales.
Entomophthoromycotina Consisting part of Fungi incertae sedis, Entomophthoromycotina (with one order Entomophthorales)
(Zygomycota) are pathogens of insects, nematodes, and tardigrades, as well as free-living saprotrophs.
(continued)

TABLE 1.1 (continued)

Classification of the Kingdom Fungi
Subphylum (Former
Phylum (Subkingdom) Classification) Brief Description
Zoopagomycotina Consisting part of Fungi incertae sedis, Zoopagomycotina (with one order Zoopagales) are
(Zygomycota) pathogens of microscopic animals such as amoebae.
Kickxellomycotina Consisting part of Fungi incertae sedis, Kickxellomycotina include four orders: Asellariales,
(Zygomycota) Kickxellales, Dimargaritales, and Harpellales.
Microsporidia Microsporidia cover about 150 genera (containing >1200 species) that were previously considered
as protozoa, of which 12 species (representing 8 genera) have been shown to cause opportunistic
infections in humans. Microsporidia lack mitochondria and motile structures (e.g., flagella) and
produce highly resistant spores, the morphology (oval or pyriform, occasionally rod-shaped or
spherical) of which is often used for their differentiation.
Sources: James, T.Y. et al., Nature, 443, 818, 2006; Hibbett, D.S. et al., Mycol. Res., 111, 509, 2007.
1.2.2 Biology reproduce both asexually and sexually. During the budding
process, a small bud (or daughter cell) forms on the parent
Filamentous fungi are characterized by the production of cell, and the nucleus of the parent cell splits into a daughter
hyphae, which are cylindrical, thread-like structures of nucleus which migrates into the daughter cell. The growing
2–10 μm in diameter and up to several centimeters in length. bud eventually separates from the parent cell to become a
Hyphae can be either septate (with two or more compart- new cell.
ments separated by right-angled internal cell walls called Fungi are widespread in all environments and habitats,
septa) or aseptate (or coenocytic, with each compartment including soil, plants, insects, animals, humans, air, deserts,
containing one or more nuclei). Septa have pores that facili- and deep-sea sediments [14]. Most fungi are saprophytes that
tate passage and interchange of cytoplasm, organelles, and at play an essential environmental role in the decomposition and
times nuclei. Hyphae are important for penetration/invasion recycling of organic matters, and form symbiotic relationship
into the host cells and for the uptake of nutrients from liv- with plants and animals; some have the capacity to cause
ing hosts and other substrates. New hyphae typically emerge diseases in plants, animals, and humans. Furthermore, some
from hyphal tips (apices), arise from existing hyphae by a fungi have other properties that can be exploited for bread/
process called branching, or occasionally grow hyphal tips beverage making, insect pest control, medicine, and scien-
bifurcate (fork) giving rise to two parallel-growing hyphae. tific research. For instance, yeasts have been employed in (i)
The combined effects of apical growth and branching/fork- the two-hybrid screening systems for the general detection of
ing result in the formation of an interconnected network of protein–protein interactions; (ii) the yeast artificial chromo-
hyphae (with high surface area to volume ratios) known as somes (YACs) for cloning large fragments (200–800 kb) of
mycelium (plural mycelia), which is also commonly called DNA; and (iii) expression systems for heterologous proteins.
mold. Mycelia grown on solid agar media are referred to as
colonies, which may exhibit a variety of sizes, shapes, and
1.2.3 Genetics
colors (pigmentations) [9].
In general, fungi reproduce by means of microscopic Relative to other higher level eukaryotes (e.g., mam-
propagules called spores (conidia) as a result of an asex- mals), fungal genomes are simple and compact, with sizes
ual process. Near a third of all fungi reproduce by differ- ranging from 12,068 kb in Saccharomyces cerevisiae,
ent modes of propagation, showing two well-differentiated 22,540 kb in Trichophyton verrucosum HKI 0517 (GenBank
stages (i.e., the teleomorph or sexual stage and the anamorph ACYE00000000), 28,467 kb in Penicillium marneffei ATCC
or asexual stage) within the life cycle of a species. Achieved 18224 (GenBank ABAR00000000), 32,228 kb in Penicillium
via vegetative spores or mycelial fragmentation, asexual chrysogenum Wisconsin 54-1255 to 51,230 kb in Nectria
reproduction helps clonal populations to adapt to a specific haematococca (anamorph Fusarium solani) (GenBank
niche and allows more efficient dispersal than sexual repro- ACJF00000000).
duction. Sexual reproduction through meiosis involves vari- The 12 Mb genome of baker’s yeast Saccharomyces cere-
ous sexual structures (e.g., fruiting bodies) and reproductive visiae is clustered into 16 chromosomes (of 200–2200 kb
strategies. Compatible fungi may fuse their hyphae into an in size), with a total of 6183 open-reading frames (ORFs),
interconnected network in a process known as anastomosis, of which 5885 are predicated to be protein-coding genes.
which is required for the initiation of the sexual cycle. Its ribosomal RNA (rRNA) genes are coded by about 140
Yeasts commonly undergo asexual reproduction (mitosis) genes of a single tandem array on chromosome XII; small
by budding or fission, although some have the capacity to nuclear RNAs are coded by 40 genes; and transfer RNAs

(tRNAs) are coded by 275 genes. S. cerevisiae mitochon- to Acremonium, Madurella, and Pseudallescheria; subcuta-
drial DNA encodes components of the mitochondrial trans- neous zygomycosis due to Basidiobolus and Conidiobolus;
lational machinery and about 15% of the mitochondrial rhinosporidiosis due to Rhinosporidium; and lacaziosis (or
proteins [15]. lobomycosis) due to Lacazia loboi.
Whereas the 22 Mb genome of Penicillium marnef- Systemic mycoses: Some fungi, especially dimorphic
fei ATCC 18224 harbors 10,136 ORF; the 32 Mb genome fungi, have the capacity to breach the physical and immu-
of Penicillium chrysogenum Wisconsin 54-1255 contains nological defenses of the human host, causing pulmo-
13,911 ORF, with 12,791 being protein-coding genes [16]. nary and other infections after the inhalation of conidia.
As a member of the “Fusarium solani species complex” Examples of such systemic mycoses include histoplas-
that encompasses >50 species, Nectria haematococca mosis due to Histoplasma capsulatum; coccidioidomy-
MPVI (anamorph Fusarium solani) has been shown to cosis due to Coccidioides immitis; blastomycosis due to
possess a 51 Mb genome, which is organized in 17 chromo- Blastomyces dermatitidis; and paracoccidioidomycosis due
somes (of 530 kb–6.52 Mb in size) with 15,707 predicted to Paracoccidioides brasiliensis [7].
genes [17].
On the other hand, microsporidia possess extremely 1.3 Phenotypic Characterization
reduced eukaryotic genomes, which may be as small as
2.6 Mb with 2000 genes. These organisms have remnant Due to the fact that clinical presentations of human mycoses
mitochondria and show unique morphologies related to para- caused by various fungal species are nonspecific and indis-
sitism, including polar tube to penetrate host cells and initiate tinguishable, and that different fungal pathogens demonstrate
infection. varied resistance to commonly used antifungal drugs, there
is a need to identify the causative agents to genus and species
1.2.4 Clinical Presentation level in order to implement effective control and prevention
strategies.
Although most fungal species are saprophytic organisms Traditionally, laboratory identification and characteriza-
with a very low inherent virulence, some have the ability to tion of fungi rely mainly on morphological (e.g., the size and
take advantage of the weakened host defense (e.g., trauma shape of spores or fruiting structures), biochemical (e.g., the
and immunosuppression) and invade the host cells, caus- ability to metabolize certain biochemicals, or the reaction
ing a variety of clinical diseases, ranging from (i) super- to chemical tests), biological (e.g., the ability to mate), and
ficial, (ii) cutaneous, (iii) subcutaneous to (iv) systemic other phenotypic criteria. Apart from some mycotic/hyphal
mycoses [18]. elements, most fungi present in the clinical samples are
Superficial mycoses: As cosmetic fungal infections of the impossible to distinguish upon direct microscopic examina-
skin or hair shaft, superficial mycoses do not invade the liv- tion. Therefore, in vitro cultivation is vital for isolation of the
ing tissue nor elicit cellular response from the host. Patients fungal pathogens of interest, permitting subsequent deter-
with superficial mycoses seeking medical advices are largely mination on the basis of distinct colonial (macroscopic) and
for social or cosmetic reasons. Examples of such superficial microscopic features [19–21].
mycoses include pityriasis versicolor and seborrhoeic der-
matitis due to Malassezia furfur, tinea nigra due to Hortaea 1.3.1 Sample Collection and Processing
werneckii, white piedra due to Trichosporon species, and
black piedra due to Piedraia hortae. 1.3.1.1 General Guidelines for Specimen Handling
Cutaneous mycoses: Being another form of superficial Fungal pathogens are capable of spreading through spores
fungal infections of the skin, hair, or nails, cutaneous myco- and may pose danger to laboratory personnel if sufficient
ses do not invade the living tissue but may cause a variety caution is not heeded. Therefore, when dealing with fungal
of pathological changes in the host due to the presence of specimens in laboratory, it is essential to (i) wear a protec-
the infectious agent and its metabolic products. Examples of tive gown or laboratory coat while in the laboratory, (ii) wear
such cutaneous mycoses comprise dermatophytosis due to gloves when handling clinical and culture materials, (iii)
Epidermophyton, Microsporum, and Trichophyton; candidi- transport cultures in a rack or canister, (iv) disinfect speci-
asis (of skin, mucous membranes, and nails) due to Candida men containers contaminated on the outside by wiping with
species; and dermatomycosis due to non-dermatophyte molds gauze before opening, (v) open specimens in laminar flow
such as Onychocola, Scopulariopsis, and Scytalidium. safety cabinet, (vi) use mechanical pipetting devices for any
Subcutaneous mycoses: As chronic, localized infections material or reagent, and (vii) clean the work area with a 2%
of the skin and subcutaneous tissue following the traumatic amphyl solution when work is completed.
implantation of a soil saprophyte, subcutaneous mycoses A diverse range of clinical and environmental samples
may present a variety of clinical symptoms. These range can be utilized for fungal testing. In order to ensure accu-
from sporotrichosis due to Sporothrix; chromoblastomy- rate and consistent results, samples intended for mycological
cosis due to Cladosporium, Fonsecaea, and Phialophora; investigations need to be collected and processed in such a
phaeohyphomycosis due to Bipolaris, Cladosporium, way that offers the best chance for isolation and identification
Curvularia, Exophiala, and Exserohilum; eumycetoma due of causative fungal agents.

1.3.1.2 Sputum, Bronchial Washings, (i) Centrifuge the urine for 10–15 min at 2000 rpm. Decant
and Throat Swabs the supernatant and pool the sediment if necessary. (ii)
Collection: (i) Collect sputum (5–10 mL, as a result of a deep Prepare a direct smear of the sediment in KOH for direct
cough not saliva) in sterile container in the early morning. microscopy. PAS, Gram, or India ink preparations may also
Patients are advised not to eat before sputum collection. be helpful.
Thick sputum can be emulsified by the addition of l2–20
1.3.1.7 Cerebrospinal Fluid
sterile glass beads and 3–5 mL of sterile distilled water fol-
lowed by shaking. (ii) Collect bronchial washings (tracheal Collection: (i) Collect 2–5 mL CSF aseptically by clinician.
lavage or bronchial lavage) aseptically by physicians. (iii) (ii) Leave CSF at room temperature or incubate at 30°C if
Obtain throat specimens by rolling a moist sterile swab there is a delay in processing. (iii) Centrifuge CSF and pro-
over the affected area. For suspected Candida specimen, cess the sediment as follows. Processing: (i) Use 1 drop of
scrape the affected area with a sterile tongue depressor. (iv) the sediment to make an India ink mount. (ii) Resuspend the
Store samples at 4°C in case of short delays in processing. remaining sediment in 1–2 mL of CSF and inoculate onto
Processing: (i) Make wet mounts in KOH (l drop) and Gram Sabouraud’s dextrose agar with chloramphenicol and genta-
stained smears (l drop) for direct microscopy. Use periodic micin and incubate at 26°C and 35°C. (iii) Inoculate sediment
acid-Schiff (PAS) stain if KOH preparation is unsatisfac- onto BHIA supplemented with 5% sheep blood and incubate
tory. (ii) Inoculate sample onto Sabouraud’s dextrose agar at 35°C. Maintain cultures for at least 4 weeks.
with chloramphenicol and gentamicin and incubate dupli-
1.3.1.8 Tissue Biopsies from Visceral Organs
cate cultures at 26°C and 35°C. (iii) Inoculate sample onto
brain heart infusion agar (BHIA) supplemented with 5% Collection: (i) Collect tissue from the center and edge of
sheep blood and incubate at 35°C. Maintain cultures for 4 the lesion aseptically by clinician. Include normal tissue
weeks. for comparison. (ii) Keep a portion of the tissue in forma-
lin for rapid frozen sectioning with staining by hematoxy-
1.3.1.3 Blood, Bone Marrow, and Body Fluids lin and eosin (H&E), Grocott’s methenamine silver (GMS),
Collection: (i) Collect blood (8–10 mL aseptically using and PAS. (iii) Keep tissue samples moist with sterile water,
vacutainer tube [#4960, containing 1.7 mL of 0.35% sodium saline, or BHI broth. Do not refrigerate at 4°C for more than
polyanethol sulfonate (SPS) as an anticoagulant]). Clean the 8–10 h. Processing: (i) Tease apart tissue specimens asepti-
collection site with a disinfectant at the time of collection. cally in a sterile Petri dish. (ii) Perform a smear for direct
(ii) Collect bone marrow and body fluids (pleural, synovial, microscopic examination with staining by H&E, GMS,
and peritoneal) aseptically by physicians. Add SPS or hepa- and PAS (as well as Gram stain, Ziehl Neelsen stain, and
rin as an anticoagulant. Processing: (i) Prepare smears for modified Ziehl Neelsen stain if necessary) and inoculate
Giemsa, Gram, and PAS staining. (ii) Inoculate 0.5–1.0 mL directly onto the isolation media, if areas of pus and necro-
of buffy coat (after centrifugation of 5–8 mL of blood) onto sis are present. (iii) Mince tissue specimen with a sterile
the surface of culture media (Sabouraud’s dextrose agar scalpel blade, or grind in a sterile glass tissue grinder, if
with chloramphenicol and gentamicin, and BHIA supple- no areas of pus or necrosis are present, and inoculate the
mented with 5% sheep blood) with a loop, or 1 part blood to minced or homogenized material onto the isolation media.
10–20 parts brain/heart infusion broth. (iii) Inoculate bone (iv) Inoculate onto Sabouraud’s dextrose agar with chloram-
marrow and body fluids (pleural, synovial, and peritoneal) phenicol and gentamicin and incubate duplicate cultures at
on culture media. Maintain cultures at 26°C and 35°C for 4 26°C and 35°C; (v) Inoculate onto BHIA supplemented with
weeks. 5% sheep blood and incubate at 35°C. Maintain cultures for
4 weeks.
1.3.1.4 Pus, Exudate, and Drainage
Collection: (i) Aspirate material from undrained abscesses 1.3.1.9 Nail, Hair, Skin Scraping, and Swabs
using a sterile needle and syringe. (ii) Express pus using a Collection: For nail, (i) clean nail with 70% alcohol. (ii)
sterile, sharp-pointed scalpel. (iii) Place the material in a Scrape outer dorsal plate surface and discard. (iii) Scrape the
sterile container. deeper portion and remove a portion of debris from under
the nail with a scalpel. (iv) Collect whole nail or nail clip-
1.3.1.5 Vaginal Swabs pings. (v) Place all material in a clean envelope labeled with
Collection: (i) Collect material from the vagina using several the patient’s data. For hair, (i) Select infected areas and with
sterile swabs. (ii) Insert swabs into a sterile tube. Processing: forceps, epilate at least 10 hairs. (ii) Use a scalpel or a blade
Smear swab onto heat-sterilized glass slide for Gram stain. knife for hairs broken off at the scalp level. (iii) Place hairs
between two clean glass slides or in a clean envelope labeled
1.3.1.6 Urine with the patient’s data. For skin scraping and swabs, (i) wipe
Collection: (i) Collect an early morning, mid-stream catch lesions and interspaces between the toes with alcohol sponge
of >2.0 mL (do not process more than 50.0 mL) in a sterile or sterile water. (ii) Scrape the entire lesion and both sides of
container when aspiration or cystoscopy cannot be done. (ii) interspaces with a sterile scalpel and place scrapings between
Store urine at 4°C for up to 12–14 h if necessary. Processing: two clean glass slides or place in a clean envelope labeled

with the patient’s data. (iii) Swab the lesion with a sterile the adhesive glue holding the flag to the applicator stick, (iv)
swab (wetted in distilled water if necessary) and place the placing the flag onto a small drop of lactophenol cotton blue
swab into a clean tube. Processing: (i) For nail, hair, and skin on a clean glass slide, removing the applicator stick and dis-
scrapings, make a wet mount preparation in KOH for direct carding, and (v) adding another drop of stain, covering with
microscopy. Calcofluor-stained mount may also be useful. a coverslip, gently pressing and moping up any excess stain
(ii) For skin swabs, smear swab onto heat-sterilized glass before microscopy [7].
slide for Gram stain. (iii) Inoculate skin scraping and swab For macroscopic examination of colonial morphology,
specimens onto Sabouraud’s dextrose agar slopes contain- attention should be paid to (i) surface texture (e.g., glabrous,
ing chloramphenicol and gentamicin, but NO cycloheximide suede-like, powdery, granular, fluffy, downy, cottony, and
and incubate at 35°C. (iv) For suspected secondary bacterial velvety); (ii) surface topography (e.g., flat, raised, heaped,
infection, inoculate the swab onto a blood agar plate, fol- folded, domed, radial, and grooved); (iii) surface pigmen-
lowed by the Sabouraud’s agar containing the antibiotics and tation (e.g., white, cream, yellow, brown, green, grey, and
then place into brain heart infusion broth. Incubate at 35°C. black); (iv) reverse pigmentation (e.g., none, yellow, brown,
Maintain the cultures for 4 weeks. red, and dark); (v) growth rate (e.g., fast, moderate, and
slow); and (vi) growth temperature (e.g., 25°C, 37°C, 40°C,
and 45°C). For microscopic assessment of cultured isolates,
1.3.2 Microscopic Examination
the morphologic characteristics of conidia are recognized in
In addition to macroscopic assessment of colonial size, shape, terms of septation, shape (e.g., spherical, pyriform, clavate,
and color, direct microscopic observation of mycotic ele- and ellipsoidal), size, color (hyaline and darkly pigmented),
ments in clinical specimens and subsequent examination of surface texture (e.g., smooth, rough, verrucose, and echinu-
fungal structures of cultured isolates are critical for correct late), type (microconidia and macroconidia), and arrange-
identification of causal fungal agents and accurate diagnosis ment (e.g., single, in mass) [7].
of mycoses. Direct microscopy not only facilitates the selec-
tion of the proper portion of clinical specimen, the appropri-
1.3.3 In Vitro Cultivation
ate media, and inoculation techniques for enhanced recovery
of the fungus, but also alerts the physician as to the likely eti- In vitro cultivation remains a critical step for the phenotypic
ology of the disease. In general, all fungal specimens of suf- characterization of fungi. Macroscopic examination of colo-
ficient quantity are examined microscopically and inoculated nial size, shape, and color followed by microscopic inves-
on culture media. However, when the quantity of a fungal tigation of final structures of fungal isolates allows proper
sample is insufficient, culture should take priority over direct determination of fungal organisms implicated in human
microscopy due to its higher sensitivity. mycoses in most cases. Further characterization of fungal
A number of stains (e.g., KOH and its derivatives, lacto- isolates is possible by using various biological and biochemi-
phenol cotton blue, India ink, and Southgate’s mucicarmine cal techniques as well as antifungal drug resistance testing.
stain) can be applied for improved identification and recogni- The composition, preparation, and application of common
tion of mycotic elements in clinical samples and structural mycological media are summarized in Table 1.3.
details of fungal isolates (Table 1.2) [22,23]. Ocular lens Slide culture preparation allows observation of the precise
containing a micrometer disc may be employed on light arrangement of the conidiophores and conidial ontogeny (the
microscope for the measurement of hyphae, conidia, and way the conidia are produced). This is conducted by (i) using
other fungal structures. For detection of fungal elements a sterile blade to cut out an agar block (7 × 7 mm) from a plate
in tissue biopsies, PAS stain, GMS stain, Fontana-Masson of cornmeal agar or Czapek dox agar that is small enough
stain, Gridley’s stain, and H&E may be utilized. In particu- to fit under a coverslip, (ii) flipping the block up onto the
lar, GMS stain represents an essential stain for detection of surface of the agar plate, (iii) inoculating the four sides of the
fungal elements in tissue sections. Fontana-Masson stain is agar block with spores or mycelial fragments of the fungus,
indispensable for detection of melanin in the cell walls of (iv) placing a flamed coverslip centrally upon the agar block,
dematiaceous fungi. Besides standard light or phase-contrast (v) incubating the plate at 26°C until growth and sporula-
microscopy, transmission electronic microscopy (TEM) tion occur, (vi) removing the cover slip from the agar block,
enables visualization of fine structural details (e.g., the outer (vii) applying a drop of 95% alcohol as a wetting agent, (viii)
wall layers of conidia and ascospores) of fungal organisms, gently lowering the coverslip onto a small drop of lactophe-
leading to more accurate speciation. nol cotton blue on a clean glass slide, (ix) leaving the slide
Cellotape flag preparation may be utilized for rapid overnight to dry and resealing later with fingernail polish,
mounting and keeping intact of the reproductive structures and (x) applying a coat of clear polish followed by one coat of
of sporulating fungi. This is performed by (i) using clear red-colored polish before microscopy [7].
2 cm wide cellotape and a wooden applicator stick (orange Apart from in vitro culture, in vivo animal models (e.g.,
stick) to make a small cellotape flag (2 × 2 cm), (ii) using ster- rodents and rabbits) have been occasionally applied to com-
ile technique to gently press the sticky side of the flag onto pare diagnostic procedures for the estimation of fungal bur-
the surface of the culture, (iii) applying a drop of 95% alco- dens in blood, bronchoalveolar lavage (BAL), and tissue
hol to the flag to act as a wetting agent and also to dissolve samples.

8
TABLE 1.2
Composition, Preparation, and Application of Common Mycological Stains
Stain Composition Preparation Application
15% KOH KOH 15 g 1. Place a portion of specimen onto a clean glass microscope slide. For direct microscopic examination of
Glycerol 20 mL 2. Add a drop of 15% KOH to specimen and mix. skin scrapings, hairs, nails, and other
Distilled water 80 mL 3. Pace a cover glass over the preparation. clinical specimens for fungal
4. Leave slide at room temperature until the material is cleared. The slide may be elements.
warmed for speedy clearing.
5. Observe slide under microscopy.
6. Slides that appear negative for fungi may be reexamined the following day.
10% KOH with Solution A 1. Mix one drop of solution A and a drop of solution B on the center of a clean For direct microscopic examination of
Calcofluor white KOH 10 g microscope slide. skin scrapings, hairs, nails, and other
Glycerin 10 mL 2. Place specimen in the solution and cover with a coverslip, squash the preparation clinical specimens for fungal
Distilled water 90 mL with the butt of the inoculation needle and then blot off the excess fluid. elements.
Solution B 3. Gently heat the slide and examine microscopically for the presence of fungal Calcofluor white (or Uvitex 2B,
Calcofluor white (fluorescent brightener 28, F6259, elements that fluoresce a chalk-white or brilliant apple green color, depending on Blankophor) binds to cellulose and
Sigma) 0.1 g the filter combination. chitin and fluoresces blue-white or
Distilled water 100 mL apple-green when exposed to
ultraviolet light.
KOH with Parker Ink KOH 10 g 1. Remove a portion of specimen with an inoculation needle and mount in a drop of For direct microscopic examination of
Molecular Detection of Human Fungal Pathogens

Glycerol 10 mL KOH on a clean microscope slide. skin scrapings, hairs, nails, and other
Parker Quink permanent blue ink 10 mL 2. Cover with a coverslip, squash the preparation with the butt of the inoculation clinical specimens for fungal elements.
Distilled water 80 mL needle, and then blot off the excess fluid. Keep negative specimens and
(dissolve the KOH in water; add glycerol and Parker 3. Gently heat by passing through a flame two or three times. reexamine the next day to avoid
ink. The glycerol prevents crystallization of the 4. When the specimen is cleared (from 20 min for skin scrapings to several hours for reporting false-negative. Preparations
reagent and prevents the specimen from drying out). nail scrapings), examine microscopically for faintly blue-stained fungal elements. may be kept until culture result is
known.
KOH-DMSO Dimethyl sulfoxide (DMSO) 40 mL 1. Remove a small portion of the specimen with an inoculation needle and mount in For direct microscopic examination of
preparation Distilled water 60 mL KOH 10 g a drop of KOH-DMSO on a clean microscope slide. skin scrapings, hairs, and nails for
2. Cover with a coverslip, squash the preparation with the butt of the inoculation fungal elements.
needle, and then blot off the excess fluid. Do not heat the preparation. DMSO gives more rapid maceration
3. Examine the mount within 20 min microscopically for unstained refractile fungal and clearing, but preparations do not
elements. keep long.
Lactophenol cotton Cotton blue (Aniline blue) 0.05 g 1. Dissolve the cotton blue in the distilled water overnight; For direct microscopic examination of
blue Phenol crystals (C6H5O4) 20 g 2. Add phenol crystals to the lactic acid in a glass beaker, mix to dissolve, and then skin scrapings, hairs, nails, and other
Glycerol 40 mL add glycerol; clinical specimens for fungal
Lactic acid (CH3CHOH COOH) 20 mL 3. Filter the cotton blue and distilled water solution into the phenol/glycerol/lactic elements.
Distilled water 20 mL acid solution, mix, and store at room temperature.
Introductory Remarks
India Ink India Ink (colloidal carbon) Place a drop of India Ink on the specimen, mix well with a sterilized loop, and cover For direct microscopic examination of
with a coverslip. Cryptococcus neoformans and other
encapsulated fungi in a cell
suspension (e.g., CSF sediment).
Southgate’s Carmine 1 g 1. Take sections to water. For detection of capsular material in
Mucicarmine stain Aluminium hydroxide 1 g 2. Stain nuclei with hematoxylin. Cryptococcus neoformans.
50% alcohol 100 mL (mix above by shaking) 3. Differentiate in acid-alcohol and blue in tap water. Mucicarmine stains acidic mucins
Aluminium chloride (anhydrous) 0.5 g (boil in water 4. Stain with Southgate’s mucicarmine solution for 30 min and rinse in distilled pink.
bath for 2–3 min, cool, make up to original volume water.
with 50% alcohol and filter. The stain is stable for a 5. Dehydrate, clear, and mount.
few months).
Periodic acid-Schiff 1% periodic acid (50%) 1. Take sections to water. For demonstration of glycogen and
(PAS) and PAS Periodic acid 2 mL 2. For PAS digest only, digest glycogen with saliva for 15 min, wash in water. neutral mucins, and for detection of
digest stain Distilled water 98 mL 3. Treat with 1% periodic acid for 5 min, rinse in water. fungal elements in tissue sections. As
Schiff’s reagent 4. Treat with Schiff’s reagent for 10 min, wash in running tap water for 5 min (to a counter stain, PAS reveals the
Basic fuchsin (C.I. 42500) 1 g help develop the color). background host cellular detail, tissue
Potassium metabisulfite 2 g 5. Counterstain nuclei lightly with Mayer’s hematoxylin for 1 min, wash, and “blue architecture, and inflammatory
Distilled water 200 mL up” in Li2Co3. response.
HCl concentrate 2 mL 6. Dehydrate, clear, and mount. PAS-positive material appears
Deactivated charcoal 1–2 g (add basic fuchsin slowly magenta; nuclei appear blue; PAS
to boiling distilled water, mix, and cool to 50°C. digest material appears colorless.
Add potassium metabisulfite, mix, and cool to room
temperature before adding HCl. Keep in the dark
overnight. Add charcoal and filter through coarse
filter paper, then fine filter paper. Store in fridge).
Gomori’s 5% aqueous chromic acid 1. Take sections to water. An essential stain for detection of
methenamine silver 1% aqueous sodium bisulfite 5% aqueous borax 2. Oxidize in 5% chromic acid for 1 h and wash in running tap water for 10 min. fungal elements in tissue sections. As
stain (GMS) [or 0.1% aqueous gold chloride 3. Treat with sodium bisulfite for 1 min to remove any residual chromic acid, and a counter stain, GMS removes the
Grocott-Gomori 2% aqueous sodium thiosulfate wash in tap water then distilled water. fine details of background host cells
silver stain] Stock methenamine silver solution 4. Place section in the working silver solution at 60°C in a water bath and rinse in and tissues, but provides a more
(add 5 mL of 5% silver nitrate to 100 mL of 3% distilled water. sensitive stain for detecting small
hexamine, mix, and keep at 4°C for 2 months). 5. Tone in 0.1% gold chloride for 5 min and rinse in distilled water. fragments of cell wall.
Working silver solution (filter before use). 6. Treat with 2% sodium thiosulfate for 1–2 min to remove unreduced silver and Fungi stain black; cell walls stain
(stock methenamine silver solution 25 mL, distilled wash thoroughly. brown to black; background stains
water 25 mL, 5% borax 1–2 mL) 7. Counterstain with light green. pale green.
8. Dehydrate, clear, and mount.
Fontana-Masson 10% silver nitrate 1. Deparaffinize and hydrate to distilled water. For staining cell walls of dematiaceous
stain 0.1% gold chloride 2. Add 10% silver nitrate, place in 60°C oven for 1 h, and rinse in distilled water. fungi and Cryptococcus neoformans
5% hypo Nuclear-Fast Red 3. Add 0.1% gold chloride, leave for 10 min, and rinse in distilled water. in tissue sections.
4. Add 5% hypo, leave for 5 min, wash in tap water, and rinse in distilled water. Melanin, argentaffin cells stain black;
5. Add nuclear-Fast Red, leave for 5 min, and wash in tap water. nuclei stain red; background stains
6. Dehydrate, clear, and coverslip. pale pink.
(continued)
9
10
Composition, Preparation, and Application of Common Mycological Stains
Stain Composition Preparation Application
Gridley’s stain Chromic acid 1. Bring sections to water via xylene and ethanol. For enhanced visualization of fungi
Metabisulfite bleach 2. Place in chromic acid for 1 h, wash with tap water. and their morphology in tissue
Schiff’s reagent Aldehyde fuchsin 3. Treat with the metabisulfite bleach for 1 min, wash with tap water, and rinse with sections.
Metanil yellow distilled water. Cell walls stain purple to magenta;
4. Place in Schiff’s reagent for 20 min, wash with tap water, and rinse with 70% yeasts stain rose to purple; capsules
ethanol. stain deep purple; background stain
5. Place in aldehyde fuchsin 30 min, rinse off excess with 95% ethanol, and wash yellow.
with tap water.
6. Counterstain with metanil yellow for 1 min, and rinse with distilled water.
7. Dehydrate, clear and mount in a resinous medium.
Hematoxylin and Alum hematoxylin 1. Bring sections to distilled water. For visualization of host response to
eosin 0.3% acid alcohol 2. Stain nuclei with alum hematoxylin, rinse in running tap water. fungus in tissue sections.

Scott’s tap water substitute 3. Differentiate with 0.3% acid alcohol, rinse in running tap water, then in Scott’s tap Nuclei stain blue, cartilage; calcium
Eosin/phloxine water substitute, and again in tap water. deposits stain dark blue; cytoplasm
4. Stain with eosin/phloxine for 2 min. and other components stain shades of
5. Dehydrate, clear, and mount. red; erythrocytes stain bright red.
Sources: McGinnis, M.R., Laboratory Handbook of Medical Mycology, Academic Press, New York, 1980; Schwarz, J., Human Pathol., 13, 519, 1982; Koneman, E.W. and Roberts, G.D., Practical Laboratory
Mycology, The Williams and Wilkins Co., Baltimore, MD, 1985.
TABLE 1.3
Composition, Preparation, and Application of Common Mycological Media
Medium Composition Preparation Application
Potato dextrose agar Potato infusion 200 g 1. Mix ingredients in 1000 mL water. For routine cultivation and identification of fungi.
(PDA) Dextrose 20 g 2. Heat with frequent stirring, bring to boil for 1 min.
Bacto agar 15 g Distilled water 1000 mL 3. Dispense for slopes as required.
(potato infusion, dextrose, and Bacto agar may be 4. Autoclave at 121°C for 15 min; slope or pour for plates as
replaced with 39 g PDA, Oxoid CM139). required.
Cornmeal agar (CA) CA (BD) 8.5 g Distilled water 500 mL 1. Mix agar in 500 mL water. For routine cultivation and identification of fungi.
2. Heat with frequent stirring, bring to boil for 1 min.
3. Dispense for slopes as required.
4. Autoclave at 121°C for 10 min; slope or pour for plates as
required.
Oatmeal agar (OA) Oatmeal (Difco) 60 g 1. Mix oatmeal and agar in 1000 mL water. For routine cultivation and identification of fungi.
Agar 12.5 g 2. Heat with frequent stirring, bring to boil for 1 min.
Distilled water 1000 mL (oatmeal and agar may be 3. Dispense for slopes as required.
replaced with 72.5 g OA, Difco 255210). 4. Autoclave at 121°C for 15 min; slope or pour for plates as
required.
Malt extract agar Malt extract 20 g 1. Mix malt extract, peptone and dextrose in 1000 mL water (adjust For routine cultivation and identification of fungi.
(MEA) Peptone 1 g to pH 6.5 with NaOH if necessary). MEA is a useful alternative to bread and BHI broth for
Dextrose 20 g 2. Heat with frequent stirring, bring to boil for 1 min. recovery of zygomycetes.
Bacto agar 15 g 3. Dispense for slopes as required.
Distilled water 1000 mL 4. Autoclave at 121°C for 10 min, slope or pour for plates as
(malt extract, peptone and dextrose may be replaced required.
with 20 g malt extract, Oxoid L39)
Inhibitory mould Pancreatic digest of casein 3.0 g IMA is an enriched medium with inorganic salts, chloramphenicol, For isolation of fungi and inhibition of bacteria.
agar (IMA) Sodium phosphate 2.0 g and gentamicin and can be obtained commercially.
Peptic digest of animal tissue 2.0 g
Magnesium sulfate 0.8 g
Yeast extract 5.0 g
Ferrous sulfate 0.04 g
Dextrose 5.0 g
Sodium chloride 0.04 g
Starch 2.0 g
Manganese sulfate 0.16 g
Dextrin 1.0 g
Agar 15.0 g
Chloramphenicol 0.125 g
Distilled water to 1000 mL
(continued)
11
12
1% Peptone agar Bacto peptone (BD) 5 g 1. Mix agar and peptone in 500 mL water. For cultivation and differentiation of fungi.
(PA) Bacto agar (BD) 10 g 2. Heat with frequent stirring, bring to boil for 1 min.
Distilled water 500 mL 3. Dispense for slopes as required.
required.
Sabouraud’s dextrose SDA (Oxoid CM41) 65 g 1. Mix SDA and chloramphenicol in 1000 mL water. For primary isolation and cultivation of yeasts and
agar (SDA) with Chloramphenicol 1× 250 mg capsule 2. Heat with frequent stirring, bring to boil for 1 min. moulds.
chloramphenicol Distilled water 1000 mL 3. Add gentamicin, mix, dispense for slopes as required.
and gentamicin Gentamicin (40 mg/mL) 0.65 mL 4. Autoclave at 121°C for 10 min; slope, or pour for plates as
required.
SDA with SDA (Oxoid CM41) 65 g 1. Mix the first four ingredients in 1000 mL water. For primary isolation and cultivation of dermatophytes.
cycloheximide, Cycloheximide (actidione) 0.5 g 2. Heat with frequent stirring, bring to boil for 1 min. Mycosel (BBL) and mycobiotic (Difco) agars
chloramphenicol, Chloramphenicol 1× 250 mg capsule 3. Autoclave at 121°C for 10 min and cool to 50°C. (containing SDA, 1% glucose, chloramphenicol, and
gentamicin, and Yeast extract 5 g 4. Add gentamicin, dispense for slopes, or plates as required. cycloheximide) are commercially available.
yeast extract. Distilled water 1000 mL
Gentamicin (40 mg/mL) 0.65 mL
SDA with 5% NaCl SDA (Oxoid CM41) 32.5 g 1. Mix agar and NaCl in 500 mL water. For the cultivation and differentiation of dermatophytes
NaCl 25 g 2. Heat with frequent stirring, bring to boil for 1 min. especially T. rubrum from T. mentagrophytes.
Distilled water 500 mL 3. Dispense for slopes as required.
required.

Dermatophyte test Papaic digest of soybean meal 10.0 g 1. Mix ingredients to 1000 mL water. For recovery of dermatophytes from heavily
medium (DTM) Dextrose 10.0 g 2. Heat with frequent stirring, bring to boil for 1 min. contaminated clinical specimens and for presumptive
Phenol red 0.2 g 3. Autoclave at 121°C for 15 min. indication of the presence of a dermatophyte.
Cycloheximide 0.5 g Dermatophytes and a few other fungi/bacteria turn
Agar 20.0 g medium from pink to red.
Distilled water 1000 mL
Trichophyton agar Trichophyton agar nos. 1–7 (BD) 11.8 g 1. Mix agar in water. For differentiation of Trichophyton species.
nos. 1–7 Distilled water 200 mL 2. Heat with frequent stirring, bring to boil for 1 min. (i) Trichophyton mentagrophytes grows well on agar
3. Dispense for slopes, autoclave at 118°C for 10 min, and slope. nos. 1,2,3, and 4
(ii) Trichophyton tonsurans grows well on agar nos. 3
and 4; poorly on agar nos. 1 and 2. (iii)
Trichophyton verrucosum grows well on agar nos.
2 and 3; poorly on agar nos. 1 and 4.
Lactrimel agar (LA) Dutch Jug skimmed milk powder 7 g 1. Mix milk powder with 150 mL water, add other ingredients, and For production of pigment by Trichophyton rubrum.
Glycine 10 g Honey 10 g 850 mL water.
CA (BD) 17 g 2. Heat with frequent stirring, bring to boil for 1 min.
Chloramphenicol 1× 250 mg capsule 3. Dispense for slopes as required.
Distilled water 1000 mL 4. Autoclave at 115°C for 10 min, slope, or pour plates as required.
Hair perforation test Autoclaved pre-pubital hair (blonde if available) cut 1. Place autoclaved hair in a vial containing 5 mL sterile water. For differentiation of dermatophytes.
(HPT) into short pieces (1 cm) 2. Inoculate with small fragments of the test fungus and incubate at Trichophyton mentagrophytes and its variants produce
Sterile distilled water 5 mL in vial room temperature. marked localized areas of pitting and marked erosion
3. Remove individual hairs at intervals up to 4 weeks and examine whereas T. rubrum does not.
microscopically in lactophenol cotton blue.
Urease agar (UA) Bacto agar (BD) 1.5 g 1. Autoclave agar in 100 mL bottle at 115°C for 20 min and cool to For differentiation of dermatophytes.
with 0.5% glucose Distilled water 91 mL 50°C.
Sterile urea solution (Oxoid SR20) 5 mL 2. Add aseptically 5 mL of sterile urea solution and 4 mL of 10%
10% sterile glucose solution 4 mL sterile glucose solution.
3. Dispense for slopes.
Rice grain slopes Rice 1/2 teaspoon Distilled water 8 mL 1. Add 1/2 teaspoon rice grains into wide neck 20 mL vials For induction of sporulation and for differentiation of
(RGS) containing 8 mL distilled water to each vial. M. audouinii, M. canis, and M. distortum.
2. Close lid, autoclave at 121°C for 15 min, and slope.
Littman oxgall agar LOA (BD) 27.5 g 1. Mix agar in 500 mL water. For routine inoculation of specimens from skin, nails,
(LOA) Distilled water 500 mL 2. Heat with frequent stirring, bring to boil for 1 min. and hair, etc.
required.
CGB (l-canavanine, Solution A Solution A For differentiation between Cryptococcus neoformans
glycine, 2 Glycine 10 g 1. Dissolve ingredients in beaker and adjust to pH 5.6. var. neoformans and Cryptococcus neoformans var.
bromthymol blue) KH2PO4 1 g 2. Filter sterilize solution using 0.22 μm filter; store in refrigerator. gattii.
agar MgSO4 1 g Solution B
Thiamine HCl 1 mg 1. Dissolve the bromthymol blue in 64 mL of 0.01 N NaOH.
l-canavanine sulfate 30 mg 2. Add 36 mL water.
Distilled water 100 mL CGB agar
Solution B 1. Mix 20 g agar and 20 mL solution B, autoclave to 121°C for
Bromthymol blue 0.4 g 15 min, and cool to 48°C.
0.01 N NaOH 64 mL 2. Add 100 mL of the filtered solution A, mix, and dispense in
Distilled water 36 mL plates.
CGB agar
Bacto agar 20 g
Solution B 20 mL
Solution A 100 mL
Brain-heart infusion BHIA (Oxoid) 52 g 1. Mix BHI agar and chloramphenicol in 1000 mL water. For recovery of Cryptococcus neoformans from sterile
agar (BHIA) with Chloramphenicol 1× 250 mg capsule 2. Heat with frequent stirring, bring to boil for 1 min. specimens such as CSF, and for yeast-mould
5% sheep blood Distilled water 1000 mL 3. Autoclave at 121°C for 15 min, cool to 50°C. conversions of Sporothrix and Paracoccidioides.
Sheep blood 50 mL 4. Add sheep blood and gentamicin, dispense for slopes, or plates as BHIA is an alternative to chocolate agar for isolation
Gentamicin (40 mg/mL) 0.65 mL required. of fungi and bacteria, and used routinely by some
ophthalmologists for corneal scrapings. Ready-to-use
BHIA is commercially available.
Brain heart infusion BHI agar (Oxoid) 52 g Aseptic addition of sterile penicillin to BHI broth inhibits bacteria. BHI broth with penicillin is useful for isolation of
(BHI) broth Distilled water 1000 mL zygomycetes.
Penicillin G (20 U/mL) 1 mL
(continued)
13
14
Dixon’s agar (DA) Malt extract (Oxoid L39) 18 g 1. Mix ingredients in 500 mL water. For primary isolation and cultivation of Malassezia
Peptone (BDH 44075) 18 g 2. Heat with frequent stirring, bring to boil for 1 min. furfur.
Bacto agar (BD) 7.25 g 3. Dispense for slopes.
Ox-bile desiccated (Oxoid L50) 10 g 4. Autoclave at 121°C for 10 min and then slope.
Tween 40 5 mL
Glycerol monooleate 2.5 mL
Czapek dox agar CDA (Oxoid CM97) 45.4 g 1. Mix agar in 1000 mL water. For routine cultivation of fungi, especially Aspergillus,
(CDA) Distilled water 1000 mL 2. Heat with frequent stirring, bring to boil for 1 min. Penicillium, and non-sporulating moulds.
4. Autoclave at 121°C for 10 min, slope or pour for plates as
required.
Bird seed agar Niger seed (Guizotia abyssinica) 50 g 1. Grind Guizotia abyssinica seeds finely and add to 1000 mL For selective isolation of Cryptococcus neoformans.
Glucose 1 g distilled water.
KH2PO4 1 g 2. Boil for 30 min, pass through filter paper and adjust volume to
Creatinine 1 g 1000 mL.
Bacto agar (BD) 15 g 3. Add the remaining ingredients except Bacto agar to filtrate and
Distilled water 1000 mL dissolve.
Penicillin G (20 U/mL) 0.5 mL per 500 mL 4. Cool to room temperature, adjust to pH 5.5, and dispense into
Gentamicin (40 mg/mL) 0.5 mL per 500 mL 500 mL bottles.

5. Add 7.5 g Bacto agar to each 500 mL reagent bottle and autoclave
at 110°C for 20 min.
6. Cool to 48°C, add 0.5 mL Penicillin G and 0.5 mL gentamicin to
each 500 mL, mix, and pour into 90 mm plastic Petri dishes.
Cornmeal glucose CA (BD) 17 g 1. Mix dry ingredients into 1000 mL water. For sporulation of some zygomycetes, such as
sucrose yeast Dextrose (glucose) 2 g 2. Heat with frequent stirring, bring to boil for 1 min. Saksenaea and Apophysomyces.
extract agar Sucrose 3 g Yeast extract (Difco) 1 g 3. Dispense for slopes.
Distilled water 1000 mL 4. Autoclave at 120°C for 10 min and slope.
Sterile bread Bread Glass Petri dish 1. Sterilize a piece of bread in a humidified glass Petri dish. Sterile bread without preservatives is superior to other
2. Inoculate specimens from non-contaminated sites directly; treat media for recovery of zygomycetes from clinical
contaminated specimens with antibacterial agents before specimens.
inoculation.
Sources: McGinnis, M.R., Laboratory Handbook of Medical Mycology, Academic Press, New York, 1980; Schwarz, J., Human Pathol., 13, 519, 1982; Koneman, E.W. and Roberts, G.D., Practical Laboratory
Mycology, The Williams and Wilkins Co., Baltimore, MD, 1985.
1.3.4 Biochemical and Antifungal Testing Grinding lyophilized or fresh mycelia in liquid nitrogen
with a mortar and pestle represents a common way to dis-
Many fungi demonstrate varied tolerance to temperature, rupt the fungal cell walls. Because of its time-consuming
which can be exploited as a complementary tool in the dif- and laborious nature and its potential for cross-contam-
ferentiation of dematiaceus fungi. Examination of primary ination between samples, this technique is not suitable for
metabolites such as ubiquinones (coenzyme Q) has proven dealing with a large number of samples. Another means to
useful for the taxonomy of black yeasts and filamentous mechanically break the fungal cell walls is through the use
fungi. Secondary metabolites (e.g., steroids, terpenes, alka- of glass beads with a vortex mixer. In addition, sonicator may
loids, cyclopeptides, and coumarins) produced by fungal be employed for disruption of fungal cell walls. Alternative
organisms may be examined by thin-layer chromatography. methods to disrupt the fungal cell walls include enzymatic
The resulting pattern of secondary-metabolite production digestion (using a combination of lyticase, zymolase, chitin-
provides a reliable approach for identification and classifica- ase, gluculase, and proteinase K), acid, and alkali treatments.
tion of lichens. Using pyrolysis gas chromatography, pyroly- Subsequent treatment with organic solvents (e.g., phenol/
sis mass spectrometry, gas chromatography, and partition chloroform) and detergents (e.g., sodium dodecyl sulfate,
aqueous polymer two-phase systems, the cellular fatty acid SDS; hexadecyltrimethylammonium bromide, CTAB; and
composition of fungi can be determined, which represents N-lauroylsarcosine) denatures cytosolic proteins and lipid
another useful means for differentiating filamentous fungi. membranes and inactivates endogenous DNase/RNAse,
In addition, the structure and composition of the cell wall facilitating their removal. Subsequent precipitation using
may be targeted for the definition of fungi. For example, chi- ethanol or isopropanol results in the isolation of high-purity
tin and glucan are present in ascomycetes and basidiomyce- nucleic acids.
tes whereas chitosan and polyglucuronic acid are found in The development of various easy-to-use commercial
zygomycetes. The presence or absence of polysaccharides kits has eliminated the use of hazardous organic solvents
(e.g., fucose, galactose, rhamnose, and xylose) in the walls in the isolation of DNA/RNA from fungi. For the prepa-
of yeast cells allows their differentia on. Further, isoenzyme ration of fungal DNA, Qiagen DNeasy Plant Kit (Qiagen),
patterns generated by electrophoretic techniques (zymo- UltraClean™ Microbial DNA kit (Mo Bio Laboratories),
grams) enable the determination of generic relationships of DNAzol® (Invitrogen), and Whatman FTA cards (Whatman)
fungi [9]. Various serological tests have been also described are highly efficient [39]. Furthermore, automated DNA
for specific detection of fungal antigens in clinical speci- extraction systems have become increasingly sophisticated
mens. Matrix-assisted laser desorption/ionization time-of- and affordable, contributing to the reduction of potential
flight intact cell mass spectrometry (MALDI-TOF ICMS) cross-contamination during manual handling.
has been used to identify fungal organisms, including (i) the
terverticillate penicillia, (ii) aflatoxigenic, black, and other
aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting 1.4.2 Target Genes
fungi (e.g., Serpula lacrymans), and (v) dermatophytes [24]. For accurate and efficient identification of fungal organisms,
Moreover, assessment of the sensitivity of fungal isolates to a number of gene regions have been proven valuable. The
various antifungal drugs offers an additional way to their dis- most versatile and widely used target is rRNA gene [40–42].
crimination as well as treatment [25–27]. This is followed by rpb1, rpb2, tef1a, and atp6 genes. Other
genes of interest include those encoding β-tubulin, actin, chi-
tin synthase, acetyl coenzyme A synthase, glyceraldehyde-3-
1.4 Genotypic Characterization
phosphate dehydrogenase, isoepoxydon dehydrogenase (idh),
Phenotypic characterization of fungal organisms on the basis lignin peroxidase, and orotidine 5′-monophosphate decar-
of morphological, biological, and biochemical features suf- boxylase genes [43,44].
fers from the drawbacks of laborious, time-consuming, and Ribosome is an essential cellular organelle that is involved
variable, especially for poorly differentiated filamentous in protein synthesis in all living organisms. Being the key
fungi. For improved taxonomic resolution and epidemiologi- component of the ribosome, rRNA molecules consists of two
cal study of fungal organisms, molecular techniques detect- complex folded subunits of differing sizes (small and large),
ing the nucleic acids have been increasingly utilized [28–38]. whose main functions are to provide a mechanism for decod-
ing messenger RNA (mRNA) into amino acids (at the center
of small ribosomal subunit) and to interact with tRNA during
1.4.1 Nucleic Acid Purification
translation by providing petidytransferase activity (large sub-
Due to the presence of a tough cell wall in fungi, it is often nec- unit [LSU]). The two rRNA subunits in eukaryotes includ-
essary to undertake several steps to purify the nucleic acids ing fungi have sedimentation coefficiency values of 40S
before molecular testing becomes feasible. These include (i) (Svedberg units) and 60S. The small rRNA subunit (40S) in
disruption of cell walls, (ii) denaturation of nucleoprotein eukaryotes contains a single RNA species (i.e., 18S rRNA),
complexes, (iii) inactivation of endogenous DNase/RNAse, whereas the large rRNA subunit (60S) in eukaryotes com-
and (iv) removal of contaminating proteins, polysaccharides, prises three RNA species (5S, 5.8S, and 25S–28S rRNA).
polyphenolic pigments, and other compounds. On the other hand, the two rRNA subunits in prokaryotes

18S RNA ITS1 5.8S ITS2 25S–28S RNA IGS1 5S IGS2

RNA RNA

Figure 1.1 Organization of fungal ribosomal RNA (rRNA) genes. Considerable variations exist in the small and large subunits of rRNA
genes among different fungal groups. Notably, the small subunit (SSU) of rRNA gene in filamentous fungi and yeasts is 18S in size, while
that in microsporidia is 16S. Similarly, the large subunit (LSU) of rRNA gene in filamentous fungi is 28S in size, that in yeasts is 25S and
that in microsporidia is 23S. ITS, internal transcribed spacer; IGS, intergenic spacer.
measure 30S and 50S, respectively. While the small rRNA the end of 18S RNA) and ITS4 (located at the beginning of
subunit (30S) in prokaryotes contains a single RNA species 28S RNA). The resulting product is sequenced with prim-
(i.e., 16S rRNA), the large rRNA subunit (50S) in prokaryotes ers ITS1, ITS2, ITS3, and ITS4 (see Table 1.4) [43,51]. The
contains two RNA species (5S and 23S rRNA). Interestingly, identity of the testing fungus is defined by its ITS sequence
despite their recent redesignation as a phylum in the king- similarity (%) to the type strain or control isolate: species,
dom Fungi, microsporidia possess rRNA subunits (30S and ≥99%; genus, 93%–99%; and inconclusive, ≤93%.
50S) and RNA species (16S and 23S) that are characteris- Similar to ITS1 and ITS2 regions, the rRNA IGS regions
tic of prokaryotes, with a notable absence of 5.8S rRNA in (of 2–5 kb in length, depending on fungal taxa) also experi-
microsporidia. ence more genetic drift and consequently are not as highly
The fungal rRNA genes exist as a family of multiple-copy conserved. This makes the rRNA IGS regions another poten-
genes that are arranged in a head-to-toe manner, with each tial target for fungal identification. The rRNA IGS regions
copy (of 8–12 kb in size) consisting of 18S RNA (small sub- can be amplified with primers LR12R (located at the end
unit [SSU]), ITS 1 (internal transcribed spacer 1), 5.8S RNA, of 28S RNA) and invSR1R (located at the beginning of
ITS 2, 25S–28S RNA (LSU), IGS 1 (intergenic spacer), 5S, 18S RNA); the resulting product is sequenced with prim-
and IGS 2 (Figure 1.1). The tandemly repeated copies of ers LR12R, 5SRNA, 5SRNAR, and invSR1R for all basid-
rRNA genes have been homogenized by concerted evolution iomycetes and some ascomycetous yeasts, and with primers
and contain highly similar nucleotide sequence. Therefore, LR12R and invSR1R plus other internal primers for filamen-
they are almost always treated as a single-locus gene. Along tous ascomycetes (as 5S RNA is nonexistent in filamentous
with ITS, the 18S, 5.8S, and 25S–28S rRNAs are transcribed ascomycetes) (see Table 1.4) [43,52].
as a 35S–40S precursor, with all spacers being later spliced
out of the transcript. A nontranscribed or IGS region exists
1.4.3 Template Amplification
between the copies of the 18S, 5.8S, and 25S–28S rRNA
repeats, serving to separate the repeats from one another on Prior to the advent of polymerase chain reaction (PCR) in the
the chromosome. A 5S RNA gene takes a position within mid-1980s, molecular procedures for fungal identification
the IGS region and is transcribed in the opposite direction. were insensitive and cumbersome. With the development of
In filamentous ascomycetes, the 5S RNA gene is absent PCR and other nucleic acid amplification technologies (such
[45–49]. as ligase chain reaction [LCR], nucleic acid sequence-based
Much of the 25–28S RNA (LSU) gene is conserved across amplification [NASBA], transcription-mediated amplifica-
widely divergent taxa, and only the first 600–900 bases con- tion [TMA], strand displacement amplification [SDA], roll-
tain three divergent domains (D1, D2, and D3) that are use- ing circle amplification [RCA], cycling probe technology
ful for phylogenetic study of fungal organisms. Typically, [CPT], branched DNA [bDNA], and loop-mediated isother-
the first 900 bases of LSU are amplified with primers 5.8SR mal amplification [LAMP]), it has become possible to rapidly
(located in the 5.8S RNA) and LR7 (located in the 28S RNA), and specifically detect a single copy of nucleic acid in a mat-
and the resulting amplicon is sequenced with primers LR5, ter of hours [38].
LR16, LR0R, and LR3R (see Table 1.4) [43]. In yeasts, the Due to their efficiency, simplicity, robustness, and ver-
D1 and D2 variable regions of 25S rRNA regions are often satility, PCR and its derivatives have been widely adopted
targeted [50]. in both research and clinical laboratories for identification
The 18S rRNA (SSU) gene also includes alternating and determination of fungi and other microbial organisms.
regions of sequence conservation and heterogeneity. The con- From the original version using a pair of primers and gel-
served regions are often targeted for phylogenetic analysis of based detection, improvements have been made in the forms
higher taxonomic orders (e.g., phylum, family, and genus), of nested PCR, multiplex PCR, reverse transcription-PCR
while the regions of sequence diversity are valuable for char- (RT-PCR), real-time PCR, quantitative PCR (Q-PCR), and
acterization of isolates to the genus or species level (with iso- arbitrarily primer PCR (or random amplified polymorphic
lates showing sequence identity >97% in the 18S rRNA gene DNA, RAPD), etc. [38,53]. These developments have not
being considered as the identical species). only enhanced the assay sensitivity (nested PCR) and ver-
Compared to the rRNA genes, the rRNA ITS1 and ITS2 satility (multiplex PCR detecting multiple genes and/or
regions are not as essential, and thus tend to be more variable, organisms and RT-PCR detecting RNA instead of DNA),
offering extremely valuable targets for fungal speciation and but also enabled the accurate quantitation of fungal organ-
identification. Frequently, the ITS1 and ITS2 regions together isms (Q-PCR) and elimination of manual handling post-
with 5.8S RNA are amplified with primers ITS1 (located at amplification (real-time PCR).

TABLE 1.4
Identity and Sequence of Common rRNA, ITS, and IGS Primers for PCR Amplification and Sequencing
Analysis of Fungal Organisms
Nucleotide Positions in M. grisea
Gene Region Primer Sequence (5′–3′) Orientation (Nucleotide Positions in S. cereviseae)
18S rRNA NS1 GTA GTC ATA TGC TTG TCT C Forward 413–422
18S rRNA NS1R GAG ACA AGC ATA TGA CTA C Reverse 413–422
18S rRNA NS2 GGC TGC TGG CAC CAG ACT TGC Reverse 943–963
18S rRNA NS3 GCAAGTCTGGTGCCAGCAGCC Forward 943–963
18S rRNA NS4 CTT CCG TCA ATT CCT TTA AG Reverse 1525–1544
18S rRNA NS5 AAC TTA AAG GAA TTG ACG GAA G Forward 1523–1544
18S rRNA NS6 GCA TCA CAG ACC TGT TAT TGC CTC Reverse 1806–1829
18S rRNA NS7 GAG GCA ATA ACA GGT CTG TGA TGC Forward 1806–1829
18S rRNA NS8 TCC GCA GGT TCA CCT ACG GA Reverse 2162–2181
18S rRNA NS17 CAT GTC TAA GTT TAA GCA A Forward 447–465
18S rRNA NS18 CTC ATT CCA ATT ACA AGA CC Reverse 887–906
18S rRNA NS19 CCG GAG AAG GAG CCT GAG AAA C Forward 771–792
18S rRNA NS20 CGT CCC TAT TAA TCA TTA CG Reverse 1243–1262
18S rRNA NS21 GAA TAA TAG AAT AGG ACG Forward 1193–1210
18S rRNA NS22 AAT TAA GCA GAC AAA TCA CT Reverse 1687–1706
18S rRNA NS23 GAC TCA ACA CGG GGA AAC TC Forward 1579–1598
18S rRNA NS24 AAA CCT TGT TAC GAC TTT TA Reverse 2143–2162
18S rRNA SR1R TAC CTG GTT GAT TCT GC Forward 394–410 (1–21)
18S rRNA SR1 ATT ACC GCG GCT GCT Reverse (578–564)
18S rRNA SR2 CGG CCA TGC ACC ACC Reverse (1277–1263)
18S rRNA SR3 GAA AGT TGA TAG GGC T Reverse 696–711 (318–302)
18S rRNA SR4 AAA CCA ACA AAA TAG AA Reverse (838–820)
18S rRNA SR5 GTG CCC TTC CGT CAA TT Reverse (1146–1130)
18S rRNA SR6 TGT TAC GAC TTT TAC TT Reverse (1760–1744)
18S rRNA SR6R AAG WAA AAG TCG TAA CAA GG Forward (1744–1763); similar to ITS 1
18S rRNA SR7 GTT CAA CTA CGA GCT TTT TAA Reverse (617–637)
18S rRNA SR7R AGT TAA AAA GCT CGT AGT TG Forward (637–617)
18S rRNA SR8R GAA CCA GGA CTT TTA CCT T Forward (732–749)
18S rRNA SR9R QAG AGG TGA AAT TCT Forward (896–910)
18S rRNA SR10R TTTG ACT CAA CAC GGG Forward (1181–1196)
18S rRNA SR11R GGA GCC TGA GAA ACG GCT AC Forward 779–798
18S rRNA SSU1Fd CTG CCA GTA GTC ATA TGC TTG TCT C Forward 407–431
18S rRNA SSU1Rd CTT TGA GAC AAG CAT ATG AC Reverse 416–435
18S rRNA SSU2Fd GAA CAA YTR GAG GGC AAG Forward 930–947
18S rRNA SSU2Rd TAT ACG CTW YTG GAG CTG Reverse 974–991
18S rRNA SSU3Fd ATC AGA TAC CGT YGT AGT C Forward 1389–1407
18S rRNA SSU3Rd TAY GGT TRA GAC TAC RAC GG Reverse 1397–1416
18S rRNA SSU4Fd CCG TTC TTA GTT GGT GG Forward 1670–1686
18S rRNA SSU4Rd CAG ACA AAT CAC TCC ACC Reverse 1682–1699
18S rRNA SSU5Fd TAC TAC CGA TYG AAT GGC Forward 2037–2054
18S rRNA SSU5Rd CGG AGA CCT TGT TAC GAC Reverse 2148–2165
18S rRNA SSU6Fm GCT TGT CTC AAA GAT TAA GCC ATG CAT GTC Forward 423–452
18S rRNA SSU6Rm GCA GGT TAA GGT CTC GTT CGT TAT CGC Reverse 1707–1733
18S rRNA SSU7Fm GAG TGT TCA AAG CAG GCC TNT GCT CG Forward 1153–1178
18S rRNA SSU7Rm CAA TGC TCK ATC CCC AGC ACG AC Reverse 1921–1943
18S rRNA SSU8Fm GCA CGC GCG CTA CAC TGA C Forward 1848–1866
18S rRNA V9G TTA CGT CCC TGC CCT TTG TA Forward 2002–2021
ITS ITS1 TCC GTA GGT GAA CCT GCG G Forward 2162–2180
ITS ITS1F CTT GGT CAT TTA GAG GAA GTA A Forward 2124–2145; similar to ITS 1
ITS ITS1Fd CGA TTG AAT GGC TCA GTG AGG C Forward 2043–2064
ITS ITS1Rd GAT ATG CTT AAG TTC AGC GGG Reverse 2671–2691
(continued)


ITS ITS2 GCT GCG TTC TTC ATC GAT GC Reverse
ITS ITS3 GCA TCG ATG AAG AAC GCA GC Forward
ITS ITS4 TCC TCC GCT TAT TGA TAT GC Reverse 2685–2704
ITS ITS4B CAG GAG ACT TGT ACA CGG TCC AG Reverse
ITS ITS4S CCT CCG CTT ATT GAT ATG CTT AAG Reverse 2680–2703
ITS ITS5 GGA AGT AAA AGT CGT AAC AAG G Forward 2138–2159; similar to ITS 1
ITS ITS5R CCT TGT TAC GAC TTT TAC TTC C Reverse
5.8S rRNA 5.8S CGC TGC GTT CTT CAT CG Forward (51–35)
5.8S rRNA 5.8SR TCG ATG AAG AAC GCA GCG Reverse (34–51)
5.8S rRNA 5.8S1Fd CTC TTG GTT CBV GCA TCG Forward 2333–2350
5.8S rRNA 5.8S1Rd WAA TGA CGC TCG RAC AGG CAT G Reverse 2451–2472
28S rRNA F377 AGA TGA AAA GAA CTT TGA AAA GAG AA Forward 3005–3030
28S rRNA LR0R GTA CCC GCT GAA CTT AAG C Forward 2668–2686
28S rRNA LR1 GGT TGG TTT CTT TTC CT Reverse (73–57); similar to ITS 4
28S rRNA LR2 TTT TCA AAG TTC TTT TC Reverse 3009–3025
28S rRNA LR2R AAG AAC TTT GAA AAG AG Forward 3012–3028
28S rRNA LR3 GGT CCG TGT TTC AAG AC Reverse 3275–3291
28S rRNA LR3R GTC TTG AAA CAC GGA CC Forward 3275–3291
28S rRNA LR5 TCC TGA GGG AAA CTT CG Reverse 3579–3595
28S rRNA LR5R GAA GTT TCC CTC AGG AT Forward 3580–3596
28S rRNA LR6 CGC CAG TTC TGC TTA CC Reverse 3756–3772
28S rRNA LR7 TAC TAC CAC CAA GAT CT Reverse 4062–4078
28S rRNA LR8 CAC CTT GGA GAC CTG CT Reverse 4473–4489
28S rRNA LR8R AGC AGG TCT CCA AGG TG Forward 4473–4489
28S rRNA LR9 AGA GCA CTG GGC AGA AA Reverse 4799–4815
28S rRNA LR10 AGT CAA GCT CAA CAG GG Reverse 5015–5031
28S rRNA LR10R GAC CCT GTT GAG CTT GA Forward 5013–5029
28S rRNA LR11 GCC AGT TAT CCC TGT GGT AA Reverse 5412–5431
28S rRNA LR12 GAC TTA GAG GCG TTC AG Reverse 5715–5731
28S rRNA LR12R CTG AAC GCC TCT AAG TCA GAA Forward 5715–5735
28S rRNA LR13 CAT CGG AAC AAC AAT GC Reverse 5935–5951
28S rRNA LR14 AGC CAA ACT CCC CAC CTG Reverse 5206–5223
28S rRNA LR15 TAA ATT ACA ACT CGG AC Reverse 2780–2796
28S rRNA LR16 TTC CAC CCA AAC ACT CG Reverse 3311–3327
28S rRNA LR17R TAA CCT ATT CTC AAA CTT Forward 3664–3681
28S rRNA LR20R GTG AGA CAG GTT AGT TTT ACC CT Forward 5570–5592
28S rRNA LR21 ACT TCA AGC GTT TCC CTT T Reverse 3054–3072
28S rRNA LR22 CCT CAC GGT ACT TGT TCG CT Reverse 2982–3001
28S rRNA LSU1Fd GRA TCA GGT AGG RAT ACC CG Forward 2655–2674
28S rRNA LSU1Rd CTG TTG CCG CTT CAC TCG C Reverse 2736–2754
28S rRNA LSU2Fd GAA ACA CGG ACC RAG GAG TC Forward 3280–3299
28S rRNA LSU2Rd ATC CGA RAA CWT CAG GAT CGG TCG Reverse 3379–3402
28S rRNA LSU3Fd GTT CAT CYA GAC AGC MGG ACG Forward 3843–3863
28S rRNA LSU3Rd CAC ACT CCT TAG CGG ATT CCG AC Reverse 3876–3898
28S rRNA LSU4Fd CCG CAG CAG GTC TCC AAG G Forward 4469–4487
28S rRNA LSU4Rd CGG ATC TRT TTT GCC GAC TTC CC Reverse 4523–4545
28S rRNA LSU5Fd AGT GGG AGC TTC GGC GC Forward 3357–3373
28S rRNA LSU5Rd GGA CTA AAG GAT CGA TAG GCC ACA C Reverse 5355–5379
28S rRNA LSU6Fd CCG AAG CAG AAT TCG GTA AGC G Forward 5499–5520
28S rRNA LSU6Rd TCT AAA CCC AGC TCA CGT TCC C Reverse 5543–5564
28S rRNA LSU7Fd GTT ACG ATC TRC TGA GGG TAA GCC Forward 5943–5966
28S rRNA LSU7Rd GCA GAT CGT AAC AAC AAG GCT ACT CTA C Reverse 5927–5954


28S rRNA LSU8Fd CCA GAG GAA ACT CTG GTG GAG GC Forward 3469–3491
28S rRNA LSU8Rd GTC AGA TTC CCC TTG TCC GTA CC Reverse 4720–4742
28S rRNA LSU9Fm GGT AGC CAA ATG CCT CGT CAT C Forward 4882–4903
28S rRNA LSU9Rm GAT TYT GCS AAG CCC GTT CCC Reverse 4979–4999
28S rRNA LSU10Fm GGG AAC GTG AGC TGG GTT TAG A Forward 5543–5564
28S rRNA LSU10Rm CGC TTA CCG AAT TCT GCT TCG G Reverse 5499–5520
28S rRNA LSU11Fm TTT GGT AAG CAG AAC TGG CGA TGC Forward 3753–3776
28S rRNA LSU12Fd GTG TGG CCT ATC GAT CCT TTA GTC C Forward 5355–5379
IGS LR12R GAA CGC CTC TAA GTC AGA ATC C Forward
IGS 5SRNA ATC AGA CGG GAT GCG GT Reverse
IGS 5SRNAR ACQ GCA TCC CGT CTG AT Forward
IGS invSR1R ACT GGC AGA ATC AAC CAG GTA Reverse
Sources: White, T.J. et al., Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in Innis, M.A. et al. (eds.), PCR
Protocols: A Guide to Methods and Applications, Academic Press, New York, 1990, pp. 315–322; Bruns, T.D. et al., Ann. Rev. Ecol. Syst.,
22, 525, 1991; Bruns, T.D. et al., Mol. Phylog. Evol., 1, 231, 1992; Gardes, M. and Bruns, T.D., Mol. Ecol., 2, 113, 1993; Vilgalys, R. et al.,
Mycol. Helvet., 6, 73, 1994; Gargas, A. and DePriest, P.T., Mycologia, 88, 745, 1996; Crous, P.W. et al., Stud. Mycol., 64, S17, 2009.
Notes: Primer names with a “d” ending denote degenerate primers, whereas those with an “m” ending denote specific primers. The nucleotide posi-
tions of the primers refer to the rRNA gene sequence of Magnaporthe grisea (GenBank accession AB026819) or that of Saccharomyces cere-
viseae in the 5′–3′ direction [43,61].
1.4.4 Product Detection (either in the form of oligonucleotides or amplified DNA

fragments with specificity for unique portions of the 18S
In its standard form, the products generated by PCR are sepa- rRNA gene) are affixed to a solid surface (e.g., glass, plastic,
rated by agarose gel electrophoresis with or without modifica- or silicon chip) as probes, forming an array for simultaneous
tion (e.g., enzymatic digestion), stained with a DNA-binding identification of fungal organisms. Universal 18S rRNA gene
dye (e.g., ethidium bromide or gel star), and visualized under primers (one of which contains a fluorescent label) are used in
UV light. For PCR products <100 bp or for distinction of PCR to amplify all the 18S rRNA genes present in a sample.
products with minor size differences, polyacrylamide gel The resulting PCR products are added to the array and will
electrophoresis and its derivatives (e.g., single-strand con- only bind to the probes for which they have a complementary
formational polymorphism [SSCP] analysis, denaturing gra- sequence. Pathogens are identified by the pattern of fluoresc-
dient gel electrophoresis [DGGE], and temperature DGGE ing spots in the array. Line probe assay (LiPA) is another
[TGGE]) may be used. nucleic acid hybridization test that is modified from DNA
To improve the sensitivity of PCR product detection, microarray. Instead of a glass, silicon, plastic chip, LiPA is
enzymatic signal amplification (e.g., ELISA and flow cytom- conducted on a nitrocellulose strip, on which specific oligo-
etry) can be applied. In a common version of PCR-ELISA, nucleotide probes are attached at known positions as parallel
streptavidin-coated microtiter plate is incubated with a bio- lines and are hybridized with biotin-labeled PCR products.
tinylated capture probe (or oligonucleotide) with specificity Recent advances in instrument automation and fluores-
for a fungal gene. Aliquots of a PCR products generated cent dye chemistry permit instant monitoring of PCR ampli-
with digoxigenin-labeled primers (derived from the same cons (so-called real-time PCR) without additional manual
gene) are denatured in NaOH and subsequently hybridized handling. In one form of real-time PCR, a double-stranded
to the capture probe. Specific hybridization products are then DNA intercalating dye (e.g., SYBR Green) is used. SYBR
visualized by a colorimetric detection system based on an Green increases its emission spectrum by 50- to 100-fold
anti-digoxigenin horseradish peroxidase conjugate in the when binding to double-stranded DNA. As the double-
presence of a chromogenic substrate solution. After stopping stranded DNA is synthesized during PCR, an increase in flu-
the enzyme reaction with H2SO4, the absorbance is measured orescence correlates to an increasing concentration of PCR
in an ELISA reader with a 450 nm filter. products, which can be determined real time with reference
PCR amplicons can be also detected by DNA microar- to a standard sample. Discrimination of amplicons gener-
ray (also known as DNA chip, gene or genome chip, or gene ated by multiplex PCR from different genes is also possible
array). Typically, a collection of microscopic DNA spots if these gene products have sufficiently different Tm values.

A melting curve analysis is performed post-PCR, using the time period; and (ii) reproducibility—the variation arising
SYBR Green as a fluorescent marker. As the melting point is using the same measurement process among different instru-
reached, the DNA denatures and the fluorescence decreases ments and operators from one run to another (i.e., inter-assay
sharply. The plotting of fluorescence versus temperature in a precision) or over longer time periods. Linearity refers to the
graph assists calculation of the melting temperature for each tendency of measurements by a quantitative assay to form
product. Other forms of real-time PCR employ specifically a straight line when plotted on a graph. Data from linearity
designed probes that target a region of amplicon and incor- experiments may be subjected to linear regression analysis
porate a fluorescent dye. Examples of these probes include with an ideal regression coefficient of 1. In case of a nonlin-
hydrolysis dual-labeled probes (TaqMan®), hybridization ear curve, other objective, statistically valid methods may be
probes (LightCycler), molecular beacons, peptide nucleic utilized.
acid (PNA) probes, TaqMan minor groove-binding (MGB™)
probes, locked nucleic acid (LNA®) primers and probes, and
1.5.2 Result Interpretation
scorpions™ [38].
DNA sequencing analysis provides a most accurate way A positive result by a molecular assay for a given patho-
to ascertain the identity of PCR amplicons generated from gen normally confirms the etiologic relationship if the clini-
fungi and other organisms. The classical chain termination cal syndrome is compatible with the pathogen identified.
sequencing method (or Sanger method) utilizes primers or Considering the sensitive nature of the amplified methods
dideoxynucleotides that are labeled with radioactive isotope such as PCR, it is important to rule out the possibility of a
or fluorescent tag, and the sequencing products are detected false-positive result. Occasionally, false-positive results may
by exposure to x-rays or UV light. More recently, pyrose- originate from the low diagnostic specificity of the assay, in
quencing, Roche 454, and Illumina Solexa platforms have which primers bind to irrelevant sequences and occasion-
been adopted for high-throughput sequencing analysis of ally a homologous sequence that is shared among related
PCR products. The nucleotide sequences of the PCR ampli- or unknown bacteria. More often, false-positive results in
con are then compared with those stored at reference data- the molecular testing come from contamination, which may
bases such as GenBank, and the phylogenetic relationships of arise during manual handling of the samples in the testing
related fungi are displayed in the form of trees, constructed laboratory either at the pre- or post-extraction (while set-
with distance matrix methods (resulting in phenograms) and ting up the PCR) stages. This risk is heightened when a
maximum-parsimony methods (resulting in cladograms) [54]. high copy-number polynucleotide (or plasmid) is used as a
quantification standard and distributed around the labora-
tory, contaminating reaction source. Additionally, contami-
1.5 Result Interpretation,
nation may be attributable to samples that are referred from
Standardization, Quality other laboratories, which do not utilize manipulation tech-
Control, and Assurance niques that are mandatory for the molecular testing. These
may include the use of unplugged pipette tips, infrequent
1.5.1 Key Performance Characteristics
changing of gloves, and using pipette for long periods with-
The performance of a diagnostic assay is often evaluated out decontamination. Another cause of contamination is by
by using several key parameters, including detection limit, amplification products from previous tests. Contamination
sensitivity, specificity, accuracy, intra-assay precision, inter- may also occur by leakage from tubes or microtiter plates
assay precision, and linearity (as in the case of a quantitative with lids not tightly closed or by breakage of glass capillar-
assay). Detection limit (or limit of detection) is the lowest ies leading to spillage of the amplification mixture. Besides
concentration or quantity of bacteria that can be detected by a the adoption of stringent laboratory practice, the risk of con-
given assay. Sensitivity is the percentage of samples contain- tamination with PCR products may be reduced by replac-
ing bacteria of interest that are identified by the assay as posi- ing nucleotide dTTP with dUTP in PCR and implementing
tive for the bacteria. Specificity is the percentage of samples a digestion step with uracil-DNA-glycosylase (UNG) to
without bacteria of interest that are identified by the assay as remove previous PCR products containing dUTP prior to
negative for the bacteria. Accuracy (or trueness) is the degree each amplification reaction. Furthermore, inclusion of mul-
of conformity of an assay’s measurements to the actual (true) tiple negative controls, such as no-template controls (NTC)
value. It is often estimated by analyses of reference materials and no-amplification controls (NAC), may help identify the
or comparisons of results with those obtained by a reference likely source of contamination and prevent false-positive
method. The closer an assay’s measurements to the accepted results. Moreover, microbial DNA may come with PCR
value, the more accurate the assay is. Precision is the degree reagents.
of mutual agreement among a series of assay’s individual Similarly, a negative result by a molecular assay for a
measurements, values, or results. Usually characterized in given pathogen normally indicates the absence of the patho-
terms of the standard deviation of the measurements, pre- gen. However, it is equally important to rule out the possibil-
cision can be stratified into (i) repeatability—the variation ity of false-negative results. One possible cause is due to the
arising using the same instrument and operator in a single low sensitivity of the assay employed. Alternatively, insuf-
rune (i.e., intra-assay precision) or repeating during a short ficient amount of bacteria may be present in the sample (due

to sample degradation or prior antibiotic treatment). Another accuracy, repeatability (intra-assay precision), reproducibil-
may be due to the impurity of the processed sample. Enzymes ity (inter-assay precision), detection limit, and linearity (if
(e.g., DNA polymerase, reverse transcriptase) used in PCR quantitative) of molecular tests.
and RT-PCR are impeded by components in blood and feces Before validating a method, it is important to have all
(e.g., heme, hemoglobulin, lactoferrin, immunoglobulin G, instruments calibrated and maintained throughout the testing
leukocyte DNA, polysaccharides, and urea), in foods (e.g., process. The validation process may involve a series of steps
phenolics, glycogen, calcium ions, fat, and other organic including (i) testing of dilution series of positive samples (or
substances), in environmental specimens (e.g., phenolics, plasmid construct) to determine the limits of detection of
humic acids, and heavy metals), and in added anticoagulants the assay and their linearity over concentrations to be mea-
(e.g., EDTA and heparin) as well as nucleic acid purification sured in quantitative test (using minimal number of refer-
reagents (e.g., detergents, lysozyme, NaOH, alcohol, EDTA, ence calibrators such as previously tested patient samples or
EGTA, phenol, and high salt concentrations). Any impuri- pooled sera); (ii) evaluating the sensitivity and specificity of
ties and contaminations present in the samples after nucleic the assay, along with the extent of cross-reactivity with other
acid isolation may contribute to false-negative results. A use- genomic material; (iii) establishing the day-to-day variation
ful way to determine the effective of nucleic acid purifica- of the assay’s performance; (iv) assuring the quality of assem-
tion procedure for removing inhibitory substances is to spike bled assays using quality control procedures that monitor the
samples with well-defined DNA or RNA prior to and after performance of reagent batches; and (v) aligning the in-house
sample preparation (as process and amplification internal primer and probe sequences with a genome sequence data-
controls). In light of the high sensitivity of PCR, the occur- bank to avoid extended specificity testing [25–27,58].
rence of false-negative results is probably a truly underesti-
mated problem [55]. 1.5.4 Quality Control and Assurance
Because few species-specific molecular assays are avail-
able for fungal organisms, PCR amplification and DNA 1.5.4.1 Quality Control
sequencing analysis of the rRNA genes, ITS and other gene Quality control strategies for nucleic acid-based tests include
regions have remained a most useful tool for fungal identifi- (i) designation of a “clean” area for reaction setup (e.g., room
cation. As this approach is contingent on sequence compari- under negative air pressure; positive-displacement pipettes;
son, inaccuracy of data deposited in reference databases may aerosol-block pipette tips; UV-equipped PCR cabinet); (ii)
lead to incorrect identification. For example, a lack of pig- use of personal protective equipment (PPE) (e.g., dispos-
mentation in Alternaria infectoria may contribute to its mis- able gloves and laboratory coats to prevent the introduction
identification using macroscopic characteristics. Sequence of contaminating DNA or nucleases); (iii) use of uracil-N-
data from the incorrectly identified isolates that are stored glycosylase (UNG) in real-time PCR (to eliminate cross-over
in reference databases may result in erroneous determination amplicon contamination); (iv) use of a “hot-start” method (to
of testing isolates. Indeed, de Hoog and Horré [56] demon- minimize false priming events by withholding a crucial reac-
strated that about 14% of the Alternaria infectoria sequences tion component until appropriate temperature is reached); (v)
deposited in GenBank were found to be misidentified. In a use of external positive and negative controls (to monitor
separate study, Nilsson et al. [57] reported that about 20% reaction performance and contamination) and homologous
of the entries related to fungi in the International Nucleotide or heterologous internal controls (to monitor presence of
Sequence Database might have been incorrectly identified to inhibitors).
species level. A variety of test controls may be considered for diagnostic
PCR. These include (i) internal amplification control (IAC)
(negative sample spiked with sufficient pathogen and pro-
1.5.3 Standardization and Validation
cessed throughout the entire protocol); (ii) processing posi-
As molecular tests such as PCR and sequencing offer tive control (PPC) (negative sample spiked with sufficient
improved sensitivity, specificity, accuracy, precision, and closely related, but non-target, strain processed throughout
result availability for fungal identification and diagnosis, the entire protocol.); (iii) reagent control (blank) (contain-
they have been increasingly adopted and applied in routine ing all reagents, but no nucleic acid apart from the primers.);
diagnostic laboratories. Considering the possibility of false- (iv) premise control (tube containing the master mixture left
positive and false-negative results that may occur in these open in the PCR setup room) to detect possible contaminat-
highly sensitive tests, it is essential to properly standardize ing DNA in the environment (carried out at regular intervals
and validate them prior to their adoption, and to put in place as part of the quality assurance program); (v) standard (three
appropriate quality control measures to ensure their consis- to four samples containing 10-fold dilution series of known
tent performance. number of target DNA copies in a range) [31,59].
Standardization of molecular tests addresses the need
for standardized reagents and common units, contamina- 1.5.4.2 Quality Assurance
tion control mechanisms, inhibition control mechanisms, One way to assess preparedness of the diagnostic labora-
clinically relevant dynamic ranges and internal controls, tories is through the conduct of an external quality assur-
etc. Validation helps to verify the sensitivity, specificity, ance (EQA) program providing characterized specimens

containing pathogens of interest. The design of a quality changes for many fungal organisms in the past as well as
assurance program has the following components: (i) internal in recent times represent another potential source of mis-
quality control (IQC) materials are distributed every month identification if care is not taken. Therefore, future identi-
and comprising three pools of clinical samples of known fication and characterization of novel taxon-specific gene
pathogen status (typically one negative, one positive contain- markers will contribute to the increased accuracy in the
ing 1 log 10 over the lower limit of detection of the assay, molecular determination of fungal species/varieties impli-
and one low positive containing up to 1 log 10 of the lower cated in human mycoses. These gene markers may come in
limit of detection of the assay). These are incorporated in the forms of previously uncharacterized, uniquely present
test runs on a weekly basis. The purpose of IQC is to provide genes, or of taxon-specific probes recognizing distinct por-
samples of known status for repeated testing in parallel with tions of the shared genes. The latter category is exemplified
clinical samples to ensure reproducibility of the test system by the development of species-specific probes from the inter-
in an individual laboratory. (ii) EQA distributions of panels nal transcribed spacer (ITS) regions that are common to all
of five unknown samples distributed quarterly. Results are dermatophytes [62].
returned to the QA laboratory for assessment. EQA compares
the performance of different testing sites using specimens of
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30. Bretagne, S. et al. 1995. Detection of Aspergillus species Innis et al. (eds.), PCR Protocols: A Guide to Methods and
DNA in bronchoalveolar lavage samples by competitive PCR. Applications. Academic Press, New York, pp. 315–322.
J. Clin. Microbiol. 33:1164–1168. 52. Vilgalys, R. and D. Gonzalez. (1990). Organization of ribo-
31. Bretagne, S. and J.-M. Costa. 2006. Towards a nucleic acid- somal DNA in the basidiomycete Thanatephorus praticola.
based diagnosis in clinical parasitology and mycology. Clin. Curr. Genet. 18:277.
Chim. Acta 363:221. 53. Liu, D. et al. 2000. Application of PCR to the identification of
32. de Hoog, G.S. and A.H.G. Gerrits van den Ende. 1998. dermatophyte fungi. J. Med. Microbiol. 49:493.
Molecular diagnostics of clinical strains of filamentous 54. Shenoy, B.D., R. Jeewon, and K.D. Hyde. 2007. Impact of
Basidiomycetes. Mycoses 41:183–189. DNA sequence-data on the taxonomy of anamorphic fungi.
33. Bougnoux, M. et al. 1999. Serum is more suitable than whole Fungal Divers. 26:1–54.
blood for diagnosis of systemic candidiasis by nested PCR. J. 55. Wilson, I.G. 1997. Inhibition and facilitation of nucleic acid
Clin. Microbiol. 37:925–930. amplification. Appl. Environ. Microbiol. 63:3741–3751.
34. Chen, S.C., C.L. Halliday, and W. Meyer. 2002. A review of 56. de Hoog, G.S. and R. Horré. 2002. Molecular taxonomy of
nucleic acid-based diagnostic tests for systemic mycoses with the Alternaria and Ulocladium species from humans and their
an emphasis on polymerase chain reaction-based assays. Med. identification in the routine laboratory. Mycoses 45:259.
Mycol. 40:333–357. 57. Nilsson, R.H. et al. 2006. Taxonomic reliability of DNA
35. Yeo, S.F. and B. Wong. 2002. Current status of nonculture sequences in public sequence databases: A fungal perspective,
methods for diagnosis of invasive fungal infections. Clin. PLoS One 1:e59.
Microbiol. Rev. 15:465. 58. Kontoyiannis, D.P. and R.E. Lewis. 2002. Antifungal drug
36. Balajee, S.A., L. Sigler, and M.E. Brandt. 2007. DNA and the resistance of pathogenic fungi. Lancet 359:1135–1144.
classical way: Identification of medically important molds in 59. Paterson, R.R. 2007. Internal amplification controls have not
the 21st century. Med. Mycol. 45:475. been employed in fungal PCR hence potential false negative
37. Borman, A.M. et al. 2008. Molecular identification of patho- results. J. Appl. Microbiol. 102:1.
genic fungi. J. Antimicrob. Chemother. 61:S7. 60. Koneman, E.W. and G.D. Roberts. 1985. Practical Laboratory
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169:445–449. species in clinical material. Clin. Microbiol. Infect., 16, 704.

Part I
Ascomycota
Pezizomycotina: Dothideomycetes

2 Alternaria
Giuliana Lo Cascio and Marco Ligozzi
Contents
2.1 Introduction........................................................................................................................................................................ 27
2.1.1 Classification, Morphology, and Biology............................................................................................................... 27
2.1.2 Clinical Features..................................................................................................................................................... 28
2.1.2.1 Ocular infections...................................................................................................................................... 28
2.1.2.2 Rhinosinusitis.......................................................................................................................................... 28
2.1.2.3 Onychomycosis........................................................................................................................................ 28
2.1.2.4 Cutaneous and Subcutaneous Infections................................................................................................. 28
2.1.3 Pathogenesis............................................................................................................................................................ 29
2.1.4 Laboratory Diagnosis............................................................................................................................................. 30
2.1.4.1 Conventional Diagnosis........................................................................................................................... 30
2.1.4.2 Molecular Techniques.............................................................................................................................. 30
2.2 Methods.............................................................................................................................................................................. 31
2.2.1 Sample Preparation................................................................................................................................................. 31
2.2.2 Detection Procedures.............................................................................................................................................. 33
2.3 Conclusion and Future Perspectives................................................................................................................................... 33
References.................................................................................................................................................................................... 34
2.1 Introduction those affected by drought, insect infestation, or senescence.

Alternaria spores are passively dispersed from infected
2.1.1 Classification, Morphology, and Biology leaves by moderate to strong gusty wind, with velocities of
Alternaria is a genus of asexual or imperfect fungi assigned 2–3 m/s required for spore release. As a component of the dry
with the class hyphomycetes. Fungi in this genus are ana- air spora, dispersal typically occurs during dry weather that
morphs of ascomycetes, including members of the genus immediately follows periods of rain or heavy dew.
Pleospora. Alternaria is a dematiaceous mold, which Alternaria is also commonly isolated from soil, food, and
includes, at present, about 50 species and varieties. However, indoor air environment, in particular on carpets and house
only eight species have been implicated as human patho- dust.6,7
gens: A. alternata, A. brassicicola, A. chartarum, A. stem- Alternaria spp. grow rapidly and the colony size reaches
phylioides, A. dianthicola, A. infectoria, A. pluriseptata, a diameter of 3–9 cm following incubation at 25°C for 7 days
and A. tenuissima. A. iridis is mentioned only as an allergic on potato glucose agar. The colony is flat, downy to woolly,
strain.1,2 and is covered by grayish, short, aerial hyphae in time. The
In 1817 Nees described this telluric fungus is character- surface is grayish white at the beginning, which later dark-
ized by chains of spores with apical beak under the name ens and becomes greenish black or olive brown with a light
of Alternaria tenuis. Subsequently Fries noted in 1832 border. The reverse side is typically brown to black due to
described that Alternaria species are characterized by very pigment production.8–10
distinctive large multicellular dictyospores that have a beak On microscopic examination, Alternaria spp. have sep-
and are produced in chains. Species of Alternaria occur as tate, brown hyphae. The conidiophores arise singly or in
parasites on a number of crop plants, causing early blight or small groups, simple or branched, straight or flexible, some-
leaf spot diseases, or as saprobes on a wide variety of organic times geniculate, pale to mid-olivaceous or golden brown,
substrates. This genus is prominent in aerobiological litera- smooth, up to 50 μm long, 3–6 μm thick with one or several
ture because it is recognized as an important aeroallergen as conidial scars. They bear simple or branched large conidia
well as plant pathogen.3,4 (7–10 × 23–34 μm) that have both transverse and longitudinal
Many pathogenic species of Alternaria have a worldwide septations. These conidia may be observed singly or in acrop-
distribution and are infective to a variety of plants includ- etal chains and may produce germ tubes. They are ovoid to
ing potato, tomato, onion, and members of Brassicacee.5 In obclavate, darkly pigmented, muriform, and smooth or rough-
general, Alternaria attacks plants under stress, especially ened. The end of the conidium nearest to the conidiophore is
27

round while it tapers toward the apex. This gives the typical striking progress has been made in the development of better
beak or club-like appearance of the conidia.8–10 techniques to detect fungi in nasal secretion or in ethmoid
sinus. Polymerase chain reaction (PCR) with specific fungal
primers has been used to find fungal DNA in polypoid nasal
2.1.2 Clinical Features
tissue,38 in ethmoid sinus mucus, and nasal fluids, and it has
The genus Alternaria comprises a large number of mostly provided greater sensitivity in detecting fungal elements.
saprobic or plant-pathogenic species and infects mainly Alternaria DNA was detected in surgical specimens from
immunocompromised hosts. Although infections in immu- the mucosa of the middle meatus and the paranasal sinuses
nocompetent hosts have also been reported, these rarely or in ethmoid sinus mucosal specimens in almost all patients
involve invasive disease.11 Alternaria can be found on normal with CRS but in none of the normal controls.38,39 It has been
human and animal skin12 and conjunctiva.13 This fungus has postulated that fungi act as non-IgE-mediated immunologic
been associated frequently with hypersensitivity pneumoni- targets which initiate and maintain the inflammatory reac-
tis, bronchial asthma, and allergic sinusitis and rhinitis.14–21 tion in paranasal sinuses.40 It has been shown that patients
It can also cause several different types of human infections, with CRS have exaggerated cytokine and humoral immune
for example, paranasal sinusitis, ocular infections, onycho- response to fungi, particularly to Alternaria.41 The fungal
mycosis,22 cutaneous, and subcutaneous infections,23–25 and DNA detected in the mucosa by PCR has been assumed to be
in some cases also soft palate perforation26 and disseminated related to an antigen-presenting process involving cells such
disease.27,28 as dendritic cells.38
The portal of entry of the infection is usually through cor-
neal trauma or breakdown of the skin barrier. 2.1.2.3 Onychomycosis
Different epidemiological studies reported Alternaria as a
2.1.2.1 Ocular infections causative agent of onychomycosis only in the 0.08%–2.5%
The incidence of Alternaria spp. in oculomycosis ranges of the cultured-proved clinical cases of nail infections.42–47
from 2.3% to 10.4%29–32 but it can vary according to the As with onychomycosis caused by other fungi, a history
geographical location and is probably related to the risk of of contact with soil or trauma in the nail exists in most of the
trauma caused by organic matter. The majority of cases are cases. Nails appear dystrophic and distal subungual hyper-
keratitis, although endophthalmitis is frequently associated.32 keratosis and onycholysis are frequent manifestations. More
Patients, in general, are farmers or gardeners who have been than one nail could be affected, and in some cases both fin-
exposed to soil and garbage. Accidental or surgical ocular gernails and toenails show infections.42–47 Identification to
traumas are the predisposing factors.33,34 The causative agent the species level is not always reported, although A. alter-
was generally identified as A. alternata or A. infectoria, nata is the prevalent identified species, with some reports of
although identification to the species level has not been per- A. humicola, A. pluriseptata and A. chlamydospora as caus-
formed on many occasions. ative agents.47
2.1.2.2 Rhinosinusitis 2.1.2.4 Cutaneous and Subcutaneous Infections

Sinusitis, or more accurately rhinosinusitis (RS), is a common Most of clinical presentations concerned localized cutaneous
disorder affecting approximately 20% of the population.35 A infections resulting from direct, traumatic inoculation, even
report by the Centers for Disease Control and Prevention has if in a compromised host a systemic spread is possible. Most
estimated that the prevalence of sinusitis is up to 14.1% of of Alternaria infections are in patients with immunosuppres-
the U.S. adult population, and the fact that there is no treat- sion, and transplant recipients are one of the groups at risk for
ment for this disorder approved by the U.S. Food and Drug developing cutaneous alternariosis.23–25 Phaeohyphomycosis
Administration emphasizes the profound impact of this dis- caused by Alternaria may be difficult to recognize because
ease.36 Acute RS is well categorized and is often attributed to lesions appear variable in size and aspect, ranging from
bacterial or viral causal agents. However, controversies sur- crusted lesions, papules to erythematous macules or subcuta-
round chronic rhinosinusitis (CRS) and the role of fungi in neous nodules, and, rarely, as cellulitis with secondary ulcer-
this condition. CRS accounts for >90% of all cases of RS, ation. Lesions are located on exposed areas, mainly on upper
and the correct diagnosis of each category of CRS is impor- or lower limbs. Histopathologically, Alternaria infection is
tant for optimum therapy and predicting the course. Patients sometimes misdiagnosed as yeast infection or blastomyco-
with CRS suffer from long-term nasal congestion, thick sis and in vitro distinction of species may be problematic,
mucus production, loss of sense of smell, and intermittent since clinical isolates may remain degenerate and sterile.
acute exacerbations secondary to bacterial infections. An inflammatory diffuse infiltrate with mixed cellularity
CRS is characterized histologically by an intense eosino- (lymphocytes, neutrophils, plasma cells, and histiocytes)
philic infiltration into the nasal mucosa.37 The role of fungi mixed with a sarcoid-like and suppurative granulomatous
in CRS is noninvasive, and it is not a fungal infection. It inflammation is usually observed. Biopsy specimens from
needs to be differentiated from other forms of fungal sinus- early lesions (<3 months evolution) are often characterized
itis, such as fungus balls (noninvasive) and invasive fun- by the presence of epidermal changes (pseudoepithelioma-
gal sinusitis (acute, fulminant, or chronic form). Recently, tous hyperplasia, acanthosis) and a diffuse dermal mixed

Alternaria 29
inflammatory infiltrate with lymphocytes, plasma cells, his- of this pigment may protect the fungi from diverse environ-
tiocytes, and neutrophils. Histiocytic cells often presented mental insult. The microbiota at the site of the Chernobyl
with intracytoplasmic hyphae that adopted a typical aspect of nuclear reactor accident provides one of the most striking
round-to-oval, thick refractile walls (spore-like structures). associations between fungal melanogenesis and the ability of
In biopsy specimens from more advanced lesions (>3 months these organisms to survive in an extreme environment. The
evolution), the observation of an inflammatory granuloma- extreme conditions present in the Chernobyl environment,
tous infiltrate is a constant feature. Round, yeast-like struc- radiation levels over 10,000 times the lethal human dose,
tures and branched hyphae are found within the granulomas select a radiation tolerant A. alternata clone.57
and microabscesses. Melanin synthesis is associated with virulence for mam-
Two different routes of cutaneous infection have been mals in several pathogenic fungi including C. neoformans
reported: (1) from an exogenous source either as a conse- and Exophiala dermatitidis by comparing the relative patho-
quence of traumatic inoculation of fungal elements (after genicity of wild-type strains to mutants incapable of melani-
injury by a plant spine) or to colonization of pathologically zation, and it seems that melanin formation occurs in vivo.
altered skin, or (2) from an exogenous focus (inhalation of Melanins contribute to virulence with different mechanisms:
fungal conidia and systemic spread) resulting in secondary they give protection against oxidants because they are highly
cutaneous involvement. Some authors have also defined a effective scavengers of free radicals and have electron trans-
“dermatopathic” cutaneous alternariosis, consisting of sec- fer properties that can facilitate redox cycling. Moreover
ondary colonization by Alternaria of preexisting lesions such DHN melanin in A. alternata protects against permanganate
as steroid-treated eczema of the face.48 and hypoclorite and protects against neutrophil oxidative
An altered host resistance appears to be the prerequi- burst. Melanization interferes with phagocytosis and protects
site in the majority of affected patients. The majority of the against killing by macrophages.
reported patients with cutaneous alternariosis are solid-organ Melanin induces resistance to antimicrobial compounds
transplant recipients, although many of these patients were in Cryptococcus neoformans, binds amphotericin B and
receiving systemic corticosteroids. Some authors have pos- caspofungin, and prevents them from reaching their target
tulated that cutaneous fragility induced by corticosteroids sites.58
may increase the risk of percutaneous inoculation from the Melanins are immunologically active compounds; they
environment.49 can elicit a vigorous inflammatory reaction inducing granu-
Rarely, Alternaria species may cause a systemic infection loma formation. It is conceivable that cell-wall-associated
involving other organs apart from the skin. This endogenous melanin in tissue provides an indigestible material that serves
cutaneous variant is manifested by multilocular involvement as “foreign body”-like material that interferes with clear-
without having a site of predilection. Visceral and mucosal ance of infection while at the same time stimulating intense
infection is more common among patients with HIV infec- inflammation.
tion.50 The observation of cutaneous alternariosis progress- Factors other than 1,8-DHN melanin contribute to the viru-
ing to systemic involvement seems to be an exceedingly rare lence of dematiaceous fungi, although most of the pathogenic
phenomenon.25,51 virulence factors are studied in Wangiella dermatitidis used
Differentiation down to the species level is recommended, as a model for black molds.59 Among the many possibilities,
because species may have different virulence or resistance chitin is receiving the most attention. The cell wall content of
to antimycotic therapy due to the occurrence of chlamydo- chitin is significantly enriched in most of the alternative mor-
spores.52,53 Moreover blastomycosis, sporotrichosis, cryp- photypes of W. dermatitidis compared to that of its budding
tococcosis, and other subcutaneous mycosis that may yeast.59 Furthermore, additional chitin is delocalized from
present similar clinical features require different therapeuti- predominantly yeast septal regions to all cell wall locations
cal approaches. in hyphae and sclerotic forms. Chitin could act as a virulence
factor by adding additional strength to the cell walls of black
molds, whenever conditions are encountered in hosts that
2.1.3 Pathogenesis
temporarily retard rapid yeast growth or induce transition of
Melanins play an important role in the evasion of host yeast cells to other morphotypes. Many studies using chitin
defence by fungal pathogens, including dematiaceous synthase gene disruption showed morphological changes like
molds. Melanins are believed to be composed of polymer- abnormal hyperpigmentation or pseudohyphal-like growth
ized phenolic and/or indolic compounds. Melanins are dark with defective septa. Tissue sections from patients and ani-
in color, insoluble in aqueous or organic fluids, resistant to mals with chronic cutaneous and subcutaneous infections
concentrated acid, and susceptible to bleaching by oxidiz- frequently show yeast and hyphae with abnormally thickened
ing agents.54 At this moment these pigments remain poorly walls. Even brain and other tissue from systemically infected
characterized because current biochemical and biophysical humans and animals with rapidly progressing disease often
techniques are unable to provide a chemical structure for this show many types of thickwalled forms, which are variously
complex polymer. Melanin in dematiaceous molds such as described as yeast, chain and clusters of yeast, sclerotic cells,
Alternaria alternata is synthesized through the dihydroxyn- and cells with internal septa. Whether these different in vivo
apththalene (DHN) polyketide pathway.55,56 The production tissue forms are actually enriched with chitin is not known,

but chemical and cytochemical analyses of identical morpho- Heavily melanized fungal cells are not reliably detected by
types produced in vitro indicate that they are.60 using calcofluor white. Fluorescent-antibody-specific conju-
gates are not available for Alternaria sp.
2.1.4 Laboratory Diagnosis Colonies of Alternaria grow rapidly and appear flat,
downy to woolly, and are covered by grayish, short, aerial
2.1.4.1 Conventional Diagnosis hyphae in time. The surface is grayish white at the begin-
The collection, transport, and processing of clinical speci- ning, which later darkens and becomes greenish black or
mens encompass some of the most important considerations olive brown with a light border. The reverse side is typically
in determining the etiology of fungal disease. Only with the brown to black due to pigment production.
appropriate handling of specimens can the recovery of fun- Until recently, the identification of Alternaria rested upon
gal organisms be clearly associated with a disease process. microscopic morphology, with the most significant charac-
As with all disease processes, the best specimen for deter- teristics being the morphology of the conidia and the for-
mining the etiologic agent is the one taken from the active mation of conidial chains. Morphology evaluations normally
infection site. The collection details for fungal cultures are are based upon cultures that have been grown on a medium
very similar to those for bacterial cultures, although the vol- such as potato dextrose agar or cornmeal dextrose agar at
ume of material required for recovery of fungi may exceed 25°C–30°C for approximately 2 weeks. Slide culture prepara-
those necessary for bacteria. The presence of more material tion using cornmeal dextrose agar is ideal for conidiogenesis
for primary inoculum and concentration of large volumes because these nutritionally minimal media usually stimulate
of fluid greatly increases the likelihood of recovery of fun- the formation of spores.61
gal species. Appropriate transport and storage of specimens The morphological characteristics useful for distinction
are necessary for fungal elements to remain viable. Fungal among the three species more frequently isolated are the fol-
viability may be affected by excessive heat and cold; room lowing: A. alternata shows medium-brown conidia with a
temperature transport and storage, within 2 h of collection, short, cylindrical beak, forming long and profusely branched
are recommended. chains, usually 10 or more conidia; in A. tenuissima the
The most frequently submitted specimens for the recov- conidia are golden-brown, frequently tapering gradually into
ery of dematiaceous fungi include aspirates, scrapings, and a beak that is up to half the length of the conidium, and usu-
surgical tissue. Transport media should not be used, but ally occur in unbranched chains of three to five conidia; A.
specimens must not be allowed to become desiccated prior to infectoria species group comprises more than 30 named ana-
processing. Specimen portions that are necrotic, purulent, or morph taxa. Morphologically, the A. infectoria species group
caseous should be selected for microscopic examination and differs from other Alternaria species in the three-dimen-
inoculation onto isolation media. Tissue specimens should be sional sporulation pattern and has more scarce conidia, as
minced with scalpels into 0.5 mm pieces and used to directly this species usually sporulates poorly in common media, and
inoculate culture media. The use of tissue homogenizer for its small conidia (up to 70 μm in length) occur in branched
fungal cultures is discouraged because some molds do not chains with long, geniculate multiseptate secondary conidio-
have regularly septate hyphae and thus can be killed easily phores (up to 120 μm) between conidia.62
during homogenizing.
Media for primary isolation must support fungal growth 2.1.4.2 Molecular Techniques
while inhibiting bacteria, especially with specimens from Identification of pathogenic dematiaceous fungi such as
nonsterile sites. The most commonly used media are brain Alternaria spp. is typically done by morphological and physi-
heart infusion (BHI) plus antibiotics, Sabouraud dextrose ological procedures.63,64 However, these procedures are time-
agar alone or plus antibiotics, and selective media contain- consuming, require technical expertise, and are ineffective
ing cycloheximide, a eukaryotic protein synthesis inhibitor, for identification of species with poor conidia production and
as suppressor of many saprobic fungi. In facilities where a a wide diversity in anamorphic life cycles.65
large portion of the patient population may be neutropenic The use of molecular techniques facilitates the identifica-
or immunocompromised, the rapid identification of oppor- tion of rare pathogenic fungi, such as Alternaria spp., and the
tunistic fungi, which may quickly disseminate in these indi- most immediate need for nucleic acid detection methods is
viduals, is critical. The addition of cycloheximide and/or for the immunocompromised patient group. In this context,
antibiotics to potato dextrose agar may be more appropriate rapid diagnosis of mycological infection by in vitro ampli-
in this setting, since it facilitates the identification of these fication and detection of fungal DNA is a common method
organisms by providing plates for tape mounts of diagnos- used in clinical laboratories.
tic structures from primary media. Sheep blood on BHI may
inhibit conidiation, so is useful for primary isolation but not 2.1.4.2.1 Selection of Target DNA
for identification. Two avenues are available for this application; first is the
Microscopic examination of a clinical specimen is essen- selection of the target DNA using specific sequence informa-
tial to detect fungal elements. In laboratories that use fluo- tion from databases, allowing primers to be designed across
rescence microscopy, bright-field examination of a positive conserved and variable regions, and second is cloning and
field must be done to evaluate the amount of melanin present. sequencing of arbitrary parts of the fungal genome.

Alternaria 31
Ribosomal RNA is an essential component of protein syn- 2.2 Methods

thesis, thus ubiquitous. The sequence of rRNA has highly
conserved as well as variable regions. Examination of this 2.2.1 Sample Preparation
sequence reveals the relatedness between the species or the A key step in the detection of fungi using PCR is the ability
genetic distance between the organisms in question. An addi- to efficiently extract DNA from hyphae and/or conidia. The
tional advantage of this is that these genes are not horizon- efficiency of this step is even more critical when attempt-
tally transferred, like other prokaryotic genes, for example, ing to detect small quantities of fungal material in biological
drug resistance genes. samples such as human blood, mucus, and tissues.
Nuclear ribosomal RNA is coded by ribosomal DNA The detection of fungal pathogens in clinical samples by
(rDNA), which is organized in ribosomal operons (usually PCR requires the use of extraction methods that efficiently
100–200 identical copies in fungi) located in chromosomes. lyse fungal cells and recover DNA suitable for amplifica-
The nuclear-encoded rRNA genes of fungi exist as a mul- tion. The DNA extraction stage of the sample is critical
tiple-copy gene family consisting of highly similar DNA processing because fungi have cell walls that impede lysis
sequences (typically from 8 to 12 kb each) arranged in a and the recovery of nucleic acids. Because of the structure
head-to-toe manner. Each operon codes for the large subunit and composition of the fungal cell wall, consisting of thick
(LSU-rRNA; 28S rRNA), small subunit (SSU-rRNA; 18S layers of chitin, (1–3)-β-d-glucan, (1,6) β-glucans, man-
rRNA), 5.8S rRNA and 5S rRNA. The position of 5S rRNA nan, mannoproteins, lipids, and peptides, and the pres-
varies, but the organization of the rest of the genes is the ence of a melanin complex in the cell wall, the release of
same in all fungi (Figure 2.1). fungal DNA usually requires additional lysis steps, such
The fungal identification is based on detection of con- as mechanical disruption and enzymatic digestion or use
served sequences in 5.8S and 28S rDNA that enable the of toxic chemicals.54 In general, the sample preparation
amplification of the ITS region between these two regions method should release intracellular DNA from the fungal
and detection of D1/D2 domain contained in 28S rDNA. cell wall and/or thick capsule; it must concentrate DNA tar-
Then the tools for molecular investigation are represented by gets that may be present in very small amounts; and it must
(i) DNA sequencing of the full length internal transcribed eliminate protein debris, contaminants, potential inhibi-
spacer (ITS) region ITS1–2 and (ii) DNA sequencing of the tors, and other extraneous materials without degrading the
D1/D2 region of LSU gene in 28S rDNA. target DNA.
The main area for the development of fungal diagnostics Alternaria molecular detection test can be used indirectly
is ribosomal genes,66 present in all organisms and at high on molds isolated from a patient’s specimen or directly on
copy numbers aiding detection and the sensitivity of PCR. clinical samples. Several commercial kits are available for
The fungal nuclear rDNA consists of three genes: the large mold DNA extraction in a clinical microbiology laboratory.67
subunit gene (28S), the SSU gene (18S), and the 5.8S gene, Many extraction methods use a bead matrix and lysis buffer
separated by ITS regions, in a unit repeated many times. to destroy mycelial cells, followed by adsorption of DNA to a
The ITS region is an area of particular importance to fungal spin filter, a wash step, and the elution of DNA in buffer, prior
diagnostics. It has areas of high conservation and areas of to amplification steps.
high variability and is an ideal starter for the development
of specific PCR primers for identification of fungal spe- (i) Clinical sample pretreatment—Step 1
cies. Universal primers66 are available for fungi that isolate Tissue: For DNA extraction, all tissue samples are
the regions of the ITS. Once cloned these sequences can be incubated for ≥2 h in proteinase K and digestion
compared to the wealth of other sequences in the sequence buffer at 55°C, and the DNA is extracted using the
database and diagnostic primers developed for a particular miniMAG™ or easyMAG instrument (bioMérieux)
fungus. The MicroSeq D2 large-subunit rDNA sequencing according to the manufacturer’s instructions. The
kit appears to be accurate and useful for the identification of DNA could be stored at −20°C prior to use.
filamentous fungi, even those that are relatively uncommon (Note: Digestion buffer: 50 mM Tris–HCl,
that are seen in the clinical laboratory. However, the library 100 mM EDTA, 100 mM NaCl, 1% SDS, pH 8.0.
includes more of the common Alternaria spp. and other envi- Proteinase K: 0.5 mg/mL in digestion buffer).
ronmental flora that cause disease in immunocompromised 1. Place 20–25 mg of tissue into a polypropylene
patients. microfuge tube.
ITS1 NL1
18S ITS
5.8S ITS 28S
1 2
ITS4 NL4 200
bp
Figure 2.1 Map of fungal rRNA from the 3′ end of the 18S rRNA gene to the 3′ end of the 28S rRNA gene. PCR primers for sequencing
and PCR assay development (arrows).

2. Add 0.5 mL of DNA digestion buffer with pro- 7. Centrifuge the MicroBead tubes for 2 min at
teinase K. 10,000 × g.
3. Incubate for a minimum of 3 h to overnight at 8. Add 100 μL of solution MD2 to a clean 2.0 mL
55°C with gentle shaking. microcentrifuge tube. Solution MD2 contains
Corneal scrapings are collected from all of the ammonium acetate, a reagent to precipitate non-
patients while they are under local anesthesia DNA organic and inorganic material including
by use of a slit-lamp microscope and a flame- cell debris and proteins.
sterilized Kimura spatula.68 One part of each 9. Transfer the supernatant from step 7 to the
scraping is directly transferred to lysis buffer tube containing solution MD2. Vortex briefly.
for DNA extraction. Incubate at 4°C until tubes are completely
Cutaneous swab is immersed in lysis buffer chilled (at least 15 min).
in a microcentrifuge tube and incubated at room 10. Centrifuge tubes for 1 min at 10,000 × g.
temperature for 5 min with occasional agitation. 11. Add 900 μL of solution MD3 to a clean 2.0 mL
(ii) Clinical sample pretreatment—Step 2 microcentrifuge tube. Solution MD3 contains
1. Up to 1 mL of lysis buffers each pre-treated guanidine hydrochloride/isopropanol which is
sample containing fungal cells or conidia. a highly concentrated salt solution necessary to
2. Add 300 mg of MicroBeads in each sample bind DNA to the spin filter membrane in the fol-
tube. lowing step.
3. Continue DNA purification as described below 12. Transfer the supernatant in step 10 to the tube
in step 3 for Method UC (UltraClean DNA iso- containing solution MD3. Vortex briefly and
lation kit [MoBio, Inc., Solana Beach, CA]) of spin down. During transfer of supernatant, do
DNA extraction from culture. not touch the protein pellet with the pipette tip.
The supernatant solution (above the beads) is 13. Transfer 650 μL to a clean spin filter. Centrifuge
transferred to a mini-MAG for a semi-auto- for 30 s at 10,000 × g. Discard the flow through.
mated magnetic extraction or an easy MAG for Replace the filter into the same collection tube.
automated magnetic extraction. 14. Repeat step 13 with remaining supernatant
(iii) DNA extraction from culture MD3.
Method UC (UltraClean DNA isolation kit [MoBio, 15. Add 300 μL of solution MD4 to the spin filter
Inc., Solana Beach, CA]) uses a bead matrix and and centrifuge for 30 s at 10,000 × g. Solution
lysis buffer to pulverize cells by horizontal shaking MD4 is an ethanol based wash solution used to
on a vortex mixer, followed by adsorption of DNA further clean the DNA that is bound to the silica
to a spin filter, a wash step, and the elution of DNA filter membrane in the spin filter. Discard the
in buffer.69 Microcentrifuge tubes with sample and flow through. Replace the filter into the same
bead matrices were attached to a horizontal plat- collection tube.
form on a vortex mixer and agitated vigorously for 16. Centrifuge the empty spin filter for 60 s at
40 min. Each sample is eluted in a TE1X buffer. In 10,000 × g. Discard the collection tube.
detail the method: (Note: Alcohol is a PCR inhibitor. Make sure
1. Add 300 μL of MicroBead solution to a no residual alcohol remains near the filter
MicroBead tube. MicroBead Solution contains basket).
salts such as guanidine thiocyanate and a buffer 17. Place the spin filter into a clean 2.0 mL micro-
which stabilizes and homogeneously disperses centrifuge tube.
the microbial cells prior to lysis. 18. Add 35 μL of solution MD5 onto the center
2. Use an inoculation loop to collect approximately of the filter. Solution MD5 is 10 mM Tris pH
half of the mycelia covering the cellophane 8. Incubate for 2 min. Centrifuge for 30 s at
square. Transfer mycelia into the MicroBead tube. 10,000 × g.
3. Add 50 μL of solution MD1 to the MicroBead 19. Be careful not to touch the filter with the pipette
tube. Solution MD1 contains sodium dodecyl tip.
sulfate (SDS) and other disruption agents 20. Discard the spin filter. Extracted DNA is con-
required for cell lysis. tained in the flow through and is ready for use.
4. Place MicroBead tubes in a liquid nitrogen Store DNA at −20°C.
safe container (such as a Styrofoam cup) and (iv) Semi-automated extraction
cover with liquid nitrogen. Incubate for 1 min. DNA could be isolated using a semi-automated mag-
Remove MicroBead tubes from the liquid nitro- netic extraction method, NucliSens® miniMAG, and
gen using forceps. Immediately vortex tubes for the NucliSens Magnetic Extraction Reagents (bio-
10 min. Mérieux S.A.).
5. Repeat previous step two times. In the first step, 2 mL of NucliSens lysis buffer
6. Vortex MicroBead tubes an additional 10 min. containing guanidine thiocyanate (5 mol/L) is added

Alternaria 33
to each 1.5 mL tube containing a single pretreated by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for
sample. 1 min, followed by a final extension at 72°C for 10 min.
The tubes are pulse vortexed for 15 s, and then To validate the presence of amplifiable DNA and absence
incubated for 10 min at room temperature. The of inhibitory substances a PCR is performed using the
NucliSens lysis buffer contains a chaotropic salt for primer set GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′)
efficient lysis and inactivation of nucleases. In the and PC04 (5′-CAACTTCATCCACGTTCACC-3′) targeting
second step, 50 μL of Magnetic Silica Particles (bio- the human β globin gene.71 The conditions are as described
Mérieux) are added to allow DNA binding during a above except that 5 μL plasmid DNA containing partial β glo-
10 min incubation period at room temperature. bin gene are used, in noncellular sample. When the internal
Next, the silica particles are washed twice with control result is negative, DNA extraction must be repeated if
400 μL of wash buffer 1 (5 mol/L guanidine thio- enough material is available.
cyanate, Tris/HCl, Triton X 100, EDTA), twice with Following amplification, aliquots (10 μL) are removed
400 μL of wash buffer 2 (MES hydrate), and once from each reaction mixture and examined by electrophoresis
with 500 μL of wash buffer 3 (component: disodium (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco,
tetraborate) using the NucliSens miniMAG instru- United Kingdom) in TAE buffer (40 mM Tris, 20 mM acetic
ment to remove any contaminants from the biologi- acid, 1 mM EDTA pH 8.3), stained with ethidium bromide
cal specimen. (5 μg/100 mL).
Removal of wash buffer is done by vacuum The two most important clinical species, A. alternata and
aspiration. A. infectoria, can easily be differentiated according to the
Finally, the DNA is recovered from the particles length of the ITS1-4 amplicon, with the ITS spacer domain
using 40 μL of wash buffer 3 during a 10 min incu- being ca. 570 bp in the former species and ca. 600 bp in the
bation period at 70°C and under constant shaking latter. A. tenuissima cannot reliably be distinguished from
(1400 rpm). Thirty microliter of each supernatant is A. alternata using this method.
used as template for PCR amplification. All PCR products are purified before DNA sequence
(v) Automatic extraction analysis particularly to remove dNTPS, polymerases, salts,
Extraction with the easyMAG (bioMérieux S.A.) and primers using a QIAquick PCR purification kit (Qiagen)
is done according to the manufacturer’s recom- according to the manufacturer’s instructions. Purified ampli
mendations. Up to 1 mL of each pretreated sample cons were then sequenced on both strands using the same
is placed in the disposable sample vessel and it is primers as described above. BigDye terminator cycle sequenc
loaded onto the extractor. ing Ready Reaction kits (Applied Biosystems) were employed
After the initial lysis incubation, 100 μL of mag- as recommended by the manufacturer. All cycle sequencing
netic silica particles, prepared as recommended by reactions were performed on a MJ PTC200 thermal cycler
the manufacturer, are added to each sample, and the using an initial denaturation at 96°C for 5 s, followed by
extractor restart. 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min.
Samples are eluted in 55–110 μL. All samples are Products are purified using a Dye-Ex spin kit (Qiagen), dried
transferred to a 1.5 mL microcentrifuge tube and in a vacuum centrifuge, and resuspended in either tem-
stored at 4°C for 24 h, or −20°C for no more than 2 plate suppression reagent (Applied Biosystems; D1/D2R
months. products) or formamide (Applied Biosystems; ITS prod-
The internal control is added after the initial ucts). Products are then analyzed on an automated capillary
incubation step, immediately before the magnetic DNA sequencer (ABI Prism 310 genetic analyzer; Applied
silica is added. Biosystems). Comparative sequence analysis and GenBank
searches are assisted by the Genetics Computer Group
software package (FASTA, BESTFIT, STRETCHER, and
2.2.2 Detection Procedures
PILEUP algorithms; University of Wisconsin, Madison), the
PCR mixes (50 μL) are set up as follows: 10 mM Tris–HCl Clustal W alignment program,72 and the nucleotide–nucleo-
pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 μM (each) dATP, tide Basic Local Alignment Search Tool (BLAST) algorithm
dCTP, dGTP, and dTTP; 1.25 U of Taq DNA polymerase (blastn).73
(Amplitaq Gold (Applied Biosystems), 0.2 μM (each) of the
appropriate primers targeting both the D1/D2 region NL1
2.3 Conclusion and Future Perspectives
(forward) 5-GCATATCAATAAGCGGAGGAAAAG-3 and
NL4 (reverse) 5-GGTCCGTGTTTCAAGACGG-3′, as well Alternaria is a cosmopolitan mold, found in soil or more
as the larger 18S-ITS1-5.8SITS2-28S (ITS1/ITS4), ITS1 often on living and dead plants. This mold is a well-known
(forward) 5-TCCGTAGGTGAACCTGCGG-3′ and ITS4 phytopathogen fungal genus, but numerous published cases
(reverse) 5-TCCTCCGCTTAT TGATATGC-3, as previously of human alternarioses are reported. The most frequent
described,70 and 5 μL of DNA template. The reaction mix- clinical presentations are cutaneous and subcutaneous infec-
tures are subjected to the following thermal cycling param- tions, followed by oculomycosis, RS, and onychomycosis.
eters in a MJ PTC 20 thermocycler: 95°C for 3 min followed Immunosuppression is frequently associated with clinical

manifestations, and solid organ transplantation is the most References

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3 Aureobasidium
Miia Pitkäranta and Malcolm D. Richardson
Contents
3.1 Introduction........................................................................................................................................................................ 37
3.1.1 Classification, Morphology, and Environmental Sources...................................................................................... 37
3.1.1.1 Classification............................................................................................................................................ 37
3.1.1.2 Morphology.............................................................................................................................................. 39
3.1.1.3 Environmental Sources............................................................................................................................ 39
3.1.2 Pathogenesis and Clinical Features........................................................................................................................ 40
3.1.2.1 Pathogenesis............................................................................................................................................. 40
3.1.2.2 Hypersensitivity Pneumonitis (Extrinsic Allergic Alveolitis)................................................................. 40
3.1.2.3 Mycotic Keratitis...................................................................................................................................... 40
3.1.2.4 Disseminated Infections, Fungemia and Peritonitis................................................................................ 40
3.1.2.5 Antifungal Treatment............................................................................................................................... 41
3.1.3 Diagnosis................................................................................................................................................................ 41
3.1.3.1 Conventional Techniques......................................................................................................................... 41
3.2 Methods.............................................................................................................................................................................. 44
3.2.1.1 In Vitro Pure Culture............................................................................................................................... 44
3.2.1.2 DNA Extraction....................................................................................................................................... 44
3.2.2.1 Morphological Identification................................................................................................................... 44
3.2.2.2 PCR Amplification of rDNA................................................................................................................... 45
3.2.2.3 DNA Sequencing..................................................................................................................................... 45
3.2.2.4 Phylogenetic Analysis of Obtained Sequences........................................................................................ 45
3.2.2.5 RFLP Analysis of PCR Products............................................................................................................. 45
3.3 Conclusions and Future Perspectives................................................................................................................................. 45
References.................................................................................................................................................................................... 46
3.1 Introduction the hyphomycetes are the black yeasts1 that are defined as
asexual fungi potentially able to produce melanized budding
Aureobasidium is a saprophyte distributed widely throughout cells (a yeast phase) in any stage of their life cycle. Often
the environment, commonly isolated from plant debris, soil, strictly filamentous relatives of pathogenic black yeasts are
wood, textiles, and wet areas of bathrooms. In particular, it is included in this group. Here the term “black yeast-like fungi”
found in osmotic environments, such as in food and on plant is applied. Aureobasidium pullulans, the only well-known
leaves. It is a frequent colonizer of damp stone and glass and species in the genus, is a dematiaceous fungus, characterized
commonly found in the clinical laboratory as a contaminant. by the presence of melanin pigment in the cell wall and the
Only exceptionally is Aureobasidium involved in human production of hyaline blastoconidia that develop darkly pig-
disease. mented chains of arthroconidia in culture.2
According to present taxonomy, Aureobasidium belongs
3.1.1 C
lassification, Morphology, and to Ascomycota, order Dothideales, family Dothioraceae.3
Environmental Sources More than 20 species are listed under this genus in Mycobank
(http://www.mycobank.org). As such, the genus is polyphy-
3.1.1.1 Classification letic and many species are better known by their synonyms
Hyphomycetes form the major part of the moulds encoun- (Table 3.1). Most species are saprophytes on various sub-
tered in the medical mycology laboratory. They are composed strates or are plant pathogens causing leaf spots.2 The species
of regularly septate hyphae and produce asexual propagules of Aureobasidium and closely related genera Hormonema
directly on the hyphae, without fruit bodies.1 Included in and Kabatiella are difficult to distinguish in culture by
37

Table 3.1
Members of the Genus Aureobasidium and Availability of Molecular Data
Species Currently Used Synonym Pathogenicity Sequence Data
Aureobasidium aleuritidis Plant N/A
(Vassiljevsky) Herm.-Nijh. 1977
A. apocryptum Plant N/A
(Ellis & Everh.) Herm.-Nijh. 1977
A. australiense N/A N/A
McAlpine 1896
A. bolleyi Microdochium bolleyi Plant 18S, ITS
(Sprague) Arx 1957 (Sprague) de Hoog & Herm.-Nijh.
1977
A. caulivorum Kabatiella caulivora Plant 18S, 28S, ITS, ELO1
(Kirchn.) Cooke 1962 (Kirchn.) Karak. 1923
A. dalgeri Plant N/A
(Morelet) Herm.-Nijh. 1977
A. foliicola Lecythophora hoffmannii Human14–16 18S, 28S, ITS, Cytb
(Oudem.) Muell. 1964 (Beyma) Gams & McGinnis 1983
A. harposporum Kabatiella harpospora Plant N/A
(Bres. & Sacc.) Herm.-Nijh. 1977 (Bres. & Sacc.) Arx 1957
A. indicum N/A N/A
Pande & Ghate 1985
A. lilii Plant N/A
Crisan & Hodisan 1964
A. lini Kabatiella lini Plant 18S, 28S, ITS, EF1, TUB, ELO1
(Laff.) Herm.-Nijh. 1977 (Laff.) Karak. 1950
A. mansonii Exophiala castellanii Human17 18S, 28S, RPB1, Cytb
(Castell.) Cooke 1962 Iwatsu, Nishim. & Miyaji 1999
A. microstictum Kabatiella microsticta Plant 18S, 28S, ITS, EF1, TUB, ELO
(Bubák) Cooke 1962 Bubák 1907
A. microstromoides N/A N/A
(Moesz) Cooke 1962
A. nigricans Kabatiella nigricans Plant N/A
(Atk. & Edgerton) Cooke 1962 (Atk. & Edgerton) Karak. 1923
A. nigrum Torula dematia N/A N/A
(Marpmann) Cif. & Dalla Torre 1963 Berkhout 1923
A. prunicola Plant N/A
A. prunorum Hormonema prunorum Saprophytic 18S, ITS
Dennis & Buhagiar 1973 (Dennis & Buhagiar)
Herm.-Nijh. 1977
A. pullulans Human, other 18S, 28S, ITS, EF1, TUB, ELO,
(de Bary) Arnaud 1918 mammalian, saprophytic other
A. ribis Plant N/A
(Vassiljevsky) Herm.-Nijh. 1977
A. salmonis Exophiala salmonis Fish 18S, 28S, ITS, mt ssu rRNA,
(Carmich.) Borelli 1969 Carmich. 1966 RPB1, RPB2, EF1, Cytb
A. sanguinariae Plant N/A
A. slovacum Chmelia slovaca Human18 N/A
Svob.-Pol., Chmel & Bojan. 1966 (Svob.-Pol., Chmel & Bojan.)
Svob.-Pol. 1966
A. thujae-plicatae Plant N/A
Morelet 1978
A. umbellulariae Plant N/A
(Harv.) Herm.-Nijh. 1977

Aureobasidium 39
Table 3.1 (continued)

Members of the Genus Aureobasidium and Availability of Molecular Data
Species Currently Used Synonym Pathogenicity Sequence Data
A. vaccinii Plant N/A
Richit. & Teodoru 1989
A. zeae Kabatiella zeae Plant ITS
(Narita & Hirats.) Dingley 1973 Narita & Y. Hirats. 1959
Abbreviations: 18S, nuclear small subunit rRNA gene; 28S, nuclear large subunit rRNA gene; ITS, nuclear internal transcribed spacer region;
ELO1, fatty acid elongase gene; Cytb, mitochondrial gene for cytochrome b; EF1, translation elongation factor 1-α-like gene; TUB, β-tubulin
gene; RPB1, RNA polymerase II largest subunit gene; RPB2, RNA polymerase II second largest subunit gene; N/A, data not available. Synonym
data: http://www.mycobank.org
morphology or nutritional physiology.4 Several studies have

assessed the taxonomy of the group using molecular methods.
The genera Aureobasidium and Kabatiella are overlapping
based on common micromorphological, physiological, and
molecular features.2,5,6 The taxonomy of the group remains
largely uncertain.7
A. pullulans is a clonal anamorph species without a
confirmed teleomorph. It has got no close relatives among
human pathogenic fungi, but certain other species, that is,
plant pathogenic Kabatiella spp., as well as Selenophoma
mahoniae, a pycnidial fungus with Aureobasidium-like cul-
tural synanamorph, and Discosphaerina (Columnosphaeria)
fagi, a potential teleomorph of A. pullulans, show high
genetic affinity with it. Hormonema dematioides resembles
Aureobasidium pullulans phenotypically and these species
are often mixed up.1,4,6–11 Phytopathogenic species A. bolleyi,
A. prunorum, and A. zeae, as well as A. salmonis, a patho- Figure 3.1 Septate, hyaline hyphae with groups of synchronous
gen of fish, show considerable genetic distance to A. pullu- conidia of Aureobasidium pullulans. (From Andreoni, S. et al.,
lans complex (see Table 3.1 for synonyms in use). Unknown Medical Mycology Atlas, 2004.)
Aureobasidium-like strains have been isolated from various
environments.7,12 DNA sequence analysis is a powerful tool
for characterizing these isolates, and novel entities belonging chlamydospores, and expanding hyphae with irregular
to Aureobasidium will probably be described in the future.7,13 dichotomous branching. The conidia-bearing cells appear
For most species in the genus Aureobasidium, pheno- and undifferentiated and mostly intercalary in hyaline hyphae.
genotypic data is not available or is available only from a Conidia are initially produced synchronously in dense
restricted number of strains. groups from small denticles. They are smooth, oval, and
non-pigmented. Subsequently, the conidia are produced
3.1.1.2 Morphology percurrently and adhere to one another in slimy heads.
On malt extract agar (MEA) or glucose peptone agar, The conidia are hyaline, ellipsoidal, vary considerably in
Aureobasidium grows rapidly when incubated at 30°C, shape and size, 7.5–16.0 × 3.5–7.0 μm, and are one celled,
reaching 30 mm in 1 week, appearing flat and smooth, and often with an indistinct hilum. Polar budding of the conidia
soon covered with a slimy exudate. The colonies are cream is often observed. An additional feature is the presence of
or pink and then later becoming brown or black. The reverse endoconidia (asexual propagules formed inside a cell) in
of the colony is cream, turning brown or black with age. The intercalary cells.
varieties show different pigmentation behavior, the entire
colonies of var. melanigenum turning green to black usu- 3.1.1.3 Environmental Sources
ally within 1 week, while other varieties stay mainly light The environmental sources of Aureobasidium are varied.
colored for at least 1–2 weeks. Bright color variants have It is a ubiquitous microorganism that can be easily isolated
been isolated from the tropics.13 The microscopical appear- from the phyllosphere and from plant residues, flowers, soil,
ance (Figure 3.1)70 is vegetative hyphae 3–12 μm wide, hya- wood, indoor and outdoor air, and even stone (reviewed in
line, locally converting into blackish-brown, thick-walled Cooke and Taylor et al.).19–21

3.1.2 Pathogenesis and Clinical Features phaeohyphomycosis in both HIV-infected and non-HIV-
infected individuals. Systemic infections have been reported
3.1.2.1 Pathogenesis including peritonitis and invasive disease in AIDS patients.
In Aureobasidium, A. pullulans is the most important spe-
cies with respect of pathogenicity, but A. foliicola (L. hoff- 3.1.2.2 Hypersensitivity Pneumonitis
mannii), A. mansonii (E. castellanii), and A. slovacum (C. (Extrinsic Allergic Alveolitis)
slovaca) have occasionally been implicated as causative Aureobasidium pullulans is present in the environment and
agents of clinical conditions in compromised patients.16–18 exposure may result in sensitization to the organism and is
The latter three species are not related to other species of associated with the development of allergic diseases.20,29,30 A
Aureobasidia,8,22 and, regarding their close affinity to other study in a cohort of 405 children suggested that specific dust-
genera these species are discussed in detail elsewhere. borne fungi (Aspergillus and Aureobasidium pullulans) found
Individual cases of cutaneous phaeohyphomycosis, peritoni- at home in the first 3 months of life might be associated with
tis, and fungemia caused by H. dematioides in immunocom- an increased risk of developing allergic rhinitis by 5 years of
promised patients have been described.23–25 age.29 A further study showed that sensitivity to A. pullulans
Aureobasidium pullulans is an opportunistic pathogen was significantly associated with more severe asthma.31
able to cause disease in compromised patients (BSL-1). It A. pullulans has been associated with an outbreak of
may cause phaeohyphomycosis, keratomycosis, pulmonary hypersensitivity pneumonitis (extrinsic allergic alveolitis).32
mycosis with sepsis, peritonitis, and other opportunistic Hypersensitivity pneumonitis is a syndrome that is caused by
infections, as well as cutaneous mycoses such as eumycotic a broad spectrum of inhaled organic dusts or chemical prod-
dermatitis. Aureobasidium may also colonize hair, skin, and ucts that cause an immunologically mediated inflammatory
nails in humans, but its ability to penetrate healthy tissues response of the alveoli and bronchioles, frequently accompa-
is limited. Patients with an Aureobasidium blood stream nied by systemic symptoms. This expression depends on sev-
infection as a result of major trauma such as road traffic eral factors including immunological responsiveness of the
accidents exemplify the possibility of accidental inocula- host, intensity of the exposure, and antigenicity of the inhaled
tion of the pathogen into the host. Although the mechanism biological dust. Despite extensive studies, the exact immu-
of infection remains unknown, it is likely that inoculation nological mechanisms are not known. Antigen exposure is
occurs as open fractures come into contact with contaminat- associated with the presence of circulating IgG antibodies in
ing material like soil during the accident. After entering the exposed individuals. Various forms of hypersensitivity pneu-
body, the fungus may be capable of survival or dissemina- monitis have been described including ventilation hypersen-
tion, depending on the general condition and immunological sitivity pneumonitis caused by thermophilic actinbacteria,
status of the patient and the virulence of the fungal strain. A moulds such as Aspergillus fumigatus, and Aureobasidium
similar mechanism of infection is possible in immunocom- pullulans. Cases of hypersensitivity pneumonitis secondary
promised patients with peritoneal dialysis catheter, central to residential exposure to A. pullulans and other fungi pro-
venous line, or other piece of indwelling synthetic material. liferating in wet, contaminated building constructions have
A. pullulans has affinity for synthetic materials, for exam- also been described.32–34
ple, Silastic devices and indwelling catheters which, when
contaminated, may serve as the primary source and focus
3.1.2.3 Mycotic Keratitis
of infection. Removal of the contaminated device is gener-
ally needed to resolve the infection.26 The pathogenicity in Rare cases of keratomycosis (corneal and scleral ulcer)
healthy subjects is low and the fungus is commonly consid- caused by Aureobasidium pullulans have been described.
ered as a contaminant in clinical specimens. An illustrative case report describes a patient who devel-
Aureobasidium pullulans is an agent of phaeohyphomy- oped Aureobasidium pullulans keratitis following refractive
cosis.27 Clinically, it has been reported to cause a variety of laser epithelial keratomileusis (LASEK).35 The patient was
localized infections, including peritonitis (among patients on referred to a tertiary care center 1 month after LASEK for
peritoneal dialysis), cutaneous infection, pneumonia, men- the treatment of a corneal ulcer that was unresponsive to con-
ingitis, corneal and scleral infection, and abscesses in the ventional therapy. Mycology culture and direct microscopy
spleen and jaw (reviewed in Hawkes et al. and Richardson identified Aureobasidium as the infectious organism. The
and Warnock).26,28 The underlying conditions of patients infection responded well to treatment with topical natamycin
with reported Aureobasidium infection include carcinoma, and systemic itraconazole.
leukemia, chronic renal failure, diabetes mellitus, dissemi-
nated lymphoma, multiple traumatic injuries, organ trans- 3.1.2.4 Disseminated Infections,
plantation, congenital heart lesion repaired with intracardiac Fungemia and Peritonitis
prosthetic material, and pregnancy. Many human infections A. pullulans is a very rare cause of systemic infection in
by Aureobasidium have followed traumatic inoculation. humans. Disseminated systemic infection has been reported
Published reports have included keratitis, onychomycosis, in only four cases so far (reviewed in Joshi et al).36 Of the
cutaneous and subcutaneous phaeohyphomycosis, osteomy- four cases, one had acute myeloid leukemia, the second
elitis of the mandible after tooth extraction, and systemic patient had ovarian carcinoma (both had Hickman catheters

Aureobasidium 41
in situ), the third patient had met with a road traffic accident lesions, or other clinical specimens.28 Often, however, patients
with accidental inoculation of pathogen, and the fourth was have died and the infection has remained unrecognized until
a child with congenital heart disease who had undergone postmortem material obtained has been investigated.
closure of an atrial septal defect with a Goretex patch. Very
occasionally, cutaneous involvement by Aureobasidium 3.1.3.1 Conventional Techniques
in patients with systemic infection has been reported, for There are no descriptions of direct microscopy being applied
example, in kidney and liver transplant patients.36 to primary clinical specimens from cases of presumed
Nosocomial infections caused by A. pullulans are rare Aureobasidium infection. Aureobasidium can be isolated
and have been described only in some cases of peritonitis from clinical and environmental specimens using various
involving patients undergoing peritoneal dialysis and in one media (see Section 3.2). Histopathological features are simi-
case of severe infection where the fungus was isolated from lar to those seen for other agents of phaeohyphomycosis. The
a splenic abscess (reviewed in Bolignano and Criseo37). Even pigment produced by these fungi is also produced in vivo.
more uncommon are infections where A. pullulans can be The pigment is often a complex phenol-derived substance.
isolated from blood cultures. Catheter-related fungemia has Dark-walled, short, septate hyphae may be observed in H&E-
been reported.38 stained sections. If the pigment production is not prominent,
Cases of A. pullulans fungemia after allogeneic stem the use of periodic acid-schiff stain (PAS), Gömöri methe-
cell transplantation have been reported.36 In one case, the namine silver stain (GMS), or the Fontana-Masson silver
blood culture initially grew yeast-like colonies suggestive stain may be advantageous.39 There are no serological tests
of Candida species.36 However, on subculture, the charac- for the diagnosis of Aureobasidium infection.
teristic colony morphology (moist and creamy colonies in 2
days, which matured into shiny brownish black colonies with 3.1.3.2 Molecular Techniques
a gray fringe and pigment production) was suggestive of A. Diagnostics, taxonomic placement, and intraspecies variabil-
pullulans fungemia. The source of fungemia in this patient ity of Aureobasidium pullulans have been assessed by various
was the central venous catheter. molecular methods in addition to traditional morphological
A rare case of disseminated nosocomial fungal infection and physiological studies. These include fingerprinting tech-
was seen in a patient who was severely traumatized follow- niques like restriction enzyme analysis of polymerase chain
ing a road traffic accident.37 The patient was diagnosed with reaction (PCR)-amplified ribosomal DNA target area (PCR-
diffuse cerebral edema, hemoperitoneum, multiple fractures ribotyping or restriction fragment length polymorphism
of the left femur and tibia, a suspected pelvic fracture, and (RFLP)-PCR),4,40,41 universally primed PCR (UP-PCR),40,42
multiple ruptures of the liver’s right lobe, which made imme- arbitrary primed PCR (ap-PCR),43 random amplified poly-
diate surgery mandatory. After surgery, the patient underwent morphic DNA (RAPD-PCR),43,44 fluorescent amplified frag-
mechanical ventilation and prolonged total parenteral nutri- ment length polymorphism (fAFLP),45 and repetitive-element
tion. Aureobasidium pullulans was isolated from blood and PCR (rep-PCR) method,41 as well as sequence analysis of
urine samples. Isolation from urine was not achieved ini- rRNA genes and internal transcribed spacer (ITS) regions,
tially and became possible only after isolation from blood, and protein coding genes.6,7,9,41 Intraspecies variability has
thus suggesting a progressive colonization of the patient’s also been assessed using whole-cell protein analysis with
organs. The patient was treated with fluconazole 400 mg/day. sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Subsequently, all cultures became negative. The isolates from (SDS-PAGE) technique.41 Strain-specific sequence-charac-
this case were further identified as A. pullulans var. melanige- terized amplification region (SCAR) primers,46,47 species-
num on the basis of early black pigmentation of the colonies.2 specific oligonucleotide probes,48 and PCR primers49 have
This appears to be the first reported case of disseminated nos- been developed for the detection of A. pullulans, as well
ocomial fungal infection by A. pullulans var. melanigenum. as a quantitative PCR (qPCR) assay,50 see http://www.epa.
gov/nerlcwww/moldtech.htm. The applicability of some of
3.1.2.5 Antifungal Treatment these methods for clinical identification and strain typing of
There is no standard treatment for infection caused by A. pul- A. pullulans is discussed below.
lulans because of the paucity of human cases reported in lit- Apart from qPCR and other specific PCR protocols, the
erature. Amphotericin-B alone or in combination with azoles methods described here require the isolation of the microbe
has been tried with variable success. Combination therapy is in pure culture. Also a monoculture (in respect of fungi) in
probably the treatment of choice. The duration of treatment is normally sterile medium can serve as identification target. At
not certain, though most patients received antifungal treatment present there are no commercial tools for molecular diagnos-
for 4–8 weeks. Aureobasidium is susceptible to the majority tics of Aureobasidium.
of antifungal drugs with the exception of 5-fluorocytosine.17,26
3.1.3.2.1 Molecular Identification of
Aureobasidium pullulans
3.1.3 Diagnosis
Both DNA sequencing-independent and sequencing-based
The diagnosis of Aureobasidium infection is seldom sus- methods can be used to identify A. pullulans. Due to the
pected until the fungus is isolated from blood, cutaneous genetic distance between A. pullulans and other pathogens,

DNA-based separation is usually straightforward. This well as from related fungi. Most data is retrieved from ribo-
applies to the separation from other melanized hyphomyce- somal genes and spacers, the ITS region being most widely
tes, as well as from yeasts like Candida albicans, Candida represented (more than 150 sequences), followed by partial
parapsilosis, and Cryptococcus albidus, which resemble lsu (more than 85 seqs) and full or partial ssu rRNA genes
A. pullulans in young culture. Distinguishing A. pullulans (more than 45 seqs). DNA sequences from the genes elongase
from nonpathogenic relatives is more challenging, but sev- (ELO, 39 seqs), translation elongation factor (EF1α, 29 seqs),
eral unique genetic markers are nevertheless available. RFLP and β-tubulin (TUB, 29 seqs) are available, but reference data
analysis of a PCR amplicon containing partial large subunit from other fungal species is limited compared to ribosomal
(lsu or 28S) rRNA gene and ITS2 region with DdeI restric- targets. The disadvantage of the abundance of published ribo-
tion endonuclease creates unique banding pattern for A. pul- somal sequences is that public databases harbor also misan-
lulans, distinguishing it from phytopathogenic relatives.4,41,51 notated A. pullulans sequences as well as non-A. pullulans
For further identification of non-A. pullulans strains, sequences annotated as A. pullulans, and hence interpretation
enzymes RsaI, HhaI, MspI, and AluI can be used according of database query results must always be done with delibera-
to the scheme presented by Yurlova et al.4 Since restricted tion. (Data from EMBL Nucleotide database in April 2009.)
amount of reference information for this region is available, Several studies point out the potential of ITS region
the use of RFLP in diagnostics calls for further validation sequencing for fungal identification in clinical labora-
and may become obsolete for identification along with the tory.54–59 Due to the varying degree of inter- and intraspecies
reducing prices of more unambiguous DNA sequencing- variability of ITS region among different fungal clades, few
based methods. It may, however, be used for screening large general rules have been suggested concerning the interpreta-
numbers of isolates for epidemiologic purposes. When more tion of sequence comparison results. The intraspecies varia-
precise results of the identity of studied isolates are wanted, tion of the whole ITS region is below 3% in A. pullulans,
the ITS2 region of the obtained undigested PCR products can whereas the distance to closest relatives is 5% or more; ITS
be sequenced. region sequencing readily distinguishes A. pullulans from all
Species specific oligonucleotide probe for small subunit known pathogenic fungi, as well as from Hormonema spp.,
(ssu or 18S) rRNA gene was published for A. pullulans in Selenophoma mahoniae, Pringsheimia sp., Dothiora sp., and
1996.48 The probe has been used to detect A. pullulans by Kabatiella spp. apart from K. lini.7 ITS2 region is more vari-
fluorescent in situ hybridization on microscope slides and able than ITS1 in Aureobasidium and relatives,6 and can be
leaf surfaces,52 but it has not been reportedly used in clini- used for the identification alone or in combination with 5.8S
cal applications. Along with increasing amount of available rRNA gene and ITS1. Recently, a detailed ITS sequencing-
sequence data, the probe seems to be to some extent unspe- based protocol was presented for the wide-spectrum iden-
cific and the use of this probe in clinical settings calls for tification of fungi in clinical laboratory. This included fast
validation. Other species-wide A. pullulans-specific probes DNA extraction from pure culture, universal PCR, and ITS
have not been published so far in the scientific literature, yet sequencing followed by comparison against a novel com-
molecular data for the species suggests that this approach mercial database as well as against public DNA database
would be feasible. Recently, a PCR assay targeting ITS2 (GenBank).58 In evaluation study, the protocol provided an
region was described for species wide detection of endo- identical or more accurate identification compared to tra-
phytic A. pullulans in grapewine.49 Based on publicly avail- ditional identification in case of 84% of tested 244 random
able sequence data, the primer pair is prone to amplify most clinical isolates. The study included isolates that could not
strains in A. pullulans var. melanigenum with lowered effi- be identified to species level within 5 days by standard phe-
ciency compared to var. pullulans due to suboptimal match- notypic criteria, including two strains of A. pullulans. The
ing of the primers. database and data handling platform are commercially avail-
The U.S. Environmental Protection Agency (EPA) has able from SmartGene (SmartGene, Zug, Switzerland). The
developed validated qPCR assays for more than 130 major authors represent acceptable limits for sequence similarity
indoor air fungi, including A. pullulans. The assays are between analyzed and database sequences for identification
based on TaqMan chemistry.53 The primer-probe set tar- to species and genus level. The species-level limits can be
gets the ITS1 region, matching well with presently available applied to A. pullulans (see Section 3.2). ITS sequence com-
A. pullulans nucleotide data, and has been used to detect and parison has been used to identify a pathogen cultivated from
enumerate A. pullulans among other fungi in indoor envi- septic patient as A. pullulans.38 The authors report the identi-
ronments.21,50 The patented assays are available through a fication of two yeast-like isolates yielded from blood cultures
license. of two septic patients. For one isolate, an initial identification
Along with lowering costs of sequencing technology and using VITEK system (Yeast Biochemical Card, bioMerieux,
an increasing amount of reference data in public databases, France) revealed 90% similarity with Cryptococcus lauren-
DNA sequencing-based identification is becoming one of the tii. ITS sequencing revealed 100% similarity with A. pullu-
golden standards in mycology. At present, a relatively well lans for both of the strains.38
covering set of molecular data exists from A. pullulans in A quick, culture-independent detection of fungal patho-
public DNA databases, including DNA sequence information gens directly from clinical samples is achievable by a broad-
from about 200 distinct strains or isolates of the species, as range fungal real-time PCR reaction recently described by

Aureobasidium 43
Vollmer et al.60 The assay utilizes TaqMan chemistry and is and metabolic characteristics into the above-mentioned
designed for the variable D1/D2 region of lsu rRNA gene. varieties with high confidence.7 Even more precise sepa-
Species identification is attainable by sequencing the posi- ration is achieved by comparison of partial sequences of
tive PCR product, yet only genus-level identification can be the ELO gene.7 The genetic relationships of the four vari-
achieved in case of some species belonging to genera with eties as depicted by alignment of D1/D2 region of the lsu
little genetic variation within the region. Aureobasidium pul- rRNA gene are shown in Figure 3.2. Potentially novel enti-
lulans is detected by the assay with slightly lowered sensitiv- ties comprising tropical isolates of Aureobasidium were
ity due to suboptimal primer matching. The authors report recently characterized from Thailand using sequence
results consistent with cultivation-based identification and analysis of five gene loci. Combined data analysis of BT2,
a diagnostic turnaround time of 9 h for the identification RPB2, EF1-1α, and ITS sequences produced 12 well-
of fungal pathogens in cervical swabs and tracheal secre- supported phylogenetic clades. The studied strains shared
tion samples.60 The method is not quantitative. Traditional a good level of congruence in respect of colony characteris-
PCR targeting the same region has commonly been applied tics and level of pullulan production and xylanase activity
to identifying numerous ascomycetous yeasts as well as within each clade. The clades are separate from the var.
medically important zygomycetes and dematiaceous fungi, pullulans, but their relation to other entities remains to be
including A. pullulans.10,61–63 solved.13 Certain strains of A. pullulans are of commercial
interest due to their biocontrol abilities and production of
3.1.3.2.2 Molecular Subtyping of A. pullulans specific metabolites like the water-soluble polysaccharide
A. pullulans shows considerable variability in its vegetative pullulan and β1.3–1.6 glucans with reported beneficial
morphology and physiological properties.5 There is also a immunomodulatory properties.13,46
remarkable amount of genetic diversity inside A. pullulans Several papers describe the design of strain-specific
that differentiates between distinct varieties, and can also primer and probing systems for tracking biocontrol strains of
be used for strain-specific monitoring.7,41,45–47 Redefinition A. pullulans in agricultural environment, especially in phyl-
of A. pullulans and its varieties has recently been presented loplane.46,47,52,64 Meanwhile, the genetic properties of clinical
by Zalar et al.7 Data from multigene analysis supports the Aureobasidium isolates have not been assessed, and there is no
division of A. pullulans into two well-known entities, A. information of genotypic differences between clinical strains
pullulans var. pullulans and var. melanigenum, as well as and the rest of A. pullulans. According to available geno- and
into two novel varieties, var. subglaciale and var. namib- phenotypic data, practically all clinically significant strains
iae.7 Sequence comparison of variable D1/D2 region of of A. pullulans cluster to the var. melanigenum.10,37,38,56,60,65
lsu rRNA gene clusters strains with shared morphological This is consistent with the thermophilic nature and strong
A. pullulans var. pullulans CBS 584.75 NT [FJ150942] (+11)

A. pullulans var. pullulans CBS 109810 [FJ1509531]
A. pullulans var. pullulans dH 12637 [FJ150948]
A. pullulans MT 5 [AJ876763]
var. pullulans
A. pullulans var. pullulans EXF-1668 [FJ150949]
A. pullulans HA1556 [AJ507454]
97
A. pullulans MT 7 [AJ876762]
Aureobasidium pullulans Kabatiella lini CBS 125.21 [FJ150946]
79
80 A. pullulans var. nov. EXF-2481/CBS H-20186 [FJ150913] (+6) var. nov. subglaciale
A. pullulans var. nov. CBS 147.97 [FJ150937] var. nov. namibiae
A. pullulans var. Melanigenum dH 12740 [FJ150920]
67 87 A. pullulans var. Melanigenum CBS 105.22 NT [FJ150926] (+15)
var. melanigenum
A. pullulans SN22 [FJ515219] (+1)
53 96
A. pullulans UM16 [FJ515253]
88 Selenophoma mahoniae CBS 388.92 [EU754213]
Kabatiella caulivora CBS 242.64 [FJ150944]
94
Kabatiella microsticta CBS 114.64 [FJ150940]
Selenophoma linicola CBS 468.48 [EU754212]
100 Dothichiza pithyophila dH 12609 [FJ150969]
100 Pringsheimia smilacis CBS 873.71 [FJ150970]
Dothiora cannabinae AFTOL-ID 1359 [DQ470984]
0.005 Sydowia polyspora CBS 215.50 [FJ150968] (+2)
Figure 3.2 Neighbor joining tree of Dothioraceae showing relationships among varieties of Aureobasidium pullulans and relatives.
The taxon and strain name, sequence id (in square brackets), and number of additional strains with identical sequence presently available
(in brackets) are given for representative strains. Bootstrap values above 50% are shown.

pigmentation of A. pullulans var. melanigenum that may play at 14,000 rpm for 5 min. Transfer the supernatant
a role in increased pathogenicity of the variety.2,7 to clean Eppendorf tube, and add two volumes of
cold (−20°C) 96% ethanol. Mix gently. Incubate
in −20°C for 30 min to overnight. Centrifuge at
3.2 Methods
14,000 rpm for 5 min, discard supernatant, and wash
Like many fungi of low virulence, A. pullulans is considered the pellet twice with 500 μL cold 70% ethanol. Dry
a contaminant when isolated from a healthy host. It may be in room temperature until no liquid is visible on the
considered a pathogen when isolated from a normally sterile pellets (~5–30 min). Resuspend in 50 μL TE buffer
site, from multiple samples, in the presence of clinical signs (10 mM Tris–HCl, pH 7.5, 0.1 mM EDTA) contain-
of infection, or with pathological evidence of tissue-invasive ing 20 mg/mL RNase A. Incubate for 5–30 min at
disease.26 37°C to dissolve DNA. Adapted from Gerrits vd
Ende and de Hoog.67
3.2.1 Sample Preparation 2. Fastprep extraction. Collect 1 cm2 of mycelium
from plate culture with a disposable loop into 1 mL
3.2.1.1 In Vitro Pure Culture of TE buffer, pH 8.0. Add about 0.5 mL volume of
Aureobasidium can be isolated from clinical and environ- glass beads (0.5 mM Zirconia/Silica beads, BioSpec
mental specimens using various media, for example, half- Products, Inc., Bartlesville, OK). Disrupt the myce-
strength corn meal agar (CMA), rose bengal agar, dichloran lium using FastPrep instrument (QBiogene), set at
rose bengal chloramphenicol (DRBC), MEA, sabourad dex- number 4, for 30 s. Isolate DNA from the lysate
trose agar (SDA), or brain heart infusion agar with gentami- using QIAquick PCR purification kit (Qiagen)
cin and chloramphenicol, and identified on yeast malt extract according to manufacturer’s instructions. Adapted
agar (YMA), MEA, glucose peptone agar, SDA with gen- from Manitchotpisit et al.13
tamicin and chloramphenicol, potato dextrose agar (PDA), 3. FTA® filter extraction. Collect and suspend fun-
or blakeslee’s MEA.7,13,58 On MEA or glucose peptone agar, gal mycelium by scraping from culture plate to
Aureobasidium grows rapidly when incubated at 30°C, 0.2–1 mL of sterile water yielding a mixture of
reaching 30 mm in 1 week, appearing flat and smooth, and fungal cell mass and water in proportion of ca.
soon covered with slimy exudates. The optimum tempera- 1:10–20 v/v and vortex briefly to suspend. Transfer
ture for growth is 25°C, maximum near 35°C. The var. mela- 125 μL of the solution directly to a FTA Microcard
nigenum has higher optimum temperature for growth (30°C) (Whatman International Ltd.). Place the damp cards
than other varieties, and some strains can grow in 37°C. open in a microwave and subject to two cycles of
Aureobasidium does not grow on media containing cyclo- heating at 800 W for 30 s, with a pause of at least
heximide. It tolerates salt concentrations up to 10%–15%. In 30 s between cycles. Place a pyrex beaker contain-
the setting of fungemia, Aureobasidium can be isolated in ing 50 mL of sterile water to dissipate excess heat.
blood culture systems, for example, the Bactec 9240, using Store the treated filters in sterile plastic bags con-
pediatric aerobic culture media.26 taining desiccant. For use in PCR, remove a 2 mm
punch from FTA filter with Harris micro-punch
3.2.1.2 DNA Extraction (Whatman International Ltd.) into a PCR reaction
Alternative DNA extraction protocols suitable for putative tube and wash the filter twice for 1 min with 100 μL
Aureobasidium and other pathogenic fungal isolates are of Whatman FTA wash reagent and twice for 1 min
presented here. They differ in the utilization of in-house with 100 μL of TE buffer. Dry tubes with filters for
methods and reagents, or fast but more costly commercial 5 min at 55°C on a dry heat block. Add PCR reac-
products and equipment. All used equipment must be DNA tion mixture directly to the washed and dried filter
free. The methods below are for fungal pure cultures. For punch. The punches can be reused at least five times
DNA extraction from clinical samples and direct PCR detec- in a new PCR reaction with same or different prim-
tion of fungi, see Vollmer et al.60 For additional information ers without carryover of primers or products from
about DNA extraction procedures, see Hebart et al.66 the first reaction, after washing and drying the filter
as described before. Adapted from Borman et al.68
1. Cetyltrimethylammonium bromide (CTAB) extrac-
tion. Collect 1 cm2 of mycelium from plate culture 3.2.2 Detection Procedures
with disposable loop and transfer to 2 mL Eppendorf
tube containing 2:1 mixture of silicagel and Celite 3.2.2.1 Morphological Identification
545 and 300 μL CTAB buffer (Tris–HCl 200 mM, The colonies are cream or pink and then later becoming
pH 7.5, 200 mM Na-EDTA, 8.2% NaCl w/v, 2% brown or black (see Section 3.1.1.2). The microscopical mor-
CTAB w/v). Grind with micropestle for 1–2 min. phology of Aureobasidium, particularly the synchronous
Add 200 μL of CTAB buffer. Shake to homoge- blastoconidial formation, is best studied utilizing the Dalmau
nize. Incubate in 65°C water bath for 10 min. Add plate method for demonstrating chlamydospores in Candida
500 μL of chloroform and vortex briefly. Centrifuge albicans. Aureobasidium is differentiated from Hormonema

Aureobasidium 45
dematiodes by synchronous blastoconidia produced only ITS4 for ITS region and NL4 for lsu D1/D2, or with both
from hyaline hyphae. However, Yurlova et al. have noted forward and reverse primers to get sequence from both DNA
that it is often difficult to distinguish between these modes of strands, for best fidelity.
conidiogenesis and they suggested differences in the number
of conidiogenesis loci and physiological tests as methods for 3.2.2.4 Phylogenetic Analysis of Obtained Sequences
differentiation.40 These differences, however, are also small Compare the obtained sequences with published DNA
and variable. A. pullulans and H. dematioides are easily dis- sequences using online Blast or Fasta alignment tools (http://
tinguishable by genotype, for example, by means of DNA blast.ncbi.nlm.nih.gov/Blast.cgi and http://www.ebi.ac.uk/
sequencing or RFLP analysis.6 Tools/fasta33/nucleotide.html) against GenBank or EMBL
DNA database. Note that Blast alignment easily “drops out”
3.2.2.2 PCR Amplification of rDNA the perimeters of the alignment if lower similarity occurs on
Perform amplification of ribosomal DNA target in 50 μL the area, thus giving a partial alignment with high percentage
reaction volume in 250 μL reaction tubes or plates. Prepare identity. Fasta is slower compared to Blast but performs full
a PCR reaction mastermix for samples and negative con- alignment for the target sequence. High similarity percent-
trol reaction, containing 1 U of DNA polymerase (e.g., ages (>99%) with reference sequences from authenticated
DyNAzyme™ II DNA polymerase, Finnzymes, Espoo, strains should be emphasized in interpretation of the results.
Finland), 1× solution of matching reaction buffer, 250 μM See Figure 3.2 for reference strains. An ITS sequence from
of each dNTP, and 50 pmol of each primer per reaction. A. pullulans is supposed to have a similarity of 97%–100%
Divide into 49 μL aliquots and add 1 μL of genomic DNA with preferably two or more independent A. pullulans data-
extract for template. Use following thermal conditions: ini- base sequences. The D1/D2 lsu rRNA sequences of isolates
tial denaturation for 2 min in 95°C followed by 40 cycles belonging to same variety generally show 99%–100% simi-
of denaturation at 95°C for 30 s, annealing at Tann for 45 s larity, whereas the distance between varieties is generally
and elongation at 72°C for 1 min, followed by a final elon- 2%–3%. For ITS-sequencing-based identification of non-A.
gation in 72°C for 10 min in thermocycler apparatus with pullulans strains, guidelines given by Ciardo et al.58 are rec-
heated lid. For amplification of ITS region, use prim- ommended to be followed.
ers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
(5′-TCCTCCGCTTATTGATATGC-3′)69 and Tann of 50°C. 3.2.2.5 RFLP Analysis of PCR Products
For the D1/D2 region of lsu rDNA gene, use primers NL Digest the obtained 5.8SR-LR7 PCR products (10 μL per
1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL digestion) with DdeI and optionally with HhaI, MspI, and
4 (5′-GGTCCGTGTTTCAAGACGG-3′)61 and Tann of 52°C. RsaI restriction endonucleases according to manufacturer’s
For PCR-RFLP analysis, perform PCR protocol with prim- instructions. Amplify and digest corresponding fragment in
ers 5.8SR (5′-TCGATGAAGAACGCAGCG-3′) and LR7 parallel from representative, authenticated reference strains.
(5′-TACTACCACCAAGATCT-3′).51 Use elongation time of Separate the digests in 2% agarose gel with appropriate
2 min instead of 1 min at 72°C and Tann of 50°C. Visualize size standard. Stain with ethidium bromide. DdeI produces
the products by electrophoresis in 1% agarose gel with ethid- unique digestion pattern for A. pullulans, whereas other
ium bromide staining (0.5 μg/mL). The expected products enzymes distinguish between Hormonema sp. and other rela-
sizes are 581–582 bp for ITS1–ITS4, 614 bp for NL1–NL4, tives of Aureobasidium.4
and ∼1900 bp for 5.8SR-LR7. The primers are universal fun-
gal, that is, amplify the target region from all fungal species. 3.3 C
onclusions and Future
Purify the products for sequencing and RFLP application
Perspectives
with, for example, QIAquick PCR purification kit (Qiagen).
In case of failed amplification, try diluting the template With increasing survival of immunocompromised patients,
DNA in 1:100 and 1:1000 in sterile water, use more efficient the significance of rare and weakly pathogenic fungi
DNA polymerase (e.g., Phusion® high fidelity DNA poly- becomes more obvious. During past years, several species
merase, Finnzymes) or repeat DNA extraction with different with some pathogenicity to humans have been affiliated to
protocol. the genus Aureobasidium, but apart from A. pullulans, these
have later been shown to be more closely related to other
3.2.2.3 DNA Sequencing genera. Aureobasidium pullulans represents an opportunis-
Comparison of ITS region sequence is suitable for spe- tic fungus that only occasionally causes disease, but it has
cies identification of putative A. pullulans. Varieties are been reported to be involved in serious conditions includ-
most reliably resolved using the variable D1/D2 region of ing pneumonia, meningitis, and systemic fungemia. The
lsu rRNA gene or ELO gene.7 The most popular method diagnosis of Aureobasidium traditionally relies on observ-
for sequencing is enzymatic Sanger sequencing, using, for ing characteristic macro- and micromorphology, yet these
example, BigDye™ 3.1 chemistry of Applied Biosystems characteristics vary by strains and Aureobasidium is often
(Life Technologies, Carlsbad, CA). Sequencing is available mixed up with other black yeast-like fungi or even yeasts.
as commercial service. The obtained PCR products can be A. pullulans is of commercial interest due to its biocon-
sequenced using one of the primers used in PCR, that is, trol abilities and production of various metabolites. In this

perspective, the fungus is relatively well-characterized and 17. de Hoog, G.S., Guarro, J., Gené, J., and Figueras, M.J., Atlas
the taxonomy, identification, and subspecific typing of agri- of Clinical Fungi, a pilot CD-ROM version of the 3rd edi-
cultural, biotechnological, and environmental strains have tion, Centraalbureau voor Schimmelcultures, Baarn, the
Netherlands, 2009.
been assessed in numerous studies. In contrast, there are
18. Svobodova, Y., Chmelia slovaca gen. nov. a dematiaceous
few studies that involve both clinical aspects, and phylog- fungus, pathogenic for man and animals, Biologia (Bratisl),
eny or development of molecular identification methods. 21, 81, 1966.
No commercial products are presently available for specific 19. Cooke, W.B., An ecological life history of Aureobasidium
detection of A. pullulans. Molecular methods like prob- pullulans (De Bary) Arnaud, Mycopathologia, 12, 1, 1959.
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21. Vesper, S.J. et al., Relative moldiness index as predic-
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4 Bipolaris and Drechslera
Dongyou Liu and Joanna Gray
Contents
4.1 Introduction........................................................................................................................................................................ 49
4.1.1 Classification and Morphology............................................................................................................................... 49
4.1.2 Clinical Features and Pathogenesis........................................................................................................................ 50
4.1.2.1 Cutaneous infections................................................................................................................................ 50
4.1.2.2 Mycotic keratitis...................................................................................................................................... 50
4.1.2.3 Rhinosinusitis.......................................................................................................................................... 50
4.1.2.4 Other Infections....................................................................................................................................... 51
4.2 Methods.............................................................................................................................................................................. 52
4.2.2.1 Sequence Analysis of ITS Regions.......................................................................................................... 52
4.2.2.2 Sequence Analysis of D1–D2 Region of 28S rRNA . ............................................................................. 53
4.3 Conclusion.......................................................................................................................................................................... 53
References.................................................................................................................................................................................... 53
4.1 Introduction Dothideomycetes, subphylum Pezizomycotina, phylum

Ascomycota, and kingdom Fungi. The family Pleosporaceae
The term “dematiaceous fungi” refers to a heterogeneous covers the genera of Brachycladium, Cochliobolus, Crivellia,
group of darkly pigmented fungal organisms, which derive Decorospora, Edenia, Lewia, Macrospora, Macroventuria,
their coloration from melanin in the cell walls, and produce mitosporic Cochliobolus, mitosporic Pleosporaceae, Pleos
brown yeast-like cells, pseudohyphae, and irregular true pora, Pyrenophora, Setosphaeria, and some unclassified
hyphae in tissues. Being commonly present in the soil, plants, Pleosporaceae; the mitosporic Cochliobolus group is made
and other environments, dematiaceous fungi are introduced up of the genera of Bipolaris and Curvularia. The genus
into human hosts through inhalation, or after injury and other Bipolaris (teleomorph: Cochliobolus) is further divided into
traumatic events, causing a range of superficial and deep 33 recognized species and 36 unassigned species [1].
infections that are collectively known as chromoblastomyco- Bipolaris spp. are primarily plant pathogens; how-
sis, eumycetoma, and phaeohyphomycosis. ever, several (i.e., Bipolaris spicifera [obsolete synonyms:
Currently, over 130 dematiaceous fungal species belong- Curvularia spicifera, Brachycladium spiciferum, Drechslera
ing to 70 genera have been implicated in human infections. spicifera, and Helminthosporium spiciferum], Bipolaris
While both chromoblastomycosis and eumycetoma are asso- hawaiiensis [obsolete synonyms: Drechslera hawaiiensis
ciated with a relatively small group of dark-walled fungi, the and Helminthosporium hawaiiensis] and Bipolaris aus-
former is characterized by the formation of sclerotic bodies in traliensis [obsolete synonyms: Drechslera australiensis and
tissue and is usually seen in tropical areas, and the latter is a Helminthosporium australiensis]) have been implicated in
deep tissue infection characterized by the presence of mycotic human infections [2].
granules, usually of the lower extremities. On the other hand, Bipolaris colonies grow moderately fast and are effuse,
phaeohyphomycosis is generally reserved for the remainder grey to blackish brown, suede-like to floccose with a black
of clinical syndromes (ranging from superficial infections, reverse. Pale brown pigmented, pseudoseptate conidia
allergic disease, pneumonia, cerebral infection to dissemi- are produced through pores (poroconidia) in a sympodi-
nated disease) caused by a large number of dematiaceous ally elongating geniculate (or zigzag) conidiophore and
fungi including members of Bipolaris and Drechslera genera. are straight, fusiform to ellipsoidal, rounded at both ends,
smooth to finely roughened, and germinating only from the
ends (bipolar) [3].
4.1.1 Classification and Morphology
At the species level, B. spicifera conidia are cylindri-
The genus Bipolaris belongs to the mitosporic Cochliobolus cal with a broadly rounded base, have three septa and
group, family Pleosporaceae, order Pleosporales, class a distinctly raised hilum, which shows a zone of dark
49

pigmentation. B. australiensis conidia are ellipsoidal with The curvature in Bipolaris conidia involves a slight change
a more or less tapering base, which becomes truncated in the central cell, whereas those in Curvularia conidia are
because of the hilum, and 20% of its conidia are with four due to the enlargement of the central cell. Thus, Bipolaris
or five septa. In addition, B. spicifera conidia are slightly is distinguishable from Curvularia by the absence of an
broader than those of B. australiensis, and B. spicifera enlarged cell in its curved conidia.
isolates have a small hyaline area just above the hilum,
which is absent in B. australiensis. B. hawaiiensis conidia
are approximately 6.8 by 23.3 μm and have predominantly 4.1.2 Clinical Features and Pathogenesis
four or five septa [2]. Moreover, B. spicifera is differenti- Bipolaris and Drechslera are dematiaceous (dark-walled)
ated from B. australiensis by its thinner cell wall, from B. fungi that are occasionally involved in human infections
hawaiiensis by its production of swollen conidia with 3 to including cutaneous infections, sinusitis, keratitis, peritoni-
7 septa, and from B. papendorfii by its being curved and tis, allergic bronchopulmonary disease, brain abscess, men-
broadest at the second cell. ingitis, fatal fungal endarteritis, fungemia, and disseminated
The genus Drechslera belongs to the mitosporic Pleospo fungal disease [5–29].
raceae group in the family Pleosoraceae, and contains Bipolaris spicifera is the most commonly reported human
the genera Alternaria, Dendryphiella, Dendryphion, pathogenic Bipolaris species, especially in subtropical and
Drechslera, Embellisia, Exserohilum, Nimbya, Pithomyces, tropical regions (e.g., Texas, South Carolina, Arizona, and
Pyrenochaeta, Stagonospora, Stemphylium, Ulocladium, Georgia in the United States; Brisbane, Australia; Pakistan;
and Unifilum. At present, there are 20 recognized species and India). Other less common Bipolaris spp. associated
and 11 unassigned species within the genus Drechslera (tele- with human disease are Bipolaris hawaiiensis and Bipolaris
omorph: Pyrenophora) [1]. australiensis.
Drechslera spp. are generally present in soil and plants
although one of its members, i.e., Drechslera biseptata, has 4.1.2.1 Cutaneous infections
been reported recently from a brain abscess. Many previous Bipolaris spicifera has been reported as the causative agent
Drechslera or Helminthosporium isolates from human and of cutaneous infections in both immunocompetent and
animal cases have been shown to actually belong to the gen- immunocompromised individuals [30–34]. Bilu et al. [35]
era Bipolaris or Exserohilum [2]. documented a case of Bipolaris spicifera-related cutaneous
Drechslera colonies grow rapidly and are suede-like to fungal infection on the left cheek of a 5-year-old boy with
downy, brown to blackish brown with a black reverse. Conidia B-precursor-cell acute lymphoblastic leukemia.
are pale to dark brown, usually cylindrical or subcylindrical,
straight, smooth walled, and are formed apically through a 4.1.2.2 Mycotic keratitis
pore (poroconidia) in a sympodially elongating geniculate Bipolaris spp. have also been shown to cause mycotic
conidiophore. Conidia are transversely septate (phragmoco- keratits [36–43]. Bashir et al. [44] described a Bipolaris
nidia), and the hilum is not protuberant [3]. hawaiiensis-related corneal ulcer with abscess and hypo-
As closely related members of the mitosporic Cochliobolus pyon in an immunocompetent male following trauma with
group and the mitosporic Pleosporaceae group within the rice stalk to the left eye. Direct examination of the corneal
family Pleosporaceae, Bipolaris (teleomorph: Cochliobolus), scrapings showed septate, branched fungal hyphae. Culture
Curvularia (teleomorph: Cochliobolus), Drechslera (tele- of the scrapings on blood agar and Sabouraud dextrose agar
omorph: Pyrenophora), and Exserohilum (teleomorph: yielded Bipolaris hawaiiensis.
Setoshaeria) are differentiated through a combination of
characters such as conidial shape, the presence or absence 4.1.2.3 Rhinosinusitis
of a protruding hilum, the contour of the basal portion of Allergic fungal sinusitis (AFS) is a noninvasive form of fun-
the conidium and its hilum, the point at which the germ tube gal rhinosinusitis accounting for 6%–9% of all rhinosinusitis
originates from the basal cell, and the sequence and location requiring surgery. Patients with AFS typically show chronic
of the first three conidial septa. rhinosinusitis with nasal polyps, inhalant atopy, elevated
Specifically, Bipolaris conidia are fusiform ellipsoi- total serum immunoglobulin E (IgE), and sinus-obstructing
dal and central cells are not much darker and broader than inspissates of a characteristic extramucosal “peanut buttery,”
the distal ones; hilum is not protuberant and germination viscoelastic, eosinophil-rich, hyphae-containing material
is bipolar. Drechslera conidia are cylindrical, germinating called “allergic mucin.” Sinus computer tomography (CT)
from any cell, and hilum is not protuberant. Curvularia shows findings of chronic rhinosinusitis that often include
conidia have two to three broader and darker central cells, central areas of increased contrast (“hyperattenuation”)
often curved, with or without a prominent hilum; germina- within abnormal paranasal sinuses due to the presence of
tion is bipolar. Exserohilum conidia are fusiform cylindri- fungal-containing allergic mucin. AFS allergic mucin cul-
cal to obclavate, with a protuberant hilum; germination is tures are often positive for either dematiaceous fungi such
bipolar [4]. as Bipolaris spicifera or Curvularia lunata, or Aspergillus
Furthermore, in common with Curvularia, some Bipolaris species such as A. fumigatus, A. flavus or A. niger [45,46].
spp. also produce curved conidia with hyaline apical cells. Indeed, Bipolaris spp. are increasingly recognized as the

Bipolaris and Drechslera 51
cause of fungal sinusitis in humans with the sphenoid and Gadallah et al. [14] documented a case of Bipolaris
posterior ethmoid sinuses being most often involved followed hawaiiensis peritonitis in a 73-year-old female on continu-
by the anterior ethmoid sinus, frontal sinus, and maxillary ous cyclic peritoneal dialysis (CCPD) with a nonfunctioning
sinus involvement [47–58]. peritoneal catheter. The catheter harbored characteristic dark
Buzina et al. [59] presented a detailed description of gray particles consisting of a fungal ball within the lumen of
Bipolaris spicifera-associated rhinitis in a 19-year-old the catheter. Microscopic examination confirmed the organ-
immunocompetent man after the patient presented with ism attached to the inner wall of the catheter. The patient was
restricted nasal breathing. A CT scan showed massive treated by removing the catheter and the administration of a
sinusitis with significant decalcification and destruction of 2 week course of oral itraconazole 100 mg twice daily (with-
the bone at skull base. An endoscopic examination revealed out using amphotericin B or ketoconazole).
a total obstruction of both nostrils with glassy polyps. The In combination with surgical debridement Bipolaris infec-
immunological examination of the patient’s blood dem- tions are often treated with amphotericin B, which is the
onstrated a highly elevated level of total IgE. Histological antifungal drug of choice for chronic invasive sinusitis [7].
examinations of formalin-fixed and paraffin-embedded Ketoconazole, itraconazole, voriconazole, and natamycin may
tissue and mucus samples resected/collected from sinus also be utilized [8,23,41]. Itraconazole is a highly effective
showed inflammatory sinonasal polyps and clusters of antifungal agent for chromoblastomycosis and subcutaneous
eosinophilic granulocytes within the mucus. Gomori’s phaeohyphomycosis; ketoconazole is useful for mycetoma.
methenamine silver (GMS) staining uncovered septate fun- Postoperative oral corticosteroids and antiallergic inflamma-
gal hyphae in the fungal masses (fungus balls) as well as tion therapy may also be prescribed for AFS [62,63].
within the mucus, although a fungal invasion of the tissue
was not observed. Culture of the mucus on Sabouraud dex-
4.1.3 Laboratory Diagnosis
trose agar at 25°C showed fast-growing colonies, reaching a
diameter of 4 cm in 7 days. Colonies were velvety, brown- Dematiaceous (brown- or dark-pigmented) fungi are a het-
ish, and flat. Microscopic observation showed septate, pig- erogeneous group of molds that are classified in the gen-
mented hyphae and unbranched, zigzagged conidiophores era Alternaria, Bipolaris, Cladophialophora, Curvularia,
with thick-walled, darkly pigmented, cylindrical conidia Drechslera, Exserohilum, Exophiala (Wangiella), Fonsecaea,
(predominantly with three septate). The fungus was iden- Madurella, Phialophora, Scedosporium, and Scytalidium.
tified as Bipolaris spicifera, which was further confirmed These fungi are widespread in soil, wood, and decomposing
by sequencing analysis of the internal transcribed spacer plant debris and are opportunistic human pathogens causing
(ITS) region of the ribosomal gene cluster using fungus- phaeohyphomycosis, chromoblastomycosis, and eumycotic
specific primers. Indeed, the resulting 575 bp amplicon mycetoma.
demonstrated a 100% identity with Cochliobolus spiciferus, Traditionally, dematiaceous fungi are identified on the
which is the teleomorph of Bipolaris spicifera. In a separate basis of morphological characteristics such as the presence
study, Castelnuovo et al. [53] described a phaeohyphomy- of annellides (Phaeoannellomyces, Exophiala), phialides
cotic sinusitis due to Bipolaris hawaiiensis in an immuno- (Phialophora, Wangiella), or adelophialides (Phialemonium
logically competent patient. The patient was successfully without collarettes, Lecythophora with collarettes); dif-
treated by surgical drainage and amphotericin B. ferentiation of conidiophores (Xylohypha, Cladosporium)
Dyer et al. [60] reported a Bipolaris australiensis–asso- and conidial hilum, septation and germination (Bipolaris,
ciated allergic bronchopulmonary disease in a 40-year-old Drechslera, Exserohilum) [2]. Fontana-Masson stain is help-
white male with past history of allergic rhinitis and asthma. ful for distinguishing the pigmented dematiaceous organisms
The patient presented with back pain and cough. CT scan of from other septated fungal forms [45]. For histological exam-
the chest showed a mass in the lower lobe of the right lung. ination, GMS and hematoxylin and eosin (H&E) stains may
Culture of the bronchial biopsy and lavage yielded a pure be employed. Enhancement of sporulation by growth of spe-
isolate of Bipolaris australiensis that was confirmed by DNA cies of Bipolaris, Curvularia, Drechslera, and Exserohilum
technique. on cellulose substrates may facilitate their identification in
culture and production of spores at relatively high concentra-
4.1.2.4 Other Infections tions [64].
Kobayashi et al. [61] described a Bipolaris spicifera-related Biochemical tests (e.g., 12% gelatin test, nitrate assimila-
case of disseminated infection in an immunocompetent tion) and determination of growth temperature (e.g., 37°C for
male. Histopathological studies of lymph node, lung, and Exophiala jeanselmei, 40°C for Exophiala [Wangiella] der-
liver biopsy specimens revealed a dark pigmented, granu- matitidis and Bipolaris spicifera, 42°C for Xylohypha ban-
lar fungal structure inside the granuloma. Culture of lymph tiana, and 45°C for Dactylaria constricta var. gallopava and
node specimen grew a fungal isolate that was identified as Scedosporium inflatum) may also be useful for their differen-
Bipolaris spicifera on the basis of morphology and molecular tiation [65]. Fluorescent halogen immunoassay (fHIA) may
analysis. The patient responded to combination therapy with be utilized for examination of mycoaerosols from individuals
intravenous amphotericin B and voriconazole, but the infec- with exposure and sensitization to fungal allergens in indoor
tion reoccurred during oral itraconazole. environments. Indeed, Bipolaris conidia were commonly

detected from these individuals by this immunostaining pro- the supernatant is decanted. The pellet is washed with ice-
cedure [66]. cold 70% ethanol twice and dried by using a vacuum dryer.
In recent years, molecular techniques have been applied The powder is resuspended in 48.5 μL of Tris-EDTA buffer
for identification of dematiaceous fungi including Bipolaris with 1.5 μL of 10 mg of RNase/mL, incubated at 37°C for
and Drechslera [58,67]. Polymerase chain reaction (PCR) 15–30 min, and stored at −20°C until use [71].
amplification and sequencing analysis of rRNA internal tran-
scribed spacer (ITS) regions allow rapid and precise diagno-
sis of fungal infections in humans [67–69]. 4.2.2 Detection Procedures
4.2.2.1 Sequence Analysis of ITS Regions
Pounder et al. [70] described a real-time PCR with SYBR
4.2 Methods green DNA-binding dye and amplicon melting temperature
4.2.1 Sample Preparation analysis for fungal detection using pan-fungal primers ITS1
forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
Specimens may be stained with lactophenol cotton blue stain reverse (5′-TCCTCCGCTTATTGATATGC-3′) [72]. The iden-
or periodic acid-Schiff (PAS) stain and examined under the tity of the fungi is verified by subsequent sequencing analysis.
microscope for mycotic elements. Portions of samples are
grown on inhibitory mold agar, modified Sabouraud agars, Procedure
or potato dextrose agar at 25°C and 35°C.
After growth of 1–7 days on potato dextrose agar slants, 1. PCR mixture is composed of 1× Lightcycler FastStart
mycelia are collected by scraping the slant with a sterile DNA Master Hybridization Probes mixture (Roche
stick in 1 mL of sterile water. The material is transferred to Applied Science) (containing deoxynucleoside tri-
a 2 mL screw-cap tube. The tubes are centrifuged for 1 min phosphates, FastStart Taq DNA polymerase, and
at 6000 × g. If the mycelia do not pellet, the material is con- 1 mM MgCl2, additional MgCl2 is added to a final
tained with a pediatric blood serum filter (Porex Corp). The concentration of 4.6 mM), 0.4 μM each of ITS1 for-
supernatant is removed and the material is resuspended in ward and ITS4 reverse primers, 1× SYBR green
200 μL of IDI sample buffer and transferred to the lysis tube (Molecular Probes), and 3 μL template DNA.
containing glass beads. The lysis tubes are vortexed on the 2. Thermal cycling parameters include 95°C for
highest setting for 5 min. The tubes are then placed in a boil- 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and
ing water bath for 15 min. The tubes are centrifuged for 5 min 76°C for 30 s; and a final extension at 72°C for 2 min.
at 16000 × g. The supernatant is stored at −20°C until ampli- 3. The quality of the amplicon is determined using the
fication [70]. derivative of the melt analysis curve (55°C–99°C,
Alternatively, about 1 cm2 of fungal material is trans- 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
ferred to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) (Corbett Robotics, Inc).
mixture of silica gel and Celite (silica gel H, Merck 7736/ 4. The amplified product is purified for bidirectional
Kieselguhr Celite 545; Machery) and 300 μL of TES buffer sequencing using ExoSAP-IT (USB Corp). Five
(2 g Tris [hydroxymethyl]-aminomethane, 0.38 g Na-EDTA microliters of Big Dye Terminator Ready Reaction
[ethylenediaminetetraacetic acid], and 2 g sodium dodecyl Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
sulfate in 80 mL of ultrapure water [pH 8]). The fungal mate- each primer (0.8 pmol/μL) and 3 μL of purified PCR
rial is ground with a micropestle for 1–2 min. The volume product. Cycle sequencing is performed with a 9700
is adjusted by adding 200 μL of TES buffer. After vigorous thermal cycler (ABI), using 25 cycles of 96°C for
shaking and the addition of 10 μL of a 10 mg/mL concentra- 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
tion of proteinase K to the tube, the mixture is incubated reaction products are passed through a Sephadex
at 65°C for 10 min. With the addition of 140 μL of 5 M G-50 fine column to remove unincorporated dye ter-
NaCl solution, the mixture is combined with 1/10 volume minators. Purified sequencing reaction products are
(∼65 μL) of 10% CTAB (cetyltrimethylammonium bromide) run on an ABI Prism 3100 Genetic Analyzer with a
buffer, followed by incubation for another 30 min at 65°C. 50 cm capillary array.
One volume (∼700 μL) of chloroform-isoamyl alcohol (vol/ 5. Sequences are analyzed with the SmartGene
vol = 24/L) is added and mixed by inversion. After incuba- Integrated Database Network software version
tion for 30 min at 0°C (on ice water) and centrifugation at 3.2.3 vr. SmartGene is a web-based software and
14,000 rpm at 4°C for 10 min, the top layer is transferred to database system with reference sequences derived
a clean Eppendorf tube. The sample is added with 225 μL from the National Center for Biological Information
of 5 M NH4-acetate and incubated for at least 30 min (on (NCBI) GenBank repository.
ice water) and centrifuged. The supernatant is transferred
to a clean sterile Eppendorf tube and mixed with a 0.55 Note. In case that real time PCR instrument is not avail-
volume (∼510 μL) of ice-cold isopropanol. After centrifuga- able, standard PCR may be performed with primers ITS1
tion for 7 min at 14,000 rpm and 4°C (or room temperature), and ITS4, and the resulting amplicon is sequenced with the

same primers. Sequence-based identifications are defined 3. Kwon-Chung, K.J. and Bennett, J.E. Medical Mycology. Lea
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69. Bagyalakshmi, R. et al. Newer emerging pathogens of ocu- 72. White, T.J. et al. Amplification and direct sequencing of fun-
lar non-sporulating molds (NSM) identified by polymerase gal ribosomal RNA genes for phylogenetics. In M.S. Innis and
chain reaction (PCR)-based DNA sequencing technique tar- D.H. Gelfand (Eds.), PCR Protocols: A Guide to Methods and
geting internal transcribed spacer (ITS) region. Curr Eye Res. Applications. Academic Press, New York, pp. 315–322. 1990.
2008;33:139–147. 73. Leaw, S.N. et al. Identification of medically important yeast
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fungi identified as nonsporulating molds. J Clin Microbiol. regions. J Clin Microbiol. 2006;44:693–699.
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5 Botryomyces
Contents
5.1 Introduction........................................................................................................................................................................ 57
5.1.3 Diagnosis................................................................................................................................................................ 58
5.2 Methods.............................................................................................................................................................................. 59
5.3 Conclusions......................................................................................................................................................................... 59
References.................................................................................................................................................................................... 59
5.1 Introduction by the formation of black cauliflower-like colonies consisting

of densely aggregated thick-walled cells. Although colonies
‘Dematiaceous fungi’ is a colloquial, nontaxonomic term of microcolonial fungi belonging to the different taxa are
used to describe a heterogeneous group of fungal organisms indistinguishable morphologically, upon isolation on suit-
that have melanized cell walls. The presence of melanins in able growth media, microcolonial fungi readily develop into
these fungi not only accounts for their dark-green, brown, various morphologies, allowing genus- and species-specific
or black color, but also enhances their survival under stress- identification.
ful conditions. Further morphological, physiological, and
biological adaption has endowed these organisms with the
ability to tolerate desiccation, temperature, and osmolar- 5.1.1 Classification and Morphology
ity changes. Because of their distinct morphological and The genus Botryomyces (Botrys, Greek for bunch of grapes,
biological features, dematiaceous fungi are separated into + mykes, Greek for fungus) is a meristematic black fun-
black yeasts, meristematic fungi, microcolonial fungi, and gus belonging to the mitosporic Dothideomycetes group,
other darkly pigmented fungi, with each of these categories class Dothideomycetes, subphylum Pezizomycotina,
encompassing multiple fungal taxa of its own. phylum Ascomycota, and kingdom Fungi [1]. The mito-
Black yeasts are characterized by the production of daugh- sporic Dothideomycetes group is divided into nine gen-
ter cells through yeast-like multilateral or polar budding. era: Asteromella, Botryomyces, Cenococcum, Cryomyces,
Many black yeasts also demonstrate mycelial growth and Cyclothyrium, Cystocoleus, Racodium, Sclerostagonospora,
generate conidia (either unseptated or containing three trans- and Seifertia. The genus Botryomyces contains a single spe-
versal septa) from phialides (ranging from simple phialides, cies Botryomyces caespitosus, which was first described
phialides with collarettes, annelated phialides, to rhachides) from human skin lesions [2]; Botryomyces angioformans is
or undifferentiated conidiogenous cells. a doubtful species from an unconfirmed disease of which no
Meristematic fungi form aggregates of thick-walled, material has been preserved [3].
densely melanized cells and grow by isodiametric cell wall B. caespitosus colonies are pink when young and form
expansion and division. Propagules are released by break- restricted, meristematic cell clumps that disarticulate irregu-
ing apart of aggregates or by endogenous conidiogenesis that larly. Meristematic growth is characterized by the produc-
subsequently disrupts the mother cell wall. Some may form tion of swollen isodiametric cells with thick cell walls, in
blastic conidia from yeast-like budding cells and thus may which melanin is deposited. The fungus produces multi-
be regarded as black yeasts. Meristematic fungi are found on celled, irregularly septate, thick-walled spores, which may
the exposed surfaces of desert rock, outdoor statues, leathery be regarded by some to be vegetative structures [2].
plant leaves, and Antarctic rock, as well as in hypersaline The genus Botryomyces is one of the 25 genera of meriste-
coastal ponds. They have the potential to erode and destroy matic black fungi that inhibit cracks in marble and rock sur-
marble, sandstone, and glass. faces in the Mediterranean Basin (Italy, Spain) and Ukraine.
Microcolonial fungi refer to the in situ growth pattern of They are associated with biodeterioration of monuments,
the meristematic fungi and some black yeasts on mineral sculptures, and archaeological objects. Because meriste-
substrates (e.g., rock, glass, or metal), which is characterized matic black fungi present characteristics comparable to those
57

of fungi isolated from deserts rocks, they are occasionally dissemination of propagules. The fungus is responsible
referred to as “microcolonial fungi.” In addition, the term occasionally for chromoblastomycosis-like subcutaneous
“black yeast” is sometimes used to describe black fungi that infections after trauma. Clinical symptoms may range from
have yeast-like stages of reproduction and a meristematic dermatomycosis (mycoses), cutaneous phaeohyphomycosis
growth pattern. to mycotic granuloma. Skin lesions appear on arms and legs,
Meristematic black fungi are classified under four fami- usually in immuno compromised patients or in patients with
lies within the Ascomycota: (i) Herpotrichellaceae (order chronic renal failure, transplants, and immunosuppressive
Chaetothyriales) covers Exophiala and Sarcynomices petri- therapy [8,9]. Human infection of tonsils with B. caespito-
cola; (ii) Dothideaceae (order Dothideales) consists of some sus may exhibit recurrent tonsillitis, sore throat, dysphasia,
epiphytic species occasionally isolated from rocks such as high temperature, and enlarged tonsils. Upon examination,
Trimmatostroma abietis, Aureobasidium pullulans, and the tonsils may show “grains” in the crypts [10].
Hortaea werneckii; (iii) Capnodiaceae (order Capnodiales)
includes C. renispora, which was isolated from a tile; and
5.1.3 Diagnosis
(iv) Pleosporaceae (order Pleosporales) includes B. caespito-
sus, which is closely related to Alternaria (which is also fre- As meristematic fungi lack pronounced diagnostic features,
quently found on stones) based on internal transcribed spacer species-specific identification on the basis of microscopic
(ITS) sequence analysis [4,5]. morphology and reproductive structures (e.g., conidiophores,
Stone-inhabiting meristematic black fungi tend to show conidia, and conidial ontogeny) is often difficult. This is fur-
intercrystalline growth by colonizing the weakest parts along ther exacerbated by the fact that many meristematic fungal
marble crystals, leading to the detachments of crystals. They species are highly pleomorphic, with anamorph life cycles
also grow preferentially in cavities and in already-formed and widely divergent methods of propagation. Some species
cracks and fissures, often producing a deepening of the display meristematic growth as the only type of reproduc-
fissures. tion, consisting of isodiametrically dividing cells and endo-
conidiation, which do not allow delimitation of taxa [11].
Thus, morphology gives only a presumptive identification at
genus level, and the use of physiological characteristics (e.g.,
Meristematic black fungi such as B. caespitosus are rec- nitrogen and carbohydrate assimilation tests, growth at dif-
ognized agents of phaeohyphomycosis [6] as distinct from ferent temperatures and proteolytic activity) are helpful for
chromoblastomycosis. Readers should be aware of the term their identification.
“botryomycosis,” which, surprisingly, refers to a bacterial B. caespitosus may be detected from surfaces by tape
infection (affecting the skin, and sometimes the viscera lifts or tease mounts from bulk samples. The laboratory
due to Staphylococcus aureus and several other bacteria) diagnosis of chromoblastomycosis is performed by the
[7] and should not be confused with the current disease. demonstration of sclerotic bodies upon direct microscopi-
Phaeohyphomycosis is cosmopolitan although patients are cal examination of wet KOH mounts of aspirated pus, skin
usually adults and approximately half are immunocompro- scrapings, or biopsy material. However, B. caespitosus
mised; however, this figure has probably increased substan- causes occasionally a chromoblastomycosis-like infection;
tially until the present time. Lesions may occur on almost it is in fact a phaeohyphomycosis.
any part of the body, often on exposed areas, with the upper Colonies on malt extract agar are restricted, cauliflower-
arm lesions being most prevalent. The most typical and like, heaped, pale brown initially becoming brown-black
common lesions are cutaneous or subcutaneous abscesses with age. Microscopy reveals that hyphae and budding cells
or cysts. Primary lesion is a single, discrete, asymptomatic are absent. The thallus is composed of clumps of irregularly
small nodule and this evolves gradually to an encapsulated, septate, thick-walled cells, which are subhyaline, becoming
fluctuant abscess with a liquefied center. However, the over- dark brown with age. These disarticulate into smaller cell
lying epidermis is hardly affected. Occasionally, a slightly packets. Blastic conidia are in fact occasionally present. A
elevated, granulomatous plaque appears when the main site series of approximately 50 physiological tests (e.g., growth
of the lesion is in the dermis and epidermis. Infrequently, it is on glucose, arabinose, salicin) are also available [3]. Hence,
observed as a small verrucous nodule or a verrucous plaque some useful characters are present in the case of this species
comprising a coalescent nodule, which actually resembles at least. B. caespitosus differs from Sarcinomyces phaeomu-
chromoblastomycosis. Phaeohyphomycosis may involve the riformis by young colonies being pink.
central nervous system or other internal organs (e.g., liver, Molecular techniques have been applied for the identifi-
lungs, and pancreas) and may appear as a hematogenous cation of meristematic black fungi including B. caespitosus.
metastasis from cutaneous or subcutaneous infections or Polymerase chain reaction (PCR), restriction fragment length
with no visible lesions. polymorphism (RFLP) and analyses of amplified small
Specifically, B. caespitosus may gain entry into human (SSU, 18S rRNA) and large (LSU, 5.8S rRNA and internal
hosts by traumatic inoculation, through prolonged con- transcribed spacers ITS1, ITS2) subunit ribosomal genes are
tact with domestic animals, and presumably via airborne employed [4,11–14].

Botryomyces 59
5.2 Methods thermal cycler (ABI), using 25 cycles of 96°C for

10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
5.2.1 Sample Preparation reaction products are passed through a Sephadex
Molds from a specimen are grown on inhibitory mold G-50 fine column to remove unincorporated dye ter-
agar, modified Sabouraud agars, or potato dextrose agar. minators. Purified sequencing reaction products are
Microscopic structures are observed on tease or tape prepa- run on an ABI Prism 3100 Genetic Analyzer with a
rations and slide cultures for up to 21 days. 50 cm capillary array.
After growth for 1–7 days on potato dextrose agar slants, 5. Sequences are analyzed with the SmartGene
lysates are prepared from approximately 1 cm2 of mycelia Integrated Database Network software version
with IDI lysis kits (GeneOhm Sciences, San Diego, CA). 3.2.3 vr. SmartGene is a web-based software and
Briefly, in a biological safety cabinet, mycelia are collected by database system with reference sequences derived
scraping the slant with a sterile stick in 1 mL of sterile, molec- from the National Center for Biological Information
ular-grade H2O. The material is transferred to a 2 mL screw- (NCBI) GenBank repository.
cap tube. The tubes are centrifuged for 1 min at 6000 × g. If
the mycelia does not pellet, the material is contained using Note. In case that real time PCR instrument is unavail-
a pediatric blood serum filter (Porex Corp., Fairburn, GA). able, standard PCR may be performed with primers ITS1
The supernatant is removed. The material is resuspended in and ITS4, and the resulting amplicon is sequenced with the
200 μL of IDI sample buffer and transferred to the lysis tube, same primers. Sequence-based identifications are defined
which contains glass beads. Lysis tubes are vortexed on the by percent identity: species, ≥99%; genus, 93%–99%; and
highest setting for 5 min. The tubes are placed in a boiling inconclusive, ≤93%. For strains producing discrepant iden-
water bath for 15 min and centrifuged for 5 min at 16,000 × g. tification between the methods based on phenotypic char-
The supernatant is stored at −20°C until amplification [13]. acteristics and ITS sequence analysis, the D1–D2 region of
the large-subunit RNA gene is amplified with primers NL1
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4
5.2.2 Detection Procedures (5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for
Pounder et al. [13] described a real-time PCR with SYBR species clarification [15].
green DNA-binding dye and amplicon melting temperature
analysis for fungal detection using pan-fungal primers ITS1 5.3 Conclusions
reverse (5′-TCCTCCGCTTATTGATATGC-3′) that cover the B. caespitosus is a black fungus in the mitosporic Dothideo
internal transcribed spacer 1 (ITS1)–5.8S–ITS2 rRNA gene mycetes group that is responsible for a phaeohyphomycosis
cluster. The identities of the fungi are verified by subsequent in humans. The organism is also associated with biodeterio-
sequencing analyses. ration of monuments. Given the close morphological simi-
larity among black fungi, use of molecular techniques such
Procedure as PCR and sequencing is critical for accurate and specific
1. PCR mixture is composed of 1 × Lightcycler FastStart identification of the fungus.
DNA Master Hybridization Probes mixture (Roche
Applied Science) (containing deoxynucleoside tri- References
phosphates, FastStart Taq DNA polymerase, and 1. The UniProt Consortium. Available at http://www.uniprot.
1 mM MgCl2, additional MgCl2 is added to a final org/, accessed on January 27, 2011.
concentration of 4.6 mM), 0.4 μM each of ITS1 for- 2. de Hoog, G.S. and Rubio, C., A new dematiaceous fungus
ward and ITS4 reverse primers, 1 × SYBR green from human skin. Med. Mycol., 20, 15, 1982.
(Molecular Probes), and 3 μL template DNA. 3. de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn.,
2. Thermal cycling parameters include 95°C for 1126pp, Centraalbureau voor Schimmelculture, Utrecht, the
Netherlands, 2000.
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and
4. Sterflinger, K., de Hoog, G.S., and Haase, G., Phylogeny and
76°C for 30 s; and a final extension at 72°C for 2 min. ecology of meristematic ascomycetes. Stud. Mycol., 43, 5,
3. The quality of the amplicon is determined using the 1999.
derivative of the melt analysis curve (55°C–99°C, 5. de Hoog, G.S. and Horré, R., Molecular taxonomy of the
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 Alternaria and Ulocladium species from humans and their
(Corbett Robotics, Inc). identification in the routine laboratory. Mycoses, 45, 259,
4. The amplified product is purified for bidirectional 2002.
6. Matsumoto, T. and Ajello, L., Agents of phaeohyphomycosis.
sequencing using ExoSAP-IT (USB Corp). Five
In: Topley & Wilson’s Microbiology and Microbial Infections,
μL of Big Dye Terminator Ready Reaction Mix v. 9th edn., Medical Mycology (Eds.: Ajello, L. and Hay, R.J.),
1.1 (Applied Biosystems) is added to 4 μL of each pp. 503–524, Chap. 27, Arnold, London, U.K., 1998.
primer (0.8 pmol/μL) and 3 μL of purified PCR 7. Kwon-Chung, K.J. and Bennett J.E., Medical Mycology,
product. Cycle sequencing is performed with a 9700 866pp., Lea & Febiger, Philadelphia, PA, 1992.

8. Carapeto, F.J. et al., Dermatomycosis in plaques caused by 13. Pounder, J.I. et al., Discovering potential pathogens among
Botryomyces caespitosus. A new causative agent. Med. Cutan. fungi identified as nonsporulating molds. J. Clin. Microbiol.,
Ibero. Lat. Am., 13, 71, 1985. 45, 568, 2007.
9. Benoldi, D. et al., Botryomyces caespitosus as an agent of cuta- 14. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
neous phaeohyphomycosis. J. Med. Vet. Mycol., 29, 9, 1991. non-sporulating molds (NSM) identified by polymerase chain
10. Martins, R.H. et al., Actinomycosis and botryomycosis of the reaction (PCR)-based DNA sequencing technique targeting
tonsil. Auris Nasus Larynx., 18, 377, 1991. internal transcribed spacer (ITS) region. Curr. Eye Res., 33,
11. de Hoog, S. et al., Relationships of dothideaceous black 139, 2008.
yeasts and meristematic fungi based on 5.8S and ITS2 rDNA 15. Leaw, S.N. et al., Identification of medically important yeast
sequence comparison. Stud. Mycol., 43, 31, 1999. species by sequence analysis of the internal transcribed spacer
12. White, T.J. et al., Amplification and direct sequencing of fungal regions. J. Clin. Microbiol., 44, 693, 2006.
ribosomal RNA genes for phylogenetics. In: PCR Protocols: A
Guide to Methods and Applications (Eds.: Innis, M.A. et al.),
pp. 315–322, Academic Press, San Diego, CA, 1990.

6 Botryosphaeria and Lasiodiplodia
Dongyou Liu
Contents
6.1 Introduction........................................................................................................................................................................ 61
6.2 Methods.............................................................................................................................................................................. 62
6.2.2.1 ITS1–5.8S–ITS2 rRNA Gene Cluster Sequencing Analysis................................................................... 63
6.2.2.2 Partial EF1α Gene Sequencing Analysis................................................................................................. 63
6.3 Conclusion.......................................................................................................................................................................... 63
References.................................................................................................................................................................................... 63
6.1 Introduction clavate, with a well-developed apical chamber, forming in

a basal hymenial layer, intermixed among hyaline pseudo-
The genus Botryosphaeria consists of a large number of cos- paraphyses that are constricted at the septa. Ascospores are
mopolitan, plant-infecting fungi that are commonly associ- hyaline, aseptate, fusoid to ellipsoid or ovoid, bi- to triseri-
ated with dieback and cankers of woody hosts in tropical ate, without a mucoid sheath or appendages; ascospores turn
and subtropical regions. Of these, Botryosphaeria rhodina brown and become septate and slightly verruculose upon
and its anamorph Lasiodiplodia theobromae are occasion- germination.
ally involved in human phaeohyphomycosis, producing clini- The genus Lasiodiplodia is classified in the mitosporic
cal diseases of varying severity (e.g., subcutaneous abscess, Botryosphaeriaceae group, family Botryosphaeriaceae.
keratitis, pneumonia, and death). The mitosporic Botryosphaeriaceae group contains 17 spe-
cies: Aplosporella, Chaetoconis, Diplodia, Dothiorella,
6.1.1 Classification and Morphology Endomelanconiopsis, Fusicoccum, Lasiodiplodia, Macro
phomina, Microdiplodia, Neofusicoccum, Neoscytalidium,
The genus Botryosphaeria belongs to the family Botryos Phyllosticta, Pseudofusicoccum, Sphaeropsis, Stenocarpella,
phaeriaceae, order Botryosphaeriales, class Dothideomy Taeniolella, and Tiarosporella. In turn, the genus
cetes, subphylum Pezizomycotina, phylum Ascomycota, and Lasiodiplodia consists of nine recognized (Lasiodiplodia
kingdom Fungi. The family Botryosphaeriaceae covers 11 crassispora, Lasiodiplodia gonubiensis, Lasiodiplodia mar-
recognized genera: Barriopsis, Botryosphaeria, Guignardia, garitacea, Lasiodiplodia parva, Lasiodiplodia plurivora,
Melanops, mitosporic Botryosphaeriaceae, Neodeightonia, Lasiodiplodia pseudotheobromae, Lasiodiplodia rubro-
Otthia, Phaeobotryon, Phaeobotryosphaeria, Saccharata, purpurea, Lasiodiplodia theobromae, and Lasiodiplodia
and Spencermartinsia as well as some unclassified venezuelensis) and eight unassigned species, of which ana-
Botryosphaeriaceae [1]. morph Lasiodiplodia theobromae (obsolete synonyms
The genus Botryosphaeria consists of 16 recognized and Botryodiplodia tubericola, Diplodia theobromae, Diplodia
58 unassigned species, of which Botryosphaeria rhodina tubericola, and Lasiodiplodia tubericola) has been associ-
(synonym Botryodiplodia theobromae) is a teleomorph of ated with mycotic keratitis, lesions on nail and subcutaneous
Lasiodiplodia theobromae [1]. Botryosphaeria rhodina/ tissue in humans [2,3].
Lasiodiplodia theobromae is a common endophyte and Lasiodiplodia theobromae colonies are grayish sepia to
opportunistic pathogen affecting >500 tree species in the mouse grey to black, fluffy with abundant aerial mycelium,
tropics and subtropics, leading to stem-end rot and dieback and fuscous black to black on reverse. Pycnidia (up to 5 mm
as well as rot in fruits in most species it infects [21]. in width) are simple or compound, often aggregated, stro-
Botryosphaeria spp. tend to form uni- to multilocular matic, ostiolate, frequently setos. Conidiophores are hyaline,
ascomata with multi-layered walls, occurring singly or in simple, sometimes septate, rarely branched cylindrical, aris-
clusters, and intermixed with pycnidial conidiomata. Asci ing from the inner layers of cells lining the pycnidial cav-
are bitunicate, with a thick endotunica, stalked or sessile, ity. Conidiogenous cells are hyaline, simple, cylindrical to
61

subobpyriform, holoblastic, annellidic. Conidia are initially was speculated that the patient may have acquired the infec-
unicellular, hyaline, granulose, subovoid to ellipsoid oblong, tion through consumption of longan (Dimocarpus longan),
thick walled, base truncate; mature conidia (of 20–30 × a very popular fruit in southern China during summertime,
10–15 μm) are two celled (with one septate), cinnamon to which is often infected with L. theobromae. Alternatively,
fawn or dark brown, often longitudinally striated. Paraphyses the fungus may have been acquired from the cadaveric
when present are hyaline, cylindrical, sometimes septate, up transplanted liver and may have invaded the lung via the
to 50 μm long [4]. bloodstream.
6.1.2 Clinical Features 6.1.3 Laboratory Diagnosis

Botryosphaeria rhodina/Lasiodiplodia theobromae is a com- Traditionally, laboratory identification of molds is based
mon plant pathogen in tropical Asia (India, Cambodia, Hong on detection of fungal elements in clinical specimens and
Kong, Japan, and the Philippines), Australia, Central and South further examination of macroscopic and microscopic fea-
America (Columbia, Guyana, Jamaica, and United States), tures of cultured isolates [20]. To augment sporulation spe-
where Lasiodiplodia theobromae has been shown to infect the cifically for identification, mold may be grown on oatmeal
eyes, skin, and soft tissues of humans, with clinical manifesta- agar in sunlight. Despite being time consuming, phenotypic
tions ranging from keratitis, corneal ulcers and abscess, ony- identification of molds and other fungi requires expertise
chomycosis, subcutaneous abscess and phaeohyphomycosis, for the recognition of characteristic microscopic features of
endophthalmitis, sinusitis, pneumonia to death [5–18]. various fungi.
Summerbell et al. [15] documented a case of subcutane- More recently, molecular techniques such as polymerase
ous phaeohyphomycosis caused by Lasiodiplodia theobro- chain reaction (PCR) and sequencing analysis of the inter-
mae in a 50-year-old Canadian woman who stumbled on an nal transcribed spacer (ITS) regions have been employed for
outdoor wooden staircase and sustained an injury to the right more precise and objective determination of fungi [21–24].
leg while visiting Jamaica. Five weeks later, the patient pre- However, for accurate identification of L. theobromae to the
sented with an ulcer at the injury site. An excisional biopsy species level, partial EF1α sequencing appears to be more
revealed broad, septate, melanized fungal filaments penetrat- reliable than ITS sequencing [19]. The ITS sequence of the
ing into the tissue and its culture generated a nonsporulating patient isolate differed by six bases from that of another
melanized mycelium. After 16 weeks cultivation on modi- strain of L. theobromae but differed by only eight bases
fied Leonian’s agar at 25°C, the fungus developed pycnidia from that of a strain of L. gonubiensis [19]. In addition, the
characteristic of Lasiodiplodia theobromae. The ulcer was Lasiodiplodia spp. are clearly distinguishable from Diplodia
excised during the biopsy procedure and ultimately resolved spp. and Dothiorella spp. based on morphology and ITS and
after treatment with saline compresses. EF1α nucleotide sequences [2,3].
Woo et al. [19] described a case of Lasiodiplodia theo-
bromae pneumonia in a 45-year-old patient with hepatitis
B virus-related hepatocellular carcinoma. Analysis of a 6.2 Methods
direct KOH smear of the bronchoalveolar lavage (BAL) fluid
6.2.1 Sample Preparation
revealed septate hyphae. Culture of BAL fluid specimens
on Sabouraud dextrose agar (SDA) yielded a dematiaceous Molds from a specimen are grown on inhibitory mold
mold, which grew as cottony colonies that became dark agar, modified Sabouraud agars, or potato dextrose agar.
gray within 7 days at 37°C. Microscopic examination of the Microscopic structures are observed on tease or tape prepa-
mold after lactophenol cotton blue staining revealed septate rations and slide cultures for up to 21 days.
brown hyphae of 6 μm in width. The mold was also grown on After growth for 1–7 days on potato dextrose agar slants,
oatmeal agar in sunlight to stimulate sporulation. Colonies lysates are prepared from approximately 1 cm2 of mycelia
produced hairy, dark brown structures (pycnidia). Conidia with IDI lysis kits (GeneOhm Sciences, San Diego, CA).
released from the pycnidia were hyaline and nonseptate Briefly, in a biological safety cabinet, mycelia are col-
when young but were septate and brown, with longitudinal lected by scraping the slant with a sterile stick in 1 mL of
striations, when mature. Pycnidia were sometimes produced sterile, molecular-grade H2O. The material is transferred to
in aggregates as shown in periodic acid-Schiff-stained his- a 2 mL screw-cap tube. The tubes are centrifuged for 1 min
tological sections of the mold on oatmeal agar. Examination at 6000 × g. If the mycelia does not pellet, the material is
under higher magnification demonstrated a pycnidium with contained with a pediatric blood serum filter (Porex Corp.,
an obvious neck and conidiogenous cells and paraphyses Fairburn, GA). The supernatant is removed. The material is
(sterile filaments among conidia) lining the internal wall. resuspended in 200 μL of IDI sample buffer and transferred
The fungus was identified as Lasiodiplodia theobromae by to the lysis tube, which contains glass beads. Lysis tubes
microscopic morphology and elongation factor 1α (EF1α) are vortexed on the highest setting for 5 min. The tubes are
gene sequencing. The patient died 14 days after cadaveric placed in a boiling water bath for 15 min and centrifuged for
liver transplantation. Because the conidia of this fungus are 5 min at 16,000 × g. The supernatant is stored at −20°C until
borne inside pycnidia and are released in a slimy mass, it amplification [22].

Botryosphaeria and Lasiodiplodia 63
6.2.2 Detection Procedures 6.2.2.2 Partial EF1α Gene Sequencing Analysis

Woo et al. [19] utilized primers EF1–728F
6.2.2.1 ITS1–5.8S–ITS2 rRNA Gene
(5′-CATCGAGAAGTTCGAGAAGG-3′) and EF1–986R
Cluster Sequencing Analysis
(5′-TACTTGAAGGAACCCTTACC-3′) for PCR amplifica-
Pounder et al. [22] described a real-time PCR with SYBR tion and DNA sequencing of a 289 bp fragment of the EF1α
green DNA-binding dye and amplicon melting temperature gene from Lasiodiplodia theobromae isolate. The sequence
analysis for fungal detection using pan-fungal primers ITS1 was compared with those of related species listed in the
forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 GenBank database by using ClustalX 1.83. Phylogenetic
reverse (5′-TCCTCCGCTTATTGATATGC-3′) [24]. These relationships were determined using the neighbor-joining
primers generate a 500 bp fragment from the internal tran- method.
scribed spacer 1 (ITS1)–5.8S–ITS2 rRNA gene cluster (ITS).
Subsequent sequencing analysis of the amplicon helps verify
the identity of the fungal isolates. 6.3 Conclusion
Botryosphaeria rhodina/Lasiodiplodia theobromae is a
Procedure
ubiquitous plant pathogen in tropical regions that may infect
the eyes, skin, and soft tissues of humans, leading to kerati-
1. PCR mixture is composed of 1 × Lightcycler tis, corneal ulcers and abscess, onychomycosis, subcutaneous
FastStart DNA Master Hybridization Probes mix- abscess and phaeohyphomycosis, endophthalmitis, pneumo-
ture (Roche Applied Science) (containing deoxy- nia, and death. As phenotypic techniques for mold identifica-
nucleoside triphosphates, FastStart Taq DNA tion are time consuming and variable, molecular methods are
polymerase, and 1 mM MgCl2, additional MgCl2 increasingly utilized. PCR and sequencing analysis of the ITS
to a final concentration of 4.6 mM), 0.4 μM each of regions have proven valuable for determination of a variety
ITS1 forward and ITS4 reverse primers, 1 × SYBR of molds including Lasiodiplodia theobromae. Furthermore,
green (Molecular Probes), and 3 μL template DNA. examination of the EF1α gene sequence offers an additional
2. Thermal cycling parameters include 95°C for tool for differentiation among Lasiodiplodia spp.
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s,
and 76°C for 30 s; and a final extension at 72°C for
2 min. References
3. The quality of the amplicon is determined using the 1. The UniProt Consortium. Available at http://www.uniprot.org/,
derivative of the melt analysis curve (55°C–99°C, accessed on August 1, 2010.
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 2. Pavlic, D. et al., Lasiodiplodia gonubiensis sp. nov., a new
(Corbett Robotics, Inc). Botryosphaeria anamorph from native Syzygium cordatum in
South Africa. Stud. Mycol., 50, 313, 2004.
4. The amplified product is purified for bidirectional 3. Burgess, T.I. et al., Three new Lasiodiplodia spp. from the
sequencing using ExoSAP-IT (USB Corp). Five tropics, recognized based on DNA sequence comparisons and
microliters of Big Dye Terminator Ready Reaction morphology. Mycologia, 98, 423, 2006.
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of 4. Mycology online, School of Molecular & Biomedical Science,
each primer (0.8 pmol/μL) and 3 μL of purified PCR The University of Adelaide, Australia. Available at http://
product. Cycle sequencing is performed with a 9700 www.mycology.adelaide.edu.au/, accessed on August 1, 2010.
thermal cycler (ABI), using 25 cycles of 96°C for 5. Laverde, S. et al., Mycotic keratitis: 5 cases caused by unusual
fungi. Sabouraudia, 11, 119, 1973.
6. Valenton, M.J., Rinaldi, M.G., and Butler, E.E., A corneal
reaction products are passed through a Sephadex abscess due to the fungus Botryodiplodia theobromae. Can. J.
G-50 fine column to remove unincorporated dye ter- Ophthalmol., 10, 416, 1975.
minators. Purified sequencing reaction products are 7. Rebell, G. and Forster, R.K., Lasiodiplodia theobromae as a
run on an ABI Prism 3100 Genetic Analyzer with a cause of keratomycoses. Sabouraudia, 14, 155, 1976.
50 cm capillary array. 8. Restrepo, A. et al., The isolation of Botryodiplodia theobro-
5. Sequences are analyzed with the SmartGene mae from a nail lesion. Sabouraudia, 1, 41, 1976.
9. Slomovic, A.R., Forster, R.K., and Gelender, H., Lasiodiplodia
Integrated Database Network software version
theobromae panophthalmitis. Can. J. Ophthalmol., 20, 225,
3.2.3 vr. SmartGene is a web-based software and 1985.
database system with reference sequences derived 10. Vélez, H. and Díaz, F., Onychomycosis due to saprophytic
from the National Center for Biological Information fungi. Report of 25 cases. Mycopathologia, 91, 87, 1985.
(NCBI) GenBank repository. 11. Marasas, W.F. and Van Rensburg, S.J., Mycotoxicological
investigations on maize and groundnuts from the endemic
area of Mseleni joint disease in Kwazulu. S. Afr. Med. J., 69,
Note: In case that real time PCR instrument is not available, 369, 1986.
standard PCR may be performed with primers ITS1 and 12. Maslen, M.M., Collis, T., and Stuart, R., Lasiodiplodia theo-
ITS4, and the resulting amplicon is sequences with the same bromae isolated from a subcutaneous abscess in a Cambodian
primers. immigrant to Australia. J. Med. Vet. Mycol., 34, 279, 1996.

13. Borderie, V.M. et al., Endophthalmitis after Lasiodiplodia theo- 21. Denman, S. et al., Circumscription of Botryosphaeria species
bromae corneal abscess. Graefe’s Arch. Clin. Exp. Opthalmol., associated with Proteaceae based on morphology and DNA
235, 259, 1997. sequence data. Mycologia, 95, 294, 2003.
14. Thomas, P.A., Current perspectives on ophthalmic mycoses. 22. Pounder, J.I. et al., Discovering potential pathogens among
Clin. Microbiol. Rev., 16, 730, 2003. fungi identified as nonsporulating molds. J. Clin. Microbiol.,
15. Summerbell, R.C. et al., Subcutaneous phaeohyphomycosis 45, 568, 2007.
caused by Lasiodiplodia theobromae and successfully treated 23. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
surgically. Med. Mycol., 42, 543, 2004. non-sporulating molds (NSM) identified by polymerase chain
16. Bhartiya, P. et al., Fungal keratitis in Melbourne. Clin. Exp. reaction (PCR)-based DNA sequencing technique targeting
Ophthalmol., 35, 124, 2007. internal transcribed spacer (ITS) region. Curr. Eye Res., 33,
17. Thew, M.R. and Todd, B., Fungal keratitis in far north 139, 2008.
Queensland, Australia. Clin. Exp. Ophthalmol., 36, 721, 2008. 24. White, T.J. et al., Amplification and direct sequencing of fun-
18. Kindo, A.J. et al., Maxillary sinusitis caused by Lasiodiplodia gal ribosomal RNA genes for phylogenetics, pp. 315–322. In
theobromae. Indian J. Med. Microbiol., 28, 167, 2010. Innis, M.A. et al. (Eds.), PCR Protocols: A Guide to Methods
19. Woo, P.C.Y. et al., Lasiodiplodia theobromae pneumonia in a and Applications. Academic Press, San Diego, CA, 1990.
liver transplant recipient. J. Clin. Microbiol., 46, 380, 2008.
20. Thomas, O.A. et al., Use of lactophenol cotton blue mounts of
corneal scraping as an aid to the diagnosis of mycotic keratitis.
Diagn. Microbiol. Infect. Dis., 14, 219, 1991.

7 Corynespora
Dongyou Liu and Po-Ren Hsueh
Contents
7.1 Introduction........................................................................................................................................................................ 65
7.2 Methods.............................................................................................................................................................................. 67
7.2.2.1 Pan-Fungal Real-Time PCR and Sequencing Analysis........................................................................... 67
7.2.2.2 Sequencing Analysis of ITS1 and ITS2................................................................................................... 67
7.3 Conclusion.......................................................................................................................................................................... 68
References.................................................................................................................................................................................... 68
7.1 Introduction cucumber, papaya, pepper, rubber, soybean, and tomato,

primarily found in the tropics and subtropics [2–4]. Rubber
Dematiaceous fungi are characterized by their production trees infected with C. cassiicola develop necrotic lesions on
of dark-pigmented hyphae or yeast-like cells. Being widely leaves, as well as chlorosis and darkening of the veins, lead-
distributed in the soil, plants and other environments, these ing to defoliation and loss of crop [5]. C. cassiicola has also
dark-walled fungi have the ability to cause opportunistic been associated occasionally with human infections [6,7]
infections in humans and animals, affecting the skin, subcu- (Figure 7.1).
taneous tissues and internal organs. Among the 130 species On potato dextrose agar (PDA), C. cassiicola colonies
of 70 genera of dematiaceous fungi that have been implicated are effuse, gray or brown in the front and gray or black on
in human infections, Corynespora cassiicola is a common reverse. Conidiophores (measuring 4–11 μm by 110–850 μm)
pathogen of grasses and other herbaceous plants in the trop- are pale to light brown, 3–10 septate, cylindrical, straight
ics, and has been confirmed as an etiologic agent of subcuta- or curved, unbranched, and smooth. Conidia (of 5–10 μm
neous phaeohyphomycosis in humans. by 27–192 μm in size) develop at the conidiophore apex and
appear smoky to olivaceous, smooth, obclavate to cylindri-
cal, subhyaline to pale olivaceous brown, 4–20 pseudosep-
7.1.1 Classification, Morphology, and Biology
tate, singly or in acropetal chains, with a rounded apex and
The genus Corynespora is a mold belonging to the mito- truncated base [8,9].
sporic Pleosporales group, order Pleosporales, subclass Phylogenetic analysis in the ribosomal DNA internal
Pleosporomycetidae, class Dothideomycetes, subphylum transcribed spacer (ITS) regions of ribosomal RNA (rRNA)
Pezizomycotina, phylum Ascomycota, kingdom Fungi [1]. gene, random hypervariable loci (caa5 and ga4), and the
The mitosporic Pleosporales group encompasses 16 genera: actin-encoding locus act1 identified six phylogenetic lineages
Ascochyta, Camarosporium, Chaetodiplodia, Chaetophoma, in C. cassiicola, corresponding to host of origin, pathogenic-
Chalastospora, Cheirosporium, Coniothyrium, Corynespora, ity, and growth rate but not to geographic location [10]. In
Monascostroma, Ochrocladosporium, Phialophorophoma, addition, restriction fragment length polymorphism (RFLP)
Plenodomus, Pleurophoma, Pseudodiplodia, Pseudorobillarda, analysis of ITS regions of rRNA and random amplified poly-
and Zeloasperisporium. In turn, the genus Corynespora is sep- morphic DNA (RAPD) studies revealed significant genetic
arated into seven recognized species: Corynespora cassiicola, variation among C. cassiicola isolates collected from differ-
Corynespora citricola, Corynespora olivacea, Corynespora ent host plants [2–5,10,11].
melongenae, Corynespora proliferata, Corynespora sesa-
mum, and Corynespora smithii, in addition to an unassigned
7.1.2 Clinical Features and Pathogenesis
species Corynespora sp. SXZ-01 [1].
Within the Corynespora genus, Corynespora cassi- Corynespora cassiicola is a pathogenic fungus associated
icola is a well-known plant pathogen causing leaf spot dis- with leaf spot disease in plants. The involvement of this fun-
ease in more than 70 host plant species including cowpea, gus in human disease was first noted in 1969, when a case of
65

(A) (C)
(B) (D)
Figure 7.1 (A) Necrotic subcutaneous lesions over the dorsal aspects of both hands with purulent discharge in a patient with chronic sub-
cutaneous infection caused by Corynespora cassiicola. (B) Hyphae with septa (black arrow) in the edematous papillary dermis (PAS stain,
400× magnification). (C) Confluent growth with velvety and gray–black colonies after 7 days of incubation on PDA at 28°C. (D) Straight
and septate conidiophores and catenate conidia (white arrow) (lactophenol cotton blue stain, 400× magnification). (From Huang, H.K. et al.,
J. Infect., 60, 188, 2010.)
maduromycetoma of the foot was attributed to C. cassiicola the isolation and characterization of a 28 kDa, 27 residue
[6]. Recently, C. cassiicola was shown to cause chronic subcu- O-glycosylated protein (known as cassiicolin) in this fungus
taneous infection in a 69-year-old female farmer with diabe- [12,13]. Cassiicolin induces host-selective cellular damages
tes mellitus and on long-term oral antihyperglycemic therapy resembling those observed in leaf fall disease of rubber trees
[7]. The patient presented erythematous lesions on the bilat- with C. cassiicola infection [12,13].
eral forearms and dorsal aspect of both hands with purulent
discharge, which was accompanied by pain, heat, swelling,
and tenderness. Multiple bullae developed on the erythem-
atous lesions, which ruptured with milk-colored, pus-like Traditional methods for identification of fungi are based on
discharge. The patient underwent debridement and culture examination of their characteristic macroscopic and micro-
of the debrided tissue and discharged pus on PDA (Becton scopic features. Typically, clinical specimens (e.g., discharge
Dickinson) formed velvety and gray-black colonies after 7 and scrapings) are examined under microscope to identify
days of incubation at 28°C. Microscopically, the organism hyphae and other mycotic elements, with the help of 10%
showed long, straight, septate conidiophores and elliptic, cat- KOH or lactophenol cotton blue stain. Biopsies are exam-
enate conidia using lactophenol cotton blue stain. Histological ined for mycotic elements and pathological changes using
examination of biopsy using periodic acid-Schiff (PAS) stain PAS stain or other specific fungal stains. Portions of clinical
revealed tissue necrosis with the presence of septate hyphae specimens are cultured on Sabouraud dextrose agar (SDA) or
in the edematous papillary dermis. Upon sequence analysis potato dextrose agar (PDA) at 25°C, 35°C, respectively. The
of the region comprising the ITS1 and ITS2 regions of the resulting fungal isolates are further characterized on their
rRNA genes using fungus-specific universal primers ITS1 colony morphology and microscopic features as well as bio-
and ITS4, the fungus demonstrated a nucleotide identity of chemical properties.
98% (527/534) with the published C. cassiicola sequences in Given the close morphological and biochemical similar-
the GenBank (accession number FJ852715). The mold was ity among many pathogenic fungal species, laboratory diag-
thus identified as C. cassiicola according to the characteris- nosis of fungal infections using phenotypic criteria can be
tic phenotypes and relevant molecular methods. The patient challenging and time consuming. With the development and
recovered fully after amphotericin B treatment [7]. application of nucleic acid amplification techniques, rapid
The exposure to the fungus in plants such as betel nut and precise identification of fungal organisms has become
and banana through trivial wounds (farmer) and the immu- possible. In particular, PCR amplification and sequencing
nocompromised status (old age, diabetes mellitus, and adre- analysis of the small subunit (SSU) and large subunit (LSU)
nal insufficiency) might have contributed partly to this case as well as ITS1 and ITS2 regions of the rRNA genes provide
of C. cassiicola infection [7]. The ability of C. cassiicola a useful tool for confirmation of fungal identity including
to cause cellular damages was demonstrated recently with C. cassiicola [7,14–17].

Corynespora 67
7.2 Methods analysis for fungal detection using pan-fungal primers ITS1
7.2.1 Sample Preparation reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
Specimens are stained with 10% KOH, lactophenol cotton blue of the fungi is verified by subsequent sequencing analysis.
stain or PAS stain and examined under microscope for mycotic Procedure
elements. Portions of samples are grown on inhibitory mold
agar, modified Sabouraud agars, or PDA at 25°C and 35°C. 1. PCR mixture is composed of 1× Lightcycler
After growth for 1–7 days on PDA slants, mycelia are col- FastStart DNA Master Hybridization Probes mix-
lected by scraping the slant with a sterile stick in 1 mL of ture (Roche Applied Science) (containing deoxy-
sterile water. The material is transferred to a 2 mL screw- nucleoside triphosphates, FastStart Taq DNA
cap tube. The tubes are centrifuged for 1 min at 6000 × g. polymerase, and 1 mM MgCl2, additional MgCl2 is
If the mycelia do not pellet, the material is contained with added to a final concentration of 4.6 mM), 0.4 μM
a pediatric blood serum filter (Porex Corp). Supernatant is each of ITS1 forward and ITS4 reverse primers, 1×
removed, and the material is resuspended in 200 μL of IDI SYBR green (Molecular Probes), and 3 μL template
sample buffer and transferred to the lysis tube, which con- DNA.
tains glass beads. Lysis tubes are vortexed on the highest set- 2. Thermal cycling parameters include 95°C for
ting for 5 min. The tubes are placed in a boiling water bath 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s,
for 15 min. Tubes are centrifuged for 5 min at 16,000 × g. The and 76°C for 30 s; and a final extension at 72°C for
supernatant is stored at −20°C until amplification [15]. 2 min.
Alternatively, about 1 cm2 of fungal material is trans- 3. The quality of the amplicon is determined using the
ferred to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) derivative of the melt analysis curve (55°C–99°C,
mixture of silica gel and Celite (silica gel H, Merck 7736/ 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
Kieselguhr Celite 545; Machery) and 300 μL of TES buffer (Corbett Robotics, Inc).
(2 g Tris [hydroxymethyl]-aminomethane, 0.38 g Na-EDTA 4. The amplified product is purified for bidirectional
[ethylenediaminetetraacetic acid], and 2 g sodium dodecyl sequencing using ExoSAP-IT (USB Corp). Five
sulfate in 80 mL of ultrapure water, pH 8). The fungal mate- microliters of Big Dye Terminator Ready Reaction
rial is ground with a micropestle for 1–2 min. The volume Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
is adjusted by adding 200 μL of TES buffer. After vigorous each primer (0.8 pmol/μL) and 3 μL of purified PCR
shaking and the addition of 10 μL of a 10 mg/mL concentra- product. Cycle sequencing is performed with a 9700
tion of proteinase K to the tube, the mixture is incubated thermal cycler (ABI), using 25 cycles of 96°C for
at 65°C for 10 min. With the addition of 140 μL of 5 M 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
NaCl solution, the mixture is combined with 1/10 volume reaction products are passed through a Sephadex
(∼65 μL) of 10% cetyltrimethylammonium bromide (CTAB) G-50 fine column to remove unincorporated dye ter-
buffer, followed by incubation for another 30 min at 65°C. minators. Purified sequencing reaction products are
One volume (∼700 μL) of chloroform-isoamyl alcohol (vol/ run on an ABI Prism 3100 Genetic Analyzer with a
vol = 24/l) is added and mixed by inversion. After incuba- 50 cm capillary array.
tion for 30 min at 0°C (on ice water) and centrifugation at 5. Sequences are analyzed with the SmartGene
14,000 rpm at 4°C for 10 min, the top layer is transferred to Integrated Database Network software version
a clean Eppendorf tube. The sample is added with 225 μL 3.2.3 vr. SmartGene is a web-based software and
of 5 M NH4 –acetate and incubated for at least 30 min (on database system with reference sequences derived
ice water) and centrifuged. The supernatant is transferred to from the National Center for Biological Information
a clean sterile Eppendorf tube and mixed with a 0.55 vol- (NCBI) GenBank repository.
ume (∼510 μL) of ice-cold isopropanol. After centrifugation
for 7 min at 14,000 rpm and 4°C (or room temperature), the Note. In case that real time PCR instrument is not available,
supernatant is decanted. The pellet is washed with ice-cold standard PCR may be performed with primers ITS1 and
70% ethanol twice and dried by using a vacuum dryer. The ITS4, and the resulting amplicon is sequenced with the same
powder is resuspended in 48.5 μL of Tris–EDTA buffer primers [18]. Sequence-based identifications are defined
with 1.5 μL of 10 mg of RNase/mL, incubated at 37°C for by percent identity: species, ≥99%; genus, 93%–99%; and
15–30 min, and stored at −20°C until use [16]. inconclusive, ≤93%.
7.2.2 Detection Procedures 7.2.2.2 Sequencing Analysis of ITS1 and ITS2

Leaw et al. [14] utilized fungus-specific universal prim-
7.2.2.1 P
an-Fungal Real-Time PCR and ers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and
Sequencing Analysis ITS2 (5′-GCATCGATGAAGAACGCAGC-3′) to
Pounder et al. [15] described a real-time PCR with SYBR amplify the ITS1 region, and universal primers ITS3
green DNA-binding dye and amplicon melting temperature (5′-GCATCGATGAAGAACGCAGC-3′) and ITS4

(5′-GCATATCAATAAGCGGAGGA-3′) to amplify the C. cassiicola causes leaf spot disease in >70 plant species,
ITS2 region. Subsequent sequencing analysis of the result- and is occasionally associated with human infections [6,7].
ing amplicons facilitated identification of a range of fungal As shown in a recent case of C. cassiicola-related chronic
pathogens. subcutaneous infection, the fungus may be acquired through
exposure to plants and it has the ability to take advantage of
Procedure
the weakened host immune defense such as old age, diabe-
1. PCR mixture (50 μL) is made up of 10 mM Tris–HCl tes mellitus, and adrenal insufficiency to establish in human
pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each of hosts [7,18].
deoxynucleoside triphosphates, 1.2 U of Taq DNA As conventional laboratory procedures for identification
polymerase, 0.4 μM (each) of the ITS1 region prim- of Corynespora and other fungal pathogens are slow and
ers (ITS1/ITS2) or the ITS2 region primers (ITS3/ demand specialized skills, molecular techniques are increas-
ITS4), 2 μL (1–5 ng) of DNA template. A negative ingly utilized for rapid and precise determination of these
control is included in each run by replacing the tem- organisms. Indeed, PCR and sequencing analysis of the SSU
plate DNA with sterile water in the PCR mixture. and LSU as well as ITS1 and ITS2 regions of the rRNA genes
2. Amplification is conducted at an initial 94°C for offer a reliable supplemental approach for accurate speciation
3 min; 30 cycles of 94°C, 60°C, and 72°C for 1 min of fungal organisms including C. cassiicola.
each; and a final 72°C for 3 min.
3. After checking the PCR products on 1.5% agarose
gel, the amplicons are purified using a commercial References
PCR cleanup kit. The DNA fragments are sequenced 1. The UniProt Consortium. Available at http://www.uniprot.
using an ABI Prism 377 automated DNA sequencer org/, accessed on August 1, 2010.
(Applied Biosystems) with a BigDye Terminator cycle 2. Silva, W.P.K., Deverall, B.J., and Lyon, B.R., Molecular, phys-
sequencing kit (version 3.1; Applied Biosystems). iological and pathological characterization of Corynespora
All amplicons are sequenced on both strands using leaf spot fungi from rubber plantations in Sri Lanka. Plant
Pathol., 47, 267, 2002.
primers ITS1 and ITS2 for the ITS1 region and prim- 3. Silva, W.P. et al., Genetic variation in Corynespora cassiicola:
ers ITS3 and ITS4 for the ITS2 region. A possible relationship between host origin and virulence.
4. After sequencing, portions of the 18S, 5.8S, and Mycol. Res., 107, 567, 2003.
26S rRNA gene sequences of the PCR products 4. Silva, W.P.K. et al., RFLP and RAPD analyses in the iden-
are removed to obtain the exact ITS1 and ITS2 tification and differentiation of isolates of the leaf spot
sequences. fungus Corynespora cassiicola. Aust. J. Botany, 43, 609,
5. For all yeasts, the sequences of the 3′ ends of the 1995.
5. Nghia, N.A. et al., Morphological and inter simple sequence
18S and 5.8S rRNA genes are GCGGAAGGA
repeat (ISSR) markers analyses of Corynespora cassiicola
TCATTA and GTTTGAGCGTCATTT, respec- isolates from rubber plantations in Malaysia. Mycopathologia,
tively, and the sequences of the 5′ ends of the 5.8S 166, 189, 2008.
and 26S rRNA genes are AAACTTTCAACAA and 6. Mahgoub, E., Corynespora cassiicola, a new agent of madu-
GACCTCAAATCAG, respectively. romycetoma. J. Trop. Med. Hyg., 72, 218, 1969.
6. The ITS1 or ITS2 sequence is compared with 7. Huang, H.K. et al., Subcutaneous infection caused by
those available at the GenBank databases using the Corynespora cassiicola, a plant pathogen. J. Infect., 60, 188,
BLAST sequence analysis tool (http://www.ncbi. 2010.
8. Zaher, E.A. et al., Leaf spots of ornamental foliage plants
nlm.nih.gov/BLAST/) (blastn) with default settings in Egypt with special reference to Corynespora cassiicola
except that sequences are not filtered for low com- [(Berk. & Curt.)Wei] as a new causal. Egypt. J. Phytopathol.,
plexity. Species identification is determined from 33, 87, 2005.
the lowest expected value of the BLAST output. 9. Jayasuriya, K.E. and Thennakoon, B.I., First report of
Corynespora cassiicola on Codiaeum variegatum (Croton) in
Note. For strains producing discrepant identification Sri Lanka. J. Sci. (Bio. Sci.), 36, 138, 2007.
10. Dixon, L.J. et al., Host specialization and phylogenetic diver-
between the methods based on phenotypic character- sity of Corynespora cassiicola. Phytopathology, 99, 1015,
istics and ITS sequence analysis, the D1–D2 region 2009.
of the LSU RNA gene is amplified with primers NL1 11. Qi, Y. et al., Molecular and pathogenic variation identified
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 among isolates of Corynespora cassiicola. Mol. Biotechnol.,
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for 41, 145, 2009.
species clarification. 12. Barthe, P. et al., Structural analysis of cassiicolin, a host-
selective protein toxin from Corynespora cassiicola. J. Mol.
Biol., 367, 89, 2007.
7.3 Conclusion 13. de Lamotte, F. et al., Purification and characterization of cas-
siicolin, the toxin produced by Corynespora cassiicola, causal
The genus Corynespora is composed of eight mold species agent of the leaf fall disease of rubber tree. J. Chromatogr. B
that are distributed ubiquitously in the environment of which Analyt. Technol. Biomed. Life Sci., 849, 357, 2007.

Corynespora 69
14. Leaw, S.N. et al., Identification of medically important yeast 17. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
species by sequence analysis of the internal transcribed spacer non-sporulating molds (NSM) identified by polymerase chain
regions. J. Clin. Microbiol., 44, 693, 2006. reaction (PCR)-based DNA sequencing technique targeting
15. Pounder, J.I. et al., Discovering potential pathogens among internal transcribed spacer (ITS) region. Curr. Eye Res., 33,
fungi identified as nonsporulating molds. J. Clin. Microbiol., 139, 2008.
45, 568, 2007. 18. Lv, G.X. et al., Phaeohyphomycosis caused by a plant patho-
16. Zeng, J.S. et al., Spectrum of clinically relevant Exophiala gen, Corynespora cassiicola. Med. Mycol., posted online on
species in the United States. J. Clin. Microbiol., 45, 3713, January 31, 2011.
2007.

8 Curvularia
Audrey N. Schuetz
Contents
8.1 Introduction........................................................................................................................................................................ 71
8.1.1 Classification and Epidemiology............................................................................................................................ 71
8.1.1.1 Classification............................................................................................................................................ 71
8.1.1.2 Epidemiology and Risk Factors for Infection.......................................................................................... 71
8.1.2 Pathogenesis............................................................................................................................................................ 72
8.1.3.1 Ocular Disease......................................................................................................................................... 73
8.1.3.2 Allergic Fungal Sinusitis.......................................................................................................................... 73
8.1.3.3 Cutaneous and Subcutaneous Infections................................................................................................. 74
8.1.3.4 Cerebral Infections................................................................................................................................... 74
8.1.3.5 Peritonitis................................................................................................................................................. 74
8.1.3.6 Miscellaneous Infections......................................................................................................................... 74
8.1.3.7 Curvularial Infections, Other Than C. lunata......................................................................................... 74
8.1.4 Diagnosis................................................................................................................................................................ 75
8.2 Methods.............................................................................................................................................................................. 76
8.3 Conclusions......................................................................................................................................................................... 79
References.................................................................................................................................................................................... 79
8.1 Introduction lesions (such as phaeohyphomycosis and chromoblastomy-

cosis), ocular infections (such as keratitis and endophthal-
8.1.1 Classification and Epidemiology mitis), peritonitis, onychomycosis, and other less common
8.1.1.1 Classification clinical entities. Dissemination to the brain and to other
organs rarely occurs. However, isolation in culture is not
Curvularia comprises part of the Dematiaceae family of
always clinically significant, as Curvularia may occasion-
thermally monomorphic dematiaceous molds, which pos-
ally contaminate a culture. Infections due to Curvularia can
sess brown melanin or melanin-like pigments in their hyphal
occur in immunocompetent hosts as well. In their review
and/or conidial cell walls. Related to the sexual teleomorph
of human Curvularia infection, Rinaldi et al. reported that
ascomycete Cochliobolus (family Pleosporaceae, order
only two patients were immunosuppressed in their series of
Pleosporales), Curvularia is a member of the Hyphomycete
24 patients [3].
class of fungi. The genus is comprised of approximately 40
species, including C. lunata (the most commonly isolated
species in humans), C. clavata, C. brachyspora, C. genic- 8.1.1.2 Epidemiology and Risk Factors for Infection
ulata, C. inaequalis, C. pallescens, C. senegalensis, and Most Curvularia spp. are saprobic soil agents and live on
others. dead plant or grain material as weak pathogens. Curvularia
Increasingly recognized as an emerging cause of human phytopathogens cause plant diseases, ranging from seed
infection, particularly in immunocompromised persons, failure to leaf blight and grass “fade-out” during hot and
Curvularia causes a wide range of human diseases [1]. humid weather [4]. The spores of Curvularia are large (i.e.,
First reported from a mycetoma by Baylet et al. in 1959 [2], 20–30 μm × 8–12 μm) [5].
Curvularia spp. have been commonly associated with cer- Risk factors for human infection include soil contact asso-
tain diseases, such as infections of the cornea and sinuses, as ciated with outdoor recreational or occupational activities.
well as allergic fungal sinusitis (AFS). Other diseases asso- Persons are usually exposed through the respiratory tract;
ciated with Curvularia include cutaneous and subcutaneous however, skin injury is a second important route of exposure [6].
71

Lawn-tool injuries due to projectiles have caused ocular or Fungal rhinosinusitis predominates in hot, humid cli-
cutaneous lesions [7]. Curvularia has been cultured from mates, such as the southwestern United States, India, and the
rose bush thorns on plants in the Oklahoma City area [8]. In Sudan [5]. The number of aerially borne Curvularia varies
one study from the United States of 43 cases of Curvularia seasonally and rises just after a wet, warm month [17]. The
keratitis, half of the cases were associated with trauma due to relative prevalence of filamentous fungal keratitis appears
plant or dirt inoculation, or metal injury [4]. to increase near tropical latitudes [18]. At a Texas institu-
Environmental contamination has been documented in tion [4], Wilhelmus and Jones reported that two-thirds of the
many cases of Curvularia infection, such as the case of a Curvularia keratitis cases occurred during the summer, cor-
patient with Curvularia peritonitis who worked in his garden responding to the peak of airborne spores along the Texas
prior to receiving dialysis exchanges [9]. Canon et al. report a coast [19]. In a retrospective ecological study of 52 episodes
case of peritonitis in a 13-year-old boy undergoing peritoneal of dematiaceous fungal keratitis along the Gulf of Mexico
dialysis who played in a wooded area and wore his dialysis spanning the years 1972 to 2004, researchers reported an
tubing and transfer set in his underpants, predisposing him to increase in the diagnosis of ocular phaeohyphomycosis dur-
infection [10]. Cutaneous inoculation due to skin injury may ing the late summer, which persisted throughout the autumn
cause severe disease, especially in an immunocompromised months [20]. Specifically, new diagnoses of Curvularia kera-
host. Tessari et al. report a 69-year-old heart transplant recip- titis clustered in warm, humid months.
ient who died from overwhelming sepsis due to cutaneous
C. lunata infection, from a splinter introduced while work-
8.1.2 Pathogenesis
ing in his garden [11]. At autopsy, Curvularia was recov-
ered in numerous organs, including the skin, lungs, mouth, Relatively little is known regarding the pathogenic mecha-
upper third of the esophagus, and right lung. Rohwedder et nisms by which Curvularia and other dematiaceous fungi
al. report the case of an immunocompetent football player lead to disease, especially in immunocompetent individuals.
with disseminated C. lunata who developed cervical lymph- Curvularia produces several mycotoxins, such as curvular-
adenopathy, pleural effusion, and a paraspinous abscess ins, brefeldins, and radicinin, which can act as both cytotox-
after presumptive cutaneous inoculation from skin scrapes ins and antivirals [4,21]. Curvularia also metabolizes certain
and cuts during football practice [12]. Other cases have been steroids and produces other toxins and enzymes, including
documented in gardeners and children who played outdoors, anthroquinones, curvapallides, cytochalasins, neocoprogen,
including Curvularia phaeohyphomycosis in 22-year-old triticones, pyrenocenes, spirostaphylotrichins, pectinases,
male who had overdosed on cocaine, was wandering nude zaragozic acid, lipid phosphatase, galactosidase, glucosidase,
in a wooded area, and subsequently developed infection of endoglucanase, chloroperoxidase, and cellulases [22]. The
the lacerations on his extremities [3,13,14]. Finally, Grieshop roles of these enzymes and toxins have not yet been fully
et al. describe acute, localized cutaneous and subcutaneous described.
phaeohyphomycosis due to C. lunata, after explosion at a There is increasing evidence that melanin, present in the
chemical plant, which was believed to have inoculated organ- cell walls of dematiaceous fungi, may act as a virulence
isms into body tissues due to the force of the explosion [15]. factor [23–25]. Melanin binds to and inactivates hydrolytic
Environmental contamination with Curvularia spp. is not enzymes and scavenges free radicals and hypochlorite [23].
restricted to outdoor, wooded areas but has been documented Some authors have postulated that Curvularia shows a pre-
in a healthcare institution as well. Kainer et al. reported a dilection for organic silicone or inorganic silicon substrate,
hospital-associated outbreak of Curvularia spp., which con- which may predispose to infection in those patients who have
taminated saline-filled silicone shell prosthetic breast implants silicone-containing foreign bodies such as catheters [26].
in women who had undergone cosmetic breast augmentation Reports have demonstrated that Curvularia may colonize
surgery [16]. In their series, five patients who underwent sur- silicon-containing stone churches [27] and silicone voltage
gery at their institution developed Curvularia contamination insulators [28]. Likewise, Curvularia has been implicated in
of their breast implants; one patient’s breast implants con- cases of silicone peritoneal dialysis catheters, silicone breast
tained macroscopically apparent black specks, proven to be implants [16], and silicone lenses [16,29]. Silicone appears to
microcolonies of Curvularia. An epidemiologic investiga- be associated not only with the presence of Curvularia, but
tion revealed that Curvularia was isolated from the sterile low levels of silicone also appear to enhance the growth of
supply room, in which saline bottles were stored underneath Curvularia and Fusarium in an alkaline medium, such as the
a water-damaged ceiling, as well as from the corridor out- alkaline environment of fresh peritoneal dialysis fluid [30].
side of the operating room. The authors postulated that the Curvularia is an important inhalant allergen associated
saline used to fill the silicone implants was likely contami- with respiratory allergic disorders, such as rhinitis. Patients
nated while sitting in an open bowl before or during surgery, with nonspecific nasobronchial symptoms demonstrate
when fungal spores circulated in air drafts introduced into positive skin reactions to C. lunata extract [31]. A study
the operating room, which maintained a positive air pres- of asthma and rhinitis patients in Singapore showed that
sure differential. Risk factors associated with contamination patients demonstrated a high prevalence of fungal sensitiza-
included increased length of time spent in the operating room tion to Curvularia (26%–32%) [32]. In addition, approxi-
and longer duration of surgery. mately 60% of rural allergic patients in agricultural India

Curvularia 73
showed sensitization to Curvularia extracts [33]. Cross- Curvularia has been reported as the most frequent pig-
reactivity of Curvularia allergens has been demonstrated to mented fungal pathogen in corneal ulcers by other investi-
Aspergillus, Alternaria, and Epicoccum [34]. Studies have gators as well [48–51]. In Thailand, Curvularia was second
identified that proteins of 26, 31, 38, 45, 50, and 78 kDa from only to Fusarium as a cause of severe mycotic keratitis [52].
C. lunata share IgE and IgG cross-reactivity within species Curvularia was also a fairly common cause of fungal kerati-
[35]. Two major C. lunata allergens have been identified and tis in far north Queensland, Australia, where a retrospective
cloned: Cur 1 1 was identified as a serine protease [36], while review was performed of all cases of fungal keratitis over a
rCur 1 2 proved to be an enolase, a glycolytic enzyme that 10-year period [53]. A higher incidence of fungal keratitis
has shown allergic potential in individuals allergic to molds was demonstrated during the monsoon and winter seasons
[37]. In addition, C. lunata enolase shows 90% homology than during the drier summer months.
with a Cladosporium allergen [37]. Although most research Risk factors for ocular Curvularia infections include
on Curvularia allergens is recent and has been performed soft contact lens wear [54] and laser in situ keratomileusis
at the gene recombinant level, recent mouse models have (LASIK) [55,56]. Tuli and Yoo report the case of a 50-year-
also shown that the serine protease Cur 1 1 plays an impor- old male who had recently undergone LASIK but devel-
tant role in the exacerbation of airway inflammation [38]. oped Curvularia keratitis postsurgically, likely linked to a
Allergens of this and other fungi should be further studied fungal skin infection of the patient’s pet cat [55]. Although
to contribute toward a greater understanding of Curvularia- Curvularia may be isolated from unwashed skin, it is an
related allergy and immunotherapy of allergy-mediated uncommon colonizer, present on less than 1% of normal
d isorders [39]. human eyelids [57]. Therefore, ocular infection is usually
related to injury or manipulation of the eye. In their ret-
rospective review of fungal keratitis, Iyer et al. reported
that, in 2005, contact lens use surpassed trauma as the most
The clinical manifestations are diverse and range from common risk factor for fungal keratitis, which remains a
noninvasive disease to severe, disseminated infection. common cause of corneal ulcers in the southern United
Furthermore, as molecular studies are becoming more avail- States [58]. In their study, Curvularia was the most com-
able and easier to perform, speciation of Curvularia has mon of the filamentous fungi cultured from corneal ulcers,
broadened our understanding of the spectrum of disease comprising 12%.
caused by this genus. Reports of infection in various sites are Ocular Curvularia infections may also present as con-
described below. junctivitis, dacryocystitis [59], endophthalmitis after intra-
ocular lens implantation [60], or extension to sino-orbital
8.1.3.1 Ocular Disease cellulitis; however, the cornea is most commonly affected.
Curvularia can cause various ocular infections. In their One sign of probable dematiaceous keratitis is the presence
review of 32 patients with culture-proven Curvularia ker- of clinically pigmented corneal ulcers [45]. Keratitis due to
atitis over a 30-year period, Wilhelmus and Jones demon- Curvularia spp. commonly presents as a slowly progressing,
strated that, while dematiaceous fungi accounted for 22% feathery, superficial corneal infection, Endophthalmitis may
of all fungal corneal isolates, one-third of the isolates were present as a posterior capsule plaque [61,62].
comprised of Curvularia spp. [4]. Curvularia was the fourth
most common fungal isolate identified from cases of kerati- 8.1.3.2 Allergic Fungal Sinusitis
tis, following Candida, Fusarium, and Aspergillus, at a large A form of noninvasive fungal rhinosinusitis, AFS is caused
hospital in Texas [4]. Curvularia spp. isolated from the Texas by a hypersensitivity response to the presence of fungal
study included C. senegalensis, C. lunata, C. pallescens, hyphae in the sinuses. Accounting for up to 7% of all sinusitis
and C. prasadii. Many of the largest case series of keratitis cases requiring surgery, AFS is commonly caused by demati-
involving dematiaceous fungi originate from around India aceous molds [63,64]. The diagnosis of AFS is primarily his-
[40–42]. In these series, Curvularia caused 3.3%–7.4% of topathologic, with demonstration of scattered fungal hyphae
all mycotic keratitis cases [41–43], 8.2% of fungal corneal within the viscoelastic allergic mucin viewed on biopsy.
ulcers in North India [40], and 6% of all cases of suppura- In most instances, invasion of the mucosal surface by the
tive keratitis in Bangladesh [44]. In a large series of mycotic organism is not seen. Cultures of surgically obtained mate-
keratitis reported from India, Curvularia spp. was the most rial commonly are positive for organisms such as Curvularia
common cause of dematiaceous fungal keratitis (approxi- spp. [65,66]. Although the criteria for diagnosis of AFS have
mately 23% of all dematiaceous keratitis cases) [45]. In changed over time, the current diagnostic criteria favor the
South India, among 1352 cases of fungal keratitis reviewed, presence of characteristic “allergic mucin,” accompanied
Curvularia represented 2.8% of all fungal etiologies and by the presence of noninvasive fungal elements within the
was the most common dematiaceous fungus isolated [46]. mucin, detected by either staining or culture [67]. The aller-
Similarly, among a hospital-based study in New Delhi of gic mucin usually contains numerous eosinophils, sometimes
191 patients with mycotic keratitis presenting with corneal with Charcot-Leyden crystals, and the mucin seems to be
ulcers, Curvularia spp. was the second most common cause more commonly viewed in biopsies of patients from warm,
of ulceration, second only to Aspergillus spp. [47]. humid climates [68].

A clinical history of nasal polyposis, thick mucus, and multiple nodules and sinuses draining black granules [81,82].
abnormal sinus computed tomography scans may be dem- C. lunata also caused a deep sternal wound infection in a
onstrated in patients with AFS [69]. Other diagnostic crite- neonate who underwent operation for congenital heart dis-
ria include a positive skin test to fungal allergens [68] or the ease, likely due to direct inoculation of aerosolized spores
presence of allergies to multiple aeroallergens [67]. In a study into the open sternal wound [83].
of 67 AFS patients from the southwestern United States, all
patients demonstrated inhalant atopy and skin test positivity 8.1.3.4 Cerebral Infections
to the AFS-implicated mold [70]. The majority of patients The first reported case of cerebral infection due to Curvularia
from this study also presented with reactive airway disease, was a 13-year-old boy with multiple cerebral lesions [84].
and a minority demonstrated aspirin/nonsteroidal anti- Other reported cases include a 41-year-old male with multiple
inflammatory drug hypersensitivity. Elevated total serum cerebral and pulmonary lesions [85] and C. lunata present-
IgE in AFS is also common, and serial monitoring of total ing as a pituitary mucocoele associated with optic atrophy
serum IgE over time reflects patient clinical status, as can [86]. Carter and Boudreaux report the case of fatal cerebral
changes in antigen-specific IgG [71]. C. lunata infection, which presented as a cerebral mass in an
Manifestations of AFS are due to type I immediate hyper- immunocompetent 21-year-old male [87].
sensitivity response to the extramucosal hyphae. Although
much remains unknown about the immunopathology of the 8.1.3.5 Peritonitis
disease, certain features have been consistently demonstrated In 1985, DeVault et al. reported catheter malfunction and
in AFS patients, including an eosinophilic/lymphocytic obstruction due to C. lunata in a continuous ambulatory peri-
mucosal inflammatory response [70], elevated total serum toneal dialysis (CAPD) patient, who presented without signs
IgE, and potential roles for IgG, and perhaps Staphylococcus or symptoms of peritonitis [88]. Several other reports of C.
aureus superantigens, in disease development [71]. lunata peritonitis have since been reported from peritoneal
Curvularia can also cause colonization of the tracheo- dialysis patients. Many of the peritonitis cases are reported
bronchial passage and sinus cavities, leading to recurrent or from the southern United States, such as Georgia, North
persistent hypersensitivity in certain individuals. True para- Carolina, Tennessee, and Louisiana [20,88–90]. A case of
nasal sinus infections may occur [72]. Intrasinus mycoses Curvularia peritonitis has also been reported from Brazil in
may lead to extension to contiguous structures such as the a 63-year-old male with end-stage renal failure due to diabe-
orbital contents or intracranial area. Chronic invasive sinus- tes mellitus who presented with turbid dialysis effluent with
itis is progressive and may spread to the orbit and brain. At gelatinous brown exudates discovered on exploratory lapa-
least one case report has documented C. lunata pansinusitis rotomy [91]. Several cases have documented the presence of
extension to the brain in an immunocompetent host [73]. black flakes in the dialysate fluid or the Tenckhoff catheter,
corresponding to Curvularia contamination of the fluid. In
8.1.3.3 Cutaneous and Subcutaneous Infections one peritonitis case, black material obstructed the lumen of
Curvularia has been associated with both cutaneous and deep the catheter, preventing flow [9]. CAPD patients with perito-
subcutaneous infections. Hiromoto et al. describe the case neal infection due to Curvularia may not always be symp-
of a 67-year-old immunocompetent male with Curvularia tomatic. In many reported cases, there were no problems
infection of an ulcerating lesion on his arm, after having with the catheters, such as site infection or mishandling of
been pricked and injured by a tree [6]. Curvularia has also the tubing, leading to contamination. However, Diskin et al.
been associated with an ecthyma gangrenosum-like lesion suggest that the risk factor for acquisition of Curvularia may
on the palm of a bone marrow transplant recipient [74], der- be the silicone dialysis tubing itself [26].
matomycosis of the toe web mimicking intertriginous tinea
pedis [75], and invasive fungal dermatitis, characterized by 8.1.3.6 Miscellaneous Infections
erosive, crusting lesions, in extremely low birth weight neo- Infections of internal organs are rare. A urinary tract infec-
nates [76]. Onychomycosis can also occur; in India, 4.5% of tion (UTI) has been reported in a 5-year-old girl, who pre-
onychomycosis was due to Curvularia [77–79]. sented with recurrent UTIs and black specks in her urine
Deeper infections include chromoblastomycosis, myce- [92]. Additionally, Shigemori et al. report a 10-year-old girl
toma, and phaeohyphomycosis. Chromoblastomycosis refers with acute monocytic leukemia, who presented with neu-
to subcutaneous and soft tissue infections that are character- tropenia and developed a hepatosplenic abscess caused by
ized on histopathology by pigmented, thick-walled sclerotic Curvularia boedijn [93].
bodies. Phaeohyphomycosis, on the other hand, is a broader
term and includes infections ranging from localized to dis- 8.1.3.7 Curvularial Infections, Other Than C. lunata
seminated invasive disease. Phaeohyphomycosis is differen- Although rarely isolated from humans, C. clavata has been
tiated from mycetoma by the absence of sulfur granules and reported to cause infections in various sites. Ebright et al. [89]
from chromoblastomycosis by the absence of sclerotic bodies described the first known case of C. clavata implicated in
within the biopsy [80]. Curvularia spp. have been associ- invasive sinusitis of a 46-year-old female, with bony destruc-
ated with many types of deep infections, including eumyce- tion of the cribiform plate and bifrontal cerebritis. Another
toma, as illustrated by the case of a 27-year-old farmer with case of C. clavata causing cutaneous, red plaque-like lesions

Curvularia 75
on the foot of a 64-year-old farmer with no known immu- cutaneous infections due to C. pallescens have been reported
nosuppression has recently been described [1]. None of as well [103].
the three case patients described above were known to be C. senegalensis has been associated with mycotic kerati-
immunocompromised at the time of presentation. Therefore, tis [51,104]. In at least one of these keratitis cases, there was
immunosuppression does not appear to be a necessary state no evidence of trauma to the eye to explain fungal inocula-
for infection with this species. tion; however, one of the patients was diabetic and had used
C. brachyspora was documented to cause cutaneous infec- steroid-containing eye drops [104].
tion in an immunocompetent 58-year-old male [94]. A month In summary, various Curvularia species have been
prior to presentation, the patient had cryotherapy performed associated with a wide variety of disease presentations. As
on some pigmented thigh lesions, which were suspected molecular techniques become more available, it is likely that
to be seborrheic keratoses. After cryotherapy, the lesions newer disease states associated with certain Curvularia spe-
worsened, developing into necrotizing ulcers, which demon- cies will continue to be discovered.
strated necrotizing cellulitis on biopsy and C. brachyspora
on culture.
8.1.4 Diagnosis
Occasional reports of C. geniculata have emerged in the
literature. C. geniculata has been reported as a cause of 8.1.4.1 Conventional Techniques
mycotic keratitis [95] and endocarditis associated with an 8.1.4.1.1 Microbiologic Culture
aortic valve graft [96]. Disseminated disease due to C. genic-
In the laboratory, colonies of Curvularia spp. display a
ulata was reported in a football player with B- and T-cell
brownish-black to dark olive-green surface and a dark-
hypofunction, after an initial infected skin lesion dissemi-
colored to black reverse. The colonies are filamentous and
nated to lymph nodes, vertebrae, and the pulmonary cavity
expanding. Colonies grow rapidly on plates, generally reach-
[97]. The first known case of C. geniculata peritonitis was
ing maturity within 5 days. The optimal temperature for
described by Vachharajani et al. in a 45-year-old male on
growth is 28°C, although growth may occur from 25°C to
CAPD, despite no macroscopic evidence of catheter infection
35°C [105,106]. Microscopically, a lactophenol cotton blue,
[98]. C. geniculata has also been shown to cause central ner-
or other preparation of the colony, will demonstrate that the
vous system infection in a 35-year-old male who presented
conidiophores are brown, erect, and multicellular, producing
with obstructive hydrocephalus due to a large cranial base
conidia sympodially (bent at points of conidium formation).
lesion, which initially appeared on imaging and biopsy to
Conidia are dark, thin-walled, ellipsoidal, and large (up to
be a meningioma [99]. C. geniculata was isolated in culture,
14 μm in width and 35 μm in length), with a variable num-
and speciation was confirmed by molecular sequencing. An
ber of true transverse septae that differ according to the spe-
underlying plasma cell dyscrasia was subsequently diagnosed
cies. As the central cell of the conidium is larger and darker
in this patient, which likely predisposed him to development
than the others, the conidia show a characteristic curve, or
of this infection. Despite aggressive therapy with two anti-
boomerang-like bend, which becomes more pronounced with
fungal agents, the patient died 4 months after initial biopsy
age (Figure 8.1).
from multiorgan failure.
Immature Curvularia spp. may be mistaken for Bipolaris
C. inaequalis was recently reported to cause human dis-
spp., which possess straight, multicellular conidia. Conidia
ease in 2005 by Pimentel et al., who described the case of an
of some Curvularia species may show a prominent hilum. In
85-year-old CAPD patient with peritonitis [100]. The peri-
the mycology laboratory, speciation can be performed with
toneal fluid grew C. inaequalis, which was confirmed by
the use of conidial morphology and growth patterns [105].
sequencing of the internal transcribed spacer (ITS) region. As
the patient reported no recent contact with soil, risk factors
for acquiring infection with this organism were unknown.
C. inaequalis was also reported to cause eosinophilic fun-
gal rhinosinusitis in a 45-year-old male, manifesting as bony
erosions of the nasal cavity [101]. Although the patient was
deemed to be immunocompetent in this case report (human
immunodeficiency virus negative and no evidence of malig-
nancy), the patient did receive steroids for his long-standing
nasal obstruction, which may have led to some degree of
immunosuppression.
The first known cerebral case Curvularia infection was
that of C. pallescens in an immunocompetent 13-year-old
boy with pulmonary and cerebral abscesses [84]. The sec-
ond reported case of C. pallescens was described in a report
of cutaneous infection in a 75-year-old woman with rheu-
matoid arthritis who presented with an ulcerated lesion on Figure 8.1 Slide culture preparation of Curvularia spp.,
her leg with no antecedent history of trauma [102]. Other d emonstrating the characteristic curved shape of the conidia.

8.1.4.1.2 Histopathology [109]. The rRNA gene is structured as a small-subunit (SSU)

On a hematoxylin and eosin (H&E)-stained slide, the fun- 18S rRNA, a large-subunit (LSU) 28S rRNA, and a 5.8S
gus appears as yeast-like cells that are solitary or in short rRNA. ITS regions I (ITSI) and ITSII show more variability
chains, or as septate hyphae or pseudohyphae (Figure 8.2). than the remainder of the ribosomal gene subunits. ITSI is
Hyphae may exhibit vesicular swelling and bizarre shapes located between SSU rRNA and 5.8S rRNA, while ITSII is
[107]. The similarity on tissue sections to Aspergillus located between 5.8S rRNA and LSU rRNA. ITSI is more
spp., as well as to other organisms, such as Fusarium and variable than ITSII and, thus, allows for better species dif-
Scedosporium, renders diagnosis almost impossible with- ferentiation [110].
out microbiologic culture results. Brown pigmentation of Researchers have used many methods to assess sequence
Curvularia on routine H&E-stained tissue is common but variation of amplified DNA such as direct sequencing of the
not invariably present [108]. Although Fontana-Masson sil- amplified product, random fragment length polymorphism,
ver staining, which highlights the melanin in the cell walls temperature gradient gel electrophoresis, and denaturing gra-
of dematiaceous fungi, may be used to differentiate dema- dient electrophoresis, as well as the single-stranded confor-
tiaceous from nonpigmented fungi, the stain is not specific, mation polymorphism (SSCP) technique [110–113].
as many nondematiaceous fungi may stain with this tech-
nique [108].
8.2 Methods
8.1.4.2 Molecular Techniques
Identification of fungal pathogens in the microbiology lab-
oratory may be challenging, as filamentous fungi may not Many methods for extraction of fungal DNA have been pro-
sporulate, or morphologic features may seem similar among posed [110,114–117]. Outlined below is one of several extrac-
species. Phenotypic assays allow differentiation among spe- tion methods that may be used, which is adopted from Kumar
cies but may take up to 12 weeks, and more rapid identifica- and Shukla [114]:
tion is often needed [105]. In addition, new species of fungi
are being discovered, which may not be familiar enough 1. For extraction of fungal DNA, it is suggested to
to the laboratory worker to be included in the differential. begin with a strain that has been inoculated in
Species differentiation may be important for appropriate 100 mL of Sabouraud dextrose broth under shaking
therapy. Although nucleic acid probes are commercially conditions at 37°C, which optimizes extraction, as
available for a limited number of fungi, such as the ther- log-phase growth is generally achieved under these
mally dimorphic fungi, molecular methods of detection conditions.
are proving to increase both the reliability and the speed of 2. The cultures should be examined microscopically
diagnosis. to insure purity.
A common target for the molecular diagnosis of filamen- 3. Centrifuge the fungus for 15 min at 6000 × g at 4°C.
tous fungi is the ITS region. ITS regions are located within 4. Wash the pellets twice with 0.8% physiologic saline.
ribosomal DNA (rDNA). rDNA is a useful fragment for fun- 5. Transfer the pellets to 200 μL extraction buffer
gal identification, as it is universally present and is the most (0.2 M Tris–HCl pH 7.6, 0.5 M NaCl, 0.1% sodium
conserved region in the genome of fungi but maintains some dodecyl sulfate, 0.01 M EDTA).
variability to allow for species separation and identification 6. Add glass beads to this mixture in a 1:1 ratio.
7. Vortex vigorously in a bead beater to achieve
60% lysis of the mycelial mass (approximately
30 min, with intermission every 5 min to avoid
overheating).
8. Fungal DNA may then be recovered with the use of
kits such as the DNeasy plant mini kit (QIAGEN).
9. In order to assess for product, electrophorese the
product on a 1% agarose gel with 1× TBE buffer
(8.9 mM Tris-borate, 0.2 mM EDTA).
10. Stain with ethidium bromide and assess for product.
11. Check for purity of the extracted DNA at 260 and
280 nm with spectrophotometric analysis.

Some detection methods that have been assessed using
Figure 8.2 Histopathologic slide of Curvularia spp. in a sinus Curvularia spp. are outlined in Table 8.1. Below is a sug-
biopsy of a patient with paranasal disease, demonstrating the swollen, gested method of detection using sequence analysis of the ITS
elongated, yeast-like cells viewed on biopsy. rRNA gene region, as described by Sanguinetti et al. [117]:

Curvularia
Table 8.1
Published Molecular Methods for Identification of Curvularia spp.
No. of Sample
Examined Curvularia Preparation and Performance of System, as
Method Target Material spp. Examined Extraction Detection Method Relates to Curvularia spp. Miscellaneous Reference
MicroSeq D2 D2 LSU Filamentous 2 Curvularia PrepMan ultra Cycle sequencing followed by ABI 3100 16 Curvularia identified Commercially [116]
LSU rDNA rDNA fungi, spp. sample preparation capillary genetic analyzer (Applied correctly. One isolate available
sequencing fragment including reagent (Applied Biosystems) identified as Bipolaris
kit dematiaceous Biosystems) hawaiiensis/Cochliobolus
ITS (internal ITS1-5.8S- Black-grain 4 Curvularia Glass bead lysis Amplification of the rRNA gene with primers Differentiated Curvularia Certain [115]
transcribed ITS2 DNA mycetoma spp. method, V9D and LS266 in ICycler thermocycler well from other fungi, but components are
spacer)1 region agents snap-frozen, then (Bio-Rad). Sequencing performed using some difficulty with commercially
sequencing DNeasy plant kit BigDye Terminator Cycle Sequencing Ready intraspecies variation available
(QIAGEN) Reaction kit, version 3.1 (Applied Biosystems,
Foster City, CA). Analysis performed with
ABI Prism 3700 automated DNA analyzer
(Applied Biosystems)
Sequencing ITSI and Direct corneal 1 case of Glass bead lysis Six fungus-specific primers (primers ITSI, One case of C. inaequalis Certain [114]
of ITSI and ITSII scrapings Curvularia method, then ITS2, ITS3, ITS4, invSR1R, and LR12R) were keratitis confirmed by components are
ITSII, sequencing keratitis DNeasy plant kit used in amplification. Sequencing of ITSI and culture commercially
confirmed (QIAGEN) ITSII was performed with primer pair ITS1 available
by SSCP and ITS4 with an ABI Prism automated DNA
sequencer. Results confirmed with SSCP
Note: ITS, internal transcribed spacer; SSCP, single-stranded conformation polymorphism.
77
1. Amplify the ITS1-5.8S-ITS2 region of the rRNA Cochliobolus (the MicroSeq system was unable to distin-
gene, using a 1:50 dilution of template DNA. guish between the two genera listed). However, the distance
2. The total reaction volume should be 50 μL (10 mM score was only mildly elevated, at 2.48. In summary, the
Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, D2 rDNA fungal library contains sequences that represent
0.8 mM deoxynucleoside triphosphates at 0.2 mM 205 genera of filamentous fungi and include type culture for
each, 1.2 U Taq DNA polymerase, 0.5 μM each many fungi, while offering rapid and accurate identification
of the fungus-specific universal primers ITS1 of organisms.
(5′-TCCGTAGGTGAACCTGCGG-3′), and ITS4 Desnos-Ollivier et al. described the accuracy of molec-
(5′-TCCTCCGCTTATTGATATGC-3′). ular identification of several dematiaceous fungi causing
3. Thermal conditions: 94°C for 3 min, 35 cycles each black-grain mycetoma, using the ITS1-5.8S-ITS2 DNA
at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min region [115]. The authors examined 54 strains of fungi from
and 30 s, and the final extension step of 72°C for the Pasteur Institute and the National Reference Center for
10 min. Mycoses and Antifungals; 78% of strains were clinical in
4. Purify amplicons (suggestion includes a Minielute origin. Four C. lunata stains were examined in their study.
PCR purification kit [QIAGEN]). Their methods are outlined briefly below. For DNA extrac-
5. Sequence with primer ITS1 or ITS4 and BigDye ter- tion, strains cultured in RPMI 1640 medium were extracted
minator cycle sequencing kit (Applied Biosystems) using a glass bead lysis method and the DNeasy plant kit
on the ABI Prism 3100 genetic analyzer (Applied (QIAGEN). The rRNA gene was then amplified by PCR
Biosystems). using primers V9D and LS266 [118]. Amplification took
6. Species may be identified by searches on the BLAST place in an ICycler thermocycler (Bio-Rad). PCR products
sequence analysis tool (http://www.ncbi.nlm.nih. were purified on P-100 Gel Fine (Bio-Rad). Strands were then
gov/BLAST/). An isolate may be assigned to a spe- sequenced using the BigDye Terminator Cycle Sequencing
cies if there is ≥99% homology in sequence to the Ready Reaction kit, version 3.1 (Applied Biosystems), with
highest entry, and if the next entry/species shows primers V9D and LS266. Products were then analyzed using
less than 95% homology when the entire length of an ABI Prism 3700 automated DNA analyzer (Applied
the sequence is examined. Biosystems). Sequences were edited with Chromas, version
2.24 (Technelysium, Helensvale, Queensland, Australia).
Hall et al. reported on their experience with the commer- Three of the four C. lunata strains from the study by
cially available MicroSeq D2 LSU rDNA fungal sequencing Desnos-Ollivier et al. showed intraspecies similarities of
kit (Applied Biosystems) [116]. The MicroSeq kit has multi- >98%. The single strain that showed sequence differences of
ple components and is comprised of PCR and cycle sequenc- more than 50 base pairs (>10% differences) was tentatively
ing modules, identification and analysis software, and a identified as Curvularia species, as the species could not
library of fungal nucleic acid sequences. The authors com- be confirmed. In comparison to other dematiaceous black-
pared nucleic acid sequencing using the MicroSeq kit with grain mycetoma agents analyzed in this study, the four C.
traditional identification of cultures by phenotypic methods. lunata strains showed sequence similarities ranging from
A total of 234 filamentous fungi from culture were evaluated, 60% to 79%, both between species and between genera. In
2 of which were Curvularia spp. In their study, DNA extrac- conclusion, although intraspecies variation was observed for
tion was performed using PrepMan Ultra sample preparation C. lunata, the wide intergenera and interspecies variation
reagent (Applied Biosystems). The D2 LSU rDNA fragment observed between C. lunata and other black-grain mycetoma
was then amplified. Once the presence of amplicon was con- agents demonstrate that ITS sequencing may be a useful tool
firmed, purification of the PCR product was performed to for reliable separation of Curvularia spp. from other com-
remove excess primers and nucleotides. Next, cycle sequenc- mon black-grain mycetoma agents examined in this study.
ing was performed using the sequence module reagents, with However, more ITS sequencing studies should be performed
the following parameters: 25 cycles of 96°C for 10 s, 50°C on Curvularia spp. to examine the system’s ability to speci-
for 5 s, and 60°C for 4 min. Labeled amplicon was placed ate this organism.
onto the ABI 3100 16 capillary genetic analyzer (Applied Kumar et al. diagnosed four cases of mycotic keratitis
Biosystems). The organism that gave the closest match to the using SSCP of the ITS1 region [110]. Clinical testing material
sequence file was considered to be the most closely related consisted of corneal scrapings from affected patients, from
organism. whom genomic DNA was directly extracted. The authors
Of the two Curvularia spp. in the study by Hall et al. found their method to be both sensitive as well as cost-
that were identified based on phenotypic methods and then effective, although no cases of Curvularia were diagnosed
sequenced, one had a distance score of ≤1% (99% similar- in this study.
ity) to the MicroSeq D2 fungal library (which contained 788 In another study, Kumar and Shukla diagnosed mycotic
species of filamentous fungi) (version 1.4.2, February 2002). keratitis in three patients using SSCP with six fungus-
The other Curvularia spp. showed a distance score of ≥1%, specific primers (primers ITSI, ITS2, ITS3, ITS4, invSR1R,
and its sequence was identified as Bipolaris hawaiiensis/ and LR12R) [114]. All three patients were positive for

Curvularia 79
fungi by this technique, one of whom had C. inaequalis treatment may be unknown. It will be necessary for micro-
keratitis. Molecular results correlated with culture results. biology laboratories and clinicians to communicate these
Complete sequencing of the ITSI and ITSII was performed potentially significant findings.
with primer pair ITS1 and ITS4 using an ABI Prism auto-
mated DNA sequencer (model 3100, version 3.0, Applied
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keratitis. J. Clin. Microbiol., 43, 662, 2005.

9 Exserohilum
K. Lily Therese and H.N. Madhavan
Contents
9.1 Introduction........................................................................................................................................................................ 83
9.1.1.1 Classification............................................................................................................................................ 83
9.1.1.2 Description and Natural Habitats............................................................................................................ 84
9.1.2 Epidemiology.......................................................................................................................................................... 84
9.1.3 Pathogenesis and Clinical Features........................................................................................................................ 85
9.1.3.1 Invasive and Noninvasive Infections....................................................................................................... 85
9.1.3.2 Signs and Symptoms................................................................................................................................ 85
9.1.4 Immune Response.................................................................................................................................................. 86
9.1.5 Diagnosis................................................................................................................................................................ 86
9.1.5.1 Phenotypic Identification......................................................................................................................... 86
9.1.5.2 Genotypic Identification.......................................................................................................................... 86
9.1.6 Treatment and Outcome......................................................................................................................................... 87
9.2 Methods.............................................................................................................................................................................. 88
9.2.2.1 Phenotypical Criteria for Identification of Molds.................................................................................... 88
9.2.2.2 Slide Culture............................................................................................................................................ 88
9.2.2.3 Lactophenol Cotton Blue Mount.............................................................................................................. 89
9.2.2.4 Lactophenol Mounts with Tease Mount Procedure................................................................................. 89
9.2.2.5 Scotch Tape Lactophenol Mount............................................................................................................. 89
9.2.2.6 Molecular Detection Method................................................................................................................... 90
9.3 Conclusion and Future Perspectives................................................................................................................................... 90
References.................................................................................................................................................................................... 91
9.1 Introduction skin infections, although a few cases of cerebral abscesses,

keratitis, osteomyelitis, prosthetic valve endocarditis, and
Phaeohyphomycosis is the term used to denote the infec- disseminated infection have been described.4–7
tions caused by more than 100 species in 60 genera belong-
ing to dematiaceous fungal pathogens (including the
common and closely related genera Bipolaris, Drechslera, 9.1.1 Classification, Morphology, and Biology
and Exserohilum). The genus Exserohilum was established1
for species having distinctly protruding hila.2 This elimi- 9.1.1.1 Classification
nated the inconsistency and permitted a more logical group- The taxonomic classification is as follows: Kingdom
ing of species into three anamorphic genera, Bipolaris, Fungi, Phylum Ascomycota, Class Euascomycetes, Order
Drechslera, and Exserohilum. Alcorn et al.3 described in Pleosporales, Family Pleosporaceae, Genus Exserohilum.
detail the concept and justification for grouping them into The distinct telemorphic state of Exserohilum is Setosphaeria.
three genera. Exserohilum species are ubiquitous but rarely Exserohilum is a terrestrial plant pathogen and is differenti-
pathogenic for human beings. They are encountered in grass ated from the closely related genera based on the conidial
or rotting wood and thrive in warm and humid climates. formation from a protruding exserted hilum. The dark pig-
The genus is characterized by its conidia, which are ellipsoi- mentation is caused by deposition of dihydroxynaphthalene
dal, distoseptate, and have a protruding and truncate hilum. melanin.4 The hilum is present as a scar on a conidium, at the
Three species of Exserohilum have been recognized as point of attachment to the conidiophore. It is distinguished
human pathogens: Exserohilum rostratum, Exserohilum lon- on the basis of conidial shape and size, hilar morphology,
girostratum, and Exserohilum mcginnisii. The most common location and sequence of the conidial septa, and the origin
infections caused by Exserohilum species are sinusitis and of the germ tubes from the conidial cells. The conidium of
83

Exserohilum species is ellipsoidal to fusiod and germinates measuring about 13–16 × 196–260 μm in size. Conidia are
by germ tubes originating from either one or both of the end broadest at the basal end with a strongly protruding truncate
cells or other intermediate cells. basal hilum, contain 13–21 distosepta, and narrow gradually
towards the apex into a long beak. They resemble the conidia
9.1.1.2 Description and Natural Habitats of E. rostratum with the pale end cells and finely roughened
Exserohilum is one among the three dematiaceous (Bipolaris, walls. E. longistratum has predominantly 9–11 septa. The
Drechslera, Exserohilum) fungi that are commonly known conidia are present in two forms—long (13–21 septa) and
to cause infections in human beings. It is cosmopolitan in short conidia (5–7 septa). The conidia have prominent dark
nature inhabiting plant material, particularly grasses, and basal and distal septa, frequently displaying a curvature in
soil. More than 20 host-specific species are known world- the middle. The size of the conidia measures between 60
wide. It has been shown that human Exserohilum infec- and 475 μm in length × 12–26 μm in width. E. longistratum
tions occur mainly in warm, tropical, and subtropical areas produce elongate conidia with a rostrate shape when peri-
such as the southern United States, India, and Israel. The odically exposed to fluorescent light, but when grown in the
two recently reported new species Exserohilum israeli5 dark, the septa separating the distal and basal conidial cells
and Exserohilum sodomii6 are from Southern Israel. The appears darkly pigmented and thickened. The obsolete names
differential identification of the common three species of for anamorph of E. longirostratum are Bipolaris longiro-
Exserohilum is based on the macroscopic and microscopic stratum, Drechslera longirostratum, and Helminosporium
morphology of the conidia. longirostratum.8
9.1.1.2.1 Exserohilum rostratum (E. rostratum) 9.1.1.2.3 Exserohilum mcginnisii (E. mcginnisii)
E. rostratum grows well on Sabouraud’s dextrose agar (SDA) The colonies on PDA are raised, wooly, and deep mouse
and potato dextrose agar (PDA). The colonies appear cot- gray. The size of the colony varies depending on the tem-
tony to woolly in texture, raised in the central area measur- perature at which it grows. When grown at 25°C the diam-
ing about 25–27 mm in diameter, at the end of 2 weeks of eter is 35–37 mm, at 37°C the diameter is 25–27 mm, and at
incubation at 25°C. The colonies are gray to blackish-brown, 40°C the diameter is only 5–6 mm at the end of 2 weeks of
suede-like to floccose in texture, and have an olivaceous incubation.7
black reverse. The conidial formation on PDA may take up The hyphae are septate, branched, and pale to mid brown
to 3 weeks. The fungus grows well at 37°C, but the growth in color. The conidiophores are simple, arising singly, erect
is very slow at 40°C with only 5–6 mm in diameter after 2 or flexuous, upper part geniculate, and brown to mid brown.
weeks. The growth is partially inhibited when grown on SDA Conidia are straight, cylindrical to ellipsoidal, broadest
containing cycloheximide.7 in the middle with rounded apices, 64–100 × 10–15 μm, and
Hyphae are septate and dematiaceous, with conidio- typically with 4–13 septa, but the mature conidia have typi-
phores up to 200 μm long, which are nonbranched, septate, cally 9–11 septa. The conidial walls are irregular with warty
geniculate, and are pale near the apex. Conidia are straight projections. The hilum is distinctly protuberant with irregular
to slightly curved, rostrate or beaklike, and olivaceous warty projections. Germination is bipolar. The pale end cells
brown, and contain typically 7–9 pseudosepta (range from of the conidia are not separated from the intercalary golden
4 to 14). The conidia have prominent, dark basal, and distal brown cells by thick walled distosepta.7 The size of the septa
septa, and a strongly protruding truncate hilum. The mature also ranges from 10 to 15 × 64 to 100 μm. The characteristic
conidia range from 8 (ranging from 6 to 13 μm) to 23 μm features useful for differential identification of E. mcginnisii
(ranging from 14 to 34) in size, straight, slightly curved or from E. rostratum are lack of darkly pigmented bands at the
bent, and cylindrical to rostrate. The end cells, often paler ends of its conidia, irregular warty projections from the outer
than the other cells, and the walls are often finely rough- conidial cell walls, and slightly concave depressions in the
ened. Conidia have a strongly protruding truncate hilum conidial cell walls with larger numbers of septa and broader
and the septum above the hilum is usually thickened and basal cells.8
dark. The mature conidia have characteristic darkly pig-
mented septum at both ends of conidium that is charac-
9.1.2 Epidemiology
teristic of E. rostratum, useful to differentiate conidia of
E. mcginnis.8 Exserohilum species are ubiquitous but rarely pathogenic for
human beings. They are encountered in grass or rotting wood
9.1.1.2.2 Exserohilum longirostratum (E. longirostratum) and thrive in warm and humid climates.9 The major predispos-
E. longirostratum is considered as a variant of E. rostratum. ing factors reported are trauma, corticosteroid therapy, diabe-
Colonies on PDA are woolly to cottony and are black to dark tes, autoimmune diseases, lymphoma, leukemia, etc. Some
gray with a black reverse. Growth is rapid at 25°C.7 are true pathogens and are kept under biosafety level two
Hyphae are septate and brown. Conidiophores are pale containment, others are opportunistic pathogens. The clinical
near the apex, septate, simple, geniculate, and measure localization of the pathogens has been seen in nails, skin, cor-
up to 200 μm in length. The mature conidia are straight, nea, lungs, sinuses, liver, heart, genitals, bone, kidneys, brain,
ellipsoidal, and strongly protruding with a truncate hilum lymph nodes, etc. The four major clinical manifestations are

Exserohilum 85
cerebral infections, subcutaneous infections, paranasal sinus- only 23 (20 E. rostratum, 2 E. longirostratum, and 1 E.
itis, and disseminated form of infections. mcginnisi) were identified to species level. The details of
The first review on 10 published reports on human all the 33 patients were grouped into three different cat-
infections caused by Bipolaris and Exserohilum species10 egories: invasive infections (10), skin and corneal infections
indicated that these genera were able to cause infections (14), and chronic sinusitis (9). All the details pertaining to
in apparently healthy hosts. Involvement of the paranasal the 33 are tabulated in their review article. Briefly, in the
sinuses, central nervous system, and other tissues was com- invasive infection category, 7 out of the 10 were children
mon to both genera. The ability of the above-mentioned below the age of 18 and the predisposing factors were acute
species to cause life threatening neurotropic disease is lymphoblastic leukemia (4), aplastic anemia (2), and acute
indicated by their ability to grow at 40°C. The authors myeloid leukemia (1). Noninvasive infections were subdi-
concluded that Bipolaris or Exserohilum species isolates vided according to the main site of infection: solitary skin
from clinical specimens should not be regarded simply as infections, corneal infections, and chronic sinusitis. The
contaminants; appropriate studies should be carried out to corneal infections followed local trauma without any sys-
determine if they play an etiological role. In yet another temic infections. The majority of the skin infections with
study by Padhye et al. (1986),7 seven cases of Bipolaris Exserohilum infections were immunocompromised patients
infection and two of Exserohilum infection were reported, (cases of sinusitis were categorized as acute-invasive or
demonstrating the capability of these two genera to cause chronic).16
invasive as well as “allergic” diseases in humans. These
two genera of fungi are to be included in the differential 9.1.3.2 Signs and Symptoms
diagnosis of the central nervous system, disseminated fun- Sinusitis, the most common form of disease caused by
gal diseases, sinusitis, keratitis, peritonitis associated with Exserohilum, occurs in otherwise healthy patients with
continuous ambulatory peritoneal dialysis, and allergic nasal polyps and allergic rhinitis. The most frequent find-
bronchopulmonary diseases. Although pathologic evidence ings in the immunocompromised patient are acute inflam-
of bone invasion may not be found, there frequently is radio- mation and with prominent vascular invasion, thrombosis
graphic evidence of invasive disease. As the analysis did not and infraction. In contrast, granulomatous inflammation
show a correlation between the regional mold counts and the and leukocytoclastic vasculitis are seen in meningoen-
incidence of infection, the geographic difference in preva- cephalitis caused by these fungi. Exserohilum is capable of
lence could be related to a climate-induced alteration in the causing central nervous system disease, osteomyelitis, and
physiology of the mold, which renders it more pathogenic.11 sinusitis and is associated with allergic bronchopulmonary
The most common infections caused by Exserohilum spe- disease. In immunocompromised patients the disseminated
cies are sinusitis and skin infections, although a few cases disease is characterized by tissue necrosis and vascular
of cerebral abscesses, keratitis, osteomyelitis, prosthetic invasion. These various entities have distinct histopatho-
valve endocarditis, and disseminated infection have been logic characteristics that are described as case reports in
described in the literature.12–15 the literature.
A PubMed search of the English-language literature Pathogenesis of the disease caused by Exserohilum is lit-
using the key words Exserohilum and Drechslera from tle understood, however, melanin, enzymes, and toxins are
1950 till 2006 by Adler et al.11 resulted in 33 cases of reported as virulent factors. Exserohilum may infect immu-
Exserohilum infection, of which 23 were reported since nocompromised as well as immunocompetent patients. The
1993. The increase may be due to improved diagnostic tech- clinical manifestations range from cutaneous infections to
niques. The majority of the Exserohilum infections were severe disseminated disease. The infiltrating inflammatory
reported from regions with hot climates—16 cases from cells include lymphocytes, macrophages, focal neutrophils
southern or southwestern states of the United States, 6 cases and eosinophils, and plasma cells. There are different types
from Israel, and 5 cases from India.11 Impaired immunity of Exserohilum infections associated with distinct clinical
was present in the majority of patients with invasive and presentations and modes of therapy. Invasive infections, most
skin infections. In cases with corneal infections and allergic commonly of the respiratory tract, affected mainly immuno-
fungal sinusitis, local trauma and atopy were the predispos- compromised patients. Apparently, the mode of transmission
ing factors. in these cases was inhalation, sometimes with hematogenous
spread to other sites (brain, bone, skin). The management of
patients with invasive disease included prolonged courses of
9.1.3 Pathogenesis and Clinical Features systemic antifungal agents and, if feasible, surgery. Outcome
appeared to be better (attributed mortality of 30% in inva-
9.1.3.1 Invasive and Noninvasive Infections sive infections) than for other mold infections and depended
Exserohilum infections are divided into two groups: inva- mainly on the underlying diseases. Surgery was required in
sive and noninvasive infections. The invasive infections the following cases: paranasal sinus infection, other invasive
occurred mostly in immunocompromised patients in the infections (bone and cardiac valve), subcutaneous infections,
respiratory tract. The possible mode of transmission could and in two of the corneal infections that were similar to other
be inhalation. Of the 33 cases reported by Adler et al.,11 mold infections.17,18

9.1.4 Immune Response the simple KOH preparation by allowing for easier and faster
detection of fungal elements using a florescent microscope.
The host’s innate immune response plays an important role The diagnosis of Exserohilum infections is based pri-
in protection against invading fungal pathogens. The first marily upon the morphologic findings. Exserohilum, like
line of defense includes acellular microbicidal components its related genera, is characterized by a sympodial conidio-
such as products of complement activation cascade, cationic phore. It has an ellipsoidal to fusoid conidium and, more spe-
proteins, and proteolytic enzyme secreted along the mucosal cifically, a protruding, truncate, pigmented hilum. However,
lining of the respiratory tract. The principal cellular com- current culture-based phenotypic methods are insensitive
ponent during this early nonantigen-specific innate immune and slow, may initially be nonspecific, and require consider-
response is mediated via phagocytic activity of alveolar mac- able expertise for correct morphological identification of less
rophages and recruited neutrophils; toll-like receptor (TLR) common or unusual fungi.
2 and TLR 4, were recently recognized as critical in the Recent efforts to improve the sensitivity and specificity of
early recognition of infection. Interestingly, TLR 4-mediated diagnostic tests have focused on culture-independent methods,
immune activation is more focused against the fungal microin particular nucleic acid-based methods, such as polymerase
conidia, whereas TLR 2 is important for eliciting immune chain reaction (PCR) assays. These can be applied to fresh and
response against microconidia and the developed fungal formalin-fixed, paraffin-embedded (FFPE) sections. Numerous
hyphae.19 Tumor necrosis factor (TNF) has also been shown studies have highlighted the advantages of using PCR technol-
to be an essential mediator of early immune activation follow- ogy to detect viable and nonviable fungal pathogens in a variety
ing ingestion of infectious fungal spores or microconidia.20 of clinical specimens. The majority of assays target multicopy
Improved understanding of the molecular pathogenesis of genes, in particular the ribosomal RNA (rRNA) genes (18S,
fungal infections and of the complexity of host antifungal 28S, and 5.8S) and the intervening internal transcribed spacer
immune responses has provided the critical information to (ITS) regions (ITS1 and ITS2), in order to maximize sensitivity
readdress existing treatment paradigms. and specificity. Sequence-based identification of PCR products
is a sensitive alternative, provided that accurate sequences have
9.1.5 Diagnosis been submitted to public databases like GenBank.
9.1.5.1 Phenotypic Identification 9.1.5.2 Genotypic Identification

The clinical microbiology laboratory should provide and The identification of fungi to species level is very important
have experience with methods used for the rapid detection in directing the treatment strategy due to the high incidence
and identification of the fungi associated with infections of of antifungal drug resistance and with regard to newly emerg-
the immunocompromised patient. The clinician must sub- ing fungal pathogens. In about 75% cases fungal identifica-
mit the specimens to the microbiology laboratory to confirm tion to species level is possible by conventional methods. In
the diagnosis of opportunistic fungal infection. Diagnosis the remaining, the identification is not possible because of
of Exserohilum infections are based on direct microscopy, atypical characters of certain fungi, ability to grow in dif-
culture, and histopathology. Early, rapid, and accurate iden- ferent morphological forms, and emergence of new species.
tification of pathogenic fungi is important for the selection Numerous targets within the fungal genome have been
of appropriate antifungal therapy. Most often stains and cul- evaluated, with much of the current work using sequence
tures of tissue biopsies are necessary to establish a primary areas within the rRNA gene complex. Multiple applica-
diagnosis. Fungal serologic tests are not helpful in making tions using this as target include direct detection from clini-
the diagnosis for Exserohilum infections. The most rapid and cal specimens, culture identification, phylogenetic research,
useful of the methods available is the microscopic detection and molecular typing for epidemiological purposes. The
of fungal elements in clinical specimens. breadth of applications of rRNA gene complex has great
The potassium hydroxide (10% KOH) preparation is the potential in characterization and identification of fungi. PCR
most widely used method for the detection of fungal elements based detection of fungal DNA sequences are proven to be
directly from any clinical specimen. Basically, samples of specific, sensitive rapid and reliable. The coding regions of
a clinical specimen, usually body fluid and 10% KOH are the 18S, 5.8S, and 28S nuclear rRNA genes are conserved
placed on a microscope slide to digest the tissue debris and among fungi as they are known to evolve slowly and provide
artifact which may interfere with the detection of fungal ele- a molecular basis of establishing phylogenetic relationships.
ments. This method may provide the earliest evidence of a Between coding regions are the ITS1 and 2 regions (ITS1 and
fungal infection for the clinician. In addition, a fluorescent ITS2, respectively) which evolve more rapidly and may there-
brightener calcofluor white, is recommended for use with the fore vary among different species within a genus. Thus, PCR
KOH preparation. This reagent fluoresces upon excitation amplification targeting ITS region may facilitate the identifi-
with ultraviolet light and stains fungi, causing them to exhibit cation of DNA sequences with sufficient polymorphism to be
fluorescence which can be detected using the florescent useful for identifying the Exserohilum to species level.
microscope. Tissue section specimens can also be stained Identification of fungi to species level is essential to direct
with calcofluor white, and results are immediately available the antifungal treatment. Development of rapid and accu-
within 15–20 min. This method has better sensitivity than rate assays to diagnose fungal infections could potentially

Exserohilum 87
impact care and improve outcome of affected patients.21,22 to screen for fungal infection, while the variable sequences
Fungi have a rRNA gene complex with comparable charac- can be exploited for species identification. Indeed, favor-
teristics. The organization of this complex in fungi includes able results targeting this ribosomal gene as a tool for fungal
a sequence coding for the 18S rRNA gene, an internal tran- detection and identification have been obtained.
scribed spacer region 1(ITS1), the 5.8 S rRNA gene coding
region, another ITS region called ITS2 and the sequence 9.1.5.2.2 Amplification and Detection Methods
coding for 28S rRNA gene. The 28S rRNA gene evolved Nucleic acid hybridization and amplification methods are
slowly and is relatively conserved among fungi providing a fundamental to molecular diagnosis. Hybridization tech-
molecular basis of establishing phylogenetic relationships. niques employ a DNA probe to determine whether a par-
Early work in molecular testing using rRNA complex as tar- ticular organism is present. PCR is the most frequently used
get concentrated in the region of 18S rRNA also referred as amplification procedure because it can readily adapt to many
the small subunit rRNA gene. The only way to identify these applications. However, when a single-copy gene is used as
fungi is by using rapid molecular techniques—PCR-based the target for PCR, special amplification steps such as nested
RFLP and PCR-based DNA sequencing. PCR-based DNA PCR (nPCR) are often necessary to achieve the necessary
sequencing on the ITS region is useful specially to identify degree of sensitivity. The PCR-generated product is usually
non-sporulating molds (NSM). analyzed by ethidium bromide-stained gel electrophoresis.
Other genes within the molecular complex having also Although gel electrophoresis is simple and inexpensive, it
been used for the molecular evaluation of fungi include the is much less sensitive than Southern blotting.23 Detection of
5S rRNA, 5.8S rRNA, and 28S like rRNA. The comparison PCR products by ethidium bromide staining and by Southern
of nucleotide sequences within this region has been success- blotting is time consuming, and the interpretation of results
ful for the separation of genera and species. The 18S gene may be subjective. Hence, PCR–enzyme immunoassay
of fungi is about 1800 bp in size with both conserved and (EIA)—a three part method that includes PCR amplification,
variable domain sequences. Sequence variation within this hybridization with the complementary labeled probe, and
region has been used to assess the taxonomic relationships detection of reaction products in an EIA that provides either
and to separate genera and species based on sequence poly- colorimetric or fluorescence readout—was developed.24 In
morphisms. However, the drawback in using this region addition, the PCR–EIA format provides further amplifica-
for the identification of the species is the relative sequence tion without losing the species-specific binding associated
homology among fungal species and the need to sequence with Southern blotting and multiple samples can be assayed
a large number of bases in order to do comparative analysis. in parallel and semiquantitation of DNA is possible.25
The 5.8S region on the other hand is only about 160 bp long
and highly conserved. Owing to its small size and conserved 9.1.5.2.3 Species Identification
nature, it is not used for phylogenetic studies. However this Studies have demonstrated that the most promising targets
sequence is used as an attachment site for primers to amplify for molecular identification of fungi are the ITS1 and/or the
flanking spacer regions. The 28S rRNA which is around ITS2 region, followed by the D1–D2 region of the large-
3400 bp in size and the variable domains of this subunit have subunit rRNA gene (28S rRNA). Most molecular diagnostic
also been used to allow comparisons from higher taxonomic methods are able to screen patients in the initial stages of
levels to the species level. Much of the 28S rRNA gene is fungal infection, but not all protocols can identify the source
conserved limiting the usefulness of this region for species of the DNA to the genus or species level. Bagyalakshmi et
identification.21 al.26 applied the PCR-based DNA sequencing to identify the
The application of PCR technology to molecular diag- NSM targeting the ITS region and one of the NSM in their
nostics holds great promise for the early identification of study was Exserohilum.
medically important pathogens. PCR has been shown to be
useful for the detection of the presence of fungal DNA in 9.1.5.2.4 Diagnostic Considerations
clinical specimens. Considerable interest has been focused The detection and identification of fungal pathogens by DNA-
on the utility of selecting universal primers that recognize based methods can yield results sooner than cultivation.
constant regions among most, if not all, medically important Although the disappearance of fungal DNA from the blood
pathogens. of patients correlates with successful therapy, no experimen-
tal data are available to explain these aspects of therapy.
9.1.5.2.1 Target Selection
Targets that have been used in molecular diagnostic tests
9.1.6 Treatment and Outcome
for fungal infections include single and multicopy nuclear
and mitochondrial genes and RNA. In general, molecular Surgical excision is reported as the treatment of choice for
diagnostic methods targeting multicopy genes have better localized infections with Exserohilum infection with or
detection thresholds than those targeting single-copy genes. without adjunctive systemic antifungal chemotherapy in the
The ribosomal genes contain conserved sequences that are immunocompromised patients.11 The modes of therapy dif-
common to all fungi and also variable domains and highly fer according to the type of infection. Invasive infections
variable ITS regions. The conserved sequences can be used are treated with systemic antifungal agents though some

of the infections require surgical procedures. The duration emphasize more on the conidial ontogeny rather than color,
of systemic antifungal therapy was prolonged, from 2 to 13 conidial septation, or the appearance of growth on natural
months. Skin infections are managed with systemic drugs, substrates.
with or without topical antifungal agents. Corneal infections Accurate identification of filamentous molds is based on
were managed with topical antifungal agents in most of the microscopic examination of the sporulating parts of a colony.
patients reported in the literature and some patients required Living cultures are necessary for preparing slide cultures and
surgical intervention. Surgical debridement was the princi- the presence of growth verifies that the slide culture has been
pal mode of therapy for allergic fungal sinusitis and cere- set up appropriately. The tease preparation from cultures is
bral infection with Exserohilum infection. Successful use of also another method for identification of the fungus but the
amphotericin B and itraconazole as systemic antifungal che- disadvantages are that if the growth is not teased apart well,
motherapeutic agents are reported in the literature.7,8,11 or if the material is taken from a non-sporulating area, it may
In case of invasive infections amphotericin B was consid- be difficult to observe the sporulating structures and, more-
ered as the drug of choice.12,27 Most patients who were treated over, the conidia and spores may also get disrupted during
initially with surgical debridement and amphotericin B were preparation of the mount.
cured, but longer follow-up were necessary in these patients.
Adler et al. (2006)11 reported in their recent review of the lit- 9.2.2 Detection Procedures
erature, that in three cases the triazoles (itraconazole and vori-
9.2.2.1 P henotypical Criteria for
conazole) seemed to have better activity against Exserohilum.
Identification of Molds
However, there is insufficient data to support recommenda-
tion of any single agent as initial therapy as it is impossible to Visual examination of the fungal colony based on color,
deduce in vitro–in vivo correlations. The prevention of local texture, diffusible pigments, exudates, growth zones, aerial
expansion, cosmetic deformity, and possible dissemination of and submerged hyphae, growth rate, and colony topogra-
infection with Exserohilum is made possible by prompt aggres- phy, is useful for identification of the fungus. It is easier
sive surgical intervention. However disseminated disease has to identify the fungi based on conidial ontogeny than keys
to be treated with systemic antifungal chemotherapy.27,28 The based on the arrangement and color of the conidiophores
minimum inhibitory concentrations (MICs) usually show sen- and conidia. The dissection microscope is very useful to
sitivity to amphotericin B, itraconazole, and terbinafine, and observe the distinct gross colony characteristics and micro-
resistance to 5-fluorocytosine and ketoconazole. No specific scopic appearance.29
MIC were found in the literature for the echinocandins. There Mycelia Sterilia is a form that contains the filamentous
is limited clinical experience reported with azoles, but voricon- fungi that remain sterile in spite of the attempts to induce
azole seems very promising from in vitro data and low MICs.8 the formation of conidia or spores due to lack of appropri-
ate environmental and nutritional needs, or both. Such fungi
can rarely act as opportunistic pathogens and it becomes
9.2 Methods
essential to identify the fungus by inducing the spore/conidia
9.2.1 Sample Preparation formation. Though there are various spore inducing culture
media available, there is no universal culture medium and
The slide culture system consists of a mini incubation cham- various media and techniques must be tried until the correct
ber designed to produce optimum conditions for sporulation. combination of variables is found.
It allows for examination of the colony in various stages of
development, and improves the chances of observing the nat- 9.2.2.2 Slide Culture
ural configuration of the spores and conidia on the sporulat- Accurate identification of filamentous molds is based on
ing structures.8 microscopic examination of the sporulating parts of a colony.
The dissecting microscope is useful for observing the The slide culture system consists of a mini incubation cham-
gross colony characteristics and microscopic observations. ber designed to produce optimum conditions for sporulation.
The criteria for identification of molds are the morphology, It allows for examination of the colony in various stages of
culture characteristics, nutritional requirements, temperature development, and improves the chances of observing the
tolerance, proteolytic activity, cycloheximide resistance, and natural configuration of the spores and conidia on the sporu-
the ability to hydrolyze urea. Characteristics like color of the lating structures. Specimen living cultures are necessary for
colony, pigment production, or spores are dependent on the preparing slide cultures.
media and environmental conditions; they are not consis-
tently to be used as the main criteria to identify molds. The Procedure
important characteristic features like texture, topography of Sterile slide culture Petri dish preparation:
the colony, diffusible pigments, color of the colony, aerial
and submerged hyphae, growth rate, and macroscopic struc- A filter paper is laid to line the bottom of a glass
tures such as ascocarps, pycnidia, sclerotia, sporodochia, Petriplate.
and synnemata can be well appreciated by visual examina- A V-shaped piece of bent glass tubing is kept on the
tion of the colony. However the recent classification schemes filter paper to keep the slide culture.

Exserohilum 89
A 22 × 22 mm cover slip is kept on the slide. A small portion of fungal growth between the col-
The lid of the Petriplate is replaced and the unit is auto- ony center and edge is removed and placed on the
claved for 25 min at 121°C. lactophenol.
A medium known to induce sporulation for the fungus The fungus is teased apart gently by using two dissect-
(PDA) is used as it stimulates sporulation. ing needles, to spread out the fungus.
A block of sterile PDA agar 2 mm2 and approximately A coverslip is placed at the edge of the lactophenol and
1 mm thick is cut and placed on the microscope slide. slowly placed on the wet preparation to avoid trap-
Using aseptic technique throughout, the material from ping air bubbles under the coverslip.
a sporulating part of the colony is removed with a The excess of lactophenol is removed from the edges of
firm needle and inoculated two to three areas of the the coverslip by blotting with a paper towel.
block. If the mount is to be preserved the edges of the cover-
The coverslip is placed on top of the block using for- slip are sealed with nail polish.
ceps and pressed gently. While tease preparations are useful, they have two
Sterile water (1–2 mL) is added to the filter paper at the main disadvantages:
bottom and covered with the Petriplate lid. If too much growth is removed, or not teased
The slide culture is incubated at 25°C and observed apart well, or if the material is taken from a non-
for sporulation at regular intervals by examining sporulating area, it may be difficult to discern
the preparation under a dissecting or compound sporulating structures.
microscope. Spores and conidia are often disrupted during prep-
The preparation is kept moistened by adding additional aration of the mount.
water when needed.
The proximity of the glass and the agar plus the humid 9.2.2.5 Scotch Tape Lactophenol Mount
atmosphere in the dish encourages hyphal develop- The Scotch tape mount is yet another easy and rapid proce-
ment along the glass. dure that is used for the identification of filamentous fungi
since most structures are intact for observation.
9.2.2.3 Lactophenol Cotton Blue Mount As with the lactophenol mount, the organism is immersed
in the solution, rendering the organism safe for handling out-
The fungus is mounted when it is sporulating (takes around
side of the biological safety hood.
3–10 days):
To mount the coverslip, remove it from the agar block Procedure
using forceps.
A drop of lactophenol is placed just off center on a clean A strip of Scotch transparent tape is cut and the ends are
microscope slide and the coverslip with the growth is placed placed between thumb and index finger, gummed
at the edge of the lactophenol and slowly placed on the wet side out.
preparation to avoid trapping air bubbles under the coverslip. Making a loop by closing fingers, open plate with
The excess of lactophenol is removed from the edges of opposite hand and press tape against the colony to
the coverslip by blotting with a paper towel. identify.
The slides are labeled using a permanent marker, with the A drop of lactophenol is kept on a labeled slide and the
identifying name or number and other pertinent details such tape is pressed against slide with lactophenol.
as the medium used, age, and the fact that it is a slide culture. The tape is smoothened back on the slide by opening
If the mount is to be preserved the edges of the coverslip fingers and using gauze.
are sealed with nail polish. Another drop of lactophenol is placed on top of the
Sporulation of fungi can be appreciated with modes of tape.
conidiogenesis being clearly visible for study. A large 20 × 40 mm coverslip is placed on top of the
slide and examined under the microscope.
9.2.2.4 Lactophenol Mounts with The limitations are that the tape will dissolve eventu-
Tease Mount Procedure ally so that it is not to be used for permanent mounts
and the procedure can only be performed on molds
A tease mount is the common and rapid method to mount
growing from plates.
fungi for microscopic examination. The disadvantages of this
method are when the growth is teased apart with dissecting
On potato glucose agar Exserohilum hyphae are septate, sub-
needles, conidia or spores get dislodged from the conidiog-
hyaline to pale to mid brown, and 3.5–5.0 μm in diameter.
enous or sporogenous cells.
The conidiophores are simple, erect or wavy, and their upper
Procedure portions are fertile and geniculate. The conidia are straight
and cylindrical to ellipsoidal, with rounded apices, measuring
A drop of lactophenol is placed just off center on a around 64–100 × 10–15 μm, and have 8–13 distosepta (having
clean microscope slide. the individual cells each surrounded by a sac-like wall distinct

from the outer wall). The hila of the conidia are black and dis- • This is followed by centrifugation at 14,000 rpm for
tinctly protuberant germination of the conidia is bipolar. 10 min and the supernatant is transferred into another
Many criteria are considered when identifying tube to which equal volume of isopropanol is added
Exserohilum species such as the morphology, culture char- and kept at room temperature for 5 min. The sam-
acteristics, temperature tolerance, and cycloheximide sensi- ple is centrifuged at 14,000 rpm for 10 min and the
tivity. It is distinguished on the basis of conidial shape and deposit is washed with 70% ethanol thrice and finally
size, hilar morphology, location and sequence of the conidial resuspended in 50 μL of tris EDTA (TE) buffer.
septa, and the origin of the germ tubes from the conidial
Amplification using ITS primers:
cells. The conidium of Exserohilum species is ellipsoidal to
Primer sequence targeting ITS region26:
fusiod and germinates by germ tubes originating from either
one or both of the end cells or other intermediate cells. The
ITS1 5′ TCC GTA GGT GAA CCT GCG G 3
conidia of Exserohilum species are ellipsoidal to fusoid and
ITS4 5′ TCC TCC GCT TAT TAT GC 3
have distinctly protruding hila with truncated bases. The
• In brief, for a 50 μL reaction, 8 μL of dNTPs
conidia germinate by germ tubes originating from either one
(200 μmol), 5 μL of 1 × PCR buffer (1.5 mM
or both of the end cells or other intermediate cells. The spe-
MgCl2, 50 mM KCl, 10 mM Tris Cl, 0.001%
cies level identification is based on the conidial structure as
gelatin), 6 μL of 25 mM MgCl2 (1 in 10 diluted
described. The fungus is identified to the species level based
to get 1.5 mM), 10 pmol of the forward primer,
on the characteristic features.7,8
reverse primers targeting ITS region, and 10 μL
9.2.2.6 Molecular Detection Method of template DNA is used.26
PCR-based DNA sequencing technique for identification PCR thermal cycling profile:
of the fungus to species level targeting internal transcriber
spacer region (ITS region).26 • Initial denaturation at 95°C for 5 min followed by
Specimen preparation can have a significant impact on 35 cycles of denaturation at 95°C for 30 s, annealing
the sensitivity and reproducibility of a molecular diagnostic at 55°C for 60 s, and extension at 72°C for 60 s, fol-
test. In general, the sample preparation method should release lowed by final extension at 72°C for 6 min.
intracellular DNA from the fungal cell wall and/or thick cap-
Sequencing in ABI Prism 310/3100 AVANT genetic analyzer:
sule; it must concentrate DNA targets that may be present in
very small amounts, and it must eliminate protein debris, con-
taminants, potential inhibitors, and other extraneous materials • PCR-based DNA sequencing targeting ITS region
without degrading the target DNA. At present, there are many is carried out in a genetic analyzer (ABI Prism 3100
protocols for sample preparation, but no universal method AVANT Applied Biosystems).
for optimally extracting, purifying, and concentrating fungal • The sequences of PCR amplified product are sub-
DNA from clinical specimens is available. The efficiencies of jected to BLAST analysis to identify the species
the molecular diagnostic methods applied to different types and/or genera of fungi.
of clinical samples might not be equivalent, as DNAs present • Fungi with 99% homology is identified to species
in different clinical samples are probably different in origin. level, and the genus level is assigned when there is a
homology of 95%–98%.26
DNA extraction from fungal growth: • The nucleotide sequences of different isolates
belonging to the same species are analyzed using
• After growth of 7 days on SDA, approximately Multalin analysis software.
1 cm 2 mycelia is cut from the fungal growth for • The variations in nucleotide sequences, single and
extraction of DNA. multiple nucleotide polymorphisms can be identi-
• The mycelia are collected by scraping the slant fied by Multalin analysis.
with a sterile stick in a biological safety cabinet
(Class II).
• The material is transferred to a presterilized 9.3 C
onclusion and Future
microfuge vial (1.5 mL) containing 1 mL of ster-
Perspectives
ile water and the tube is centrifuged for 5 min at
3000 rpm in a microcentrifuge. Exserohilum is a dematiaceious fungus that may infect both
• The supernatant is discarded and the material is immunocompromised and immunocompetent hosts. Invasive
resuspended in 200 μL of lysis buffer containing infections, most commonly of the respiratory tract, affect
200 mM Tris, 50 mM EDTA, 10% sodium dodecyl mainly immunocompromised patients. There is an increased
sulphate (SDS), and 1 M sodium chloride and incu- number of Exserohilum reported in the literature within the
bated at 56°C for 60 min. last two decades. Exserohilum infections in human beings are
• To this mixture 150 μL of sodium acetate is added manifested in various clinical forms starting from cutaneous
and stored at −20°C for 20 min. infections to fulminant disseminated disease. Local trauma

Exserohilum 91
and atopy are reported to be the predisposing factors for 13. Aquino, V.M. et al., Fatal disseminated infection due to
corneal infections and allergic fungal sinusitis respectively. Exserohilum rostratum in a patient with aplastic anemia: Case
However, majority of the affected patients with invasive report and review. Clin. Infect. Dis., 20, 176, 1994.
14. Rinaldi, M.G., Phaeohyphomycosis. Dermatol. Clin., 14, 147,
infections were reported to have impaired immunity. Surgical
1996.
debridement is considered as the principal mode of therapy 15. Lasala, P.R. et al., Invasive Exserohilum sinusitis in a patient
for most of the infections with Exserohilum. Amphotericin with aplastic anemia. Pediatr. Infect. Dis. J., 24, 939, 2005.
B is the drug of choice for most invasive diseases reported in 16. Morpeth, J.F. et al., Fungal sinusitis: An update. Ann. Allergy
the literature30 though some cases improved after the addi- Asthma Immunol., 76, 128, 1996.
tional treatment with triazole agents.3 Outcome appeared to 17. Revankar, S.G. et al., Disseminated phaeohyphomycosis: Review
be better than for other mold infections and depended mainly of an emerging mycosis. Clin. Infect. Dis., 34, 467, 2002.
on the underlying diseases.32 Studies on the growth kinetics 18. Stevens, D.A. et al., Practice guidelines for diseases caused by
Aspergillus. Clin. Infect. Dis., 30, 696, 2000.
of Exserohilum, its susceptibility patterns to the in-use and 19. Netea, M. et al., Aspergillus fumigatus evades immune recog-
emerging antifungal agents is a potential area of research nition during germination through loss of tolllike receptor-4-
in the near future. The ability to determine the nucleic acid mediated signal transduction. J. Infect. Dis., 188, 320, 2003.
sequence of the genomic DNA has revolutionized most 20. Rolides, E. et al., Tumor necrosis factor alpha enhances anti-
areas of contemporary biomedical research. Further nucleic- fungal activities of polymorphonuclear and mononuclear
based amplification techniques involving real-time PCR and phagocytesagainst Aspergillus fumigatus. Infect. Immunol.,
reverse-transcriptase PCR need to be optimized to assess the 66, 5999, 1998.
21. Iwen, P.C., Hinriche, H., and Rupp, M.E., Utilization of ITS
transcripts produced by Exserohilum. Future research needs
regions as molecular targets to detect and identify fungal
to focus on improvement in diagnostic techniques and devel- pathogens. Med. Mycol., 40, 87, 2001.
opment of new therapeutic antifungal compounds to prevent 22. Lau, A. et al., Development and clinical application of a pan-
Exserohilum infection in humans. fungal PCR assay to detect and identify fungal DNA in tissue
specimens. J. Clin. Microbiol., 45, 380, 2007.
23. Tang, C.M. et al., The detection of Aspergillus spp. by the
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(Drechsler). Mycologia, 66, 290, 1974. 24. Loeffler, J., Henke, N., and Hebart, H., Quantification of
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3. Alcorn, J.L., Generic concepts in Drechslera, Bipolaris, and and the Light cycler system. J. Clin. Microbiol., 38, 2902,
Exserohilum. Mycotaxon, 17, 1, 1983. 2000.
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soil from Timma Park (Israel), and its physiological proper- lar non-sporulating moulds (NSM) identified by polymerase
ties. Anton. Van Leeuwen., 78,153, 2000. chain reaction (PCR)-based DNA sequencing technique tar-
6. Ferguson, B.J. et al., Geographic variation in allergic fungal geting internal transcribed spacer (ITS) region. Curr. Eye
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caused by a new species of Exserohilum. J. Clin. Microbiol., phomycosis. Micologia, 72, 1094, 1980.
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J. Infect. Dis. Med. Microbiol., 18, 200, 2007. phomycosis due to Exserohilum rostratum (Drechslera
10. Rolston, K.V.I., Hopfer, R.L., and Larson, D.L., Infections rostrata) in a child with leukemia. Pediatr. Infect. Dis., 5,
caused by Drechslera species: Case report and review of the 380, 1986.
literature. Rev. Infect. Dis., 7, 525–529, 1985. 31. Sharkey, P.K. et al., Itraconazole treatment of phaeohypho-
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10 Fusicoccum and Scytalidium
Marie Machouart and Jean Menotti
Contents
10.1 Introduction........................................................................................................................................................................ 93
10.1.1 Classification........................................................................................................................................................... 93
10.1.2 Morphology, Biology, and Epidemiology............................................................................................................... 94
10.1.4 Treatment................................................................................................................................................................ 95
10.1.5 Diagnosis................................................................................................................................................................ 96
10.2 Methods.............................................................................................................................................................................. 97
10.2.2.1 PCR–RFLP.............................................................................................................................................. 97
10.2.2.2 Panfungal PCR........................................................................................................................................ 97
10.3 Conclusions and Future Perspectives................................................................................................................................. 98
Acknowledgments........................................................................................................................................................................ 99
References.................................................................................................................................................................................... 99
10.1 Introduction S. dimidiatum was first described under the name

Hendersonula toruloidea in 1933 by Nattrass from pome and
Members of the genus Scytalidium are generally found on stone fruit trees as the conidial state of Dothiorella mangif-
plants as pathogens or saprobes, or isolated from soil, with a erae that was originally described on mangoes [3].
worldwide distribution [1]. More than 15 species have been In 1971, Gentles and Evans first reported the fungus under
listed by referring to different sources: S. thermophilum, S. the name Hendersonula toruloidea from patients immigrat-
japonicum, S. multiseptatum, S. album, S. dimidiatum, S. ing to the United Kingdom but originating from tropical
hyalinum, S. lignicola, S. infestans, S. indonesiacum, S. mus- areas. Based on clinical appearance and direct microscopic
corum, S. uredinicola, S. aurantiacum, S. flavobrunneum, examination, these patients were first suspected to have der-
S. acidophilum, S. vaccinii, S. circinatum, S. fulvum, and matophytosis, but cultures of the biological samples rapidly
Neoscytalidium novaehollandiae (http://www.ncbi.nlm.nih. grew a black mold in media without cycloheximide [4].
gov/pubmed/, http://www.cbs.knaw.nl/, www.atcc.org/). A few years later, in 1977, Campbell and Mulder described
In this chapter, we focus on two species, S. dimidiatum the first case of S. hyalinum, in a patient having a mycosis
and S. hyalinum, frequently found in tropical or subtropi- similar to those of Gentles and Evans, resembling dermato-
cal countries and responsible for superficial (or rarely deep) phytosis, but caused by a fast-growing white mold [5].
infections in humans, resembling dermatophytosis [2]. Since these first descriptions, this fungus has then been
renamed several times because the production of both arthro-
conidial and pycnidial synanamorphs (form with conidia in a
cavity [pycnidia]) has led to nomenclatural controversy.
The taxonomy of S. dimidiatum and S. hyalinum is very Then, in 1989, the genus Hendersonula was revised by
confusing, with several factors having enhanced the con- Sutton and Dyko that proposed to synonymize Dothiorella
fusion over the years. Cultural variants have been isolated mangiferae and H. toruloidea under the name Nattrassia
with or without production of pycnidia, with distinct mac- mangiferae. The mycelial synanamorph was further
roscopic morphological features and different growth rates. described as Scytalidium dimidiatum [6].
The nomenclature has changed several times since the origi- In 2005, Farr et al. conducted a molecular study on inter-
nal descriptions of these molds, further to the discovery of nal transcribed spacer (ITS) and β-tubulin of Fusicoccum
new characteristics. Actually, the nomenclature continues to sp. They concluded from their analysis that Nattrassia
evolve with molecular studies and new names are still being mangiferae and Scytalidium dimidiatum belong to the
proposed. genus Fusicoccum (asexual state of Botryosphaeria), most
93

closely related to Botryosphaeria mamane. Based on these was also proposed. Nattrassia mangiferae, previously widely
olecular sequence data and on morphological similarities
m used to refer to S. dimidiatum isolates, should be considered
of the pycnidial and arthric conidial states with Fusicoccum, now as a distinct species, which differs from the latter taxon
they introduced the name Fusicoccum dimidiatum to replace in lacking an arthroconidial anamorph. Therefore, isolates
Scytalidium dimidiatum and stated that the genus Nattrassia previously described as Nattrassia mangiferae would be
mangiferae should be synonymized under the name placed in this Neofusicoccum group and synonymized under
Fusicoccum dimidiatum [7]. the name Neofusicoccum mangiferae.
Recently, Crous et al. have proposed a taxonomic revision S. hyalinum has been described as a very similar fungus
of the Botryosphaeriaceae family based on the analysis of to S. dimidiatum but nonpigmented and only reported from
28S rDNA sequences. In the resulting phylogenetic tree, iso- human skin and nail infections. According to molecular
lates producing arthroconidia typical of S. dimidiatum and data, it has been suggested that S. dimidiatum and S. hyali-
strains of Nattrassia mangiferae obtained from mangoes and num might be conspecific [10,11]. In the study of Crous et al.,
lacking synanamorphs were grouped in separate clades [8]. no synonymy has been proposed between both fungi. Very
In this study, the type species of the genus Scytalidium, S. recently, a new name based on the hypothesis that S. hyali-
lignicola (CBS 233.57), was associated with Leotiomycetes num would be an albino variant of N. dimidiatum has been
and clustered outside Botryosphaeriaceae (Dothideomycetes). suggested: Neoscytalidium dimidiatum var. hyalinum [12].
These data support that S. lignicola and S. dimidiatum would Because the taxonomy is controversial according to the
be distinct species belonging to different classes. This is in authors, in the next paragraph, both fungi still will be named
agreement with the conclusion of Sigler et al. in their litera- as Scytalidium dimidiatum and Scytalidium hyalinum.
ture review. These authors suggested the distinction between
S. dimidiatum and S. lignicola by morphological differences,
10.1.2 Morphology, Biology, and Epidemiology
leading to a confusion between both species in reports iden-
tifying the latter species as a human pathogen [9]. According On Sabouraud agar media, S. dimidiatum produces a fast
to these arguments and to DNA sequence data derived from growing, aerial mycelium, that is cottony and hyaline at
the CBS collection, Crous et al. concluded that the genus first but soon becomes dark brown to blackish, floccose,
Scytalidium would be polyphyletic and proposed the name and dense (Figure 10.1A). The reverse is black. S. hyalinum
Neoscytalidium in order to accommodate S. dimidiatum as grows whitish colonies with a colorless to beige reverse, the
Neoscytalidium dimidiatum. Moreover, they suggested the mycelium remaining flushy (Figure 10.1C) [1]. These molds
coelomycete synanamorph Hendersonula toruloidea (asex- generally grow on cycloheximide-free media, although some
ual state) as a synonym of N. dimidiatum. S. dimidiatum have already been reported not to be inhibited
Secondarily in this study, the proposition of Farr et al. to by cycloheximide [13].
place into synonymy the genera Scytalidium and Fusicoccum Microscopic examination demonstrates septate hyphae
and to synonymize Fusiccocum dimidiatum and Scytalidium with cylindrical to ellipsoidal both hyaline and darkly
dimidiatum was rejected. Thus, a new genus, Neofusicoccum p igmented arthroconidia in chains or disarticulated
20.0 µm
(A) (B)
(C) (D)
Figure 10.1 Macroscopy and microscopy of Scytalidium dimidiatum (A and B) and Scytalidium hyalinum (C and D). The black arrows
indicate conidia with characteristic septa.

Fusicoccum and Scytalidium 95
(Figure 10.1B and D). Some of these arthroconidia are (two-foot one-hand syndrome) [33]. No clinical difference is
c haracterized by the presence of two cells separated by a observed between S. hyalinum and S. dimidiatum infections.
thick septum (arrows in Figure 10.1B and D). In old cultures, According to Lacroix et al., melanonychia usually observed
pycnidial synanamorph may be produced [8,9]. in people with black skin would be a result of an inflamma-
Scytalidium dimidiatum (or under the name Nattrassia tory process more than of the pigmentation of S. dimidiatum
mangiferae) has been reported as a significant cause of [13,32]. Asymptomatic carriage has been hypothesized but
pathology in fruit trees or woody plants as Ficus, Arbutus, this remains to be demonstrated, because subclinical infec-
Citrus, Juglans, and others in tropical or subtropical areas tion may be common in certain regions [2,31,32]. No studies
[6,9,14]. This organism was first found to be responsible for have been carried out to test this hypothesis, and these should
gummosis and dieback of stone fruit trees in Egypt [3], dry- be conducted by experts in the field of mycological diagnosis
ing of grape vines in Iraq and India [15,16], death on citrus involving dermatologists together with mycologists.
trees in Iraq [17], leaf spot disease in India [18], leaf spot Rare cases of deep infections involving most of time S.
and dieback of mango in India and Niger [19,20], and tip rot dimidiatum have also been reported. Central nervous system
of banana trees in Jamaica and Hawaii [21,22]. This is also abcesses, endophtalmitis, sinusitis, osteomyelitis, mycetoma,
the cause of the branch wilt disease on the Persian walnut subcutaneous lesions, and disseminated infections have been
tree Juglans regia in California [23], dieback and canker of published, mainly affecting immunocompromised patients
citrus and eucalyptus trees of Arizona [24,25], and canker of such as renal or lung transplants, diabetic patients, or indi-
almond trees [26]. More recently, Hendersonula toruloidea viduals receiving immunosuppressive therapy [34–48]. The
has also been found to cause a foliar disease of strawberry underlying conditions appear to be similar to those of other
tree in Greece [27]. invasive opportunistic fungal infections. Until now, only two
Whereas S. dimidiatum has been widely reported from cases have been reported from immunocompetent patients
such plants, S. hyalinum has never been isolated from [45,46]: an endophtalmitis occurring after traumatic implan-
environment. tation and a cerebral phaeohyphomycosis. Approximately
As human pathogens, both fungi are endemic in tropical 50% of the patients concerned by deep-seated infections
and subtropical countries comprising South America, the died. Only a few cases involving S. hyalinum have been
Caribbean, Asia, and Africa and cause diseases called scy- described [48,49].
talidioses representing approximately 40% of dermatomyco- Virulence factors involved in the pathogenesis of
ses in these areas [2,28]. Nevertheless, cases from temperate Scytalidium sp. may be linked to a keratinase facilitating
countries are increasingly reported owing to immigration invasion of skin and production of melanin for S. dimidia-
and travels [12,28,29]. In a study examining data from 332 tum mediating resistance to phagocyte-produced oxygen
patients infected by Scytalidium and addressed to the der- radicals [2,50]. The ability of S. dimidiatum and S. hyalinum
matology department of a French hospital, the sex ratio was to degrade nail keratin has been demonstrated, although this
2 men for 1 woman (67.5% men versus 32.5% women) with phenomenon is less pronounced compared to what occurs in
a predominance in the 36- to 55-year age group (61.5%) [13]. dermatophytosis due to T. rubrum and T. mentagrophytes
Most S. hyalinum infections seem to be related to the West [50]. It is therefore possible that mechanisms operating in
Indies, South America, and West Africa, whereas patients Scytalidium spp. to degrade and digest keratin may be simi-
from Asia, the Indian Ocean region, and Central Africa are lar to those of dermatophytes.
mostly infected by S. dimidiatum. Some mixed S. hyalinum
and S. dimidiatum infections have also been described. Mixed
10.1.4 Treatment
infections with dermatophytes have been reported [13,30,31].
Infection is thought to occur through contact with con- The clinical response of Scytalidium spp. to antifungal treat-
taminated soil or plant material especially for barefooted ment is poor with the available topical or systemic agents
individuals, or possibly through direct contact with infected used for onychomycosis. Scytalidium spp. are quite resistant
nail or skin. to drugs used in dermatology, that is, griseofulvin, ketocon-
azole, fluconazole, itraconazole, and terbinafine [51]. In vitro
efficacy of antifungals has been evaluated by Etest methods
[52,53] and by using microdilution testing with CLSI M38-A
Clinically, these fungi cause mostly superficial skin infec- methods [54]. These studies resulted in MICs ranges of
tions and onychomycosis indistinguishable from dermato- <0.03–0.5 mg/L for voriconazole, 0.06–2 mg/L for posacon-
phytosis [9,32]. Most infections are generally located on nails, azole, <0.03–16 mg/L for itraconazole, 0.06–2 mg/L for ter-
toe webs, and the soles of the feet, hands being more rarely binafine, 0.06–8 mg/L for caspofungin, and 0.06–1 mg/L
involved. Infected soles show hyperkeratosis, associated or for amphotericin B. Amphotericin B, voriconazole, and
not with desquamation. Desquamation and maceration may posaconazole exhibit low MICs in these in vitro studies.
be observed for toe web infections. Paronychia is not rare. These data are related to successful in vivo treatments with
Toenail lesions are characterized by laterodistal invasion. amphotericin B alone or with a regimen of liposomal ampho-
Total nail dystrophy may also occur. Classically palm lesions tericin B followed by voriconazole in cases of invasive infec-
are unilateral whereas sole affection is generally bilateral tions [29,44].

In vivo, amphotericin B dermatologic lotion has been curette. For an onyxis, the nail should be cut and the under-
described to be effective in curing superficial scytalidiosis nail zone should be scraped, just at the edge of healthy tis-
in the toe webs, but this is no longer commercially available sues. Searching for other infected sites should be systematic
[13]. A case of Scytalidium hyalinum onychomycosis was (fold, other foot, hands, intertoe, etc.). In case of maceration,
also successfully treated with a 5% amorolfine nail lacquer swabs should be used.
[55]. Voriconazole has also been proposed as a new thera-
peutic approach for superficial infections [54]. Although the 10.1.5.1.2 Direct Examination
low minimal inhibitory concentration (MIC) ranges reported Squama are the most productive samples for direct examina-
with voriconazole give promising results for the treatment of tion and are directly examined in black chlorazol solution or
scytalidiosis and may be indicated in severe cases, the cost- by using staining with calcofluor. A 30% potassium hydrox-
effectiveness ratio is not in favor of such a strategy for super- ide solution may be used in order to soften the nail fragments.
ficial dermatomycosis. Microscopic examination at objectives 20× and 40×
As reported for onychomycosis in general, oral antifungals shows fungal septate hyphae indistinguishable at first from
combined with topical nail lacquer may result in higher cure dermatophytes by an inexperienced eye. Nevertheless, the
rate than in cases treated only per os. This combination seems appearance of characteristic sinuous irregular hyphae should
also to be judicious for scytalidiosis in regard to the high level lead to the diagnosis of scytalidiosis.
of difficulties of cure and the importance of resistance [56].
The reasons for treatment failure are not well understood. 10.1.5.1.3 Culture
One possible explanation is that nail plate infection is often S. dimidiatum and S. hyalinum grow on cycloheximide-
diagnosed after several years of evolution and is thus accom- free Sabouraud agar supplemented with antibiotics. A better
panied by such an extensive onycholysis that the subungual sensitivity of cultures is achieved if an abundant material is
fungi are not in contact with the nail bed, where the drug cultured. Plates and slants are incubated at 30°C for up to 3
presents the maximum concentration [2]. Therefore, the weeks in order to isolate other slow-growing potential patho-
optimal therapy should associate a periodic toenail mechani- gens. Scytalidium spp. are fast-growing molds that after 48 h
cal removal of the abnormal portions of the nail plate per- give pale gray colonies soon becoming olivaceous gray to
formed by podiatrists, dermatologists, or practitioners who dark brown or black for S. dimidiatum with a black reverse.
are specialized in the field with an antifungal therapy [57]. For S. hyalinum, cultures remain whitish with a colorless to
Nevertheless, therapeutic success is also dependent on the beige reverse. The mycelium is aerial, floconnose, filling in a
good compliance of patients for this treatment, often requir- week petri dishes or slants for S. dimidiatum and remaining
ing application for more than 6 months. flushy for S. hyalinum.
Microscopic morphology is observed on a cellophane tape
10.1.5 Diagnosis preparation with lactophenol blue. Characteristic features
demonstrate septate and branched hyaline to dark brown
10.1.5.1 Conventional Techniques hyphae ranging from 2 to 8 μm in width. Uni- or bicellular
The sensitivity of diagnosis is dependent on many criteria arthroconidia are observed that are sometimes larger than
such as the experience of the operator for sampling or read- hyphae (4 × 8 μm). Hyphae remain hyaline for S. hyalinum.
ing the direct examination and the quality and quantity of Presence of numerous arthoconidia in chains should not be
the samples. For onychomycosis, high rates of false-negative confused with the Geotrichum species [1].
reaching, respectively, 5%–15% and 30% still occur in direct In case of invasive infection, histopathology is necessary
examinations and cultures, even in highly experienced labo- and if positive shows pigmented or not, refractile and often
ratories [58]. The optimal method to improve the accuracy sinuous, septate hyphae on biopsies as in other deep mycosis
of dermatomycosis diagnosis in routine practice still remains [9,60].
unclear. A diagnostic algorithm including validated proce-
dures by experts is generally used and involves five main 10.1.5.2 Molecular Techniques
steps, each one needing to be carefully carried out: mycologi- Nowadays, sequencing of PCR products is widely used in
cal sampling, direct examination, cultures, macroscopic and order to precisely identify the genus and species of fungi that
microscopic observation, and interpretation [59]. are isolated in pathogenic samples. A panfungal PCR target-
ing the ITS region is performed according to the procedures
10.1.5.1.1 Mycological Sampling described by White et al. [61]. Thereafter, amplicons may
Sampling of biological material should always be accom- be sequenced by using the same primer set. The obtained
panied by precise examination of the clinical lesion and a sequences are then analyzed by using bioinformatics pro-
complete interrogation of the patient about his or her urban grams such as BLAST on dedicated Web sites and databanks
or rural lifestyle, possible travel in endemic areas, antifungal such as National Center for Biotechnology Information
treatment, duration and evolution of lesions, etc. (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Each lesion should be sampled separately, after disin- Another molecular biology-based approach has been
fection with alcohol at 70%. Samples are collected in ster- developed in order to discriminate between dermatophytes
ile plates. Squama should be collected by scraping with a and Scytalidium spp. This method combines a PCR targeting

of the 18S ribosomal DNA followed by a restriction fragment 10.2.2 Detection Procedures
length polymorphism (RFLP). This method has been evalu-
ated experimentally on cultures and on samples from patients 10.2.2.1 PCR–RFLP
with dermatomycoses [62,63]. According to Machouart et al. [62], PCR is performed in
a reaction mixture of 25 μL containing 1.5 mM MgCl2,
50 μM of each dNTP, 25 pmol of each primer, 1 U of Taq
10.2 Methods polymerase, and 25 ng of extracted DNA. A primer set,
DH2L (5′-TGTACTGGTCCGGCCGGG-3′) – DH1R
10.2.1 Sample Preparation (5′-CGGCGGTCCTAGAAACCAAC-3′), described as pref-
DNA may be extracted from fungal cultures for the identi- erentially targeting the 18S rDNA of Scytalidium spp., is used
fication or confirmation of isolated species of Scytalidium with amplification conditions consisting of 3 min at 94°C for
spp., for example, in cases of atypical morphological features the initial denaturation, followed by 30 cycles at 94°C for
occurring macroscopically or microscopically (slight sporu- 1 min, 58°C for 1 min, and 72°C for 40 s, and one final exten-
lation). Epidemiological studies may also be conducted in sion step at 72°C for 5 min. Next, 5 μL of PCR product is
order to highlight interstrain polymorphisms according to electrophoresed in 2% agarose gel in the presence of interca-
their origin (geographically or biologically). lating dye and visualized under UV light. The expected size
Direct manipulation of biological samples may be infor- is of 184 bp.
mative in cases of patients under treatment at the time of sam- Amplification products are then digested with BamHI for
pling or with an evocative lesion associated with a negative 1 h at 37°C in a total volume of 20 μL containing 5 μL of the
direct examination. This may also be performed retrospec- specific PCR product and 5 U of the enzyme. The digested
tively if the cultures remain negative with a direct examina- samples are analyzed on 3.5% Nusieve ethidium bromide
tion that was previously positive. (BET)–agarose electrophoresis gel (Figure 10.2) [62].
Two main methods have to be considered for DNA extrac-
tion. Rapid protocol and routine use will require the use of 10.2.2.2 Panfungal PCR
commercial kits. For such kits, a pre-incubation with lyticase Fungal identification by molecular biology may also
may be useful in order to efficiently digest chitin-containing use panfungal primers targeting ITS regions: ITS1
fungal cell walls. Epidemiological studies or research will (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
prefer total fungal genomic DNA extractions that may (5′-TCCTCCGCTTATTGATATGC-3′) with an annealing
involve different techniques. The advantages of such tech- temperature of 51°C [61].
niques are their low cost and obtaining of a higher amount of The PCR mixture (50 μL) is composed of 2 U of FastStart
pure DNA. The disadvantage is the delay necessary for the Taq Polymerase (Roche Diagnostics), 5 μL of 10× PCR
procedure that may take up to 1 or 2 working days. We pro- buffer–MgCl2 (20 mM), 20 pmol of each primer, 200 μM
pose here a protocol adapted from Möller et al. and slightly of dATP, dGTP, dTTP, and dCTP, and 5 μL of DNA. The
modified [64]. Approximately 1 cm2 of mycelium taken from PCR is run in a thermal cycler under the following condi-
2-week-old cultures is transferred to 2.0 mL Eppendorf tubes tions: initial denaturation at 96°C for 1 min; 35 cycles at 94°C
containing 300 μL TES buffer (Tris 1.2% [w/v], Na–ethyl- for 1 min, 57°C for 1 min, and 72°C for 1 min; and a final
enediaminetetraacetic acid [EDTA] 0.38% [w/v], sodium extension at 72°C for 10 min. Then 5 μL of PCR products are
dodecyl sulfate 2% [w/v], pH 8.0). Fungal cells are disrupted
mechanically in a tight-fitting sterile pestle for approximately
M 1 2 3 4 5
1 min. Subsequently, 10 μL proteinase K is added and incu-
bated for 10 min at 65°C. After adding 140 μL of 5 M NaCl
and 1/10 vol CTAB 10% (hexadecyltrimethylammonium
bromide) buffer, the mixture is vortexed and the material
is incubated for 30 min at 65°C. Subsequently, 700 μL of a
mixed solution of chloroform and isoamylalcohol (v/v = 24:1)
is added, vortexed, incubated for 30 min on ice water, and
centrifuged for 10 min at 14,000 rpm.
The supernatant is transferred to a new tube with 200 μL
5 M NH4 –acetate, incubated on ice water and centrifuged
again at 4°C for 10 min at 14,000 rpm. The supernatant is
transferred to a new sterile Eppendorf tube with 0.55 vol
Figure 10.2 PCR amplification of human samples containing
isopropanol, mixed and centrifuged at 4°C for 5 min at
fungi and restriction patterns with BamHI. Lane M, 100 bp ladder;
14,000 rpm. Subsequently, the pellet is washed with cold lane 1, nondigested amplicon (DH2L–DH1R); lanes 2–5; BamHI
70% ethanol. Air-dried pellet is resuspended in 100 μL digested products showing only the presence of Scytalidium (lanes
TE buffer (Tris 0.12% [w/v], Na–EDTA 0.04% [w/v]). 2, 3, and 5), and showing a partial digestion (lane 4): this is charac-
DNA is used immediately for PCR amplification or stored teristic of the presence of Scytalidium with a nondigested product
at −80°C. corresponding to another fungus in the sample.

electrophoresed in a 2% agarose gel in the presence of BET remains to be completed because the study was conducted
and visualized under UV light. The expected PCR product on few strains. However, the authors proposed to subordin S.
size is approximately 600 bp. hyalinum as a variety of S. dimidiatum with this new com-
The amplified fragments may be sequenced after purifi- bination of Neoscytalidium dimidiatum var. hyalinum by
cation with commercial kits. Sequencing is then performed referring to the work of Crous et al. [8,12]. We conducted
on both strands with the same primers as those used in the another study on ITS, 28S rDNA, actin (Act), and elongation
initial amplification. Sequencing reactions are performed factor (EF) on 80 isolates of Scytalidium species, including
by using the Big Dye Terminator v1.1 Cycle Sequencing kit S. dimidiatum, S. hyalinum, and other species [67]. Our aim
(Applied Biosystems) on an automated sequencer. was to give arguments in order to specify the classification
Briefly, in a final volume of 10 μL, 1 μL of PCR prod- of the genus Scytalidium and particularly the genetic rela-
uct is added to 2 μL of mix, 1 μL of buffer, and 1.6 pmol of tionships between the different species. We confirmed poly-
primer. Sequencing reactions are performed in a thermal morphisms in ITS and highlighted new differences on 28S,
iCycler IQ System (Bio-Rad) with an initial denaturation Act, and EF. We also discovered a different distribution of
at 96°C for 1 min, followed by 25 amplification cycles at intronic insertions between S. dimidiatum and S. hyalinum
96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The result- [67] (unpublished data). These arguments let us to conclude
ing products are purified by using Sephadex filtration, com- that S. dimidiatum is an ancestral form of the fungus that has
mercial kits, or a succession of washing and centrifugation adapted to humans in the course of evolution. This is con-
steps. For the latter procedure, the resulting product is pre- comitant with genetic evolution implying nucleotidic muta-
cipitated by adding 2 μL of 3 M NaAc (Sigma Aldrich), 2 μL tions in some genes and insertion or deletion of introns. The
EDTA 125 mM, and 50 μL of 96% ethanol. After incuba- perspective of this work is now to complete the study and to
tion on ice for 15 min and centrifugation at 13,000 rpm for compile all already known and new data and to focus on a
20 min, 125 μL of 70% ethanol is added. A final centrifuga- multilocus phylogeny in order to clarify and reclassify the
tion is carried out at 13,000 rpm for 15 min. Each sample species belonging to the genus Scytalidium. Only after this
is run on an ABI PRISM 3100 Genetic Analyzer (Applied complete study will it be possible to propose new formula-
Biosystems). tions for S. dimidiatum or S. hyalinum with regard to the
The sequences obtained are aligned by using the Bioedit other species and their position.
Software or the AliBee program on the Web site at http:// In endemic areas, S. dimidiatum and S. hyalinum are fre-
www.genebee.msu.su/genebee.html, the consensus sequence quently isolated comparing with dermatophytes. These fungi
is then analyzed with the BLASTn program (http://blast.ncbi. also have the potential to cause deep and life-threatening
nlm.nih.gov/Blast.cgi). infections mostly in patients with risk factors for develop-
ing opportunistic diseases [68]. Considering their high
10.3 C
onclusions and degree of resistance to antifungals, these molds must not be
overlooked in populations of immigrants or patients having
Future Perspectives
traveled in endemic zones. Because scytalidiosis is rare in
Almost 40 years after the first description of Scytalidium spp. temperate countries, the development of efficient treatment
in humans, knowledge is still fragmented about these molds. is obviously not a priority, even though this mycosis is very
Taxonomy of these fungi is still controversial and this has common in tropical areas. Moreover, their importance may
lead to an unstable nomenclature [65]. Because the genotyp- be underestimated because of the nonconsultation of patients
ing approach of pathogenic fungi may clarify epidemiology living in tropical and subtropical countries. Diagnosis dif-
and may be associated with the medical traits of fungi, some ficulties remain a reality with the importance of false nega-
studies have been conducted on Scytalidium spp. [8,11,12]. tive results in mycology even after appropriate sampling of
This led to place S. dimidiatum in Botryosphaeriaceae and lesions; the reasons for this are unclear. In some cases, fungal
to propose a new name for S. dimidiatum: Neoscytalidium identification is also hindered by the presence of contami-
dimidiatum. Nevertheless, the position of S. hyalinum and nants requiring subcultures, delaying the diagnosis. Correct
other Scytalidium spp. not encountered in pathology has not diagnosis of scytalidiosis is necessary to avoid unnecessary
been clearly considered. lengthy and potentially toxic antifungal treatment. Thus,
Genetic differences have been detected between S. hyali- the ability to discriminate Scytalidium species from other
num and S. dimidiatum in addition to the geographical distri- pathogens by a PCR may ease the path to correct diagno-
bution and to the epidemiological sources. Introns have been sis in difficult cases. The PCR–RFLP rapidly discriminating
found in the 18S rDNA of S. dimidiatum and were absent at among dermatophytes, Scytalidium, and other fungi has been
the homologous sites in S. hyalinum [11,66]. Some nucleotide evaluated in clinical practice and showed that this technique
polymorphisms have also been highlighted in the genes cod- might facilitate the rapid diagnosis of onychomycosis due to
ing for 18S rDNA, ITS, and tubulin. Combined analysis of dermatophytes or Scytalidium spp. with a similar sensitiv-
polymorphic loci has thus helped define five sequence types ity and specificity when compared to reference mycological
from which two were detected exclusively in isolates from techniques [46,63]. Use of such techniques may also be suit-
plants, two were found only in clinical isolates, and one was able for laboratories that are not trained to correctly sample
observed in isolates from humans and a mango tree. But this nails or inexperienced in the field [69].

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Chemother., 61, 835, 2008. phyte, agent of superficial mycoses and phaehyphomycosis,
55. Downs, A.M., Lear, J.T., and Archer, C.B., Scytalidium hyali- Med. Trop., 59, 375, 1999.
num onychomycosis successfully treated with 5% amorolfine 69. Savin, C. et al., Multicenter evaluation of a commercial
nail lacquer, Br. J. Dermatol., 140, 555, 1999. PCR-enzyme-linked immunosorbent assay diagnostic kit
56. Avner, S., Nir, N., and Henri, T., Combination of oral terbin- (Onychodiag) for diagnosis of dermatophytic onychomycosis,
afine and topical ciclopirox compared to oral terbinafine for J. Clin. Microbiol., 45, 1205, 2007.
the treatment of onychomycosis, J. Dermatolog. Treat., 16,
327, 2005.

11 Hortaea
Dongyou Liu and Larry Hanson
Contents
11.1 Introduction.......................................................................................................................................................................101
11.1.1 Classification, Morphology, and Biology..............................................................................................................101
11.1.2 Clinical Features and Pathogenesis...................................................................................................................... 102
11.1.3 Diagnosis.............................................................................................................................................................. 102
11.2 Methods............................................................................................................................................................................ 103
11.2.1 Sample Preparation............................................................................................................................................... 103
11.2.2 Detection Procedures............................................................................................................................................ 103
11.2.2.1 Specific PCR Identification.................................................................................................................... 103
11.2.2.2 Sequencing Analysis of ITS Regions..................................................................................................... 103
11.3 Conclusion........................................................................................................................................................................ 103
References.................................................................................................................................................................................. 103
11.1 Introduction to a new genus Hortaea as Hortaea werneckii due to its com-
bination of sympodial and annellidic conidiogenesis [2]. To
Dematiaceous fungi cover a large group of melanised fun- date, no teleomorph of this fungus has been identified.
gal organisms that may be plant-infecting pathogens or free- Hortaea werneckii is a dematiaceous fungus possessing a
living saprobes. As a melanised, yeast-like fungal genus, yeast-like phase as well as a hyphal phase. The fungus grows
Hortaea is a common plant pathogen in tropical and sub- on standard mycological media in 5–8 days and may take
tropical countries. Of the three recognized species within the up to 21 days to mature. From the front, H. werneckii colo-
Hortaea genus, Hortaea werneckii (previously incorrectly nies are initially pale in color, moist/mucoid, shiny black,
classified in the genera of Cladosporium, Cryptococcus, and yeast-like, and later become velvety, dark olivaceous in
Exophiala and Phaeoannlomyces) is occasionally respon- color, with abundant aerial mycelia. From the reverse, the
sible for a superficial mycosis in humans known as tinea color is black. Microscopically, H. werneckii hyphae (up to
nigra. This fungus colonizes dead keratin cells, and produces 6 μm wide) are septate, thick-walled, and brown to dark oli-
hyperchromic plaques on the skin of the palms and soles, that vaceous in color. Its conidia (2–5 × 5–10 μm) are two-celled,
may be mistaken for various types of pigmented birthmarks hyaline to pale brown, cylindrical to spindle-shaped yeast-
and moles (nevi) as well as melanomas. like cells that usually occur in aggregated masses and that
taper toward the ends to form an annellide and new annel-
loconidia. Annelloconidia are produced at intercalary and
The genus Hortaea is black yeast-like hyphomycete belong- lateral annellidic points along the hyphae. Some of the yeast-
ing to the mitosporic Dothideales group, order Dothideales, like conidia have a central septum and are bicellular. They
subclass Dothideomycetidae, class Dothideomycetes, sub- may gradually become chlamydospore-like cells. Hortaea
phylum Pezizomycotina, phylum Ascomycota, kingdom werneckii is halophilic and tolerates 3%–30% NaCl, but it
Fungi. The mitosporic Dothideales group consists of six does not grow at 37°C [5,8–10].
genera: Coleophoma, Dichomera, Hobsonia, Hortaea, Hortaea werneckii is a common saprophytic fungus that
Selenophoma, and Stigmina. The genus Hortaea is separated occurs in soil, compost, humus, and on wood in humid tropi-
into three recognized species: Hortaea acidophila, Hortaea cal and subtropical regions. It is prevalent in highly saline
werneckii, and Hortaea thailandica, in addition to an unas- water of crystallization ponds and has been isolated also
signed species Hortaea sp. F47 [1–6]. from saltwater fish [11–13]. The fungus may cause superfi-
Within the genus Hortaea, Hortaea werneckii (obsolete cial mycotic infection of stratum corneum (often known as
synonyms: Cladosporium werneckii, Exophiala werneckii, tinea nigra) in humans, although tinea nigra may be also due
and Phaeoannellomyces werneckii) is implicated in human to infection with Stenella araguata [14–16]. Humans often
diseases [7]. First described as Cladosporium werneckii acquire Hortaea werneckii infection via direct inoculation
in 1921, Hortaea werneckii was transferred to the genus of the fungus onto the skin during contact with soil, wood,
Exophiala as Exophiala werneckii in 1970, and then moved decaying vegetation, or sewage.
101

11.1.2 Clinical Features and Pathogenesis 11.1.3 Diagnosis

Hortaea werneckii causes a superficial mycosis called tinea Tinea nigra is a superficial mycosis due to the melanized,
nigra (or tinea nigra palmaris) [17]. Owing to its halophilic polymorphic, and yeast-like fungus Hortaea werneckii. This
and lipophilic behavior, H. werneckii thrives in hypersaline fungus induces flat, pigmented, brownish, irregular and
environments such as the palm of the hand [18]. The fungus asymptomatic macules on the palm of one hand or foot, which
adheres to human hands through the hydrophobic feature of are covered by fine scaling, with well-defined borders and
its yeast cells, colonizes dead keratin cells on the skin, and without inflamed margins. Differential diagnosis includes skin
its branched hyphae grow between the layers of cornified epi- lesions produced by other fungi (e.g., Stenella araguata, also
dermis, utilizing decomposed lipids in the stratum corneum a dematiaceous fungus) as well as viruses (e.g., herpesviruses
as nutrient, leading to hyperkeratosis (abnormal thickening and poxviruses). In addition, tinea nigra may be mistaken for
of the cornified epidermis) [19]. The incubation period typi- other pigmented skin diseases (e.g., Addison’s disease, mela-
cally lasts for 2–7 weeks. nocytic naevus, dysplastic naevus, acrolentiginous melanoma,
Appearing as an uncommon discoloration of the skin syphilis, pinta, and yaws) or silver nitrate stain [34–36].
(due to the accumulation of a melanin-like substance within Morphologically, Hortaea werneckii is notably pleomor-
the fungus), tinea nigra is characterized by the formation phic with a yeast-like phase as well as a hyphal phase. Its
of a flat, painless, discrete, and light brown-to-black, non- colonies are initially black with a creamy appearance (yeast-
inflammatory, nonscaling, oval-shaped patch (macule) that like phase, as a result of conidia germinating with hyphae)
typically occurs on the palmar aspects of hands and occa- and later become filamentous (hyphal phase, to complete the
sionally the plantar and other surfaces of the skin (e.g., soles anamorph life cycle). However, some strains with poor conid-
of the feet and the fingers) as well as toes and nails. In some iogenesis are occasionally encountered in clinical isolates.
rare cases, the skin of the neck, chest wall, and penis may Its relatively restricted, black primary cultures, 1 μm-wide
be affected. Occasionally, the macule is mottled or velvety annellated zones, and one-septate conidia are useful features
and may appear irregular in shape, or there may be multi- for its specific identification. Direct KOH examination of cul-
ple patches. The macules range from a few millimeters to tured isolates demonstrates the short, tortuous, thick, light
several centimeters in diameter and may show more intense brown, occasionally darkened hyphae, which sometimes
pigmentations early in the morning, and fade as the day goes present short filaments and yeast-like cells [37].
by. This possibly reflects the fact that the fungus is cleared Pathogenic dematiaceous fungi such as Exophiala salmo-
from the hands as a result of daily activities. The course of nis (Chaetothyriales) also produce two-celled conidia [5]; how-
H. werneckii infection is 1–18 months (average 3.8 months). ever, the pigmentation of hyphae unambiguously distinguishes
The developing lesions may become visible within 15–30 tinea nigra from other types of dermatophytoses or skin infec-
days of infection [20]. tions. In comparison with Cladosporium spp., Hortaea wer-
Sometimes, H. werneckii behaves as an opportunistic neckii conidia are pigmented yeast cells with a dark central
causative agent of systemic phaeohyphomycosis. It was iso- septum, and their outer wall later become thick-walled, heavily
lated from the serum and a splenic abscess of two leukemia pigmented. Furthermore, whereas the chaetothyrialean black
patient and from the house dust of a hypersensitivity pneu- yeast-like fungus Cladophialophora saturnica shows invasive
monitis patient [10]. behavior, Hortaea werneckii is strictly limited to the stratum
Due to its asymptomatic nature and the possibility of corneum. Skin biopsies of tinea nigra show fungal elements in
spontaneous cure, tinea nigra is observed rarely, account- the stratum corneum with a discrete perivascular infiltrate [37].
ing for about 1% of all mycoses. The disorder is frequently Use of physiobiochemical properties and antigen detection
reported in tropical and subtropical countries such as Latin further enhances the identification of H. werneckii [38,39].
America, Asia, as well as Europe and the United States Hortaea werneckii tolerates high concentrations of sodium
[21–25]. Predisposing factors for the illness consist of hyper- chloride (halotolerance) that may be used as a physiological
hydrosis, presence in coastal sea areas, salt-marsh, river marker for the identification of this fungus [9,40,41]. Because
estuaria, natural, or man-made saltpans hypersaline environ- H. werneckii has a carbon-source assimilation pattern identical
ments, and exposure to plants and grasses or substrata of high to that of Exophiala dermatitidis, Exophiala jeanselmei, and
salinity such as sea water, as well as to house dust [26]. The Exophiala spinifera, the commercial API 20C system does
salty property of human palm, which has an increased con- not allow differentiation among these four species [42,43].
centration of eccrine sweat glands, provides an ideal environ- The time-consuming and variable nature of morphologi-
ment for H. werneckii infection. cal, biochemical examinations creates long delays in the
Most cases of tinea nigra are resolved with keratinolytic identification of H. werneckii. Molecular techniques offer
agents (e.g., urea, undecylenic acid, salicylic acid, Whitfield a rapid and accurate alternative for determining pathogenic
ointment, retinoic acid, and 4% aspirin) and epidermal tape fungi including H. werneckii [41,44]. In particular, sequenc-
stripping [27]. Many topical antifungals (e.g., miconazole, ing analysis of small subunit (SSU, 18S) and large subunit
ketoconazole, itraconazole, bifonazole, tiabendazole, terbin- (LSU, 28S) ribosomal RNA (rRNA) genes as well as rRNA
afine, and ciclopirox olamine) are also effective against tinea internal transcribed spacer (ITS) region permits rapid and
nigra [20,28–33]. Tinea nigra does not usually recur. precise characterization of fungal genera and species. For

Hortaea 103
example, comparison of sequences from the D1/D2 region Procedure

of the LSU rRNA gene indicates that H. werneckii has more
than 100 nucleotide differences from Exophiala species. 1. PCR mixture (25 μL) is made up of 2.5 μL tem-
Located between the 18S and 28S rRNAs, the ITS region plate DNA, 2.5 μL (2 pmol) each Hor-F and Hor-R
consists of two spacers (ITS1 and ITS2) separated by a 5.8S primer, 2 μL (2.5 mM) dNTP mixture, 0.125 μL
conserved region. Nucleotide differences in the ITS1 and/or (5 U/μL) Taq polymerase (Nippon Gene), and 2.5 μL
ITS2 regions provide an effective approach for species iden- 10× reaction buffer.
tification of pathogenic fungi from other, closely related taxa 2. Amplification is conducted with 1 cycle of 95°C for
[45–47]. Sequence analysis of the ITS regions shows that H. 4 min; 30 cycles of 94°C for 1 min, 58°C for 2.5 min,
werneckii has more than 200 nucleotide differences from the and 72°C for 2.5 min; and a final extension at 72°C
Exophiala species. Exploiting the nucleotide differences in for 10 min.
the internal transcribed spacer (ITS) region, H. werneckii 3. Upon completion, 2 μL of the amplified product is
primers were designed for specific polymerase chain reaction run on a 1.5% agarose gel, stained with ethidium
(PCR) amplification of a 306 bp fragment [48]. In addition, bromide, and visualized by UV illumination.
examination of restriction fragment length polymorphisms
Note. This primer set recognizes DNA from H. werneckii only,
(RFLP) of mitochondrial DNA (mtDNA) enables assessment
but not DNA from other related dematiaceous fungal taxa.
of intraspecies diversity of H. werneckii and subsequent sep-
Thus, the PCR assay employing primers Hor-F and Hor offers
aration of seven mtDNA types (populations) [44,49].
a useful tool for rapid specific identification of H. werneckii.
11.2 Methods 11.2.2.2 Sequencing Analysis of ITS Regions

Abliz et al. [48] employed universal primers ITS5
11.2.1 Sample Preparation (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4
Biopsy specimens are obtained from patients with tinea nigra (5′-TCCTCCGCTTATTGATATGC-3′) [51] for PCR ampli-
by carefully scraping off the stratum corneum with a scalpel fication of the ITS1–5.8S–ITS2 region in H. werneckii
blade. KOH (20%) microscopic examination of cutaneous DNA. The resulting PCR product is sequenced with an ABI
scrapings reveals one- to two-celled yeasts and thick, sep- PRISM 3100 sequencer after labeling with BigDye™ termi-
tate, branching hyphae with a dark pigment in their walls. nator cycle sequencing ready reaction (Applied Biosystems).
Periodic acid-Schiff-positive septate hyphae are seen in the Two external primers ITS5 and ITS4 and two internal
stratum corneum in biopsy specimens, along with hyperkera- primers ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) and
tosis and mild acanthosis. The pigmented cell walls of the ITS3 (5′-GCATCGATGAAGAACGCAGC-3′) are used
fungus are readily observable with hematoxylin and eosin for sequencing. The nucleotide sequence of the ITS region
stain. Culture of the scrapings on Sabouraud glucose agar from H. werneckii is aligned to those from other dematia-
without or with antibiotics (Mycobiotic, Difco) at 25°C yields ceous fungal species and medically important yeasts using
growth in a week. H. werneckii isolates typically shows two- CLUSTAL W (version 1.6).
celled yeast forms (annellides) that produce one- and two-
celled conidia [20]. The isolates are identified on the basis of 11.3 Conclusion
macroscopic and microscopic characteristics [25]. Hortaea werneckii is a causative agent of tinea nigra (super-
For DNA extraction, a small amount of fungal pellet is ficial mycosis) in the tropical and subtropical environments.
suspended in 100 μL extraction buffer (100 mM Tris–HCl, The infected patients develop asymptomatic black or brown
pH 7.5, 30 mM EDTA, 0.5% [w/v] sodium dodecyl sulfate patch with well-defined border on the palm of one hand or
[SDS]) and vortexed for 15 s. The mixture is then incubated at foot. In order to implement effective therapy, it is important
100°C for 15 min, 100 μL of 2.5 M potassium acetate is added to differentiate Hortaea werneckii from other dematiaceous
and mixed, chilled on ice for 60 min, and centrifuged at fungi and pathogenic viruses that infect the skin. However, due
12,000 rpm for 5 min. The supernatant is transferred to a new to its pleomorphic nature and its morphological resemblances
tube, and the DNA is precipitated with an equal volume of to other related dematiaceous fungal taxa, laboratory identifi-
cold isopropanol (−20°C), washed with 500 μL of 99% etha- cation of H. werneckii on the basis of phenotypic characteris-
nol, air dried, and resuspended in 100 μL distilled water [50]. tics is tedious and demands considerable technical expertise.
Application of PCR and sequencing techniques facilitates
11.2.2 Detection Procedures rapid, specific, and sensitive identification of H. werneckii.
11.2.2.1 Specific PCR Identification References

Abliz et al. [48] designed an oligonucleotide primer set,
Hor-F (5′-TGGACACCTTCATAACTCTTG-3′) and Hor-R org/, accessed on August 1, 2010.
(5′-TCACAACGCTTAGAGACGG-3′) from the ITS1-5.8S- 2. Nishimura, K. and Miyaji, M., Hortaea, a new genus to
ITS2 sequence for specific amplification of a 306 bp frag- accommodate Cladosporium werneckii. Jpn. J. Med. Mycol.,
ment from H. werneckii. 25, 139, 1984.

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12 Leptosphaeria
Dongyou Liu
Contents
12.1 Introduction...................................................................................................................................................................... 105
12.1.1 Classification and Morphology............................................................................................................................. 105
12.1.2 Clinical Features and Epidemiology.................................................................................................................... 105
12.1.3 Diagnosis.............................................................................................................................................................. 106
12.2 Methods............................................................................................................................................................................ 106
12.2.2.1 Sequence Analysis of ITS Region......................................................................................................... 106
12.2.2.2 PCR-Restriction Fragment Length Polymorphism Analysis................................................................. 107
12.3 Conclusion........................................................................................................................................................................ 107
References.................................................................................................................................................................................. 107
12.1 Introduction reverse, colonies are dark olive to black in color and with a
grayish margin. After 5 weeks, a brown diffusing pigment is
Leptosphaeria is a dark-walled fungal genus that grows noticeable in the medium. Microscopic examination of blue
saprophytically and also inhabits the leaves and culms of lactic-stained cultures reveals septate hyphae. The sexual
grasses, dead herbaceous stems, and driftwood. Among the form of the fungus is produced on potato-carrot agar media
multiple members within the genus, Leptosphaeria senega- after 6 weeks at 30°C, with the presence of ascomata (cle-
lensis and Leptosphaeria thompkinsii have been identified as istothecia), asci, and ascospores. Ascomata containing asci
the agents of human black-grain mycetoma in Africa, which are globose to subglobose in shape, black in color, without
is a highly debilitating tropical disease after a subcutaneous ostioles. Asci are clavate to cylindrical and bitunicate, with
inoculation of the organisms by means of thorns, splinters, each ascus carrying eight ascospores inside. Ascospores are
snake or insect bites, farm implements, knives, or scratches, four- to nine-celled, hyaline or pigmented, fusoid to curved,
resulting in subcutaneous masses with sinuses from which and have a constriction at each septum. Leptosphaeria tomp-
drain pus and grains. kinsii differs from Leptosphaeria senegalensis in size, shape,
s eptation, and the nature of the gelatinous sheath of the asco-
12.1.1 Classification and Morphology spores. Specifically, Leptosphaeria tompkinsii ascospores
have six septa and pointed ends, while Leptosphaeria sen-
The genus Leptosphaeria is a dematiaceous (phaeoid, egalensis ascospores show four septa and rounded ends [3].
or dark-walled) filamentous fungus belonging to the
family Leptosphaeriaceae, order Pleosporales, class
Dothideomycetes, subphylum Pezizomycotina, phylum
12.1.2 Clinical Features and Epidemiology
Ascomycota, kingdom Fungi. The family Leptosphaer Leptosphaeria spp. are present in soil and several species
iaceae is composed of the genera Leptosphaeria, mitosporic are associated with mycetoma and phaeohyphomycosis in
Leptosphaeriaceae, Ophiobolus, and some unassigned humans, particularly in tropical regions [4–9].
Leptosphaeriaceae. In turn, the genus Leptosphaeria Mycetoma is a highly debilitating, chronic infection in the
is separated into 22 recognized and 23 unassigned spe- tropics that is characterized by the formation of a subcutane-
cies, of which Leptosphaeria coniothyrium (anamorph: ous mass with multiple sinuses draining pus and “grains.”
Coniothyrium fuckelii), Leptosphaeria senegalensis, and Depending on the color of the grains (of irregular shape and
Leptosphaeria tompkinsii are implicated in human dis- 1–2 μm in diameter), mycetoma is divided into black-grain
eases [1,2]. and white-grain categories. The disease begins with a sub-
Being an ascomycetous mold, Leptosphaeria grows in cutaneous inoculation (through cut or splinter) of true fungi
its teleomorphic phase. On Sabouraud glucose medium con- (eumycetoma) (accounting for 40% of mycetoma cases) or
taining chloramphenicol at 27°C and 37°C, Leptosphaeria actinomycetes (actinomycetoma) (accounting for 60% of
colonies grow slowly, showing wooly texture. From the front, mycetoma cases), which remain indolent until extension to
colonies are dark olive in color with a gray margin. From the deeper tissues and bone takes place [10].
105

Initially described in India (Madura foot), eumycetoma is For DNA extraction, isolates are grown in 20 mL
prevalent in Africa, with the area covering Sudan to Senegal of RPMI 1640 medium with l-glutamine but without
delineating the “mycetoma belt.” Leptosphaeria senegalen- sodium bicarbonate (Sigma-Aldrich) buffered to pH 7.0
sis and Leptosphaeria tompkinsii are among fungi that are with 0.165 M morpholinepropanesulfonic acid (MOPS)
associated with black-grain mycetomas in humans. Other ( Sigma-Aldrich). After 3–14 days of growth at 30°C under
black-grain causing fungi include Madurella mycetomatis, agitation (100 rpm), the mycelium is transferred into a tube
Madurella grisea, Pyrenochaeta mackinnonii, Pyrenochaeta and washed in 40 mL of sterile distilled water. Mycelium
romeroi, Exophiala jeanselmei, Phlenodomus avramii, (100 mg) is homogenized for 1 min in a tube containing
and Curvularia lunata. On the other hand, fungi causing 1 mL of lysis buffer (2% Triton X-100, 1% sodium dodecyl
white or white to yellow mycetomas consist of Acremonium sulfate, 10 mM Tris–HCl pH 8, 100 mM NaCl, 1 mM EDTA
(Fusarium) falciforme, Acremonium kiliense, Acremonium pH 8), three 0.5 cm diameter glass beads (Sigma), and
recifei, Aspergillus nidulans, Cylindrocarpon destructans, approximately 500 mg of 425–600 μm glass beads (Sigma).
Fusarium moniliforme, Fusarium solani, Neotestudina The homogenized mycelia are then snap-frozen in liquid
rosatii, and Pseudallescheria boydii. nitrogen, thawed, and refrozen once. DNA extraction is then
Mycetoma is a chronic, suppurative infection of the sub- done with the DNeasy plant kit (QIAGEN) according to the
cutaneous tissue and contiguous bone. The feet are the most manufacturer’s instructions [17].
common site for infection and account for at least two-thirds Alternatively, 4-day-old mycelium is harvested by vacuum
of cases. Other less common sites comprise the lower legs, filtration on sterile muslin, frozen at −20°C, and freeze-dried.
hands, head, neck, chest, shoulder, and arms. After entry Freeze-dried mycelium is ground with forceps in a 1.5 mL
through sites of local trauma, the organism causes a granu- microfuge tube, so that approximately 100 μL of powder is
lomatous reaction, producing a small, hard painless nodule, obtained. Two hundred microliters of lysis buffer (50 mM
which over time begins to soften on the surface and ulcer- Tris–HCl, pH 8; 50 mM EDTA; 2% sodium N-lauroyl sarco-
ate to discharge a viscous, purulent fluid containing grains. sinate; and 150 mM NaCl) and 200 μL of phenol (saturated
The infection slowly spreads to adjacent tissue including bone, with 1× Tris–HCl, pH 8) are added, and lysis takes place on
often resulting in considerable deformity. Sinuses continue to ice for 1 h 30 min with occasional vortexing. The mixture
discharge serosanguinous fluid containing granules (grains) of is centrifuged for 5 min at 13,000 × g at 4°C. The aqueous
variable size, color, and degree of hardness, depending on the phase is centrifuged an additional 15 min, the supernatant
etiologic species. These grains are the hallmark of mycetoma. transferred to a new tube, and nucleic acids are precipitated
using 0.6 volume of isopropanol at −20°C. After 70% ethanol
12.1.3 Diagnosis wash, the DNA is resuspended in 1× Tris–EDTA buffer and
residual RNA can be removed with RNase digestion [13].
Several dematiaceous fungi including Leptosphaeria spp.
are responsible for black-grain mycetoma, a chronic subcu-
taneous infection of humans with devastating consequence. 12.2.2 Detection Procedures
Identification of the dematiaceous fungi causing black-grain
mycetoma has traditionally relied on standard mycological 12.2.2.1 Sequence Analysis of ITS Region
procedures such as in vitro culture and microscopy. Species Desnos-Ollivier et al. [17] utilized universal primers
identification can be made on sexual reproductive structures V9D (5′-TTAAGTCCCTGCCCTTTGTA-3′) and LS266
observed on potato-carrot agar media. Because some dema- (5′-GTAGTCATATGCTTGTCTC-3′) for polymerase chain
tiaceous fungi display poor growth or delayed sporulation, reaction (PCR) amplification and sequence analysis of the
diagnosis of these organisms on the basis of macroscopic and ITS region for identification of dematiaceous fungi. In the
microscopic characteristics may take up to 12 weeks [11,12]. case of negative amplification with V9D and LS266, other
To improve the diagnosis, epidemiological investigations, fungal universal primers (e.g., ITS1/ITS4, ITS4/ITS5, ITS1/
and treatment evaluation of eumycetoma, molecular tech- ITS2, ITS3/ITS4) may be employed [18].
niques have been increasingly adopted in the clinical laborato- Procedure
ries [13–15]. In particular, sequence analysis of small-subunit
rRNA genes and internal transcribed spacer 1 (ITS1)-5.8S- 1. PCR mixture (20 μL) is made up of 1 μL of genomic
ITS2 region allows rapid and precise determination of vari- DNA, 1.25 U of AmpliTaq gold (Roche), 2 μL of 10×
ous fungal species causing black-grain mycetoma including PCR buffer (Roche), 2 μL of 25 mM MgCl2 (Roche),
L. senegalensis and L. tompkinsii [16,17]. 2 μL of 2.5 mM deoxynucleoside triphosphate, and
1 μL each of 10 μM concentrated primers.
12.2 Methods 2. PCR amplification is conducted in an ICycler ther-
mocycler (Bio-Rad) with a first cycle of denatur-
ation for 10 min at 95°C, followed by 30 cycles of
Clinical specimens are examined under microscope for denaturation at 94°C for 30 s, annealing at 58°C for
mycotic elements with the help of fungal stains. Portions of 30 s, and elongation at 72°C for 30 s, with a final
the samples are cultured on standard mycology media at 28°C. extension step of 10 min at 72°C.

Leptosphaeria 107
3. An aliquot of PCR products is separated on an aga- are calculated with the Molecular Analyst v. 1.4
rose gel, stained with ethidium bromide, and visual- s oftware (Bio-Rad) using the 1 kb, 100 bp, and 10 bp
ized under UV light. ladder (Life Technologies, Inc.) as references.
4. The remaining PCR products are purified on P100
Note: An alternative PCR-RFLP (restriction fragment length
Gel Fine (Bio-Rad), and sequenced on both strands
polymorphism) protocol utilizes primer pair ITS4/ITS5 for
by using the BigDye Terminator Cycle Sequencing
amplification and endonuclease SmaI for digestion (1 h at
Ready Reaction kit, version 3.1 (Applied Biosystems),
27°C). Restriction fragments are visualized on a 3% agarose
using the primers V9D and LS266. Reaction prod-
gel after ethidium bromide staining [17].
ucts are analyzed using an ABI Prism 3700 auto-
mated DNA analyzer (Applied Biosystems).
5. Sequences are edited and manually corrected with 12.3 Conclusion
Chromas, version 2.24 (Technelysium, Helensvale,
Queensland, Australia). Multiple-sequence align- Leptosphaeria spp. are dematiaceous fungi that are com-
ment is carried out using ClustalW 1.8. Phylogenetic monly distributed in soil. Several Leptosphaeria species
trees are constructed by the neighbor-joining such as L. senegalensis and L. tompkinsii are associated
method using the Phylip package (http://www.info- with black-grain mycetoma as well as phaeohyphomycosis in
biogen.fr) and visualized using Treeview. humans, particularly in tropical regions [4–10,19]. Traditional
methods for differentiation of Leptosphaeria spp. from other
mycetoma-causing dematiaceous fungi are lengthy and
12.2.2.2 P CR-Restriction Fragment Length imprecise. PCR amplification and sequencing analysis of the
Polymorphism Analysis ITS region provide a rapid and reliable approach for determi-
Balesdent et al. [13] used the 18S primer PN3 (for- nation of Leptosphaeria and other dematiaceous fungi.
ward) (5′-CCGTTGGTGAACCAGCGGAGGGATC-3′)
and the 28S rDNA for primer PN10 (reverse)
References
(5′-TCCGCTTATTGATATGCTTAAG-3′) for PCR ampli-
fication followed by digestion with restriction enzymes for 1. The UniProt Consortium. Available at http://www.uniprot.
identification and phylogenetic analysis of Leptosphaeria org/, accessed on August 1, 2010.
2. El-Ani, A.S., A new species of Leptosphaeria, an etiologic
spp.
agent of mycetoma. Mycologia 1966;58:406–411.
Procedure 3. Kwon-Chung, K.J. and Bennett, J.E., Medical Mycology. Lea
& Febiger, Philadelphia, PA, 1992.
4. Venugopal, P.V. and Venugopal, T.V., Leptosphaeria tompkin-
1. PCR mixture (25 or 50 μL) is composed of 50 ng sii mycetoma. Int J Dermatol. 1990;29(6):432–433.
of template DNA, 200 μM of each deoxyribonu- 5. Venugopal, P.V. et al., Antimycotic susceptibility testing
cleotide triphosphate, 2.5 μM of each primer, 1.5 U of agents of black grain eumycetoma. J Med Vet Mycol.
(for 25 μL mixes) or 3 U (for 50 μL mixes) of Taq 1993;31(2):161–164.
polymerase in 1× Taq DNA incubation mix (10 mM 6. Mahe, A. et al., Mycetomas in Mali: Causative agents
Tris–HCl, pH 9.0; 50 mM KCl; 1.5 mM MgCl2; 0.1% and geographic distribution. Am J Trop Med Hyg.
1996;54(1):77–79.
Triton X-100; and 0.2 mg of bovine serum albumin
7. Khatri, M.L. et al., Mycetoma in Yemen: Clinicoepi
per milliliter) (Appligene-Oncor). demiologic and histopathologic study. Int J Dermatol.
2. The reaction is carried out for 37 cycles in a 2002;41(9):586–593.
GeneAmp PCR system 9600 (Perkin-Elmer/ 8. Gioulekas, D. et al., Allergenic fungi spore records (15
Applied Biosystems), with each cycle consisting of years) and sensitization in patients with respiratory allergy
30 s at 94°C, 30 s at 58°C, and 1 min at 72°C. in Thessaloniki-Greece. J Investig Allergol Clin Immunol.
3. PCR products (6–8 μL) are digested for 2–5 h with 2004;14(3):225–231.
10 U of one of the following enzymes: AluI, BamHI, 9. Gonianakis, M.I. et al., Mold allergy in the Mediterranean
Island of Crete, Greece: A 10-year volumetric, aerobiological
EcoRII, HaeIII, MspI, RsaI, or TaqI, (Eurogentec) study with dermal sensitization correlations. Allergy Asthma
according to the manufacturer’s instructions. Proc. 2006;27(5):354–362.
4. PCR products and restriction fragments are ana- 10. Fahal, A.H., Mycetoma: A thorn in the flesh. Trans R Soc Trop
lyzed upon electrophoresis in 1.4%–1.8% agarose Med Hyg. 2004;98:3–11.
gels with 1× TBE (45 mM Tris–borate and 1 mM 11. de Hoog, G.S. et al., Diagnostic problems with imported cases
EDTA, pH 8) as a buffer and staining with 0.5 μg of mycetoma in The Netherlands. Mycoses 1993;36:81–87.
of ethidium bromide per milliliter. As an alter- 12. de Hoog, G.S. et al., Phylogeny and typification of Madurella
mycetomatis, with a comparison of other agents of eumyce-
native, 3%–3.5% MetaPhor agarose gels (FMC
toma. Mycoses 2004;47(3–4):121–130.
BioProducts, Rockland, ME) are used to separate 13. Balesdent, M.H. et al., Conidia as a substrate for internal tran-
restriction fragments. Gels are viewed and recorded scribed spacer-based PCR identification of members of the
using the Gel Doc 1000 fluorescent gel docu- Leptosphaeria maculans species complex. Phytopathology
mentation system (Bio-Rad). Molecular weights 1998;88(11):1210–1217.

14. Iwen, P.C., Hinrichs, S.H., and Rupp, M.E., Utilization of 17. Desnos-Ollivier, M. et al., Molecular identification
the internal transcribed spacer regions as molecular targets of black-grain mycetoma agents. J Clin Microbiol.
to detect and identify human fungal pathogens. Med Mycol. 2006;44(10):3517–3523.
2002;40:87–109. 18. White, T.J. et al., 1990. Amplification and direct sequencing
15. Kaczmarek, J. et al., Analyses of air samples for asco- of fungal ribosomal RNA genes for phylogenetics, in M.S.
spores of Leptosphaeria maculans and L. biglobosa by Innis and D.H. Gelfand (eds.), PCR Protocols: A Guide to
light microscopy and molecular techniques. J Appl Genet. Methods and Applications. Academic Press, New York,
2009;50(4):411–419. pp. 315–322.
16. Ahmed, A.O. et al., Molecular detection and identification of 19. Machmachi, H. et al., Black grain mycetoma caused by
agents of eumycetoma: Detailed report of two cases. J Clin Leptosphaeria tompkinsil, Medi. Mycol., 2011:49, 186.
Microbiol. 2003;41:5813–5816.

13 Macrophomina
Contents
13.1 Introduction...................................................................................................................................................................... 109
13.1.1 Classification, Morphology, and Biology............................................................................................................. 109
13.1.2 Clinical Features and Pathogenesis.......................................................................................................................110
13.1.3 Diagnosis...............................................................................................................................................................110
13.1.3.1 Conventional Techniques........................................................................................................................110
13.1.3.2 Molecular Techniques.............................................................................................................................111
13.2 Methods.............................................................................................................................................................................112
13.2.1 Sample Preparation................................................................................................................................................112
13.2.2 Detection Procedures.............................................................................................................................................113
13.3 Conclusion and Future Perspectives..................................................................................................................................114
References...................................................................................................................................................................................114
13.1 Introduction conidium, transformed into two lateral tentaculiform, apical

mucoid appendages (type C, Nag Raj6). When mature, the
13.1.1 Classification, Morphology, and Biology conidia become medium to dark brown, with a granular outer
Macrophomina phaseolina Tassi (Goid.) is the type species layer that in some cases appears pitted, without any mucoid
of the genus Macrophomina Petr., and this name has also appendages; conidial hilum frequently with a marginal frill.1,4
been applied, erroneously, to the coelomycete (pycnidial) The genus Tiarosporella Höhn is characterized by hav-
synanamorph of Rhizoctonia bataticola (Taubenh.) E.J. ing conidia formed from smooth, hyaline conidiogenous cells
Butler.1 Macrophomina phaseolina is a seed and soilborne that lack periclinal thickenings and percurrent proliferations,
plant pathogenic fungus that causes seedling blight, root and and hyaline, subcylindrical to fusiform conidia that have
stem rot of more than 500 cultivated and wild plant species irregular, apical mucoid appendages.1,6 Because conidia of
including economically important crops, forest trees, fruit, M. phaseolina have apical appendages, von Arx7 introduced
and weed species.2,3 the name Tiarosporella phaseolina (Tassi) van der Aa for this
Due to the lack of knowledge about its sexual stage fungus and also reduced the genus Macrophomina to synon-
(teleomorph), this fungus was placed in the anamorphic ymy under Tiarosporella, but this has not been followed by
Ascomycetes with no apparent affiliation to the known fami- the plant pathological and mycological communities.
lies of Ascomycetes. According to recent phylogenetic data,1 In their work, Crous et al.1 studied several isolates of
M. phaseolina is currently recognized as a member of the M. phaseolina and provided an updated description, as
family Botryosphaeriaceae even though its teleomorph is well as illustrations and photographs showing the distinc-
still unknown. The name of the synanamorph, and its taxo- tive morphological features that characterize the species and
nomic placement, has been the topic of much controversy (for genus. The authors also pointed out the differences between
details see Crous et al.1). Tiarosporella and Macrophomina. Thus, Macrophomina
The asexual structures formed by the fungus are pyc- phaseolina has conidia with apical mucoid appendages
nidia and microsclerotia. Black sclerotia, 100–1000 μm as found in Tiarosporella, but it is distinguished by having
diameter, occur in host tissue or in soil and constitute the percurrently proliferating conidiogenous cells and conidia
primary inoculum source of the pathogen. They can survive that become dark brown and slightly roughened at maturity,
several years depending on environmental conditions and appearing more Diplodia-like in morphology. Based on these
whether or not the sclerotia are associated with host resi- differences and also on phylogenetic differences between
dues.1–5 Secondary dispersal by pycnidiospores (conidia) is M. phaseolina and Tiarosporella species, Crous et al.1 retained
host- and isolate-dependent.4 The pycnidial conidiomata are the genus Macrophomina and the name M. phaseolina.
dark brown to black, up to 200 μm in diameter. The conidia This fungus is known mainly as a plant pathogen on an
are ellipsoid to obovoid, (16−)20–24(−32) × (6−)7–9(−11) extremely wide range of hosts and is distributed throughout
μm. Immature conidia are hyaline, enclosed in a mucous the world, predominantly in regions with hot and dry con-
sheath, which, upon dehiscence, encloses the top half of the ditions during the growing season. The severity of disease
109

caused by M. phaseolina in various hosts is associated with illustration of the conidia suggest that a different fungal spe-
environmental conditions such as high temperatures8 or cies was probably the etiologic agent.13
water stress.9 However, the pathogen has also been detected It is not well understood how plant pathogenic fungi
in hosts without any disease symptom when growing condi- like M. phaseolina are capable of infecting organisms from
tions were optimal for the plant. Sudden disease outbreaks members of different biological kingdoms, namely animals.
in mature plants may occur when plants are under stress and Several aspects regarding environmental conditions, popula-
cause high yield losses in previously healthy plants.10 This tion biology traits, and molecular processes may help under-
fact is a common feature among species of plant pathogenic stand how these microorganisms are capable of performing
fungi of the family Botryosphaeriaceae, which have the abil- these cross-kingdom host jumps.17 It has been speculated that
ity to live as endophytes inside healthy plants causing disease pathogenic microorganisms that have a broad host range are
following the onset of stress.11 more likely to become pathogenic on cross-kingdom hosts
than highly specialized microorganisms.17 In the case of
M. phaseolina, being an unspecialized pathogen with a very
wide host range may have enabled it to expand its pathogenic
The relevance of M. phaseolina as a human pathogen is not spectrum and become capable of infecting humans. A shift
well understood. As with other species of Botryosphaeriaceae from an environmental state into a clinical, pathogenic state
this fungus is most likely an opportunistic human pathogen inside a host requires adaptation or tolerance to the new
that is capable of causing disease under appropriate condi- environment. A crucial moment occurs when fungi enter
tions. The infection process (as with other coelomycetes) a mammalian host and experience substantially increased
occurs most commonly by trauma resulting in the implanta- temperature.17 The ability to withstand and grow at elevated
tion of the fungus from plant material or soil.12 Coelomycete temperatures (37°C) is an essential prerequisite for a micro-
fungi such as M. phaseolina may also be of concern to organism to be a human pathogen. Macrophomina phaseo-
patients under immunosuppressive therapy, namely bone lina is able to grow in vitro in a wide range of temperatures
marrow and organ transplant recipients, cancer patients, and it has been shown that optimum growth temperature is
among others.12 35°C–37°C, with some isolates being capable of growth at
The number of reports describing this species as the eti- temperatures as high as 40°C.4 However, this characteris-
ological agent of fungal infection in humans is small. The tic is isolate dependent and some isolates have lower opti-
first report of human infection by M. phaseolina concerns its mum growth temperatures. Macrophomina phaseolina (at
occurrence in a patient (an adult male) who had received a least some isolates) are clearly adapted to withstand normal
renal transplant and was receiving immunosuppressive med- human body temperature, which makes this species poten-
ication. The patient developed swelling, pain, and purulent tially a human pathogen.
discharge involving the left great toe. Swabs of the discharge
and an aspirate of the metatarsophalangeal joint yielded a 13.1.3 Diagnosis
filamentous fungus that was later identified as M. phaseo-
lina.13 Another case of human infection (a cutaneous infec- 13.1.3.1 Conventional Techniques
tion) with Macrophomina phaseolina was found in a child The identification of fungi in clinical specimens can be
with acute myeloid leukemia who had received an unrelated performed by histopathological analysis and direct micro-
hematopoietic stem cell transplant.14 The patient was under scopic examination using appropriate stains. These proce-
immunosuppressive as well as prophylactic antifungal (vori- dures, although useful for the detection and identification
conazole) and antibacterial (vancomycin, ciprofloxacin, and of some of the more common fungal pathogens, are not
trimethoprim-sulfamethoxazole) therapy. The girl developed really useful for identifying unusual fungal species causing
an erythematous, papular, nonulcerative, tender lesion above infection.18
the right medial malleolus with no induration or inguinal Fungal identification is based largely on morphological
lymphadenopathy. The lesion progressed and became indu- criteria, most specifically on the morphology of the repro-
rated, with a central necrotic eschar and a surrounding rim ductive structures.12,19 Since it is highly unlikely that fungi
of erythema, and extended into the subcutaneous tissue. A develop these reproductive structures on the human host tis-
fungal culture identified as M. phaseolina was retrieved sues, it is necessary to obtain cultures of the fungus from
from a punch biopsy performed through the central ulcer- clinical samples in order to accomplish a proper identifica-
ative lesion.14 The only other reported cases of M. phaseolina tion of the etiological agent.
infection of humans were in a study of non-sporulating molds Macrophomina phaseolina and coelomycetes, in general,
(NSMs) associated with ocular infections in India.15 The spe- are not particularly difficult to isolate from clinical speci-
cies M. phaseolina was identified as the causal agent in seven mens and have a moderate to rapid growth rate on a variety of
clinical cases. routine culture media. The main problem with these fungi is
Apart from its occurrence in human infections, there is related to induction of sporulation in order to obtain the diag-
also a description of a subcutaneous granuloma on the tail nostic reproductive structures necessary for identification
of a cat that was attributed to M. phaseolina.16 However, of the isolates. This process is not only time consuming but
the authors’ description of slow growth at 37°C and their also frequently unsuccessful.12 Macrophomina phaseolina is

Macrophomina 111
particularly recalcitrant to sporulation and commonly pro- Difficulties in the identification of M. phaseolina are
duces only dark sclerotia in routine cultures and these are not well patent in the clinical reports regarding this fungus. For
sufficient for identification, at least for someone who is not example, Tan et al.13 reported that the fungus was initially
familiar with the species. This is well illustrated by the clini- thought to represent Pseudallescheria boydii based on the
cal reports concerning infections by M. phaseolina where grayish colonies, but the identification was later revised to
the isolates of this fungus were grown on a variety of cul- Lasiodiplodia theobromae. This was due to the fact that the
ture media and under different conditions of temperature and isolate produced dark brown sclerotia, which were suspected
light. Although the fungus produced sclerotia, it invariably to represent immature pycnidia of L. theobromae. A defini-
failed to produce pycnidia and conidia, which are fundamen- tive identification was only accomplished when molecular
tal diagnostic structures.13,14 methods were used. The same occurred with Srinivasan et
It is known that the cultivation of fungi on sugar-rich al.14 who were unable to induce their M. phaseolina isolate to
media is prejudicial to sporulation and that culture of fungi sporulate and could only identify it on the basis of molecular
(especially Coelomycetes) on media containing sterilized methods.
plant tissue promotes sporulation.12,20 Our experience has Immunological methods, because of their high speci-
shown that for fungi of the family Botryosphaeriaceae, the ficity and sensitivity, provide a rapid means of diagnosing
addition of sterilized plant material such as pine needles, fungal infections.18,29 Several commercial enzyme-linked
oak, or poplar twigs, among others on water agar or half- immunosorbent assay (ELISA) techniques are available,
strength potato dextrose agar, results in good sporula- which reliably detect candidiasis, histoplasmosis, and
tion of cultures.1,21 Pycnidia of M. phaseolina have been coccidioidomycosis.18
induced on propylene oxide sterilized or autoclaved plant ELISA techniques have been developed for the specific
tissues and on groundnut meal irradiated with UV light detection and quantification of M. phaseolina in plant tis-
and Whatman filter paper treated with vegetable oil.22 sue.10,29 However, Srivastava and Arora29 reported a lack of
Also, Crous et al.1 were able to induce numerous strains specificity of the ELISA assay since the polyclonal antisera
of M. phaseolina to sporulate on sterile pine needles on exhibited strong cross-reactivity with many other nontarget
water agar. fungal isolates tested. More recently, Afouda et al.10 devel-
Considerable variation in morphology, physiology, and oped a double-antibody sandwich ELISA (DAS-ELISA),
pathogenicity has been reported for isolates of M. phaseolina which revealed high specificity and sensitivity in the detec-
even when obtained from the same plant.23 The identification of M. phaseolina. This serological approach, however,
tion of isolates of M. phaseolina is based on morphological has never been tested on clinical specimens and therefore its
criteria, but due to wide variations in the phenotype of the applicability and validity in the clinical setting have not been
isolates these criteria are often not reliable and the identifica- evaluated.
tion process may be difficult.24 Molecular (DNA-based) methods are being increasingly
Selective or semi-selective culture media have been tra- applied in the field of medical mycology for identification of
ditionally applied for detection of M. phaseolina.25,26 Such human pathogenic fungi.15,27,28,30–34 These molecular meth-
methods, although effective, especially for the enumeration ods present advantages over the traditional morphology-
of microsclerotia in the soil, require exceptional expertise, based identification. Thus, DNA-based methods are faster
are time consuming, and therefore not appropriate to use in with a turnaround time of about 24 h from the time of DNA
clinical laboratories. extraction, yield results that are objective with data portable
between laboratories, and despite the higher cost could be
13.1.3.2 Molecular Techniques more economical in the long run.15,27,28,30–34 These methods,
Identification of fungi to genus and species level is increas- especially the ones taking advantage of the polymerase chain
ingly important as the spectrum of opportunistic fungal reaction (PCR) technology, can be applied to non-sporulat-
pathogens continues to expand. Although the clinical presen- ing isolates or isolates for which identification could not be
tation of many fungal infections may be indistinguishable, attained. Also, it is possible to obviate the culture step as they
an early, fast, and accurate identification of the etiological can be applied directly to clinical specimens.15,27,28,30–33
agent is of fundamental importance for effective treatment A wide array of molecular identification techniques (non-
and management of the infection. sequence-based) is available including the commercially
The traditional morphological methods of fungal iden- available AccuProbe (Gen-Probe) assay, single-step PCR,
tification can be time consuming and laborious, may ini- RAPD-PCR, rep-PCR, nested PCR, PCR-RFLP, PCR-EIA,
tially be nonspecific, and require considerable expertise and microarray-based, Luminex technology-based, and real-
for correct identification of less common or unusual fungi. time PCR-based methods. Many of these methods have not
Further shortcomings of morphological identification are the been widely used or rigorously validated in the clinical envi-
inability of some cultures to sporulate or cultures exhibiting ronment.18,34 Some of the above-mentioned methods (e.g.,
atypical morphology making the identification difficult or RAPD, rep-PCR, PCR-RFLP) have been applied to evalu-
impossible and resulting even in misidentifications. The inci- ate the genetic diversity within isolates of M. phaseolina but
dence of NSMs is quite high in cultures derived from clinical were never tested regarding their potential for identification/
specimens.15,27,28 detection within the clinical setting.5,24,35,36

At present, the molecular method of choice for fungal Also, through the use of species-specific oligonucleotide
species identification is DNA sequence analysis, which con- primers or probes, this approach has been used to detect a
stitutes PCR amplification of a selected genomic region fol- particular species or group of species without the need for
lowed by sequencing of the resulting amplicon. The obtained DNA sequencing.39 Babu et al.24 developed specific primers
sequence can then be queried against a nucleotide sequence and an oligonucleotide probe targeting the ITS region for
database such as GenBank.15,31,33,34 the identification/detection of M. phaseolina under in vitro
The genomic region to be used must be orthologous, have conditions. ITS sequences of several isolates of M. phaseo-
a high level of interspecific variability combined with low lina were aligned and two primers (MpKR1 and MpKF1)
levels of intraspecific variability, and should not undergo were designed from selected regions. Optimization of the
recombination. Moreover, the target locus should be easy to PCR conditions and validation of primers yielded a specific
amplify and sequence, and the amplicons should be within 350 bp amplicon for M. phaseolina isolates. The specific-
the size range obtainable with the most commonly used auto- ity of this primer set was confirmed by the absence of an
mated DNA sequencers (about 600–800 bp).19,33,34 amplicon with the same size in other species of fungi, bac-
The ITS region (noncoding sequence interspaced among teria, and actinomycetes. Thus, the PCR assay with prim-
highly conserved fungal rRNA genes) complies with most of ers MpKR1 and MpKF1 could be used to rapidly identify
these requirements since this region can be reliably ampli- M. phaseolina. Additionally, an oligonucleotide probe
fied for most fungi, is conserved, is present as multiple (MpKH1) was also designed within the ITS region and was
copies in the fungal genome, yields sufficient taxonomic shown to detect the target sequences at varying concentra-
resolution for most fungi, and has the additional advantage tions. The probe was also shown to be specific for M. phase-
that the GenBank (http://www.ncbi.nlm.nih.gov), European olina and no signals were obtained with nonspecific target
Molecular Biology Laboratory nucleotide sequence data- ITS sequences. Before this method can be used in clini-
base (http://www.ebi.ac.uk/embl/), and DNA Data Bank of cal laboratories, it first needs to be rigorously tested and
Japan (http://www.ddbj.nig.ac.jp/) contain a large number of validated. Moreover, the use of a specific PCR approach
sequences from this locus, enabling a ready comparison of although fast and accurate may not be entirely feasible
the sequence from an unknown isolate.19,33,34 because in order to select the primer set to use, it is nec-
There is considerable consensus regarding the use of essary to have a suspect of the fungal species responsible
ITS sequencing in the identification of fungi. Also, the for the infection. Otherwise, a large set of primers needs to
International Subcommission on Fungal Barcoding has be tested in order to identify the etiological agent. In this
proposed the ITS region as the prime fungal barcode or the respect, a panfungal PCR assay using “universal” fungal
default region for species identification. Main disadvantages primers for amplification and sequencing of the ITS region
regarding the use of ITS are the inability of this region for is probably a better choice.31,40
differentiation of species complexes and failure to distin-
guish between closely related species or cryptic species.19,33,34
In these cases, it may be necessary to use more variable gene 13.2 Methods
regions. The locus to be used will depend on the fungal group
in question. Regarding the family Botryosphaeriaceae, when
the ITS region fails to differentiate species complexes or cryp- PCR-based methods require very small quantities of DNA.
tic species, the locus of choice has been the elongation factor Ribosomal DNA regions are present in multiple copies in the
1-alpha, which has shown successful results.37 This problem fungal genome and therefore can be easily amplified even
does not occur with M. phaseolina since it is the only spe- with minute amounts of DNA. Nevertheless, the success
cies currently known within the genus Macrophomina and of any molecular detection procedure is largely dependent
it can be easily identified on the basis of the ITS sequence. on the DNA extraction step, which must retrieve enough
Nevertheless, an important factor that must be considered quantity of DNA and simultaneously the DNA should not
is that an accurate sequence-based identification is heavily be contaminated with PCR inhibitory agents. A good DNA
dependent on the reliability of sequences deposited in refer- extraction method is crucial for PCR detection to avoid the
ence databases. possibilities of false-negative results. Also, strict precautions
The PCR-based DNA sequencing technique targeting the must be taken throughout the whole procedure, from collec-
ITS region and other ribosomal DNA regions has been found tion and processing of samples to DNA extraction and PCR
to be a rapid and reliable tool to identify M. phaseolina clini- amplification in order to avoid contaminations and conse-
cal isolates.13–15 In the clinical reports concerning M. phaseo- quently false-positive results.
lina, the fungal isolates could only be definitively identified Macrophomina phaseolina can be isolated from clinical
by DNA sequencing of the ITS region13–15 and also of the 18S samples using a variety of culture media. As soon as colonies
ribosomal DNA13 and the D1/D2 variable region of the 28S start to develop, DNA can be extracted by different methods
ribosomal DNA genes.14 already available, including commercial kits, which are usu-
PCR-based culture-independent methods have been ally faster than other methods.15,21,27,28,30,32,39 Alternatively,
developed and applied for the detection of fungi on clini- DNA can be extracted directly from the clinical samples
cal specimens, thus eliminating the need for culturing.32,38 using the same DNA extraction procedures used for fungal

Macrophomina 113
cultures or others developed specifically for human tissue amplify the target region. The complete sequences of the
samples rendering the process even faster.31,39 By eliminating ITS region is read and edited using available software such
the isolation procedure, this last approach greatly reduces the as Chromas 1.45 (http://www.technelysium.com.au/chromas.
time necessary for the molecular diagnosis of the fungal spe- html), FinchTV 1.4.0 (http://www.geospiza.com/finchtv), or
cies responsible for the infection. others. The sequences must be checked manually and nucleo-
tide arrangements at ambiguous positions clarified using both
Procedure primer direction sequences. The final step in the identification
procedure consists of performing a sequence similarity search
1. Inoculate cultures onto potato dextrose agar (Difco) of the retrieved sequence against nucleotide sequences data-
and incubate at 25°C. After 1 week the colony is base such as GenBank using the BLAST tool (http://www.
scraped from the agar surface, frozen in liquid nitro- ncbi.nlm.nih.gov/BLAST). The outcome of this similarity
gen, and ground in a porcelain mortar. Alternatively, search must be interpreted with caution due to the well-known
cultures can be grown in potato dextrose broth. problems with the reliability of the ITS sequences deposited
2. Genomic DNA can be extracted from the ground in the reference databases (e.g., GenBank/EMBL/DDBJ).33,46
mycelium using any DNA isolation protocol. In our Errors in fungal sequences within GenBank, the most widely
lab, we have good results in terms of yield and purity used database, have been found to be as high as 20%.46 In
of DNA samples following the method described by order to circumvent this problem, when performing compara-
Alves et al.21 tive sequence analyses only sequences that have been obtained
from cultures thoroughly characterized and properly identi-
Note: This same procedure can be used directly on clinical fied in morphological terms should be considered. Preferably,
samples. sequences from ex-type or authentic cultures should be cho-
sen for comparison. In the case of M. phaseolina, there are no
such sequences but nonetheless sequences from well-charac-
terized isolates have been deposited in GenBank.
PCR amplification of the ITS region as well as the ribosomal
genes (18S and 28S rRNA) can be easily amplified using Procedure
“universal” fungal primers in the vast majority of fungi. We
would suggest that for identification of M. phaseolina the ITS 1. Prepare a PCR reaction mixture (50 μL) containing
region, or alternatively the D1/D2 variable region of the 28S 1× PCR buffer with (NH4)2SO4 (MBI Fermentas,
ribosomal DNA gene, are the best options. Vilnius, Lithuania), 3 mM MgCl2, 200 mM of each
For Macrophomina the primer sets ITS1/ITS4 or ITS5/ nucleotide, 15 pmol of primer ITS1, 15 pmol of
ITS441 give a good amplification of the target ITS region primer ITS4, 1 U of Taq DNA polymerase (MBI
resulting in amplicons with a size of about 500–600 bp Fermentas, Vilnius, Lithuania), and 50 ng of tem-
and primers NL1/NL442 readily amplify the D1/D2 region plate DNA.
resulting in an amplicon with 614 bp in size. As an alterna- Note: The forward primer ITS5 can be used as an
tive, the forward primer (ITS1 or ITS5) can be combined alternative to primer ITS1.
with the reverse primer NL4, which anneals within the 28S 2. Perform the PCR amplification in a thermocycler
rRNA gene resulting in a larger amplicon (aprox. 1200 bp)43 using the following conditions: initial denaturation
and that includes the ITS region plus the D1/D2 variable of 3 min at 95°C, followed by 30 cycles of 30 s at
region of the 28S rRNA gene. The PCR mixtures and ampli- 94°C, 30 s at 50°C, and 1 min at 72°C, and a final
fication conditions have been described elsewhere.21,43,44 In extension period of 10 min at 72°C.
some cases where amplification is weak or unsuccessful, 3. Purify the PCR amplicons before sequencing. In our
good results are usually obtained by performing a second lab, we have good results with the Jet Quick PCR
PCR using as template the first PCR amplification prod- Product Purification Spin Kit (Genomed, Löhne,
uct.45 Another approach involving a nested or semi-nested Germany) but virtually any PCR product purifica-
PCR reaction can be performed. Thus, for example, an ini- tion kit can be used.
tial amplification would be performed using the primer set 4. Prepare cycle sequencing reactions (20 μL) contain-
ITS1/NL4 and in the second PCR reaction the primer set ing 30–90 ng DNA template, 3.2 pmol of primer, and
used would be ITS1/ITS4. This kind of approach is fre- 4 μL BigDye™ Terminator from the ABI PRISM®
quently used and is known to increase the sensitivity of the BigDye Terminator Cycle Sequencing Ready
PCR detection.30 Finally, from our experience, the addition Reaction Kit with AmpliTAQ DNA Polymerase (PE
of 5% DMSO to the PCR amplification reaction is useful to Applied Biosystems, Foster City, CA).
help the amplification of some difficult templates and also 5. Remove excess dye terminator by mixing the reac-
increases reproducibility.37,43–45 tion with 2 μL of 3 M sodium acetate (pH 4.6) and
The nucleotide sequence of the ITS region is then obtained 50 μL of 100% ethanol. Precipitate for 20 min at
using standard automated DNA sequencing procedures and room temperature and centrifuge for 20 min at
using in the sequencing reaction the same primers used to 16,000 g. Discard the supernatant and wash the

pellet with 250 μL of 70% ethanol. Dry the pellet in 7. von Arx, J.A., The Genera of Fungi Sporulating in Pure
a heat block at 90°C for 1 min. Culture, 3rd edn., J. Cramer, Berlin, Germany, 1981.
6. Sequence in an automated DNA sequencer such as 8. Agarwal, D.K., Gangopadhyay, S., and Sarbbhoy, A.K., Effect
of temperature on the charcoal rot disease of soybean, Indian
the ABI PRISM 310 Genetic Analyzer (PE Applied Phytopathol., 26, 587, 1973.
Biosystems, Foster City, CA). 9. Ghaffar, A. and Erwin, D.C., Effect of soil water stress
on root rot of cotton caused by Macrophomina phaseoli,
13.3 C
onclusion and Future Phytopathology, 59, 795, 1969.
10. Afouda, L., Wolf, G., and Wydra, K., Development of a sensi-
Perspectives tive serological method for specific detection of latent infec-
Invasive fungal infections caused by opportunistic fungal tion of Macrophomina phaseolina in cowpea, J. Phytopathol.,
pathogens have been increasing significantly in the last years. 157, 15, 2009.
11. Slippers, B. and Wingfield, M.J., Botryosphaeriaceae as endo-
Rapid and precise identification of the infectious agent is of
phytes and latent pathogens of woody plants: Diversity, ecol-
extreme importance for the initiation of targeted antifungal ogy and impact, Fungal Biol. Rev., 21, 90, 2007.
therapy. The emergence of a wide and taxonomically diverse 12. Sutton, D.A., Coelomycetous fungi in human disease. A
array of unconventional fungal pathogens poses considerable review: Clinical entities, pathogenesis, identification and
diagnostic and therapeutic challenges to the field of medical therapy, Rev. Iberoam. Micol., 16, 171, 1999.
mycology. 13. Tan, D.H. et al., Disseminated fungal infection in a renal
Although it is apparent that M. phaseolina has the abil- transplant recipient involving Macrophomina phaseolina
and Scytalidium dimidiatum: Case report and review of taxo-
ity to infect and cause disease in man, its importance as a
nomic changes among medically important members of the
human pathogen is not fully established yet. This species is Botryosphaeriaceae, Med. Mycol., 46, 285, 2008.
particularly difficult to identify by the traditional morpho- 14. Srinivasan, A. et al., Cutaneous infection caused by
logical procedure due mainly to the difficulty of obtaining Macrophomina phaseolina in a child with acute myeloid leu-
in culture the diagnostic reproductive structures. This fact is kemia, J. Clin. Microbiol., 47, 1969, 2009.
clearly highlighted by the clinical reports where unequivo- 15. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
cal identification could only be accomplished by molecular non-sporulating molds (NSM) identified by polymerase chain
methods. reaction (PCR)-based DNA sequencing technique targeting
internal transcribed spacer (ITS) region, Curr. Eye Res., 33,
Molecular detection methods, most specifically, PCR 139, 2008.
amplification coupled with DNA sequence analysis of the 16. Hasegawa, T. et al., Subcutaneous granuloma associated with
ITS regions or ribosomal DNA genes have proven extremely Macrophomina species infection in a cat, Vet. Rec., 156, 23,
useful in the identification of fungal infections due to 2005.
M. phaseolina. It is envisaged that in the future, with a wider 17. van Baarlen, P. et al., Molecular mechanisms of pathoge-
application of these methods in the clinical laboratory, it will nicity: How do pathogenic microorganisms develop cross-
help reveal the real extent of fungal infections caused by kingdom host jumps?, FEMS Microbiol. Rev., 31, 239,
2007.
this species. It is likely that many clinical cases caused by
18. McGinnis, M.R. and Pfaller, M.A., The laboratory and clini-
M. phaseolina may have previously passed unnoticed due cal mycology. In: Anaissie, E.J., McGinnis, M.R., and Pfaller,
to misidentifications or that this fungus has simply been M.A. (Eds), Clinical Mycology, pp. 55–78, 2nd edn., Chap. 4,
reported as an NSM as shown by Bagyalakshmi et al.15 Churchill Livingstone Inc., New York, 2009.
19. Guarro, J., Gené, J., and Stchigel, A.M., Developments in fun-
gal taxonomy, Clin. Microbiol. Rev., 12, 454, 1999.
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thesis, Wageningen University, the Netherlands, 2007. phomina phaseolina by using species-specific oligonucle-
5. Purkayastha, S. et al., Characterization of Macrophomina otide primers and probe, Mycologia, 99, 797, 2007.
phaseolina, the charcoal rot pathogen of cluster bean, using 25. Papavizas, G.C. and Klag, N.G., Isolation and quantita-
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14 Madurella
Wendy W.J. van de Sande, Ahmed H. Fahal,
G. Sybren de Hoog, and Alex van Belkum
Contents
14.1 Introduction.......................................................................................................................................................................117
14.1.1 Classification..........................................................................................................................................................117
14.1.1.1 Madurella mycetomatis (Laveran) Brumpt, 1905..................................................................................117
14.1.1.2 Madurella grisea Mackinnon, Ferrade et Montemayer, 1949................................................................118
14.1.1.3 Madurella Species..................................................................................................................................119
14.1.2 Epidemiology and Risk Factors.............................................................................................................................119
14.1.3 Clinical Presentation............................................................................................................................................ 120
14.1.4 Disease Management and Antifungal Therapy.................................................................................................... 120
14.1.5 Diagnosis.............................................................................................................................................................. 121
14.1.5.1 Conventional Techniques....................................................................................................................... 121
14.1.5.2 Molecular Techniques............................................................................................................................ 122
14.2 Methods............................................................................................................................................................................ 123
14.2.2.1 Identification of Fungi Causing Black-Grain Mycetoma by PCR-RFLP.............................................. 123
14.2.2.2 Identification of Fungi Causing Black-Grain Mycetoma by Sequencing.............................................. 123
14.2.2.3 Identification of Madurella mycetomatis by PCR................................................................................. 124
14.2.2.4 Molecular Typing of Madurella mycetomatis....................................................................................... 124
14.3 Conclusions and Future Perspectives............................................................................................................................... 125
References.................................................................................................................................................................................. 126
14.1 Introduction species, as summarized in Table 14.1. In addition to M. myce-

tomatis and M. grisea, a wide range of closely related agents
14.1.1 Classification are able to cause eumycetoma. Those have presumptively
The generic criteria for Madurella are primarily based on tis- been referred to as Madurella spp. [4–6].
sue morphology and overall sterility on mycological media,
as well as an invasive potential in human and animal hosts. 14.1.1.1 M adurella mycetomatis
The genus is composed of two formally described species, (Laveran) Brumpt, 1905
namely, Madurella mycetomatis and Madurella grisea, plus M. mycetomatis has been known under many names. The
a dozen poorly characterized species collectively named species was first described in 1902 by Laveran and was
Madurella spp. [1]. M. mycetomatis is the generic type spe- named Strepthothrix mycetomi [3,7]. In 1905, Brumpt placed
cies. All Madurella species were isolated from cases of the organism in the genus Madurella and named it Madurella
black-grain mycetoma [2]. Mycetoma is a chronic inflam- mycetomi [3]. This name was used for more than seven
matory disease, which remains localized, involves cutane- decades until it was corrected to M. mycetomatis in 1977 by
ous and subcutaneous tissues, the fascia, and bones [3]. The the British Medical Research Council [3].
disease is characterized by tumefaction, discharging sinuses, M. mycetomatis produces slow-growing colonies. These
and the presence of fungal grains [3]. colonies are whitish and woolly at first, becoming oliva-
From recent molecular phylogeny studies, it appeared ceous, yellow, or brown, generally producing a brownish,
that M. mycetomatis and M. grisea, although both placed diffusing pigment also known as pyomelanin [2,3,8]. Next
in the genus Madurella, do not share common ancestors [4]. to pyomelanin, M. mycetomatis is also able to form another
Based on ribosomal sequences, M. mycetomatis has been type of melanin, namely, 1,8-dihydroxynaphthalene (DHN)-
repositioned in the order Sordariales, whereas M. grisea is melanin, which is responsible for the black color of the
now an acknowledged member of the order Pleosporales [4]. grain [8]. Although sporulation has not been systematically
This explains the many differences found between the two observed, two types of conidiation are known to exist in vitro.
117

TABLE 14.1
Characteristics of Madurella mycetomatis and Madurella grisea
Species Madurella mycetomatis Madurella grisea
Order Sordariales Pleosporales
Grain Black Black
Cement material throughout grain Cement material on the outer edge of grain
Optimal growth temperature 37°C 30°C
Pyomelanin Present Absent
Conidia Uncommon Uncommon
Pyriform or spherical shaped Chlamyospores or pycnidia
Sugar assimilation Lactose (+) Lactose (−)
Sucrose (−) Sucrose (+)
Epidemiology Africa America
Abbott described oval or pyriform conidia of 3.5–5.0 μm in caspofungin, anidulafungin, and micafungin, with MIC90s of
diameter with truncate bases when he cultured M. myceto- >64, 128, >128, and >128 μg/mL, respectively [14,16].
matis on soil-extract agar [4,9]. These conidia were produced
on simple or branched conidiophores [2,4,9]. Chalmers and 14.1.1.2 M adurella grisea Mackinnon,
Archibald also described this type of sporulation in 1916 [9]. Ferrade et Montemayer, 1949
Another type of sporulation occurs on potato-carrot agar or M. grisea was first described as a new species in 1949 by
cornmeal agar. The spores are small, spherical, and produced Mackinnon [18]. This species belongs to the order Pleosporales
on tapered tips of flask-shaped phialides [9,10]. and hence is phylogenetically closer to Leptosphaeria spp.
M. mycetomatis has a distinctive character once present in than to M. mycetomatis [4]. Cultures of M. grisea are unable
the human body: it forms black grains. These grains can be to grow at 37°C and grow best at 30°C. M. grisea forms
firm and brittle, and the shape is usually globose, oval, or lob- slow-growing gray or olive-colored colonies [9]. In contrast
ulated [9]. Grains are usually 0.5–1 mm in size and are com- to M. mycetomatis, M. grisea does not secrete a pigment in
posed of light brown hyphae. Terminal cells at the margin of the culture medium. The hyphae are brown walled, septate,
the grains reach 12–15 μm in diameter [9]. The hyphal cells and 1–3 μm in diameter [9]. Isolates are usually sterile but
in the center of the grain are embedded in cement material, can form chlamydospores occasionally [9]. When grown on
a brown matrix supposedly consisting of debris of both fun- low-nutrient media, some isolates of M. grisea produce pyc-
gal and host cells [9,11]. When stained with hematoxylin and nidia with pycnoconidia [3,9]. The pycnidia resemble those
eosin, these grains appear uniformly rusty brown [9]. There found in Pyrenochaeta mackinnonii and P. romeroi, which
is only one exception, in the vesicular type of grain the center are also members of the order Pleosporales [3,4,9]. Since
is light colored and the hyphae in the periphery brown [9,11]. M. grisea is usually thought of as being sterile, most diagnos-
The grains of the latter type resemble those of M. grisea [9]. tic laboratories do not attempt to induce pycnidial formation
In the past it was difficult to determine the antifungal in isolates of M. grisea. Therefore, some P. mackinnonii or
susceptibility of M. mycetomatis toward common antifun- P. romeroi isolates may have been misidentified as M. grisea
gal agents. According to the CLSI M38A reference method [3]. Molecular studies demonstrated that indeed many fun-
for antifungal susceptibility testing of filamentous fungi, as gal mycetoma isolates have been misidentified as M. grisea
starting material a conidial suspension has to be used [12]. In [4–6]. Like M. mycetomatis, M. grisea forms black grains in
the case of M. mycetomatis this is not feasible, since conidia the human body. They resemble the vesicular type of grain
are only rarely obtained. Therefore, antifungal susceptibili- of M. mycetomatis. The size of the grain is up to 1 mm. The
ties were determined by using hyphal fragments as starting grain is soft at first but will become hard and brittle upon
material [13–16]. In vitro, M. mycetomatis appeared to be maturation [19]. The brown cement material is limited to
the most susceptible to the azoles ketoconazole, itracon- the outer edge of the grain. The center appears to be hollow
azole, and voriconazole, with MIC90s of 0.125, 0.064, and in histological sections and is composed of a loose, hyaline
0.125 μg/mL, respectively [14,17]. Fluconazole was the least mycelial network of small hyphae (1–3 μm) [19]. In a couple
effective azole, only inhibiting the fungal growth at a MIC90 of cases, M. grisea was found to grow in tissue without orga-
of 16 μg/mL. Amphotericin B appeared to be less effec- nization into grains [9].
tive than ketoconazole, itraconazole, and voriconazole in For M. grisea very limited in vitro antifungal susceptibil-
inhibiting M. mycetomatis growth (MIC90 of 2 μg/mL) [14]. ity data are available, and only a small number of strains of
Interestingly, M. mycetomatis was also very susceptible to antifungal studies have been published. M. grisea appeared
tea tree oil, with an MIC90 of 0.25% v/v [13]. M. mycetomatis to be less susceptible to the azoles than M. mycetomatis,
isolates appear to be intrinsically resistant to 5-flucytosine, with MIC90s of 5 μg/mL for ketoconazole, of 2.5 μg/mL for

Madurella 119
itraconazole and econazole, and of 10 μg/mL for micon- mycetoma [2]. Among these, both bacteria and fungi are
azole [17]. Only for voriconazole a lower MIC was obtained, found. The most prevalent causative agent of black-grain
namely, of 0.5 μg/mL [20]. The MIC for amphotericin B was mycetoma worldwide, and in Africa in particular, is
0.25 μg/mL [20]. Madurella mycetomatis [3]. As can be seen in Figure 14.1, M.
mycetomatis causes more than 70% of all mycetoma infec-
14.1.1.3 Madurella Species tions in some parts of central Africa, including the Sudan
Next to Madurella grisea and Madurella mycetomatis, [22,23]. M. grisea is a less common causative agent and has
about a dozen unidentified black-grain mycetoma causative been found primarily in the Americas (Figure 14.1 and Refs.
agents have been included in the genus Madurella and given [9,24]). Of the 40 cases reported worldwide, nearly 80% orig-
a number [4–6]. In older literature, numerous species have inated from South America [9].
been described, but type materials are not known to exist for Anyone living in an endemic area could become infected
most of them. Not much is known about these fungi, except with Madurella spp. Overall, however, mycetoma is more
that they were described as causative agents of black-grain common in herdsmen, farmers, and other field laborers who
mycetoma. De Hoog et al. [21] listed numerous species as are in frequent and direct contact with the natural environ-
being of doubtful identity. The more recently introduced ment. Males are five times more often affected by myce-
Madurella species which belong to the order Pleosporales toma than females, even in areas where both sexes spend a
could be grouped in three genetically distinct clades [6]. The lot of time outdoors [2,3,25]. Mycetoma is seen in all age
Madurella-like species which belong to the order Sordariales groups, but it usually affects adults between 20 and 40 years
are more heterogeneously distributed and are scattered within old [26]. The infection is not considered to be transmissible
the order. Further characterization of these new Madurella from person to person or from animal to human. In general,
species is needed to properly identify these agents and eluci- mycetoma patients are considered to have a functioning
date their phylogeny. immune system, although there are some reports which show
impairment of the cell-mediated immune (CMI) response in
patients severely infected or not responding to medical treat-
14.1.2 Epidemiology and Risk Factors
ment [27]. This finding was supported by animal studies,
In addition to M. mycetomatis and M. grisea, approxi- since mycetoma was more successfully induced in athymic
mately 50 species of microorganisms are capable of causing mice than in immunocompetent mice [28]. In another report,
(A)
America Africa Saudi Arabia India

Mexico Brazil Argentina Senegal Niger Sudan Somalia
(B)
FIGURE 14.1 Mycetoma endemic areas. These areas include both actinomycetoma and eumycetoma regions. Overall, actinomycetoma
is more often found in Middle and South America. Eumycetoma is more commonly found in Africa. (A) Dark gray colored countries are
countries with common and frequent incidence. Lighter colored areas are countries with regular to moderately common incidence. (B) The
incidence of Madurella mycetomatis (black) and Madurella grisea (gray) in a selection of countries. Mycetoma due to other organisms (both
bacteria and fungi) are colored white. The graphs are based on data obtained from Refs. [19,74–82].

some impairment in the innate immune response was noted by M. mycetomatis or M. grisea are black in nature [2]. After
[29]. But overall, still no convincing evidence exists that discharge these sinuses can close transiently. Fresh adjacent
mycetoma patients have particular immune defects. sinuses may open, whereas some of the older ones may heal
completely. The nodules are connected to each other through
deep abscesses, and to the skin surface [2,22,25]. With time,
14.1.3 Clinical Presentation
the mycetoma granuloma will increase in size (Figure 14.2).
Mycetoma is primarily a subcutaneous disease. Since both The skin of the lesion will be stretched and become smooth
M. mycetomatis and M. grisea are assumed to be soil inhab- and shiny. Areas of hypo- or hyper-pigmentation may develop
itants [30], it is believed that the etiological agent is intro- [32]. Increased local hyperhydrosis has also been seen in
duced into the subcutaneous tissue by a minor trauma, such some patients [25,33]. When mycetoma increases in size, it
as a thorn prick [26]. That is why it is not surprising that will also grow into the deeper tissues; muscles and bones will
most mycetoma lesions are usually seen in the feet (70%) be invaded [3]. For unknown reasons, the tendons and the
(Figure 14.2). Other common sites are the hands (12%), legs, nerves are spared until very late in the disease process [32].
and knee joints [3,9,25]. In highly endemic areas, other parts Even this late in the disease, the blood supply in the myce-
of the body might become affected as well. After the incuba- toma area remains adequate and in many cases it is increased
tion period, which can be several months or years, a small [34]. Although mycetoma usually remains a localized infec-
nodule will arise [3]. On the foot it can be either on the dor- tion, some reports of lymphatic or bloodstream spreading
sum or plantar surfaces [9]. The subcutaneous nodule is usu- have appeared in literature. Secondary nodules can arise in
ally a few centimeters in diameter, firm and rounded, but it the affected area, sometimes with more distant lymphatic
can also be soft and lobular. It is rarely cystic [31] and is metastases in some advanced cases [32].
often movable. The overlying skin appears normal and may
not be attached to the mass early in the course of infection.
14.1.4 Disease Management and Antifungal Therapy
The nodule is usually painless in nature, and rarely medical
advice is sought [26]. Over the ensuing months, the disease Disease management for mycetoma is always necessary since
will progress. The nodule will start to grow, and multiple spontaneous cure has never been reported [2]. As mycetoma
sinus tracts will be formed. During the active phase of the can be caused by either bacteria or fungi, the first step in
disease, the sinuses will discharge a serous, serosanguinous, proper management of mycetoma is to identify whether the
or purulent discharge containing fungal grains [3,26]. The causative agent is a bacterium or a fungus. In actinomycetoma,
color of the grains is dependent on the causative agent and chemotherapy alone results in high cure rates (60%–90%),
can give an indication of its identity [2,3]. Grains produced and surgery is only required in advanced cases and those
refractory to antimicrobial treatment [35]. Unfortunately, for
eumycetoma, the prospects are not as good. No fully effec-
tive antifungal agent has been discovered yet. For eumyce-
toma, surgery is always needed [35,36]. The aim of surgery
(C) is complete excision of the lesion. This is only possible when
the mycetoma lesion is small and well-encapsulated. In larger
lesions, only reduction of the amount of infected tissue is
possible, and occasionally multiple surgeries are needed to
(A) (D) excise most of the lesion [33,37]. Especially in large lesions,
effective surgery needs aggressive excision or debridement
under general anesthesia, which usually cripples the limb
or leads to permanent disability due to mandatory amputa-
(E) tion [37]. To prevent recurrent infections, surgery is always
combined with antifungal treatment, both before and after
[3,33,37]. Antifungal therapy for eumycetoma still depends
mainly on the azole antifungals ketoconazole and itracon-
azole. Amphotericin B is generally considered ineffective
(B) (F) [3,38,39]. Liposomal amphotericin B had somewhat better
in vivo efficacy, since treatment with liposomal amphotericin
FIGURE 14.2 Clinical presentation of mycetomaa. (A) An early B led initially to remission, but relapse occurred within 6
case of mycetoma of the foot. (B) An advanced case of mycetoma months after the end of therapy [40]. The azole class of anti-
of the foot. (C) An x-ray showing a lateral view of the elbow joint
fungal agents appeared to be more suitable to treat mycetoma.
with multiple big bone cavities with well-defined margins charac-
teristic of eumycetoma. (D) Histological slide of Madurella myce-
The initial trials with ketoconazole and itraconazole showed
tomatis surrounded by inflammatory cellular reaction (type 1 tissue a clear progress in healing of the lesions [41]. In contrast,
reaction), H&E, 40 times enlarged. (E) Macroscopic appearance of when fluconazole was used to treat mycetoma caused by M.
M. mycetomatis in a surgical specimen. Grains are surrounded by mycetomatis or M. grisea, only temporary improvement was
intense fibrous capsules. (F) Culture of Madurella mycetomatis. achieved with quick relapses [3,42]. In one case of M. grisea

Madurella 121
mycetoma, itraconazole also yielded great improvement [41]. diagnosis [11]. The color of the grain can give a first impres-
The results obtained with itraconazole in mycetoma caused sion of the causative agent but it cannot identify the causative
by M. mycetomatis were more variable. According to Hay, agent in full detail. For example, not only Madurella spp.
itraconazole resulted in improvement of 42% of the M. myce- form black grains in mycetoma lesions, but at least 14 other
tomatis mycetoma cases and in no response in 33% of the fungal species including L. senegalensis and P. romeroi are
cases [40,43]. For both M. mycetomatis and M. grisea, favor- known to cause black-grain mycetoma [2,9]. Therefore, for
able results have been reported with ketoconazole [44,45]. species identification, high-quality diagnostic methods have
Long-term ketoconazole therapy is used in the Mycetoma to be used, as have already been implemented in endemic
Research Centre. Hay reported that in 50 patients seen in the areas. These include radiology, histology, serology, and cul-
Mycetoma Research Centre, 72% showed clinical improve- ture techniques. Furthermore, new molecular diagnostic
ment or were even cured with ketoconazole, while only 8% tools have been developed to identify the mycetoma etiologic
did not respond at all [3,40]. Also Venugopal and Venugopal agents to the species level.
noticed significant therapeutic efficacy of ketoconazole [45].
Currently, although showing varying degrees of clinical effi- 14.1.5.1 Conventional Techniques
cacy, itraconazole and ketoconazole are still the best treat- 14.1.5.1.1 Imaging Methodology
ment options [33,38,46,47]. Within the Mycetoma Research To assess the extent of the mycetoma, imaging techniques
Centre (Khartoum, Sudan; http://www.mycetoma.edu.sd), can be useful. With classic radiology, multiple radiological
both ketoconazole (400–800 mg daily) and itraconazole changes are found. It can show the presence and extent of
(400 mg daily) are recommended for first-line use [26,32]. soft tissue granulomas and demonstrates when the bone is
The duration of treatment varies with the severity of the penetrated by the causative agent [32]. Magnetic resonance
infection and the general health status of individual patients. imaging (MRI) is also useful in visualization of soft tissue
Treatment may need to be continued for 18–24 months or involvement and bone destruction [32,51,52]. On MRI, grains
more, and the liver function of the patients needs regular appear as conglomerates of small (2–5 mm), round hyperin-
monitoring, especially with long-term ketoconazole treat- tense lesions, representing the granulation tissue, surrounded
ment in cases of advanced mycetoma [33,37]. However, long- by a low-signal-intensity rim [52]. The central low-signal-
term treatment may lead to antifungal resistance, which may intensity dot is caused by the grain [52]. This unique appear-
in turn complicate patient management. Both ketoconazole ance was named “dot-in-circle” and is highly suggestive of
and itraconazole are implicated in encapsulating the myce- mycetoma [52,53]. Still these radiological techniques are
toma lesion. This renders final surgical treatment a likely not always specific and cannot always differentiate myce-
option. But in advanced lesions, especially when bone tis- toma from chronic bacterial osteomyelitis, granulomas, bone
sue is involved, the response to chemotherapeutic treatment tuberculosis, and soft tissue tumors [32]. Furthermore, in
still remains very poor [48]. In the search for new, alterna- most of the endemic regions, MRI equipment is not available.
tive antifungal strategies in the treatment of mycetoma, A more specific imaging technique for mycetoma is ultra-
terbinafine has been tried in the treatment of eumycetoma. sonic imaging [54]. The mycetoma grains, its capsule, and
Twenty five percent of patients were cured and 55% showed the accompanying inflammatory granulomata have charac-
clinical improvement [49]. Recently, the effect of posacon- teristic ultrasonic appearances [54]. Ultrasound imaging can
azole was evaluated on six eumycetoma cases, of which differentiate between mycetoma and other non-mycetoma
two were caused by M. mycetomatis and three by M. grisea lesions and even between eumycetoma and actinomycetoma
[50]. In all the M. grisea cases, posaconazole resulted in a [32,54]. In eumycetoma lesions, the grains produce numer-
successful outcome with various symptomatic improve- ous sharp bright hyper-reflective echoes, which correspond
ments (lesion diameter decreased, sinuses closed, secretions to the black grains [32].
stopped, pain and swelling resolved, and motility increased)
[50]. Resolution of bone abnormalities was also observed in 14.1.5.1.2 Histology and Cytology
osteomyelitic mycetomas, suggesting that posaconazole may As stated before, the mycetoma grain is a first key feature
penetrate this tissue after oral administration [50]. The out- for the identification of the causative agent. In order to study
come of M. mycetomatis mycetoma was variable, in one case the grain more closely in the tissue, histological examination
a complete success was noted, in the other case no clinical is used. In stained sections, the grain of M. mycetomatis is
improvement was noted [50]. rounded, oval, or trilobed [9,11]. It has a compact brown cor-
tex and a lighter medulla. In some grains, the division into
cortex and medulla is not evident. The grain filaments are
14.1.5 Diagnosis
usually embedded in a hard brown cement matrix [11].
As is clear from the above section, early diagnosis may In M. mycetomatis infection, two main morphological
improve therapeutic outcome. In endemic areas, mycetoma types of grains are identified: the filamentous and the vesic-
must be considered in the differential diagnosis of all sub- ular type [9,11]. The filamentous type is the most common
cutaneous swellings. Grains present in the discharge of type and consists of brown septate and branched hyphae that
the sinuses or in biopsies taken from lesions are important may be slightly more swollen toward the periphery [11]. In
for diagnosis. Biopsies without grains are not suitable for the cortex, the filaments are arranged radially, while in the

medulla they tend to run multidirectionally [11]. Rounded or has been prepared for M. mycetomatis, none for M. grisea.
oval cells, 7–15 μm in diameter are seen, in particular in the Unfortunately the recombinant antigen, the translationally
periphery [11]. The vesicular type is composed of unusually controlled tumor protein of M. mycetomatis was not useful
large cells which look like vesicles. Both types of grain can for diagnostic purposes, since not all patients had produced
be found in the same lesion. antibodies against this protein, while in contrast some of the
The inflammation reaction around the grain is variable. healthy controls did [63]. Given the experimental ease of the
Three types of tissue reaction can be seen. In type I, there ELISA test, additional investigation in novel recombinant
is a zone of neutrophils in the vicinity of the grain [11,55]. antigens is highly recommended.
Some histocytes may also be seen among the neutrophils but
they are more numerous outside the neutrophil zone [11,55]. 14.1.5.1.4 Culture
In type II, the neutrophil zone is absent, instead a layer of his- For successful culture, a deep-seated surgical biopsy speci-
tocytes and multinucleated giant cells is seen. Some of these men is preferred to collect grains discharged with pus through
giant cells contain fragments of grain or pigmented cement the sinuses [64]. The specimen obtained is divided into two
substance [11]. At this stage, the grain is usually small and portions. One portion is used for direct microscopy, the other
fragmented. The most uncommon reaction is the type III for culture. Direct microscopy can be performed by exami-
reaction in which the grain material has largely or completely nation of crushed grains in 10% KOH [58]. Black grains
disappeared. This leaves a compact epithelioid granuloma usually are composed of hyphae of approximately 15 μm in
with or without Langerhans giant cells [11]. width. For culture, grains are washed in saline containing
Another technique in use to diagnose mycetoma is fine- chloramphenicol, grown on blood agar and Sabouraud dex-
needle aspiration cytology (FNAC). In FNAC mycetoma trose agar and incubated at 37°C and 26°C for 6–8 weeks
lesions have a distinct appearance, characterized by the pres- [58]. Identification of isolates is achieved by observation
ence of polymorphous inflammatory cells and grains [56]. In of the rate of growth, colony morphology, production of
the smear, grains are surrounded and infiltrated by neutro- conidia, and assimilation patterns [58]. Culturing these fungi
phils. Outside the neutrophil zone, monocytic cells and giant remains troublesome, often no growth is obtained or cultures
cells are seen. They are surrounded by granulation tissue rich are contaminated with bacteria. Identification of the various
in fibroblasts and blood vessels [56]. FNAC allows morpho- fungi responsible for black-grain mycetoma remains difficult
logic identification of mycetoma and its classification into with standard mycological procedures since these fungi only
eumycetoma and actinomycetoma, in a comparable manner rarely produce conidia [65,66]. Misidentification, therefore,
as with histological slides [56,57]. often occurs. Furthermore, most of these fungi have low
growth rates which may delay the identification until up to
14.1.5.1.3 Serology 12 weeks [21].
Serological assays can be used to diagnose mycetoma.
Serological assays most frequently used in the endemic areas 14.1.5.2 Molecular Techniques
are immunodiffusion (ID) and counter-immuno-electropho- 14.1.5.2.1 Species Identification
resis (CIE), although enzyme-linked immunosorbent assays In order to identify the causative agents of black-grain
(ELISAs) have been developed as well. In the past, ID was mycetoma more accurately, molecular diagnostic tools have
a popular test to diagnose mycetoma but since then CIE been developed. The identification of black-grain mycetoma
was found to be quicker and more sensitive than ID [58,59]. agents, including M. mycetomatis, is based on the internal
The ELISA used by McLaren et al. and by Taha showed transcribed spacer (ITS) sequences [5,67]. The ITS region
that both mycetoma patients and uninfected controls liv- can be either amplified with pan-fungal primers or with
ing in the endemic areas had antibodies against mycetoma M. mycetomatis-specific primers. The pan-fungal prim-
agents [60–62]. This makes these ELISAs less suitable for ers are located at the 18S ribosomal sequence and the 28S
diagnostic purposes. Therefore, in the endemic areas, only ribosomal sequence. The resulting PCR product is therefore
CIE is still used in the diagnosis of mycetoma. In addi- composed of part of the 18S ribosomal sequence, the ITS 1
tion to its diagnostic value, CIE has been used to monitor region, the 5.8S ribosomal sequence, the ITS 2 region, and
therapeutic outcome in patients. When there is a response part of the 28S ribosomal sequence. The M. mycetomatis-
to treatment, both intensity and number of precipitation specific primers are located on the ITS 1 and ITS 2 regions.
bands decrease and eventually disappear [59]. One of the The resulting PCR products can be analyzed with either a
large drawbacks of all serological techniques used to diag- restriction fragment length polymorphism (RFLP) digestion
nose mycetoma is that they rely on crude antigens prepared [67] or by sequencing [5]. The latter results in a more precise
in diagnostic laboratories in the endemic areas. The anti- identification. The M. mycetomatis-specific PCR could not
gens are prepared by grinding the mycelia in physiological only be used in diagnosis, but it was even possible to detect
saline, disintegrate them ultrasonically, and collect the cyto- M. mycetomatis DNA in soil and thorn-samples [68].
plasmatic proteins by centrifugation [58,59,62]. This rough
production of antigens may result in variation between tests. 14.1.5.2.2 Molecular Typing
A serological assay based on a recombinant antigen would Three different molecular techniques have also been used so
be preferred. Currently only a single recombinant antigen far to type individual strains of M. mycetomatis. The first

Madurella 123
technique was a restriction endonuclease assay (REA) [69]. frozen in liquid nitrogen, and ground in a porcelain
Lopes et al. performed REA on 17 isolates of M. myceto- mortar.
matis obtained from nine different countries and found two 2. DNA can be isolated from the resulting pulp with
larger clusters and seven unique genotypes [69]. The two any DNA isolation protocol. In our lab we have the
larger clusters contained isolates from Africa, the remain- best results with the Promega Wizard Kit (Promega).
ing genotypes mainly originated from other continents [69]. When using this protocol, 300 μL nuclei lysis solu-
The second technique used was random amplification of tion (Promega Wizard) is added to the ground
polymorphic DNA (RAPD). This method has been used by mycelia, and the solution is mixed by pipetting gen-
two research groups, with different outcomes. The RAPD tly. From this step onward, the yeast protocol from
performed on the 17 isolates isolated by Lopes et al. ren- the Promega Wizard protocol is used according to
dered nine different genotypes [69]. In agreement with the the manufacturer’s instructions.
REA data generated by Lopes, two larger clusters and seven
individual genotypes were again found [69]. RAPD was also
performed by Ahmed et al. [70]. In contrast to Lopes et al., 14.2.2 Detection Procedures
they did not find genetic variation when typing 38 M. myce- 14.2.2.1 Identification of Fungi Causing
tomatis isolates from Sudan, and two from Mali [70]. The
Black-Grain Mycetoma by PCR-RFLP
discrepancy between the study performed by Lopes et al. and
the one performed by Ahmed et al. could be due to multiple The molecular identification of black-grain mycetoma agents
factors. First of all, different primer sets and PCR conditions is based on the analysis of the ITS region [5,67]. Ahmed et al.
were used for both RAPD studies. Furthermore, Lopes et al. developed an RFLP method to differentiate between the vari-
[69] used a worldwide collection of strains, whereas Ahmed ous black-grain mycetoma agents [67].
et al. primarily used strains from Sudan [70]. van de Sande Procedure
et al. later added a third typing method, namely, amplified
fragment length polymorphism (AFLP) [71] analysis. AFLP 1. Prepare a PCR mixture (100 μL) containing 50 ng
proved to be more discriminatory than RAPD, and it sepa- DNA, 1× Supertaq PCR buffer 1 (HT Biotechnology,
rated the Sudanese isolates in two large and one minor cluster Cambridge, U.K.), 0.2 mM nucleotide mix, 25 pmol
[71]. In cluster I mainly isolates obtained from large lesions primer ITS 4 (5′-TCCTCCGCTTATTGATATGC-3′)
from patients living in Central Sudan were found, while the and 25 pmol primer ITS 5 (5′-GGAAGTAAA
strains from cluster II were of more heterogeneous origin AGTCGTAACAAGG-3′), and 1.2 U Supertaq (HT
[71]; Cluster III consisted only of one strain and was there- Biotechnology).
fore not analyzed further. 2. Perform the PCR amplification in a thermocycler
In contrast to M. mycetomatis, no typing studies have using a cycling program consisting of a denaturation
been performed for M. grisea or the various other Madurella step at 94°C for 4–10 min; 30 cycles of 94°C for 30 s,
spp. 58°C for 30 s, and 72°C for 30 s; and a final exten-
sion step at 72°C for 10 min [5].
14.2 Methods 3. Prepare an RFLP mixture (25 μL) containing 15 μL
PCR product, 2 U of either CfoI, MspI, HaeIII, RsaI,
14.2.1 Sample Preparation Sau3A, or SpeI, and 1× corresponding restriction
Different DNA extraction methods have been used to iso- buffer (dependent on the enzyme used and the man-
late DNA from M. mycetomatis [5,67,68,72]. Usually DNA is ufacturer). Incubate overnight at the temperature
isolated from fungi grown on Sabouraud dextrose agar, but indicated by the manufacturer.
DNA can be isolated from grains as well. For direct isolation 4. Analyze RFLP banding patterns on 3% Nusieve
of DNA from grains, the grain is frozen in liquid nitrogen GTG agarose gels. Most of the black-grain isolates
and then crushed (Abdalla Ahmed, personal communica- are identifiable by their banding patterns [67].
tion). Afterward, different DNA isolation protocols can be
used, including commercial kits. Higher DNA concentra- Note: For most of the black-grain-producing isolates, the
tions are obtained when DNA is isolated from strains grown length of the ITS region is similar between strains of a given
in vitro. species, except for some Pyrenochaeta spp. and Madurella
grisea isolates. In these isolates, the ITS sequences range
Procedure
from 951 to 1327 bp [5]. For M. mycetomatis, the length of
the ITS region is 624 bp.
1. Transfer fungal grain onto Sabouraud dextrose agar
(Difco) and culture at 37°C in case of M. myceto-
matis and at 25°C in case of M. grisea. For identi- 14.2.2.2 Identification of Fungi Causing
fication purposes, it is wise to split the sample to Black-Grain Mycetoma by Sequencing
two agar plates and culture at both temperatures. In order to analyze the ITS region more accurately, many
After 4 weeks, the colony is excised from the agar, scientists have chosen to sequence this region [4,5,67].

Procedure 2. Perform the PCR amplification in a thermocycler

using a cycling program with 40 cycles of 94°C for
1. Prepare a PCR mixture (50 μL) containing 50 ng 1 min, 58°C for 2 min, and 72°C for 1 min [67].
DNA, 1× Supertaq PCR buffer 1 (HT Biotechnology), 3. Separate the PCR products on a 1% agarose gel and
0.2 mM nucleotide mix, 25 pmol primer V9D stain with ethidium bromide.
(5′-TTAAGTCCCTGCCCTTTGTA-3′) and 25 pmol
primer LS266 (5′-GTAGTCATATGCTTGTCTC-3′), Note: With this M. mycetomatis-specific PCR, it is also
and 1.2 U Supertaq (HT Biotechnology). possible to detect M. mycetomatis DNA in soil and thorn-
2. Perform the PCR amplification in a thermocycler samples [68].
using a cycling program consisting of a denaturation
step at 94°C for 4–10 min; 30 cycles of 94°C for 30 s, 14.2.2.4 Molecular Typing of Madurella mycetomatis
58°C for 30 s, and 72°C for 30 s; and a final exten- 14.2.2.4.1 REA
sion step at 72°C for 10 min [5]. Lopes et al. developed an REA for the typing of M. myce-
3. Prepare a sequencing mixture (11 μL) containing tomatis. In this technique, DNA is digested with restriction
40 ng PCR product, 1.5 μL sequencing buffer (Applied endonucleases, and the resulting banding patterns are used to
Biosystems, Cheshire, U.K.), 1 μL sequencing reac- differentiate the various genotypes [69].
tion mix (Applied Biosystems), and 5 pmol primer
ITS 4 (5′-TCCTCCGCTTATTGATATGC-3′). For Procedure
the reverse reaction, replace primer ITS 4 by primer
ITS 5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). 1. Prepare a restriction endonuclease mixture (20 μL)
4. Perform the sequencing reaction in a thermocycler containing 100 ng of DNA, 2 U of ScaI (New
using a sequencing program consisting of 25 cycles England Biolabs), 1× NEBuffer 3 (New England
of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Biolabs), and 5 μg of M. mycetomatis DNA.
5. Precipitate the resulting product by adding 80 μL Incubate for 3 h at 37°C according to the manufac-
precipitation mix consisting of 0.11 M NaAc pH turer’s instructions [69].
4.8 (Sigma Aldrich) and 75% ethanol, incubate for 2. Electrophorese the restriction digest on a 0.7% aga-
20 min at room temperature, and centrifuge for rose gel at 2 V/cm for 16 h and visualize the banding
30 min at 2500 × g at room temperature. patterns with ethidium bromide stain [69].
6. Wash the pellet with 150 μL 70% ethanol.
7. Add 20 μL HiDi formamide (Applied Biosystems) Note: Next to performing an REA with ScaI, other restric-
to each sample and sequence the product with the tion endonucleases can be used as well. These include EcoRI,
ABI Prism 3700 automated DNA analyzer (Applied EcoRV, HindIII, and HpaI. The results obtained with ScaI
Biosystems). are the most encouraging, since all 17 isolates typed by Lopes
et al. give distinct, intense, and well-defined bands than those
with the other restriction endonucleases [69].
Note: By using these pan-fungal ITS primers, most of the
black-grain mycetoma isolates can be amplified [5,67]. Only 14.2.2.4.2 RAPD
for four new Madurella species, the complete ITS region
In the RAPD technique, usually a PCR is performed with
could not be amplified with primer pairs V9D/LS266 and
one short primer at a low annealing temperature which will
ITS 4/ITS 5. Identification of these four isolates was based
result in a banding pattern. Two different RAPD protocols
on the amplification of the ITS 2 region with primers ITS 3
have been used to type M. mycetomatis [69,70]. Since the
(5′-GCATCGATGAAGAACGCAGC-3′) and ITS 4 [5].
RAPD protocol of Lopes et al. [69] results in different geno-
types, it is presented here.
14.2.2.3 Identification of Madurella
mycetomatis by PCR Procedure
Ahmed et al. developed a M. mycetomatis species-specific
PCR, based on the ITS region. 1. Prepare an RAPD PCR mixture (50 μL) containing
25 ng isolated DNA, 0.1 mM dNTPs, 0.2 μM primer,
Procedure 2.5 U Taq polymerase (HT Biotechnology), 50 mM
KCl, 10 mM Tris–HCl (pH8.0), and 1.5 mM MgCl2.
1. Prepare a PCR mixture (50 μL) containing As primer, either one of the following five primers
50 ng DNA, 1 × Supertaq PCR buffer 1 (HT can be used: 28289 (5′-ATGGATCCGC-3′), 28290
Biotechnology), 0.2 mM nucleotide mix, 25 pmol (5′-AAGAGCCCGT-3′), 28308 (5′-AACGCG
primer 26.1A (5′-AATGAGTTGGGCTTTAAC CAAC-3′), 28309 (5′-GTTTCCGCCC-3′), and
GG-3′) and 25 pmol primer 28.3A (5′-TCCCGGT 28323 (5′-GCGATCCCCA-3′) [69].
AGTGTAGTGTCCCT-3′), and 1.2 U Supertaq (HT 2. Perform the RAPD in a thermocycler using a
Biotechnology). cycling program consisting of a denaturation step at

Madurella 125
94°C for 5 min; 45 cycles of 94°C for 1 min, 35°C for (5′-GACGATGAGTCCTGAGTAAAC-3′), 0.4 U
1 min, and 72°C for 2 min; and a final extension step Taq polymerase, 10 mM Tris–HCl pH 8.3, 1.5 mM
at 72°C for 5 min [69]. MgCl2, 50 mM KCl, and 0.2 mM dNTP mixture
3. Visualize the banding patterns on a 1.5% agarose [71]. As an alternative selective amplification mix-
gel with ethidium bromide strain [69]. ture, primer E12 can be replaced by primer E20.
8. Perform the selective amplification in a thermocy-
14.2.2.4.3 AFLP cler using a cycling program consisting of 36 cycles
van de Sande et al. developed an AFLP method for typing of 94°C for 30 s, an annealing temperature for 30 s,
M. mycetomatis isolates [71]. In this technique, DNA is digested and 72°C for 1 min. The annealing temperature in
with two restriction endonucleases after which the restriction the first cycle is 65°C, is subsequently reduced each
fragments are amplified selectively. The resulting banding pat- cycle by 0.7°C for the next 12 cycles, and is contin-
terns are used to differentiate the various genotypes. ued at 56°C for the remaining 23 cycles [73].
9. Following amplification, mix the reaction products
Procedure with an equal volume of formamide dye consisting
of 98% formamide, 10 mM EDTA pH 8.0, and bro-
1. Prepare a DNA digestion mixture (40 μL) contain- mophenol blue and xylene cyanol as tracking dyes.
ing 0.5 μg of DNA, 5 U EcoRI, 5 U MseI, 10 mM 10. Heat the resulting mixture at 90°C for 3 min and
Tris–HAc pH 7.5, 10 mM MgAc, 50 mM KAc, then cool on ice [73].
5 mM dithiothreitol (DTT), and 50 ng/μL BSA. 11. Load the samples on a 4.5% denaturing poly-
Incubate the mixture at 37°C for 1 h. acrylamide gel (4.5% acrylamide, 0.25% methy-
2. To ligate the adapters, add 10 μL of a solution lene bisacryl, 7.5 M urea in 50 mM Tris/50 mM
containing 5 pMol EcoRI adapters, 50 pMol MseI boric acid/1 mM EDTA, 0.05% APS, and 0.1%
adapters, 1 U T4 DNA-ligase, 1 mM ATP in 10 mM N,N,N′,N′-tetramethylethylenediamine [TEMED])
Tris–HAc pH 7.5, 10 mM MgAc, 50 mM KAc, 5 mM [73] and electrophorese for 2 h at 110 W in 100 mM
DTT, 50 ng/μL BSA. Incubate the reaction for 3 h at Tris/100 mM boric acid/2 mM EDTA [71,73].
37°C [73]. The structure of the EcoRI adapter is
5΄-CTCGTAGACTGCGTACC 14.3 C
onclusions and
CATCTGACGCATGGTTAA-5΄ Future Perspectives
The genus Madurella consists of many species of which only
The structure of the MseI adapter is M. mycetomatis and M. grisea are well-characterized. M. myce-
5΄-GACGATGAGTCCTGAG tomatis and M. grisea erroneously ended up in the same genus
based on the fact that they both cause black-grain mycetoma
TACTCAGGACTCAT-5΄ and that they form sterile cultures. Furthermore, in the past, and

based on these criteria many other fungi have been misidenti-
3. Prepare a pre-amplification mixture containing 30 ng fied as either M. mycetomatis or M. grisea. This demonstrates
of primer E (5′-GACTGCGTACCAATTC-3′), 30 ng that the identification of Madurella species has always been
of primer M (5′-GACGATGAGTCCTGAGTAA-3′), troublesome. Unfortunately, since the taxonomic description of
0.4 U Taq polymerase, 10 mM Tris–HCl pH 8.3, both species, little improvement has been made in the classical
1.5 mM MgCl2, 50 mM KCl, and 0.2 mM of dNTPs diagnosis of mycetoma. With imaging techniques, histopathol-
[73]. ogy, serology, and even culturing still many misidentifications
4. Perform pre-amplification in a thermocycler using a occur. Until this moment, no adequate point-of-care diagnostic
cycling program consisting of 20 cycles of 94°C for tools are available to accurately diagnose mycetoma.
30 s, 56°C for 30 s, and 72°C for 1 min [73]. The first real breakthrough in the differentiation of the
5. Dilute the pre-amplification mixture 10-fold. two Madurella species was made when molecular tools
6. Prepare an end-labeling mixture (50 μL) contain- were developed to identify these species. By characterizing
ing 500 ng EcoRI primer, 100 μCi [γ-33P] ATP, 10 U the ITS region of the Madurella species, M. mycetomatis
T4 polynucleotide kinase, 25 mM Tris–HCl pH 7.5, and M. grisea could be easily distinguished [5,6,67,68].
10 mM MgCl2, 5 mM DTT, 0.5 mM spermidine– No misidentification could occur, since both species differ
3HCl [73]. In the method developed by van de considerably in their respective ITS regions. In fact, char-
Sande et al., two different EcoRI primers were used, acterizing the ITS region revealed that both species belong
namely, E12 (5′-GACTGCGTACCAATTCAC-3′) to completely different fungal orders [4]. Another benefit
and E20 (5′-GACTGCGTACCAATTCGC-3′). of molecular diagnostic tools is that the duration of species
7. Prepare a selective amplification mixture identification can be shortened considerably. Since DNA can
(20 μL) containing 5 μL diluted pre-amplificated be isolated directly from fungal grains, species identification
product, 5 ng primer E12, 30 ng primer M12 can be obtained within 2–3 working days.

Next to identification of fungal species by molecular 13. van de Sande, W.W. et al., In vitro susceptibility of
methods, the development of molecular typing tools also Madurella mycetomatis, prime agent of Madura foot, to tea
allowed to differentiate strains within the species. For tree oil and artemisinin, J. Antimicrob. Chemother., 59, 553,
2007.
M. mycetomatis, three molecular typing methods were
14. van de Sande, W.W. et al., Testing of the in vitro sus-
developed: REA, RAPD, and AFLP [69,71]. AFLP proved ceptibilities of Madurella mycetomatis to six antifungal
to be the most discriminating method. Based on the results agents by using the sensititre system in comparison with a
obtained from these typing methods, it was established that viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-
the species M. mycetomatis, although quite clonal, can still [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)
be differentiated in various genotypes that can even be found assay and a modified NCCLS method. Antimicrob. Agents
within a single geographic region [71]. Chemother., 49, 1364, 2005.
The diagnostic molecular tools have gradually taken 15. Ahmed, A.O. et al., In vitro susceptibilities of Madurella
mycetomatis to itraconazole and amphotericin B assessed by
over the culture-based identification procedures for iden- a modified NCCLS method and a viability-based 2,3-Bis(2-
tification of black-grain mycetoma agents. Identification is methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-
now based on sequencing the ITS region of these fungi, 2H-tetrazolium hydroxide (XTT) assay. Antimicrob. Agents
and misidentifications are a phenomenon of the past. Chemother., 48, 2742, 2004.
Unfortunately, in the endemic regions, molecular diagnos- 16. van de Sande, W.W.J. et al., Madurella mycetomatis is not
tic tools have not been in use so far. Most of the endemic susceptible to the echinocandin class of antifungal agents.
countries are considered to be developing countries and Antimicrob. Agents Chemother., 54, 2738, 2010.
17. Venugopal, P.V. et al., Antimycotic susceptibility testing of
do not have the means to routinely implement these more
agents of black grain eumycetoma. J. Med. Vet. Mycol., 31,
reliable molecular tools in the frequently rural tropical set- 161, 1993.
tings. This situation may change if the expense of molec- 18. Mackinnon, J.E., New species of fungus pathogenic to man in
ular biological reagents and equipment comes down, and South America. Cienc. Invest., 7, 77, 1951.
alternative cost-effective molecular diagnostic procedures 19. Rippon, J.W., Mycetoma. In: Medical Mycology, the
are developed in future. Pathogenic Fungi and the Pathogenic Actinomycetes, 3rd edn.,
Chap. 5, pp. 80–84, W.B. Saunders Company, Philadelphia,
PA, 1988.
20. McGinnis, M.R. and Pasarell, L., In vitro testing of sus-
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15 Neodeightonia
Contents
15.1 Introduction...................................................................................................................................................................... 129
15.1.3 Diagnosis.............................................................................................................................................................. 130
15.2 Methods............................................................................................................................................................................ 132
15.3 Conclusion and Future Perspectives................................................................................................................................. 133
References.................................................................................................................................................................................. 133
15.1 Introduction Neodeightonia subglobosa is characterized by having

clavate, thick-walled bitunicate, eight-spored asci typical of
15.1.1 Classification, Morphology, and Biology the members of the Botryosphaeriaceae. Ascospores are dis-
The ascomycete genus Neodeightonia C. Booth belongs in tichous, the mature ones are brown, oval to broadly ellip-
the family Botryosphaeriaceae, order Botryosphaeriales. soidal, becoming one-septate, measuring 20–26 × 7–10 μm
Members of the family Botryosphaeriaceae are best known with a finely roughened surface. Its coelomycete anamorph
as pathogens, saprophytes, and endophytes in a wide range has spherical to subglobose conidia which are initially hya-
of plant hosts.1 Their occurrence and relevance as human line and become light to dark brown when mature, aseptate,
pathogens is low in comparison to other clinical fungi, and measuring 9–12 × 6–9 μm.8 In N. phoenicum conidia are
and only a few species such as Lasiodiplodia theobromae ovoid to ellipsoid, with the apex and base broadly rounded,
(Pat.) Griffon & Maubl.,2,3 Macrophomina phaseolina Tassi widest in middle to upper third, thick-walled, initially hya-
(Goid.),4,5 Neoscytalidium dimidiatum (Penz.) Crous & line and aseptate, becoming dark brown and one-septate
Slippers,4,6 and Neodeightonia subglobosa C. Booth7 have some time after discharge from pycnidia, with melanin
shown the potential to cause disease to man. deposits on the inner surface of wall arranged longitudinally
The genus Neodeightonia was introduced by Booth (in giving a striate appearance to conidia, and measure 18.6–
Punithalingam 1969)8 to accommodate a single species, 19.5 × 11.2–11.8 μm.11 The teleomorph of N. phoenicum has
namely N. subglobosa. von Arx & Müller (1975)9 within not been found. Detailed morphological descriptions as well
their broad concept of the genus transferred this species to as microphotographs and illustrations of the genus and spe-
Botryosphaeria. Recently, the taxonomy and systematics of cies can be found elsewhere.8,10,11
the genus Botryosphaeria sensu lato has been subjected to Neodeightonia subglobosa is a poorly studied species and
considerable revision.10,11 In their work on the dark-spored virtually nothing is known regarding its biology, geographic
telemorph genera in the Botryosphaeriaceae, Phillips et al. distribution, and ecology. Neodeightonia subglobosa (as
(2008)11 reinstated Neodeightonia on account of its phyloge- Sphaeropsis subglobosa) was first described from dead culms
netic and morphological distinction from other genera in the of Bambusa in Georgetown (Guyana) in 1879 and Belgium in
Botryosphaeriaceae. 1882. Both states (anamorph and telemorph) of this species
Thus, currently Neodeightonia is a very small genus com- were described in 1969 on dead culms of Bambusa arundi-
prising only two species, namely the type species N. sub- nacea in Sierra Leone. Since then the sexual state or teleo-
globosa and N. phoenicum A.J.L. Phillips & Crous.11 The morph was never found again and there are only two reports
last species was introduced by Phillips et al. (2008)11 so to published concerning this species. The first one deals with
accommodate isolates obtained from palms (Phoenix dac- its occurrence as the cause of keratomycosis on a man who
tylifera and Phoenix canariensis) and there is no evidence had poked a bamboo cane into his eye.7 The second and most
to date that it may incite any kind of clinical manifestation. recent one describes the occurrence of this species as an
129

endophyte on herbaceous medicinal plants from India.12 It is Coelomycetous fungi generally display a moderate to
not clear if this species is a plant pathogen or merely a sapro- rapid growth rate on a variety of routine culture media and
phyte that is able to grow on dead plant material. As occurs are not particularly difficult to recover from excised material.
with several plant pathogens in the Botryosphaeriaceae,1 The main problem lies in promoting sporulation in order to
it is likely that N. subglobosa is a plant pathogen that has obtain the diagnostic reproductive structures necessary for
an endophytic stage in its life cycle and that it is capable of identification of the isolates. Apart from the considerable
causing diseases when the host is under stress. However, this amount of time required, particularly for pycnidial species
assumption remains to be proven. (up to months in some strains), it is also necessary to employ
a medium upon which these pycnidia will develop.13
15.1.2 Clinical Features and Pathogenesis The culture of Coelomycetes on sterilized plant tissue is
deemed to produce conidiomata more representative of those
As mentioned earlier N. subglobosa is a poorly known spe- in nature. It is known that culture on nutrient-rich synthetic
cies and this applies also to the clinical environment. There is media often results in atypical characteristics and that spor-
only a single case reporting this species as a human pathogen ulation may be hindered or absent on sugar-rich media.13,15
causing keratomycosis.7 The patient was accidentally injured Our experience with cultures of botryosphaeriaceous fungi,
in the eye with a bamboo cane causing a shelving, penetrat- such as Neodeightonia, has shown that the addition of steril-
ing corneal wound, which contained small slivers of bamboo. ized plant material (e.g., pine needles, twigs of oak, poplar, or
The splinters were removed and in agreement with the fungal other plants) on water agar, or half-strength potato dextrose
sensitivity tests, a treatment with 2% clotrimazole eye drops agar, encourage the production of conidiomata. Another
was applied for 16 weeks. This resulted in the restoration of medium that is frequently used is oatmeal agar.11,16
complete visual acuity. Nevertheless, 39 months after the end As mentioned above, the identification of fungi is based
of the topical antifungal therapy the infection recurred and on their morphology. Despite the fact that the sexual stages
a keratoplasty had to be performed in order to remove the (telemorphs) are the baseline of fungal taxonomy and nomen-
fungal mass that developed. The surgery was followed by clature, medical mycologists are generally more familiar
topical steroid and clotrimazole therapy7 and there was no with the asexual stage or anamorph, which is the stage most
subsequent relapse. frequently found in cultures of clinical isolates.14 In the case
According to Kirkness et al.7 fungal keratytis due to of botryosphaeriaceous fungi the teleomorph is rarely found
N. subglobosa is difficult to treat, especially when associated in nature and it is highly improbable that sexual fruiting bod-
with a penetrating injury. These authors hypothesized that ies (pseudothecia) will be seen in cultures of clinical isolates.
dark, thick-walled hyphae or brown conidia, which could be Although Punithalingam (1969)8 reported that N. subglobosa
resistant to antifungal therapy, may have lain dormant in the is a homothallic species and its teleomorph forms in culture,
cornea resulting in a recurrent infection. we have not observed that in our work. In fact, in the closely
Neodeightonia subglobosa is probably not a primary related species N. phoenicum our isolates also did not form
human pathogen and as in most coelomycetous fungi it is the teleomorph in culture, even after long periods of incuba-
most likely an opportunistic human pathogen. In the coe- tion (>3 months).11
lomycetes causing opportunistic mycoses the infection Species identification in the Botryosphaeriaceae has
occurs most frequently by implantation of the fungus from relied heavily on morphological characters of the anamorph,
plant material (or soil) through abrasions, lacerations, punc- including size, shape, color, septation, wall thickness, and
ture wounds, or other traumas rather than by inhalation of texture of conidia. However, some morphological charac-
spores.13 These fungi may also be of concern to immunocom- ters exhibit extensive plasticity. Thus, size ranges of conidia
promised patients, such as bone marrow and organ transplant of different species overlap, while age and state of maturity
recipients, cancer patients, among others.13 affect conidial pigmentation and septation. Morphological
characters can also be influenced by the substrate on which
15.1.3 Diagnosis the fungus is growing.1,17,18 Because species may differ in
minor morphological features, identification can be a diffi-
15.1.3.1 Conventional Techniques cult task for someone who is not familiar with these fungi.
As for other mycoses the hispathological identification of The genus Neodeightonia is phylogenetically related to
hyphae is elemental to confirm a fungus as the causal agent Diplodia and Lasiodiplodia and has intermediate morpholog-
of the disease. Although the pigmentation and shape of ical features between both genera.11 Conidia are initially hya-
hyphae and the presence or absence of septa can give an line and aseptate becoming brown and one-septate (in some
idea of the identity, a fungal culture is required to accom- cases) with age, characters that are common to both Diplodia
plish an accurate and reliable diagnosis of the etiological and Lasiodiplodia. Punithalingam8 referred to a germ slit in
agent.13,14 The identification of fungi, unlike other impor- the conidia of N. subglobosa. Crous et al.10 suggested that this
tant pathogens such as bacteria or viruses, relies mainly is in fact a striation on the conidial wall, and that more than
on morphological criteria. Most specifically fungal iden- one could occur per conidium, a feature that was confirmed
tification is based on the morphology of the reproductive in a later study.11 Moreover, Phillips et al.11 described a new
structures. species in Neodeightonia, namely N. phoenicum, which has

Neodeightonia 131
clear striations in the conidial wall. Such striate walls are a mycology has definitively embraced molecular methods
typical feature of Lasiodiplodia species suggesting an affin- of identification/detection of fungi and these methods are
ity to this genus. Nevertheless, Neodeightonia can be dis- being increasingly applied.19–29 Molecular methods are rapid
tinguished from Lasiodiplodia by the absence of pycnidial with a turnaround time of about 24 h from the time of DNA
paraphyses which are also not present in Diplodia. Thus, extraction, yield results that are objective with data portable
conidial striations distinguish Neodeightonia from Diplodia, between laboratories, and despite the higher cost could be
and the absence of pycnidial paraphyses distinguishes it from more economical in the long run.19–29 These methods, espe-
Lasiodiplodia. cially the polymerase chain reaction (PCR)-based ones, can
Identification to species level is not as complicated as in be applied to cultures for which the phenotypic approach has
other genera of Botryosphaeriaceae because the genus is been found to be ineffective, and offer the additional advan-
quite small currently comprising only two species, that is, tage of eliminating the need for isolation of cultures as they
Neodeightonia subglobosa and N. phoenicum, which are can be applied directly to clinical samples.19–27
easily distinguished on the size, shape, and septation of the A myriad of DNA-based identification methods is available
conidia. Conidia of N. subglobosa are spherical to subglo- and these are being increasingly employed in clinical labora-
bose, aseptate, and smaller than the conidia of N. phoenicum tories. These nonsequence-based molecular methods include
which are oval to ellipsoid and have one transverse septum. the commercially available GenProbe assay, methods based
on the polymerase chain reaction such as single-step PCR,
15.1.3.2 Molecular Techniques RAPD-PCR, rep-PCR, nested PCR, PCR-RFLP, PCR-EIA,
Keratytis is currently recognized as the most common fun- and more recent microassay-based, Luminex technology-
gal infection of the eye, with some authors reporting up to based, and real-time PCR-based methods. Great variation
16%–37% of keratitis cases related to a mycotic aetiology.19,20 in assay complexity, targets, and detection methods can be
Early, rapid, and accurate identification of the fungal species found, and many of these methods have not been widely used
is important in order to guide the selection of appropriate or rigorously validated.29 Of the above-mentioned methods
antifungal therapy and is essential for an effective treatment rep-PCR and MSP-PCR have proven useful for differentiat-
of ocular infections such as fungal keratytis.19–22 ing Neodeightonia species,30 but their applicability in clinical
Diagnosis of fungal infections depends on recovery of settings has not been evaluated.
fungi from culture of clinical specimens, and their identifica- At present, the “gold standard” for fungal species identi-
tion requires the presence of reproductive structures. Clinical fication is DNA sequence analysis. This method is based on
diagnosis of these ocular infections is confirmed by obtaining PCR amplification of a selected region of genomic DNA, fol-
intraocular (aqueous or vitreous) specimens or corneal scrap- lowed by sequencing of the resulting amplicon. The obtained
ings. However, standard microbiological tests (Gram and sequence can then be used to perform searches against a
Giemsa stains and culture) are positive in only 50%–80% of nucleotide sequence database library.20,22,25,28
keratitis cases.19,23,24 Even in the cases where culture tests are The success of this strategy depends largely on the choice
positive an identification of the fungal species can be diffi- of the appropriate locus. The gene target must be orthologous,
cult to obtain. The culture-based phenotypic methods can be have a high level of interspecific variability combined with
time consuming and laborious, may initially be nonspecific, low levels of intraspecific variability, and should not undergo
and require considerable expertise for correct morphological recombination. Additionally, it must be easy to amplify and
identification of less common or unusual fungi. Additional sequence and the amplicons should be within the size range
drawbacks of conventional morphological identification are obtainable with the most commonly used automated DNA
the inability of some cultures to sporulate, or cultures exhib- sequencers (about 600–800 bp).14,28,29
iting atypical morphology resulting in the impossibility to The ITS region (noncoding sequence interspaced among
identify the species or even in misidentifications.25–27 In fact, highly conserved fungal rDNA genes) complies with most
the occurrence of non-sporulating moulds seems frequent in of these requirements since this region can be reliably
cultures derived from ocular infections.25,27 amplified for most fungi, is conserved, is present as multi-
For Neodeightonia isolation and morphological identifica- ple copies in the fungal genome, yields sufficient taxonomic
tion of cultures from clinical specimens has been successful resolution for most fungi, and has the additional advantage
in the single case reported to date.7 Nevertheless, one cannot that the GenBank (http://www.ncbi.nlm.nih.gov), European
discard the chance that in some cases retrieving cultures will Molecular Biology Laboratory nucleotide sequence data-
not be possible and some cultures/strains may not be able base (http://www.ebi.ac.uk/embl/), and DNA Data Bank of
to sporulate thus hindering the identification. Also, even if Japan (http://www.ddbj.nig.ac.jp/) contain a large number
successful isolation is accomplished the sporulation process of sequences from this locus, enabling a ready comparison
(development of conidiomata and conidia) may take 2–3 of the sequence from an unknown isolate.14,28,29
weeks to occur,11 a time frame that is not clinically useful. There is considerable consensus regarding the use of
Molecular methods for identification of pathogenic fungi ITS sequencing in the identification of fungi. Also, the
have been validated for use in clinical settings in order to International Subcommission on Fungal Barcoding has pro-
overcome the shortcomings of traditional morphological posed the ITS region as the prime fungal barcode or the default
identification mentioned previously. The field of medical region for species identification. The main disadvantages

regarding the use of ITS is the inability of this region for dif- including commercial kits which are usually faster than other
ferentiation of species complexes and failure to distinguish approaches.16,19,20,24–27,32 In a faster approach the DNA can
between closely related species or cryptic species.14,28,29 In be obtained directly from the ocular samples via the same
these cases, it may be necessary to use more variable gene DNA extraction procedures used for fungal cultures or oth-
regions. The locus to be used will depend on the fungal group ers developed specifically for human tissue samples.20,22,32 By
in question. For example, in the Botryosphaeriaceae when eliminating the isolation and cultivation procedure this last
the ITS region fails to differentiate species complexes or approach greatly reduces the time necessary for the molecu-
cryptic species the locus of choice has been the elongation lar diagnosis of the causal agent.
factor 1-alpha which has shown successful results.31
Procedure
Regarding Neodeightonia the molecular identification
of species can be based solely on the ITS sequence. ITS
1. Inoculate cultures onto potato dextrose agar (Difco)
sequences of N. subglobosa and N. phoenicum are suffi-
and incubate at 25°C. After 1 week the colony is
ciently different from one another to account for a reliable
scraped from the agar surface, frozen in liquid
identification at species level. In the course of recent studies
nitrogen and ground in a porcelain mortar. As an
dealing with the genus Neodeightonia and other genera in
alternative, cultures can be grown in potato dextrose
Botryosphaeriaceae10,11 sequences of the 18S and 28S rDNA
broth.
(D1/D2 variable region) and ITS region, as well as the elon-
2. Genomic DNA can be extracted from the ground
gation factor 1-alpha and beta-tubulin became available for
mycelium using any DNA isolation protocol. In our
authentic and ex-type cultures of N. subglobosa and N. phoe-
lab we have good results in terms of yield and purity
nicum. This fact is highly relevant since the success of the
of DNA samples following the method described by
DNA sequence-based identification depends on the reliabil-
Alves et al. (2004).16
ity of sequences deposited in reference databases.
PCR-based culture independent methods have been devel-
oped and applied for the detection of fungi causing kerato- 15.2.2 Detection Procedures
mycosis directly on ocular specimens.21,24 Also, through the
Once DNA is obtained it is necessary to perform a PCR
use of species specific oligonucleotide primers or probes
amplification of the ITS region. The ITS can be amplified
this approach has been used to detect a particular species or
easily using “universal” fungal primers in the vast majority
group of species without the need for DNA sequencing.32 To
of fungi. Our experience shows that with Neodeightonia (and
date, no specific primers or oligonucleotide probes have been
Botryospheriaceae in general) the primer sets ITS1/ITS4 or
developed for species of Neodeightonia and thus if molecular
ITS5/ITS433 result in good amplification of the target region
detection methods are to be applied to Neodeightonia species
resulting in amplicons with a size of about 500–600 bp.11,16,31
these will have to depend (for now) on the PCR amplification
As an alternative the ITS1 forward primer can be combined
and sequencing of the ITS region.
with the primer NL434 which anneals within the 28S rDNA
gene resulting in a larger amplicon (approx. 1200 bp) that
15.2 Methods includes the ITS region plus the D1/D2 variable region of the
28S rDNA gene.35 The PCR mixtures and amplification con-
15.2.1 Sample Preparation ditions have been described elsewhere.11,16,35 In some cases
The first and probably the most important step in any molec- where amplification is weak or unsuccessful, good results are
ular detection method is the isolation of DNA from the fun- usually obtained by performing a second PCR using as tem-
gal culture or directly from the clinical specimen. Although plate the first PCR amplification.36 Another approach involv-
for PCR-based methods the amount of DNA required is very ing a nested or semi-nested PCR can be performed. Thus, for
small it is important to use DNA extraction procedures that example, an initial amplification would be performed using
guarantee a sufficient quantity of DNA and simultaneously the primer set ITS1/NL4 and in the second PCR the primer
that it is not contaminated with PCR inhibitory agents. A set used would be ITS1/ITS4. This kind of approach is fre-
good DNA extraction method is crucial for PCR detection quently used and is known to increase the sensitivity of the
to avoid the possibilities of false negative results. Also, strict PCR detection.19 Finally, from our experience, the addition
precautions must be taken throughout the whole procedure, of 5% DMSO to the PCR amplification reaction is useful to
from collection and processing of samples to DNA extraction help the amplification of some difficult templates and also to
and PCR amplification in order to avoid contaminations and increase reproducibility.11,31,35,36
consequently false positive results. The nucleotide sequence of the ITS region is then obtained
Clinical diagnosis of keratomycosis is confirmed by using standard automated DNA sequencing procedures. The
obtaining intraocular (aqueous or vitreous) specimens or complete sequences of the ITS region are read and edited
corneal scrapings.19–21,24,25 These samples can be used to using available software such as Chromas 1.45 (http://www.
inoculate culture media in order to isolate the fungus respon- technelysium.com.au/chromas.html), FinchTV 1.4.0 (http://
sible for the infection. As soon as colonies start to develop www.geospiza.com/products/finchtv.shtml), or others.
DNA can be extracted by a number of existing methods, The sequences must be checked manually and nucleotide

Neodeightonia 133
arrangements at ambiguous positions clarified using both over the past 2 decades. The field of medical mycology has
primer direction sequences. The final step in the identifica- become an extremely challenging study of infections caused
tion procedure consists in performing a sequence similarity by a wide and taxonomically diverse array of opportunistic
search of the retrieved sequence against nucleotide sequences fungi. These opportunistic mycoses pose considerable diag-
database such as GenBank using the BLAST tool (http:// nostic and therapeutic challenges and a rapid, accurate diagno-
www.ncbi.nlm.nih.gov/BLAST). The outcome of this simi- sis is essential for the initiation of targeted antifungal therapy.
larity search must be interpreted with caution due to the well Given the general lack of knowledge about N. subglobosa
known problems with the reliability of the ITS sequences in clinical settings and the aforementioned problems with
deposited in the reference databases (e.g., GenBank/EMBL/ traditional morphological identification of fungi it is possible
DDBJ).28,37 Errors in fungal sequences within GenBank, the that infections caused by this species may have previously
most widely used database, have been found to be as high passed unnoticed due to inadequate diagnosis. The clinical
as 20%.37 However, in the case of Neodeightonia this task is significance of N. subglobosa is poorly understood. However,
facilitated since sequences from authentic, ex-type and well- as emphasized by Kirkness et al. (1991)7 this species should
characterized species are available in GenBank. be considered in ocular infections as a result of injuries
caused by plant materials. This fungus should be particularly
Procedure
considered in tropical and subtropical countries where inci-
dence of keratomycosis is higher than in temperate regions.25
1. Prepare a PCR mixture (50 μL) containing 1× PCR
Although Neodeightonia species are not especially dif-
buffer with (NH4)2SO4 (MBI Fermentas, Vilnius,
ficult to identify based on morphological characters, they
Lithuania), 3 mM MgCl2, 200 mM of each nucleo-
require considerable expertise of the taxonomy of the
tide, 15 pmol of primer ITS1, 15 pmol of primer ITS4,
Botryosphaeriaceae. Because this group of fungi is not fre-
1 U of Taq DNA polymerase (MBI Fermentas), and
quent in clinical settings this may constitute a problem when
50 ng of template DNA.
trying to obtain a correct identification. In this respect,
Note: The forward primer ITS5 can be used as an
molecular methods have the advantage that no taxonomic
alternative to primer ITS1.
expertise is required. Moreover, the molecular detection pro-
2. Perform the PCR amplification in a thermocycler
cedure can be performed directly on the clinical specimen
using the following conditions: initial denaturation
without the need to isolate the fungus, which decreases the
of 3 min at 95°C, followed by 30 cycles of 30 s at
time needed for the diagnosis and consequently for the initia-
94°C, 30 s at 50°C, and 1 min at 72°C, and a final
tion of proper antifungal therapy.
extension period of 10 min at 72°C.
3. Purify the PCR amplicons before sequencing. In
our lab we have good results with the Jet Quick PCR References
Product Purification Spin Kit (Genomed, Löhne, 1. Slippers, B. and Wingfield, M.J., Botryosphaeriaceae as endo-
Germany) but virtually any PCR product purifica- phytes and latent pathogens of woody plants: Diversity, ecol-
tion kit can be used. ogy and impact, Fungal Biol. Rev., 21, 90, 2007.
4. Prepare cycle sequencing reactions (20 μL) contain- 2. Rebell, G. and Forster, R.K., Lasiodiplodia theobromae as a
ing 30–90 ng DNA template, 3.2 pmol of primer, cause of keratomycoses, Sabouraudia, 14, 155, 1976.
4 μL BigDye? Terminator from the ABI PRISM? 3. Summerbell, R.C. et al., Subcutaneous phaeohyphomycosis
BigDye, and Terminator Cycle Sequencing Ready caused by Lasiodiplodia theobromae and successfully treated
surgically, Med. Mycol., 42, 543, 2004.
Reaction Kit with AmpliTAQ DNA Polymerase (PE
4. Tan, D.H. et al., Disseminated fungal infection in a renal
Applied Biosystems). transplant recipient involving Macrophomina phaseolina
5. Remove excess dye terminator by mixing the reac- and Scytalidium dimidiatum: Case report and review of taxo-
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50 μL of 100% ethanol. Precipitate for 20 min at Botryosphaeriaceae, Med. Mycol., 46, 285, 2008.
room temperature and centrifuge for 20 min at 5. Srinivasan, A. et al., Cutaneous infection caused by
16,000× g. Discard the supernatant and wash the Macrophomina phaseolina in a child with acute myeloid leu-
kemia, J. Clin. Microbiol., 47, 1969, 2009.
pellet with 250 μL of 70% ethanol. Dry the pellet in
6. Elinav, H. et al., Invasive Scytalidium dimidiatum infection
a heat block at 90°C for 1 min. in an immunocompetent adult, J. Clin. Microbiol., 47, 1259,
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the ABI PRISM 310 Genetic Analyzer (PE Applied 7. Kirkness, C.M. et al., Sphaeropsis subglobosa keratomycosis—
Biosystems). First reported case, Cornea, 10, 85, 1991.
8. Punithalingam, E., Studies on Sphaeropsidales in culture,
Mycol. Pap., 119, 1, 1969.
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onclusion and Future 9. von Arx, J.A. and Müller, E., A re-evaluation of the bitunicate
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11. Phillips, A.J.L. et al., Resolving the phylogenetic and taxo- 25. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
nomic status of dark-spored teleomorph genera in the non-sporulating molds (NSM) identified by polymerase chain
Botryosphaeriaceae, Persoonia, 21, 29, 2008. reaction (PCR)-based DNA sequencing technique targeting
12. Krishnamurthy, Y.L., Naik, S.B., and Jayaram, S., Fungal internal transcribed spacer (ITS) region, Curr. Eye Res., 33,
communities in herbaceous medicinal plants from the Malnad 139, 2008.
Region, Southern India, Microb. Environ., 23, 24, 2008. 26. Rakeman, J.L. et al., Multilocus DNA sequence comparisons
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review: Clinical entities, pathogenesis, identification and 3324, 2005.
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15. Sutton, B.C., The Coelomycetes, Kew, England: CMI, 1980, 28. Balajee, S.A. et al., Sequence-based identification of
696 pp. Aspergillus, Fusarium, and Mucorales species in the clinical
16. Alves, A. et al., Botryosphaeria corticola sp. nov. on Quercus mycology laboratory: Where are we and where should we go
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2004. sical way: Identification of medically important molds in the
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on morphology and ITS rDNA phylogeny, Stud. Mycol., 45, Botryosphaeriaceae by PCR fingerprinting, Res. Microbiol.,
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18. Jacobs, K.A. and Rehner, S.A., Comparison of cultural and 31. Alves, A. et al., Morphological and molecular data reveal
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typing in ocular infections, J. Clin. Microbiol., 39, 2873, 33. White, T.J. et al., Amplification and direct sequencing of fungal
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16 Phoma and Phomopsis
Dongyou Liu
Contents
16.1 Introduction...................................................................................................................................................................... 135
16.1.1.1 The Genus Phoma.................................................................................................................................. 135
16.1.1.2 The Genus Phomopsis........................................................................................................................... 136
16.1.2.1 Phoma Infections................................................................................................................................... 136
16.1.2.2 Phomopsis Infections............................................................................................................................. 137
16.1.3 Diagnosis.............................................................................................................................................................. 137
16.2 Methods............................................................................................................................................................................ 137
16.2.2.1 PCR Detection of Phoma exigua........................................................................................................... 137
16.2.2.2 Real-Time PCR Detection of Phomopsis spp........................................................................................ 138
16.2.2.3 Real-Time PCR and Sequencing Analysis of ITS Region..................................................................... 138
16.2.2.4 Sequencing Analysis of 28S rRNA Gene.............................................................................................. 138
16.3 Conclusion........................................................................................................................................................................ 138
References.................................................................................................................................................................................. 139
16.1 Introduction the genera Didymella, Leptosphaeria, Pleospora, and

Mycosphaerella [1–3].
As cosmopolitan plant-infecting pathogens or free-living Phoma spp. are cosmopolitan in nature. Some Phoma
saprobes, dematiaceous fungi have the capacity to take spp. are saprophytes on various plants (known as plurivo-
advantage of host’s temporary weakness (e.g., injury and rous fungi, which are generally saprobic or weakly parasitic,
immunosuppression), and establish infections in otherwise mainly from temperate regions in Eurasia); while others
refractory hosts. This is evidenced by the observations that are pathogens to cultivated plants and humans. For exam-
along with a growing number of immunocompromized, can- ple, Phoma beta causes heart rot and blight in beets, and
cer and post-operative patients in recent decades, there has Phoma batata causes dry rot in sweet potato. The human
been a significant increase in the incidence of unusual fungal pathogenic Phoma spp. comprise Phoma cruris-hominis,
infections. Species of Phoma and Phomopsis represent the Phoma denissii var. oculo-hominis, Phoma eupyrena,
examples of ubiquitous and plant-infective fungal organisms Phoma glomerata, Phoma herbarum, Phoma hiber-
that have emerged as unusual pathogens in weakened human nica, Phoma minutella, Phoma minutispora, and Phoma
hosts, resulting in notable morbidity and mortality. sorghina. These organisms are responsible for occasional
infections in humans, often after trauma and immunosup-
pression, leading to cutaneous, subcutaneous, corneal, or
systemic phaeohyphomycosis.
16.1.1.1 The Genus Phoma Phoma colonies grow rapidly, and are flat, spreading, gray-
The genus Phoma is a dematiaceous, filamentous fungus ish-brown, powdery to velvety (suede-like), and often largely
belonging to the family Pleosporaceae, order Pleosporales, submerged in the medium. From the front, colonies are ini-
class Dothideomycetes, subphylum Pezizomycotina, phy- tially white and later become olive grey with an occasional
lum Ascomycota, kingdom Fungi. The genus Phoma (obso- tint of pink. From the reverse, they are dark brown to black.
lete synonyms: Deuterophoma, Leptophoma, Peyronellaea, Some species (e.g., Phoma cruris-hominis and Phoma her-
Plenodomus, Polyopeus, and Sclerophomella) covers over barum) produce a reddish-purple to yellowish-brown diffus-
2000 species, which produce spores of <15 μm in size as the able pigment which is readily visible from the reverse. Phoma
larger spored forms are placed in the genus Macrophoma. hyphae are septate, hyaline to brown. Pycnidia (70–100 μm
The teleomorphs of the genus have been described in in diameter) are large, round to pyriform (globose), asexual
135

fruiting bodies. Further, pycnidia are membranous to leath- Diaporthe vexans, the teleomorph of Phomopsis vexans,
ery, dark in color and bear phialides at their inner lining, with produces pycnidia (100–300 μm in diameter) with subepi-
one to several openings (ostioles) on their surface from which dermal, erumpent, dark, thick-walled, flattened to globose
the conidia are released outside. Pycnidia may be depressed appearance and with or without a beak (to 76 μm). Phialides
in the tissues of the host. Conidia are globose to cylindrical, (10–16 μm in length) are hyaline, simple or branched, some-
unicellular, hyaline (colorless), and are produced in abun- times septate, arising from the innermost layer of cells lining
dance within the pycnidia on narrow thread-like phialides, the cavity. Alpha conidia (5–8 × 2–3 μm) are hyaline, aseptate,
which are not easily differentiated from the inner pycnidial and sub-cylindrical. Beta conidia (18–32 μm × 0.5–2.0 μm)
wall cells. Conida are usually extruded in slimy masses are filiform, curved, hyaline, septate, and non-germinating.
from the apical ostiole. Some Phoma species produce brown Hyphae (2.5–4 μm wide) are hyaline, and septate. Perithecia
chlamydospores arranged singly or in chains, and may be (130–350 μm in diameter) occur in clusters in culture, with
unicellular or multicellular and “alternarioid” (resembling sinuous, carbonaceous, irregular beaks (80–500 μm in
Alternaria) in appearance [4]. length). Asci (24–44 μm × 5–12 μm) are clavate, sessile,
eight-spored. Ascospores (9–12 μm × 3–4.5 μm) are biseri-
16.1.1.2 The Genus Phomopsis ate, hyaline, narrowly ellipsoid to bluntly fusoid, one-septate,
The genus Phomopsis (formerly Phoma subgen. Phomopsis, constricted at the septum.
and Myxolibertella) belongs to the mitosporic Valsaceae
group, family Valsaceae, order Diaporthales, subclass 16.1.2 Clinical Features and Epidemiology
Sordariomycetidae, class Sordariomycetes, subphylum
Pezizomycotina, phylum Ascomycota, kingdom Fungi. The Members of the genera Phoma and Phomopsis are widely
genus Phomopsis contains >400 described species, and distributed in soil and plants, many of which are plant patho-
the teleomorphs of the genus Phomopsis are identified in gens. Occasionally these organisms may be introduced to
the genus Diaporthe. human hosts via traumatic implantation, resulting in subcu-
Phomopsis spp. are plant pathogens, causing spots on taneous infections, keratitis, and invasive disease [6–15].
various plant parts. For example, Phomopsis juniperovora
infects junipers, representing an important pest of seedlings 16.1.2.1 Phoma Infections
and juvenile plants in the nursery industry. Phomopsis lep- Baker et al. [16] described a case of subcutaneous infection
tostromiformis is infective to lupin plants, and produces the due to Phoma minutella on the foot of a farmer undergoing
potent mycotoxins phomopsin A and phomopsin B, which corticosteroid therapy for myasthenia gravis. Microscopic
are toxic to various animals following ingestion, resulting in examination of stained smears and biopsy sections demon-
a form of mycotoxicosis called lupinosis. Phomopsis vexans strated dematiaceous fungal elements consistent with the
causes disease in aubergine, brinjal, eggplant, leading to mold. Culture of the specimens grew a fungus that was iden-
poor seed germination and damping-off of seedlings to leaf tified as Phoma minutella.
and stem lesions and to fruit rot, both in the field and after Rishi and Font [17] reported a Phoma-related nonhealing
harvest. Phomopsis viticola causes a plant disease called corneal ulcer with brownish pigmentation in a 72-year-old
dead-arm, forming lesions on shoots, leaves, and rachises, man. Histopathologic examination of the keratectomy speci-
and causing fruit rot. men showed round spherules (5–30 μm in diameter) together
Morphologically, Phomopsis mycelium is immersed, with septate hyphae at the edges of the perforated cornea.
branched, septate, hyaline to pale brown. Conidiomata are Culture of the specimen yielded a fungus belonging to the
brown to dark brown, thick-walled, immersed, stromatic genus Phoma. Polymerase chain reaction (PCR) amplifi-
(true stroma—host tissue incorporated into the pycnidium), cation of DNA extracted from five (10 μm thick) paraffin-
separate or aggregated and confluent, globose, ampulliform embedded sections using pan-fungal primers generated a
or applanate (flattened), unilocular or multilocular (single or single product of 360 bp.
several cavities), with one or several circular papillate osti- Everett et al. [18] documented a case of subcutaneous
oles. Conidiophores are branched and septate at the base, Phoma infection in an immunosupressed 50-year-old female,
occasionally short, more frequently multiseptate and fili- who undertook maintenance immunosuppression with aza-
form, hyaline, formed from inner cells of the locular walls. thioprine and prednisone after bilateral nephrectomy, sple-
Conidiogenous cells are enteroblastic, phialidic, determi- nectomy, and cadaveric renal transplant for the treatment of
nate, integrated, rarely discrete, hyaline, cylindrical, apera- end-stage renal disease secondary to familial nephritis. The
ture apical on long or short lateral and main branches of the patient presented with pain, warmth, and swelling in the
conidiophores, collarette, channel and periclinal thickening dorsum of her left wrist with a decreased motion. A syno-
minute. Phomopsis spp. produce both alpha conidia (hyaline, vectomy of the fourth dorsal compartment and debridement
fusiform, straight, usually biguttulate, and >5 μm in length) of the third extensor tendon were performed. Examination
and beta conidia (hyaline, filiform to hamate or hooked, of tissue sections with Grocott-Gomori methenamine-silver
egutullate or without guttules, aseptate, usually >15 μm in staining (GMS) uncovered branching filaments and pycnidia.
length) [5]. Culture of the specimen yielded a Phoma sp after 2 weeks

Phoma and Phomposis 137
of incubation. Treatment with amphotericin B resulted in the Conventional identification of Phoma, Phompsis and other
resolution of her symptomatology and no further recurrence fungal organisms (e.g., Pleurophoma, Pleurophomopsis,
after 2 years of follow-up. Pyrenochaeta, Chaetomium, Pseudallescheria, Conio
Balis et al. [19] reported a case of aggressive and deeply thyrium, Microsphaeropsis, and Pseudochaetosphaeroma)
invasive hand infection due to Phoma exigua in a renal trans- relies on the detection of distinct colony color, conidia mor-
plant recipient with acute myeloid leukaemia and diabetes. phology, and chlamydospore structures [21]. However, due to
Treatment required surgical debridement and the use of pro- their apparent lack of diagnostic morphological features and
longed systemic amphotericin B therapy. their tendency to show varied phenotypic characteristics on
culture media, it has been difficult and technically demanding
16.1.2.2 Phomopsis Infections to accurately determine their species identity.
Application of molecular techniques such as PCR and
Sutton et al. [5] described a case of osteomyelitis due to a
sequence analyses of the 18S rRNA (SSU) and the 28S rRNA
Phomopsis species in a 61-year-old female urban gardener,
(LSU), internal transcribed spacer (ITS), actin, and three-
who was under chronic immunosuppression (prednisone
tubulin gene regions enables rapid and precise identification
and methotrexate) for the treatment of diabetes and rheuma-
of Phoma and Phomopsis [2,3].
toid arthritis. The patient developed an erythematous, pain-
ful subcutaneous abscess on the distal phalanx of the right
fourth finger complicated by osteomyelitis. Osteomyelitis 16.2 Methods
was confirmed radiographically by the presence of lytic bone
changes on plain x-rays and abnormally increased radio-
tracer uptake in the distal phalanx on bone scans. Draining Corneal scrapings for PCR are collected in lysis buf-
of the abscess produced a large amount of purulent mate- fer (50 mM Tris–Cl pH 7.2, 50 mM EDTA, 3% SDS, and
rial that contained fungal hyphae. Culture of the specimen 1% β-mercaptoethanol in 400 μL). The specimens are
grew a mold that was initially white and woolly, but became kept at −20°C until further processing. For DNA extrac-
slightly greenish after 4 weeks at room temperature (25°C). tion, tubes are vortexed at moderate speed for 15 s, incu-
Subcultures of the isolate on potato flakes agar (PFA) were bated at 65°C for 1 h, and boiled for 10 min. Equal volume
buff and sterile with a few black sclerotia after 4 weeks at of phenol:chloroform:isoamylalcohol (25:24:1) is added to
25°C, with chains of chlamydoconidia. Subcultures onto car- the tube and then revortexed. The tubes are centrifuged at
nation leaf agar produced dark brown to black pycnidia after 14,000 rpm for 15 min. To the aqueous phase, a 1:10 volume of
2 weeks of incubation at 25°C. Periodic acid-Schiff (PAS) 3 M sodium acetate pH 5.2 and 0.6 volume of isopropanol are
staining of carnation leaf sections containing mature pyc- added. The tubes are inverted and mixed gently, and DNA is
nidia demonstrated ostiolate, thick-walled, immersed, mul- precipitated at −20°C for 1 h. The tube is again centrifuged at
tilocular conidiomata (101–106 μm × 196–274 μm). Crushed 14,000 rpm for 15 min. The supernatant is discarded, and the
conidiomata showed short, ellipsoidal alpha conidia (2.0– pellet containing the DNA is washed twice with 70% alcohol.
2.5 μm × 5.8–6.0 μm) and long, filamentous beta conidia The pellet is air dried and resuspended in 30 μL of autoclaved
(0.4–0.5 μm × 18 to 22 μm). The fungus was identified as a double-distilled water [22].
Phomopsis species. The patient was treated with a 6-month
course of itraconazole. At 16 months of follow-up, she 16.2.2 Detection Procedures
remained free of recurrence.
Mandell and Colby [20] reported a case of Phomopsis 16.2.2.1 PCR Detection of Phoma exigua
fungal keratitis in a patient after a rose thorn injury that Balis et al. [19] described a PCR assay for specific
occurred 2 months earlier while gardening. The patient pre- identification of Phoma exigua using primers Pe1
sented with deep stromal keratitis with extension of hyphae (5′-GCTTTGCCTACCATCTCTTACCCA-3′) and Pe2
through Descemet’s membrane. Culture of the surgical spec- (5′-GTTATGAGTGCAAAGCGCGAGATG-3′). These
imens yielded a Phomopsis organism. Treatment consisted primers are selected from the ITS regions 1 and 2 of the pub-
of therapeutic keratoplasty combined with oral and topical lished P. exigua sequence (GenBank AF268192).
antifungal medications.
Procedure
1. PCR mixture (50 µL) consists of 6 µL MgCl2

16.1.3 Diagnosis
(25 mM), 10 µL Mg-free buffer (100 mM Tris–
Species of the Phoma and Phomopsis genera are common HCl, 500 mM KCl, and 1% Triton-X), 2.5 U Taq
plant pathogens that may be occasionally involved in human Polymerase (Promega), 0.04 mM of each dNTP, 100
infections. Diagnosis of rare fungal pathogen infections pmol of each primer, and 5 µL of template DNA.
is challenging and demands a rational approach that takes 2. Amplification parameters include an initial 3 min at
account of clinical manifestations, histological findings, and 95°C, 38 cycles of 1 min at 95°C, 1 min at 58°C, 1
mycological observations. min at 72°C, and a final 5 min time at 72°C.

3. The PCR product (410 bp) is separated on 2% aga- product. Cycle sequencing is performed with a 9700
rose gel, stained with ethidium bromide and visual- thermal cycler (ABI), using 25 cycles of 96°C for
ized under UV transillumination. 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
reaction products are passed through a Sephadex
Note: The specific amplicon may be further verified by G-50 fine column to remove unincorporated dye ter-
sequence analysis. minators. Purified sequencing reaction products are
run on an ABI Prism 3100 Genetic Analyzer with a
16.2.2.2 Real-Time PCR Detection of Phomopsis spp. 50 cm capillary array.
Hietala et al. [23] developed a real-time PCR for detec- 5. Sequences are analyzed with the SmartGene
tion of Phomopsis spp. using primers and probe derived Integrated Database Network software version
from the ITS rRNA region. The primer pair (forward: 3.2.3 vr. SmartGene is a web-based software and
5′-GCACCCAGAAACCCTTTGTG-3′ and reverse: database system with reference sequences derived
5′-AAGAGTTGACTTGGCCGCC-3′) amplifies a 116 bp from the National Center for Biological Information
product; the probe (5′-CGGTAACGAGGAGCAGCCCGC-3′) (NCBI) GenBank repository.
is labeled with FAM at the 5′ end and TAMRA at the 3′ end.
PCR mixture (25 μL) is composed of TaqMan Universal Note: In case that real time PCR instrument is not available,
PCR Master Mix (P/N 4304437; Applied Biosystems), standard PCR may be performed with ptimers ITS1 and
300 nM each primer, 400 nM probe and 3 μL of the DNA. ITS4, and the resulting amplicon is sequenced with the same
PCR cycling parameters are 95°C for 10 min followed by primers. Sequence-based identifications are defined by per-
40 cycles of 95°C for 15 s and 60°C for 1 min. cent identity: species, ≥99%; genus, 93%–99%; and incon-
Fluorescence emissions are detected with an ABI Prism clusive, ≤93%.
7700 (Applied Biosystems). Data acquisition and analysis For strains producing discrepant identification
are performed with the Sequence Detection System software between the methods based on phenotypic characteris-
package (1.7a; Applied Biosystems). tics and ITS sequence analysis, the D1–D2 region of the
large-subunit RNA gene is amplified with primers NL1
16.2.2.3 Real-Time PCR and Sequencing (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4
Analysis of ITS Region (5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for
Pounder et al. [24] described a real-time PCR with SYBR species clarification.
green DNA binding dye and amplicon melting temperature
analysis for fungal detection also using pan-fungal primers 16.2.2.4 Sequencing Analysis of 28S rRNA Gene
ITS1 forward (5′-TCCGTAGGTGAACCTGCGG-3′) and Vengayil et al. [22] utilized universal primers (forward:
ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′). The 5′-GTG AAA TTG TTG AAA GGG AA-3′ and reverse:
identity of the fungi is verified by subsequent sequencing 5′-GAC TCC TTG GTC CGT GTT-3′) for amplification of a
analysis. 28S rRNA gene region for identification of medically impor-
Procedure tant fungi.
PCR mixture (25 μL) is made up of 1× PCR buffer without
1. PCR mixture is composed of 1 × Lightcycler FastStart MgCl2, 10 pmol each of forward and reverse primers, 1.5 U of
DNA Master Hybridization Probes mixture (Roche Taq polymerase, 2 mM MgCl2, 200 μM dNTPs, and 5 μL of
Applied Science) (containing deoxynucleoside tri- template DNA.
phosphates), FastStart Taq DNA polymerase, and Amplification is conducted with an initial denaturation at
1 mM MgCl2 (additional MgCl2 is added to a final 95°C for 5 min and 50 cycles of 94°C for 1 min, 50°C for
concentration of 4.6 mM), 0.4 μM each of ITS1 for- 1 min, and 72°C for 5 min in a thermocycler (GeneAmp PCR
ward and ITS4 reverse primers, 1× SYBR green System 9700; Applied Biosystems).
(Molecular Probes), and 3 μL template DNA. The amplified product is checked in 1% agarose gel con-
2. Thermal cycling parameters include 95°C for taining 0.5 μg/mL ethidium bromide and visualized in an
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and ultraviolet transilluminator. The nucleotide sequence of the
76°C for 30 s; and a final extension at 72°C for 2 min. amplicon is analyzed.
3. The quality of the amplicon is determined using the
derivative of the melt analysis curve (55°C–99°C,
16.3 Conclusion
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
(Corbett Robotics, Inc.). Members of the genera Phoma and Phomopsis are common
4. The amplified product is purified for bidirectional plant pathogens, some of which have been shown to cause
sequencing using ExoSAP-IT (USB Corp.). Five human subcutaneous infections, keratitis, and invasive dis-
μL of Big Dye Terminator Ready Reaction Mix v. ease on a number of occasions. Conventional laboratory
1.1 (Applied Biosystems) is added to 4 μL of each diagnosis of human infections due to these rare fungal patho-
primer (0.8 pmol/μL) and 3 μL of purified PCR gens is time-consuming and challenging, since Phoma and

Phoma and Phomposis 139
Phomopsis are extremely complex, with hundreds of species 11. Hirsh AH, Schiff TA. Subcutaneous phaeohyphomycosis
being assigned in each of these taxa. This is further exacer- caused by an unusual pathogen: Phoma species. J. Am. Acad.
bated by the fact that many of these organisms demonstrate Dermatol. 1996;34:679–680.
12. Rosen T et al. Cutaneous lesions due to Pleurophoma (Phoma)
few distinct morphological features for accurate identifica-
complex. South Med. J. 1996;89(4):431–433.
tion and determination. Use of PCR and sequence analyses 13. Zaitz C et al. Subcutaneous phaeohyphomycosis caused by
of the 18S rRNA (SSU) and the 28S rRNA (LSU), ITS, actin, Phoma cava. Report of a case and review of the literature. Rev.
and three-tubulin gene regions facilitates improved detection Inst. Med. Trop. Sao Paulo 1997;39(1):43–48.
and differentiation of Phoma and Phomopsis from other fun- 14. Oh CK et al. Subcutaneous pheohyphomycosis caused by
gal organisms. Phoma species. Int. J. Dermatol. 1999;38(11):874–876.
15. Suh MK. Phaeohyphomycosis in Korea. Nippon Ishinkin
Gakkai Zasshi 2005;46(2):67–70.
References 16. Baker J et al. First report of subcutaneous phaeohyphomyco-
1. The UniProt Consortium. Available at http://www.uniprot. sis of the foot caused by Phoma minutella. J. Clin. Microbiol.
org/taxonomy/, accessed on August 1, 2010. 1987;25:2395–2397.
2. Aveskamp MM et al. DNA phylogeny reveals polyphyly of 17. Rishi K, Font RL. Keratitis caused by an unusual fungus,
Phoma section Peyronellaea and multiple taxonomic novel- Phoma species. Cornea 2003;22(2):166–168.
ties. Mycologia 2009;101:363–382. 18. Everett JE et al. A deeply invasive Phoma species infec-
3. de Gruyter J et al. Molecular phylogeny of Phoma and allied tion in a renal transplant recipient. Transplant. Proc.
anamorph genera: Towards a reclassification of the Phoma 2003;35:1387–1389.
complex. Mycol. Res. 2009;113:508–519. 19. Balis E et al. Lung mass caused by Phoma exigua. Scand.
4. http://mycology.adelaide.edu.au/, accessed on August 1, J. Infect. Dis. 2006;38:552–555.
2010. 20. Mandell KJ, Colby KA. Penetrating keratoplasty for inva-
5. Sutton DA et al. Human phaeohyphomycotic osteomyeli- sive fungal keratitis resulting from a thorn injury involving
tis caused by the coelomycete Phomopsis saccardo 1905: Phomopsis species. Cornea 28, 1167, 2009.
Criteria for identification, case history, and therapy. J. Clin. 21. Dooley DP et al. Phaeohyphomycotic cutaneous disease
Microbiol. 1999;37(3):807–811. caused by Pleurophoma in a cardiac transplant patient.
6. Young NA, Kwon-Chung KJ, Freeman J. Subcutaneous J. Infect. Dis. 159, 503, 1989.
abscess caused by Phoma sp. resembling Pyrenochaeta 22. Vengayil S et al. Polymerase chain reaction-guided diagno-
romeroi: Unique fungal infection occurring in immuno- sis of mycotic keratitis: A prospective evaluation of its effi-
suppressed recipient of renal allograft. Am. J. Clin. Pathol. cacy and limitations. Invest. Ophthalmol. Vis. Sci. 50, 152,
1973;59:810–816. 2009.
7. Shukla N, Agarwal GP, Gupta DK. Phoma as a human patho- 23. Hietala AM, Solheim H, Fossdal CG. Real-time PCR-based
gen. Mykosen 27, 255, 1984. monitoring of DNA pools in the tri-trophic interaction
8. Rai MK. Phoma sorghina infection in human being. between Norway spruce, the rust Thekopsora areolata, and an
Mycopathologia 1989;105(3):167–170. opportunistic ascomycetous Phomopsis sp. Phytopathology
9. Montel E, Bridge PD, Sutton BC. An integrated approach to 98, 51, 2008.
Phoma systematics. Mycopathologia 1991;115(2):89–103. 24. Pounder JI et al. Discovering potential pathogens among
10. Morris JT et al. Lung mass caused by Phoma species. Infect. fungi identified as nonsporulating molds. J. Clin. Microbiol.
Dis. Clin. Pract. 1995;4:58–59. 45, 568, 2007.

17 Piedraia
Dongyou Liu
Contents
17.1 Introduction.......................................................................................................................................................................141
17.1.1 Classification and Morphology..............................................................................................................................141
17.1.3 Diagnosis.............................................................................................................................................................. 142
17.2 Methods............................................................................................................................................................................ 142
17.3 Conclusion.........................................................................................................................................................................143
References...................................................................................................................................................................................143
17.1 Introduction Piedraia hortae colonies are slow growing, small, folded,
velvety, dark brown to black in color; reverse is black.
Clinical presentations of fungal infections in humans gener- Colonies may produce a reddish brown diffusible pigment
ally fall under the following four categories: (i) superficial, and remain glabrous or covered with short aerial hyphae.
(ii) cutaneous, (iii) subcutaneous, and (iv) systemic myco- Hyphae are septate, darkly pigmented, with intercalary
ses. Superficial and cutaneous fungal infections are often chlamydoconidium-like cells. Ascostromata are pseudopar-
restricted to the stratum corneum and its adnexal structures, enchymatous structures of subglobose to irregular shape and
with little or no tissue involvement. black in color, with each ascostromata usually containing a
Black piedra (piedra, meaning “stone” in Spanish) is a single ascus. Asci (with readily dissolvable walls) are ellip-
superficial fungal infection of humans caused by a dematia- soid, solitary, or in clusters and contain eight ascospores.
ceous fungus called Piedraia hortae. This infection typically Ascospores (30–45 μm × 5.5–10 μm) are hyaline to darkly
affects the frontal scalp hair and facial hair, producing darkly pigmented, one celled (aseptate), fusoid, curved, and tapering
pigmented hair-shaft nodules as large as a few millimeters in toward both ends to form the typical whip-like appendages.
diameter. P. hortae is one of only a few pathogenic human fungi that
Black piedra differs from white piedra (trichosporosis) generate sexual spores in its parasitic phase [3,4]. Piedraia
in that white piedra is caused by basidiomycetous arthroco- quintanilhae differs from Piedraia hortae morphologically
nidial yeast Trichosporon spp., producing lightly pigmented, in that its ascospores do not have appendages [5].
white to light-brown, loosely attached hair-shaft nodules
with a soft texture on the scalp hair, pubic hair, axillary
hair, beards, moustaches, eyebrows, and eyelashes. In addi- 17.1.2 Clinical Features and Pathogenesis
tion, black piedra is found mainly in hot and humid tropical Piedraia is a filamentous fungus commonly present in soil
climate, whereas white piedra is prevalent in temperate and as well as stagnant water and crops in tropical regions of the
semitropical climates [1,2]. world. Piedraia hortae, the etiologic agent of black piedra,
has been isolated in tropical South and Central Americas,
Southeast Asia, and Africa. The hot humid environment
together with the habit of using plant oil on hair facilitates
The genus Piedraia is a dematiaceous (dark-walled) mold Piedraia hortae growth [6–8].
belonging to the family Piedraiaceae, order Capnodiales, Superficial infection of hair shafts by Piedraia hortae
subclass Dothideomycetidae, class Dothideomycetes, sub- results in the formation of asymptomatic, brown to black,
phylum Pezizomycotina, phylum Ascomycota, kingdom stone-like concretions (nodules) of up to a few millimeters
Fungi. Being the only member in the family Piedraiaceae, in diameter on the scalp (frontal, occipital, and parietal)
the genus Piedraia consists of two species: Piedraia hortae and facial hair but not beard, moustache, and pubic hair
and Piedraia quintanilhae [2]. While Piedraia hortae is a (commonly known as black piedra). Very firmly attached
keratinolytic fungus, causing black piedra in man, Piedraia to the hair shaft, the nodules are composed of ascostro-
quintanilhae has been isolated from chimpanzees in Central mata, which are the fruiting body of the fungus contain-
Africa, and its pathogenicity in humans is unknown. ing subglobose asci and aseptate ascospores in groups of
141

eight covered with a gelatinous sheath. The periphery of climate, whereas white piedra is endemic in temperate and
the nodule has regularly aligned hyphae and arthroconidia. semitropical climates [2,3,17].
The hard, darkly pigmented hair-shaft nodules of black pie- On the other hand, tinea capitis due to dermatophyte
dra have a gritty feeling and may produce a metallic sound fungi Trichophyton tonsurans, Microsporum audouinii,
when hair is brushed. They rarely produce hair breakage in and M. canis does not form nodules on hair shaft. These
severe cases. organisms affect the base of the hair shaft and the follicle.
Coimbra et al. [9] conducted an epidemiological survey of Trichophyton tonsurans grows inside hair shaft (endothrix-
black piedra among Zoró Indians in the Brazilian Amazon, growing), with arthroconidia forming within hair shaft;
who use plant oil on hair as a custom, and showed that 74 Microsporum audouinii and Microsporum canis grow out-
(56.9%) of the 130 individuals had the infection. Gip [10] side hair shaft (ectothrix-growing), with numerous arthroco-
documented a case of black piedra in a 23-year-old Swedish nidia surrounding hair shaft and destroying its cuticle.
man after his return from 4 months’ stay in India. The patient Direct microscopic examination of infected hair in 10%
presented with typical clinical signs of black piedra of his KOH allows a clear differential diagnosis of black and white
scalp. Black nodules were found around the hair shafts, and piedra, as well as eggs of pediculosis. White and black pie-
the crushed nodules revealed numerous asci and ascospores dra can also be distinguished from the color of the nodules
on microscopy. Piedraia hortae was isolated from the con- (i.e., white to light brown, pale greenish or yellowish in white
cretions. After treatment with oral terbinafine 250 mg daily piedra and black in black piedra) and by the presence of
for 6 weeks, nodules disappeared. ascospores and asci in black piedra and its absence in white
Upon examination of the ultrastructural pattern of human piedra.
hair infection by Piedraia hortae in vivo, Figueras et al. [11] In culture, Piedraia hortae grows slowly at 25°C on
demonstrated that the fungus has the capacity to destroy the Sabouraud’s dextrose agar. As Piedraia hortae is uninhibited
cuticular layers of the hair and to penetrate deeply into the by cycloheximide, it will grow in dermatophyte test media
cortex. The main reasons that guarantee the long survival of (DTM) incorporating cycloheximide (e.g., Mycosel, DTM).
the fungus, and therefore the chronic course of the disease, Trichosporon spp. grow well on Sabouraud’s dextrose agar
are possibly the slow rate of keratin degradation at the cortex at 28°C–30°C. However, they are inhibited by cyclohexi-
together with the compacted stromatic organization of the mide and will not grow in DTM containing cycloheximide.
nodules. Because P. hortae rarely produces ascospores on Sabouraud
agar, special techniques using transplantation and biotine
may be utilized to stimulate its formation of ascospores.
17.1.3 Diagnosis
Piedraia hortae and other hair invading fungal organisms
Besides Piedraia hortae, a number of other fungi are capa- can be identified in a much rapid and precise manner through
ble of infecting human scalp and facial hair. Chief among polymerase chain reaction (PCR) and sequencing analysis
them are white piedra (trichoporosis)-causing basidiomy- of internal transcribed spacer (ITS) and rRNA gene regions
cetous arthroconidial yeast Trichosporon spp. (of which [18,19].
Trichosporon ovoides, Trichosporon inkin, Trichosporon Treatment options for black piedra include shaving or
mucoides, and Trichosporon asahii are linked to white pie- cutting the hair and oral terbinafine. The compact nature of
dra, with T. inkin likely on pubic hair and T. ovoides on head black piedra nodules may compromise the effectiveness of
hair) and tinea capitis-causing dermatophytes Trichophyton antifungal therapy [10,20,21].
tonsurans, Microsporum audouinii, and Microsporum canis
[12]. Occasionally, Acremonium spp., Brevibacterium spp.,
and coryneform bacteria may also be involved in genital 17.2 Methods
white piedra [13]. Other differential consideration is hair lice
(pediculosis capitis).
Clinically, black piedra due to Piedraia hortae generates Hairs with visible nodules are plucked, sectioned, and stained
darkly pigmented, firmly attached hair-shaft nodules (con- with toluidine blue. A 10%–15% KOH solution is used to
cretions) on the scalp and facial hair, whereas white piedra stain hair-shaft nodules on a glass slide; a fungal stain (e.g.,
due to Trichosporon spp. induces lightly pigmented, white to chlorazol black E stain or Parker blue-black ink) is added
light-brown, loosely attached hair-shaft nodules (concretions) to delineate the hyphae. Nodule is crushed, taking care not
on the scalp hair as well as hair in other locations (includ- to break the coverslip. Microscopic observation of septate
ing pubic hair, axillary hair, beards, moustaches, eyebrows, brown hyphae, asci, and fusiform ascospores confirms the
and eyelashes) [11,14]. The nodules of black piedra consist diagnosis. Culture of the organism is carried out on media
of ascostromata (fruiting body) with subglobose asci and with antibacterial agents, as well as media with both antibac-
aseptate ascospores in groups of eight inside the hyphae and terial agents and cycloheximide.
arthroconidia in the periphery; the nodules of white piedra For DNA extraction, fungal isolates are cultured in
have darkly stained hyphae, blastoconidia (2–8 μm long), 20 mL of RPMI 1640 medium with l-glutamine but with-
and arthroconidia fixed to the hair shaft [15,16]. Further, out sodium bicarbonate (Sigma-Aldrich) buffered to pH
black piedra is distributed mainly in hot and humid tropical 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS)

Piedraia 143
(Sigma-Aldrich). After 3–14 days of growth at 30°C under 5. Sequences are analyzed with the SmartGene
agitation (100 rpm), mycelium is transferred into a tube Integrated Database Network software version
and washed in 40 mL of sterile distilled water. Mycelium 3.2.3 vr. SmartGene is a web-based software and
is then stored at −20°C until use. DNA extraction is carried database system with reference sequences derived
out by a glass bead lysis method. A measured quantity of from the National Center for Biological Information
100 mg of mycelium is homogenized for 1 min in a tube con- (NCBI) GenBank repository.
taining 1 mL of lysis buffer (2% Triton X-100, 1% sodium
dodecyl sulfate, 10 mM Tris–HCl pH 8, 100 mM NaCl, 1 mM Note. In case that real time PCR instrument is not available,
ethylenediaminetetraacetic acid [EDTA] pH 8), three 0.5 cm standard PCR may be performed with primers ITSI and
diameter glass beads (Sigma), and approximately 500 mg of ITS4, and the resulting amplicon is sequenced with the same
425–600 μm glass beads (Sigma). The homogenized mycelia primers. Sequence-based identifications are defined by per-
are then snap-frozen in liquid nitrogen, thawed, and refrozen cent identity: species, ≥ 99%; genus, 93%–99%; and incon-
once. DNA extraction is then done with the DNeasy plant kit clusive, ≤ 93%.
(QIAGEN) [22]. For strains producing discrepant identification
between the methods based on phenotypic characteris-
tics and ITS sequence analysis, the D1–D2 region of the
17.2.2 Detection Procedures large-subunit RNA gene is amplified with primers NL1
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for
green DNA binding dye and amplicon melting temperature
species clarification.
analysis for fungal detection also using pan-fungal primers
ITS1 forward (5′-TCCGTAGGTGAACCTGCGG-3′) and
ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′). The
identity of the fungi is verified by subsequent sequencing 17.3 Conclusion
analysis. Piedraia hortae is a keratinophilic dematiaceous fungus
Procedure causing black piedra, which is characterized by the forma-
tion of black, firmly adhered nodules on scalp and facial
1. PCR mixture is composed of 1 × Lightcycler hair. Crushed nodules mounted with KOH (10%–20%) dis-
FastStart DNA Master Hybridization Probes mix- play tightly packed, thick-walled fungal cells and asci con-
ture (Roche Applied Science) (containing deoxy- taining two to eight single-celled, fusiform, slightly curved
nucleoside triphosphates, FastStart Taq DNA ascospores with a single polar filament at each end. Black
polymerase, and 1 mM MgCl2, additional MgCl2 is piedra needs to be differentiated from white piedra (tricho-
added to a final concentration of 4.6 mM), 0.4 μM sporosis) caused by basidiomycetous arthroconidial yeast
each of ITS1 forward and ITS4 reverse primers, Trichosporon spp. as well as tinea capitis caused by der-
1 × SYBR green (Molecular Probes), and 3 μL tem- matophyte fungi Trichophyton tonsurans, Microsporum
plate DNA. audouinii, and Microsporum canis. White piedra is char-
2. Thermal cycling parameters include 95°C for acterized by the formation of white to light brown, easily
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, detachable nodules surrounding hair shaft. Apart from caus-
and 76°C for 30 s; and a final extension at 72°C for ing white piedra, Trichosporon spp. also have the capacity
2 min. to induce systemic infections (termed trichosporonosis) in
3. The quality of the amplicon is determined using the immunocompromised patients. Use of molecular techniques
derivative of the melt analysis curve (55°C–99°C, such as PCR and sequencing analysis enables accurate and
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 rapid identification of Piedraia hortae and other hair invad-
(Corbett Robotics, Inc). ing fungal organisms.
4. The amplified product is purified for bidirectional
microliters of Big Dye Terminator Ready Reaction References
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of 1. Schwartz, P.R.A., Superficial fungal infections. Lancet.
each primer (0.8 pmol/μL) and 3 μL of purified PCR 2004;364:1173–1182.
product. Cycle sequencing is performed with a 9700 2. Bonifaz, A. et al., Tinea versicolor, tinea nigra, white piedra,
thermal cycler (ABI), using 25 cycles of 96°C for and black piedra. Clin Dermatol. 2010;28(2):140–145.
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing 3. Chong, K.C., Adam, B.A., and Soo-Hoo, T.S., Morphology of
Piedra hortae. Sabouraudia. 1975;13:157–160.
4. de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn.,
G-50 fine column to remove unincorporated dye ter- vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, the
minators. Purified sequencing reaction products are Netherlands, 2000.
run on an ABI Prism 3100 Genetic Analyzer with a 5. Larone, D.H., Medically Important Fungi—A Guide to
50 cm capillary array. Identification, 3rd edn. ASM Press, Washington, DC, 1995.

6. Adam, B.A., Soo-Hoo, T.S., and Chong, K.C., Black piedra in 15. de Almeida, H.L. Jr., Rivitti, E.A., and Jaeger, R.G., White
West Malaysia. Aust J Derm. 1977;18:45–47. piedra: Ultrastructure and a new microecological aspect,
7. Venugopal, P.V. and Venugopal, T.V., Superficial mycoses in Mycoses 1990;33:491–497.
Saudi Arabia. Australas J Dermatol. 1992;33:45–48. 16. de Almeida, H.L. Jr., Salebian, A., and Rivitti, E.A.,
8. Kanitakis, J. et al., Black piedra: Report of a French case Ultrastructure of black piedra, Mycoses 1991;34:447–451.
associated with Trichosporon asahii. Int J Dermatol. 17. Ellner, K.M. et al., White piedra: Evidence for a synergistic
2006;45:1258–1260. infection, Br J Dermatol. 1990;123:355–363.
9. Coimbra, C.E.A. and Santos, R.V., Black piedra among 18. Abliz, P. et al., Identification of pathogenic dematiaceous
zoro Indians from Amazonia (Brazil). Mycopathologia fungi and related taxa based on large subunit ribosomal DNA
1989;107:57–60. D1/D2 domain sequence analysis. FEMS Immunol Med
10. Gip, L., Black piedra: The first case treated with terbinafine Microbiol. 2004;40:41–49.
(Lamisil). Br J Dermatol. 1994;130(Suppl 43):26–28. 19. Pounder, J.I. et al., Discovering potential pathogens among
11. Figueras, M.J., Guarro, J., and Zaror, L., New findings in fungi identified as nonsporulating molds. J Clin Microbiol.
black piedra infection. Br J Dermatol. 1996;135:157–158. 2007;45:568–571.
12. Youker, S.R. et al., White piedra: Further evidence of a syner- 20. Gip, L., Terbinafine for black piedra. Lancet 1993;341:1164.
gistic infection, J Am Acad Dermatol. 2003;49:46–49. 21. Drake, L.A. et al., Guidelines of care for superficial mycotic
13. McBride, M.E. et al., A new Brevibacterium species isolated infections of the skin: Piedra—Guidelines/Outcomes
from infected genital hair of patients with white piedra, J Med Committee, J Am Acad Dermatol. 1996;34:122–124.
Microbiol. 1993;39:255–261. 22. Desnos-Ollivier, M. et al., Molecular identification
14. Figueras, M.J., Guarro, J., and Zaror, L., Ultrastructural of black-grain mycetoma agents. J Clin Microbiol.
aspects of hair digestion in black piedra infection. J Med Vet 2006;44(10):3517–3523.
Mycol. 1997a;35:1–6.

18 Pyrenochaeta
Dongyou Liu
Contents
18.1 Introduction...................................................................................................................................................................... 145
18.1.2 Clinical Features................................................................................................................................................... 146
18.1.3 Diagnosis...............................................................................................................................................................147
18.2 Methods............................................................................................................................................................................ 148
18.2.1.1 Procedure for Endophthalmitis Samples............................................................................................... 148
18.2.1.2 Procedure for Keratitis Samples............................................................................................................ 148
18.2.1.3 DNA Extraction..................................................................................................................................... 148
18.2.2.1 Standard PCR and Sequencing Analysis of ITS Region....................................................................... 149
18.2.2.2 Real-Time PCR and Sequencing Analysis of ITS Region..................................................................... 149
18.3 Conclusion........................................................................................................................................................................ 149
References.................................................................................................................................................................................. 150
18.1 Introduction Nimbya, Pithomyces, Pyrenochaeta, Stagonospora,

Stemphylium, Ulocladium, and Unifilum. In turn, the
Pyrenochaeta is dark-walled, coelomycete genus in the order genus Pyrenochaeta contains eight recognized spe-
Dothideales that produces asexual fruit bodies during its cies: Pyrenochaeta acicola, Pyrenochaeta gentianicola,
life cycle. Members of this genus are widely distributed in Pyrenochaeta inflorescentiae, Pyrenochaeta lycopersici,
the environment, soil, wood, and plant debris and are also Pyrenochaeta nobilis, Pyrenochaeta romeroi, Pyrenochaeta
encountered as plant pathogens. Several Pyrenochaeta spe- terrestris, and Pyrenochaeta unguis-hominis, in addi-
cies such as P. romeroi and P. mackinnonii are involved as tion to six unassigned species [1]. The teleomorphs of the
agents of chronic, suppuratives, and subcutaneous infections Pyrenochaeta genus are found in the genus Herpotrichia
in immuno-competent patients after traumatic implantation, (obsolete synonyms: Herpotrichiopsis and Lasiophoma) and
ultimately leading to mycetoma. In addition, Pyrenochaeta possibly also in the genus Leptosphaeria, both of the family
species are occasionally implicated in onychomycosis and Leptosphaeriaceae [2,3].
keratitis as well as deep, non-mycetomatous infections. Pyrenochaeta spp. are saprophytic fungal organisms
Although Pyrenochaeta is a relatively infrequent cause that inhabit the soil and plant debris, particularly in tropi-
of human diseases in comparison with black yeast genera cal and subtropical areas. Many Pyrenochaeta spp. are
Cladophialophora, Exophiala, and Fonsecaea, in the order pathogenic to plants, and several are among the rare causes
Chaetothyriales, their similarity in morphological, biologi- of human infections. The human pathogenic Pyrenochaeta
cal, and clinical terms make correct identification of these species include Pyrenochaeta keratinophila, Pyrenochaeta
organisms essential in order to implement effective control mackinnonii, Pyrenochaeta romeroi (obsolete synonym:
and prevention strategies. Phlenodomus avramii), and Pyrenochaeta unguis-hominis
[3]. Pyrenochaeta romeroi and Pyrenochaeta mackinnonii
have been isolated from mycetoma, containing soft, irregu-
lar, and black grains with a subhyaline center. A number of
The genus Pyrenochaeta is a dematiaceous (dark- isolates previously recognized as Madurella grisea have also
walled) filamentous fungus belonging to the mito- been reidentified as Pyrenochaeta romeroi [4]. Pyrenochaeta
sporic Pleosporaceae group, family Pleosporaceae, unguis-hominis has been isolated from the infected nails of
order Pleosporales, subclass Pleosporomycetidae, class some cases. Pyrenochaeta keratinophila is a new species
Dothideomycetes, subphylum Pezizomycotina, phylum that was recently isolated from corneal scrapings of a case of
Ascomycota, kingdom Fungi. The mitosporic Pleosporaceae keratitis in Spain [3].
group covers 13 genera: Alternaria, Dendryphiella, Pyrenochaeta colonies grow moderately rapidly and
Dendryphion, Drechslera, Embellisia, Exserohilum, appear flat, woolly to cottony, white initially and becoming
145

olivaceous green to olivaceous gray with age; reverse is dark. cells (12–18 × 2–3.5 μm) are phialidic, mostly cylindri-
Hyphae are septate, hyaline to subhyaline. Pycnidia (singular cal. Conidia (2–4 × 1–2 μm) are whitish in mass, ellipsoi-
pycnidium, which is a round or flask-shaped fruiting body dal, wide, straight or slightly curved, hyaline, and smooth
containing conidia) are globose to flask shaped, ostiolate, walled [2].
brown to black, with setae (rigid hair located on the pycnidia) Although P. keratinophila and P. unguis-hominis have
arising from their upper portion. Phialides arise from the similar conidiophores and conidia, they can be distinguished
inner lining of the pycnidia. Conidia (2–4 × 1–2 μm) are one by the color of the colonies and the location of the setae on
celled, oval to cylindrical, hyaline, and straight or slightly the pycnidia. P. keratinophila colonies on OA are gray oli-
curved [5,6]. vaceous to greenish olivaceous, while P. unguis-hominis
At the species level, Pyrenochaeta keratinophila colonies colonies are brown vinaceous to fawn. The pycnidial setae of
reach a diameter of 24–30 mm on oatmeal agar (OA) at 20°C P. keratinophila are scarce and placed mainly near the ostiole,
for 10 days, with even margin, and are colorless, flat; immersed while those of P. unguis-hominis are usually more abundant
mycelium is pale gray olivaceous to greenish olivaceous, and more dispersed over the entire surface of the pycnidium.
aerial mycelium is concolorous, diffuse, woolly, floccose; P. keratinophila also produces conidia from the mycelium, a
reverse is olivaceous gray to olivaceous black. Colonies reach feature unknown in P. unguis-hominis or any other species
a diameter of 18–23 mm on malt extract agar at 20°C for 10 of the genus Pyrenochaeta. P. mackinnonii is distinguished
days, with even margin and buff color; immersed mycelium from P. keratinophila by having more restricted, raised, or
is olivaceous black or brown vinaceous; aerial mycelium is wrinkled colonies. P. romeroi is distinguished by its grayish-
dense, felty to woolly, pale to olivaceous gray; reverse is dark sepia to fuscous-black colonies and the presence of discrete
hazel. Superficial pycnidia are abundant. On OA, pycnidia conidiogenous cells [3].
(100–400 μm in diameter) are olivaceous brown to almost
black, globose or flask shaped, single or confluent, with one
to three ostioles (10–25 μm in diameter), displaying hyaline,
thick-walled periphyses; pycnidial walls of textura angula- The term “dematiaceous fungi” refers to a heterogeneous
ris comprise cells (4–9 mm in diameter) with dark-brown group of fungal organisms with black filaments (dematiaceae)
intercellular material. The outer surface of the pycnidial wall that may cause opportunistic superficial or deep infections
displays scarce, brown, slightly roughened, septate setae in humans, producing a diverse range of clinical syndromes
(20–35 × 2.5–4 μm), with a blunt apex, mostly positioned (collectively known as phaeohyphomycosis, chromoblasto-
near the ostiole. Conidiogenous cells (12–23 × 2–3.5 μm) mycosis, and mycetoma). To date, about 130 species belong-
arise from the entire inner surface of the pycnidial wall and ing to 70 dematiaceous fungal genera have been shown to
are rarely discrete, ampuliform to doliform, mostly cylindri- induce these clinical diseases.
cal, and integrated in conidiophores, which are branched at Mycetoma is a highly debilitating disease that is char-
the base, acropleurogenous (i.e., having terminal and lateral acterized by a subcutaneous mass with multiple sinuses
apertures), phialidic, with a distinct periclinal thickening and draining pus and grains (<0.5–2 mm in diameter), which
sometimes with a short collarette. In older cultures, conidiog- may appear white (i.e., white-grain mycetoma), black (black-
enous cells often show several distinct percurrent prolifera- grain mycetoma), yellow, or red. This disease can be caused
tions. Conidia (2–4 × 1–2 μm) are whitish in mass; ellipsoid, by fungi (eumycetoma) or actinomycetes (actinomycetoma).
straight, or slightly curved; hyaline, continuous, smooth, While eumycetoma usually produces either white or black
with granular contents or few guttules; and rounded at both grains, actinomycetoma may show white, yellow, or red
ends. Phialoconidia arising directly from solitary apertures grains [7].
in the aerial mycelium in 3-week-old cultures are initially The most common fungal species involved in white-grain
indistinguishable from conidia originating from pycnidia but eumycetoma include Acremonium (Fusarium) falciforme,
later become inflated unilaterally, globose or subglobose, Acremonium kiliense, Acremonium recifei, Cylindrocarpon
3–4 μm in diameter, pale brown, and slightly thick walled. destructans, Fusarium moniliforme, Fusarium solani,
Chlamydospores-like structures (8–10 μm in diameter) also Neotestudina rosatii, and Pseudallescheria boydii, whereas
form on old OA cultures and are terminal, hyaline, globose, common fungal species responsible for black-grain eumyce-
smooth, and thick walled. Molecular data suggest that the toma are Exophiala jeanselmei, Madurella grisea, Madurella
closest sexual state of P. keratinophila is Leptosphaeria mycetomatis, Leptosphaeria tomkinsii, Leptosphaeria sene-
(family Leptosphaeriaceae) [3]. galensis, Pyrenochaeta mackinnonii, Pyrenochaeta romeroi,
Pyrenochaeta unguis-hominis pycnidia (100–400 μm and Phlenodomus avramii.
in diameter) are olivaceous brown to black, globose or As saprophytes commonly occurring in soil and plants,
flask shaped, with one to three ostioles, and with several Pyrenochaeta spp. may gain entry to human hosts through
setae positioned near the ostiole. The pycnidial wall is of direct traumas by a plant or a soiled object. With kerato-
textura angularis with dark brown intercellular material. mycosis, a number of factors may predispose the hosts to
The conidiophores emerging from all over the inner sur- Pyrenochaeta and other fungal infections. These include
face of the pycnidial wall are branched at the base, bearing chronic ocular surface disease, dry eye, contact lens wearing,
terminal and lateral conidiogenous cells. Conidiogenous atopic disease, topical steroid use, long-term treatment with

Pyrenochaeta 147
broad-spectrum antibiotics, and trauma (particularly with Ferrer et al. [2] described a Pyrenochaeta unguis-
vegetable material or soil) [8]. In addition, patients with sup- hominis-related case of keratitis in a 77-year-old diabetic
pressed immune functions are vulnerable to Pyrenochaeta woman with pain and decreased vision in her left eye. Slit
and other fungal diseases [9,10]. lamp examination showed conjunctival hyperemia, periph-
First identified in 1959, Pyrenochaeta romeroi has been eral corneal pannus, corneal edema, and a central corneal
reported in mycetoma cases involving mainly immunosup- ulcer (4 mm in diameter) with white stromal infiltrates
pressed individuals [11–13]. In recent years, it has been inferiorly. Corneal scrapings were plated on Columbia agar
shown that besides eumycetoma, P. romeroi may also be plates supplemented with 5% sheep blood, Columbia choc-
responsible for non-mycetoma phaeohyphomycosis. Girard olate agar, MacConkey agar, thioglycolate broth, and brain
et al. [14] reported a case of non-mycetoma deep cutaneous heart infusion broth (BioMérieux); and incubated at 37°C.
infection due to Pyrenochaeta romeroi in an immunocom- Corneal scraping and biopsy taken from the ulcer edge
promised 45-year-old man who originated from Senegal. were also inoculated onto Sabouraud dextrose agar with
The patient had a multibacillary leprosy treated by sequential chloramphenicol and incubated at 30°C. Direct examina-
antibiotics for more than 20 years, and presented with non- tion of the corneal scrapings and biopsy using Gram and
inflammatory, painful and deep lesions of both lower limbs calcofluor white stains showed the presence of numerous
reminiscent of cold abscesses. Clinical examination revealed septate and branched hyphae. After 6 days, a filamentous
a large subcutaneous, flaccid lesion of the lateral side of the fungus grew on chocolate agar, and a panfungal polymerase
left leg, with an occasional purulent white-yellow discharge chain reaction (PCR) generated a 532 bp amplicon, which
through superficial sinus tracts, a 2 cm large nodule of the showed 86% homology to Leptosphaeria strain. The fun-
right tibial crest and two crusted papulo-nodules of the left gus was subcultured on malt extract agar and oatmeal agar
foot. X-rays showed no bone alteration underlying the cuta- (30 g oat flakes, 1 g MgSO 4, 1.5 g KH2PO4, 15 g agar, 1 L
neous lesions, while ultrasound examination of the left leg tap water) at 20°C in the dark. After 1 week of incubation,
uncovered a 20 mm long, 5 mm large, and 6 mm thick cav- pycnidia typical of the coelomycetous genus Pyrenochaeta
ity with multiple internal septa, extending to the subcutane- developed, and the fungus was identified as Pyrenochaeta
ous fat but without obvious involvement of the underlying unguis-hominis.
muscle or bone. A deep biopsy sample from the larger cuta- Recently, Verkley [3] described the first case of
neous lesion showed a chronic granulomatous dermal infil- Pyrenochaeta keratinophila-associated keratitis in a
trate with giant cells and microabscesses and the presence of 77-year-old diabetic woman in Spain. The patient presented
septate filaments scattered within the infiltrate upon periodic with conjunctival hyperaemia, peripheral corneal pannus,
acid-Schiff (PAS) staining. Direct examination of purulent corneal edema, and a central ulcer with white infiltrates in her
material from leg sinus tract showed no granules but fun- left eye. Calcofluor white staining of corneal biopsy revealed
gal, septate filaments. Culture of the lesion material grew a fungi invading the cornea. Culture of the corneal biopsy on
fungus, which was identified as P. romeroi. After surgical several media grew a fungus that was identified morpho-
excision, drainage of the largest abscess, treatment with oral logically as a Pyrenochaeta species. Sequencing analysis of
itraconazole (100 mg daily) and anti-bacillary antibiotics (clo- the ITS region in the rRNA gene showed it belonged to an
fazimine, rifampicine, dapsone), all cutaneous lesions slowly unknown new species Pyrenochaeta keratinophila. Despite
resolved within 1 month with minimal scarring. Itraconazole treatment with topical natamycin and oral ketoconazole for
was interrupted after 4 months, and no relapse occurred dur- 1 month with considerable lesion improvement, the cornea
ing a 1-year follow-up. could not be saved and the patient underwent an optical pen-
In a separate study, Badali et al. [15] documented another etrating keratoplasty.
case of non-mycetomatous infection (subcutaneous pha-
eohyphomycotic cyst) due to Pyrenochaeta romeroi in a
18.1.3 Diagnosis
45-year-old Indian female. The patient presented with a ver-
rucous plaque and a swelling (30 mm in diameter) on the Over 70 dematiaceous fungal genera containing 130 or so
right forearm that gradually increased in size over a period distinct species have been implicated in human phaeohypho-
of 3 months. Direct microscopic examination with 10% KOH mycosis, chromoblastomycosis, and mycetoma. Given that
and histopathological investigation of exudates showed sep- the clinical symptoms caused by these fungi are largely indis-
tate hyphae without granules. A fungus was isolated from tinguishable and nonspecific, and that different fungal taxa
the exudates in culture and identified as Pyrenochaeta often display varied sensitivity to antifungal drugs, there is
romeroi on the basis of morphologic features and sequence a necessity to precisely determine the species identity of the
homology in the internal transcribed spacer (ITS) regions suspected fungal organisms for tailor-made, cost-effective
of ribosomal RNA (rRNA). Treatment consisted of surgi- treatment.
cal excising of the cyst without any antifungal therapy, with Together with clinical and epidemiological information,
no relapse during a 1-year follow-up. It is possible that the identification of dematiaceous fungi including Pyrenochaeta
apparent immune dysfunction in this patient might contrib- species relies on the observation of mycotic elements in
ute partially to the lack of granule formation characteristic biopsy and other specimens by microscopy. Subsequent
of mycetoma. isolation of suspected fungal organisms in culture media

facilitates detection of characteristic colonial and micro- 18.2.1.3 DNA Extraction

scopic features, leading to the confirmation of the species Pyrenochaeta isolates are grown on potato dextrose agar
identity. Unfortunately, this testing scheme can take up to 12 on 9 cm diameter petri dishes at 25°C until mycelium com-
weeks to complete. With cultures that are negative or con- pletely covers the agar surface. Mycelia are collected by add-
taminated with bacteria, culturing with fresh samples can ing sterile distilled water containing 0.05% (v/v) Tween 80 to
further delay the diagnosis. the surface of the culture and gently scrubbing with a sterile
To improve the efficiency and specificity of laboratory spatula. The mycelial suspension is transferred to a 1.5 mL
diagnosis of fungal pathogens, molecular methods have tube and centrifuged at 3000 × g at 4°C for 5 min. The super-
been developed and applied in recent years [16–21]. PCR natant is discarded and the pellet (100 mg mycelia) is stored
amplification and sequencing analysis of the ITS1–5.8S– at −80°C until further use.
ITS2 rRNA have enabled precise identification of the fungi Extraction buffer (0.2 mL) (3% sodium dodecyl sulfate
responsible for black-grain mycetoma [4,22,23]. In addition, [SDS] [w/v] containing 0.5 mM ethylenediaminetetraace-
sequencing examination of the D1–D2 regions in the large tic acid [EDTA], 1.0 M NaCl, and 0.1 mM hydroxymethyl-
subunit (LSU) rRNA D1–D2 region offers another poten- hydrochloride Tris–HCl pH 8.0) is added to 10 mg of each
tial avenue for taxonomical determination of dematiaceous fungal mycelium and the suspension shaken vigorously for
fungi. Furthermore, combination of a modified FTA extrac- 15 s. Next, 0.2 mL chloroform-phenol mix (1:1) is slowly
tion protocol with pyrosequencing of ITS2 region makes added and incubated at 65°C for 5 min. The mixture is cooled
it possible to identity a fungal culture within half a day to room temperature and centrifuged at 10,000 × g at 4°C
[24,25]. for 5 min. The supernatant is transferred to a new microtube,
and an equal volume of cold absolute isopropanol or ethanol
is added and the contents mixed thoroughly for precipitating
18.2 Methods total DNA at −20°C for 20 min; the mixture is then centri-
18.2.1 Sample Preparation fuged at 10,000 × g for 10 min. The pellet is washed twice
with 75% ethanol and centrifuged at 10,000 × g at 4°C for
18.2.1.1 Procedure for Endophthalmitis Samples 5 min.
The extraocular environment is sterilized with 5% povi- The supernatant is discarded and the pellet is resuspended
done iodine solution before surgery. About 100–200 μL of in 0.03 mL MiniQuantum (deionized) water, and stored at
aqueous fluid is withdrawn using a 30 gauge needle with a −80°C until further use. Concentration, yield, and quality
limbal paracentesis. Vitreous samples (200 μL) are taken at control indices based on absorbance readings at 230, 260,
the time of three-port pars plana vitrectomy. The samples and 280 nm (A260/280 and A260/230 ratios) are carried out
are divided into two aliquots and transported at 4°C. One with 2 μL resuspended total DNA [26].
portion is immediately examined by conventional microbio- Alternatively, fungal strains are cultured in 20 mL of
logical diagnostic tests and the other is frozen at −20°C until RPMI 1640 medium with l-glutamine but without sodium
processed by PCR. bicarbonate (Sigma-Aldrich) buffered to pH 7.0 with
For the microbiological diagnostic test, 50 μL of aqueous 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma-
humor or 50 μL of vitreous is cultured at 30°C in Sabouraud’s Aldrich). After 3–14 days of growth at 30°C under agitation
dextrose agar or at 37°C in thioglycolate broth, blood agar, (100 rpm), mycelium is transferred into a tube and washed
chocolate agar, cystine lactose electrolyte deficient (CLED) in 40 mL of sterile distilled water. Mycelium is then stored
agar, or MacConkey agar. Filamentous fungi are differentiat −20°C until use. DNA extraction is carried out by a glass
ated by isolation in Sabouraud dextrose agar plus chloram- bead lysis method. Approximately 100 mg of mycelium is
phenicol and morphological examination of macroscopic and homogenized for 1 min in a tube containing 1 mL of lysis
microscopic characteristics. Microscopic structures may also buffer (2% Triton X-100, 1% SDS, 10 mM Tris–HCl pH 8,
be observed on tease or tape preparations and slide cultures 100 mM NaCl, 1 mM EDTA pH 8), three 0.5 cm diameter
for up to 21 days [18]. glass beads (Sigma), and approximately 500 mg of 425–
600 μm glass beads (Sigma). The homogenized mycelia are
18.2.1.2 Procedure for Keratitis Samples then snap-frozen in liquid nitrogen, thawed, and refrozen
Upon completion of the ocular examination and after instil- once. DNA extraction is then done with the DNeasy plant
lation of topical anesthetic, a sterile Kimura spatula is used kit (QIAGEN) [4].
to scrape the area of infection. Scrapings are inoculated into Furthermore, Whatman FTA filter may be used for DNA
thioglycolate broth, Roiron broth, and Löwenstein-Jensen extraction. Whatman FTA filter matrices are fibrous cards
medium and are placed onto glass slides for staining with pretreated with chelators and denaturants that lyse and inac-
Gram and Giemsa stains. The PCR sample is obtained by tivate most microorganisms on contact. The large nucleic
scraping and stirring the spatula for a few seconds in 100 μL acids released after lysis become physically entangled in the
of sterile water in a 1.5 mL sterile Eppendorf tube. Two ali- fibers of the FTA matrix, whereas cellular debris is rapidly
quots of 50 μL are taken from each sample and stored at removed by washing the inoculated card. Fungal hyphae
−20°C [18]. fragments and conidia (in the case of moulds) are added

Pyrenochaeta 149
to dry FTA filters, followed by brief microwave treatment, mixture (Roche Applied Science) (containing
allowed liberation of amplifiable DNA with a total prepara- deoxynucleoside triphosphates, FastStart Taq DNA
tion time of ∼15 min [25]. polymerase, and 1 mM MgCl2, additional MgCl2 is
added to a final concentration of 4.6 mM), 0.4 μM
each of ITS1 forward and ITS4 reverse primers, 1×
18.2.2 Detection Procedures SYBR green (Molecular Probes), and 3 μL template
18.2.2.1 Standard PCR and Sequencing DNA.
Analysis of ITS Region 2. Thermal cycling parameters include 95°C for
Ferrer et al. [18] utilized universal primers ITS1 (5′-TCC
and 76°C for 30 s; and a final extension at 72°C for
GTA GGT GAA CCT GCG G-3′, which hybridizes at the
2 min.
end of 18S rRNA), and ITS4 (5′-TCC TCC GCT TAT TGA
3. The quality of the amplicon is determined using the
TAT GC-3’, which hybridizes at the beginning of 28S rRNA)
[27] to amplify a region covering ITS/5.8S rRNA for identifi-
cation of pathogenic fungi.
(Corbett Robotics, Inc).
Procedure 4. The amplified product is purified for bidirectional
1. PCR mixture (50 μL) is made up of 10 μL of DNA microliters of Big Dye Terminator Ready Reaction
template, 6 μL of 25 mM MgCl2, 5 μL of PCR buffer Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
without MgCl2, 200 μM each deoxynucleoside tri- each primer (0.8 pmol/μL) and 3 μL of purified PCR
phosphate, 25 pmol of each primer, and 1 U of Taq product. Cycle sequencing is performed with a 9700
DNA polymerase (Biotools B&M Labs). thermal cycler (ABI), using 25 cycles of 96°C for
2. Amplification is performed with 1 cycle at 95°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
5 min; 35 cycles of 95°C for 30 s, 55°C for 1 min, and reaction products are passed through a Sephadex
72°C for 1 min; and 1 cycle at 72°C for 6 min. G-50 fine column to remove unincorporated dye ter-
3. Aliquots (10 μL) of each amplified product are minators. Purified sequencing reaction products are
e lectrophoretically separated in a 2% agarose gel in run on an ABI Prism 3100 Genetic Analyzer with a
1× Tris–borate–EDTA buffer and visualized using 50 cm capillary array.
ethidium bromide under UV illumination. 5. Sequences are analyzed with the SmartGene
4. PCR amplicon is purified using the GeneClean II Integrated Database Network software version
kit (Bio 101) and directly cycle sequenced in both 3.2.3 vr. SmartGene is a web-based software and
directions using the BigDye terminators Ready database system with reference sequences derived
Reaction Kit (PE Applied Biosystems) on an ABI from the National Center for Biological Information
Prism automated DNA sequencer (model 377, ver- (NCBI) GenBank repository.
sion 2.1.1; Applied Biosystems), using primers ITS4
and ITS86. Note. Sequence-based identifications are defined by percent
5. The 550 bp ITS2/5.8S rRNA sequences are ana- identity: species, ≥99%; genus, 93%–99%; and inconclusive,
lyzed by using the BLAST alignment program of the ≤93%.
GenBank database. Multiple-sequence alignment is For strains producing discrepant identification
carried out using ClustalW 1.8. Phylogenetic trees between the methods based on phenotypic character-
are constructed by the neighbor-joining method using istics and ITS sequence analysis, the D1–D2 region
the Phylip package and visualized using Treeview. of the LSU RNA gene is amplified with primers NL1
18.2.2.2 Real-Time PCR and Sequencing (5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for
species clarification.
Analysis of ITS Region
Pounder et al. [23] described a real-time PCR with SYBR green
DNA binding dye and amplicon melting temperature analysis 18.3 Conclusion
for fungal detection also using panfungal primers ITS1 for-
ward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 reverse The genus Pyrenochaeta consists of eight dematiaceous fun-
(5′-TCCTCCGCTTATTGATATGC-3′). The identity of the gal species that are soil saprophytes with potential to cause
fungi is verified by subsequent sequencing analysis. infections in plants. Four Pyrenochaeta species have been
associated with occasional infections in humans, producing
Procedure clinical presentations ranging from mycetoma, subcutane-
ous infections, keratitis, and other diseases, especially in
1. PCR mixture is composed of 1× Lightcycler immunocompromised individuals. Because clinical diseases
FastStart DNA Master Hybridization Probes induced by Pyrenochaeta spp. are indistinguishable from

those caused by other 70 dematiaceous fungal genera (and 12. Rinaldi, M.G., Phaeohyphomycosis. Dermatol Clin.
130 distinct species), and because individual dematiaceous 1996;14:147–153.
fungal taxa often demonstrate differing resistance to com- 13. Cerar, D. et al., Isolation, identification and susceptibility of
Pyrenochaeta romeroi in a case of eumycetoma of the foot in
monly used antifungal drugs, it is important to determine the
the UK. Int J Antimicrob Agents 2009;34(6):617–618.
species identity of the suspected fungal organisms. 14. Girard, C. et al., Subcutaneous phaeohyphomycosis due to
Although it is possible to correctly identify dematia- Pyrenochaeta romeroi in a patient with leprosy. Acta Derm
ceous fungi including Pyrenochaeta species on the basis Venereol. 2004;84(2):154–155.
of macroscopic and microscopic criteria, the whole process 15. Badali, H. et al., Subcutaneous phaeohyphomycotic cyst caused
is notoriously time consuming and technically demanding. by Pyrenochaeta romeroi. Med Mycol. 2010;48(5):763–768.
Application of molecular methods such as PCR amplification 16. Chen, Y.-C. et al., Identification of medically important yeasts
and sequencing analysis of the ITS1-5.8S-ITS2 rRNA and using PCR-based detection of DNA sequence polymorphisms
in the internal transcribed spacer region 2 of the rRNA genes.
other gene regions offers the opportunity to vastly enhance J Clin Microbiol. 2000;38:2302–2310.
the specificity and speed of fungal detection and determi- 17. Chen, Y.-C. et al., Polymorphic internal transcribed spacer
nation. In addition, these techniques also enable improved region 1 DNA sequences identify medically important yeasts.
phylogenetic analysis and epidemiological tracking of future J Clin Microbiol. 2001;39:4042–4051.
outbreaks due to dematiaceous fungi, leading to the imple- 18. Ferrer, C. et al., Detection and identification of fungal patho-
mentation of highly effective treatment and control against gens by PCR and by ITS2 and 5.8S ribosomal DNA typing in
these emerging infective organisms. ocular infections. J Clin Microbiol. 2001;39:2873–2879.
19. Ahmed, A.O. et al., Molecular detection and identification of
agents of eumycetoma: Detailed report of two cases. J Clin
References Microbiol. 2003;41:5813–5816.
20. Schwarz, P. et al., Molecular identification of zygomyce-
1. The UniProt Consortium. Available at http://www.uniprot. tes from culture and experimentally infected tissues. J Clin
org/, accessed on August 1, 2010. Microbiol. 2006;44:340–349.
2. Ferrer, C. et al., New Pyrenochaeta species causing keratitis. 21. Linton, C.J. et al., Molecular identification of unusual patho-
J Clin Microbiol. 2009;47:1596. genic yeast isolates by large ribosomal subunit gene sequenc-
3. Verkley, G.J.M., Pyrenochaeta keratinophila sp. nov., iso- ing: 2 years experience at the UK Mycology Reference
lated from an ocular infection in Spain. Rev Iberoam Micol. Laboratory. J Clin Microbiol. 2007;5:1152–1158.
2010;27:22–24. 22. Iwen, P.C., Hinrichs, S.H., and Rupp, M.E., Utilization of
4. Desnos-Ollivier, M. et al., Molecular identification the internal transcribed spacer regions as molecular targets
of black-grain mycetoma agents. J Clin Microbiol. to detect and identify human fungal pathogens. Med Mycol.
2006;44(10):3517–3523. 2002;40:87–109.
5. Kwon-Chung, K.J. and Bennett, J.E., Medical Mycology. Lea 23. Pounder, J.I. et al., Discovering potential pathogens among
& Febiger, Philadelphia, PA, 1992. fungi identified as nonsporulating molds. J Clin Microbiol.
6. de Hoog, G.S. et al., Atlas of Clinical Fungi. Centraalbureau 2007;45(2):568–571.
voor Schimmelcultures, Utrecht, the Netherlands, 2000. 24. Borman, A.M. et al., Molecular identification of pathogenic
7. de Hoog, G.S. et al., Diagnostic problems with imported cases fungi. J Antimicrob Chemother. 2008;61:S7.
of mycetoma in The Netherlands. Mycoses 1993;36:81–87. 25. Borman, A.M. et al., An improved protocol for the preparation
8. Thomas, P.A., Fungal infections of the cornea. Eye of total genomic DNA from isolates of yeast and mould using
2003;17:852–862. Whatman FTA filter papers. Mycopathologia 2010;169:445.
9. Andre, M. et al., Fungal mycetoma with black grains due to 26. González-Mendoza, D. et al., A rapid method for isolation of
Pyrenochaeta romeroi in Cambodia. Bull Soc Pathol Exot Fil. total DNA from pathogenic filamentous plant fungi. Genet
1968;61:108–112. Mol Res. 2010;9(1):162–166.
10. English, M.P., Infection of the finger-nail by Pyrenochaeta 27. White, T.J. et al., Amplification and direct sequencing
unguis-hominis. Br J Dermatol. 1980;103:91–93. of fungal ribosomal RNA genes for phylogenetics. In
11. Thammayya, A., Sanyal, M., and Basu, N., Pyrenochaeta M.S. Innis and D.H. Gelfand (Eds.), PCR Protocols: A
romeroi causing mycetoma pedis in India. J Indian Med Guide to Methods and Applications. Academic Press, New
Assoc. 1979;73:66–67. York, 1990, pp. 315–322.

19 Ramichloridium
Dongyou Liu
Contents
19.1 Introduction...................................................................................................................................................................... 151
19.1.3 Diagnosis.............................................................................................................................................................. 153
19.2 Methods............................................................................................................................................................................ 153
19.3 Conclusion........................................................................................................................................................................ 154
References.................................................................................................................................................................................. 154
19.1 Introduction (→ Rhinocladiella basitona), Ramichloridium cerophi-

lum (→ Rhinocladiella aquaspersa), Ramichloridium fas-
Ramichloridium is an anamorph genus that comprises
ciculatum (→ Rhinocladiella fasciculata), Ramichloridium
multiple species of saprobes, human and, plant pathogens.
mackenziei (formerly Ramichloridium obovoidea and
Morphologically, these organisms possess erect, dark,
Ramichloridium obovoiedum) (→ Rhinocladiella macken-
branched or unbranched conidiophores and predominantly
ziei), Ramichloridium musae (formerly Veronaea musae)
aseptate conidia produced on a sympodially proliferating
(→ Periconiella musae), and Ramichloridium subulatum
rachis. Currently, Ramichloridium schulzeri is the main
(→ Radulidium subulatum) (http://www.indexfungorum.
Ramichloridium species involved in human disease, and
org/) [5–9].
other previous human pathogens within the genus, that is,
Ramichloridium colonies are flat to raised with entire
Ramichloridium mackenziei, Ramichloridium basitonum,
margin; surface is olivaceous-green to olivaceous-black.
and Ramichloridium musae have been now reclassified as
Mycelium consists of submerged and aerial hyphae: sub-
Rhinocladiella mackenziei, Rhinocladiella basitonum, and
merged hyphae are hyaline to subhyaline and thin walled
Periconiella musae, respectively.
whereas aerial hyphae are smooth or verrucose. Conidiophores
are straight, unbranched, rarely branched, thick walled, dark
brown (darker than the subtending hyphae), and continuous
or with several additional thin septa. Conidiogenous cells
The genus Ramichloridium is a dematiaceous (dark- are integrated, terminal, polyblastic, smooth, thick walled,
walled) fungus belonging to the mitosporic Capnodiales golden-brown, apical part subhyaline, with sympodial prolif-
group, order Capnodiales, class Dothideomycetes, sub- eration, straight or flexuose, geniculate or nodose, with con-
phylum Pezizomycotina, phylum Ascomycota, kingdom spicuous conidiogenous loci. Scars are crowded or scattered,
Fungi. Currently, the genus Ramichloridium consists of unthickened, unpigmented to faintly pigmented, or slightly
nine recognized species: Ramichloridium apiculatum (the prominent denticles. Conidia are solitary, with 0–1 septum,
type species of the genus), Ramichloridium australiense, subhyaline to pale brown, smooth to coarsely verrucose, thin
Ramichloridium biverticillatum, Ramichloridium brasilia- walled, obovate, obconical or globose to ellipsoidal, fusiform,
num, Ramichloridium epichloes, Ramichloridium indicum with a slightly pigmented hilum; conidial secession is schizo-
(basionym: Chloridium apiculatum), Ramichloridium pini, lytic. The type species of the genus is R. apiculatum [7,8].
Ramichloridium schulzeri (obsolete synonyms: Acrotheca At the species level, Ramichloridium apiculatum (basi-
acuta, Chloridium schulzeri, Pleurophragmium acu- onym: Chloridium apiculatum) colonies reach a diameter of
tum, Psilobotrys schulzeri, Rhinocladiella schulzeri and 35 mm after 14 days at 24°C on malt extract plates (MEA),
Rhinotrichum multisporum), and Ramichloridium strelitziae, with minimum growth temperature of above 6°C, opti-
in addition to five unassigned species [1–4]. No teleomorph mum temperature at 24°C, maximum temperature at 30°C.
has been linked to the species of Ramichloridium so far. Colonies are raised, velvety, dense, with entire margin; sur-
Other former species in the genus include face is olivaceous-green, reverse is olivaceous-black, often
Ramichloridium anceps (formerly Veronaea parvispora) with a diffusing citron-yellow pigment. Submerged hyphae
(→ Rhinocladiella anceps), Ramichloridium basitonum (1–2.5 μm wide) are hyaline to subhyaline, thin walled; aerial
151

hyphae are slightly darker, smooth walled. Conidiophores Ramichloridium brasilianum colonies are slow-growing,
(up to 100 μm long) generally arise at right angles from reaching a diameter of 6 mm after 14 days at 24°C on MEA,
creeping aerial hyphae, straight, unbranched, thick walled, and appear velvety to hairy, with entire margin; surface is
dark brown, continuous or with 1–3 additional thin septa; dark olivaceous-gray; black gelatinous exudate droplets pro-
intercalary cells are 10–28 μm long. Conidiogenous cells duced on oatmeal agar (OA). Submerged hyphae (1.5–2 μm
(25–47 × 2–3.5 μm) are integrated, terminal, smooth, thick wide) are pale olivaceous, smooth or slightly rough; aer-
walled, golden-brown, straight, cylindrical, proliferating ial hyphae are olivaceous, smooth or rough, narrower
sympodially, resulting in a straight rachis with conspicu- and darker than the submerged hyphae. Conidiophores
ous conidiogenous loci. Scars (less than 1 μm in diam- (2–2.5 μm × 70 μm) are unbranched, arising vertically from
eter) are prominent, crowded, slightly pigmented. Conidia creeping aerial hyphae, straight or flexuose, dark brown,
(3–7.5 × 2–4 μm) are solitary, obovate to obconical, pale with up to 10 additional septa, thick walled, and cylindri-
brown, finely verrucose. Hilum (about 1 μm in diameter) is cal. Conidiogenous cells (10–30 μm long) are integrated,
conspicuous, slightly pigmented [7,8]. terminal, proliferating sympodially, giving rise to a long,
Ramichloridium australiense colonies are slow growing, straight rachis with crowded, slightly darkened minute scars
reaching a diameter of 8 mm after 14 days at 24°C on MEA, (about 0.5 μm in diameter). Conidia (4–8.5 μm × 2–3 μm)
with entire, smooth margin. Mycelium is flat, olivaceous- are solitary, obovoid to fusiform with the widest part below
gray, becoming granular, with gelatinous droplets at the the middle, thin walled, verruculose, aseptate, pale brown,
margin developing with age; reverse is pale olivaceous-gray. slightly rounded at the apex, truncated at the base, with a
Submerged hyphae (1–2 μm wide) are hyaline, smooth, thin slightly thickened and darkened hilum (1–1.5 μm in diam-
walled; aerial hyphae are pale brown, warted. Conidiophores eter) [7,8].
arise vertically and clearly differentiate from creeping aerial Ramichloridium indicum (basionym: Chloridium indi-
hyphae, up to 400 μm tall, with several additional thin septa. cum; synonyms: Veronaea indica and Veronaea verrucosa)
Intercalary cells (8–40 μm × 2–5 μm) arise from the broad- colonies reach a diameter of 35 mm after 14 days at 24°C
est part at the base tapering toward the apex, are subhya- on MEA. Colonies are velvety, rather compact, slightly ele-
line, later become pale brown and warted in the lower part. vated, with entire, smooth, whitish margin, dark olivaceous-
Subtending hyphae are thick walled, warted. Conidiogenous green in the central part. Submerged hyphae (1–2.5 μm
cells (10–18 μm long) are integrated, terminal, proliferating wide) are smooth, thin walled, hyaline, with thin septa;
sympodially, giving rise to a short rachis with conspicuous aerial hyphae (2–2.5 μm wide) are coarsely verrucose, oli-
conidiogenous loci. Scars (about 1 μm in diameter) are slightly vaceous-green, thick walled, with thin septa. Conidiophores
thickened and darkened. Conidia (10–23 μm × 2.5–3 μm) are (250 μm × 2–4 μm) arise vertically from creeping hyphae at
solitary, aseptate, thin walled, smooth, subhyaline, subcy- right angles, are straight, unbranched, thick walled, smooth,
lindrical to obclavate, with a truncated base and a slightly dark brown, with up to 10 thin septa, often with inflated basal
darkened and thickened hilum (1.5–2 μm in diameter), rarely cells. Conidiogenous cells (up to 165 μm long) are terminally
fusing at the basal part [7,8]. integrated, smooth, dark brown, sympodially proliferating,
Ramichloridium biverticillatum (basionym: Periconiella rachis straight or flexuose, geniculate or nodose, subhyaline;
musae) is named after its biverticillate conidiophores. scars (about 0.5 μm in diameter) are thickened and darkened,
Colonies are slow growing, reaching a diameter of 16 mm clustered at nodes. Conidia (5–10 μm × 4–9 μm) are solitary,
after 14 days at 24°C on MEA, with entire, smooth, sharp with 0–1 septum, not constricted at the septum, subhyaline to
margin, compact, and velvety. Surface is vinaceous-buff to pale brown, smooth or coarsely verrucose, rather thin walled,
olivaceous-buff; reverse is buff. Submerged hyphae (1–2 μm broadly ellipsoidal to globose, with truncated base; hilum
wide) are smooth, hyaline, thin walled; aerial hyphae (about 1 μm in diameter) is conspicuous, slightly darkened,
are subhyaline, smooth, slightly darker. Conidiophores not thickened [7,8].
(2–3 μm × 250 μm) arise vertically from creeping aerial Ramichloridium schulzeri colonies grow moderately
hyphae, pale brown, profusely branched, biverticillate, rapidly, and appear compact, flat; submerged hyphae are
with up to three levels of main branches, which taper dis- pale orange; aerial hyphae are powdery, brownish; reverse
tally. Conidiogenous cells (15–50 μm long) are terminally is pink to orange. Conidiophores (up to 250 μm long) are
integrated, cylindrical, variable in length, rachis straight or erect, straight, unbranched, thick walled, reddish-brown,
geniculate, pale brown, as wide as the basal part, elongating gradually becoming paler toward the apex, elongat-
sympodially, forming a rachis with crowded, slightly dark- ing sympodially during conidiogenesis, with scattered,
ened and thickened minute scars (<0.5 μm wide). Conidia pimple-shaped conidium bearing denticles, which have
(2–5 × 1.5–2.5 μm) are solitary, aseptate, hyaline to sub- unpigmented scars. Conidia (6.5–37 μm × 3–4 μm) are sub-
hyaline, dacryoid to pyriform, smooth, thin walled, with hyaline, smooth walled or slightly rough walled, ellipsoi-
an inconspicuous hilum. Ramichloridium biverticillatum is dal, obovoidal or fusiform, usually with an acuminate base
a new name based on Periconiella musae. It is differenti- and unpigmented scars. Ramichloridium schulzeri, includ-
ated from Periconiella musae by having profusely branched ing its varieties, is phylogenetically as well as morphologi-
conidiophores, and smaller conidia (2–5 μm × 1.5–2.5 μm) cally distinct from the other genera in the Ramichloridium
than those of Periconiella musae (5–11 μm × 2–3 μm) [7,8]. complex [7,8].

Ramichloridium 153
Ramichloridium strelitziae (named after its host, Strelitzia) 19.1.3 Diagnosis

colonies are slow growing, reaching a diameter of 5 mm after
14 days at 24°C on MEA, with entire margin; aerial myce- The genus Ramichloridium is characterized by the presence
lium is compact, raised, dense, olivaceous-gray; reverse is of erect, dark, differentiated, branched or unbranched conid-
olivaceous-black. Submerged hyphae (2–2.5 μm wide) are iophores and predominantly aseptate conidia produced on a
smooth, hyaline, thin walled; aerial hyphae are pale brown, sympodially proliferating rachis [2].
verrucose. Conidiophores (40 μm × 2 μm) arise vertically Morphologically and phylogenetically, Ramichloridium
from creeping aerial hyphae, clearly differentiated from the demonstrates close relation to Rhinocladiella and Veronaea.
vegetative hyphae, subhyaline, later becoming pale brown, Ramichloridium and Rhinocladiella are separated mainly
thick walled, smooth, or verruculose, with 1–3 additional on the basis of (i) macronematous conidiophores in
septa. Conidiogenous cells (10–35 μm long) are integrated, Ramichloridium versus micronematous conidiophores in
terminal, cylindrical, subhyaline, later turning pale brown, Rhinocladiella and (ii) presence of a yellow or an orange
fertile part as wide as the basal part, proliferating sympo- diffusible pigment in Ramichloridium and absence in
dially, forming a straight rachis with slightly thickened and Rhinocladiella [3].
darkened, circular, somewhat protruding scars (about 0.5 μm Other useful features for differentiation among
in diameter). Conidia (3–5.5 μm × 1–2.5 μm) are solitary, Ramichloridium, Rhinocladiella, and Veronaea include
aseptate, smooth or verruculose, subhyaline, oblong, ellip- (i) the absence of exophiala-type budding cells in
soidal to clavate, with truncated base and unthickened, non- Ramichloridium and Veronaea and its presence in
pigmented hilum [7,8]. Rhinocladiella; (ii) production of largely two-celled conidia
Phylogenetically, the genus Ramichloridium is related to (one-septate) by Veronaea and one-celled conidia (aseptate)
the genera Periconiella, Rhinocladiella and Veronaea. The by Ramichloridium and Rhinocladiella [10].
main morphological feature to distinguish Ramichloridium Nonetheless, identification of Ramichloridium from
from Rhinocladiella is the presence of exophiala-type bud- Rhinocladiella and Veronaea on the basis of macroscopic
ding cells in the species of Rhinocladiella [3] (see also and microscopic characteristics are not only time consum-
Chapter 43 of this book). Although both Ramichloridium and ing, but also technically challenging. For these reasons,
Veronaea lack exophiala-type budding cells, Veronaea is dif- molecular techniques targeting the sequence diversity of the
ferentiated from Ramichloridium by having predominantly internal transcribed spacer (ITS) region of ribosomal RNA
1-septate conidia (i.e., two-celled conidia) (in comparison (rRNA), as well as small subunit (SSU) and large subunit
with one-celled or aspetate conidia of Ramichloridium). The (LSU) rRNA, have been developed and applied for improved
largely aseptate conidia are also found in Rhinocladiella [10]. differentiation of Ramichloridium from Rhinocladiella and
Periconiella differs from Veronaea chiefly by its dark brown, Veronaea [18–20].
apically branched conidiophores [11].
Members of the genus Ramichloridium are largely soil 19.2 Methods
saprobes. However, Ramichloridium pini is a known plant
pathogen, causing a needle disease on Pinus contorta [3], and
Ramichloridium schulzeri may be occasionally involved in Biopsy samples are examined under microscope for mycotic
human infection. elements using various stains. Samples are also cultured on
inhibitory mold agar, modified Sabouraud agars, or potato
dextrose agar (PDA). Colony colors (surface and reverse) are
assessed after 2–4 weeks on different media at 25°C in the
The genus Ramichloridium represents 1 of 70 dematiaceous dark. Isolates are cultured on 2% MEA by obtaining single
filamentous fungal genera that are largely saprophytes, but conidial colonies. Colonies are subcultured onto fresh MEA,
have the potential to cause phaeohyphomycosis, chromo- OA, PDA, and synthetic nutrient-poor agar (SNA) and incu-
blastomycosis, and eumycetoma in humans, especially those bated at 25°C under continuous near-ultraviolet light to pro-
with suppressed immune functions [12–16]. Ramichloridium mote sporulation [6,8].
schulzeri was reported in a case of “golden tongue” involving Microscopic structures are observed on tease or tape prep-
a 54-year-old woman with acute lymphocytic leukemia. The arations and slide cultures for up to 21 days. Slide cultures are
patient was neutropenic, with an erosive lesion on the left set up in Petri dishes containing 2 mL of sterile water, into
side of the tongue, which extended over the dorsum of the which a U-shaped glass rod is placed, extending above the
tongue and appeared golden orange. Microscopic examina- water surface. A block of freshly growing fungal colony, about
tion of surface scrapings revealed branching septate myce- 1 cm2, is placed onto a sterile microscope slide, covered with a
lia, and culture of a biopsy specimen of the tongue grew a somewhat larger, sterile glass cover slip and incubated in the
few colonies of a fungus. The fungus was golden orange on moist chamber. Fungal sporulation is monitored over time,
Sabouraud’s glucose agar and brown-gray on corn-meal agar and when optimal, images are captured by means of a Nikon
and was identified as Ramichloridium schulzeri. The lesion camera system (Digital Sight DS-5M, Nikon Corporation,
was resolved after the patient underwent a week therapy with Japan). Structures are mounted in lactic acid and 30 measure-
amphotericin B [17]. ments (×1000 magnification) are determined [10].

Genomic DNA is isolated from fungal mycelium grown 7. Other measures calculated include tree length, con-
on MEA, using the UltraClean™ Microbial DNA Isolation sistency index, retention index, and rescaled con-
Kit (Mo Bio Laboratories) according to the manufacturer’s sistency index (TL, CI, RI, and RC, respectively).
protocols. Alternatively, genomic DNA is extracted follow- The robustness of the obtained trees is evaluated
ing the cetyltrimethylammonium bromide (CTAB)-based by 1000 bootstrap replications. Bayesian analysis is
protocol [6,8]. performed. The best nucleotide substitution model
is determined using MrModeltest v. 2.2. MrBayes v.
3.1.2 is used to perform phylogenetic analyses, using
a general time-reversible (GTR) substitution model
Arzanlou et al. [10] utilized the universal primers ITS1 and with inverse gamma rates, dirichlet base frequen-
ITS4 [21] to amplify the ITS region of the nuclear rRNA cies and the temp value set to 0.5.
operon, including: the 3′ end of the 18S rRNA gene, the first
internal transcribed spacer region (ITS1), the 5.8S rRNA Note. Part of the LSU 28S rRNA gene may be also ampli-
gene, the second internal transcribed spacer region (ITS2) fied with primers LR0R and LR5 followed by sequencing
and the 5′ end of 28S rRNA gene. Subsequent sequencing analysis.
analysis allows identification of fungal organisms including
Chaetomium species.
Procedure 19.3 Conclusion
The genus Ramichloridium consists of nine dematiaceous
1. PCR mixture (25 μL) is composed of 0.5 U Taq polyfungal species of which Ramichloridium schulzeri is involved
merase (Bioline), 1× PCR buffer, 0.5 mM MgCl2, in human infection. Due to the fact that Ramichloridium spp.
0.2 mM of each dNTP, 5 pmol of each primer, closely resembles Rhinocladiella (which contains a signifi-
approximately 10–15 ng of fungal genomic DNA. cant human pathogenic species Rhinocladiella mackenziei)
2. Amplification is performed on a GeneAmp PCR and Veronaea morphologically, it is important to determine
System 9700 (Applied Biosystems) with primary these organisms to species level. As phenotypical methods
denaturation at 96°C for 5 min; 36 cycles of 96°C for for identification of Ramichloridium, Rhinocladiella, and
30 s, 52°C for 30 s, and 72°C for 60 s; a final exten- Veronaea lack desired speed, accuracy, and convenience,
sion at 72°C for 7 min. molecular techniques such as PCR and sequencing analysis
3. The amplicons are sequenced using BigDye of the ITS region of rRNA, as well as SSU and LSU rRNA,
Terminator v. 3.1 (Applied Biosystems,) or DYEna offer a valuable alternative for improved differentiation of
micET Terminator (Amersham Biosciences) Cycle these organisms.
Sequencing Kits and analyzed on an ABI Prism
3700 (Applied Biosystems).
4. Newly generated sequences are subjected to a Blast References
search of the NCBI databases, sequences with high
similarity are downloaded from GenBank and com- 1. The UniProt Consortium. Available at http://www.uniprot.
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weight. Branches of zero length are collapsed and all 8. Crous PW et al., Unravelling Mycosphaerella: Do you believe
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Mycol. 2010;48(3):546–556. Ramichloridium schulzeri. Arch Dermatol. 1985;121:892–894.
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20 Ulocladium
Dongyou Liu
Contents
20.1 Introduction...................................................................................................................................................................... 157
20.1.1 Classification......................................................................................................................................................... 157
20.1.2.1 Ulocladium chartarum.......................................................................................................................... 158
20.1.2.2 Ulocladium botrytis............................................................................................................................... 158
20.1.2.3 Ulocladium atrum.................................................................................................................................. 158
20.1.3 Diagnosis.............................................................................................................................................................. 158
20.2 Methods............................................................................................................................................................................ 159
20.2.2.1 Standard PCR Amplification and Sequencing Analysis of ITS Region................................................ 159
20.2.2.2 Real-Time PCR Amplification and Sequencing Analysis of ITS Region............................................. 159
20.3 Conclusion........................................................................................................................................................................ 160
References.................................................................................................................................................................................. 160
20.1 Introduction Ulocladium subcucurbitae, and Ulocladium tuberculatum, as

well as 25 unassigned species [1].
The genus Ulocladium covers a number of saprotrophic, As saprophytes inhabiting the soil and decaying her-
darkly pigmented hyphomycetes that share similar morpho- baceous plants, Ulocladium spp. are widely distributed
logical characteristics with some saprotrophic Alternaria in nature and have been isolated from paper, textiles, and
species. The morphological separation between the genera wood. Although often considered as contaminants, some
Ulocladium and Alternaria hinges on whether develop- Ulocladium spp. (e.g., Ulocladium chartarum, Ulocladium
ing conidia are ovoid or obovoid, in addition to their subtle botrytis, and Ulocladium atrum) are occasionally implicated
differences in the pigmentation and verrucosity of mature in human disease process, causing subcutaneous phaeohy-
conidia. Although Ulocladium spp. have been often consid- phomycosis, onychomycosis, keratitis, and other infections.
ered as contaminants in medical microbiology laboratories, Ulocladium grows moderately rapidly. Colonies are wooly
there is increasing evidence that supports the role of these (suede-like) to cottony (floccose), brown to olivaceous-black
organisms in the pathogenesis of human mycoses. or grayish on potato dextrose agar (PDA) at 25°C. Hyphae
are brown, septate. Conidiophores are simple or branched,
smooth, strongly geniculate (bent at the points where the
conidia are produced, leading to a zigzag or bent knee appear-
The genus Ulocladium (obsolete synonym: Pseudo ance) and bear the conidia. Conidia (13–30 μm × 6–19 μm)
stemphylium) is a dematiaceous (dark-walled) filamentous are brown to black, typically obovoid (narrowest at the base),
fungus belonging to the mitosporic Pleosporaceae group, fam- smooth or rough and verrucous. These conidia are typi-
ily Pleosporaceae, order Pleosporales, class Dothideomycetes, cally muriform with transverse and longitudinal septations,
subphylum Pezizomycotina, phylum Ascomycota, kingdom appear solitary (Ulocladium botrytis) or form short chains
Fungi. The mitosporic Pleosporaceae group consists of 13 (Ulocladium chartarum). Solitary, multicelled conidia (dic-
genera: Alternaria, Dendryphiella, Dendryphion, Drechslera, tyoconidia) are formed through a pore (poroconidia) by
Embellisia, Exserohilum, Nimbya, Pithomyces, Pyrenochaeta, a sympodially elongating geniculate conidiophore. When
Stagonospora, Stemphylium, Ulocladium, and Unifilum. In chains are produced, a tubular, short outgrowth is formed
turn, the genus Ulocladium is separated into 14 recognized spe- on the conidia at the point of secondary conidium formation
cies: Ulocladium alternariae, Ulocladium atrum, Ulocladium [2,3].
botrytis, Ulocladium capsicum, Ulocladium chartarum, Previously, Ulocladium chartarum had been included in
Ulocladium consortiale, Ulocladium cucurbitae, Ulocladium the genus Alternaria as A. chartarum and A. stemphylioides.
dauci, Ulocladium multiforme, Ulocladium obovoid- Ulocladium differs from Alternaria by its strongly geniculate
eum, Ulocladium oudemansii, Ulocladium septosporum, (zigzag) conidiophores and the absence of beak-like tapered
157

apex of conidia. It differs from Bipolaris, Curvularia, and 6 months of antifungal therapy, the surgical wound had fully
Drechslera by producing muriform conidia. Ulocladium is healed with no relapse of the lesion.
differentiated from Stemphylium by having geniculate, sym-
podial conidiophores. 20.1.2.2 Ulocladium botrytis
Romano et al. [8] reported a Ulocladium botrytis-related case
of disto-lateral onychomycosis of the third toe of the right
foot in a 45-year-old man is reported. Culture of pathologi-
Ulocladium spp. are dematiaceous fungi that exist as sap- cal material grew a mold that was identified as Ulocladium
robes on rotten plant material and soil. These organisms botrytis based on the macro and microscopic characteris-
appear to have low pathogenicity and are occasionally asso- tics of the colonies. After 3 months of topical therapy with
ciated with subcutaneous infection, keratitis, and onychomy- ciclopirox olamine, the lesion was completed resolved.
cosis in humans, especially those under immunosuppressive
therapies or after a local trauma. 20.1.2.3 Ulocladium atrum
Badenoch et al. [9] described a case of Ulocladium atrum
20.1.2.1 Ulocladium chartarum keratitis in a 43-year-old man. The patient presented with
Several cases of cutaneous infections due to U. chartarum have corneal ulcer in his right eye, showing photophobia, marked
been reported in the literature. Because U. chartarum was for- conjunctival injection, corneal edema, Descemet’s membrane
merly regarded as a member of the genus Alternaria, most of folding, a central stromal infiltrate with a feathery edge, and
these cases were described as being alternariosis [4–6]. a reduced visual acuity (VA) (for hand motions only) in the
Duran et al. [7] documented a cutaneous mycoses caused right eye. Gram staining of corneal scrapings showed sep-
by Ulocladium chartarum in a 62-year-old male heart trans- tate hyphae, and culture of the material on Sabouraud’s dex-
plant recipient under immunosuppressive therapy (consisting trose agar (without antibiotics) grew a visible colony at 28°C
of tacrolimus 2 mg/day, azathioprine 100 mg/day, and predni- after 4 days, which reached a diameter of 2 cm by day 8 and
sone 10 mg/day). The patient noticed a painless, flesh-colored appeared grayish brown and powdery. Under microscope,
cutaneous lesion on his right toe over the previous 4 weeks, conidia were dark brown, coarsely verrucose with transverse
with no fever, malaise, or sweating and no history of trauma. and longitudinal septa. Subcultures of the isolate on corn-
The lesion appeared as a 6 × 6 cm sharply demarcated plaque meal agar at 23°C or 28°C revealed long, flexuous, or simple
on the dorsal area of his right big toe, with a granular surface to short, geniculate, and branched conidiophores. Conidia
and a vermiculate consistency. In cutaneous biopsy tissue appearing within 48 h of subculture were spherical to ellip-
sections stained with hematoxylin–eosin, numerous rounded, soidal and mostly single. By 72 h, septate conidia became
refringent, hyaline or slightly eosinophilic thick-walled fun- dark brown, verrucose at either temperature, with the septa
gal structures, together with a few elongated budding yeast- often intersecting at right angles. The isolate was identified
like forms and branched septate hyphae, were observed in as a Ulocladium species on morphological grounds, most
the granuloma and within the giant cells present. Fungal closely resembling U. atrum, and internal transcribed spacer
elements were also revealed by periodic acid-Schiff (PAS) (ITS) sequence analysis confirmed its U. atrum identity.
and Grocott-Gomori methenamine-silver nitrate stains, After receiving hourly eye drops of natamycin (5%) and flu-
but brown pigment was detected in fungal cell walls with conazole (0.2%; the neat intravenous preparation), the patient
Masson-Fontana stain. The lesion was completely removed was comfortable, with clinical signs disappearing in 4 weeks
by surgery, and culture of the lesion tissue homogenate on and an improved VA (20/20).
bacteriologic (blood, chocolate, and MacConkey agars and
thioglycolate broth) and mycologic media (Sabouraud dex-
20.1.3 Diagnosis
trose agar [SDA] with and without chloramphenicol and gen-
tamicin and brain heart infusion agar with 5% of blood) grew Ulocladium represents one of the dematiaceous mold gen-
a mold after 2 days at 30°C and 35°C, respectively. Subculture era that are ubiquitously distributed in the environment and
of the mold on PDA generated powdery to lanose and black to that may transiently colonize the respiratory, integument, and
olivaceous black colonies of 6 cm in diameter after 7 days at gastrointestinal systems of human hosts, with the potential
30°C, which grew more slowly at 35°C. Microscopic exami- to take advantage of the weakened host defense mechanisms
nation of slide cultures on PDA demonstrated a mycelium (or injury), causing a diverse range of clinical diseases (often
with pale yellow or brown septate hyphae. Conidiophores referred to as phaeohyphomycosis, chromoblastomycosis,
were erect, geniculate, simple or branched and golden brown. and eumycetoma) [10–12]. In the case of Ulocladium spp.,
Conidia were brown and verrucose with transverse and longi- the most common clinical presentations are subcutaneous
tudinal septa, solitary or in chains through apical production infections in addition to keratitis and onychomycosis.
of short conidiophores (false beaks). The fungus was thus Preliminary diagnosis of phaeohyphomycoses includ-
identified as U. chartarum. Treatment with oral itraconazole ing Ulocladium infections are through demonstration of
(400 mg/day) began after complete surgical removal of the brownish hyphal and/or yeast-like elements in tissue, often
lesion, with a reduced intake of tacrolimus and prednisone with melanin-specific Fontana-Masson stain. However, the
and switch of azathioprine to mycophenolate mofetil. After dark pigmented elements of some dematiaceous fungi (e.g.,

Ulocladium 159
Alternaria infectoria and Ulocladium chartarum) may not be 20.2.2 Detection Procedures
visible histologically by Fontana-Masson staining [9,13]. This
is clearly demonstrated by Badenoch et al. [9] in their dealing 20.2.2.1 Standard PCR Amplification and
of Ulocladium chartarum, whose dematiaceous nature was Sequencing Analysis of ITS Region
only evident after examination of a cultured isolate. Badenoch et al. [9] utilized universal fungal primers
Because of the close morphological and clinical similari- ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
ties between Ulocladium and Alternaria, including the for- (5′-TCCTCCGCTTATTGATATGC-3′) to amplify the ITS
mation of brown multiseptate conidia and the induction of region of the rRNA gene complex, incorporating ITS1, the
nonspecific cutaneous infections, Ulocladium needs to be 5.8S gene, and ITS2 [22].
distinguished from Alternaria. Morphologically, Ulocladium Procedure
differs from Alternaria by having obovoid, coarsely ver-
rucose conidia with tapered and narrow bases and with no 1. PCR mixture (25 μL) is made up of 1× GeneAmp
or short, spindle-shaped apices (false beaks). Furthermore, PCR buffer (Applied Biosystems), 5% glycerol,
Ulocladium conidia are in short chains or non-catenate. In 125 μM each deoxynucleoside triphosphate, 0.5 μM
addition, Ulocladium species should not be confused with each primer (ITS1 and ITS4), 1.25 U of Taq DNA
other poroconidial genera such as Stemphylium, Bipolaris, polymerase (Applied Biosystems), and 10 μL of DNA.
Exserohilum, Dreschlera, and Curvularia [14,15]. 2. Amplification is conducted with an initial denatur-
Polymerase chain reaction (PCR) amplification and ation at 94°C for 2 min; 30 cycles of 94°C for 15 s,
sequencing analysis of the rRNA internal transcribed spacer 55°C for 30 s, and 72°C for 30 s; and a final exten-
(ITS) regions allows clear separation of Ulocladium from sion of 72°C for 6 min.
Alternaria and other fungal genera [9,13,15–19]. 3. The amplified product is purified using a GFX PCR
DNA and gel band purification kit (Amersham
Biosciences) and then sequenced using the ITS1
20.2 Methods primer and a BigDye Terminator v. 3.1 cycle
20.2.1 Sample Preparation sequencing kit in an ABI PRISM 3100-Avant
genetic analyzer (Applied Biosystems).
Clinical specimens are examined microscopically with fun- 4. The sequence is edited using Chromas v. 2.23
gal stains and also inoculated onto SDA for fungal culture software (Technelysium Pty. Ltd.), and a 520 base
[20]. Microscopic structures are observed on tease or tape sequence is compared with other sequences at
preparations and slide cultures for up to 21 days. GenBank (02/06) using FASTA.
Susceptibility of Ulocladium isolates to amphotericin B,
flucytosine, fluconazole, itraconazole, ketoconazole, vori-
20.2.2.2 Real-Time PCR Amplification and
conazole is performed using the National Committee for
Sequencing Analysis of ITS Region
Clinical Laboratory Standards M38-A method for molds.
For Ulocladium susceptibility to natamycin and terbinafine, Pounder et al. [18] described a real-time PCR with SYBR green
an agar disk method adapted from M38-A is carried out. DNA binding dye and amplicon melting temperature analysis
Namely, Neo-Sensitabs tablets (Rosco Diagnostica, Taastrup, for fungal detection also using pan-fungal primers ITS1 for-
Denmark) containing either diffusible natamycin (50 μg) or ward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 reverse
terbinafine (30 μg) are incorporated into RPMI 1640 agar (5′-TCCTCCGCTTATTGATATGC-3′). The identity of the
supplemented with glucose (0.2%) and buffered with MOPS fungi is verified by subsequent sequencing analysis.
(morpholinepropanesulfonic acid; 0.165 M). The inoculum Procedure
is standardized between 0.4 × 104 and 5 × 104 CFU/mL using
a spectrophotometer. The plates are incubated at 35°C and 1. PCR mixture is composed of 1× Lightcycler FastStart
examined at 48 and 72 h. The zone sizes indicate the sensitiv- DNA Master Hybridization Probes mixture (Roche
ity of the fungus to natamycin and terbinafine [9,21]. Applied Science) (containing deoxynucleoside tri-
For DNA extraction, the isolate is cultured on PDA at phosphates, FastStart Taq DNA polymerase, and
30°C for 5 days. A suspension to a McFarland standard of 1 mM MgCl2, additional MgCl2 is added to a final
2.0 is prepared in saline (2 mL) and centrifuged. The pellet concentration of 4.6 mM), 0.4 μM each of ITS1 for-
is resuspended in 200 μL of sorbitol buffer containing 200 U ward and ITS4 reverse primers, 1× SYBR green
of lyticase (Sigma-Aldrich) and incubated at 37°C for 60 min (Molecular Probes), and 3 μL template DNA.
and centrifuged (5400 × g; 5 min). Spheroplasts are resus- 2. Thermal cycling parameters include 95°C for
pended in 180 μL of lysis solution T and 20 μL of protein- 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and
ase K (GenElute Mammalian Genomic DNA Miniprep kit; 76°C for 30 s; and a final extension at 72°C for 2 min.
Sigma-Aldrich) and then incubated at 55°C for 60 min. DNA 3. The quality of the amplicon is determined using the
is extracted according to the manufacturer’s instructions with derivative of the melt analysis curve (55°C–99°C,
a final elution volume of 200 μL. Samples are stored at −20°C 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
until use [9]. (Corbett Robotics, Inc).

4. The amplified product is purified for bidirectional 2. Simmons, E. G. 1997. Multiplex conidium morphol-
sequencing using ExoSAP-IT (USB Corp). Five ogy in species of the Ulocladium atrum group. Can J Bot.
microliters of Big Dye Terminator Ready Reaction 76:1533–1539.
3. de Hoog, G. S. et al. (eds.). 2000. Atlas of Clinical Fungi,
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
2nd edn. Centraalbureau voor Schimmelcultures, Utrecht, the
each primer (0.8 pmol/μL) and 3 μL of purified PCR Netherlands.
product. Cycle sequencing is performed with a 9700 4. Srinivasan, M. et al. 1997. Epidemiology and aetiological
thermal cycler (ABI), using 25 cycles of 96°C for diagnosis of corneal ulceration in Madurai, south India. Br J
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing Ophthalmol. 81:965–971.
reaction products are passed through a Sephadex 5. Magina, S. et al. 2000. Cutaneous alternariosis by Alternaria
G-50 fine column to remove unincorporated dye ter- chartarum in a renal transplanted patient. Br J Dermatol.
minators. Purified sequencing reaction products are 142:1261–1262.
6. Williamson, E. C. et al. 2000. Diagnosis of invasive asper-
run on an ABI Prism 3100 Genetic Analyzer with a gillosis in bone marrow transplant recipients by polymerase
50 cm capillary array. chain reaction. Br J Haematol. 108:132–139.
5. Sequences are analyzed with the SmartGene 7. Duran, M. T. et al. 2003. Cutaneous infection caused by
Integrated Database Network software version Ulocladium chartarum in a heart transplant recipient: Case
3.2.3 vr. SmartGene is a web-based software and report and review. Acta Derm Venereol. 83:218–221.
database system with reference sequences derived 8. Romano, C. et al. 2004. Onychomycosis due to Ulocladium
from the National Center for Biological Information botrytis. Mycoses 47:346–348.
9. Badenoch, P. R. et al. 2006. Ulocladium atrum keratitis. J Clin
(NCBI) GenBank repository.
Microbiol. 44, 1190–1193.
10. Ajello, L. 1986. Hyalohyphomycosis and phaeohyphomyco-
Note. Sequence-based identifications are defined by percent sis: Two global disease entities of public health importance.
identity: species, ≥99%; genus, 93%–99%; and inconclusive, Eur J Epidemiol. 2:243–251.
≤93%. 11. Rinaldi, M. G. 1996. Phaeohyphomycosis. Dermatol Clin.
For strains producing discrepant identification 14:147–153.
between the methods based on phenotypic characteris- 12. Thomas, P. A. 2003. Current perspectives on ophthalmic
mycoses. Clin Microbiol Rev. 16:730–797.
13. de Hoog, G. S. and R. Horré. 2002. Molecular taxonomy
large-subunit RNA gene is amplified with primers NL1 of the Alternaria and Ulocladium species from humans
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 and their identification in the routine laboratory. Mycoses
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for 45:259–276.
species clarification. 14. Simmons, E. G. 1967. Typification of Alternaria, Stemphylium,
and Ulocladium. Mycologia 59:67–92.
15. Pryor, B. M. and D. M. Bigelow. 2003. Molecular character-
20.3 Conclusion ization of Embellisia and Nimbya species and their relation-
The genus Ulocladium consists of a large number of sap- ship to Alternaria, Ulocladium, and Stemphylium. Mycologia
95:1141–1154.
rotrophic, darkly pigmented hyphomycetes that occur as
16. Pryor, B. M. and R. L. Gilbertson. 2000. Molecular phylo-
saprophytes in soil and decaying herbaceous plants. Several genetic relationships amongst Alternaria species and related
Ulocladium spp. (e.g., Ulocladium chartarum, Ulocladium fungi based upon analysis of nuclear ITS and mtSSU rDNA
botrytis, and Ulocladium atrum) are occasionally implicated sequences. Mycol Res. 104:1312–1321.
in human disease process, causing subcutaneous phaeohy- 17. Meklin, T. et al. 2004. Quantitative PCR analysis of house
phomycosis, onychomycosis, keratitis, and other infections. dust can reveal abnormal mold conditions. J Environ Monit.
Because Ulocladium spp. often appear in laboratory cul- 6:615–620.
tures for dermatophytes and are regarded as contaminants, 18. Pounder, J. I. et al. 2007. Discovering potential pathogens
among fungi identified as nonsporulating molds. J Clin
confirmation of the causative role of Ulocladium species in Microbiol. 45(2):568–571.
human disease relies on the presence of the clinical appear- 19. Bagyalakshmi, R. et al. 2008. Newer emerging pathogens
ance that are consistent with a fungal infection, the observa- of ocular non-sporulating molds (NSM) identified by poly-
tion of hyphae in biopsy tissue, and the culture isolation of merase chain reaction (PCR)-based DNA sequencing tech-
the organism with characteristic colonial and microscopic nique targeting internal transcribed spacer (ITS) region. Curr
morphology. Application of molecular techniques especially Eye Res. 33(2):139–147.
sequence analysis of the ITS region of rRNA genes provides 20. Thomas, O. A. et al. 1991. Use of lactophenol cotton blue
mounts of corneal scraping as an aid to the diagnosis of
a rapid and precise means of identifying Ulocladium spp.
mycotic keratitis. Diagn Microbiol Infect Dis. 14:219–224.
from Alternaria and other fungal taxa that may potentially 21. Pujol, I. et al. 2000. In vitro antifungal susceptibility of
confuse the diagnosis based on phenotypic features. Alternaria spp. and Ulocladium spp. J Antimicrob Chemother.
46:337–338.
References 22. White, T. J. et al. 1990. Amplification and direct sequencing
of fungal ribosomal RNA genes for phylogenetics. In Innis,
1. The UniProt Consortium. Available at http://www.uniprot.org/, M.A. et al. (eds.), PCR Protocols: A Guide to Methods and
accessed on August 1, 2010. Applications. Academic Press, New York, pp. 315–322.



21 Acrophialophora
Dongyou Liu
Contents
21.1 Introduction...................................................................................................................................................................... 163
21.1.1 Classification and Biology.................................................................................................................................... 163
21.1.3 Diagnosis.............................................................................................................................................................. 164
21.2 Methods............................................................................................................................................................................ 165
21.3 Conclusion........................................................................................................................................................................ 166
References.................................................................................................................................................................................. 166
21.1 Introduction gray-brown on PFA. Conidiophores (15 μm × 2–5 μm) arise

singly, terminally, and laterally from thin-walled, hyaline-
With an expanding spectrum of high-risk immunocompro-
to-pale brown septate hyphae (1.5–3.5 μm wide), and appear
mised patients in recent years, clinical cases of human myco-
erect, straight or slightly flexuose, tapering toward the apex,
ses due to saprobic and opportunistic fungi have shown a
pale brown, echinulate, with whorls of phialides on the upper
dramatic increase. Acrophialophora fusispora in the genus
part. Phialides (6–10 μm × 3.5–6 μm) are flask-shaped with a
Acrophialophora represents a poorly known, thermotolerant
swollen base and bear long chains of limoniform-to-fusi-
fungus that has been documented as the cause of human eye
form, one-celled, smooth to finely echinulate, pale brown
and lung infections as well as cerebral brain abscess.
conidia (4–9 μm × 2–6 μm) with indistinct spiral bands [2].
The three species of the genus Acrophialophora are dif-
ferentiated mainly by conidial ornamentation and the degree
21.1.1 Classification and Biology
of development of brown conidia, although they display over-
The genus Acrophialophora belongs to the mitosporic lapping conidial dimensions. A. nainiana produces mature
Ascomycota group, class Dothideomycetes, subphylum conidia (4–10.5 μm × 2–5 μm) that are hyaline and finely echi-
Pezizomycotina, phylum Ascomycota, kingdom Fungi. First nulate; A. fusispora has mature conidia (4–9 μm × 2–6 μm)
described in 1959, the genus currently contains three recog- that are brown, thick walled, and with echinulations in spi-
nized species: Acrophialophora fusispora, Acrophialophora ral bands; and A. levis (4.5–8 μm × 2–3.5 μm) shows mature
levis, and Acrophialophora nainiana, with A. fusispora conidia that are smooth to slightly roughened and hyaline.
(obsolete synonym: Paecilomyces fusisporus) being the type Some considered A. nainiana and A. levis as being synony-
species. Members of the genus Acrophialophora are widely mous for A. fusispora.
distributed in tropical and temperate regions and have been
isolated from soil. Of the three Acrophialophora species,
A. fusispora (originally placed in the genus Paecilomyces) is
a plant pathogen that may cause occasional infections (pha- Acrophialophora fusispora is a thermotolerant soil fungus
eohyphomycosis) in humans in India, the Middle East, Spain, that grows well at 45°C or higher temperatures. The fungus
and the United States [1]. is neurotropic and has been reported as the etiologic agent
Acrophialophora fusispora grows moderately to rapidly for a variety of human infections including brain abscess,
(with optimum growth temperature of 40°C and maximum keratitis/keratouveitis, pulmonary infection, and other inva-
growth temperature at 50°C). A. fusispora colonies attain sive diseases [2–5]. In addition, A. fusispora may be involved
diameters of 5–8 cm at 37°C after 7 days (with the average in transient airway colonization without inducing apparent
hourly growth rate of 0.46 mm/h after 3 days, compared with clinical symptoms [6,7].
0.38 mm/h after 7 days), and diameter 3.1–5.2 cm at 30°C Al-Mohsen et al. [2] reported a case of A. fusispora CNS
after 7 days. Colonies are white to buff with darker con- infection in a 12-year-old Sudanese girl with acute lym-
centric circles on Sabouraud dextrose agar (SDA) and are a phoblastic leukemia, who received four courses of induc-
darker gray-brown on potato flakes agar (PFA). The reverse tion chemotherapy with vincristine and prednisone for four
is brownish with centrally darker areas on SDA and a solid weeks. The patient was febrile with no focus of infection.
163

A computed tomography (CT) scan of the chest revealed The second case involved a 33-year-old Portuguese male
multiple nodular densities; and CT and magnetic resonance with bronchiectasis after measles, who underwent cortico-
imaging of the brain demonstrated a brain abscess in the steroid therapy after developing transplant rejection therapy
parieto-occipital area. Thick yellowish pus (consisting of 2 years earlier. The patient presented with severe dyspnea,
multiple tiny pieces of grayish soft and necrotic white tissue) fever, leukocytosis, C-reactive protein elevation, hypoxemia,
was drained from the abscess. Gomori methenamine silver and bilateral interstitial infiltrate on the chest x-ray. A single
(GMS) stain of the brain abscess aspirate revealed numer- fungus was isolated from several sputum and bronchoal-
ous branching septate hyphae, and culture of this material on veolar lavage (BAL) samples in routine mycological culture
SDA yielded several colonies of a mold that was identified media. Oral treatment with voriconazole (VRC) markedly
initially as a Paecilomyces species based on its formation of improved the existing leukocytosis and C-reactive protein
conidia in short chains. Subculture of the isolate on PFA at normalization.
room temperature in ambient air with alternating daylight The third case was a 67-year-old Spanish male with pul-
and darkness allowed its identification as Acrophialophora monary fibrosis and bullous emphysema who underwent
fusispora. The fungus grew rapidly at both 35°C and 42°C. immunosuppressive therapy after transplantation of the left
Following treatment with liposomal AMB (LAMB) and then lung previously. On day 10 posttransplantation, a filamentous
itraconazole (ITRA), the patient demonstrated clinical and fungus was isolated from respiratory secretions in routine
radiological improvement, with concomitant reduction in mycological culture media. Treatment with nebulized ampho-
size of the brain abscess and resolution of the lung nodules. tericin B (AMB) and itraconazole (ITC) resulted in negative
Arthur et al. [5] described a case of Acrophialophora cultures of the mucous secretions. However, the patient devel-
fusispora keratitis involving a 76-year-old female, which oped pneumonia and died on day 163 posttransplantation
was depicted mistakenly as Scedosporium prolificans in the because of progressive worsening clinical status. Necrotic
original report [8,9]. The patient presented with mild swell- samples from lung, pleural liquid, and myocardium were
ing of the left upper eyelid, and a necrotic mass was visible, positive for E. faecalis and the same fungus isolated before.
which contained numerous, variously sized, cauliflower-like The isolates from the above cases were characterized
growths covered by a whitish membrane on the superior sur- by the presence of conidiogenous cells basally inflated and
face. Hematoxylin-and-eosin-stained sections of the mass with an elongated neck, arising usually singly on vegetative
revealed extensive areas of necrotic tissue surrounding the hyphae or along the length of pale brown, verrucose, and
long-lost contact lens, and septate hyphae within the necrotic thick-walled conidiophores. Limoniform, pale brown, echi-
tissue were visualized with Grocott methenamine silver nulate conidia, often with ornamentation in spiral bands,
nitrate stain. Inoculation of a portion of the mass on SDA forming long chains were also common.
slant grew two colonies of a white mold with hyaline septate In a separate study, Cimon et al. [6] reported four
hyphae after 4 days of incubation at 25°C in air. Subculture cases of transient or chronic airway colonization by
of the mold on potato dextrose agar (PDA) and Mycosel agar Acrophialophora fusispora in patients with cystic fibrosis
at 25°C yielded after 5 days yielded whitish colonies that (CF). In each of these cases, A. fusispora was isolated from
turned dark with age. Lactophenol aniline blue stain of the sputum specimens along with Aspergillus fumigatus. The
isolate showed hyaline septate hyphae bearing flask-shaped A. fusispora isolates obtained were thermotolerant, show-
conidiogenous cells with elongated necks and conidia. After ing good growth at 42°C; microscopically, they exhibited
removal of the mass along with ciprofloxacin treatment, the pale brown hyphae (1.5–3.5 μm wide), and basally inflated
clinical signs of keratitis resolved rapidly. The patient has phialides with elongated necks, arising mostly singly on
demonstrated no further episodes of microbial keratitis, and vegetative hyphae or along the length and near the tip of
the vision in her left eye has improved to the level that existed pale brown echinulate conidiophores; and they showed long
prior to wearing contact lenses. chains of limoniform-to-fusiform single-celled conidia
Guarro et al. [10] documented three cases of Acrophialo (6–10 μm × 3.5–5.0 μm) that were pale brown, and smooth
phora fusispora infections, with one from India and two or finely echinulate with ornamentations often arranged in
from Europe. The first case concerned a 55-year-old Indian distinct spiral bands.
female agricultural worker. The patient complained of pain,
discharge, watering, redness, and blurring of vision after
21.1.3 Diagnosis
an injury with a wood chip in her left eye a month and a
half before. On examination, the patient had a central cor- The diagnostic features for A. fusispora consist of the fol-
neal ulcer of 7 × 4 mm with grayish white plaque, a thick lowing: (1) isolates are thermotolerant, showing good growth
hypopyon, and presence of infiltration. Culture of corneal at 42°C or higher; (2) colonies are initially buff or tan, and
scrapings on SDA, with chloramphenicol and gentamicin usually turn grayish brown with an uncolored or dark grayish
for fungal isolation grew a single fungus after 5 days. Gram brown reverse; (3) basally swollen phialides are borne mostly
staining and 10% KOH preparation demonstrated some singly on the vegetative hyphae or along the length and near
hyphae. After therapeutic keratoplasty and treatment with the tip of brown, echinulate conidiophores; (4) limoniform-
natamycin, fluconazole, and ciprofloxacin, the patient was to-fusiform or ellipsoidal conidia are borne in long chains
discharged with the healing of the lesion. and are smooth or finely to coarsely echinulate, sometimes in

Acrophialophora 165
spiral bands; (5) phialides do not curve away from the main PDA plates incubated at 30°C and 37°C. PFA slant cultures
axis; and (6) phialides sometimes proliferate to form a sec- are evaluated for growth at 25°C, 35°C, and 42°C.
ond opening. Phialides producing more than one opening not After growth for 1–7 days on PDA slants, approximately
delimited by a septum are also referred to as polyphialides. 1 cm2 of mycelia are collected by scraping the slant with a
The long, dark, echinulate conidiophores that form at matu- sterile stick in 1 mL of sterile, molecular-grade H2O. The
rity often appear to have “fallen down” on the plate and give material is transferred to a 2 mL screw-cap tube. The tubes
the colony its darker appearance. are centrifuged for 1 min at 6000 × g. If the mycelia do not
The genus Acrophialophora was originally placed in the pellet, the material is contained with a pediatric blood serum
genus Paecilomyces due to the fact that both genera form filter (Porex Corp., Fairburn, GA). Supernatant is removed,
chains of ellipsoidal to fusiform conidia from basally swol- and the material is resuspended in 200 μL of IDI sample buf-
len phialides borne either on conidiophores or directly from fer and transferred to the lysis tube, which contained glass
the vegetative hyphae. However, Acrophialophora is differ- (IDI lysis kits, GeneOhm Sciences). Lysis tubes are vortexed
entiated from Paecilomyces by (1) unbranched, erect, brown, on the highest setting for 5 min. The tubes are placed in a
echinulate conidiophores that are fertile only near the apex; boiling water bath for 15 min, and then centrifuged for 5 min
(2) a basal cell anchoring the conidiophores to the vegetative at 16,000 × g. The supernatant is stored at −20°C until ampli-
hyphae; (3) phialides with a distinct swelling at the base, not fication [13].
curving or bending away from the main axis; and (4) colonies
that become dark. Not surprisingly, in the case reported by 21.2.2 Detection Procedures
Al-Mohsen et al. [2], the causative agent of the brain abscess
was initially identified as Paecilomyces species on the basis Pounder et al. [13] described a real-time PCR with SYBR
of its formation of conidia in short chains. Only after detailed green DNA binding dye and amplicon melting temperature
examination of other characteristics of the organism, the cor- analysis for fungal detection using pan-fungal primers ITS1
rect identity of A. fusispora was made. forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
Other fungal organisms that may be confused with reverse (5′-TCCTCCGCTTATTGATATGC-3′) [14]. The iden-
Acrophialophora fusispora are Scopulariopsis charta- tity of the fungi is verified by subsequent sequencing analysis.
rum [2,11] and Scedosporium prolificans [5,8,9] as well as Procedure
Acremonium. Acrophialophora differs from Scopulariopsis
in forming conidia from phialides instead of annellides. 1. PCR mixture is composed of 1× Lightcycler
Furthermore, Scopulariopsis conidia are spherical and usu- FastStart DNA Master Hybridization Probes mix-
ally with a wide truncate base, while Acrophialophora conidia ture (Roche Applied Science) (containing deoxy-
are fusiform or lemon-shaped, pale brown, finely echinulate, nucleoside triphosphates, FastStart Taq DNA
and often with distinct spiral bands. The confusion of A. fusis- polymerase, and 1 mM MgCl2 (additional MgCl2 is
pora with S. prolificans is due probably to the fact that both added to a final concentration of 4.6 mM), 0.4 μM
form flask-shaped conidiogenous cells. However, the flask- each of ITS1 forward and ITS4 reverse primers, 1×
shaped conidiogenous cells of Scedosporium prolificans are SYBR green (Molecular Probes), and 3 μL template
often in brush-like arrangement, not single on hyphae as of DNA.
A. fusispora. In addition, A. fusispora conidia are limoni- 2. Thermal cycling parameters include 95°C for
form, and their walls are usually ornamented in spiral bands 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s,
and form chains, and A. fusispora phialides arise singly on and 76°C for 30 s; and a final extension at 72°C for
vegetative hyphae or on conidiophores, whereas S. prolifi- 2 min.
cans conidia are clavate, smooth, and not arranged in chains 3. The quality of the amplicon is determined using the
but in slimy heads [12]. Acremonium spp. also have single derivative of the melt analysis curve (55°C–99°C,
conidiogenous cells (phialides) emerging from ropes of veg- 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
etative hyphae, but they rarely produce dark colonies [8,9]. (Corbett Robotics, Inc).
Use of molecular techniques such as PCR and sequencing 4. The amplified product is purified for bidirectional
analysis of rRNA genes and internal transcribed spacer (ITS) sequencing using ExoSAP-IT (USB Corp). Five
regions provides a valuable approach for accurate determi- microliters of Big Dye Terminator Ready Reaction
nation of A. fusispora and other morphologically confusing Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
fungal organisms [13]. each primer (0.8 pmol/μL) and 3 μL of purified PCR
product. Cycle sequencing is performed with a 9700
thermal cycler (ABI), using 25 cycles of 96°C for
21.2 Methods 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
G-50 fine column to remove unincorporated dye ter-
Mycotic elements in clinical specimens are examined directly minators. Purified sequencing reaction products are
under microscope with the help of various stains. Cultural run on an ABI Prism 3100 Genetic Analyzer with a
and microscopic features are examined on SDA, PFA, and 50-cm capillary array.

5. Sequences are analyzed with the SmartGene differences among these fungi, application of molecular
Integrated Database Network software version methods such as PCR and sequencing analysis of rRNA
3.2.3 vr. SmartGene is a web-based software and genes and ITS regions should help clarify the confusion in a
database system with reference sequences derived rapid and decisive manner.
from the National Center for Biological Information
References
Note: In case that real time PCR instrument is unavail- 1. The UniProt Consortium. Available at http://www.uniprot.
able, standard PCR may be performed with primers ITS1 org/, accessed on August 1, 2010.
and ITS4, and the resulting amplicon is sequenced with the 2. Al-Mohsen, I. Z. et al., 2000. Acrophialophora fusispora
same primers. Sequence-based identifications are defined brain abscess in a child with acute lymphoblastic leuke-
mia: Review of cases and taxonomy. J Clin Microbiol.
by percent identity: species, ≥99%; genus, 93%–99%; and
38:4569–4576.
inconclusive, ≤93%. 3. Shukla, P. K. et al., 1983. Clinical and experimental keratitis
For strains producing discrepant identification between caused by the Colletotrichum state of Glomerella cingulata
the methods based on phenotypic characteristics and and Acrophialophora fusispora. Sabouraudia. 21:137–147.
ITS sequence analysis, the D1–D2 region of the large- 4. Sutton, D. A. et al., 1997. Pulmonary Acrophialophora
subunit RNA gene is amplified with primers NL1 fusispora: Case history, literature, review and mycology. In:
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 Proceedings of the 13th International Society for Human and
Animal Mycology Congress, 1997, Salsomaggiore Terme,
Italy, Abstract 363.
species clarification. 5. Arthur, S. et al., 2001. Scedosporium prolificans keratouveitis
in association with a contact lens retained intraocularly over a
21.3 Conclusion long term. J Clin Microbiol. 39(12):4579–4582.
6. Cimon, B. et al., 2005. Airway colonization by
Acrophialophora fusispora (obsolete synonym: Paecilomyces Acrophialophora fusispora in patients with cystic fibrosis.
fusisporus) is a filamentous fungus that is widely distributed J Clin Microbiol. 43(3):1484–1487.
in tropical and temperate regions. Besides causing plant dis- 7. Pihet, M. et al., 2009. Occurrence and relevance of filamen-
tous fungi in respiratory secretions of patients with cystic
ease, this organism is occasionally associated with human
fibrosis—A review. Med Mycol. 47(4):387–397.
phaeohyphomycosis, with clinical manifestation ranging 8. Guarro, J. and J. Gene, 2002. Acrophialophora fusispora
from brain abscess, keratitis/keratouveitis, pulmonary infec- misidentified as Scedosporium prolificans. J Clin Microbiol.
tion, and other invasive diseases. 40:3544.
Morphologically, A. fusispora is characterized by (1) 9. Sigler, L. and D. A. Sutton, 2002. Acrophialophora fusispora
isolates are thermotolerant, showing good growth at 42°C misidentified as Scedosporium prolificans. J Clin Microbiol.
or higher; (2) colonies are initially buff or tan, and usually 40:3544–3545.
10. Guarro, J. et al., 2007. Acrophialophora fusispora: An emerg-
turn grayish brown with an uncolored or dark grayish brown
ing agent of human mycoses. A report of 3 new clinical cases.
reverse; (3) basally swollen phialides are borne mostly sin- Diagn Microbiol Infect Dis. 59(1):85–88.
gly on the vegetative hyphae or along the length and near 11. Welsh, R. D. and R. W. Ely, 1999. Scopulariopsis chartarum
the tip of brown, echinulate conidiophores; (4) Limoniform- systemic mycosis in a dog. J Clin Microbiol. 37:2102–2103.
to-fusiform or ellipsoidal conidia are borne in long chains 12. de Hoog, G. S. et al., 2000. Atlas of Clinical Fungi, 2nd edn.,
and are smooth or finely to coarsely echinulate, sometimes in vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, the
spiral bands; (5) phialides do not curve away from the main Netherlands.
axis; (6) phialides sometimes proliferate to form a second 13. Pounder, J. I. et al., 2007. Discovering potential pathogens
among fungi identified as nonsporulating molds. J. Clin
opening.
Microbiol. 45(2):568–571.
Fungal organisms that may be confused with Acrophialo 14. White, T. J. et al., 1990. Amplification and direct sequencing
phora include Paecilomyces species, Scopulariopsis charta- of fungal ribosomal RNA genes for phylogenetics. In Innis,
rum, Scedosporium Prolificans, and possibly Acremonium M. A. et al. (eds.), PCR Protocols: A Guide to Methods and
species. Besides paying attention to the finer structural Applications. Academic Press, New York, pp. 315–322.

22 Arthrographis
Dongyou Liu
Contents
22.1 Introduction...................................................................................................................................................................... 167
22.1.3 Diagnosis.............................................................................................................................................................. 169
22.2 Methods............................................................................................................................................................................ 169
22.3 Conclusion.........................................................................................................................................................................170
References...................................................................................................................................................................................170
22.1 Introduction while others reach diameters of only 0.5–1 cm at 25°C for 7
days on potato glucose agar. Colonies are glabrous initially,
The genus Arthrographis consists of several filamentous fun- and become downy, velvety, or powdery by maturation, and
gal species that are commonly isolated from soil and compost. may form radial ridges or folds. The surface color is creamy
Within this genus, Arthrographis kalrae is an uncommon white to pale yellow or tan; the reverse side is pale yellow
human pathogen, and has been shown as a causative agent for to tan. The ability of some Arthrographis species to grow at
onychomycosis, mycetoma, sinusitis, endophthalmitis, kera- 45°C is useful for identification. Colonies on potato dextrose
titis, endocarditis, and other diseases, especially in immuno- agar (PDA) at 25°C are buff to tan initially, usually becoming
compromised patients. This fungus produces cream-colored darker with age; the reverse is centrally dark at maturity [4].
colonies that can be mistaken for those of Candida albicans. Hyphae are hyaline, and septate. Conidiophores are hya-
line, simple or branched, short. Arthroconidia are formed
22.1.1 Classification and Morphology either at tips of the conidiophores or at intercalary position
The genus Arthrographis belongs to the mitosporic along the hyphae. Arthroconidia formed at the tips are one-
Eremomycetaceae group, family Eremomycetaceae, class celled, cylindrical, smooth, and in chains; and those that are
Dothideomycetes, subphylum Pezizomycotina, phylum intercalary and arise from the undifferentiated hyphae are
Ascomycota, kingdom Fungi. Being the only member of the longer and narrower. Conidia are released by fission through
mitosporic Eremomycetaceae group, the genus Arthrographis double septa. Arthrographis may occasionally produce aleu-
contains five recognized species: Arthrographis alba, riconidia on submerged hyphae. Primary cultures may yield
Arthrographis cuboidea (obsolete synonyms: Briosia yeast cells, particularly when the colony is young. By aging,
microspora, Coremiella cuboidea, and Geotrichum cuboi- intercalary arthroconidia predominate while conidiophores
deum), Arthrographis kalrae (obsolete synonyms: A. lange- disappear.
roni and Oidiodendron kalrae), Arthrographis lignicola, At the species level, Arthrographis kalrae is characterized
and Arthrographis pinicola, as well as an unassigned spe- by the presence of one-celled, hyaline, smooth-walled, and
cies Arthrographis sp. Sl-2007a [1–3]. The type species of cylindrical arthroconidia. These arthroconidia are formed
the Arthrographis genus is A. kalrae; and the teleomorphs directly by fragmentation of undifferentiated hyphae or by
of the genus are found in the genus Pithoascus (synonym: disjunction and segmentation of hyaline fertile branches
Eremomyces). borne at the apex of the conidiophore in fresh cultures.
Arthrographis spp. are cosmopolitan filamentous fungi, Mature arthroconidia become bigger and elongated. In addi-
with common occurrence in soil and compost. Of the five tion, single-celled, hyaline, smooth, spherical blastoconidia
Arthrographis spp., Arthrographis kalrae is clinically signif- occurred directly on the sides of undifferentiated hyphae or
icant, causing mycetoma, photophobia in contact lens wearer, on short pedicels. A. kalrae grows at 42°C and is urease posi-
sinusitis and meningitis in an AIDS patient, and sinusitis and tive. Pithoascus langeronii (synonym Eremomyces langero-
ophthalmitis in healthy individual after trauma to the eye. nii) was initially described as the teleomorph of A. kalrae,
Arthrographis grows moderately to rapidly. Some but the connection between A. kalrae and E. langeronii is not
Arthrographis strains produce colonies of 3–9 cm in diameter, supported by molecular data [5].
167

Among the Arthrographis species, A. cuboidea displays sphenoidotomy revealed hyphal forms. Cultures of the puru-
more rapid growth and cube-shaped arthroconidia; A. lig- lent material from the left sphenoid on SDA (Emmons) and
nicola has broader yellow arthroconidia; A. pinnicola pro- inhibitory mold agar at 30°C for 48 h grew cream-colored,
duces floccose conidiomata and fails to grow on media glabrous colonies, which became velvety after 5 days due
containing cycloheximide; and A. alba shows a white colony to formation of hyphae and developed a pale yellow reverse.
that fails to grow at 37°C and lacks a Trichosporiella yeast Elongated oval budding yeast cells were seen under micro-
synanamorph. scope. Subculture on potato flakes agar (PFA) slants at 25°C
Arthrographis is a genus linked to Malbranchea. Due were initially cream and moist colonies with a yeast-like
to the presence of arthroconidia, Arthrographis resembles appearance, but colonies became beige, flat, and powdery to
Oidiodendron, but the conidiophores of Arthrographis lack granular in texture after 7 days. The fungus showed growth
the characteristic pigmentation. Furthermore, the arthro- on SDA at 25°C, 35°C, and 42°C, with moist colonies pres-
conidia of Arthrographis are smooth walled and lack the ent at 42°C. Microscopically, the isolate produced hyaline,
connectives between maturing conidia that are prominent septate hyphae; unbranched, and irregularly branched (tree-
in Oidiodendron. A. kalrae is similar to Trichosporon and like) conidiophores; chains of one-celled, rectangular arthro-
Geotrichum species by the formation of yeast-like colo- conidia (2 μm × 4 μm) not separated by disjunctor cells; and
nies and arthroconidia. However, unlike Arthographis occasional hyaline, sessile, subglobose conidia (4 μm × 5 μm)
and Trichosporon species, Geotrichum species are urease along the sides of the hyphae. The fungus was thus identi-
negative; Trichosporon species lack conidiophores, and fied as Arthrographis kalrae. The patient was treated with
Trichosporon assimilates a lot of carbon sources, which is itraconazole for approximately 5 months, but died secondary
not the case for Arthrographis. to enterococcal sepsis and Pneumocytis carinii pneumonia.
Postmortem examination revealed invasive fungal sinusitis
that involved the sphenoid sinus and that extended through
the cribiform plate into the inferior surfaces of the bilateral
Arthrographis is widely distributed in soil and air and has frontal lobes. There was also an associated fungal menin-
only rarely been reported as an opportunistic pathogen in gitis and vasculitis with fungal thrombosis and multiple
humans. This fungus enters the body through the respira- recent infarcts that involved the frontal lobes, right caudate
tory tract or by traumatic inoculation. The first case of ony- nucleus, and putamen. Postmortem cultures were positive for
chomycosis due to A. kalrae (A. langeronii) was described A. kalrae.
in France in 1939. Since then, A. kalrae has been shown to Xi et al. [9] documented a case of Arthrographis kalrae-
cause cutaneous and subcutaneous infections, eumycetoma, related panophthalmitis and invasive sinusitis in 39-year-old
lung infection, keratitis, corneal ulcer, panophthalmitis, farmer after receiving trauma to his left eye. He complained
sinusitis, onyxis, meningitis, and cerebral vasculitis [6]. of pain, loss of visual acuity in the injured eye, bleeding and
Degavre et al. [7] described a eumycetoma caused by edema of the upper eyelid, and discharge of pus. A CT scan
Arthrographis kalrae in an 80-year-old man in southern of the left eye revealed involvement of the maxillary and
France. The patient was admitted to hospital with pneumo- ethmoid sinuses. Potassium hydroxide mounts of purulent
nia caused by Pseudomonas aeruginosa and treated with material from the left eye orbit revealed filamentous hyaline
ceftazidime, ciprofloxacin, and systemic corticosteroids and septate hyphae, and SDA cultures at 25°C grew maize-
(methylprednisolone, 120 mg daily). Within 2 days, a well- colored, powdery colonies. The colony was initially cream
demarcated erythematous, indurated, painless tumor and colored and glabrous, gradually became maize or yellow
scattered nodules appeared on his left hand. Microscopic in color and velvety, and ultimately formed a powdery, yel-
examination of skin biopsy demonstrated septate, narrow lowish colony after 7 days. The texture of the colony was
(2.5 μm wide) hyphae. Culture of the biopsy sample on brain- soft and frangible. Slide cultures of the isolate on SDA and
heart infusion (BHI) agar at 25°C yielded a fungus after 3 potato dextrose agar (PDA) at 25°C and 37°C, produced
month incubation. Subcultures in Sabouraud dextrose agar unbranched or irregularly branched hyphae, with dendritic or
(SDA) tubes (without cycloheximide) grew round, cream- tree-like conidiophores; chains of single-celled, rectangular
colored, and velvety colonies. Microscopically, hyphae were arthroconidia not separated by disjunction cells; and occa-
narrow (2.5 μm) and septate with tufted conidiophores; sional hyaline, sessile, subglobose conidia. Lactophenol cot-
spores measured 1.5–2 μm × 2.5–4 μm. The fungus was ton blue (LPCB)-stained mounts of cells grown on BHIA at
identified as A. kalrae. Treatment with itraconazole resulted 37°C showed mainly oval to elongated yeast cells (2.5–4 μm
in complete clearing of lesion. in diameter) and short hyphae. The isolate was capable of
Chin-Hong et al. [8] reported a case of Arthrographis growth at 42°C. The sequences of regions D1 and D2 (604 bp)
kalrae pansinusitis and meningitis in a 33-year-old man and the ITS (508 bp) of the case isolate were identical to those
with AIDS after receiving an 8-week course of nafcillin of the control A. kalrae UAMH 3616 type strain. The isolate
for osteomyelitis. The patient presented with a 4-day his- was thus identified as A. kalrae. The patient received ampho-
tory of increasing bilateral retroorbital pain, and computed tericin B intravenously, itraconazole orally, and atomized
tomography (CT) scan showed severe maxillary, ethmoid, allitridum by nebulizing allitridum therapy. The patient’s
and sphenoid sinusitis. Biopsy obtained by endoscopic wound healed following surgical removal of the maxillary,

Arthrographis 169
ethmoid, and paranasal sinuses and the skin fistula, but the Arthrographis species is based on microscopic observa-
patient lost the use of his left eye. tion of fungal elements (e.g., filamentous hyaline and sep-
Pichon et al. [10] reported a case of fatal invasive fungal tate hyphae) in clinical samples using a variety of fungal
cerebral vasculitis due to Arthrographis kalrae in a 39-year- stains (e.g., KOH and LPCB). Isolation of fungal organisms
old cattle breeder. The patient presented with a 1-month from clinical specimens is conducted with several mycology
history of acute sinusitis, fever (temperature up to 39°C), dys- media (e.g., Sabouraud agar and PDA). A slide culture on
pnea, cough with purulent sputums, and weakness. Magnetic 2% malt agar may be helpful. The colonial and microscopic
resonance imaging (MRI) demonstrated an extended right features of the cultured isolates are assessed. Arthrographis
frontal stroke and showed a right frontal sinusitis. Culture of kalrae colonies are initially cream and glabrous, gradually
the CSF sample on chocolate agar plates supplemented with become maize or yellow in color and velvety, and finally
Polyvitex (BioMérieux) at 37°C with 5% CO2 revealed a mod- powdery, yellowish after 7 days. Arthrographis kalrae
erate growth of yeast-like colonies after 9 days. Subculture of hyphae appear unbranched or irregularly branched; conid-
the isolate on Sabouraud agar showed a slow growth of the iophores are tree-like; arthroconidia are single-celled, rect-
isolate at 25°C, 30°C, 37°C, and 42°C. The colonies were angular; conidia are hyaline, sessile, and subglobose; oval to
initially mucoid, with a yeast-like appearance, and became elongated yeast cells may be observed. DNA sequence anal-
hairy due to formation of hyphae after 3 days of incubation ysis of LSU rRNA region D2 (604 bp) and the ITS (508 bp)
on Chromagar BBL and Sabouraud medium at 30°C. After makes precise and efficient determination of Arthrographis
5 days of incubation at 30°C, the colonies became pale yel- species possible [13,14].
low (Sabouraud agar and PDA). The isolate was resistant to
cycloheximide, and the urease activity was positive. Slide 22.2 Methods
culture on 2% malt agar at 30°C for 6 days showed charac-
teristic one-celled, hyaline, smooth-walled, and cylindrical, 22.2.1 Sample Preparation
elongated arthroconidia formed by differentiation of undif-
Besides used for direction microscopic examination, clini-
ferentiated hyphae. Lateral spherical blastoconidia formed
cal samples are cultured on inhibitory mold agar, modified
directly on hyphae or on short pedicels using the description
Sabouraud agars, or PDA. Microscopic structures of the fun-
[Sigler and Carmichael]. The identification of Arthrographis
gal isolates are observed on tease or tape preparations and
kalrae was confirmed by sequencing the internal transcribed
slide cultures after incubation for up to 21 days [13].
spacer 1 (ITS1) region of the ribosomal DNA gene using uni-
After growth for 1–7 days on PDA slants, approximately
versal primers ITS1 and ITS2. Despite medical intervention,
1 cm2 of mycelia are collected by scraping the slant with a ster-
the patient died from fatal-stroke syndrome. Postmortem
ile stick in 1 mL of sterile, molecular-grade H2O. The material
examination of the brain tissues revealed a necrotizing arte-
is transferred to a 2 mL screw-cap tube. The tubes are centri-
ritis and confluent fungal hyphae, sometimes dividing into
fuged for 1 min at 6000 × g. If the mycelia do not pellet, the
arthroconidia, massively invading the meningeal space and
material is contained with a pediatric blood serum filter (Porex
infiltrating the brain arteries.
Corp., Fairburn, GA). Supernatant is removed, and the mate-
Biser et al. [11] described a case of keratomycosis caused
rial is resuspended in 200 μL of IDI sample buffer and trans-
by Arthrographis kalrae in a 23-year-old female contact lens
ferred to the lysis tube, which contained glass (IDI lysis kits,
wearer, with photophobia and pain in her amblyopic eye.
GeneOhm Sciences). Lysis tubes are vortexed on the highest
Culture of eye sample grew a fungus, which was determined
setting for 5 min. The tubes are placed in a boiling water bath
as Arthrographis kalrae. Treatment with amphotericin 0.5%
for 15 min, and then centrifuged for 5 min at 16,000 × g. The
drops led to moderately rapid resolution of the active kera-
supernatant is stored at −20°C until amplification [13].
titis. In a recent study, Sugiura and Hironaga [12] reported
a case of onychomycosis due to Arthrographis kalrae in a
63-year-old Japanese man with markedly dystrophic finger- 22.2.2 Detection Procedures
nails. Microscopic examination of fingernail sample showed
Pounder [13] described a real-time PCR with SYBR green
hyaline hyphae and arthroconidia. The causal agent was
DNA binding dye and amplicon melting temperature analysis
identified as A. kalrae based on morphological characteris-
for fungal detection using pan-fungal primers ITS1 forward
tics and ITS region sequencing. Treatment with oral terbin-
(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 reverse
afine, 125 mg/day and topical 1% miconazole for 7 months
(5′-TCCTCCGCTTATTGATATGC-3′) [15]. The identity of
led to a complete cure of the ungual lesion. Since the patient
the fungi is verified by subsequent sequencing analysis.
worked with soil that harbored A. kalrae, it was possible that
the infection was acquired from this source. Procedure
1. PCR mixture is composed of 1 × Lightcycler

22.1.3 Diagnosis
FastStart DNA Master Hybridization Probes mixture
Accurate and rapid identification of Arthrographis spe- (Roche Applied Science) (containing deoxynucleo-
cies is important for improving the clinical outcome of side triphosphates, FastStart Taq DNA polymerase,
the infections. Conventional laboratory identification of and 1 mM MgCl2 (additional MgCl2 is added to a

final concentration of 4.6 mM), 0.4 μM each of ITS1 unbranched or irregularly branched; conidiophores are tree-
forward and ITS4 reverse primers, 1× SYBR green like; arthroconidia are hyaline, single-celled, smooth-walled,
(Molecular Probes), and 3 μL template DNA. and cylindrical; conidia are hyaline, sessile, and subglobose;
2. Thermal cycling parameters include 95°C for oval to elongated yeast cells may be observed. The fungus
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and grows at 42°C and is urease positive. DNA sequence analysis
76°C for 30 s; and a final extension at 72°C for 2 min. of LSU rRNA region D2 and the ITS allows rapid and accu-
3. The quality of the amplicon is determined using the rate speciation of Arthrographis species.
(Corbett Robotics, Inc). References
4. The amplified product is purified for bidirectional 1. The UniProt Consortium. Available at http://www.uniprot.
sequencing using ExoSAP-IT (USB Corp). Five org/, accessed on August 1, 2010.
microliters of Big Dye Terminator Ready Reaction 2. Sigler, L. and J. W. Carmichael. 1983. Redisposition of some
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of fungi referred to Oidium microspermum and a review of
Arthrographis. Mycotaxon. 18:495–507.
each primer (0.8 pmol/μL) and 3 μL of purified PCR
3. Sigler, L., Y. Yamaoka, and Y. Hiratsuka. 1990. Taxonomy and
product. Cycle sequencing is performed with a 9700 chemistry of a new fungus from bark beetle infected Pinus
thermal cycler (ABI), using 25 cycles of 96°C for contorta var. latifolia. Part 1. Arthrographis pinicola sp. nov.
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing Can J Microbiol. 36:77–82.
reaction products are passed through a Sephadex 4. de Hoog, G. S. et al. 2000. Atlas of Clinical Fungi, 2nd edn.,
G-50 fine column to remove unincorporated dye ter- vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, the
minators. Purified sequencing reaction products are Netherlands.
5. Gené, J. et al. 1996. Studies on keratinophilic fungi. X.
Arthrographis alba sp. nov. Can J Microbiol. 42:1185–1189.
50-cm capillary array. 6. Perlman, E. M. and L. Binns. 1997. Intense photophobia
5. Sequences are analyzed with the SmartGene caused by Arthrographis kalrae in a contact lens-wearing
Integrated Database Network software version patient. Am J Ophthalmol. 123(4):547–549.
3.2.3 vr. SmartGene is a web-based software and 7. Degavre, B., J. M. Joujoux, M. Dandurand, and B. Guillot.
database system with reference sequences derived 1997. First report of mycetoma caused by Arthrographis
from the National Center for Biological Information kalrae: Successful treatment with itraconazole. J Am Acad
(NCBI) GenBank repository. Dermatol. 37:318–320.
8. Chin-Hong, P. V. et al. 2001. Invasive fungal sinusitis and
meningitis due to Arthrographis kalrae in a patient with
Note: In case that real time PCR instrument is not avail-
AIDS. J Clin Microbiol. 39:804–807.
able, standard PCR may be performed with primers ITS1 9. Xi, L. et al. 2004. First case of Arthrographis kalrae ethmoid
and ITS4, and the resulting amplicon is sequenced with the sinusitis and ophthalmitis in the People’s Republic of China.
same primers. Sequence-based identifications are defined J Clin Microbiol. 42:4828–4831.
by percent identity: species, ≥99%; genus, 93%–99%; and 10. Pichon, N. et al. 2008. Fatal-stroke syndrome revealing fungal
inconclusive, ≤93%. cerebral vasculitis due to Arthrographis kalrae in an immuno-
For strains producing discrepant identification competent patient. J Clin Microbiol. 46:3152.
between the methods based on phenotypic characteris- 11. Biser, S. A. et al. 2004. Arthrographis keratitis mimicking
Acanthamoeba keratitis. Cornea. 23(3):314–317.
tics and ITS sequence analysis, the D1–D2 region of the 12. Sugiura, Y. and M. Hironaga. 2010. Arthrographis kalrae, a rare
large-subunit RNA gene is amplified with primers NL1 causal agent of onychomycosis, and its occurrence in natural
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 and commercially available soils. Med Mycol. 48(2):384–389.
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for 13. Pounder, J. I. et al. 2007. Discovering potential pathogens
species clarification. among fungi identified as nonsporulating molds. J Clin
Microbiol. 45(2):568–571.
14. Bagyalakshmi, R. et al. 2008. Newer emerging pathogens
22.3 Conclusion of ocular non-sporulating molds (NSM) identified by poly-
merase chain reaction (PCR)-based DNA sequencing tech-
Arthrographis spp. are cosmopolitan filamentous fungi, which nique targeting internal transcribed spacer (ITS) region. Curr
are commonly isolated from soil, compost and air, etc. Of Eye Res. 33(2):139–147.
the five recognized species within the genus Arthrographis, 15. White, T. J. et al. 1990. Amplification and direct sequencing
Arthrographis kalrae is occasionally involved in human of fungal ribosomal RNA genes for phylogenetics. In Innis,
infections, leading to mycetoma, photophobia in contact lens M. A. et al. (Eds.), PCR Protocols: A Guide to Methods and
wearer, onychomycosis, endocarditis, meningitis, sinusitis Applications. Academic Press, New York, pp. 315–322.
and ophthalmitis after trauma to the eye [5–12,16,17]. 16. de Diego Candelo, J. et al. 2010. Endocarditis caused by
Arthrographis kalare. The Annals of Thoracic surgery.
Morphologically, Arthrographis kalrae colonies are 90:e4–e5.
initially cream in color and glabrous, turning to maize or 17. Thomas, B. C. et al. 2011. Severe Arthrographis kalrae
yellow in color and velvety, and eventually powdery, yel- Keratomycosis in an Immunocompetent Patient. Cornea.
lowish. Under microscope, Arthrographis kalrae hyphae are 30(3):364–366.

23 Aspergillus
Maiken Cavling Arendrup, Yanan Zhao, and David S. Perlin
Contents
23.1 Introduction.......................................................................................................................................................................171
23.1.1 Epidemiology and Morphology.............................................................................................................................171
23.1.1.1 Epidemiology..........................................................................................................................................171
23.1.1.2 Morphology.............................................................................................................................................171
23.1.3 Diagnosis...............................................................................................................................................................173
23.1.3.2 Molecular Diagnostics............................................................................................................................175
23.2 Methods............................................................................................................................................................................ 177
23.2.1.1 Aspergillus Culture.................................................................................................................................178
23.2.1.2 Blood.......................................................................................................................................................178
23.2.1.3 Tissue......................................................................................................................................................178
23.2.2.1 Primers, Probes, and Platforms............................................................................................................. 179
23.2.2.2 Secondary Drug Resistance................................................................................................................... 179
23.2.2.3 Assay Conditions................................................................................................................................... 182
23.3 Summary and Perspective................................................................................................................................................ 182
References.................................................................................................................................................................................. 183
23.1 Introduction species distribution dependent on geographical differences,

antifungal selective pressure, anatomical site, and host group
23.1.1 Epidemiology and Morphology involved.
23.1.1.1 Epidemiology
Filamentous fungi belonging to the genus Aspergillus are 23.1.1.2 Morphology
found widespread in nature. They are commonly isolated Macromorphology: The growth rate of aspergilli is rapid to
from soil, decaying plants and vegetables, and from indoor moderately rapid (1–9 cm in diameter after 7 days of cul-
air environment.1–5 Aspergillus conidia are widely dispersed ture at 25°C), with the exception of the slow growing species
and are inhaled on a daily basis unless special air filtration A. nidulans and A. glaucus. Colonies are powdery, granular,
measures are taken. Aspergillus species may cause disease or cottony with a variety of colors depending on the species
due to inhalation of conidia or ingestion of mycotoxins and the culture medium used (Table 23.1). Only A. fumigatus
resulting in allergic or toxic disease, chronic infection, and/ is thermo tolerant with the ability to grow up to 50°C, a fea-
or acute infections.6 Allergic aspergillosis occurs largely in ture that is helpful to separate this species from the others,
patients with asthma, atopy, or cystic fibrosis (CF),7,8 while including the newly defined Aspergillus section Fumigati.
invasive pulmonary disease usually only occurs in immu- Micromorphology: Characteristic features for this genus
nocompromised patients with inhalation being the primary include hyphae that are hyaline and septate with dichotomous
route of infection. Over recent decades, the number of branching. Conidiophores arise from a basal foot cell and
patients with invasive aspergillosis (IA) has increased due germinate into a vesicle at the apex. Conidiogenic phialides
largely to advances in the treatment of malignant diseases arise directly from the vesicle (uniseriate) or from inter-
with an increasing number of patients undergoing severe sected metulae (biseriate) depending on the species. Conidia
immunosuppression, as part of intensive chemotherapeuti- are round (2–5 μm) and formed into radial basipetal chains
cal regimes, hematopoietic stem cell or organ transplanta- (Figure 23.1). Species-specific features include the presence
tion.9–15 A. fumigatus is the most common species involved or absence of sclerotia, cleistothecia, Hülle cells, aleurico-
in infections (85%–90%), but A. flavus, A. niger, A. terreus, nidia, chlamydoconidia, and morphology of the vesicles and
and A. nidulans are also regularly recovered, with varying arrangements of phialides (Table 23.1).
171

172
TABLE 23.1
Colony and Microscopic Features of Common Aspergillus Species
Cleisto- Hülle
Species Surface Reverse Conidiophorea Vesicle Phialides Sclerotia thecia cells Aleuriconidia Comments
A. fumigatus Blue-green-gray White-tan Short, smooth, Round columnar U, upper half/ − − − − Growth at 50°C–55°C Whitish
colorless/greenish head two-thirds of variants have been described
the vesicle
A. flavus Yellow-green Goldish- Long, colorless, Round radiate U/B + − − − Conidiophore particularly
redbrown rough head rough near the vesicle
Radial heads tend to separate
into columns
A. niger Black White-yellow Long, smooth, Round radiate U/B − − − − Max. temp. 48°C
colorless or brown head
Radial heads tend to separate
into columns
Rough conidia
A. nidulans Green, buff-yellow Purplish Short, smooth, Round columnar B, upper half − + (red) + − Slow growth. Very evident foot
red-olive brown head of the vesicle cell
A. terreus Cinnamon-brown White/pale Short, smooth, Round compact B, upper − − − + (solitary, Clamydoconidia may be
yellow-brown colorless columnar head two-thirds of directly on present. Elevated AmB MICs

the vesicle hyphae)
A versicolor White-yellow/tan/ White-yellow/ Long, smooth, Round loosely B, upper − − +/− − Whitish-dark red exudate may
pale green or pink purplish red colorless radiate head three-quarters be present
of the vesicle
A. clavatus Blue-green White-brownish Very long, smooth Huge, elongated, U − − − − Heads tend to separate into
clavate shaped several columns
A. glaucus Green with yellow Yellow-brown Variable length, Round, Radiate U − + (yellow/ − − Phialides may appear
areas smooth, colorless to very loosely orange) rudimentary
columnar head
U, uniseriate; B, biseriate (with metulae).

a Short: <300 μm.
Aspergillus 173
skin, the valves and myocardium, and the gut may be pri-
mary sites of infection.
In the non-neutropenic host, Aspergillus may cause
chronic forms of pulmonary aspergillosis (chronic necrotiz-
ing aspergillosis, chronic cavitary aspergillosis, and chronic
fibrosing aspergillosis) and non-angioinvasive IA.18,24 Host
factors for the invasive form include GVHD, corticosteroid
therapy, CGD, and solid organ transplantation, and in the
majority of the chronic cases, preexisting architectural lung
tissue changes are present including emphysema, prior myco-
(a) (b)
bacterial infection, asthma, sarcoidosis, etc. The lesions are
characterized by pyogranulomatous infection and inflamma-
Figure 23.1 Wet mount of (a) A. fumigatus and (b) A. flavus. tory necrosis without angioinvasion.16,17 Typically, the fungal
The vesicle bearing phialides on the upper half to two-thirds of the burden in tissue is less, which in combination with a lack of
surface is clearly seen for A. fumigatus. The entire surface of the angioinvasion and the ability of the host to mount an antibody
vesicle of A. flavus is covered with metulae from which conidia pro- response may be factors involved in the lower sensitivity of
ducing phialides arise. Also note the chains of conidia surrounding
antigen tests.25 Clinically, the course of infection is weeks to
the A. flavus head as well as the rough appearance of the distal part
of the conidiophore. months in contrast to the rapidly progressive infection in the
neutropenic host and symptoms include cough, shortness of
breath, fatigue, weight loss, and hemoptysis. Aspergilloma,
23.1.2 Clinical Features and Pathogenesis which is formation of an Aspergillus fungal ball of myce-
lium in a preexisting cavity without tissue or angioinvasion
Aspergillus is a multifaceted pathogen associated with a is included in the term chronic aspergillosis, but is reserved
continuum of clinical presentations depending on the com- to the singularity of the infection. The disease progresses
plex interaction of the fungus the host. Thus, the isolation over months to years and the typical presentation on CT is a
of Aspergillus may represent transient or established colo- mobile fungus ball in a preexisting cavity. For recent reviews,
nization, fungal sensitization and allergy, localized infec- please consult Refs. [18,26–30].
tion, or acute or chronic invasive infection. The terminology
used over time has changed and the disease entities are not
always entirely distinct, which add to the complexity of the
23.1.3 Diagnosis
field. In the following, a short description of the main char-
acteristic features concerning pathogenicity and clinical fea- Successful management of invasive fungal infections depends
tures for most acute and chronic Aspergillus infections will on timely and appropriate treatment.22,31–33 Early diagnosis of
be described, although allergic manifestations are considered IA remains a major challenge for high-risk patients because
outside the scope of this chapter. the signs and symptoms of diseases are nonspecific, blood
In the neutropenic host, Aspergillus infections are charac- cultures are usually negative, and biological markers can be
terized by a hemorrhagic infection due to hyphal invasion of unreliable, particularly in the non-neutropenic host or the
parenchyma and vessels, scattered inflammatory cells, and patient receiving mold active therapy.25,34 Diagnostic imag-
coagulative necrosis.16,17 The lungs are the most common ing is not disease-specific and often comes too late in the
site of infection and depending on the site and size of vessels course of disease. Regrettably, invasive mold infections are
invaded, lesions may be spherical, nodular lesions, or wedge- too often positively diagnosed at autopsy, which has led to
shaped lesions with the base abutting the pleura.18–20 The cen- the widespread use of prophylaxis, empiric, and preemptive
ter of the lesion also called the sequestrum is a necrotic area therapies.35–38 Disease confirmation requires identification
surrounded by a hemorrhagic rim of hyphal invaded, con- of hyphae in blood (rare), respiratory specimens or biopsy
gested, and hemorrhagic tissue. On computed tomography tissue, which can be difficult to discern as morphological
(CT) scanning, this typically present as a nodule surrounded features, reproductive structures, and biochemical properties
by ground glass opacity—the so-called halo sign.21,22 During can take days to weeks to properly evaluate.
neutrophil recovery, the sequester contracts, resulting in an Drug resistance is a confounding factor for the treatment
air cap formation presenting on CT scanning as the air-cres- of IA. Yet, acquired resistance in Aspergillus is still relatively
cent sign.23 No clinical signs are diagnostic but rapidly pro- uncommon. Thus, species identification is a valuable tool
gressive infection with symptoms including fever, dyspnea, guiding choice of treatment as Aspergillus spp. characteris-
cough, chest pain, and hemoptysis is characteristic. Due to tically display specific susceptibility patterns. For example,
the angioinvasion, hematogenous dissemination may occur A. terreus is resistant to amphotericin B, while A. lentilus
involving almost any organ including the CNS, eye, heart, shows pleiotropic reduced susceptibility to several antifungal
bone, and skin. The second most common primary sites of classes. Recently, it was reported in the United Kingdom that
infection in the neutropenic host are the sinuses, potentially azole resistance in A. fumigatus has emerged as a significant
leading to direct invasion of the orbita and CNS, but also the factor in patients undergoing chronic or repeated therapy,39

and, in the Netherlands, multi-azole resistant A. fumigatus patient has already been exposed to antifungal treatment.53,54
with a specific resistance mechanism (L98H alteration and Over the years, the sensitivity, specificity, and speed have
a tandem repeat in the promoter region) has been found in been improved by the use of fluorescent brighteners such as
azole naïve patients and the environment.4,40,41 On this back- calcofluor white or blankophor.55–57 Important is to evaluate
ground, microscopy and culture allowing species identifica- hyphae with respect to regularity, size, presence of septae
tion and subsequent susceptibility testing remain a corner and branching.55 Use of special stains (Grocot Silver or PAS)
stone in the diagnosis of aspergillosis despite associated limi- increases the sensitivity and is particularly recommended for
tations including lack of sensitivity, labor and time intensive tissue biopsies.
procedures, and the difficulties in obtaining representative
specimens from thrombocytopenic hematological patients, 23.1.3.1.3 Culture
neonates, and children. Blood cultures are almost always negative in disseminated
aspergillosis. Relevant sample material depends on the ana-
23.1.3.1 Conventional Techniques tomic site involved but will in most cases be airway sam-
The diagnosis of Aspergillus infections typically involves ples. Bronchoalveolar lavage (BAL) is superior to a tracheal
the combination of clinical signs and symptoms, imaging, aspirate, which again is superior to sputum. However, as
and microbiological investigations including specimens for Aspergillus species may be present in air, dust, etc., transient
microscopy and culture, antigen or antibody detection. No colonization of the airways or contamination of the sample
test is perfect in the way that a positive test proves infec- is possible and should be kept in mind, especially, if growth
tion and a negative test rules it out. Thus, a combination of is observed distant from the region of the agar surface that
the available tools and interpretation of the results taking the has been inoculated with the clinical material. Growth of
clinical situation (underlying disease and other host factors) Aspergillus may be suppressed by concomitant growth of bac-
into account is necessary. Diagnostic criteria for stratification teria. Therefore selective media inhibiting the growth of such
of patients into proven, probable, and possible aspergillosis should be included and agars incubated for a minimum of 5
has been developed and revised and represent a tool for clas- days. Still sensitivity is around 25%–60%.58–60 Species iden-
sification of patients in clinical trials and for evaluation of tification is traditionally performed according to macro- and
new diagnostics.42,43 In the following, the individual tests and micromorphology and thermotolerance (see above).
their pros and cons are briefly summarized.
23.1.3.1.4 Susceptibility Testing
23.1.3.1.1 Imaging Reference methodologies using micro-broth dilution have
IA typically involves the lung.6,10 Chest radiographs gener- been established61,62 but susceptibility testing of molds is
ally show nonspecific infiltrates (alveolar syndrome, intersti- in general not performed in routine clinical microbiology
tial syndrome, or nodules) with lesions more characteristic laboratories. Whilst the endpoint reading is straight forward
for aspergillosis, such as cavitated nodules, only occasion- for azoles and amphotericin B due to the growth versus no
ally observed.44 Chest CT may detect lung involvement at growth pattern of inhibition, the endpoint determination
an early stage of infection in neutropenic patients.22,23 In for the echinocandins is more difficult to determine due to
particular, the halo sign, that is, a macronodule (≥1 cm in significant but partial growth inhibition resulting in altered
diameter) surrounded by a perimeter of ground-glass opac- hyphal morphology.63,64 No resistance breakpoints have been
ity, has been shown to be an early indicator of invasive pul- established but epidemiological cut off values defining mini-
monary aspergillosis (IPA), while the air-crescent sign (a mum inhibitory concentration (MIC) ranges for the wild-type
pulmonary cavitation) is a later sign appearing with the bone population of A. fumigatus are available allowing interpreta-
marrow recovery.23 In the non-immunocompromised host, tions of MICs as being within the wild-type range or above,
CT imaging is less typical. Thus, while 71% of patients with in which case acquired resistance mechanisms should be
neutropenic hematological malignancy had either halo- or considered.65,66 The commercial Etest strip based upon the
air-crescent sign,32 this was only the case in 5% or less of incubation of a plastic strip containing a concentration gradi-
patients with IPA in the ICU setting.34,45 Neuroradiological ent of the antifungal compound on an inoculated agar surface
studies are helpful in the diagnosis of CNS infections.46–48 has been evaluated and shows acceptable agreement if per-
Both CT and MRI are useful, but MRI may be more accurate formed by experienced technicians.64,67,68
and reveal more lesions than CT.49 MRI and CT appearances
of CNS aspergillosis have been studied in detail by several 23.1.3.1.5 Antigen Detection
investigators.49–51 A validated antigen detection test nowadays exists for indi-
vidual detection of Aspergillus galactomannan antigen (GM).
23.1.3.1.2 Microscopy In addition, yeast and mold infections (with the exception
Direct microscopic examination is important for two rea- of Cryptococcus and zygomycetes) can be detected using
sons.52 It provides rapid, easy, and cheep information about (1,3)-β-d-glucan detection assay.
the presence of fungi and other pathogens and may allow suf- The performance of the Aspergillus galactomannan test
ficient identification to guide management and it is more sen- has been extensively discussed in numerous publications.
sitive than culture for a number of samples, especially if the Best performance is seen if the test is used as a screening

Aspergillus 175
test twice or three times weekly in patients with neutrope- detection of small numbers of infecting organisms in a pri-
nic fever unresponsive to antibiotics.36,59,69 The sensitivity is mary specimen. PCR primers designed to conserved regions
lower in patients receiving antifungal treatment and in non- of the rRNA targets are used to amplify the sequence-variable
neutropenic patients.34,70 Positive results are available before fragments, which are then detected by a variety of end point
a positive CT scan or culture in two-thirds of the patients.59 chemistries. Molecular diagnostic approaches with improved
In the neonatal population, false-positive results may sensitivity and specificity are evolving rapidly, which can be
occur more frequently in part due to gut colonization with used with other markers and clinical symptoms to confirm
Bifidobacterium spp.71–73 Interestingly, it has recently been the presence of invasive disease.
shown that galactomannan detection on BAL fluids and tis- The Holy Grail for molecular detection is the accurate
sue biopsies may increase sensitivity.34,74,75 In recent studies and reproducible detection of small amounts of Aspergillus
of (1,3)-β-d-glucan detection in adults, the performance has hyphal fragments or circulating nucleic acid in blood. This
been encouraging76–78 but direct comparisons with the galac- has not been easy and a wide variability has been observed
tomannan assay for detection of aspergillosis do not yield a in the field. An important issue is that Aspergillus growing
consensus as to which test is best.79–82 A practical drawback in the lung is not likely to be blood-borne unless it becomes
is that the test requires incubation and reading with a kinetic angioinvasive or cells die and nucleic acid is released into the
ELISA reader and that the test plates cannot be divided into bloodstream.
separate strips, which lead to a high cost when running single
samples. 23.1.3.2.1 PCR Detection Formats
Standard PCR-based amplification is often insufficient for
23.1.3.1.6 Antibody Detection diagnostic use because of relatively high levels of false-
While antibody detection plays an important role in the positive results, since in the various target regions, some spe-
diagnosis of allergic aspergillus diseases, including aller- cies vary by as little as single nucleotide.100 Amplified DNA
gic bronchopulmonary aspergillosis in CF patients or aller- products can be detected in real time using intercalating dyes
gic rhinosinusitis, and in diagnosing chronic infections such as cyber green, although they provide no sequence-
and aspergillomas, its role in diagnosing acute infection is specific information. A wide range of endpoint detection
limited.18,83,84 technologies have been used to distinguish between fungal
species including single-strand conformational polymor-
23.1.3.1.7 Innate Immunity phism (SSCP),103,104 random amplified polymorphic DNA
Innate immune molecules play a vital role in host defence (RAPD) PCR (RAPD-PCR),105–107 reverse-hybridization
against A. fumigatus. Mannose-binding lectin (MBL) is line probe assay (LiPA),108 restriction enzyme analysis109,110
C-type serum lectin that acts as an opsonin and lectin com- enzyme immunoassay (PCR-EIA) and real-time (RT) molec-
plement pathway activator and inducer of proinflammatory ular probes, such as TaqMan™,111–113 light-cycler,114–116 and
cytokines, which helps confer innate immunity against sev- molecular beacons.92,117–119 PCR amplification used in con-
eral pathogenic organisms including A. fumigatus. Most indi- cert with self-reporting, high-fidelity hybridization probes
viduals have low levels of circulating MBLs, and diminished has helped to improve accuracy. This approach is quantita-
levels have been linked with Aspergillus infections.85,86 MBL tive and has a large dynamic range, which have been used to
variant alleles in patients with CF have been associated with rapidly identify fungal pathogens in both single target and
a poor prognosis.87 multiplex formats.92,117–119 Typically, these techniques can
detect 1–10 genomic equivalents under optimal conditions.
23.1.3.2 Molecular Diagnostics These approaches provide an opportunity to diagnose fungal
Molecular techniques are ideally suited for low abundance infections at an early stage and in a timeframe that can influ-
and difficult to culture pathogens like the Aspergillus spp. ence patient management. Yet, as the technology pushes the
because they provide for faster, more sensitive, and accurate limit of detection, it must take into account prior coloniza-
detection of infecting pathogens from a wide range of speci- tion with environmental spores of Aspergillus and the risk of
mens. Unlike classical methodologies, they can rapidly detect contamination during sampling or processing. Such baseline
in a matter of several hours a single genomic copy from blood factors require reliable quantitative data and that is linked to
or other specimens. Nucleic-based diagnostic assays utiliz- other clinical markers. Ultimately, validation must be cor-
ing amplification by the polymerase chain reaction (PCR) or related with clinical outcome.
nucleic acid sequence based assays (NASBA) are highly sen-
sitive and can positively detect fungal infections at an early 23.1.3.2.2 The Evolving PCR Experience
stage of infection.88–92 The fungal ribosomal genes 18 S, 5.8 S, The detection of fungal nucleic acid in blood and/or respira-
and 28 S, and their intervening noncoding regions, ITS1 and tory specimens at an early stage of invasive disease has been
ITS2, provide molecular sequence signatures for panfungal, a major priority. PCR can be highly sensitive and predictive
genera, and species-specific identification.93–101 These signa- for detection of Candida and/or Aspergillus.116,120 A meta-
tures can be easily and accurately resolved by high fidelity, analysis on the use of PCR for the diagnosis of IA from more
allele-specific molecular probes. Ribosomal genes are pres- than 10,000 blood, serum, or plasma samples obtained from
ent in multiple (38–91) copies per genome,102 which facilitates 1,618 patients showed a sensitivity and specificity of PCR

of 0.75 (95% CI 0.54–0.88) and 0.87 (95% CI 0.78–0.93), hyphal fragments, and spores. (The value of detecting spore
respectively.121 Yet, despite these highly promising results, nucleic acid is questionable, since they are not normally asso-
home-brew PCR systems to detect Aspergillus are highly ciated with invasive disease.) Aspergillus hyphae or hyphal
variable. The presence of nucleic acid, as detected by RT fragments can be extracted with reasonable efficiency with
PCR with Taq-man or Light Cycler probes, varies from 30% mechanical and/or enzymatic pre-lysis. For this reason, it is
to 100% when compared to circulating blood markers such as critical that the extraction system is highly efficient.
GM, diagnostic imaging, or clinical criteria.122 Many reports
indicate that PCR is superior to GM with respect to sensi- 23.1.3.2.3 Improved Extraction of Aspergillus
tivity rates.123 Yet, some labs report more reliability detect- Nucleic Acid from Blood
ing pathogen nucleic acid from respiratory specimens (e.g., The type of blood sample used for the molecular detection of
BAL) relative to blood, suggesting that fungal burden and DNA is also important, as whole blood, plasma, or serum have
extraction efficiency in the specimen is important. For these been analyzed. A comparison of different specimen types
reasons, the reliability of PCR as diagnostic tool for invasive showed that serum is generally the preferred source for the
fungal infections, especially those due to Aspergillus spp. is diagnosis of IA by PCR,121,125,126 most likely because nucleic
uncertain. acids are recovered more efficiently with routine extraction
The major problems contributing to PCR variability are procedures. Recently, several promising reports have dem-
a lack of assay standardization and inefficient extraction of onstrated a high degree of efficiency in isolating nucleic acid
nucleic acids, especially from blood. However, significant in whole blood by increasing the sample volume with an
methodological advancements in sample preparation and automated processing system (e.g., Roche MagnaPure™).125
improved amplification/detection should greatly enhance When combined with other well-established assays,116,120,121
the reliability of PCR as a diagnostic tool for invasive fungal PCR appears poised to have an important impact on the diag-
infections. First, the lack of a standardized assay is significant nosis of invasive fungal infections. Suarez et al.,125 in a 13
as nearly every lab has a different approach. In addition, the month prospective study, compared small (100 μL) and large
use of positive controls for extraction and amplification are (1000 μL) extraction volumes from blood utilizing automated
variable and quality control standards for probe and primer extraction on a MagNaPure, and evaluated it relative to GM
sets are rarely ever mentioned. The EU recognized this quan- detection by an enzyme immunoassay. A total of 938 samples
dary for IA and has organized a consortium to adopt uniform were screened for Aspergillus DNA following large volume
standards.121,124 Presently, only two standardized commercial extraction. The sensitivity, specificity, positive, and negative
PCR assays are available. The Roche SeptiFast RT PCR test predictive values were 100%, 96.7%, 81%, and 100%, respec-
detects and identifies 25 important bacterial and fungal spe- tively, with larger serum volume. The assay sensitivity sur-
cies causing bloodstream infections including C. albicans, passed GM, produced a positive result with two cases of IA
C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and A. where the GM assay result remained negative, and in four IA
fumigatus. The overall assay is reliable but labor intensive cases, DNA was detected earlier than GM. In another newly
and complex. Myconostica has recently developed molecu- published study,120 a total of 2244 samples were analyzed by
lar assays for detecting common respiratory fungal patho- real-time quantitative PCR. Taking two consecutive positive
gens of Aspergillus genus (MycAssay™ Aspergillus) and results as the diagnostic criterion, PCR detected gave sensi-
Pneumocystis jirovecii (MycAssay Pneumocystis). These are tivity, specificity, positive, and negative predictive values of
molecular beacon-based RT assays that are highly robust and 91.6%, 94.4%, 73.3%, and 98.5%, respectively. A combina-
easily expandable, and it has standardized internal controls tion of serial PCR and GM detected 100% of aspergillosis
for extraction and amplification. Presently, there is insuffi- cases, with a positive predictive value of 75.1%. These new
cient data with the commercial assays to fully assess their studies highlight the importance of extraction methodol-
impact. ogy and PCR standardization for improved performance of
The second most important issue for PCR assays is extrac- RT-PCR. There is still room for improvement in extraction
tion and recovery of pathogen-specific nucleic acid, espe- efficiency, but the new approaches illustrate the value of a
cially from blood. Unlike viremias and bacteremias, which highly efficient RT-PCR assay.
have high burdens in blood, invasive fungal infections have
low circulating levels of pathogens or naked DNA. In IA, only 23.1.3.2.4 Allele Discrimination and Multiplex Assays
low burdens of circulating nucleic acid or hyphal fragments One of the major advantages of RT-PCR assays with self-
are ever present. In fact, since Aspergillus is rarely cultured reporting probes is that they are allele discriminating, which
from blood, naked nucleic acid released from the respiratory improves accuracy and facilitates detection of single nucleo-
tract may be most important for molecular detection. It is tide changes that are needed to discriminate between closely
essential that extraction of fungal specific nucleic acid from related species or may result from mutations conferring
sterile fluids is efficient, since it is critical to even the most drug resistance.122,127 Molecular beacons, scorpion primers,
sensitive detection modality. Fungal diagnostics require effi- TaqMan and several other types of commercially available
cient extraction of nucleic acid from 1 to 50 genomic equiv- probes have allele-discriminating capacities. Ultra-sensitive
alents or ~40–2000 target molecules. This is more likely melt-curve analysis can also be used for allele discrimina-
with respiratory samples of IA, which often contain DNA, tion, but polymorphisms outside the target sequence can

Aspergillus 177
be a limiting factor for specificity.128 Innovations in nucleic probes recognizing specific mutations associated with tri-
acid amplification and detection have increased the number azole resistance.119 In a single molecular RT assay, it was pos-
of targets that can be simultaneously detected in a single sible to distinguish between triazole susceptible (wild-type
assay. The ability to detect multiple targets in a single mul- sequence) and triazole resistant alleles. The new assay takes
tiplex assay is an important application of RT detection sys- advantage of allele-discriminating probes in a multiplex
tems, which increases throughput and reduces assay costs. assay format to evaluate drug resistance and assess differ-
A wide range of approaches involving multiplexed probes, ences between triazole drugs. It is suitable for primary speci-
beads, and arrays can be employed to simultaneously detect mens and can be incorporated into routine molecular testing
a large number of targets facilitating rapid evaluation of regimens. Thus, molecular technology is now available for
multiple pathogens or drug resistance alleles. The advantage routine drug susceptibility testing of Aspergillus isolates to
of these approaches is that they combine powerful nucleic azole drugs. These targets cover a vast majority of Cyp51A
acid amplification strategies with massive screening capabil- mutations causing resistance. Yet, it is important to recognize
ity. However, a factor limiting the use of technology such as that other resistance mechanisms (e.g., pump overexpression)
microarrays has been inadequate assay sensitivity and quan- can contribute to clinical resistance, albeit at a much lower
tification.129 Direct hybridization of DNA or RNA not only frequency. The percentage of isolates with specific resistance
provides the least bias in gene detection, but also the lowest mechanisms is under investigation in several large epidemio-
level of analytic sensitivity. This is largely due to practical logical studies.
limitations of efficient hybridization of linear probe-target
hybrids. Ultimately, cost and limited sample throughput 23.1.3.2.7 Species Identification within
make conventional large planar microarrays less attractive in Aspergillus fumigatus Complex
pathogen detection schemes. Alternative microarray formats As A. fumigatus continues to be the most common etiological
such as bead arrays may circumvent cost and throughput agent of IA, identification of species within the Aspergillus
limitations. species complex (section Fumigati) may be important as
previous studies have shown the existence of cryptic spe-
23.1.3.2.5 PanProbes Systems cies whose antifungal susceptibility profile differs from A.
A variety of panGenus probes for Candida and Aspergillus fumigates.142 Aspergillus section Fumigati contains 33 dif-
species have emerged in recent years. These probe systems ferent species, with 10 strict anamorphs and 23 teleomorphs.
utilize PCR amplification from common primers recogniz- Morphological observation is not sufficient to distinguish
ing highly conserved regions of either 18S or 28S rRNA between them,143 thus molecular typing methods mostly
genes.130,131 For many years, the biggest limitation of these DNA-sequence based, is a valuable tool for species identifica-
assays was the presence of contaminating nucleic acid tion. As mentioned above, the ITS region was found to be use-
found in amplifying enzymes (Taq polymerase), as well as ful for Aspergillus identification to the species complex level,
in reagents and collection tubes, which led to false-positive but it does not always resolve very closely related phyloge-
results. Changes in manufacturing processes, as well as netic species, whereas intron-rich protein coding genes gen-
new techniques to remove contaminating nucleic acid from erally do much better.144 Therefore, the International Society
enzyme preparations, has made the use of pan-Genera probes for Human and Animal Mycology-sponsored Aspergillus
more reliable. Such broad fungal tests can be used to either Working Group has recommended the use of ITS for the spe-
identify or exclude fungal involvement, and in more com- cies complex level identification and protein-coding locus
plicated disease states, increase the certainty of invasive for the identification of species within the complex.145 Due
disease.132 to their prevalence in public databases, universality of appli-
cation, and relative resolving power, the use of β-tubulin or
23.1.3.2.6 Drug Resistance and Molecular Diagnostics calmodulin sequences has been recommended for such spe-
A wide variety of mechanisms account for triazole resistance cies identification.144 Molecular tools other than single locus
in Candida spp. including mutations of the Erg11 target, DNA sequencing, such as restriction fragment length poly-
overexpression of the target, and overexpression of multi- morphism (RFLP), RAPD, and multilocus sequence typing
drug efflux transporters of the ABC and MDS families133,134 (MLST) have also been applied to the same aim,146,147 but
induced by global transcriptional regulators.135–139 The vari- they were either having limited discriminatory power within
ety and vast array of mutations makes molecular detection the complex147–149 or low reproducibility150 and not used as
triazole resistance in Candida species difficult. In contrast, widely and effectively as β-tubulin/calmodulin sequencing.
triazole resistance in A. fumigatus is more limited and
involves largely mutations in Cyp51A (equivalent to Erg11).
Triazole resistance mechanisms in Aspergillus can involve 23.2 Methods
both overexpression of drug efflux transporters and/or modi-
fication of the target site sterol 14-α demethylase, encoded
by cyp51A and cyp51B.140,141 However, clinical resistance Besides the lack of standardization and validation of PCR,
is mostly correlated with mutations in Cyp51A, which has another major obstacle limiting the wider use of nucleic acid
been exploited to design a multiplex panel of allele-specific detection of Aspergillus is the inefficient extraction of nucleic

acids.151 As for the best extraction method, a consensus is dif- although the lysis buffer used for the disruption has to be
ficult to achieve because it varies with target type (DNA or replaced with that matching the automatic extraction machine.
RNA) and specimen source (blood, tissue, respiratory fluid). For instance, the easyMAG® system (bioMerieux) requires
As extraction methods often vary widely from lab to lab, lysis buffer with proteinase K (20 μg/mL) added to the conidia
only the most commonly used approaches for different types suspension or ground mycelia powder in the lysing matrix to
of samples will be described below. Nucleic acid extraction process the FastPrep cleared lysate. The suspension is incu-
from Aspergillus cells tends to be difficult and inefficient. bated at 55°C for 1 h followed by a 10 min centrifugation at
This is largely due to the fact that the complex cell wall of full speed using a countertop centrifuge. The cleared lysates
filamentous fungi is hard to break. Thus, rigorous extrac- are transferred into a sample cartridge and mixed with 2 mL
tion methods that utilize either enzymatic digestion such as of easyMAG lysis buffer. The silica solution is added, sample
recombinant lyticase or mechanical disruption by bead-beat- ID login and the program setting are performed by follow-
ing are required. However, the extra steps of cell lysis not only ing the user’s instructions. Other automatic extraction instru-
prolong the extraction procedure, but also may increase the ment such as MagNA Pure systems (Roche Applied Science)
possibility of fungal nucleic acid contamination especially can also be used, except the lysis buffer should be adjusted
when enzymatic lysis is performed,152,153 since the sources accordingly as stated above.
of contamination are everywhere, including lytic enzymes,
buffers, spin columns (the silica membranes), tubes, dispos- 23.2.1.2 Blood
ables, and even water. For this reason, the chosen extraction The pathogenesis of IA is poorly understood and so is our
method represents a compromise between efficiency, lack of knowledge of the optimal blood fraction required for the
exogenous contamination, and the applicability to routine molecular detection of Aspergillus nucleic acid. While some
high-throughput laboratories.83 High-speed cell disruption researchers suggest that the yield of DNA from serum,
incorporating chaotropic reagents and lysing matrices is one plasma, and the white cell pellet is similar,155 others find that
combination, which provides relatively high extraction effi- whole blood is a better source for fungal DNA extraction
ciency from Aspergillus and other filamentous fungi.154 Either than plasma.156 We recommend the collection of whole blood
manual or automated methods can be adopted, depending on for the nucleic acid detection, because the use of whole blood
the number of samples needed to be processed and the avail- allows the detection of hyphal fragments and circulating
ability of the automatic extraction instruments. nucleic acid while the use of serum only detects circulating
nucleic acid.151,157 The starting volume of whole blood var-
23.2.1.1 Aspergillus Culture ies from 200 μL to 10 mL in different clinical settings,157–160
Both types of Aspergillus culture, conidia and mycelia although it is expected that larger volumes produce greater
(hyphae), may be the starting material for extraction, although yield, which promotes higher sensitivity.161 Different extrac-
the extractions starting with mycelia are more efficient than tion protocols have been widely investigated around a com-
that with conidia.111 Conidia are usually harvested from 3-to mon principle, which is enrichment of fungal nucleic acid
5-day-old Aspergillus cultures grown on Sabouraud dextrose by removing the bulk of human nucleic acid to increase the
agar (SDA) or potato dextrose agar (PDA) by washing with sensitivity of the molecular assay. Currently, the hypotonic
sterile saline—0.1% Tween 20. Compared to conidia, myce- lysis of blood cells has been used very often for this purpose.
lia harvest takes more efforts. The overnight Aspergillus Any in-house brewed or commercial blood cell lysis buffer
culture grown in liquid media are filtrated, washed, dried, can be used, and the main components usually consist of
and thoroughly ground in liquid nitrogen using mortar and Tris buffer, MgCl2, or with Triton X-100. By centrifugation,
pestle. After collecting fungal cells, mechanical disruption fungal cells are trapped in the pellet formed by blood cell
takes place under the chaotropic condition of the lysis buffer debris, and the supernatant containing human nucleic acid
(buffer AP1 or buffer RLT added with β-Mercaptoethonal is decanted. The pellets are resuspended in lysis buffer with
if using DNeasy or RNeasy kit from QIAGEN). FastPrep proteinase K and transferred into lysing matrix as described
instrument (MP Biomedical) with the matching lysing matrix for the extraction from Aspergillus culture above. Manual or
is used in author’s lab (Perlin lab) and also many other labs automated extraction should be decided before the extraction
for the high-speed disruption. The velocity of 6 m/s for 45 s starts since different lysis buffers will be used based on the
with a 5 min rest period between runs is the chosen setting. different method.
The lysis buffer is added to the conidia suspension or the fine
powder of ground mycelia above, then transferred into the 23.2.1.3 Tissue
lysing matrix and processed on FastPrep instrument. After Histological examination and culture techniques applied to
centrifugation, the cleared lysates (supernatants) are trans- tissue are considered as the reference diagnostic standard for
ferred from the lysing matrix and are ready to proceed to the IA.42 With more and more data showing that qPCR is more
rest of extraction by simply following instructions from the sensitive than culture for the detection of Aspergillus spp.
commercial kits. in tissue,162,163 the possibility of accepting a positive PCR
Automated nucleic acid extraction can take place with result in tissue as the reference standard for IA deserves
the cleared lysates obtained from the FastPrep procedure, increasing attention. For preparation of fresh tissue samples,

Aspergillus 179
homogenization should be done in a sterile, nuclease-free simultaneously.170,171 A LightCycler-based RT-PCR approach

environment. High-speed tissue homogenizer and crushing to detect common pathogenic Aspergillus species includ-
of tissues in a plastic bag are the most often used methods ing A. fumigatus, A. flavus, A. niger, and A. terreus simul-
to prepare a tissue homogenate prior to nucleic acid extrac- taneously was recently reported.170 High-throughput DNA
tion.164 The tissue homogenates are then mixed with lysis buf- sequencing of ribosomal internal transcribed spacers (ITS1,
fer and proteinase K and processed on a FastPrep instrument, ITS2) is ideal and is fundamentally the most accurate, but
after which the protocols described for culture and blood it is not as fast or as convenient as RT detection following
are applied. For formalin-fixed, paraffin-embedded tissue nucleic acid amplification.
samples, it has been reported that the extensive cross-linking
of tissue proteins after fixation in formaldehyde can cause 23.2.2.2 Secondary Drug Resistance
nucleic acid fragmentation and inhibition of the PCR pro- As for secondary drug resistance detection, different strate-
cess.165 Sample preparation is more complicated than fresh gies can be applied depending on drug class and the resistance
tissue because of the extra dewaxing by xylene and wash- mechanisms. Our knowledge of drug resistance mechanisms
ing by ethanol; the universal DNA extraction is applicable in Aspergillus is still emerging. Secondary triazole resistance
to formalin-fixed, paraffin-embedded tissue samples.166 For in Aspergillus predominantly involves mutations in genes
detailed information, please consult Refs. [101,167]. associated with the drug target. Limited mutations in Cyp51A
confer resistance, making them ideal targets for molecular
detection. These mutations confer different susceptibil-
23.2.2 Detection Procedures ity profiles: (1) resistance to itraconazole and posaconazole
An efficient way to detect and identify the clinically impor- is associated with amino acid substitutions at Gly 54; (2)
tant Aspergillus species is using multitiered nucleic acid itraconazole-resistance and high MICs for voriconazole,
detection panels to recognize increasing complexity from ravuconazole, and posaconazole at Met 220; (3) azole cross-
pan-genera to species-specific and drug resistance-associated resistance with cyp51A overexpression produced by a tandem
targets. Thus, different sets of primers and probes aimed at a repeat (TR) of a 34 bp sequence in the cyp51A gene promoter
variety of detection targets will be discussed. As representa- in combination with an amino acid substitution at Leu 98
tives of DNA and RNA detection, RT-PCR and RT NASBA (TR-L98H); and (4) triazole cross-resistance related to a Gly
will be described below. 138 to cysteine substitution. They were used as the detection
targets for the development of a RT-PCR assay to assess tri-
azole resistance in A. fumigatus. When combined in a multi-
23.2.2.1 Primers, Probes, and Platforms plex platform, the assay provides a rapid evaluation of drug
Among the extensive studies (>200) of detecting Aspergillus resistance in A. fumigatus. By taking advantage of the single
by PCR, most utilize ribosomal RNA genes as detection tar- nucleotide difference detection capability of probes like
gets, as they are multicopy genes, contain highly conserved molecular beacons in a high-throughput, multiplex format,
sequences as well as variable regions, which facilitate the a comprehensive two-tiered molecular diagnostic assay was
genus and species-specific detection. Meanwhile, some stud- developed for triazole-resistant A. fumigatus detection.119
ies have developed new assays using different targets such Molecular probes corresponding to wild-type and triazole-
as mitochondrial DNA target.115,155 As rapidly developing resistance mutations of Aspergillus Cyp51A (Gly54, Gly138,
RT detection probes (e.g., TaqMan, molecular beacons) are Met220, and L98/Tandem Promoter repeat) can be used to
incorporated into PCR assays, RT-PCR formats are likely to profile resistance in a two-tier drop out and positive iden-
dominate the molecular diagnosis of Aspergillus in the near tification format. Other mutations have been identified but
future. A summary of the most commonly used RT nucleic not validated as a source of resistance. Nevertheless, the sys-
acid assays and the corresponding primer and probe sets for tem is robust and can be expanded to include new validated
Aspergillus detection is listed in Table 23.2. targets. Table 23.3 shows the primers and beacon sequences
In addition, newly emerged nucleic acid amplification proposed for this approach.
technology, NASBA offers some advantages over PCR. It is Resistance to echinocandin drugs remains relatively low,
an RNA-directed isothermal amplification and as such there but it has been well recognized that genetic modifications
is no need for thermal cycling. Its robust RNA amplifica- of Fks1 subunit is the universal mechanism for echinocan-
tion is more sensitive than PCR. It hasn’t been used widely din resistant fungi.172 Up to now, the widely reported amino
for Aspergillus diagnosis, largely due to cost and complex- acid substitutions in FKS1 and FKS2, which are responsible
ity of quantification. Nevertheless, good performance has for Candida species resistance to echinocandin drugs,173,174
been reported from several studies using NASBA assays to has not been observed from clinical Aspergillus isolates.
detect Aspergillus spp.91,92,168 For more detailed information, Nonetheless, the over-expression of FKS1 has been linked to
please consult Refs. [91,92,168,169]. Several other platforms the clinical A. fumigatus isolates with reduced susceptibility
including LightCycler with post-amplification (mostly PCR to caspofungin.64 An RT TaqMan PCR assay to detect FKS1
product) melting profile analysis and Luminex xMAP tech- gene developed by Costa and colleagues demonstrated good
nology have been shown to identify different Aspergillus spp. performance of being able to detect 1 fg of A. fumigatus DNA

180
TABLE 23.2
Real-Time Nucleic Acid Assays for Aspergillus Detection
Sensitivity
Target Gene Assay Format Primers and Probes Sequences (5′–3′) Specificity Determined Determined Reference
18S rRNA TaqMan PCR Forward primer 5′-TTGGTGGAGTGATTTGTCTGCT-3′ Aspergillus spp. 20 copies of Aspergillus [158]
Reverse primer 5′-TCTAAGGGCATCACAGACCTG-3′ DNA per assay
Probe 5′-FAM-TCGGCCCTTAAATAGCCCGGTCCGC-3′-
TAMRA
18S rRNA FRET PCR Forward primer 5′-ATTGGAGGGCAAGTCTGGTG-3′ Aspergillus spp. 5 CFU/mL of blood [175]
Reverse primer 5′-CCGATCCCTAGTCGGCATAG-3′
Probe (LC) 5′-Red640- 15 CFU/assay, 20 CFU/ [159]
TGAGGTTCCCCAGAAGGAAAGGTGCAGC-3′ mL of blood

Probe (FL) 5′-GTTCCCCCCACAGCCAGTGAAGGC-3′flu
28S rRNA TaqMan PCR Forward primer (ASF1) 5′-GCACGTGAAATTGTTGAAAGG-3′ Aspergillus spp. 5 copies/assay, 10 CFU/ Primers
mL of blood from [176]
Reverse primer (ADR1) 5′-CAGGCTGGCCGCATTG-3′
Probe (ASP28P) 5′-FAM-CATTCGTGCCGGTGTACTTCCCCG-TAMRA-3′ Probe from
[177]
28S rRNA NASBA-Molecular Sense primer (P2) 5′-CAGCAGTTGGACATGGGTTA-3′ Aspergillus spp. cross 0.1–1 CFU/assay [168]
Beacon react with Penicillium
chrysogenum
Antisense primer (P1) 5′-AATTCTAATACGACTCACTATAGGGGAGAATCCACA
TCCAGGTGC-3′
Probe (MB) 5′-CGCGA GTGCGCCGTGTGCCGAAA TCGCG -3′
5.8S rRNA TaqMan PCR Forward primer 5′-TTGGTTCCGGCATCGA-3′ Aspergillus spp. also 200 fg/assay [178]
amplify Fusarium spp.
Reverse primer 5′-GCAGCAATGACGCTCGG-3′
Probe 5′-FAM-TGAAGAACGCAGCGAAATGCGATAA-
TAMRA-3′
Aspergillus
Mitochondrial FRET PCR Antisense primer 5′-AACACCTGACCTTTCGCGTGTA-3′ Aspergillus fumigatus 3 fg/assay [155]
DNA (rRNA)
Sense primer/probe 5′-CTGTTAGTGCGGGAGTTCAAAXTCT-3′ (X represents
internal labeling of the T with LC-Red 640)
Probe 5′-CTGAGCTAATTTCTTTCAACCCAAGGGA-3′
Mitochondrial FRET PCR Forward primer (AfLC2s) 5′-AATGCACGATACTGTAGGATCTG-3′ Aspergillus fumigatus, 10 copies of Aspergillus [115]
DNA (cytochrome also amplify Aspergillus DNA, or 1–5 CFU/mL
b gene) clavatus of blood
Reverse primer 5′-TGCATTGGATTAGCCATAACA-3′
(AfLC2as)
Probe (Cyt3A) 5′-Red640-TAATCTATCATAATTACCAGAAATACCTAAA
GGA-3′
Probe (Cyt3B) 5′-AATCTTTAAATACAAAGTAAGGAGCGAAAG-3′flu
18S rRNA LightCycler (PCR Forward primer (ASP_F) 5′-ATTGGAGGGCAAGTCTGGTG-3′ A. fumigatus/A. flavus 5 CFU/mL of blood for Primers
+ melting curve specific A. fumigatus, and A. from [157]
analysis) flavus
Reverse primer (ASP_R) 5′-CCGATCCCTAGTCGGCAT-3′
A. terreus specific
50 CFU/mL of blood for
A. terreus and A. niger
Probe (Asp.fum) LC640Red-5′- A. niger specific Probes
TGAGGTTCCCCAGAAGGAAAGGTCCAGC-3′/5′- from [170]
GTTCCCCCCACAGCCAGTGAAGGC-3′FL
Probe (Asp.nig) LC640Red-5′-
TGAGATTCCCCAGAAGGAAAGGTCCAGC-3′/5′-
GTTCCCCCCACAGCCAGTGAAGGC-3′FL
Probe (Asp.terr) LC705-5′-TGGGATTCCCCAGAAGGAAAGGTCCAGC-
3′/5′-GTTCCCCCCACAGCCAGTGAAGGC-3′FL
Notes: The copy numbers of ribosomal DNA were found variable, from 38 to 91 copies per genome.102 The constant used for correlating the weight of genomic DNA of A. fumigatus to the number of genome was
also different among studies. It was assuming 50 fg of DNA equals to 1 conidia by some studies,157,179 and some study was calculating 33 fg of DNA as one genome.178 For mitochondrial genes, Spiess et al.
described as 10 copies of mitochondrial A. fumigatus cytochrome b gene corresponds to 13.2 fg of genomic DNA or 1–5 CFU.115
181
TABLE 23.3
Primers and Molecular Beacons for Triazole Resistance
Target
regiona Sequence (5′–3′)b Purpose
G54-F TCATTGGGTCCCATTTCTG Real-time PCR primer
G54-R GCACGCAAAGAAGAACTTG Real-time PCR primer
L98-F GCGTTCAGGGAAACGAGT Real-time PCR primer
L98-R AAACGGGGGTCGTCAATG Real-time PCR primer
G138-F CAAGCTGATGGAGCAGAAAA Real-time PCR primer
G138-R CAATAAGTGGCACATGAGAC Real-time PCR primer
M220-F TCATGACCTGGACAAGGGC Real-time PCR primer
M220-R GCCGCATAACAAGAAGCGA Real-time PCR primer
TR-F ATCGCAGCACCACTCCAG Real-time PCR primer
TR-R CTTTCATTCGGCTCAGCAC Real-time PCR primer
G54-MB CGCGATCATCAGTTACGGGATTGATCCATCGCG WT allele probe
G54W-MB CGCGATCATCAGTTACTGGATTGATCCATCGCG G54W mutant allele probe
L98-MB CGCGACAACGGCAAGCTCAAGGATGGTCGCG WT allele probe
G138-MB CGCGACGTACGGCTTGACTCAGTCTGGTCGCG WT allele probe
G138C-MB CGCGACGTACTGCTTGACTCAGTCTGGTCGCG G138C mutant allele probe
M220-MB CGCGATCATCAATTTATGCTAACCGTGGGATCGCG WT allele probe
TR-MB CGCGTCGCTGAGCCGAATGAATCACGGACGCG Tandem repetition probe
Gly 54-T AAAAGGATCAATCCCGTAACTGATGAAAA G54 allele target
G54W-T AAAAGGATCAATCCAGTAACTGATGAAAA G54W mutant allele target
Leu 98-T TTTTTCATCCTTGAGCTTGCCGTTTTT L98 allele target
Gly 138-T AAAAACAGACTGAGTCAAGCCGTACTAAAA G138 allele target
Gly 138-T AAAAACAGACTGAGTCAAGCAGTACTAAAA G138C mutant allele
Met 220-T AAAAGCGCACGGTAGCATAAAATTGATGAAAA M220 allele target
TR-T AAAAACGTGATTCATTCGGCTCAGCAAAAA Tandem repetition target
a F, sense; R, antisense; MB, molecular beacon; T, target.

b Probe and target domains are underlined.
in one PCR reaction. In this assay, the forward primer was in Table 23.2 for broad-spectrum and species-specific detec-
5′-GCCTGGTAGTGAAGCTGAGCGT-3′, the reverse primer tion and those mentioned in Section 23.2.2.2 for drug resis-
is 5′-CGGTGAATGTAGGCA TGTTGTCC-3′, and the probe tance identification.
was 5′-FAM-TCACTCTCTACCCCCATGCCCGAGCC-
3′-TAMRA.112 Other RT formats can also be used for FKS
detection, depending on the availability of different RT 23.3 Summary and Perspective
detecting instruments.
The challenges posed to positively and effectively diagnose
IA at an early stage of disease are daunting. With current
23.2.2.3 Assay Conditions technology, no single assay, apart from direct culture, can
There is no concordant condition for Aspergillus nucleic deliver a definitive diagnosis. Rather, a combination of diag-
acid tests described above. It varies when different plat- nostic approaches are required that utilize various biomarker
forms, primers and probes, and different commercial prod- endpoints, radiographic and clinical presentations. Molecular
ucts are applied. It is important to make a best fit of every technology holds great promise for rapid and sensitive detec-
parameter to improve the probability for reliable detection. tion of nucleic acids from hyphal elements, and may be
Regardless of the target used for detection, the working particularly helpful in identifying drug resistance markers
conditions always have to be optimized, especially in mul- against triazole drugs. As RT detection platforms become
tiplex assays. Intricate interactions among primers, probes, more robust, they need to detect infection at an early stage,
and templates in multiplex format decrease the sensitivity prior to the onset of major clinical symptoms. Furthermore,
of the assay as well as enhance the difficulty of working they need to address common issues of spore contamination
condition optimization. Therefore, whenever possible, con- in non-sterile compartments, such as the upper respiratory
sensus primers should be designed for multiplex detection. tract, and the monitoring of therapeutic efficacy including
For detailed information, please consult the references listed emerging drug resistance.

Aspergillus 183
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24 Blastomyces
Mark D. Lindsley
Contents
24.1 Introduction...................................................................................................................................................................... 189
24.1.1.1 Classification.......................................................................................................................................... 189
24.1.1.2 Morphology............................................................................................................................................ 189
24.1.1.3 Serotypes................................................................................................................................................ 190
24.1.1.4 Genotypes.............................................................................................................................................. 190
24.1.1.5 Virulence Factors................................................................................................................................... 190
24.1.1.6 Animal Infections...................................................................................................................................191
24.1.1.7 Epidemiology..........................................................................................................................................191
24.1.2 Clinical Features and Treatment........................................................................................................................... 192
24.1.2.1 Clinical Features.................................................................................................................................... 192
24.1.2.2 Treatment............................................................................................................................................... 192
24.1.3 Diagnosis.............................................................................................................................................................. 192
24.1.3.2 Molecular Diagnostic Techniques......................................................................................................... 194
24.2 Methods............................................................................................................................................................................ 195
Disclaimer.................................................................................................................................................................................. 197
References.................................................................................................................................................................................. 197
24.1 Introduction 24.1.1 Classification, Morphology, and Biology

Blastomycosis is a systemic fungal disease caused by the 24.1.1.1 Classification
thermally dimorphic mould Blastomyces dermatitidis and is B. dermatitidis is an ascomycete belonging to the family
one of the major endemic mycoses that infect humans and Onygenaceae in the order Onygenales. Within the genus
animals. If left untreated, blastomycosis can spread sys- Blastomyces, B. dermatitidis is the only known species. Other
temically and may become fatal, regardless of immune sta- medically important members of the family Onygenaceae
tus. B. dermatitidis was first described in 1894 by Thomas include the systemic pathogens Histoplasma capsulatum,
Caspar Gilchrist, a dermatologist, who observed numerous Paracoccidioides brasiliensis, and Emmonsia sp., the lat-
10–16 μm, round, doubly contoured refractile bodies in a skin ter an uncommon cause of pulmonary disease in humans.4,5
lesion of a patient thought to be suffering from chronic scrof- All, including B. dermatitidis, are thermally dimorphic,
uloderma.1 The organism was believed to be a Blastomycete growing as a mould at 25°C and as a yeast-like form at
yeast by Gilchrist and others until 1896 when Gilchrist dem- 37°C. Phylogenetic analysis of the members of the family
onstrated in culture the organism’s ability to produce hyphae Onygenaceae, comparing sequences from the 18S,6 28S,7,8
and conidia and described its classic growth characteristics and internal transcribed spacer regions9 of ribosomal RNA
in vitro.1 Observing that this organism was unique from other (rRNA) genes, have shown that Blastomyces is most closely
yeasts, he named the disease Blastomycetic Dermatitis and the related to Emmonsia sp. and Paracoccidioides brasiliensis.
organism Blastomyces dermatitidis.2 The first report of inva-
sive blastomycosis was in 1902 by Walker and Montgomery3 24.1.1.2 Morphology
in a patient believed to be suffering from disseminated mili- When cultured at ambient temperature, the filamentous
ary tuberculosis. Initially referred to as “Gilchrist’s disease,” mould form of B. dermatitidis produces a white, downy to
blastomycosis has also been referred to as “North American wooly colony that may become tan to brown with age. The
blastomycosis” and “Chicago disease” after the location reverse side of the culture is typically non-pigmented but
where a large number of early cases were observed. may also darken with age. Microscopically, Blastomyces
189

dermatitidis is a hyaline mould that produces septate hyphae polymorphic DNA (RAPD) fingerprint analysis. McCullough
which give rise to oval to round aleuriosporic conidia directly et al.22 analyzed 59 isolates from 15 regions in the United
from the hyphae or on short stalks arising at right angles States, India, Africa, and Canada using PCR-RFLP. The
to the hyphae. When disturbed, the infectious conidia are authors amplified the approximately 5.5 kb intergenic spacer
aerosolized and enter the body by inhalation. After entering (IGS) region of the ribosomal DNA (rDNA) with PCR prim-
the body B. dermatitidis converts to a yeast-form, typically ers BD28S and NS2 followed by digestion with restriction
8–15 μm in diameter. The yeast cell is often multinucleate endonucleases. Three genotypic groups (A–C) were also
and may contain up to 8–12 nuclei.10 The yeast reproduces observed. Of the 59 isolates analyzed, 17 belonged to group
through budding, forming a single daughter cell from the A, 23 to group B, and 19 to group C. Interestingly, while
mother cell by way of a broad-based bud with a septum which groups B and C contained isolates from national and interna-
may be as wide as the daughter cell prior to separation. The tional regions, 16 of 17 Group A isolates were from the upper
yeast form of B. dermatitidis is not believed to be directly mid-west region of the United States and Canada. RAPD fin-
infectious and therefore blastomycosis cannot be spread by gerprinting could further subdivide the three groups into 5,
person-to-person transmission. 15, and 12 subtypes, respectively. Recently, Meece et al.23
Blastomyces dermatitidis is the anamorphic or asexual likewise performing PCR-RFLP on 106 human, animal, and
form of the ascomycete Ajellomyces dermatitidis (teleo- environmental isolates by amplifying the IGS region and
morph). The teleomorphic or sexual form was first described digesting with restriction endonuclease Dde I, found that the
in 1968 by McDonough and Lewis11,12 after mating dif- majority of specimens fell into one of three groups corre-
ferent strains of B. dermatitidis.13,14 Large, 200–350 μm, sponding to those of McCullough et al., A (50 isolates), B
thick-walled cleistothecia ornamented with coiled spiral (51 isolates), and C (3 isolates). However, two isolates were
hyphae15,16 are produced. Lateral hyphae give rise to asci that unable to be placed into one of the three groups and thus two
contain eight ascospores measuring 1.5–2.0 μm in diameter. new groups were formed, groups D and E, with one isolate
B. dermatitidis was determined to be heterothallic, requir- each. Another approach for genotyping B. dermatitidis iso-
ing two separate mating types (a+ and a– mating types) to lates used PCR and sequence analysis of an approximately
produce sexual structures. Therefore, single ascospores from 360 bp portion of the promoter region of the virulence fac-
two different strains were able to produce the sexual struc- tor, Blastomyces adhesion 1 (BAD-1).23 Using this approach
tures whereas ascospores of the same strain could not.12 The the B. dermatitidis isolates were placed into four haplotypes.
teleomorph of B. dermatitidis is sufficiently similar to the Interestingly, a subset of the isolates contained two inserts of
teleomorph of Histoplasma capsulatum17 and Emmonsia cre- 35 and 251 bp, respectively. The significance of this differ-
scens4 in that all have been placed in the same teleomorphic ence remains to be determined.
genus Ajellomyces as Ajellomyces dermatitidis, Ajellomyces PCR-RFLP genotypic analysis,22 performed on 14 patient
capsulatus, and Ajellomyces crescens, respectively. isolates obtained from one of the largest point source out-
breaks of blastomycosis (Eagle River, WI), revealed that
24.1.1.3 Serotypes 10 were typed as group A, 2 were typed as group B, and
Serologic examination of B. dermatitidis isolates demon- 2 were typed as group C. Furthermore, the environmental
strated that B. dermatitidis can be divided into two sero- isolates associated with the outbreak were also genotypically
types.18–20 Kaufman et al.18 using antisera specific for the different—group B and group C, respectively. These results
immunodominant “A” antigen of B. dermatitidis revealed suggest that even within a small geographical area B. derma-
that of 88 isolates from North American and international titidis can display a range of heterogeneity. The significance
sources, all but 11 isolates that were recovered from Africa of these results remains to be determined.
reacted with the antisera. The B. dermatitidis isolates reac-
tive with the antisera were placed into serogroup 1. African 24.1.1.5 Virulence Factors
isolates failing to react with the antisera were placed into Some virulence factors expressed by Blastomyces are also
serogroup 2. This finding was confirmed when all isolates shared with the other thermally dimorphic moulds. Thermal
originating from Africa failed to react with a monoclonal dimorphism may be considered a virulence factor in that the
antibody specific for the antigen WI-1 (analogous to A anti- initial conversion from the smaller conidia, produced by the
gen).19 Further studies using Southern blot analysis demon- free living environmental mould form, results in larger thick-
strated that the African strains lacked the WI-1 gene.19 walled yeast form that may be more difficult to phagocytose,
or may even be inhibitory to phagocytosis.24
24.1.1.4 Genotypes Other virulence factors are only expressed after conversion
B. dermatitidis can be divided into at least three genotypic to the yeast form. During the process of conversion to the yeast
groups. Using restriction fragment length polymorphism form construction of the cell wall changes from a primarily
(RFLP) of genomic DNA followed by Southern blot analysis β-1,3-glucan dominant cell wall to an α-1,3-glucan dominant
using mitochondrial DNA probes, Yates-Siilata et al.21 placed cell wall. Whereas the mycelial cell wall before conversion
all but 4 of 19 B. dermatitidis isolates from Arkansas into contains 60% β-1,3-glucan and 40% α-1,3-glucan, after con-
group 1 whereas groups 2 and 3 contained two isolates each. version, the yeast-form cell wall contains approximately 95%
Group 1 could be further subdivided using random amplified α-1,3-glucan and 5% β-1,3-glucan.25 Using a monoclonal

Blastomyces 191
antibody specific for α-1,3-glucan (MOPC 104e), the amount other organs of the body including lymph nodes, skin, bones,
of α-1,3-glucan present in the cell wall of three different and eyes, and is best treated with itraconazole.39
strains of B. dermatitidis was measured both microscopically Symptomatic infection in other animals is much less com-
and through flow cytometric analysis.26,27 B. dermatitidis mon. However, an outbreak of blastomycosis was observed
strains with increased amounts of α-1,3-glucan detected in in Chicago, IL where five suburban cats were found to have
the yeast cell wall also demonstrated an increased ability to developed blastomycosis within a 3–4 month time span.40
cause disease.26 The importance of α-1,3-glucan as a viru- While all lived within 300 m of a water source, only one
lence factor has also been demonstrated in Histoplasma cap- of the five cats was an outdoor cat. All were diagnosed by
sulatum28 and Cryptococcus neoformans.29 In those fungi, observing typical yeast forms in either cytologic smears or by
inhibiting the production of α-1,3-glucan by gene disruption histopathology. Non-domesticated cats are also susceptible to
or interference with the α-1,3-synthase (AGS1) gene substan- blastomycosis. Two Asian lions, an African lion, a Siberian
tially reduced virulence in mouse infection models and in tiger, a cheetah, and a snow leopard, all housed at a Tennesse
vitro killing assays. A possible role of α-1,3-glucan in pro- zoo, were diagnosed with blastomycosis through cytology or
moting virulence is its ability to block the innate immune a combination of serology and radiographic analysis.44
response to the immune stimulating β-1,3-glucan and inhibit
the production of tumor necrosis factor (TNF-α).30 24.1.1.7 Epidemiology
BAD-1, a virulence factor specific for B. dermatitidis, is The endemic range of blastomycosis is difficult to define due to
a cell surface molecule expressed only on yeast form cells.31 the lack of sensitive environmental culturing methods.45 Also
Formerly known as WI-1, BAD-1 is a 120 kDa adhesion pro- an adequate skin test antigen, an important epidemiologic tool
tein found both on the surface of the yeast cell and secreted used to determine exposure and subclinical infection rates,
in a cell-free soluble form.32 After being secreted extracel- is lacking for blastomycosis.46,47 Endemic regions of blasto-
lularly, BAD-1 binds to chitin on the yeast cell wall, which mycosis have been estimated primarily through case reports
facilitates binding to macrophages and lung tissue33 through and locating areas of increased incidence of symptomatic
both the CD-14 receptor and complement receptor 3(CR3). disease. Endemic areas include the regions around the Great
This interaction, along with the secretion of soluble BAD-1, Lakes and the Saint Lawrence Seaway in North America, and
modulates the immune response by stimulating phagocytes the Mississippi and Ohio River valleys in the United States.
to produce transforming growth factor-β (TGF-β), an antago- Incidence of reported disease in the endemic region averages
nist of TNF-α expression.34–36 Interestingly the expression of about 1 case per 100,000 population.48 However, hyperen-
BAD-1 on the yeast cell surface is inversely correlated with demic areas of blastomycosis are found in parts of Wisconsin
virulence in that increased expression of BAD-1 is associ- and parts of eastern Canada, particularly in the Province
ated with decreased virulence.32 While avirulent strains may of Ontario.49 Annual rates of infection in these areas are as
express more BAD-1 on the cell surface, they secrete signifi- high as 2–7 cases per 100,000 population in and around the
cantly less soluble form. In this situation the avirulent strains town of Kenora, Ontario49 and a mean of 5.1–41.9 cases per
bind to phagocytes more rapidly and in greater numbers than 100,000 population in the 10 counties that make up the north-
the more virulent wild type strains.37 ern half of Wisconsin.50 Naturally acquired infections have
been observed outside the known endemic region. Infections
24.1.1.6 Animal Infections in two patients in Colorado who developed fungal pneumonia
B. dermatitidis has also been demonstrated as an impor- after working on a project to relocate prairie dogs51 and two
tant pathogen in animals. Other than humans, B. derma- patients who developed osseous lesions from dermal inocu-
titidis also infects dogs,38,39 cats,40 horses,41 and sea lions.42 lation of soil in Nebraska52 suggest that the endemic range
Dogs are by far the most common animal infected. In the may be broader than once believed, or may be increasing in
state of Louisiana, blastomycosis is the most common sys- size. Internationally, B. dermatitidis is also found in localized
temic fungal infection in dogs.38 Dogs that spend extended areas of Central and South America and Africa.
time outdoors near bodies of water, such as hunting dogs, The ecologic niche in which B. dermatitidis is most likely
are most commonly infected. Believed to be either more sus- found is near sources of water such as rivers and lakes and in
ceptible or more often exposed to infected soil due to their moist acidic soil enriched with decomposing organic debris.48
proximity, dogs are at a 10-fold greater risk of infection than In one of the largest outbreaks of blastomycosis, 48 cases of
humans.38,43 Dogs are often considered sentinels of disease blastomycosis, with an attack rate of 51%,48 occurred when a
in that they are often the first to be sick, before humans, large party of adults and children visited a beaver dam and
when exposed to the same environmental sources of conidia. lodge. However, only 2 of 47 environmental samples col-
Through mating studies of human and dog B. dermatitidis lected on and around the beaver dam grew B. dermatitidis.
strains, McDonough and Lewis12 established that a single A third specimen from the beaver dam grew B. dermatiti-
species of Blastomyces was responsible for infections in both dis upon retesting. Exposure to soil near waterways also has
dogs and humans. Whether certain subtypes of B. dermatiti- been implicated in other outbreaks.53,54 Often, locations of
dis are responsible for canine infection compared to human endemic foci are realized only after observing an increase in
infection remains to be determined. As with humans, ani- incidence of blastomycosis over an extended period of time.55
mal infection is primarily pulmonary, but can disseminate to In these cases, the source of infection is often not determined.

Geographic analysis of areas associated with infection disseminated infection can also present at the same time as
show that infections are more likely when exposures take the onset of pulmonary symptoms.77 Treatment can be effec-
place in areas of sandy soil near waterways located at eleva- tive but infection may recur after discontinuation of ther-
tions of less than 500 m.56 In vitro, B. dermatitidis grows best apy.77 While rare in HIV-infected persons,78 the incidence
on media containing by-products of plant and animal waste.57 of blastomycosis may be increasing in certain geographic
Specifically, ammonia, a by-product of animal waste, sup- areas. Pappas reported that the number of immunosup-
ported the growth of B. dermatitidis, even at concentrations pressed patients with blastomycosis seen at the University
that are normally toxic to most fungi.58 Increased levels of of Alabama hospitals increased almost 10-fold over several
these byproducts in the soil, allowing for selective growth decades.79 Dissemination in immunosuppressed patients
of B. dermatitidis, may only be present for a short period more often involves the central nervous system, and has a
of time, which may account for the difficulty of isolating much greater mortality rate than dissemination in immuno-
B. dermatitidis from environmental sources. competent patients. Even though blastomycosis may be rare
it remains important to consider B. dermatitidis in the dif-
24.1.2 Clinical Features and Treatment ferential diagnosis in immunosuppressed patients presenting
with influenza-like illness.
24.1.2.1 Clinical Features
Blastomycosis occurs as a result of inhalation of infectious 24.1.2.2 Treatment
conidia released into the environment after the disruption Guidelines and specific treatment regimens for managing
of soil containing B. dermatitidis. Many of those infected blastomycosis have recently been updated by the Infectious
will remain asymptomatic.59 The median incubation period Diseases Society of America.80 Briefly, treatment may
is 45 days from exposure to onset of symptoms.60 Whereas not be required in immunocompetent patients with mild,
symptomatic patients may present with signs and symptoms self-limited pulmonary infection unless there is reason to
similar to those with bacterial pneumonia,61 many acute suspect that disseminated disease may occur. Mild to mod-
infections often go unrecognized as a generalized influenza- erate disease can be treated with itraconazole monotherapy.
like illness that resolves spontaneously.62–64 Most commonly, Moderately severe to severe disease, whether limited to pul-
however, infected individuals display a slow indolent process monary infection or disseminated extrapulmonary infection,
of cough, weight loss, and fever that may develop over weeks, and including central nervous system disease, should be
months, or even years.65 The development of this chronic dis- treated with amphotericin B (deoxycholate or lipid formula-
ease is insidious and can mimic other more “well-known” tions). After improvement, the treatment may be switched to
diseases such as tuberculosis, delaying the diagnosis of itraconazole. For immunosuppressed patients, treatment with
blastomycosis.61,66 amphotericin B may be later switched to itraconazole and
As a further manifestation of the disease, B. dermatitidis continued indefinitely if the immunosuppression is irrevers-
can spread to extrapulmonary sites. Skin62,67 is the most com- ible. Pregnant women should be treated with amphotericin B
mon site of extrapulmonary spread and provides an accessible as itraconazole is not recommended in this patient popula-
specimen source for culture and histopathologic diagnosis.10 tion due to the drug’s teratogenic potential. Pediatric patients
Besides skin, B. dermatitidis can infect any organ system with severe blastomycosis should be treated with amphoteri-
of the body including the central nervous system,68,69 bones cin B, followed by itraconazole after clinical improvement.
and joints,70,71 and the genitourinary system.72 Because of Recent reports have demonstrated that voriconazole and the
the non-specific and generalized symptoms, the diagnosis experimental azole isavuconazole are also active against
of blastomycosis is often not attained until skin lesions or B. dermatitidis.81,82
symptoms of other extrapulmonary infections occur. Skin
infections have also been noted to occur through accidental 24.1.3 Diagnosis
needle-sticks occurring in a laboratory or veterinary setting
that may introduce the organism subcutaneously resulting in 24.1.3.1 Conventional Techniques
localized disease.73,74 Dermal inoculation of blastomycosis by Direct specimen examination, in vitro culture, serology, and
way of a dog bite has also been reported.75 However, dissemi- histopathology continue to be standard methods in the clini-
nation from this mode of infection has not been documented. cal laboratory for the diagnosis of blastomycosis. Respiratory
Whereas other fungal infections such as cryptococcosis, specimens such as sputum, bronchoalveolar lavage fluid,
histoplasmosis, and coccidioidomycosis have been found tracheal secretions, and pleural fluid are appropriate for the
important causes of opportunistic infections in immunodiagnosis of acute localized infections. B. dermatitidis can
compromised patients, blastomycosis has not. When blas- disseminate to virtually any organ and may be recovered
tomycosis does occur in this patient population, however, from skin,67 blood,83 bone,71 CNS,68 and the genitourinary
these patients usually have more severe disease with a much tract.84 When present, skin lesions are an excellent source for
greater mortality rate than that seen in immunocompetent isolation of B. dermatitidis as the lesions are readily avail-
patients.76,77 Transplant patients develop blastomycosis as able for biopsy and cultures are frequently positive. In male
early as 3 weeks and as late as 4 years post-transplant.77 Most patients, B. dermatitidis may disseminate to the prostate72,84
infections disseminate from an initial pulmonary focus but and may cause dysuria. In these patients prostatic fluid is an

Blastomyces 193
excellent specimen for recovery of B. dermatitidis. In its yeast B. dermatitidis which they named WI-1. While immuno-
form, B. dermatitidis is classified a biosafety level (BSL)-2 logically similar to the A antigen,93 WI-1 is a 120 kDa non-
pathogen85 and therefore specimens from potential blastomy- glycosylated protein whereas A antigen is estimated to be a
cosis patients as well as yeast cultures grown at 37°C may be 135 kDa protein with 37% carbohydrate. An EIA using the
handled using BSL-2 containment. On the other hand, soil or WI-1 antigen produced an assay that had a sensitivity of 77%
other environmental specimens that may contain infectious and specificity of 92%.94
conidia and sporulating mould cultures should be handled A Western blot assay using a culture filtrate antigen was
under BSL-3 conditions.85 developed and evaluated to detect serum antibodies.95 This
test increased the sensitivity to 91% in a panel of sera from
24.1.3.1.1 Direct Detection blastomycosis patients, as compared to 88% using the A anti-
Sputum, skin scrapings, and fine needle aspirates are pre- gen EIA. A 98 kDa extracellular protein antigen was shown
pared in 10% potassium hydroxide (KOH) with or without to be most highly reactive with patient sera. In an immuno-
lactophenol cotton blue stain, and examined using either light diffusion assay, the 98 kDa antigen was closely related to if
or phase contrast microscopy. Observation of the character- not identical with the A antigen and the WI-1 antigen.
istic yeast form cells (8–15 μm in diameter) with daughter Antigen detection: An EIA for the detection of
yeast buds with a wide septum provides presumptive diagno- Blastomyces antigen in urine was evaluated for the diagno-
sis of blastomycosis. The use of the fluorescent dyes such as sis of blastomycosis.96 The assay has a reported sensitivity
Calcofluor white or Blankophor increases the sensitivity of of 92.9% and specificity of 79.3%. Urine specimens from
detection, and requires a fluorescence microscope to observe healthy volunteers and patients with coccicioidomycosis
bright yellow to white fluorescent staining. Due to the non- and candidiasis were negative, but cross-reactions occurred
specific nature of the symptoms of blastomycosis, fungus with urine from patients with histoplasmosis (96.3%), para-
culture is not always ordered on specimens from patients coccidioidomycosis (100%), and penicilliosis marneffei
later shown to have blastomycosis. Stains used for the exami- (70%). Likewise, reciprocal cross-reactivity was also dem-
nation of specimens for other microbiological agents, such onstrated with specimens from blastomycosis patients when
as the Gram stain, do not stain B. dermatitidis well and the assayed using the Histoplasma antigen assay.97 In a case of
yeast-forms may not be easily visualized. Therefore, when culture-positive blastomycosis,98 antigen was demonstrated
viewing specimens stained for other microorganisms it is in the urine using the Blastomyces antigen EIA whereas the
important to be alert to the chance observation of B. derma- Blastomyces ID and CF antibody tests were negative. Urine
titidis. The diagnosis of a case of blastomycosis was delayed antigen levels were monitored during treatment in four pedi-
after a Gram stain-negative specimen later grew B. dermatit- atric Blastomyces culture- and antigen-positive patients.99
idis. Subsequent review of the Gram stain showed numerous Elevated urine antigen levels returned to normal in three
poorly stained yeast cells resembling B. dermatitidis in the patients after treatment with itraconazole; the fourth was
“background” (Dr. Glenn Roberts, personal communication). not compliant with the treatment regimen and elevated urine
antigen levels persisted. The Blastomyces antigen EIA was
24.1.3.1.2 Serodiagnosis also used to detect antigen in the urine (93.5%) and serum
Antibody detection: The presence of antibodies in a patient (87%) of 46 dogs with confirmed blastomycosis.100 Antigen
suspected of having blastomycosis signifies recent or past titers decreased over time during treatment with itraconazole.
infection. Serologic conversion from negative to positive or The Blastomyces antigen EIA appears to correlate well when
a fourfold or greater rise in titer between acute and conva- tested with samples from confirmed cases of blastomycosis.
lescent sera, are necessary for a definitive serodiagnosis of The general usefulness of this assay for the definitive diag-
acute blastomycosis. Complement fixation (CF) and immu- nosis of blastomycosis, however, remains uncertain because
nodiffusion (ID) are the methods routinely used for the of its low specificity and cross-reactivity with the other major
serologic diagnosis of blastomycosis. The use of a purified fungal pathogens.
antigen (A antigen)86 improved the assay over the originally
unpurified culture filtrate concentrate87 and purified A anti- 24.1.3.1.3 Culture
gen is now used in most commercial kits for CF and ID. Culture is still considered the gold standard for the diag-
Antibody determination provides a specificity of 87%–100% nosis of blastomycosis. For the detection of B. dermatiti-
and 100% for the CF and ID tests, respectively.88,89 The sen- dis fungemia, blood cultures should be processed using the
sitivity of ID and CF assays was reported to be 33% during lysis-centrifugation method for optimal sensitivity.83 Liquid
localized infection53 and 85% (ID) and 50% (CF), in dissemi- specimens (bronchoalveolar lavage fluid, cerebrospinal fluid,
nated blastomycosis.48 Incorporation of the A antigen into an etc.) should be centrifuged and the resulting pellet used for
enzyme immunoassay (EIA) format improved sensitivity to culture. Tissue should be homogenized using a laboratory
between 86% and 100% with a specificity of 87%–92%.48,53,90 paddle blender or minced in sterile water or saline before
A commercial version of the A antigen EIA had a reported plating on appropriate media. Processed specimens should
100% sensitivity with a specificity of 85.6%.91 However be inoculated onto enriched media containing antibiotics
this assay is no longer commercially available. Klein and such as chloramphenicol and/or gentamicin to inhibit bacte-
Jones92 described an immunodominant surface antigen of rial growth and cycloheximide to inhibit susceptible moulds,

and incubated at 25°C–30°C. Growth of B dermatitidis may 24.1.3.2 Molecular Diagnostic Techniques
be observed within the first 2 weeks of incubation. However, Commercially available molecular techniques for the diag-
cultures should be held for 4–6 weeks before being discarded nosis of blastomycosis are used for confirmation of culture
as negative. isolates. The Accuprobe Blastomyces Dermatitidis Culture
Positive cultures show an initial growth of a glabrous Identification Test (Gen-Probe) uses a chemiluminescent,
(waxy) colony, becoming fluffy white in color turning beige acridinium ester-labeled DNA probe in a hybridization
to tan with age. Microscopic morphology demonstrates hya- protection assay (HPA). The probe, specific for the B. der-
line mycelia with single round to pyriform conidia, 2–10 μm matitidis rRNA molecule, binds to the rRNA target incor-
in diameter, originating directly from the hyphae or on short porating a chemiluminescent label into the double stranded
stalks. These microscopic features may be confused with molecule formed between the probe and target protecting
other pathogenic (e.g., Scedosporium apiospermum) and non- the bound label from degradation by a hydrolyzing solu-
pathogenic (e.g., Chrysosporium species) fungi, thus requir- tion and available to emit light when the detection reagent is
ing further confirmatory testing before making the final added. Although needing specialized equipment, the assay
identification. The identification of the culture can be con- is quick to perform and requires a small amount of myce-
firmed by (1) demonstrating the conversion of the mould form lium or yeast to perform the assay. The assay has excellent
to the yeast form by growth on an enriched medium such as sensitivity (97%–100%)104,105 and specificity (99%) with only
Kelley’s at 37°C,101,102 (2) immunological identification using P. brasiliensis the only major pathogenic fungi to cross-react
the exoantigen agar gel immunodiffusion method104, or (3) use in this assay. Other cross-reactions have been noted with
of a commercial molecular probe, Accuprobe® Blastomyces Gymnascella hyalinospora.106 The assay has also been evalu-
Dermatitidis Culture Identification Test (GenProbe). The ated using 72 Blastomyces strains isolated from a variety
Accuprobe assay is more expensive than the exoantigen or of non-human mammalian isolates including those from 62
thermal conversion methods, and requires special equipment dogs, 3 cats, and 1 each from a polar bear and sea lion.107 The
and expertise. The decision as to which assay to perform probe assay confirmed the identity of all isolates with 100%
depends on the number of cultures received by the laboratory, sensitivity. Twenty eight isolates from 21 genera other than
the technical expertise available to perform the assay, and the Blastomyces were all negative (100% specificity). However,
desired turn-around time. isolates of P. brasiliensis was not included for evaluation.
Another commercially available, molecular based assay
24.1.3.1.4 Histopathology for the identification of B. dermatitidis is based on the
Hematoxylin and eosin (H&E) staining of tissue sections repetitive-sequence-based PCR (rep-PCR)108,109 recently
from blastomycosis patients reveals an initial polymorpho- developed by DiversiLab and now marketed by bioMérieux
nuclear leukocyte infiltration in early lesions.103 This early Clinical Diagnostics. PCR primers bind to multiple non-
pyogenic reaction is not typical for fungal infections and coding regions in the microbial genome amplifying DNA
diagnosis may be delayed if yeast forms are not present in the fragments of varying sizes. Through a microfluidic electro-
lesion. Later in infection, a granulomatous reaction appears phoresis separation step in a chip format, the DNA fragments
with the formation of multinucleate giant cells. The yeast are measured in size and intensity using laser technology and
cells may be located free or inside giant cells. Abscess for- plotted in a DNA electrophoresis pattern. The resulting DNA
mation may be observed. fingerprint is compared to a library of DNA fingerprints,
B. dermatitidis yeast forms may not stain well in the H&E and a dendrogram is plotted for identification. Identification
stained section, but the presence of hyaline 8–15 μm in diam- of B. dermatitidis110 using rep-PCR demonstrated the abil-
eter, thick-walled, broad-based budding yeast cells is diagnos- ity of this technology to differentiate B. dermatitidis from
tic. The yeast cells can be multinucleated with up to 12 nuclei other dimorphic fungi and placed clinical strains with the
per cell.10 Periodic acid Schiff (PAS) and Grocott methena- database strains. This assay also provides the user the capa-
mine silver (GMS) stain the fungal cell walls a bright pink bility to genetically compare multiple isolates which may be
and brown to black, respectively, providing a more defini- particularly useful in an outbreak setting. Whether rep-PCR
tive demonstration of the typical morphology. In instances can differentiate individual isolates will need further evalua-
where budding is not observed, B. dermatitidis may resemble tion using greater number of Blastomyces isolates from more
Cryptococcus neoformans and can be differentiated using diverse, worldwide sources.
the mucicarmine stain in which the capsule of Cryptococcus Non-commercial methods for the molecular diagnosis
stains a bright pink color. B. dermatitidis is typically nega- of B. dermatitidis employ the standard procedures used
tive with the mucicarmine stain but the cell wall may display for most fungi and have been recently reviewed.111,112 These
a faint reaction. Larger and smaller forms of B. dermatitidis methods have been used for confirmation of isolate identifi-
may resemble empty Coccidioides spherules or Histoplasma cation, identification directly from clinical specimens, and
yeast forms, respectively, but can be differentiated by observ- the detection of B. dermatitidis in environmental samples.
ing the broad based budding characteristic of B. dermatitidis. Universal fungal primers that amplify the 28S113 or the
Mycelial forms of B. dermatitidis in tissue are extremely rare. internal transcribed spacer (ITS)114 regions of the rRNA gene
When present, hyphae are short, hyaline, and septate.103 have been used for confirming the identification of fungal

Blastomyces 195
isolates resembling B. dermatitidis. Species-specific probes eight soil samples obtained at a dog kennel near Lexington,
generated against the 28S region and used in a slot blot assay KY where several dogs had developed blastomycosis. Of
were 100% specific.113 ITS region probes were developed for the eight samples processed, three were positive, producing
the species-specific identification of yeast-like fungi found amplicons with the Blastomyces specific primers and con-
in tissue including thermally dimorphic fungi, Cryptococcus firmed by sequence analysis.
neoformans, and Pneumocystis carinii.114 These probes were
evaluated using an EIA method after PCR amplification of
the ITS region using universal primers ITS1 and ITS4. All 24.2 Methods
probes were 100% specific except for a slight, but not sig-
nificant, cross-reactivity of the Blastomyces probe with DNA
from Coccidioides immitis; the corresponding Coccidioides Methods for the molecular diagnosis of B. dermatitidis have
immitis probe did not react with the B. dermatitidis DNA. not been standardized and are still considered experimental.
In situ hybridization and PCR have been employed for As with most fungi, the rigid fungal cell wall consisting of
the molecular identification of B. dermatitidis from forma- α- and β-glucans and chitin can make extraction of DNA
lin-fixed paraffin-embedded tissue samples. Hayden et al. from B. dermatitidis difficult. Lysis of the fungal cell usually
designed probes to identify B. dermatitidis and the other includes a form of mechanical disruption such as series of
yeast-like fungi in tissue using in situ hybridization.115 Two freeze/thaw steps,6 freezing the sample in liquid nitrogen118
probes, one from the 18S and one from the 28S region of or dry ice,119 followed by grinding the frozen tissue in a mor-
the rRNA gene, were used in combination to probe fungi- tar and pestle, and use of a mechanical homogenizer120 or
containing tissue including a total of 20 tissues containing vigorous agitation in a tube containing glass beads or sil-
B. dermatitidis. In situ hybridization for the detection of ica.121 One protocol uses a combination of a freeze–thaw step
B. dermatitidis was 90% sensitive and 97.4% specific when prior to using a commercial bead beating method extending
compared to GMS staining. the bead beating time from 10 to 30 min.110 Addition of lyti-
Tissue from dogs with histopathologically confirmed case121,122 may facilitate the disruption of the fungal cell wall.
blastomycosis was used to evaluate the extraction and PCR Extraction of DNA after the mechanical disruption phase can
amplification of B dermatitidis DNA in paraffin-embedded be performed using one of several commercial DNA extrac-
tissue.116 Two sets of nested primers were evaluated; one set tion kits.6,110,117
was directed to a portion of the 18S region of the rRNA gene Non-commercial methods for the extraction of Blastomyces
and the other to a portion of the promoter region of the BAD-1 DNA include boiling in a guanidine thiocyanate/phenol
gene. Primers in the 18S region amplified DNA from 10 of extraction buffer113 or the use of cetyl trimethylammonium
25 tissues from B. dermatitidis infected dogs but also ampli- bromide (CTAB) lysis buffer.123 The latter reagent has been
fied DNA from 20 of 20 Histoplasma positive human tissues used extensively for the extraction of DNA from plants,124,125
and 9 of 33 tissues from uninfected dogs. Amplification of soil,126 bacteria,127 and fungi.123,128 Extraction of DNA using
an approximately 360-bp portion of the BAD-1 promoter CTAB lysis buffer provides a source of clean, high molecular
region using primers Blasto I and Blasto II was demonstrated weight DNA free of nucleases and inhibitors of proteinases
to be as sensitive but more specific in that only B. derma- and restriction enzymes,125 and is also efficient in removing
titidis DNA was detected. A limitation in using the BAD-1 polysaccharides that may inhibit PCR.129
primers is that African strains of B. dermatitidis which do CTAB lysis buffer (100 mM Tris pH 8.0, 1.4 M NaCl,
not express BAD-1 have also been demonstrated not to have 20 mM EDTA, 2% cetyl trimethylammonium bromide
the BAD-1 gene19 and would be negative using these prim- [CTAB], 0.2% β-mercaptoethanol) has been used to extract
ers. It is uncertain how frequently BAD-1 negative strains are DNA from a variety of mechanically disrupted samples from
found in cases of blastomycosis. tissue ground in liquid nitrogen130 or dry ice,119 and mechani-
Molecular methods for the detection of Blastomyces in cal homgenization.120 CTAB lysis buffer can also be used in
soil have been evaluated117 also using primers Blasto I and conjunction with glass bead disruption of fungal cells using
Blasto II to amplify the BAD-1 virulence gene promoter. a Bead Beater® system. A benefit of this method is that the
The primers are specific for B. dermatitidis in that they were mechanical disruption phase of the extraction process is self-
unable to amplify DNA isolated from other onygenalean contained, providing a greater margin of safety when work-
fungi (Histoplasma capsulatum, Coccidioides immitis, and ing with pathogenic fungi.
Emmonsia crescens), other environmental fungi (Aspergillus Approximately 600–800 μL of CTAB lysis buffer is
flavus, Penicillium chrysogenum, and Candida albicans) as added to a 1.8 mL screw cap Bead Beater tube that is three
well as soil inhabiting bacteria (Streptomyces griseus and fourths full of glass beads (0.5 mm in diameter). The CTAB
Nocardia brasiliensis). Isolation of DNA from natural soil lysis buffer should entirely wet and cover the glass beads to
samples not believed to contain B. dermatitidis were nega- approximately 5 mm above the glass beads. Approximately
tive using Blasto I and Blasto II primers but were reactive 10–20 mg of fungal mycelium is added to the tube contain-
using fungal universal primers ITS1 and ITS4 demonstrating ing the glass beads and CTAB lysis buffer. The mould can
the presence of natural soil fungi. DNA was isolated from be grown in either solid or liquid medium. However, mould

~650 bp is resuspended in 50 μL Tris/ EDTA (TE) buffer at 4°C for

~350 bp 1–2 h to overnight.
ITS5 ITS1 ITS3 ITS4
18S ITS1 5.8S ITS2 28S

FIGURE 24.1 Diagram of the rRNA gene with the location of the The commonly used loci for PCR amplification and identi-
binding sites of the universal fungal primers.
fication employ universal fungal primers that are directed
to portions of the rRNA gene.112 Each of the rRNA gene
grown submerged in liquid medium by incubation on a regions, the 18S, 28S, and the ITS regions, has been used
shaker apparatus does not normally sporulate and therefore in the molecular identification of B. dermatitidis and have
infectious conidia are not produced. Furthermore, the myce- varying discriminating power.113,114,116 The ITS region of the
lium of moulds grown in liquid disperses into the CTAB lysis rRNA gene, particularly the ITS2 region between the 5.8S
buffer more readily. After the mycelium is added to the Bead and 28S regions of the rRNA gene, demonstrates sufficient
Beater tube, the screw cap is securely tightened and the tube diversity to provide an accurate identification of B. der-
is placed onto the Bead Beater shaker and the Bead Beater is matitidis and is recommended by the Clinical Laboratory
run on the maximum setting for 1 min. The tubes are placed Standards Institute (MM18-A) for identification by DNA
into a 65°C waterbath for 1–2 h, with inversion periodically sequencing.131
to ensure complete digestion. Next, the tubes are centrifuged PCR amplification of the ITS region of the rRNA gene
in a microcentrifuge at maximum speed for 10 min to pel- using primer pair ITS1 and ITS4 produces an approximately
let the glass beads. The supernatant is removed and placed 650 bp product that spans the entire ITS1, 5.8S, and ITS2
into a 1.5 mL conical microcentrifuge tube. An equal vol- regions (Figure 24.1). Another primer, ITS5, located just
ume of chloroform is added to the supernatant, mixed vigor- upstream from the ITS1 binding site, may be substituted for
ously, and centrifuged at maximum speed for 10 min. The ITS1 and in some instances may provide better discrimina-
upper aqueous layer is removed without disturbing the debris tion.132,133 The amplification product is generally the same
layer at the interface and placed into a clean 1.5 mL conical size as that using ITS1 and ITS4. ITS3 combined with ITS4
microcentrifuge tube. The DNA is precipitated by adding an amplifies the ITS2 region of approximately 350 bp. Use of
equal volume of cold isopropanol and mixed immediately this primer set may be desirable if the extracted DNA has
with a vortex mixer. The tubes are placed on ice for at least been damaged, containing only short stretches of amplifiable
10 min; incubation at 4°C overnight may increase the yield DNA, which may occur when extracting DNA from forma-
of DNA. DNA is pelleted by centrifugation in a microcen- lin fixed tissue.134 Table 24.1 describes the primers and their
trifuge at maximum speed for 15 min. The isopropanol is sequences.
removed, 500 μL of cold 70% ethanol is added, and the tube PCR is performed using 1 ng of fungal DNA added to
is centrifuged at maximum speed for 15 min. The ethanol is the PCR buffer mix consisting of 10 mM Tris–HCl buf-
removed and the DNA pellet is allowed to dry. The pellet fer with 50 mM KCl and 1.5 mM MgCl2 pH 8.0; 0.2 mM
TABLE 24.1
Primers Used for the PCR Amplification of B. dermatitidis DNA
Primer Use Direction Sequence (5′–3′)
ITS1 Universal fungal primer: ITS Forward TCCGTAGGTGAACCTGCGG
region of rRNA genea
ITS3 Universal fungal primer: ITS Forward GCATCGATGAAGAACGCAGC
ITS4 Universal fungal primer: ITS Reverse TCCTCCGCTTATTGATATGC
ITS5 Universal fungal primer: ITS Forward GGAAGTAAAAGTCGTAACAAGG
Blasto I Blastomyces specific primer: Forward AAGTGGCTGGGTAGTTATACGCTAC
promoter region for the
BAD-1 geneb
Blasto II Blastomyces specific primer: Reverse TAGGTTGCTGATTCCATAAGTCAGG
promoter region for the
BAD-1 geneb
a From White et al.141

b From Bialek et al.116

Blastomyces 197
deoxynucleoside triphosphate, each; 1.25 U of Taq poly- with adequate sensitivities and specificities that could deter-
merase; and 0.2 μM of each oligonucleotide primer. mine true infection would allow for earlier treatment, reduc-
Conditions for thermocycling begin with an initial denaturing morbidity and mortality. Also, an accurate diagnosis of
ation of 95°C for 10 min, followed by 30 cycles of 95°C for blastomycosis would aid in determining the true incidence
1 min, 53°C for 1 min, 72°C for 1 min, and a final extension of disease.
of 72°C for 10 min with a final hold temperature of 4°C. The The environmental niche of B. dermatitidis remains elu-
PCR product can then be visualized by electrophoresis using sive. Continued advancement in determining the nutritional
a 1%–2% agarose gel. If amplification of the DNA produces needs and environmental conditions for the optimal growth
a weak band, the annealing temperature of 53°C may be of B. dermatitidis may aid in predicting where exposure may
reduced to 51°C. occur and help reduce unnecessary morbidity. Accurate and
PCR primers Blasto I and Blasto II, specific for B. derma- reliable methods to detect B. dermatitidis in the environ-
titidis (Table 24.1), amplify an approximately 360 bp portion ment will help confirm the environmental requirements for
of the promoter region for the BAD-1 virulence gene116,117 B. dermatitidis. Molecular assays have the ability to provide
although some B. dermatitidis strains contain an insert that sensitive and specific results but determining the accuracy of
increases the size to approximately 660 bp. Thermocycling these assays is difficult when the “gold standard” assay is less
conditions for these primers require a denaturing step of sensitive than that being evaluated.
94°C for 5 min followed by 30 cycles of 94°C for 30 s, 50°C It has been over 100 years since Gilchrist first discovered
for 30 s, and 72°C for 1 min with final extension step of 72°C B. dermatitidis; yet there is still much to learn and many
for 5 min. challenges to overcome to better understand the biology,
Identification of the ITS generated amplicon can be epidemiology, and environmental niche of this organism.
accomplished through the use of Blastomyces-specific Newer, more sensitive, and specific diagnostic technologies
probes using slot blot113 or EIA114 methodologies or through combined with improved molecular methods may allow for
DNA sequence analysis.135 The original Sanger sequenc- rapid advancement of our current understanding of this fas-
ing method has been continually improved upon since its cinating dimorphic fungus.
original description with most of the reagents and instru-
mentation commercialized and automated. However, prior
to sequence analysis, primers and dNTPs must be removed Disclaimer
from the PCR product either through column- or enzyme- The findings and conclusions in this chapter are those of the
based methodologies.136,137 When available, sequence author and do not necessarily represent the views of the CDC.
analysis provides the most flexibility when identifying an
unknown pathogen.
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25 Chrysosporium
Contents
25.1 Introduction...................................................................................................................................................................... 203
25.1.3 Diagnosis.............................................................................................................................................................. 205
25.2 Methods............................................................................................................................................................................ 205
25.3 Conclusions....................................................................................................................................................................... 206
References.................................................................................................................................................................................. 206
25.1 Introduction [1–3]. The teleomorphs of Chrysosporium spp. are found in

the genera Aphanoascus, Nannizziopsis, and Uncinocarpus,
Chrysosporium is an anamorph genus whose members are family Onygenaceae, and order Onygenales [4,5].
common soil saprobes, with sexual states (teleomorphs) The obsolete species in the genus include Chrysosporium
found in the ascomycete order Onygenales. Of the 28 rec- dermatitidis (→ Blastomyces dermatitidis), Chrysosporium
ognized species within the genus Chrysosporium, two spe- pannorum (→ Geomyces pannorus), Chrysosporium pru-
cies (i.e., C. zonatum and C. tropicum) have been reported as inosum (→ Sporotrichum pruinosum), and Chrysosporium
causal agents for skin disorder, sinusitis, and other diseases thermophilum (→ Myceliophthora thermophila) [6]. Not dis-
in both immunocompromised and immunocompetent hosts. cussed here are Chrysosporium parvum (or Chrysosporium
parvum var. parvum) and Chrysosporium parvum var. cre-
scens, which are considered as synonymous for Emmonsia
parva and Emmonsia crescens, respectively. To simplify the
The genus Chrysosporium (obsolete synonym: Gleno presentation and reduce confusion, this chapter focuses on
sporella) belongs to the mitosporic Onygenales Chrysosporium spp. that are keratinophilic, causing subcuta-
group, order Onygenales, class Eurotiomycetes, sub- neous infections, whereas Chapter 29 deals with Emmonsia
phylum Pezizomycotina, phylum Ascomycota, and spp. that mainly cause pulmonary adiaspiromycosis.
kingdom Fungi. The group consists of nine genera: Chrysosporium colonies grow moderately at 25°C, and
Blastomyces, Chrysosporium, Coccidioides, Emmonsia, may appear granular, woolly, or cottony, flat, or raised, and
Geomyces, Locazia, Malbranchea, Myriodontium, and folded. Colonies are white cream, yellow, or tan to pale brown
Paracoccidioides. In turn, the genus Chrysosporium con- on the top, and white to brown on the reverse. Hyphae are
sists of 28 recognized species: Chrysosporium articu- septate. Conidia (aleuriconidia) are hyaline, broad based, one
latum, Chrysosporium carmichaelii, Chrysosporium celled, and smooth or rough walled. Conidia are broader than
chiropterorum, Chrysosporium europa, Chrysosporium vegetative hyphae and occur terminally on pedicels, along the
evolceanui, Chrysosporium filiforme, Chrysosporium flu- sides of the hyphae, or in intercalary positions. Conidia usu-
viale, Chrysosporium fluviale, Chrysosporium indicum, ally have an annular frill that is the remnant of the hyphal wall
Chrysosporium keratinophilum, Chrysosporium lobatum, that remains after detachment from the hypha. Arthroconidia
Chrysosporium lucknowense, Chrysosporium mephiticum, are abundant and larger than parent hyphae in diameter.
Chrysosporium merdarium, Chrysosporium minutispo- C. tropicum colonies are moderately fast growing, flat,
rosum, Chrysosporium ophiodiicola, Chrysosporium white to tan to beige, often with a powdery or granular sur-
pannicola, Chrysosporium pilosum, Chrysosporium face texture. Reverse pigment is absent or pale brownish
pseudomerdarium, Chrysosporium queenslandicum, yellow with age. Ameroconidia are hyaline, one celled, and
Chrysosporium siglerae, Chrysosporium submersum, produced directly on vegetative hyphae by nonspecialized
Chrysosporium sulfureum, Chrysosporium synchronum, conidiogenous cells. Conidia (6–7 μm × 3.5–4 μm) are typi-
Chrysosporium tropicum, Chrysosporium undulatum, cally pyriform to clavate with truncate bases and are formed
Chrysosporium vallenarense, Chrysosporium xerophilum, either intercalary (arthroconidia), laterally (often on pedi-
and Chrysosporium zonatum, and 18 unassigned species cels), or terminally.
203

C. zonatum (synonym: Chrysosporium gourii) colo- presence of droplets of colorless exudate at the center. They
nies on potato dextrose agar (PDA) at 37°C are flat and are light brown on the reverse side. On oatmeal agar (30 g
coarsely powdery, and appear yellowish white initially but oat flakes, 1 g MgSO4·7H2O, 1.5 g KH2PO4, 15 g agar, 1 L
darken by 14–21 days to buff (grayish or brownish orange) water), colonies are similar to those on PCA, with a very
with a light brown reverse. Colony topography and color restricted growth at 15°C (5 mm in diameter in 14 days). At
on PDA are similar at 37°C and 25°C, but darkening of 37°C, there is no growth. Colonies produce a strong, pun-
the colony obverse and growth rate are slightly faster gent (skunk-like) odor after 1 month of incubation in all
at 37°C (73 mm in diameter after 14 days) than at 25°C the media tested. The fungus shows a strong keratinolytic
(66 mm in diameter). Microscopically, the fungus forms activity. Phenotypically, C. ophiodiicola is separated from
solitary aleurioconidia that are borne at the ends of short, the Chrysosporium anamorph of Nannizziopsis vriesii by
typically curved stalks or that are sessile (borne on the the absence of asperulate fertile hyphae and globose-to-pyr-
sides of the hyphae). Conidia (3.5–13 μm × 2.5–5 μm) are iform conidia sometimes grouped in clusters and the pres-
single celled, rarely two celled, smooth to slightly rough- ence of an odor from C. ophiodiicola [3].
ened, and clavate (club shaped) to broadly obovoid (egg Chrysosporium species are differentiated from each other
shaped) and have a rounded tip and a broad, flat basal scar. by the texture of the colony and the morphology, location, and
Intercalary arthroconidia may be formed but are uncom- size of the conidia. C. zonatum differs from other species by
mon. Racquet hyphae (hyphae showing swellings near the its faster growth at 37°C than at 25°C and by forming darken
septa) are common [4]. The teleomorph of C. zonatum to buff colonies, and clavate, broadly truncate aleurioco-
is a heterothallic ascomycete Uncinocarpus orissi (syn- nidia typically borne on short, curved stalks. Chrysosporium
onyms, Pseudoarachniotus orissi and Gymnoascus arxii) queenslandicum (teleomorph: Uncinocarpus queenslandi-
(family Onygenaceae). U. orissi ascocarps are solitary, cus, or Apinisia queenslandica and Brunneospora reticu-
globose, and reddish brown and are composed of pale red- lata) is similar, but its colonies do not darken, and intercalary
dish brown ascospores surrounded by thin-walled hyaline arthroconidia are common [4].
racket hyphae and conidia. Pairing of C. zonatum with U. C. ophiodiicola is separated from C. mephiticum by the
orissi produces ascoma-containing ascospores that are narrow, cylindrical-to-slightly clavate conidia of C. ophiodi-
oblate (like flattened disks) with t runcate ends and appear icola, and the pyriform-to-subglobose conidia of C. mephiti-
smooth to slightly pitted (punctate) [4]. cum; C. ophiodiicola is differentiated from Aphanoascus
Chrysosporium ophiodiicola colonies attain diameters of mephitalis by the production of the telemorphic form or
27–29 mm in 14 days at 25°C on potato carrot agar (PCA; during the culture of A. mephitalis; and C. ophiodiicola is
20 g potato, 20 g carrot, 15 g agar, 1 L water). Colonies distinguished from Chrysosporium europae by the charac-
are white from top and uncolored on reverse, felty, plane, teristic vinaceous, buff-pigmented colonies on phytone-yeast
and fimbriate, with a poorly defined margin. Sparse tufts extract agar and the absence of a strong, pungent odor from
of aerial mycelium are present on the submarginal zone. C. europae. Some species such as Chrysosporium pan-
Vegetative hyphae (1.5–2.5 μm wide) are hyaline, branched, nicola do not grow at 37°C. Chrysosporium differs from
septate, smooth, and thin walled. They are often disarticu- Blastomyces by being nondimorphic, from Microsporum and
lated at maturity to form cylindrical arthroconidia (7.5– Trichophyton by lacking macroconidia, from Geomyces by
10 μm × 2–3 μm) adjacent to each other. Fertile hyphae arise lacking branched, fertile hyphae on erect conidiophores, and
as lateral branches. Terminal and lateral conidia are borne from Sepedonium by having hyaline conidia.
on straight or flexuous side branches of variable length (4.5–
16 μm) or are sometimes sessile. Conidia (4–9 μm × 2–3 μm)
are unicellular, solitary, thin walled, smooth, hyaline to pale
yellow, and cylindrical to slightly clavate. They are released Chrysosporium spp. are soil saprophytes with broad distri-
by rhexolytic dehiscence, leaving broad and long basal scars. bution. They have been isolated in soil, plant material, dung,
Intercalary, solitary conidia are often present, similar to the and birds [6–8]. These organisms may enter hosts through
terminal and lateral ones. Racquet hyphae are scarce, and airborne conidia and exposure to soil. Many Chrysosporium
chlamydospores are not observed. On potato flake agar at spp. are keratinophilic filamentous fungi involved in the
23°C, colonies are white to pale yellow, with a similarly breakdown of shed keratinous substrates, and may cause
colored reverse side, velvety to granular with age. They skin infections and onychomycosis in humans [9–12].
produce a strong, pungent odor. Conidia borne on stalks In addition, Chrysosporium species have been occasion-
are arthroconidia. On PDA (Difco Laboratories), C. ophio- ally associated with disseminated human mycosis, affecting
diicola grows more quickly and produces denser colonies the brain, lungs, sinuses, liver, and kidneys and leading to
of 31–35 mm in diameter in 14 days at 25°C. Colonies are sinusitis, pneumonia, pleuritis, pericarditis, and osteomyeli-
white to pale yellow, buff after 1 month, and powdery, with tis [13–15].
droplets of colorless or light yellow exudates at the periph- Roilides et al. [4] reported the first case of Chrysosporium
ery. On phytone-yeast extract agar (BBL), colonies measure zonatum infection in a 15-year-old boy with X-linked
32–39 mm in diameter in 14 days at 25°C and are white chronic granulomatous disease. He developed a lobar pneu-
and light yellow at the center, powdery, and dense, with the monia and tibia osteomyelitis after a 2 month prophylactic

Chrysosporium 205
therapy with γ-interferon. He presented with pain in his 25.2 Methods

right shoulder and distal part of his right tibia, infrequent
cough, and fever. A computed tomography (CT) scan of 25.2.1 Sample Preparation
the chest showed a well-demarcated large mass in the right Chrysosporium and other fungal isolates are revived from
lower lobe, enlarged left hilar lymph nodes, and lingular either freeze-dried or frozen (vapor phase of liquid nitro-
pneumonitis. A tibia x ray was diagnostic for osteomyelitis. gen) stock and grown at 25°C on petri plates containing
A biopsy of the tibia lesion revealed granulomatous tissue pablum cereal agar for 14–21 days. Blocks (1 × 1 cm) of
and a few short and thick hyphae. Chrysosporium zona- mycelium and agar from cultures of Chrysosporium spe-
tum was grown from sputum and biopsy specimens, which cies are excised from the culture plates and transferred to
was identified by its thermotolerance, darkening colonies sterile snap-cap polypropylene tubes (12 mm × 75 mm; Fisher
(yellowish white to buff) and club-shaped terminal aleurio- Scientific). The mycelial blocks are freeze-dried by using an
conidia borne at the ends of short, curved stalks. Therapy Edwards Moduylo freeze-dryer. Freeze-dried blocks of agar
with amphotericin B, itraconazole, and then liposomal and Chrysosporium mycelium (100 mg) are placed in 1.5 mL
amphotericin B subdued the osteomyelitis, pneumonia, tubes and ground to a fine powder by using a 200 μL capacity
pericarditis, and pleuritis. pipettor tip. The fungal material is rehydrated with 500 μL of
Guerrero Palma et al. [15] described a case of inva- DNA extraction buffer (50 mM Tris, 10 mM EDTA, 1% sarco-
sive sinusal mycosis caused by Chrysosporium tropicum syl, pH 8.0) with gentle agitation for 10 min. An equal volume
in a patient with acquired immunodeficiency. Levy et al. of 1:1 chloroform–phenol is added to each tube, and mixed
[16] also reported a case of aggressive fungal rhinosinus- by shaking for 20 min. Then the aqueous and organic phases
itis caused by Chrysosporium sp. in a patient with acute are separated in a microcentrifuge for 5 min at 14,000 × g.
lymphocytic leukemia. Histopathological and microbio- The aqueous phase is pipetted into a clean tube, and 0.1 vol-
logical studies permitted the identification of the culprit ume of 3 M sodium acetate (pH 6.0) and 1.3 volumes of etha-
organism. In addition, Warwick et al. [17] presented an nol are added. The tube is sealed, and the contents are mixed
invasive Chrysosporium infection in an 18-year-old patient by inverting the tube several times. Precipitated nucleic acids
after allogeneic sibling bone marrow transplant for T lin- are pelleted by centrifugation at 14,000 × g for 1 min. Ethanol
eage acute lymphoblastic leukemia. The infection started is decanted, and the pellet is dried by inverting the tube over
as a facial swelling and extended into the central nervous absorbent paper for 5 min. Nucleic acids are dissolved in
system. Despite antifungal treatment, the disease spread 100 μL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and
rapidly, and an autopsy revealed fungal invasion of brain, 250 μL of a saturated NaI solution and 10 μL of glass milk
lungs, liver, and kidneys. (Gene Clean kit, Bio 101) are added to the tube. The tube is
inverted periodically, and DNA is adsorbed to the glass milk
25.1.3 Diagnosis for 20 min. The glass milk is pelleted and rinsed, and the
genomic DNA is eluted into 50 μL of 1/10-strength TE. DNA
Conventional techniques for the identification of is stored at −20°C until used [18].
Chrysosporium spp. and other fungi rely on microscopic Alternatively, a loopful of Chrysosporium mycelium is
observation of mycotic elements in clinical specimens fol- transferred from SDA to a 1.5 mL tube containing 2.5 mg
lowed by in vitro culture. Chrysosporium colonial mor- zymolase (Sigma–Aldrich) in 250 μL zymolyase lysis buf-
phologies and growth rates are assessed on PDA at 25°C fer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA] and incubated
and 37°C. Tolerance to cycloheximide is tested by mea- for 45 min at 37°C. Subsequently, 200 μL is transferred to
suring the growth rate at 25°C on Mycosel agar (Becton another tube for DNA extraction using a Qiagen kit, resulting
Dickinson). Microscopic features of Chrysosporium iso- in 100 μL of DNA extract [20].
lates are examined in slide culture preparations with pab-
lum cereal agar (pablum precooked mixed cereal 10%, agar
1.5%). The ability of Chrysosporium isolates to digest hairs 25.2.2 Detection Procedures
in the hair perforation assay is diagnostic.
Molecular methods for the determination of
Chrysosporium identity include sequence analysis of small
analysis for fungal detection using pan-fungal primers ITS1
subunit (SSU) and large subunit (LSU) of ribosomal RNA
genes as well as internal transcribed spacer (ITS) regions
reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
[3,18,19]. PCR amplification of ITS2 using primers ITS86
of the fungi is verified by subsequent sequence analysis.
(5′-GTGAATCATCGAATCTTTGAAC-3′) and ITS4
(5′-TCCTCCGCTTATTGATATGC-3′), restriction digestion Procedure
with BstUI (CG/CG), and restriction fragment length poly-
morphism (RFLP) assessment by capillary electrophoresis 1. PCR mixture is composed of 1× Lightcycler
permit discrimination of Chrysosporium from dermatophyte FastStart DNA Master Hybridization Probes mix-
fungi such as Arthroderma, Epidermophyton, Microsporum, ture (Roche Applied Science) (containing deoxy-
and Trichophyton [20]. nucleoside triphosphates), FastStart Taq DNA

polymerase, and 1 mM MgCl2 (additional MgCl2 is sinusitis, pneumonia, pleuritis, pericarditis, osteomyelitis, to
added to a final concentration of 4.6 mM), 0.4 μM other disseminate infections.
each of ITS1 forward and ITS4 reverse primers, 1× Microscopic observation of mycotic elements in clini-
SYBR green (Molecular Probes), and 3 μL template cal specimens and in vitro isolation of fungal strains have
DNA. been the main approaches for identification and diagnosis of
2. Thermal cycling parameters are 95°C for 10 min; 50 Chrysosporium infections. More recently, molecular tech-
cycles of 95°C for 5 s, 60°C for 20 s, and 76°C for niques such as PCR, sequencing, and RFLP analysis have
30 s; and a final extension at 72°C for 2 min. been developed and applied for improved determinations.
3. The quality of the amplicon is determined using the Through examination of nucleotide sequences in the SSU
derivative of the melt analysis curve (55°C–99°C, and LSU of ribosomal RNA genes as well as ITS regions, the
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 identity of Chrysosporium spp. can be rapidly and unequivo-
(Corbett Robotics, Inc.). cally ascertained.
4. The amplified product is purified for bidirectional
sequencing using ExoSAP-IT (USB Corp.). Five
μL of Big Dye Terminator Ready Reaction Mix v. References
1.1 (Applied Biosystems) is added to 4 μL of each 1. The UniProt Consortium. Available at http://www.uniprot.
primer (0.8 pmol/μL) and 3 μL of purified PCR org/, accessed on August 1, 2010.
product. Cycle sequencing is performed with a 9700 2. Al-Musallam A, Tan CS, Chrysosporium zonatum, a new
thermal cycler (ABI), using 25 cycles of 96°C for keratinophilic fungus. Persoonia. 1989;14:69–71.
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing 3. Rajeev S et al., Isolation and characterization of a new fungal
reaction products are passed through a Sephadex species, Chrysosporium ophiodiicola, from a mycotic granu-
loma of a black rat snake (Elaphe obsoleta obsoleta). J Clin
Microbiol. 2009;47(4):1264–1268.
minators. Purified sequencing reaction products are 4. Roilides E. et al., Disseminated infection due to Chrysosporium
run on an ABI Prism 3100 Genetic Analyzer with a zonatum in a patient with chronic granulomatous disease and
50 cm capillary array. review of non-Aspergillus fungal infections with this disease.
5. Sequences are analyzed with the SmartGene J Clin Microbiol. 1999;37:18–25.
Integrated Database Network software version 5. Abarca ML et al., Cutaneous hyalohyphomycosis caused
3.2.3 vr. SmartGene is a web-based software and by a Chrysosporium species related to Nannizziopsis vrie-
database system with reference sequences derived sii in two green iguanas (Iguana iguana). Med Mycol.
2008;46:349–354.
from the National Center for Biological Information 6. Chabasse D, De Gentile L, Bouchara JP, Pathogenicity of some
(NCBI) GenBank repository. Chrysosporium species isolated in France. Mycopathologia.
1989;106(3):171–177.
Note: In case that real time PCR instrument is not available, 7. Qiu WY et al., Fungal spectrum identified by a new slide cul-
standard PCR may be performed with primers ITS1 and ture and in vitro drug susceptibility using Etest in fungal kera-
ITS4, and the resulting amplicon is sequenced with the same titis. Curr Eye Res. 2005;30(12):1113–1120.
primers. Sequence-based identifications are defined by per- 8. Singh I, Mishra A, Kushwaha R, Dermatophytes, related
keratinophilic and opportunistic fungi in indoor dust of houses
cent identity: species, ≥99%; genus, 93%–99%; and incon-
and hospitals. Indian J Med Microbiol. 2009;27(3):242–246.
clusive, ≤93%. 9. Gan GG et al., Non-sporulating Chrysosporium: An oppor-
For strains producing discrepant identification based on tunistic fungal infection in a neutropenic patient. Med J
phenotypic characteristics and ITS sequence analysis, the Malaysia. 2002;57(1):118–122.
D1–D2 region of the large-subunit RNA gene is amplified 10. Maghazy S, Incidence of dermatophytes and cyclohexamide
with primers NL1 (5′-GCATATCAATAAGCGGAGGAA resistant fungi on healthy children hairs and nails in nurseries.
AAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) Mycopathologia. 2002;154(4):171–175.
11. Stebbins WG, Cutaneous adiaspiromycosis: A distinct derma-
and sequenced for species clarification [3].
tologic entity associated with Chrysosporium species. J Am
Acad Dermatol. 2004;51(5 Suppl):S185–S189. Erratum in:
25.3 Conclusions J Am Acad Dermatol. 2004;51(6):1040.
12. Manzano-Gayosso P et al., Onychomycosis incidence
The genus Chrysosporium contains a large number of fila- in type 2 diabetes mellitus patients. Mycopathologia.
mentous saprophytic fungi classified in the mitosporic 2008;166(1):41–45.
Onygenales group, order Onygenales. The teleomorphs of the 13. Stillwell WT, Rubin BD, Axelrod JL. Chrysosporium, a new
Chrysosporium genus are found in the genera Aphanoascus, causative agent in osteomyelitis. A case report. Clin Orthop
Relat Res. 1984;(184):190–192.
Nannizziopsis, and Uncinocarpus, family Onygenaceae,
14. Tomsiková A. Causative agents of nosocomial mycoses. Folia
and order Onygenales. Many members of the genus Microbiol (Praha). 2002;47(2):105–112.
Chrysosporium are thermotolerant and keratinophilic, and 15. Guerrero Palma MA et al., Invasive sinusal mycosis due
occasionally implicated in human diseases, producing clini- to Chrysosporium tropicum. Acta Otorrinolaringol Esp.
cal symptoms ranging from skin infections, onychomycosis, 2007;58(4):164–166.

Chrysosporium 207
16. Levy FE et al., Invasive Chrysosporium infection of the 19. Pounder JI et al., Discovering potential pathogens among
nose and paranasal sinuses in an immunocompromised host. fungi identified as nonsporulating molds. J Clin Microbiol.
Otolaryngol Head Neck Surg. 1991;104(3):384–388. 2007;45(2):568–571.
17. Warwick A et al., Presumptive invasive Chrysosporium infec- 20. De Baere T et al., Evaluation of internal transcribed spacer
tion in a bone marrow transplant recipient. Bone Marrow 2-RFLP analysis for the identification of dermatophytes.
Transplant. 1991;8(4):319–322. J Med Microbiol. 2010;59:48–54.
18. Peterson SW, Sigler L. Molecular genetic variation in
Emmonsia crescens and Emmonsia parva, etiologic agents
of adiaspiromycosis, and their phylogenetic relationship
to Blastomyces dermatitidis (Ajellomyces dermatitidis)
and other systemic fungal pathogens. J Clin Microbiol.
1998;36(10):2918–2925.

26 Cladophialophora
Rubén Lopez-Martínez and Francisca Hernández-Hernández
Contents
26.1 Introduction...................................................................................................................................................................... 209
26.1.1.1 Classification.......................................................................................................................................... 209
26.1.1.2 Morphology............................................................................................................................................ 209
26.1.1.3 Biology....................................................................................................................................................210
26.1.2.1 Chromoblastomycosis.............................................................................................................................210
26.1.2.2 Phaeohyphomycosis................................................................................................................................211
26.1.2.3 Superficial and Subcutaneous Infections................................................................................................211
26.1.2.4 Systemic Forms.......................................................................................................................................211
26.1.2.5 Mycetoma................................................................................................................................................211
26.1.3 Diagnosis.............................................................................................................................................................. 212
26.2 Methods............................................................................................................................................................................ 213
26.2.1.1 Sample Collection.................................................................................................................................. 213
26.2.1.2 Culture of Clinical Specimens............................................................................................................... 213
26.2.1.3 Treatment of PE Sections........................................................................................................................214
26.2.1.4 DNA Extraction......................................................................................................................................214
26.2.1.5 Commercial Kits for DNA Extraction....................................................................................................214
26.2.2.1 PCR Identification of C. carrionii..........................................................................................................214
26.2.2.2 Sequencing Analysis of Cladophialophora spp.................................................................................... 215
References.................................................................................................................................................................................. 215
26.1 Introduction 26.1.1.1 Classification

The teleomorphic state has not yet been described; however,
26.1.1 Classification, Morphology, and Biology phylogenetic studies performed on its small subunit rDNA
The genus Cladophialophora has recently attracted great suggest that it possibly belongs to the phylum Ascomycota
interest in the scientific community. Because of the many of the genus Capronia, of the Chaetothyriales order [2].
studies performed on its morphological, taxonomic, physi- Other references indicate that Cladophialophora as well as
ological, and molecular characteristics, today there is other fungi that produce the clinical picture of chromoblas-
increased knowledge regarding its biodiversity, pathoge- tomycosis belong to the Herpotrichiellaceae family, order
nicity, and natural behavior. Thus far, around 16 differ- Ophiostomatales, phylum Ascomycota [3]. The most frequent
ent species have been identified, many of which have the Cladophialophora species as human pathogen is C. carrio-
capacity to produce infectious diseases in humans and nii (de Hoog, Kwon-Chung, and McGinnis) firstly known as
other mammals. The most recent studies published con- Cladosporium carrionii (Trejos).
cerning this most interesting genus of fungi have described
their behavior as pathogens of various clinical mycoses, 26.1.1.2 Morphology
such as chromoblastomycosis, phaeohyphomycosis, eumy- Cladophialophora colonies grown on Sabouraud dextrose
cetoma, deep tissue abscess formation, skin lesions, among agar medium are seen as compact, smooth, or folded colonies
others [1]. with a dark olive green color, which measure around 4–6 cm
209

and around their border show hyphae submerged in the agar from semiarid to humid and warm climates that lead to the
medium. These colonies can be grown in a wide tempera- conclusion that it belongs to one of the most ubiquitous and
ture range, which varies from 25°C to 35°C. Its microscopic cosmopolitan genus of fungi [6,7]. Some of the species in
morphology is characterized by a Cladosporium type of this genus survive many sterilization methods and can resist
conidiation, which shows long conidiophores that hold the temperatures from below 0°C up to 40°C. Due to its high
shape of a shield, and some may show disjunction scars. transmissibility and its thermotolerance (up to 43°C), some
From the conidiophores, elliptical chains of conidia that species of Cladophialophora, such as C. bantiana, have
are 1.5–3.0 × 2.5–7.0 μm long are formed. These chains of been catalogued as having a Biosafety Level 3 containment
conidia have a branched, flexible appearance and can be long requirement [8].
or short.
C. carrionii colonies grown on Sabouraud dextrose
agar have a dark green olive color, with a velvety appear-
ance. On microscopic examination, long conidiophores Approximately 16 species have been described in this group,
can be seen, with long, branched, flexible conidia chains, 10 of which have been isolated as a cause of different dis-
1.5–3.0 × 2.0–7.5 μm long. eases in humans and some others as agents of mycosis in ani-
C. bantiana shows olive green to gray colonies with a slow mals, such as dogs and cats; the rest of these are considered
growth rate and fluffy appearance that on its hind surface can free-living, nonpathogenic species.
show a gray to black color. Through a microscope, dema- Two of the most important Cladophialophora species that
tiaceous hyphae that are smooth and branched can be seen, can cause clinical disease are C. carrionii as an etiological
along with conidiophores with long elliptical conidia chains, agent of chromoblastomycosis and C. bantiana as an agent of
2.5–4.0 × 5.0–9.0 μm long and acropetal pigmentation. brain abscess formation.
26.1.1.3 Biology 26.1.2.1 Chromoblastomycosis

The different species of Cladophialophora have a great Chromoblastomycosis is the most frequent mycosis caused
diversity of habitats in nature, and can be isolated either from by the Cladophialophora species. It is a chronic mycosis
organic or from inorganic substrates including soil, wood, that is acquired from the inoculation by the agent through
vegetable detritus, eucalypt trees [4], sports drinks, iced tea, a wound in the skin, a frequent occurrence in field work-
air filters, and concrete floors [5]. Table 26.1 shows the patho- ers. Chromoblastomycosis often arise on a lower extremity;
genic species for human and their habitats in nature. There lesions start as papules, nodules, and scaly. In chronic forms,
are reports from almost every country in the world, ranging they become large tumoral lesions with cauliflower-like
TABLE 26.1
Cladophialophora Species Associated with Human Pathologies and Their Habitats
Species Clinical Manifestations Sources
C. arxii Tracheal abscess
C. australiensis Sports drink
C. bantiana Brain abscess, eumycetoma
C. boppii Cutaneous phaeohyphomycosis
C. carrionii Chromoblastomycosis, cutaneous phaeohyphomycosis Soil, pine, eucalyptus fence posts
C. chaetospira Roots of fir tree (Picea abies)
C. devriesii Disseminated lesion Litter, vegetable cover/soil
C. emmonsii Subcutaneous phaeohyphomycosis
C. immunda Subcutaneous phaeohyphomycosis Decaying vegetable matter, biofilter, soil
C. minourae Decaying wood, rotting wood
C. mycetomatis Eumycetoma Cultural contaminant
C. potulenturum Sports drink
C. samöensis Chromoblastomycosis
C. saturnica Cutaneous phaeohyphomycosis Litter, vegetable cover/soil, biofilter, trunk, cut tree
C. subtilis Iced tea
C. yegresii Pitaya (Stenocereus griseus)
Source: Modified from Badali, H. et al., Stud. Mycol., 61, 175, 2008; Padhye, A.A., Identification of the etiologic agents of chromo-
blastomycosis, Scientific Publication No. 479, p. 87, Pan American Health Organization, Washington, DC, 1986; Guía
técnica para la evaluación y prevención de los riesgos relacionados con la exposición a agentes biológicos. Instituto Nacional
de Seguridad e Higiene en el Trabajo. España, Real Decreto 664/1997 del 12 de mayo 1994, B.O.E. No. 124 y adaptación
contenida en la Orden de 25 de marzo de 1998, 1994; Queiroz-Telles, F. et al., Med. Mycol., 47, 3, 2009.

Cladophialophora 211
infected individuals may even have a genetic predisposition

for the development of this mycosis. Its geographical dis-
tribution is very broad. Compared with the cases of chro-
moblastomycosis produced by Fonsecaea pedrosoi and
Phialophora verrucosa, which are found in damp tropical
climates, Cladophialophora carrionii (Cladosporium car-
rionii) is found in arid or subtropical climates with annual
average rainfall of 500–600 mm, such as Madagascar, where
the majority of cases are attributed to this species [10]. Other
species that produce chromoblastomycosis, with a much
lower frequency, are C. arxii and C. samöensis [1].
26.1.2.2 Phaeohyphomycosis
Phaeohyphomycosis is an infection generated by many pig-
mented fungi, and is classified as dematiaceous. These can
be observed inside the tissues in the form of hyphae, yeast
cells, and vesicular cells. According to the site of presenta-
tion, phaeohyphomycosis can be classified as cutaneous, sub-
cutaneous, and systemic, the later usually manifested as lung
FIGURE 26.1 Chromoblastomycosis in leg showing extensive
or central nervous system (CNS) infection [11]. The clinical
cauliflower-like lesions with some black dots and ulcers. manifestations are very diverse, and vary according to the
organ or tissue affected; in most cases, the superficial and
appearance (Figure 26.1). In some cases, ulcers are present subcutaneous forms have a benign course and are observed
with exudates composed of blood-serous and caseous in immunocompetent individuals; on the other hand, lung
material. Fungal cells are expelled through the epidermis and CNS infections are usually very severe, frequently fatal,
expressed by small dark aggregations, known as black dots. and are characterized for their presentation as pyogenic
Other cases of chromoblastomycosis resemble the clinical abscesses.
aspect of mycetoma, but the differential diagnosis is deter-
26.1.2.3 Superficial and Subcutaneous Infections
mined for the presence of muriform cells or for the actinomy-
cetes or eumycetes grains (Figure 26.2). Usually, the infection The main etiological agents are C. carrionii, C. emmonsii,
occurs in individuals with no apparent factors for immuno- C. boppii, C. saturnica, and C. immunda. Traumatic inoc-
suppression [9]; there apparently exists the possibility that ulation of the skin is the mechanism of transmission most
common to these infections. These fungi grow at an ideal
temperature of 27°C, even when they can tolerate tempera-
tures up to 37°C. It seems that superficial and subcutaneous
phaeohyphomycosis is a disease most frequently found in
immunocompetent individuals.
26.1.2.4 Systemic Forms

Systemic forms are produced by three species of
Cladophialophora: C. arxii, C. devriesii, and C. ban-
tiana; the latter is a neurotrophic fungus, which causes
brain abscesses that are usually fatal in immunocompetent
patients. The mechanism of transmission is through inhala-
tion of conidia and it is able to grow in temperatures of up to
40°C. Therefore, this species is classified as an organism that
should be handled under Biosafety Level 3 containment [8].
26.1.2.5 Mycetoma
Mycetoma is a syndrome caused by exogenous sources,
belonging to at least one or two groups of microorgan-
isms: fungus (Eumycetoma) or bacteria (Actinomycetoma).
Mycetoma results from the traumatic implantation of soil
organisms into the dermis, followed by tumefaction, sup-
FIGURE 26.2 Chromoblastomycosis in leg with a mycetoma- purating abscesses, fibrosis, granulomata, and sinuses that
like appearance. Note many nodules and some ulcers with scarring drain the parasitic structures of the agents, so called “grains.”
areas. Nocardia, Actinomadura, and Streptomyces are the three

genera causing actinomycetoma; the principal etiological

agents are N. brasiliensis and A. madurae. Eumycetoma
is caused by numerous species belonging to the genera
Madurella, Pyrenochaeta, Leptosphaeria, Scedosporium,
and others. The most frequent species are M. mycetomatis
and M. grisea. Nevertheless, it has been reported that excep-
tional cases of other mycoses are caused by other fungi, such
as dermatophytes, including Aspergillus and agents of chro-
moblastomycosis [12,13]. Some species of Cladophialophora
have been discovered as agents of eumycetoma, among them
C. mycetomatis [1]. Recently, a case located in the dorsum
of the foot of a 57-year-old man, manifested with edema and
the formation of fistulas, was reported. Direct microscopy of
the exudate revealed the presence of black granules. Both a
culture and a test of the rDNA sequence determined the etio- FIGURE 26.4 Histopathology of infected tissue showing muri-
logical agent as being C. bantiana [14]. form bodies into giant cells. H & E. ×1000.
a dark olive green color and velvety texture (Figure 26.5).

26.1.3 Diagnosis
Microscopically, single conidiophores can be observed, from
26.1.3.1 Conventional Techniques which chains of elliptical conidia are shed, which are usually
Direct examination. In chromoblastomycosis, the para- small (Figure 26.6).
sitic morphology of Cladophialophora is known as muri- In some cases, to differentiate the species, it is necessary
form bodies or fumagoid cells, which are pigmented forms to implement certain physiological techniques [3]. The effects
that can be observed by a direct microscopic examination of temperature (25°C or 37°C) and calcium (0.1 mM Ca2+)
(Figure 26.3). In phaeohyphomycosis, hyphae, yeast cells, under acid conditions (pH 2.5) determine the transformation
and vesicular cells can be observed in the tissues.
Histopathology. This procedure is also important to
confirm the diagnosis of Cladophialophora infections.
In chromoblastomycosis, it is common to observe dermal
hyperkeratosis and pseudoepitheliomatous hyperplasia,
and abscesses containing muriform cells are frequent [15]
(Figure 26.4).
The growth of colonies inoculated in culture in Sabouraud
dextrose agar, incubated in the dark at 28°C–30°C or in many
cases at room temperature, can be seen in an average of
10–15 days. Cultures can be maintained on slants of 2% malt
extract agar and on oatmeal agar [16]. The colonies show
FIGURE 26.5 Colonies of Cladophialophora carrionii grown on
Sabouraud dextrose agar at 26°C during 2 weeks.
FIGURE 26.3 Direct examination of scales from a chromoblas- FIGURE 26.6 Microscopic examination of Cladophialophora
tomycosis patient, showing dematiaceous, septated muriform cells. carrionii forming conidia branched chains in acropetal disposition.

of the hyphae at muriform cells. Under these conditions, C. Ecological information with phylogenetic and taxonomic
carrionii and C. subtilis produce abundant muriforms forms. data in addition to morphological description have been orga-
On the other hand, no muriform cells are produced by C. nized to describe the presently known Cladophialophora
australiensis, C. immunda, C. mycetomatis, C. humicola, or species [1].
others [1]. As for many other fungal pathogens, the PCR-based
Immune response is not constant in these infections; sero- amplification and sequencing of specific DNA regions until
logical tests such as enzyme-linked immunosorbent assay now appears to be the most promising method to identify
(ELISA) using the antigen of C. carrionii (AgSPP) have Cladophialophora species. Thus, two recent studies have
been employed for the detection of antibodies in patients been performed by Abliz et al. [26,27]. The first one identi-
with chromoblastomycosis [17–20]. In healthy patients, this fies C. carrionii by PCR and the second one is an approach to
serological test is negative, and the criteria of remission are differentiate some Cladophialophora species by PCR ampli-
based on this test. The cell-mediated immune reaction is not fication and sequencing of amplified fragments.
standardized [21].
Although chromoblastomycosis is a difficult-to-treat dis- 26.2 Methods
ease because of its chronicity and the presence of melanine-
like fungal pigment, there are some therapeutic approaches This section begins with the general management of bio-
such as physical therapy, surgery, and several antifungal logical specimens or cultures, whose required conditions
drugs (5-fluorocytosine, amphotericin B, and some triazols are similar to those required for other potentially infectious
as voriconazol and posaconazol) that have shown efficiency materials. Preparation of paraffin-embedded (PE) tissue is
[22]. added in cases where this will be the material to be studied.
DNA extraction procedures are those used in original papers,
26.1.3.2 Molecular Techniques but we added some footnotes for those who need to make
Molecular tests are now the optimal methods for the iden- some changes according to their individual laboratory condi-
tification and differentiation of Cladophialophora species. tions. Finally, we describe two molecular protocols.
By contrast with other more common fungal pathogens
such as the opportunistic fungi Candida spp. or Aspergillus 26.2.1 Sample Preparation
fumigatus, few molecular studies of Cladophialophora spp.
have been developed. Most of these studies have focused on 26.2.1.1 Sample Collection
defining the phylogenetic relationship among highly related All specimens should be presumed to contain transmissible
species, a very laborious but determinant work for reliable agents and therefore should be collected and handled using
identification of human pathogenic species. standard precautions including the use of gloves, gown,
Thus, Caligiorne et al. [23] performed an analysis of ribo- mask, and protective eyewear when there is a risk of coming
somal DNA by polymerase chain reaction (PCR)—restric- in contact with the specimen. A special area must be desig-
tion fragment length polymorphism. By this method, it was nated for processing clinical samples. Ideally, all specimens
possible to differentiate Fonsecaea spp. from other dematia- submitted for the detection of fungi should be performed in a
ceous fungi, but it does not differentiate Cladophialophora class II biological safety cabinet.
species. Concerning fungal cultures from the skin and subcutane-
Among the earliest and most important studies applying ous lesions, ideally the infected material is aspirated with a
molecular approaches to the genus is that of Masclaux et al. needle and syringe, and the material is expelled and trans-
[24], who based them on partial LS rRNA sequences that ported into a sterile container that is tightly capped and
established the phylogenetic relationships of human patho- promptly delivered to the laboratory. If an aspirate cannot
genic Cladosporium species. This study was crucial to future be obtained, swab specimens of exudates collected from the
works regarding the Cladophialophora genus, and unam- deep portion of the lesion are acceptable.
biguously established that the melanized human pathogens For optimal evaluation of tissues, enough material should
belong to a single ascomycete family, the Herpotrichiellaceae. be collected to allow the culture, the histopathological exam-
In spite of the advances in molecular biology to define ination, and eventually the molecular processing of the speci-
fungal species, the morphological approaches and the physi- men. After collection, tissues should be placed in a sterile
ological tests are valuable tools supporting the identification container and transported rapidly to the mycological labora-
and taxonomy of many fungi as demonstrated by De Hoog tory to prevent drying.
et al. [3], who based them on nutritional physiology tests
that reclassified the human pathogen Cladosporium species 26.2.1.2 Culture of Clinical Specimens
in the Cladophialophora genus, as well as other taxonomic As for other purposes in medical mycology, we recommend
changes in related species. the growth of clinical specimens in triplicate in isolation
The complexity of C. carrionii strains has been explored media such as Sabouraud dextrose agar with and without
by sequencing of three loci, internal transcribed spacers cycloheximide and with chloramphenicol.
(ITS), β-tubulin (BT2), and translation elongation factor 1-α The Cladophialophora isolates can be grown in or on
(EF1) supported by morphological studies [25]. YMPG broth or agar (yeast extract 0.3% [w/v], malt extract

0.3% [w/v], peptone 0.5% [w/v], glucose 1% [w/v] with or case more material is needed, the fungal culture can be made
without agar 1.5% [w/v]) at 27°C for 1–2 days, as recom- in a 15 mL centrifuge tube. After the mycelia are pelleted
mended by Makimura et al. [28] for other medically impor- and washed in a benchtop centrifuge, they are transferred to
tant fungal strains. a 1.5 mL tube and crushed in the manner described above.
Other solid or liquid media can be used, depending on This culture yields about 25–35 μg of DNA, enough for at
availability of starting materials in the mycology laboratory. least 500 PCR reactions.
We use potato dextrose agar for filamentous fungi growth In modifications made by Abliz et al., approximately
with highly satisfactory results. The most important goal is 50 mg of fungal elements are suspended in 600 μL of extrac-
to obtain a sufficient fungal mass to be submitted at the next tion buffer (200 mM Tris–HCl [pH 7.5], 25 mM EDTA, 0.5%
step consisting of DNA extraction. [w/v] SDS, 250 mM NaCl) and then incubated at 100°C for
15 min. The solution is extracted with phenol–chloroform–
26.2.1.3 Treatment of PE Sections isoamyl alcohol (25:24:1 [v/v/v]).
For each PE tissue sample, 10 sections (thickness, 10 μm)
are cut using a sterile microtome blade and transferred to a 26.2.1.5 Commercial Kits for DNA Extraction
10 mL centrifuge tube. To remove paraffin wax, 5 mL of his-
Other alternative easier and faster DNA extraction methods
tolene is added, mixed by inversion, incubated at room tem-
consist of the use of several available commercial kits, most
perature overnight, and centrifuged at 3838 × g for 15 min.
of which could possibly be used for fungal culture or clinical
The supernatant is removed, and the pellet is washed with
samples. Several authors have proposed the use of particu-
5 mL of 100% ethanol, mixed by inversion, and centrifuged
lar kits [32], methods using minimal equipment (generally a
at 3838 × g for 15 min. The tissue pellet is transferred to a
hot block and microcentrifuge), and obtaining results within
2 mL centrifuge tube and washed in 1 mL of 100% ethanol,
1–4 h. Fredricks et al. [33] found the MPY (MasterPure
followed by 1 mL of 70% ethanol. The ethanol is removed
yeast DNA purification kit, Epicenter, Madison, WI) and YL
by centrifugation at 5900 × g for 10 min, and the pellet is air
GNOME (yeast cell lysis preparation kit plus GNOME kit,
dried at room temperature in preparation for DNA extrac-
Qbiogene, Irvine, CA) as the best kits for DNA extraction
tion. For DNA extraction, all tissue samples are incubated for
from yeast (Candida) and filamentous fungi (A. fumigatus),
at least 3 h in proteinase K and lysis buffer at 55°C, and the
respectively; the latter method being the most costly because
DNA is extracted using the MagNAPure LC DNA isolation
it uses two kits to lyse fungal cells and purify DNA.
kit II (for tissue) (Roche Diagnostics, Mannheim, Germany)
We have experience with fungal DNA extraction meth-
according to the manufacturer’s instructions. The DNA is
ods using three commercial kits: the UltraClean Fecal DNA
stored at −20°C prior to use [29].
kit (MoBio Laboratories), QIAamp DNA mini kit (Qiagen),
26.2.1.4 DNA Extraction and High Pure PCR Template Preparation kit (Roche
Diagnostics), all of them giving good-quality DNA for PCR
The following DNA extraction protocol includes the culture
protocols including qPCR. For any of these kits, the mycelial
conditions. It is a faster procedure and less prone to contami-
mass is collected from a culture on agar with a scalpel in
nation than culturing the fungus directly in a tube as devel-
order to obtain about a 0.25 g sample, which is put in a 2 mL
oped by Cenis [30], and avoids the first phenol-treatment step
tube. Then the manufacturer’s instructions are followed for
suggested in other protocols for plant DNA extraction [31].
all methods. The final volume is from 50 to 200 μL depend-
A 1.5 mL tube is filled with 500 μL of liquid potato dex-
ing on the kit used, enough to quantify and run several PCR
trose medium, inoculated with some hyphal threads, and
reactions.
incubated for 72 h at 25°C. The mycelial mat is pelleted by
Other interesting methods for DNA extraction have been
centrifugation for 5 min at 13,000 rpm, washed with 500 μL
proposed by several authors and consist of using FTA filters
of TE buffer and repelleted. The TE is decanted and 300 μL
giving a molecular grade DNA [34–36].
of extraction buffer is added (200 mM Tris–HCl (pH 8.5),
250 mM NaCl, 25 mM EDTA, 0.5% sodium dodecyl sulfate
[SDS]). The mycelia are crushed with a conical grinder, pre- 26.2.2 Detection Procedures
cisely fitting the tube and manipulated by hand or electric
potter at 200 rpm for some minutes. Subsequently, 150 μL of 26.2.2.1 PCR Identification of C. carrionii
3 M sodium acetate (pH 5.2) is added and the tube is placed The PCR mixture (25 μL) consists of 2.5 μL of template
at −20°C for about 10 min. The tube is then centrifuged and DNA, 2.5 μL (2 mol) of each primer (5′-TAA ACC TCA
the supernatant transferred to another tube. An equal vol- TGT TGC TTC G-3′ [Car-F] and 5′-TCG AGA M(A/C) CAC
ume of isopropanol is added, and, after at least 5 min at room TCG ACC AA-3′ [Car-R]), 2 μL (2.5 mM) of dNTP mixture,
temperature, the precipitated DNA is pelleted by centrifuga- 0.125 μL (5 U/μL) of Taq polymerase, and 2.5 μL of 10×
tion. After a wash with 70% ethanol, the pellet is vacuum reaction buffer. Amplification is performed under the follow-
dried for some minutes and resuspended in 50 μL of TE. The ing conditions: one cycle at 95°C for 4 min, followed by 30
amount of DNA obtained in this way is around 3–6 μg. This cycles at 94°C for 1 min, at 62°C for 1.5 min, and at 72°C
is enough for at least 50 PCR reactions, as determined by for 1.5 min, with a final extension at 72°C for 10 min. After
titration of the DNA concentration in several reactions. In thermal cycling, 2 μL of each amplified product is separated

by electrophoresis on a 1.5% agarose gel, stained with ethid- when the phenol–chloroform method is used. The approaches
ium bromide, and visualized with UV light. This primer set using the FTA filters [34–36] are particularly interesting on
is designed based on the sequence of the ITS regions and to this topic in order to obtain a high-quality DNA.
amplify DNAs from C. carrionii giving a 362-bp fragment. Therefore, the challenge for future research in the molec-
PCR bands are obtained within 7 h from initiating the DNA ular diagnosis of infection or in fungal identification will
extractions: 3 h for the preparation of the template DNAs; 3 h be to find genetic regions that can be highly discrimina-
for PCR; and 1 h for electrophoresis, staining, and detecting tive, to differentiate at least the most frequent pathogenic
the band profiles. The sequences determined by this study Cladophialophora species. It is desirable that this region
were registered in the DNA Data Bank of Japan, where they be amplified by PCR as only identification method and be
can be consulted under the accession numbers shown in the sufficiently discriminative without resorting to sequencing
original work [26]. procedures. For researchers identifying these species, it will
be imperative to select the best DNA extraction method, par-
26.2.2.2 Sequencing Analysis of ticularly if real-time PCR is considered for performing the
Cladophialophora spp. identification in the future because this method is highly sen-
sitive to inhibitors associated with DNA [38].
This protocol is based on nucleotide sequences of the D1/D2
Until now, molecular identification has been an expensive
domains of large subunit (26S) ribosomal DNA developed by
procedure. However, as the number of steps in the different
Abliz et al. [27].
procedures is reduced, costs and reliable results timing will
PCR is carried out in a 50 μL volume containing 5 μL of
be also reduced. Considering that molecular methods for the
template DNA, 5 μL (2 pmol) of each primer (NL-1, 5′-GCA
identification and differentiation of Cladophialophora spe-
TAT CAA TAA GCG GAG GAA AAG-3′ and NL-4m,
cies are very limited, morphological study remains a very
5′-GGT CCG TGT TTC AAG ACG-3′), 4 μL (2.5 mM)
useful tool to support identification.
dNTP mixture, 0.25 μL (5 U/μL) Taq polymerase, and 5 μL
10× reaction buffer. Amplification is performed under the
following conditions: one cycle of 95°C for 4 min followed by References
30 cycles of 94°C for 1 min, 55°C for 2.5 min, and 72°C for
2.5 min, with a final extension at 72°C for 10 min. The ampli- 1. Badali, H. et al., Biodiversity of the genus Cladophialophora.
Stud. Mycol., 61, 175, 2008.
fied products are purified and subjected to direct sequenc-
2. Haase, G. et al., Phylogenetic inference by SSU gene analysis
ing. The external primers, NL-1 and NL-4m, and the internal of members of the Herpotrichiellaceae, with special reference
primers, N L-2m, 5′-TTG TGC GCT ATC GGT CTC-3′, and to human pathogenic species. Stud. Mycol., 43, 80, 1999.
NL-3m, 5′-GAG ACC GAT AGC GCA CAA G-3, are used 3. De Hoog, G.S. et al., Nutritional physiology and taxonomy of
to sequence each DNA sample. human-pathogenic Cladosporium-Xylohypha species. J. Med.
The length of the nucleotide sequence for Clado Vet. Mycol., 33, 339, 1995.
phialophora spp. is 613 bp. The number of nucleotide differ- 4. Ridley, M.F., Soil as a Source of Pathogenic Fungi. Recent
Advances in Botany, p. 312, University of Toronto, Toronto,
ences among members of Cladophialophora species range
Ontario, Canada, 1961.
from 5 between C. devriesii, C. minourae, and C. arxii to 26 5. Esterre, P., Chromoblastomycosis, in Microbiology and
between C. devriesii, C. minourae, and C. bantiana. These Microbial Infections, 10th edn., p. 356, Merz, W.G. and Hay,
data support the sequences of D1/D2 domains as useful for R.J. Eds., Medical Mycology, Topley and Wilson’s, London,
the identification of Cladophialophora species. U.K., 2005.
6. Padhye, A.A., Identification of the etiologic agents of chro-
moblastomycosis. Scientific Publication No. 479, p. 87, Pan
26.3 C
onclusions and American Health Organization, Washington, DC, 1986.
7. Okeke, C.N. and Gugnani, H.C., Studies of pathogenic demati-
Future Perspectives aceous fungi: Isolation from natural sources. Mycopathologia,
The identification, taxonomy, and epidemiological analyses 94, 19, 1986.
of fungal pathogens have become increasingly dependent 8. Guía técnica para la evaluación y prevención de los riesgos
relacionados con la exposición a agentes biológicos. Instituto
on molecular techniques [37]. However, for dematiaceous Nacional de Seguridad e Higiene en el Trabajo. España. Real
species such as Cladophialophora, relatively few molecular Decreto 664/1997 del 12 de mayo 1994. B.O.E. No. 124 y
studies are available for their rapid, sensitive, and specific adaptación contenida en la Orden de 25 de marzo de 1998,
identification or diagnosis. One of the bigger problems that 1994.
has limited advances in this area is that most studies on spe- 9. Yegres, F. et al., Virulence and pathogenicity of human and
cies of medical interest have until now examined regions environmental isolates of Cladosporium carrionii in new born
(ITS or D1/D2) highly similar in sequence, in conditions that ddY mice. Mycopathologia, 114, 71, 1991.
10. Queiroz-Telles, F. et al., Chromoblastomycosis: An overview
do not differentiate them. A second very important aspect to
of clinical manifestations, diagnosis and treatment. Med.
be considered is the presence of the melanin-like pigment in Mycol., 47, 3, 2009.
dematiaceous fungi that potentially inhibits PCR amplifica- 11. Díaz, J.M. et al., Pleurophomopsis lignicola y
tion. This pigment and the DNA have similar solubility and Cladophialophora bantiana. Dos nuevos agentes de micosis
in consequence are frequently extracted together, particularly oportunistas en Chile. Bol. Micol., 17, 95, 2002.

12. Mehta, B. et al., Aspergillus nidulans. A rare etiological agent 26. Abliz, P. et al., Specific oligonucleotide primers for identi-
of eumycetoma. Int. J. Infect. Dis., 12, e282, 2008. fication of Cladophialophora carrionii, a causative agent of
13. Zoutman, D.E. and Sigler, L., Mycetoma of the foot caused chromoblastomycosis. J. Clin. Microbiol., 42, 404, 2004.
by Cylindrocarpon destructans. J. Clin. Microbiol., 29, 1855, 27. Abliz, P. et al., Identification of pathogenic dematiaceous
1991. fungi and related taxa based on large subunit ribosomal DNA
14. Bonifaz, A. et al., Eumycetomas caused by Cladophialophora D1/D2 domain sequence analysis. FEMS Immunol. Med.
bantiana successfully treated with itraconazole. Med. Mycol., Microbiol., 40, 41, 2004.
47, 111, 2009. 28. Makimura, K., Murayama, S.Y., and Yamaguchi, H.,
15. Lee, M.W. et al., Spores and mycelia in cutaneous chromomy- Detection of a wide range of medically important fungi by
cosis. J. Am. Acad. Dermatol., 39, 850, 1998. the polymerase chain reaction. J. Med. Microbiol., 40, 358,
16. Gams, W. et al., CBS Course of Mycology, 4th edn. 1994.
Centraalbureau voor Schimmelcultures, Utrecht, the 29. Lau, A. et al., Development and clinical application of a pan-
Netherlands, 1998. fungal PCR assay to detect and identify fungal DNA in tissue
17. Oberto-Perdigon, L. et al., An ELISA test for the study specimens. J. Clin. Microbiol., 45, 380, 2007.
of the therapeutic evolution of chromoblastomycosis by 30. Cenis, J.L., Rapid extraction of fungal DNA for PCR amplifi-
Cladophialophora carrionii in the endemic area of Falcon cation. Nucleic Acids Res., 20, 2380, 1992.
State, Venezuela. Rev. Iberoam. Micol., 22, 39, 2005. 31. Edwards, K., Johnstone, C., and Thompson, C., A simple and
18. Esterre, P. et al., Humoral immune response in chromoblasto- rapid method for the preparation of plant genomic DNA for
mycosis during and after therapy. Clin. Diagn. Lab. Immunol., PCR analysis. Nucleic Acids Res., 19, 1349, 1991.
7, 497, 2000. 32. Maaroufi, Y., Comparison of different methods of isolation
19. Vidal, M.S. et al., Immunoprecipitation techniques and of DNA of commonly encountered Candida species and its
ELISA in the detection of anti-Fonsecaea pedrosoi antibodies quantitation by using a real-time PCR-based assay. J. Clin.
in chromoblastomycosis. Rev. Inst. Med. Trop. Sao Paulo, 45, Microbiol., 42, 3159, 2004.
315, 2003. 33. Fredricks, D.N., Smith, C., and Meier, A., Comparison of
20. Vidal, M.S. et al., Highly specific and sensitive, immunob- six DNA extraction methods for recovery of fungal DNA as
lot-detected 54 kDa antigen from Fonsecaea pedrosoi. Med. assessed by quantitative PCR. J. Clin. Microbiol., 43, 5122,
Mycol., 42, 511, 2004. 2005.
21. Correa, G.P. et al., The cell-mediated immune reaction in the 34. Borman, A.M. et al., Ultra-rapid preparation of total genomic
cutaneous lesion of chromoblastomycosis and their correla- DNA from isolates of yeast and mould using Whatman FTA
tion with different clinical form the disease. Mycopathologia, filter paper technology—A reusable DNA archiving system.
156, 51, 2002. Med. Mycol., 44, 389, 2006.
22. López-Martínez, R. and Méndez-Tovar, L.J., Chromo 35. Borman, A.M. et al., Molecular identification of pathogenic
blastomycosis. Clin. Dermatol., 25, 188, 2007. fungi. J. Antimicrob. Chemother., 61 (Suppl. 1), 7, 2008.
23. Caligiorne, R.B. et al., Dematiaceous fungal pathogens: 36. Nuchprayoon, S. et al., Flinders Technology Associates
Analysis of ribosomal DNA gene polymorphism by poly- (FTA) filter paper-based DNA extraction with polymerase
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27 Coccidioides
Rossana de Aguiar Cordeiro, Raimunda Sâmia Nogueira Brilhante,
Marcos Fábio Gadelha Rocha, and José Júlio Costa Sidrim
Contents
27.1 Introduction.......................................................................................................................................................................217
27.1.1 The Genus Coccidioides........................................................................................................................................217
27.1.2 Ecology..................................................................................................................................................................218
27.1.3 Epidemiology.........................................................................................................................................................218
27.1.4 Pathogeny, Immunity, and Virulence....................................................................................................................219
27.1.5 Clinical Forms...................................................................................................................................................... 220
27.1.6 Treatment and In Vitro Antifungal Susceptibility................................................................................................ 221
27.1.7 Laboratory Diagnosis........................................................................................................................................... 222
27.1.7.1 Mycological Diagnosis........................................................................................................................... 222
27.1.7.2 Histopathological Diagnosis.................................................................................................................. 222
27.1.7.3 Immunological Diagnosis...................................................................................................................... 222
27.1.7.4 Molecular Diagnosis.............................................................................................................................. 223
27.2 Methods............................................................................................................................................................................ 223
27.2.1.1 DNA Extraction from Cultures.............................................................................................................. 223
27.2.1.2 DNA Extraction from Clinical Samples................................................................................................ 224
27.2.2 Detection Methods................................................................................................................................................ 226
27.2.2.1 PCR Amplification................................................................................................................................. 226
27.2.2.2 Hybridization......................................................................................................................................... 226
27.3 Conclusions....................................................................................................................................................................... 227
References.................................................................................................................................................................................. 227
27.1 Introduction arthroconidium is converted into a multinucleated cell that

undergoes enlargement of the cell wall and cytoplasmatic
27.1.1 The Genus Coccidioides compartmentalization during its isotropic growth, originat-
The genus Coccidioides includes the species C. immitis and ing a multinucleated spherule with a diameter of 60–100 μm.3
C. posadasii, which exhibit very similar morphologic char- After reaching cell maturity, the spherule may release more
acteristics but have molecular polymorphism and different than 800 endospores4 and each of them, after germinating,
ecological preferences.1,2 Although phylogenetically differ- will prompt the development of a new spherule, thus result-
ent,2 C. immitis and C. posadasii cause coccidioidomycosis ing in an exponential reproduction of the fungal population.
in both people and animals, which to date are apparently When the endospores reach the soil under adequate condi-
indistinct diseases when considering clinical manifestations tions, they grow into the filamentous form, thus guaranteeing
and host immune response. The species are classified as bio- the continuity of the microorganism’s biological cycle. Even
safety level 3 agents and are on the Select Agent List, accord- though they are classified as thermally dimorphic species,
ing to the U.S. Departments of Health and Human Services their transition from the filamentous to the yeast form under
(HHS) and Agriculture (USDA). laboratory conditions also depends on nutritional factors
They are telluric organisms that form hyaline hyphae and increased CO2 concentration (10%–20%). Experimental
during the filamentous phase of the biological cycle, which observations showed that the addition of Tamol N® detergent
throughout the maturation originate arthroconidia inter- also stimulates spherulation.3
spaced with cells lacking cytoplasmatic material, called Coccidioides is an ascomycete fungus that belongs to the
disjunctor cells. The arthroconidia are easily detached from order Onygenales, which includes other dimorphic fungi such
the vegetative mycelium and contain in their extremities cell as Blastomyces dermatitidis, Paracoccidioides brasiliensis,
wall remnants of disjunctor cells, facilitating their air disper- and Histoplasma capsulatum. Although the sexual phase of
sion.4 Within 72 h after inhalation by a susceptible host, each Coccidioides is not known in nature, previous experimental
217

data have revealed the presence of a recombinant genetic Until now, little is known about the ecological character-
structure, thus suggesting the existence of a sexual phase.5 istics of Coccidioides outside the endemic areas of North
Recent experimental data confirm that C. immitis and C. America. The environmental isolation of the fungus has
posadasii are heterothallic species that have MAT1-1 and already been performed in Argentina,15,16 but few ecological
MAT1-2 idiomorphs with conserved sequences.6 Therefore, data on the fungus are available in this country.
the possibility of the occurrence of sexual reproduction
within this genus may be important for the epidemiology of
27.1.3 Epidemiology
coccidioidomycosis, since ascospores and conidia may differ,
considering survivability in the soil.7 The occurrence of coccidioidomycosis in human beings has
been determined through detection of clinical cases, envi-
ronmental analysis during outbreaks, and performance of
27.1.2 Ecology
epidemiological inquiries. Such studies have shown that
Knowledge on the ecological preferences of Coccidioides autochthonous cases of coccidioidomycosis come exclusively
comes from studies with microorganism and/or genetic from the Americas, occurring in arid or semiarid regions
material isolation from soil. Although Coccidioides does located between 40°N 120°W in Northern California and
not present nutritional requirements in vitro, it is rarely 40°S 65°W in the Patagonia, Argentina.17 In these areas, the
isolated from the environment.7 Apparently, the distribu- distribution of the etiologic agents of coccidioidomycosis is
tion of Coccidioides in these environments is irregular and very peculiar: C. immitis is limited to San Joaquin Valley
sporadic.7 A study performed by Elconin et al.,8 who ana- in California, while C. posadasii presents wider distribu-
lyzed more than 5000 soil samples from endemic areas for tion, being found in the United States, Mexico, and South
the disease during 8 years, showed that the isolation rates for America.1,2 So far, endemic areas of the disease are known
Coccidioides ranged from 0% to 43%. Greene et al.7 reported in 10 countries: the United States, Mexico, Honduras,
an isolation rate lower than 1% after analyzing 720 soil sam- Guatemala, Colombia, Venezuela, Bolivia, Paraguay,
ples from hyperendemic areas for the disease through the Argentina, and Brazil.17 However, the most endemic regions
double-pour method, followed by specific PCR. are located in the southwestern United States and along the
Several biotic and abiotic factors, still not completely known, border with Northern Mexico. Cases of coccidioidomycosis
influence the discontinuous distribution of Coccidioides in have also been reported outside these areas, in tourists who
soil. Apparently, the most important factors are temperature, were contaminated in endemic areas of the United States or
amount and timing of rainfall, and conditions that are directly Mexico.18–21
related to soil physicochemical characteristics, such as low Since coccidioidomycosis is mainly acquired through
humidity, texture, alkaline pH, and salinity. In addition, levels inhalation,22 activities that involve exposure to dust particles
of exposure to sun and/or ultraviolet light, competition with in endemic areas increase the risks of acquiring the disease.23
other telluric microorganisms and/or vegetal species, and Even though farming activities are considered a risk factor
the presence of rodent burrows seem to influence the main- for coccidioidomycosis,24 the disease is not frequent among
tenance of Coccidioides in the soil.9 However, recent studies individuals who perform only farm work, since Coccidioides
have questioned the dominant role of these environmental is not a common saprophyte of agriculturable soils. Increases
variables in disease incidence in endemic areas of California in the number of immunocompromised patients in risk areas,
and have proposed the role of the anthropogenic actions in the as well as increases in the number of immigrants from non-
occurrence of coccidioidomycosis in these areas.10 endemic areas to endemic regions, have been considered
Studies of the correlation between the natural fungal dis- important issues for the rising incidence of coccidioidomyco-
tribution and local biota have shown irreproducible results. sis in the United States.23,25
Apparently, small mammals play an important role in the In the United States, approximately 150,000 new infec-
dispersion of Coccidioides throughout the environment and tions are estimated per year,22 with time-related variations
they can be implicated as temporary reservoirs of the fun- in endemic areas. In that country, besides anthropogenic
gus in soil.11 Nevertheless, the exact role of animals in the actions, the incidence of coccidioidomycosis is also influ-
ecology of Coccidioides still remains unclear. Emmons12 enced by environmental conditions, such as earthquakes26
suggested small rodents to be sentinel animals for coccidi- and sand storms27 Possibly, outside the southwestern United
oidomycosis in the United States and considered them to be States, the areas with the greatest numbers of disease cases
the main reservoirs of the fungus in nature, since carcasses, are northern Mexico and northeastern Brazil.17,28,29
urine, and feces of these animals were the main source of soil International demographic data show that coccidioidomy-
contamination. Undoubtedly, small mammals such as kan- cosis occurs at any age, but age extremes present greater risk
garoo rats, squirrels, shrews, and rabbits have an important of severe complications, including chronic and disseminated
role for fungal dissemination in North America.1 In Brazil, pulmonary diseases.23,30,31 Pulmonary coccidioidomycosis
armadillos (Dasypus novemcinctus) have been implicated as occurs more frequently in men, which probably is related to
important agents for environmental dispersion of C. posada- the occupational character of the disease.30,31 Also, men seem
sii, since the microorganism has been isolated from naturally to be more resistant to disseminated disease, which suggests
infected animals13 and their burrows.14 the existence of hormonal and/or genetic protective factors in

Coccidioides 219
women.23 Although the risk of acquiring coccidioidomycosis are also infrequent in horses, and apparently, they are at
is not related to any racial group, it is known that the dis- greater risk of developing disseminated disease with poor
seminated form of the disease occurs 10–175 times more outcome. Primates of different species are susceptible to
frequently in the Philippines and in African Americans.30,31 Coccidioides.35
Apparently, host genetic characteristics, in particular HLA
class II and the ABO blood group genes, influence the indi-
27.1.4 Pathogeny, Immunity, and Virulence
vidual susceptibility to the occurrence of grave forms of the
disease.32 Although Coccidioides infection is more commonly acquired
Underlying diseases that affect cellular response function, through respiratory route, the disease can also occur after
such as AIDS, especially in patients with CD4 < 100 cells/ traumatic inoculation. The occurrence of the disease has
mL, and cancer, particularly in Hodgkin’s disease, increase also been reported after pulmonary transplant37 and fol-
the risk of disease dissemination.31 Due to the use of immu- lowing intrauterine infection,38 even though microorganism
nosuppressant medication, transplant patients are considered transmission to the newborn is not frequent in some pregnant
at increased risk of coccidioidomycosis, which is the most women with disseminated disease.39
prevalent endemic mycosis among solid-organ transplant The risk of acquiring the disease from animals is practi-
patients in the United States.33 It is estimated that half of cally absent. Indeed, there are only two reports describing
coccidioidomycosis cases in these patients may originate this form of contamination: inhalation of fungal parasitic
from reactivation of previously acquired coccidioidal infec- structures during necropsy of a horse40 and after being bit by
tion, which occurs within the first year after the transplant. a cat with disseminated coccidioidomycosis.41
In spite of the opportunistic character of coccidioidomycosis After being inhaled by a susceptible host, the arthroco-
in HIV-positive individuals, the disease incidence in these nidia rapidly reach terminal bronchi, since their reduced
patients has declined in recent years due to the advent of size, usually smaller than 10 μm, allows them to evade
potent antiretroviral therapy.23 Additionally, the gestational certain innate defense mechanisms of the host respiratory
age, especially the third trimester, and the postpartum period tract, such as filtration, mucocilliary transport, and chemi-
are also considered risk factors for the development of dis- cal inhibitors.3,31 From that moment, these structures can
seminated coccidioidomycosis.23,31 be phagocytized by alveolar macrophages and/or neutro-
Through epidemiological surveys with coccidioidin and/ phils. However, experimental data show that only 50% of
or spherulin, important epidemiological data on coccidioido- infective arthroconidia are phagocytized by neutrophils,
mycosis have been obtained in the past years. Several studies probably due to antiphagocytic properties of the outer wall
have shown that the incidence of coccidioidomycosis infec- layer.42 After arthroconidia phagocytosis, neutrophil respi-
tion in endemic areas varies greatly depending on the stud- ratory burst occurs, and even though Coccidioides conidia
ied population. The estimated coccidioidomycosis infection are sensitive to reactive oxygen species and defensins, it is
through intradermic tests have also shown variable indexes estimated that fewer than 20% of the phagocytized arthro-
in Mexico (10%–93%), Honduras (2.5%–50%), Guatemala conidia are eliminated through this mechanism.3 Then, the
(0.5%–42%), Paraguay (0.9%–50%), and Argentina (7.3%– initial response caused by Coccidioides infective structures
40%).17 In the other endemic areas, the few studies that have in the lungs is characterized by influx of neutrophils, which
been performed to date show positivity of 12% in Brazil34 seem to respond to chemotaxins formed after the activation
and 46% in Venezuela.17 Although the reactivity indexes to of the complement system.3,30
the cutaneous tests show great variation, these studies indi- Following the cellular transformation, endospore release
cate higher positivity among males. The reactivity increases also causes the influx of neutrophils, which are possibly
with age until the age of 65, when it declines due to physi- attracted to substances released by fungal parasitic struc-
ological alterations in cellular response. Similarly to the tures.30 Nevertheless, intracellular death of these structures
epidemiological characteristics of the groups affected by the is even less efficient than that observed for arthroconidia.3
disease, the percentage of reactivity to Coccidioides antigens The complete parasitic cycle lasts for 4–6 days and depends
also varies depending on the investigated population group on the specific characteristics of each strain.42 As for other
and environmental alterations, such as deforestation, earth- intracellular pathogens, once phagocytized, Coccidioides
quakes, and sand storms. can inhibit phagosome–lysosome fusion and survive within
As for data regarding animals, apparently, domestic dogs the phagocyte.42 Even though they are capable of inducing
are the most frequently affected species. Although precise a potent host inflammatory response,4 Coccidioides spher-
epidemiological data are unavailable, it is estimated that in ules cannot be phagocytized because of their dimensions.30,43
endemic areas the disease incidence in dogs is similar to that Hence, the resulting inflammatory response is characterized
in humans.35 Coccidioidomycosis has also been described by cellular infiltration that persists throughout the infectious
in other animal species such as cats, llamas, bats, sea lions, process, which leads to granuloma formation.3,30
dolphins, wallabies and kangaroos, tapirs, Przewalski’s The clinical course of coccidioidomycosis can be modi-
horses,35 and reptiles.36 Even though Coccidioides has been fied if the host response is directed to Th1- or Th2-specific
visualized in biopsies from cattle, goats, and hogs, these ani- cytokine pattern. Processing and presentation of impor-
mals rarely develop the disease.35 Symptomatic infections tant antigens by macrophages and/or dendritic cells induce

production of interferon-γ, tumor necrosis factor-α, and forms of the disease.43 Some studies show that Coccidioides
interleukin (IL)-12 by Th1 lymphocytes and these cytokines has developed specific mechanisms to evade host immune
induce macrophage recruiting and activation, which may response. Expression of SOWgp reaches its peak during
isotropic growth of spherules, which are poorly phagocy-
inhibit or kill fungal structures, contributing to infection con-
trol.30 On the other hand, IL-4, IL-5, IL-10, and IL-13, pro- tized because of their size, and decreases during endospore
duced by Th2 lymphocytes, activate humoral response and formation, thus guaranteeing that this structure will not be
induce downregulation of functions induced by Th1 lympho- opsonized by anti-SOWgp antibodies.43 In addition, experi-
cytes,44 increasing individual susceptibility to Coccidioides mental data show that the fungus secretes a specific metal-
infection.45 Thus, asymptomatic patients or those with benign loproteinase (Mep1), responsible for degrading the SOWgp
forms of the disease present a strong intradermic reaction antigen, during endospore differentiation, which prevents
when stimulated by fungal antigens, and low levels of serum- host recognition of endospores.48
specific antibodies. On the other hand, patients with chronic Another virulence mechanism described for Coccidioides
pulmonary or disseminated coccidioidomycosis present involves the modulation of arginase I synthesis by macro-
T-cell anergy and high titers of anti-Coccidioides IgE and phages.43 Arginase I catalyzes l-arginine hydrolysis into
IgG.3 Cell immunity, manifested by delayed hypersensitivity, l-ornithine, which serves as a substrate for the enzyme orni-
is long lasting and protects individuals against fungal rein- thine decarboxylase (ODC) of Coccidioides, considered to be
fections. Thus, reactivation of coccidioidomycosis after clin- a key enzyme for the metabolism of polyamine and a putative
ical resolution has only been described in individuals who regulator of fungal parasitic differentiation.49 In macrophages,
had previously suffered serious cell immunity suppression.45 l-arginine is the substrate for another enzyme, inducible
Currently, it is clear that not only the host cellular immune nitric oxide synthase (iNOS), whose activity is regulated by
response pattern but also fungal virulence factors are deter- cytokines secreted by T lymphocytes. Experimental studies
minants of establishment of the disease.43 suggest that host exposure to Coccidioides SOWgp antigen
Even though Coccidioides pathogenicity is multifactorial, induces a Th2 response, which leads to an increase in argi-
some specific products from fungal metabolism have been nase I expression and a reduction in iNOS expression. This
associated with virulence. Proteases secreted by the fungus, process decreases nitric oxide production by macrophages,
which are able to degrade antibodies and other opsonins, have contributing to fungal intracellular survival.43 In addition,
been suggested as important virulence factors.43 Other prote- increase in arginase I expression also leads to a higher urea
ases with collagenasic and elastasic activity have also been production within infection sites.
implicated as virulence factors, considering their potential
to degrade the connective tissue matrix of the lungs, which
27.1.5 Clinical Forms
facilitates the destruction of pulmonary parenchyma and
subsequent fungal dissemination.46 Recent studies show the Coccidioidomycosis can be classified as an asymptomatic,
role of the enzyme urease in the virulence of Coccidioides. acute pulmonary, chronic pulmonary, disseminated, or pri-
The enzyme causes alkalinization of the microenvironment mary cutaneous disease.22,30 It is estimated that up to 60%
where the pathogen is found and possibly contributes to pul- of individuals exposed to infective arthroconidia are asymp-
monary parenchyma damage and exacerbation of disease tomatic, being detected only through serological inquiry or
symptoms.43 cutaneous tests. Practically all of these individuals develop
Laboratory studies have shown the presence of melanin permanent immunity and are therefore protected against sec-
or melanin-like compounds in arthroconidia, spherules, and ond primary infections.22 In approximately 40% of individu-
endospores of C. posadasii from in vitro cultivation, as well als, a symptomatic disease can occur within 1–3 weeks after
as in spherules produced during experimental infection.47 infection.22 However, during epidemic outbreaks, the index
Since melanin is an important virulence factor for many fun- of symptomatic individuals can reach up to 90%.50,51
gal species, it is also likely to contribute significantly to the The pulmonary forms of the disease are the most fre-
virulence of Coccidioides. quent, causing several clinical manifestations such as pneu-
Besides the direct action of products from metabolism, monias of different radiological patterns, pleural effusion,
several virulence mechanisms have been recognized for hilar lymphoadenopathy, and pulmonary nodules. The most
Coccidioides. Previous experimental data have shown that common clinical syndrome is pneumonia,52 which usu-
the fungus is capable of immunomodulating host response ally begins within 1–3 weeks after arthroconidia inhala-
through the deposition of a spherule outer wall (SOW) dur- tion and is characterized by cough, dyspnea, and thoracic
ing the growth of parasitic spherules. This material is also pain, besides constitutional symptoms such as fever, fatigue,
released to the extracellular environment as membranous anorexia, and arthralgia.53 In endemic areas, coccidioi-
sheets and is phagocytized by macrophages in infection domycosis can be considered a common cause of commu-
sites. Subsequent studies have shown that a fraction of this nity-acquired pneumonia.52 The acute pulmonary primary
material that is rich in glycoproteins (SOWgp) presented an form may regress spontaneously, after a few months, even
immunodominant antigen, which is recognized by specific without specific therapy.31 Among patients with pulmonary
antibodies from sera of coccidioidomycosis patients, who disease, approximately 5% develop specific manifestations
usually presented elevated titers of anti-SOWgp IgG in severe like nodous erythema or multiform erythema, resulting

Coccidioides 221
from delayed hypersensitivity,3 which are more frequent in 27.1.6 Treatment and In Vitro
females.3 However, other cutaneous manifestations, such as Antifungal Susceptibility
acute exanthema, Sweet’s syndrome, and interstitial granulo-
matous dermatitis, can be observed. The therapeutic approach for the treatment of coccidioido-
Approximately 5% of patients with primary pneumonia do mycosis basically depends on three factors: severity of the
not present spontaneous cure and may evolve to chronic pul- pulmonary infection, presence of disseminated disease, and
monary infection, which is characterized by nodular lesions the patient’s individual risk factors.22 The majority of cases
or fibrocavitary pulmonary disease.31 Clinically, patient of pulmonary coccidioidomycosis progress as a self-limited
presents nocturnal sudoresis, muscular fatigue, weight loss, disease, and according to some authors, there is no need for
chronic cough, and hemoptysis.30,31 Due to clinical, radio- therapeutic intervention, but the patients should be clinically
graphic, and histopathological resemblances, progressive and radiographically monitored for up to 2 years to docu-
pulmonary coccidioidomycosis may be mistaken for pulmo- ment infection resolution.30 In contrast, experts recommend
nary tuberculosis. In Mexico and Brazil, endemic areas for the institution of systemic antifungal therapy for all symp-
coccidioidomycosis and tuberculosis overlap, which makes tomatic patients, with fluconazole or itraconazole v.o. for up
clinical diagnosis of these diseases extremely difficult.23,54 to 6 months.58
Particularly in Brazil, where tuberculosis is the most preva- More severe, chronic or disseminated clinical cases
lent infectious disease, our experience has shown that some require a specific therapeutic approach. The drug choice
cases of coccidioidomycosis were mistaken for pulmonary depends on clinical manifestations and host immunological
tuberculosis and antituberculosis therapy was instituted.54 status.59 Currently, amphotericin B and its lipid formula-
The incorrect diagnosis influences coccidioidomycosis epi- tions are saved for severe cases of the disease. Azole deriva-
demiology in this country, since it is believed that drugs used tives, especially fluconazole and itraconazole, are used as
to treat pulmonary tuberculosis may somehow promote the sequential or combined treatment. In cases of meningitis,
recovery of patients with coccidioidomycosis, compromising fluconazole is preferred because of its good penetration in
detection of disease cases.54 the cerebrospinal fluid (CSF). However, due to its fungistatic
The disseminated form of coccidioidomycosis can affect activity, it demands long-lasting therapy, possibly throughout
1%–5% of infected individuals31 and it can occur even life, especially in immunocompromised patients.60
when there are no clinical or radiological signs of pulmo- Recently obtained data show that voriconazole and posacon-
nary infection.3 This form of the disease most frequently azole are efficient for the treatment of chronic pulmonary coc-
affects adult immunocompromised individuals.22 Fungal cidioidomycosis and coccidioidal meningitis61–63 respectively.
dissemination occurs through hematogenic or lymphatic The clinical efficacy of echinocandin antifungal caspofungin
viae and can reach several sites, such as lymph nodes, skin, was reported in two cases of disseminated coccidioidomyco-
bones, central nervous system, joints, and genitourinary sis.64,65 The therapeutic efficacy of this drug was analyzed in
apparatus.22,31 Coccidioidal meningitis is the more severe murine models. The data showed an increase in survival of
form of the disseminated disease and has a high mortality infected animals and a reduction of fungal parasitism in the
index when it is not diagnosed and properly treated.22 The treated animals, when compared to the control group. In addi-
most common complications associated with coccidioidal tion, a synergistic effect of the combination of caspofungin
meningitis are hydrocephalus, vasculitis, and parenchymal and amphotericin B was observed.66 Until now, the available
abscesses.55 data suggest the potential of caspofungin in the therapy of coc-
Although not common, pericardic involvement in coccidi- cidioidomycosis. Azole chemoprophylaxis must be considered
oidomycosis can result from contiguous pleural-pulmonary for organ transplant recipients and HIV patients who live in
lesions or from lympho-hematogenic dissemination. In these endemic areas, due to high risk of acquiring the disease.67
patients, clinical complaints are not very different from those Even though there are no data that allow establishing a
of patients with pulmonary involvement, which are thoracic precise correlation between therapeutic response and anti-
pain, cough, and dyspnea. However, some clinical findings fungal susceptibility profile,68 several studies have shown
are more characteristic for this clinical presentation, such the in vitro response of Coccidioides strains to antifungal
as orthopnoea, paradoxical pulse, and pericardic attrition. drugs. Nearly all of the results have shown that Coccidioides
Coccidioidal pericarditis usually presents unfavorable prog- is susceptible, in vitro, to most available antifungal agents,
nosis, with high indexes of morbidity and mortality.56,57 including amphotericin B, azole derivatives, and echinocan-
Primary cutaneous forms are rare and result from trau- dins,68–70 despite the absence of official endpoints. Until now,
matic inoculation of the microorganism from an envi- only one report describes the resistance of Coccidioides to
ronmental source or through laboratory manipulation.3,58 azole derivatives.71 Antifungal susceptibility tests must be
Dermatologic manifestations include papules, nodules, and performed in reference laboratories and are appropriate for
verrucose plaques that can evolve to the formation of ulcers surveillance studies and possibly for follow-up of patients
and abscesses.31 Due to the clinical diversity of the lesions, exhibiting therapeutic refractoriness.72 The same approach
cutaneous coccidioidomycosis can be mistaken for several has also been applied to the prospection of drugs with anti-
other diseases, and therefore, laboratory procedures are nec- fungal activity. For example, studies have shown inhibitory in
essary to confirm diagnosis.31 vitro effect of antituberculosis drugs against C. posadasii.54,73

27.1.7 Laboratory Diagnosis heart infusion (BHI) agar, and potato dextrose agar. Fungal
recovery from clinical specimens can sometimes take 3
As for other systemic mycoses, coccidioidomycosis does not weeks or longer, especially when the patient is using antimi-
have pathognomonic clinical-radiological findings, and it can crobial drugs.57,68 Usually, young colonies, with up to 5 days,
be mistaken for other infectious diseases.28,72 Thus, labora- are glabrous, white-colored and do not exhibit arthroconidia.
tory confirmation is essential for the definite diagnosis of As the colonies mature, they become velvety or cottony,
coccidioidomycosis. The success of laboratory diagnosis, white, cream, or gray colored and, under microscopy, exhibit
however, depends on the use of adequate techniques during septate hyaline hyphae having arthroconidia interspaced by
the pre-analytical phase, which involves clinical specimen disjunctor cells lacking cytoplasmatic material, which are
collection, transportation, and processing. 5–7.5 μm and 2.5–5 μm in length and width, respectively.14
27.1.7.1 Mycological Diagnosis 27.1.7.2 Histopathological Diagnosis

Direct examination of the clinical specimen must be per- In histopathological examinations, Coccidioides can be
formed through optical microscopy with slide preparations identified through several staining techniques, such as
and smears. Depending on the clinical presentation, several hematoxylin–eosin (HE), which, although not allowing
biological specimens may be sent to laboratories for the anal- easy visualization of fungal structures, continues to be the
ysis of Coccidioides, such as respiratory samples, CSF, aspi- most frequent technique used in several laboratories. Other
rates of osteoarticular lesions, pleural fluid, and biopsies.28,72 options include periodic acid-Schiff (PAS), which allows
Under direct examination, using wet mounts on glass better spherule, endospore, and hyphal individualization,
slides, clinical specimens are clarified by KOH 10%–40% or and Grocott-methenamine silver, which is considered the
calcofluor white fluorescent stain (CFW) to facilitate spher- most sensitive technique.72 Other options such as Giemsa,
ule visualization; smears and imprints can also be stained by Papanicolau, mucicarmine, and Gram present low efficiency
Grocott-methenamine silver (GMS). Microscopic examina- and should not be employed as primary stains for laboratory
tion shows spherules of up to 100 μm diameter, with bire- diagnosis of coccidioidomycosis.72
fringent wall, containing endospores of 2–5 μm.43,72 This
image is considered pathognomonic for the parasitic phase 27.1.7.3 Immunological Diagnosis
of Coccidioides. Some authors describe the morphological Cellular immune response during coccidioidomycosis can
similarity between coccidioidic spherules and those probe demonstrated through positive results for the cutaneous
duced in vivo by Rhinosporidium seeberi, Emmonsia spp.,42 test with coccidioidin or spherulin. In this test, the fungal
and Prototheca spp.74 Free endospores can be mistaken for antigen is intradermally inoculated in the forearm of the
conidia of Histoplasma capsulatum, Cryptococcus spp., or individual. The observation within 72 h of induration of an
Candida spp.68,72 However, these findings should not repre- area of at least 5 mm is considered a positive response.77 The
sent obstacles for the establishment of mycological diagnosis, use of these tests to establish diagnosis and prognosis of coc-
since infections caused by these agents have clinical-epidemi- cidioidomycosis is very limited, because positive results can
ological characteristics that differ from coccidioidomycosis. be associated with current or past infections or even cross
Although cheap and feasible when performed by experi- reactions. However, the conversion of a negative result can be
enced mycologists, direct examination has low sensitivity. In useful for identifying recent fungal infections. Patients with
patients with acute pulmonary disease, positive rates ranging positive intradermal reaction for coccidioidin and/or spheru-
from 30% to 40% have been described.75 lin have a better prognosis, while cases that cutaneous tests
Rarely, arthroconidiated filamentous structures can be are negative during the infectious process generally evolve to
formed in CSF, during CNS infections68,72 and in pleural disseminated disease.
fluid.74 Recently, it was demonstrated that the presentation of In coccidioidomycosis, produced antibodies do not have
atypical forms in respiratory specimens can be four times protective activity, but serve as tools for diagnosis, progno-
more frequent in patients with cavitary lesions who have sis, and follow-up of patients.78 The search for specific anti-
type-2 diabetes when compared with patients who are not bodies can be performed in various clinical specimens, such
affected by this comorbidity.76 This finding is of great impor- as serum, CSF, pleural, peritoneal, and synovial fluids,78
tance since these atypical parasitic structures can cause depending on the standardization of the technique used.
infection and thus require more caution when manipulating Tests can also be used in the immunocompromised patients,
clinical specimens. though in some cases it is necessary to combine more than
Despite the advances provided by molecular and immuno- one procedure.79
logical techniques in recent years, the isolation and growth of After symptom appearance, early detection of IgM can be
Coccidioides is still the gold standard for laboratory diagno- performed within the first and the third weeks, with positivity
sis of coccidioidomycosis.68 Primary cultures obtained from of 50% and 90% of the cases, respectively.80 Approximately
clinical specimens develop quickly, within approximately 90% of the individuals show reactivity to tests aimed at
5 days with incubation at 25°C–30°C. The microorganism detecting IgG, around the fourth week, after the appearance
grows on most of the media routinely used in mycology of symptoms.72,81 For IgM detection, the most frequently used
laboratories, such as 2% Sabouraud dextrose agar, brain methods in routine laboratories are gel immunodiffusion

Coccidioides 223
(IDTP), latex agglutination, and immunoenzymatic tests. Although the distinction of the species C. immitis and
IgG detection is performed through gel immunodiffusion C. posadasii was originally performed through analysis of
(IDCF), complement fixation reaction, and immunoenzy- microsatellite loci GAC2 and 6212 and single-nucleotide
matic tests. Recently, a quantitative enzyme immune assay polymorphisms,1 these methods are of limited value for rou-
for antigen detection in urine became available.82 tine diagnosis since they are expensive and require labori-
ous techniques.89 In addition, apparently, the oligonucleotide
27.1.7.4 Molecular Diagnosis primers used for microsatellite amplification are not spe-
Diagnosis of coccidioidomycosis based on molecular tech- cific for the recognition of Coccidioides directly from clini-
niques is very promising, since it allows fungal detection from cal samples.4 Currently, the identification of the etiological
contaminated clinical samples and from mixed colonies.4,83,84 agent can be performed through a single PCR reaction89 or
In fact, it is the only strategy so far that allows distinguishing amplification of the ITS region and posterior sequencing or
between the species C. immitis and C. posadasii. Besides restriction fragment length polymorphism of the complete
this, the use of molecular techniques for diagnosis reduces sequence.90 The main techniques used for the detection of
the inherent risk of handling Coccidioides cultures in their Coccidioides in cultures and clinical specimens, as well as
filamentous phase. However, few studies have investigated for molecular identification of C. immitis and C. posadasii,
the potential of molecular techniques for the diagnosis of are described as follows.
coccidioidomycosis. Although the first techniques used for
diagnosing the disease were based on hybridization reac-
tions, currently, PCR-based protocols are preferred because 27.2 Methods
they are easily executed.
The first tests for the detection of Coccidioides were Fungal DNA extraction from cultures and clinical specimens
based on hybridization reactions of nucleic acids and were is considered to be a critical step for molecular diagnosis.
performed by Stockman et al.,85 who used an acridinium Some techniques for Coccidioides DNA extraction from cul-
ester-labeled chemiluminescent DNA probe against rDNA tures and clinical specimens are described in the following
sequences. Currently, commercial chemiluminescent sections.
probes (“AccuProbe”) for the identification of C. immitis
are produced by GenProbe and are commercially available.
Although having high sensitivity and specificity, the detec- 27.2.1 Sample Preparation
tion of Coccidioides based on such hybridization probes is 27.2.1.1 DNA Extraction from Cultures
subject to false-negative results when formaldehyde-killed
cultures are used.86 However, to date, there are molecular 27.2.1.1.1 Protocol 1
probes for the detection of Coccidioides directly from clini- Most methods of DNA preparation from fungi are time-
cal specimens, as well as for the differentiation between the consuming or require special equipment, such as fast prep
species C. immitis and C. posadasii. homogenizers and liquid nitrogen suppliers. We have per-
The first studies involving molecular detection of formed a simple method for total DNA preparation, yield-
Coccidioides based on PCR were performed by Pan and ing a product of good quality, suitable for PCR reactions and
Cole,87 who established a protocol for the amplification of restriction digestion analysis. The procedure has been tested
the CSA gene, directly from fungal cultures. Subsequently, for suspicious Coccidioides cultures grown on mycological
several PCR-based tests were developed to confirm agar at least for 10 days.84
Coccidioides from cultures and clinical specimens. In this
context, the gene clusters from ribosomal DNA and partial 1. Add 5–7 mL of sterile phosphate-buffered saline to
sequences from internal transcribed spacers (ITS) of rDNA the agar slant.
have been used to detect Coccidioides in cultures,7 as well as 2. Scrape the culture surface gently with a swab or
in blood samples from patients and experimentally infected microbiological loops to remove hyphae and arthro-
animals.83 conidia without removing agar. Handle the tube
The detection of Coccidioides in filamentous cultures or carefully to prevent spillage.
in biopsies can also be performed through amplification of 3. Transfer the suspension to a plastic screw-cap tube
the partial sequence of the codifying gene for the molecule and autoclave at 100°C for 15 min.
“antigen 2” or “PRA” following protocols based on nested 4. After cooling, seed a loopful of mycelial debris into
PCR.4 The same target sequence was used for the identifica- BHI broth and incubate at 37°C for 1 week. Store
tion of cultures isolated from pleural fluid,57 as well as for the remaining suspension at −20°C and continue the
the diagnosis of coccidioidomycosis directly from sputum DNA extraction procedure if no fungal growth is
samples.84 Through real-time PCR, partial sequences from detected in the BHI broth.
the ITS regions can also be used as targets for Coccidioides 5. Let the cultures thaw at room temperature and then
detection in several clinical specimens, such as respiratory collect fragments of mycelia and arthroconidia by
samples and fresh biopsy material fixed in formaldehyde or centrifuging at 10,000 × g for 10 min; wash the pel-
paraffinized.88 let twice with phosphate-buffered saline.

6. Incubate the pellet with 1 mL of lyticase 3 mg/mL 27.2.1.2 DNA Extraction from Clinical Samples
at 37°C for 2 h, and then wash with phosphate- 27.2.1.2.1 Protocol 1
buffered saline.
Although many protocols for Coccidioides detection in suspi-
7. Resuspend cellular debris in lysis buffer (10 mM
cious cultures have been standardized before, few procedures
Tris–HCl [pH 8.0], 1 mM EDTA, Triton X-100 2%
have focused on fungal detection directly on clinical samples.
[v/v]), and then expose it to three cycles of heating at
Here we describe an easy protocol for Coccidioides detection
100°C for 10 min and freezing at −80°C for 10 min.
directly on sputum samples.84 After the DNA extraction, spe-
8. Centrifuge at 8000 × g for 15 s and store the super-
cific PCR reaction must be performed to detect any sequence
natant at −20°C.
of diagnostic value.
9. Check DNA degradation by 0.7%–1.0% agarose gel
electrophoresis.
1. Mix a 1 mL aliquot of sputum sample with 200 μL
10. Verify DNA purity from the A260/A280 ratio in a
of NaOH 5% (v/v) and incubate at 37°C for 1 h.
spectrophotometer.
2. Centrifuge at 14,000 × g for 5 min and wash the
resulting pellet twice with phosphate-buffered
This is a very simple protocol for DNA extraction that can be
saline.
performed even in small laboratories. The DNA yield varies
3. Incubate the pellet with 100 μL of lyticase (3 mg/
depending on the amount of mycelium harvested. The qual-
mL) at 37°C for 2 h and then incubate again at 56°C
ity of the DNA is good and usually our results have shown an
for 1 h with lysis buffer (10 mM Tris–HCl [pH 8.0],
A260/A280 ratio of circa 1.8.
1 mM EDTA, SDS 2% [w/v]) containing proteinase
27.2.1.1.2 Protocol 2 K (150 mg/L).
4. Centrifuge the lysed cells at 8000 × g for 15 s and
A safe DNA extraction protocol previously described by Burt
use the supernatant for PCR reactions.
et al.91 is as follows:
Small amounts of DNA are expected, as the quantity of par-
1. Grow Coccidioides cultures at 37°C in 100 mL of 2×
asitic structures in the clinical sample is frequently small.
GYE medium (20 g/L glucose; 10 g/L yeast extract)
However, by this method, we have succeeded in obtaining
in 500 mL plastic Nalgene flasks with plastic foam
DNA suitable for rapid diagnosis of coccidioidomycosis even
stoppers.
in sputum samples heavily contaminated with commensal
2. When there has been sufficient growth, transfer the
bacteria from the mouth and pharynx and which presented
whole container to an autoclave and steam at 1 atm
few coccidioidal spherules.84
(100°C) for 15 min.
3. Allow the culture to cool, plate 1 ml on 2× GYE or
potato dextrose agar, and freeze the rest at −20°C. 27.2.1.2.2 Protocol 2
4. If there is no growth on the plate after 1 week, the Usually clinical samples need to be prepared for DNA
culture can be removed from the containment room extraction in order to increase the amount of DNA yield, to
to proceed with the DNA extraction. remove PCR inhibitors, and to provide higher quality mate-
5. In order to prepare the samples for DNA extraction, rial. We have chosen the methods described by Binnicker et
the cultures must be filtered, frozen in liquid nitrogen, al.88 for respiratory specimens and biopsies for MagNA Pure
lyophilized, and ground with a mortar and pestle. Compact extraction (Roche Applied Sciences, USA) utilizing
6. Mix about 10 mg of dry ground tissue with 400 μL the DNA isolation kit (large volume). We believe that these
of lysis buffer (50 mM Tris, 100 μM NaCl, 5 mM protocols can be useful as preliminary steps for other com-
EDTA, 1% sodium dodecyl sulfate), incubate at mercial kits.
80°C for 10 min and cool to 40°C.
7. Add 10 μL of 10 mg/mL proteinase K for 2–3 h, and 1. Respiratory specimens (BAL fluid, bronchial
then incubate again at 80°C for 10 min. w
ashings, pleural fluid, and sputum)
8. Proceed with the DNA extraction with chloroform– a. Pipette 500 μL of raw specimen and 100 μL pro-
phenol and precipitated with isopropanol and resus- teinase K into a 1.5 mL tube containing 0.1 mm
pend the pellet in 100 μL TE. silica glass beads and 2.4 mm zirconia beads.
9. Check the yield by testing 5 μL of this material by b. Incubate samples at 55°C for 15 min in a ther-
agarose gel electrophoresis. momixer at 1400 rpm and subsequently place on
a 95°C heat block for 5 min.
Many commercial extraction kits are available for fungal c. Place the samples on a Disruptor Genie
DNA extraction and have been tested for Coccidioides detec- (Scientific Industries, Bohemia, NY) for 2 min
tion. Regardless of the method chosen, it should be borne in and then centrifuge briefly at 1000 × g to 5000 × g
mind that direct manipulation of Coccidioides cultures is a to collect the sample at the bottom of the tube.
serious hazard and should be performed only by trained tech- d. The lysate obtained can be used for further
nicians in laboratories with class 3 facilities. DNA extraction/purification.

Coccidioides 225
2. Fresh tissue b. Vortex the samples briefly and then placed

a. Place a small piece of tissue, approximately them in a thermomixer overnight at 55°C
0.5 cm2, in a sterile 1.5 mL tube containing with a mixing speed of 500 rpm; digested tis-
400 μL of 1× Tris–EDTA, 100 μL of proteinase sue can be used for further DNA extraction/
K, and 50 μL of 10% sodium dodecyl sulfate. purification.
TABLE 27.1
Summary of the Most Important PCR-Based Protocols for Coccidioides Detection
Amplicon
Target Primers (5′–3′) Cycling Program Size (bp) DNA Source Reference
CSA antigen gene AAGTTCTCACTCCTCAGCGCTATCG 1. Initial denaturation: 94°C, 519 Cultures [87]
4 min
ACATTAAGGTTCCTCCCCTTCAACC 2. 94°C, 1 min; 50°C, 1 min;
72°C, 1 min (30 cycles)
3. Final extension: 72°C, 10 min
ITS CATCATAGCAAAAATCAAAC 1. Initial denaturation: 94°C, 223 Cultures [7]
2 min
AGGCCCGTCCACACAAG 2. 94°C, 1 min; 53°C, 1 min;
PRA antigen gene First reaction First reaction First reaction: Cultures, lung and [4]
526 skin biopsy material
GTACTATTAGGGAGGATAATCGTT 1. Initial denaturation: 94°C, Second
5 min reaction: 342
GGTGTCAACTGGTGGGATGTCAAT 2. 94°C, 30 s; 5°C, 30 s; 72°C,
1 min (35 cycles)
Second reaction 3. Final extension 72°C, 5 min
ATCCCACCTTGCGCTGTATGTTCGA Second reaction
GGAGACGGCTGGATTTTTTAACATG 1. Initial denaturation: 94°C,
5 min
Hybridization probesa 2. 94°C, 30 s; 60°C, 30 s; 72°C,
1 min (30 cycles)
CCAAATTCTTGCATCTCGCCCA 3. Final extension: 72°C, 5 min
ATGGGATAAGATGAGAAGATGGAAAG
ITS CATCATAGCAAAAATCAAAC 1. Initial denaturation: 94°C, 239 Human and animal [83]
10 min sera
AGGCCCGTCCACACAAG 2. 94°C, 1 min; 53°C, 1 min;
C. immitis contig 2.2 TACGGTGTAATCCCGATACA 1. Initial denaturation: 94°C, 720 Cultures [89]
AAEC02000002 3 min
GGTCTGAATGATCTGACGCA 2. 94°C, 30 s; 60°C, 30 s, 72°C,
45 sec (30 cycles)
ITS CGAGGTCAAACCGGATA 1. Initial denaturation: 95°C, 170 Cultures, respiratory [88]
10 min specimens, biopsies
CCTTCAAGCACGGCTT 2. 95°C, 10 s; 60°C, 15 s; 72°C,
Hybridization probesa 20 s (45 cycles)
GAGCGATGAAGTGATTTCCC
TACACTCAGACACCAGGAACTCG
ITSb GCATCGATGAAGAACGCAGC 1. Initial denaturation: 94°C, ca. 169 Cultures [90]
4 min
TCCTCCGCTTATTGATATGC 2. 94°C, 30 s; 55°C, 30 s; 72°C,
90 s (38 cycles)
a Real-time PCR.
b Identification requires sequencing of the ITS region or restriction fragment length polymorphism (RFLP) of the entire ITS region with BsrI and XcmI
enzymes.

3. Paraffined samples
a. Place thin tissue sections into a 1.5 mL tube con- TABLE 27.2
taining 500 μL xylene and incubate for 5 min at Primer and Probe Sequences Used in SNPs Analysis
room temperature. for Coccidioides Distinction into Species, according
b. Centrifuge for 30 s at 20,800 × g and remove the to Castañón-Olivares et al.
xylene with a disposable pipette.
Amplicon
c. Repeat incubation with xylene and then centri-
Target Primer Sequences (5′–3′) Size (bp)
fuge again to remove xylene.
d. Add 500 μL of 95% ethanol in the tubes, vortex Glucose synthase CCGACGGCTGGCCATAT 72
192 gene
the samples gently and then incubate them for
CCAACGCCCAGTAGTATGGT
5 min at room temperature.
Hybridization probesa
e. Centrifuge for 3 min at 20,800 × g and remove
CCTGGAAAACCAC
the alcohol with a disposable pipette. CCTGGGAAACCAC
f. Add 400 μL of 1× Tris–EDTA, 100 μL of pro- Proline-rich GTCGTTGACCAGTGCTCCAA 63
teinase K, and 50 μL of 10% sodium dodecyl antigen 157 gene
sulfate. CGGCGGTGGTGTCAACT
g. Vortex the tubes and place them in a thermo- Hybridization probesa
mixer overnight at 55°C with a mixing speed of CCCAATTGAGATCCCA
500 rpm and use the digested material for fur- CCCAATTGACATCCCA
ther DNA extraction. Proline-rich CCGCTGAGCCGACTCA 50
antigen 174 gene
CGGTTGGGACGGCAGT
Note: DNA extraction from clinical samples may be performed
Hybridization probesa
with commercial kits manufactured by QIAGENiagen4,83 and
TCCTCCGTAGGCTCA
Roche Applied Sciences.88
CTCCGTGGGCTCA
Source: Castañón-Olivares, L.R. et al., Ann. N.Y. Acad. Sci., 1111, 326,
2007.
27.2.2 Detection Methods a Real-time PCR.
27.2.2.1 PCR Amplification

Many PCR-based protocols for Coccidioides detection have
protocols for Coccidioides detection (Table 27.1) and species
been described in recent years. Although only a few proto-
identification (Tables 27.2 and 27.3).
cols have been tested directly on clinical specimens, all of
them can be performed on DNA samples obtained from cul-
tures. PCR-based strategies are the most widely used tools for 27.2.2.2 Hybridization
molecular detection.92,93 Formerly, the distinction between C. A hybridization system showing high levels of sensitiv-
immitis and C. posadasii could be achieved only by microsat- ity and specificity for Coccidioides identification is cur-
ellites or single-nucleotide polymorphisms (SNPs) analysis, rently available commercially from Gen-Probe Incorporated
but now a single PCR reaction can be employed for this pur- (AccuProbe®, USA). The test can be applied to primary
pose. Recently, RT-PCR has emerged as a robust method for c ultures and, according to the manufacturer, it provides rapid
Coccidioides investigation in clinical specimens, although its and accurate results in as little as 50 min. However, the per-
use is still limited as a research tool. Here we present some formance of this test has not yet been determined on direct
TABLE 27.3
Primer Sequences in Microsatellite Analysis for Coccidioides Distinction
into Species
Target Primer Sequences (5′–3′) Allele Size (bp) References
GAC locus ATG TCTCGC CTG GAG CAC GAC C. immitis: 216–228 2, 94
GCT GTT CCA CGT TTC GAC C. posadasii: 206
621 locus ACA ATG AAC GAG CAG CAA GG C. immitis: 416–426 2, 94
TGA AAG ATG TGT AGA CCC GA C. posadasii: 397–400
KO3 locus ACCTCAAAAGGCGAGACTAC C. immitis: 243–255 2, 94
TGCCGAGTGTTTGACCACAG C. posadasii: 239–241
GA1 locus ACCTCCCGGAATATCATGTG Not informed 94
GTGCGTTGAAAGCAGAAAAT

Coccidioides 227
clinical specimens. A detailed description of the procedure samples and/or mixed cultures. The big challenge at present
can be obtained from technical documents provided by the is to make these techniques more accessible, to ensure quick,
company. Here we summarize the general steps for the test. safe, and unambiguous diagnosis. In the near future, the inte-
gration of the data generated by genomics and proteomics
1. Remove mycelia fragments from solid media with can bring great advances in the molecular diagnosis of coc-
a disposable plastic loop or needle without taking cidioidomycosis. Finally, it is expected that the analysis of
large amounts of the solid media. Transfer the mate- the data generated by sequencing the C. immitis and C. posa-
rial to a tube containing 100 μL of lysis reagent and dasii genomes will reveal molecular targets with great power
vortex briefly. In case of growth from broth media, to detect these species.
pipette a 100 μL sample from the well-mixed broth
into the lysis reagent and vortex.
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28 Cyphellophora
Dongyou Liu
Contents
28.1 Introduction...................................................................................................................................................................... 231
28.1.3 Diagnosis.............................................................................................................................................................. 232
28.2 Methods............................................................................................................................................................................ 233
28.2.2.1 Real-Time PCR and Sequencing Analysis............................................................................................. 233
28.2.2.2 Standard PCR and Sequencing Analysis............................................................................................... 233
28.3 Conclusion........................................................................................................................................................................ 233
References.................................................................................................................................................................................. 234
28.1 Introduction short lateral branches, with a prominent to indistinct collar-

ette. Conidia are sickle shaped, brown, smooth walled, 1- to
Cyphellophora is a dematiaceous fungal genus that is com- 3-septate, adhering in bundles [4]. The genus Cyphellophora
monly present in soil, plants, and the environment. Of the (incorporating the hyaline genus Kumbhamaya) is
11 Cyphellophora species identified to date, Cyphellophora d istinguished phenetically from Pseudomicrodochium by
pluriseptata has been demonstrated to be an unusual cause melanized versus hyaline thalli [8].
of subcutaneous phaeohyphomycosis involving the ear of a At the species level, Cyphellophora guyanensis (origi-
healthy individual. nally isolated from leaf litter in French Guyana) is charac-
terized by its ampulliform to flask-shaped phialides, with a
conspicuous, funnel-shaped collarette, and nearly straight to
falcate or slightly sigmoid, 2- to 6-septate conidia [4].
The genus Cyphellophora is a dematiaceous (dark-walled) Cyphellophora hylomeconis (named after its host genus
fungus belonging to the mitosporic Herpotrichiellaceae, Hylomecon) colonies are slow growing, reaching a diameter
family Herpotrichiellaceae, order Chaetothyriales, class of 20 mm after 4 weeks at 25°C in the dark on potato dex-
Eurotiomycetes, subphylum Pezizomycotina, phylum trose agar (PDA), but 12 mm on synthetic nutrient-poor agar
Ascomycota, kingdom Fungi. The mitosporic Herpo (SNA); colonies are fertile and slimy, with smooth, catenate
trichiellaceae group covers 13 genera: Blastacervulus, margins, and without aerial mycelium; surface is crumpled
Brycekendrickomyces, Cladophialophora, Cladoriella, and olivaceous black to iron gray. Hyphae (3–5 μm wide)
Cyphellophora, Exophiala, Heteroconium, Melanchlenus, are branched, greenish brown, septate, smooth, and con-
Metulocladosporiella, Phaeococcomyces, Rhinocladiella, stricted at septa. Conidiogenous cells (1 μm × 1.5–2 μm) are
Thysanorea, and Veronaea. Currently, the genus Cyphel phialidic, intercalary, appearing denticulate, with minute
lophora consists of 11 recognized species: Cyphellophora collarettes (at times proliferating percurrently). Conidia
eucalypti, Cyphellophora eugeniae, Cyphellophora fusarioi- (15–55 μm × 2.5–4 μm) are sickle shaped, smooth, medium
des (obsolete synonym: Pseudomicrodochium fusarioides), brown, guttulate, 1- to 5-septate, and constricted at septa.
Cyphellophora guyanensis, Cyphellophora hylomeconis, Apex is subacutely rounded, base subtruncate, or with a
Cyphellophora indica (obsolete synonym: Kumbhamaya slight constriction, giving rise to a subacutely rounded foot
indica), Cyphellophora laciniata, Cyphellophora pluri- cell (1 μm × 0.5–1 μm).
septata, Cyphellophora suttonii (obsolete synonym: Cyphellophora eugeniae (named after the host Eugenia)
Pseudomicrodochium suttonii), Cyphellophora taiwanensis, colonies are fertile, sporulating in slimy sporodochial
and Cyphellophora vermispora [1–7]. The type species of the masses, attaining a diameter of 15 mm after 4 weeks at 25°C.
genus is Cyphellophora laciniata [4]. Colonies on PDA are erumpent, with sparse aerial mycelium
Cyphellophora colonies are dark; hyphae (1.5–3 μm and even margins; surface is olivaceous gray, with patches
wide) are fertile, pale brown, at times constricted at septa. of iron gray; reverse is iron gray. Hyphae (3–5 μm wide) are
Conidiogenous cells are phialidic, intercalary, at times on branched, greenish brown, septate, smooth, and constricted
231

at septa. Conidiogenous cells (1 μm wide) are phialidic, diseases: (1) superficial; (2) cutaneous/corneal including der-
intercalary, inconspicuous to subdenticulate, with minute matomycosis, onychomycosis, and mycotic keratitis; (3) sub-
collarettes, with several loci aggregated at hyphal swellings. cutaneous; and (4) systemic [9,10]. Occurring in the upper
Conidia (40–90 μm × 2–3 μm) are subcylindrical, tapering and lower limbs, as well the buttocks, face, neck, and scalp,
toward obtuse ends, curved, smooth, hyaline to olivaceous, subcutaneous phaeohyphomycosis (improperly named pha-
finely guttulate, 4- to 10-septate, and prominently constricted eohyphomycotic cyst) often appears as solitary subcutaneous
at septa. cysts or abscesses, firm to fluctuant [11,12]. However, it may
Cyphellophora eucalypti (named after the host genus induce solid subcutaneous masses or red indurated lesions
Eucalyptus) colonies attain a diameter of 4 cm after 2 in patients with debilitating diseases or immunosuppres-
weeks at 25°C in the dark, and are circular, flat, medium to sive status. Other less common clinical forms of subcutane-
dark brown; margin entire, consisting of dense, immersed ous phaeohyphomycosis include keratotic plaques, pustules,
mycelium. Aerial mycelium is loose, cottony, pale gray- sinus tracts, ulcers, crusts, eczematous rash, papulonodular,
brown (surface), appearing minutely orange-brown due to exophytic, or verrucous [9]. Histopathologically, subcutane-
slimy conidial masses on mycelium, medium yellowish ous phaeohyphomycosis may show (1) suppurative and gran-
brown (reverse). Mycelium is dense, superficial, and partly ulomatous processes in the dermis associated with epidermal
immersed; hyphae (1.5–2.5 μm wide) are smooth, loosely hyperplasia and epidermal abscesses; (2) intradermal multi-
septate, predominantly thin walled, branched, and hyaline loculated cysts lined by granulomas, with abscesses, and a
to pale brown. Conidiogenous cells (5–12 μm × 3–5 μm) are normal epidermis; and (3) dermal unilocular cysts containing
intercalary or terminal on erect hyphal branches, solitary, neutrophils and lined by granulomas, with normal epidermis
subcylindrical to pyriform or lageniform, straight to slightly [13,14]. Many Cyphellophora species are involved in human
curved, pale to medium brown, thick walled, smooth, prolif- cutaneous/subcutaneous phaeohyphomycoses.
erating percurrently, with one to two annellations, and funnel- Bittencourt et al. [14] documented the first case of subcu-
shaped collarettes, constricted and somewhat darkened and taneous phaeohyphomycosis due to Cyphellophora plurisep-
thickened below the collarette. Conidia (8–25 μm × 2–3 μm) tata in a 38-year-old Brazilian male. He presented a 10-year
are clavate and 1-septate when young, becoming slightly sig- history of lesions over the left ear. Hematoxylin–eosin-stained
moid fusiform, 1- to 3-septate, hyaline to pale brown, apex sections of biopsy samples showed brown elongated or ovoid
obtusely rounded, base minutely truncate or slightly protrud- structures surrounded by a halo inside giant cells, and the
ing, thin walled, slightly thickened along the rim, refractive, Grocott stain revealed hyphae of different lengths, irregularly
and aggregating in a slimy mass. swollen to toruloid and infrequently branched. Culture of the
Among the species of the genus Cyphellophora, conidia biopsy samples on Sabouraud agar yielded rapid-growing, vel-
of C. fusarioides and C. laciniata are 1- to 3-septate, while vety, and dark gray colonies. Microscopic examination of the
those of other Cyphellophora species are >3 septate; C. fusa- isolate demonstrated smooth-walled, pale-brown hyphae with
rioides is distinguished from C. laciniata by having narrower terminal, intercalary, or lateral phialides, with a conspicuous
conidia (2.5 μm versus 5 μm). C. vermispora is closely related collarette at the tip. Conidia were pale brown, cylindrical
to C. suttonii and C. fusarioides, and has vermiform, mostly to fusiform, with rounded ends, 1- to 5-septate, straight, or
curved conidia. C. guyanensis is related to the type species slightly curved. The fungus was identified as Cyphellophora
C. laciniata. Cyphellophora hylomeconis is the first species pluriseptata. After treatment with itraconazole and ampho-
of the genus infecting a living plant host. C. hylomeconis is tericin B, the lesion showed remarkable regression.
distinguished on the basis of its conidial dimensions and sep-
tation, with its conidia (15–55 μm × 2.5–4 μm) being larger
28.1.3 Diagnosis
than those of C. fusarioides (11–20 μm × 2–2.5 μm, 1- to
2-septate) and those of C. laciniata (11–25 μm × 2–5 μm, 1- Microscopic examination of biopsy specimens for fungal
to 3-septate) [5]. C. eugeniae conidiogenous loci are similar elements (e.g., septated hyphae, irregularly swollen to toru-
to those of C. taiwanensis; however, C. eugeniae is distin- loid, branched or unbranched; yeast-like cells that bud sin-
guished by having much longer conidia [6]. C. eucalypti is gly or in chains; or spherical to oval cells [sclerotic cells]
similar to C. indica and C. pluriseptata, as well as C. lacini- that reproduce by septation in only one plane) using vari-
ata; but it is distinguished by having 1- to 3-septate conidia, ous stains represents a valuable approach for the diagnosis
with an average length of 15–20 μm [4]. of phaeohyphomycosis and chromoblastomycosis. However,
species-specific identification of the organisms involved is
not possible in most cases based on direct microscopy.
Therefore, clinical samples are routinely inoculated on
The genus Cyphellophora is one of the 70 genera (containing mycological media for the isolation of dematiaceous fungi.
about 130 species) of dematiaceous fungi linked to the etiol- Subsequent assessment of macro- and microscopic character-
ogy of phaeohyphomycosis in humans. Characterized by the istics of fungal isolates allows determination of their precise
ability of etiologic agents to develop in their host’s tissues as species identity. Because culture-based fungal identification
dark walled, septate mycelial elements, phaeohyphomycosis is notoriously time consuming and technically challenging,
represents a class of mycoses that produce four distinct clinical molecular methods such as PCR and sequencing analysis

Cyphellophora 233
have been increasingly adopted in clinical laboratories for microliters of Big Dye Terminator Ready Reaction
rapid, specific, and sensitive detection and identification of Mix v. 1.1 (Applied Biosystems) is added to 4 μL
dematiaceous fungi from cultured isolates as well as noncul- of each primer (0.8 pmol/μL) and 3 μL of the puri-
tured specimens [5,6,15,16]. fied PCR product. Cycle sequencing is performed
with a 9700 thermal cycler (ABI) using 25 cycles
28.2 Methods of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min.
Sequencing reaction products are passed through
28.2.1 Sample Preparation a Sephadex G-50 fine column to remove unincor-
porated dye terminators. Purified sequencing reac-
Biopsy samples are first examined under microscope for
tion products are run on an ABI Prism 3100 Genetic
mycotic elements using various stains. Samples are also cul-
Analyzer with a 50 cm capillary array.
tured on inhibitory mold agar, modified Sabouraud agars, or
5. Sequences are analyzed with the SmartGene
PDA. Colony colors (surface and reverse) are assessed after
2–4 weeks on different media at 25°C in the dark. Isolates
3.2.3 vr. SmartGene is a web-based software and
are cultured on 2% malt extract agar plates (MEA) by obtain-
database system with reference sequences derived
ing single conidial colonies. Colonies are subcultured onto
fresh MEA, oatmeal agar (OA), PDA, and synthetic nutrient-
poor agar (SNA), and incubated at 25°C under continuous
near-ultraviolet light to promote sporulation. Microscopic
Note: In case that real time PCR instrument is unavailable,
structures are observed on tease or tape preparations and
standard PCR may be performed with primers ITS1 and
slide cultures for up to 21 days [5,6].
ITS4, and the resulting amplicon is sequenced with the same
Genomic DNA is isolated from fungal mycelium grown
primers. Sequence-based identifications are defined by per-
on MEA, using the UltraClean? Microbial DNA Isolation Kit
cent identity: species, ≥99%; genus, 93%–99%; and incon-
(Mo Bio Laboratories) according to the manufacturer’s pro-
clusive, ≤93%.
tocols. Alternatively, genomic DNA is extracted following
the CTAB-based protocol [5,6]. 28.2.2.2 Standard PCR and Sequencing Analysis
Crous et al. [5] utilized primers V9G and LR5
28.2.2 Detection Procedures (5′-TCCTGAGGGAAACTTCG-3′) to amplify a 1700 bp
28.2.2.1 Real-Time PCR and Sequencing Analysis fragment covering part (ITS) of the nuclear rDNA operon
spanning the 3′ end of the 18S rRNA gene (small subunit),
the first ITS1, the 5.8S rRNA gene, the second ITS region,
green DNA-binding dye and amplicon melting tem-
and the first 900 bases at the 5′ end of the 28S rRNA gene
perature analysis for fungal detection using pan-fungal
(large subunit). ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)
primers internal transcribed spacer 1 (ITS1) forward
and LR0R (5′-ACCCGCTGAACTTAAGC-3′) are employed
as internal primers for sequencing to ensure good-quality
(5′-TCCTCCGCTTATTGATATGC-3′). The identity of the
overlapping sequences. Alignment gaps are treated as new
fungi is verified by subsequent sequencing analysis.
character states. The ITS sequences are compared with those
Procedure sequences available in NCBI’s GenBank nucleotide database
using a megablast search.
1. PCR mixture is composed of 1× Lightcycler FastStart
DNA Master Hybridization Probes mixture (Roche
28.3 Conclusion
Applied Science) (containing deoxynucleoside tri-
phosphates, FastStart Taq DNA polymerase, and The genus Cyphellophora consists of 11 dematiaceous fungal
1 mM MgCl2 (additional MgCl2 is added to a final species, many of which are causative agents for cutaneous/
concentration of 4.6 mM), 0.4 μM each of ITS1 for- subcutaneous phaeohyphomycosis in human hosts. Currently,
ward and ITS4 reverse primers, 1× SYBR green diagnosis of phaeohyphomycosis relies on microscopic
(Molecular Probes), and 3 μL template DNA. observation of fungal elements in biopsy specimens with the
2. Thermal cycling parameters include 95°C for help of various stains. Further species-specific determination
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, is contingent on the isolation of the fungal organisms for sub-
and 76°C for 30 s; and a final extension at 72°C for sequent assessment of their distinct macroscopic and micro-
2 min. scopic features. Due to the time-consuming and technically
3. The quality of the amplicon is determined using the challenging nature of culture-based procedures, PCR and
derivative of the melt analysis curve (55°C–99°C, sequencing analysis of small and large subunit rRNA genes
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 as well as ITS regions provides a rapid, specific, and sensitive
(Corbett Robotics, Inc.). alternative for identification of dematiaceous fungi including
4. The amplified product is purified for bidirectional Cyphellophora spp. from both cultured isolates and noncul-
sequencing using ExoSAP-IT (USB Corp.). Five tured specimens.

References 10. Rinaldi, M.G., Phaeohyphomycosis. Dermatol Clin.

1996;14:147–153.
1. The UniProt Consortium. Available at http://www.uniprot. 11. Ziefer, A. and Connor, D.H., Phaeohyphomycotic cyst. A
org/, accessed on August 1, 2010. clinicopathologic study of twenty-five patients. Am J Trop
2. De Vries, G.A., Elders, M.C.C., and Luykx, M.H.F., Med Hyg. 1980;29:901–911.
Description of Cyphellophora pluriseptata sp. nov. Anton 12. O’Donnell, P.J. and Hutt, M.S.R., Subcutaneous phaeohy-
Leeuwen. 1986;52:141–143. phomycosis: A histopathological study of nine cases from
3. Walz, A. and de Hoog, G.S., A new species of Cyphellophora. Malawi. J Clin Pathol. 1985;38:288–292.
Anton Leeuwen. 1987;53:143–146. 13. Ronan, S.G. et al., Primary cutaneous phaeohyphomycosis:
4. Decock, C. et al., A new species and three new combi- Report of seven cases. J Cutan Pathol. 1993;20:223–228.
nations in Cyphellophora, with a note on the taxonomic 14. Bittencourt, A.L. et al., Discovering potential pathogens
affinities of the genus, and its relation to Kumbhamaya and among fungi identified as nonsporulating subcutaneous pha-
Pseudomicrodochium. Anton Leeuwen. 2003;84(3):209–216. eohyphomycosis caused by Cyphellophora pluriseptata. Eur
5. Crous, P.W. et al., Opportunistic, human-pathogenic species J Dermatol. 2002;12:103–106.
in the Herpotrichiellaceae are phenotypically similar to sap- 15. Pounder, J.I. et al., Discovering potential pathogens among
robic or phytopathogenic species in the Venturiaceae. Stud fungi identified as nonsporulating molds. J Clin Microbiol.
Mycol. 2007;58:185–217. 2007;45:568–571.
6. Crous, P.W. et al., Phylogeny and taxonomy of obscure genera 16. Bagyalakshmi, R. et al., Newer emerging pathogens of ocular
of microfungi. Persoonia. 2009;22:139–161. non-sporulating molds (NSM) identified by polymerase chain
7. Cheewangkoon, R. et al., Myrtaceae, a cache of fungal biodi- reaction (PCR)-based DNA sequencing technique target-
versity. Persoonia. 2009;23:55–85. ing internal transcribed spacer (ITS) region. Curr Eye Res.
8. Kwon-Chung, K.J. and Bennett, J.E., Medical Mycology, Lea 2008;33:139–147.
& Febiger, Philadelphia, PA, 1992.
9. McGinnis, M.R., Chromoblastomycosis and phaeohyphomy-
cosis: New concepts, diagnosis, and mycology. J Am Acad
Dermatol. 1983;8:1–16.

29 Emmonsia
Contents
29.1 Introduction...................................................................................................................................................................... 235
29.1.3 Diagnosis.............................................................................................................................................................. 237
29.2 Methods............................................................................................................................................................................ 237
29.2.2.2 Panfungal PCR and Sequencing Analysis............................................................................................. 238
29.2.2.3 Sequencing Analysis of LSU rRNA Gene............................................................................................. 238
29.3 Conclusions....................................................................................................................................................................... 238
References.................................................................................................................................................................................. 239
29.1 Introduction Emmonsia crescens (an agent of adiaspiromycosis),

Blastomyces dermatitidis (the agent of blastomycosis), and
Members of the genus Emmonsia are filamentous soil Histoplasma capsulatum (the agent of histoplasmosis) are
saprophytes that have been linked to a pulmonary fungal known to form teleomorphs, associated with the ascomycete
infection termed adiaspiromycosis in small mammals and genus Ajellomyces (Onygenaceae, Onygenales). However, no
occasionally in humans including both immunocompetent sexual stage is known for Emmonsia parva [3,4]. A recent phy-
and immunocompromised individuals. Emmonsia conidia logenetic study indicated that the genus Emmonsia forms part
gain entry into the lung via inhalation, where they develop of a clade together with the genera Ajellomyces, Blastomyces,
without replication into a type of spherule termed an adi- Histoplasma, and Paracoccidioides [2]. While E. crescens
aspore, with clinical symptoms ranging from cough, dys- isolates are placed into two phylogenetic groups that correlate
pnea, low-grade fever, to negligible manifestations. Given with their continents of origin (Eurasia and North America),
their morphologic similarities, Emmonsia can be easily E. parva isolates are more diverse: B. dermatitidis strains form
misidentified as Coccidioides, Blastomyces, Histoplasma, a sister species of E. parva and Histoplasma capsulatum is also
or Cryptococcus. a close relative. Paracoccidioides brasiliensis and Histoplasma
capsulatum are ancestral to most Emmonsia isolates [4].
Emmonsia grows moderately. Colonies are glabrous to
velvety in texture, and are white, with buff to pale brown
The genus Emmonsia (obsolete synonym: centers from the top, and cream to pale brown on the reverse.
Haplosporangium) belongs to the mitosporic Onygenales Hyphae are hyaline and septate; conidiophores are simple
group, order Onygenales, class Eurotiomycetes, subphylum or occasionally branched; unicellular, thin-walled, sub-
Pezizomycotina, phylum Ascomycota, kingdom Fungi. The spherical aleurioconidia (2–5 μm × 2–4 μm) are sessile or
group consists of nine genera: Blastomyces, Chrysosporium, located on slender stalks, occurring in a solitary manner or
Coccidioides, Emmonsia, Geomyces, Locazia, Malbranchea, in two- to three-celled chains. On blood or brain–heart infu-
Myriodontium, and Paracoccidioides. Currently, Emmonsia sion agar at 37°C–40°C and in vivo, thick-walled, large, lib-
consists of three recognized species, Emmonsia cre- erated conidia are produced. These are adiaspores, which are
scens (synonyms: Emmonsia parva var. crescens and clamydospore-like and nonreproducing.
Chrysosporium parvum var. crescens), Emmonsia parva E. crescens adiaspores are multinucleate (up to a few hun-
(synonyms: Emmonsia parva var. parva, Chrysosporium dred nuclei), reaching diameter of 70 μm in vitro at 37°C and
parvum, and Haplosporangium parvum), and Emmonsia 700 μm in vivo; Emmonsia parva adiaspores are uninucle-
pasteuriana, and nine unassigned species [1]. The type spe- ate, reaching diameters of 10–25 μm in vitro at 40°C and
cies of the genus is E. parva [2]. 40 μm in vivo. In contrast, Emmonsia pasteuriana does not
235

produce adiaspores, but forms budding cell-like structures fever, and weight loss over the previous 5 months. A chest
on brain–heart infusion agar at 37°C. roentgenogram showed diffuse micronodular infiltrates.
In infected lung tissues, inhaled conidia (2–4 μm) enlarge Hematoxylin and eosin (H&E) staining of the transbronchial
in the alveoli to become adiaconidia (up to 700 μm in diam- biopsy sections revealed helminth-like structures. Sections
eter), which are surrounded by small granulomes, with con- of lung biopsy revealed granulomas and characteristic adi-
centric layers of fibrous tissue in later stages of the infection. aconidia as stained with fungal stains (periodic acid-Schiff
Adiaspores do not replicate, and become calcified at the [PAS] and Gomori-Grocott metheanamine-silver). The
primary implantation site, leading to a minimal localized patient improved symptomatically without treatment. The
reaction in the host tissue. However, the regular pulmonary second case concerned a 47-year-old man with nonproduc-
functions may be hampered by the presence of adiaspores. tive cough, dyspnea, fever, and weight loss. A chest roent-
Emmonsia is a cosmopolitan filamentous fungus that is genogram revealed diffuse interstitial infiltrates in both
present in soil and small mammals such as rodents that act as lungs. H&E-stained sections of a transbronchial biopsy
the reservoir host of the fungus. Emmonsia spp. are endemic showed two granulomas, one of which contained an adi-
in southwestern United States, Australia, and eastern Europe, aconidium; sections stained by Gomori methenamine silver
and are responsible for a pulmonary infection known as adi- demonstrated a fungal structure of 100 μm in diameter. The
aspiromycosis in rodents, other small mammals, and occa- patient recovered without treatment. The third case involved
sionally in humans [5–9]. Human aspiromycosis typically a 43-year-old man with a 1-month history of fever (39.5°C),
occurs after inhalation of conidia of Emmonsia. While cough with scant expectoration, weight loss (3 kg), dyspnea,
Emmonsia crescens is isolated primarily from humans, and thoracic pain. A chest roentgenogram revealed bilateral
Emmonsia parva is often isolated from animals. Emmonsia micronodular infiltrates. H&E-stained sections of transbron-
pasteuriana was reported in an HIV-infected patient with a chial biopsy demonstrated granulomas with characteristic
cutaneous disseminated infection. adiaconidia. The patient had disrupted two mice nests, in a
farm, 14 days before becoming ill. Treatment with itracon-
azole (200 mg/day) and prednisone (30 mg/day) alleviated the
29.1.2 Clinical Features and Epidemiology
patient’s symptoms in a week, possibly reflecting the fact that
Adiaspiromycosis is a lung disease of humans and small enlargement of Emmonsia conidia causes a localized inflam-
animals resulting from the inhalation of Emmonsia conidia, matory response and Emmonsia conidia do not replicate and
which elicit a robust, multicellular immunologic response in disseminate.
tissue against the growing conidia, leading to noncaseating Nuorva et al. [23] described a case of disseminated bilat-
granulomas and disturbance of normal lung function. The eral pulmonary adiaspiromycosis in a 2-year-old Finnish girl.
first case of human adiaspiromycosis due to Emmonsia was Electron microscopy showed that the three layers of the spore
described in France in 1964. Subsequently, cases of human wall were not typical; rather, there seemed to be a gradual
pulmonary adiaspiromycosis have been reported in Russia, transition between the main wall zones, possibly split into
the Czech Republic, Brazil, Honduras, Guatemala, and the thin layers. Varying numbers and thicknesses were seen
United States, with disseminated infection occurring in with different staining methods and in different spores. This
immunocompromised persons [10–21]. patient had become infected as a result of contact with soil
The establishment of adiaspiromycosis appears to be dust containing the spores in the yard surrounding her home,
dose dependent, as a small inoculum often produces no or and as a result of her mother’s work in a large garden shop.
mild clinical symptoms and scattered radiological pathology, She recovered from this rare infection after treatment with
while a heavy or repeated inoculum may develop an acute, amphotericin B.
sometimes severe, pulmonary disease (often referred to as Dot et al. [24] presented a case of pulmonary adiaspi-
primary progressive pulmonary adiaspiromycosis, dissemi- romycosis in a 30-year-old man with a 2-week history of
nated pulmonary adiaspiromycosis, or acute pulmonary adi- unproductive cough, fever at 39°C, progressive dyspnea, tho-
aspiromycosis), with diffuse granulomatous lesions in both racic pain, generalized weakness, and weight loss of 10 kg
lungs. Cleaning or playing in closed environments inhabited in 1 month. The patient became sick a few weeks after hav-
only by bats and mice, where conidia of Emmonsia spp. are ing played in the surroundings of an animal burrow, which
present in soil and dust, represents a risk factor for human may have functioned as a reservoir for Emmonsia. The chest
adiaspiromycosis [22]. radiography showed diffuse bilateral interstitial pneumonia
Although most human infections are attributable to with a micronodular pattern, and computerized tomography
E. crescens, a disseminated disease caused by E. parva in of the chest demonstrated disseminated pulmonary nodules.
an AIDS patient has been described. E. crescens tends to Formalin-fixed, paraffin-embedded transbronchial biopsy
form larger adiaspores and has a broader host range and geo- sections (4 μm) stained with PAS revealed round or ovoid,
graphic distribution than E. parva. occasionally elliptical adiaspores (50–100 μm) with a thick
de Almeida Barbosa et al. [22] documented three wall in the center of some granulomas. Culture of the bron-
Brazilian cases of acute pulmonary adiaspiromycosis due choalveolar fluid (BAL) and transbronchial biopsy samples
to Emmonsia crescens. The first case involved a 52-year- remained negative after 4 weeks. Sequence analysis of PCR
old man, who presented with cough, mucoid expectoration, amplicon from BAL specimen using panfungal primers

Emmonsia 237
indicated Emmonsia crescens as the culprit. Treatment with 29.2 Methods

oral itraconazole (200 mg/day) for 40 days improved his
respiratory status and stabilized the pulmonary lesions. 29.2.1 Sample Preparation
Besides pulmonary adiaspiromycosis, Mendes et al. [25] Biopsy specimens are stained with PAS stain and examined
described an outbreak of conjunctivitis involving 99 chil- microscopically for adiaconidia-containing granulomas.
dren in Brazil, with symptoms ranging from photophobia Portions of the samples are cultured on Sabouraud agar with
(57%), ocular pain (42%), to blurred vision (40%). Unlike cycloheximide (0.5 mg/mL) at 30°C [24].
conjunctivitis caused by bacterial or viral pathogens, nei- Emmonsia isolates are revived from either freeze dried
ther purulent conjunctival discharge nor hemorrhage was or frozen (vapor phase of liquid nitrogen) stock and grown
observed, and family members of case-patient households at 25°C on petri plates containing pablum cereal agar for
were not commonly affected. Furthermore, the disease was 14–21 days. Blocks (1 cm × 1 cm) of mycelium and agar from
characterized by unusual, single or multiple, white, opaque cultures of Emmonsia species are excised from the culture
scleral nodules, often with hyperemia or local edema, and plates and transferred to sterile snap-cap polypropylene tubes
in some cases with opacification extending to the limbus, or (12 mm × 75 mm; Fisher Scientific). The mycelial blocks are
angular corneal opacities and anterior uveitis with granulo- freeze dried using an Edwards Moduylo freeze-dryer [4].
mas in the anterior chamber. Microscopy of scleral biopsy Freeze-dried blocks of agar and Emmonsia mycelium
samples identified spherical shapes, thick walls, and vacu- (100 mg) are placed in 1.5 mL tubes and ground to a fine
ous central areas with lack of organized, internal structures powder by using a 200 μL capacity pipette tip. The fungal
consistent with adiaconidia of Emmonsia sp. (in contrast to material is rehydrated with 500 μL of DNA extraction buffer
Coccidiodes immitis spores that contain internal microspor- (50 mM Tris, 10 mM EDTA, 1% sarcosyl, pH 8.0) with gentle
ules). Conjunctival irritation was likely due to exposure to agitation for 10 min. An equal volume of 1:1 chloroform–phe-
Emmonsia conidia in dust caused in part by dry environ- nol is added to each tube, and mixed by shaking for 20 min.
mental conditions. Symptomatic children responded to cor- Then, the aqueous and organic phases are separated in a
ticosteroid treatment. microcentrifuge for 5 min at 14,000 × g. The aqueous phase is
pipetted into a clean tube, and 0.1 volume of 3 M sodium ace-
29.1.3 Diagnosis tate pH 6.0 and 1.3 volumes of ethanol are added. The tube is
sealed, and the contents are mixed by inverting the tube sev-
The diagnosis of acute pulmonary adiaspiromycosis is based eral times. Precipitated nucleic acids are pelleted by centrifu-
on the following criteria: (1) an acute onset of respiratory gation at 14,000 × g for 1 min. Ethanol is decanted, and the
and systemic symptoms, (2) a radiological picture of diffuse pellet is dried by inverting the tube over an absorbent paper
micronodular lesions in both lungs, and (3) histopathological for 5 min. Nucleic acids are dissolved in 100 μL of TE buffer
demonstration of the adiaconidia in the granulomas [26]. In (10 mM Tris, 1 mM EDTA, pH 8.0), and 250 μL of a satu-
particular, microscopic observation of large, round or oval, rated NaI solution and 10 μL of glass milk (GENECLEAN
thick-walled adiaspores in histopathological sections of tissue kit, Bio 101) are added to the tube. The tube is inverted peri-
using PAS or other stains provides a key diagnostic feature odically, and DNA is adsorbed onto the glass milk for 20 min.
for adiaspiromycosis. Adiaspores are thick walled, enlarged The glass milk is pelleted and rinsed, and the genomic DNA
conidia that may be uninucleate (E. parva) or multinucleate is eluted into 50 μL of 1/10-strength TE. DNA is stored at
(E. crescens). Culture of Emmonisia spp. from sputum or −20°C until used [4]. Alternatively, Emmonsia genomic DNA
BAL is not possible because adiaconidia do not multiply and can be prepared by using Whatman FTA filter papers [28].
remain trapped within lung granulomas.
Emmonisia spp. are differentiated from Blastomyces der-
matitidis, Histoplasma capsulatum, and Paracoccidioides 29.2.2 Detection Procedures
brasiliensis by their inability to convert to a yeast phase 29.2.2.1 Sequencing Analysis of ITS Regions
at 37°C. They are differentiated from Sporotrichum by
Peterson and Sigler [4] used primers ITS1
their inability to produce large chlamydospores at 25°C.
(5′-TCCGTAGGTGAACCTGCGG-3′) and D2R to amplify
Emmonisia spores in tissue (i.e., adiaspores or adiaconidia)
a 1200-nucleotide fragment including ITS1, ITS2, 5.8S
resemble the parasitic spherules of Coccidioides immitis but
rRNA, and part of the 28S rRNA for sequencing analysis of
differ by lacking internal spores.
Emmonsia and related organisms.
The variable domain D2 located in the 5′ end of large
subunit (LSU) nuclear rRNA contains sufficient nucleotide Procedure
differences for discrimination between Emmonisia and its
sibling Blastomyces species [27]. The other region of diag- 1. PCR mixture (100 μL) is composed of 1 μL of
nostic interest is the internal transcribed spacer (ITS) region genomic DNA, 10 μL of 10× buffer, 1 μL of 50 μM
of rRNA (covering the ITS1, ITS2, and 5.8S rRNA), which primer ITS1, 1 μL of 50 μM primer D2R, 0.5 μL
offers a suitable target for examining Emmonsia species vari- (2.5 U) of Taq polymerase, 10 μL of dNTP (1 mM
ability and for assessing evolutionary relationships among concentration), and 76.5 μL of sterile deionized
several related taxa. water.

2. Amplification is conducted with 30 cycles of 96°C 4. After purification, the PCR product is sequenced
for 30 s, 53°C for 30 s, and 72°C for 2.5 min followed with the same PCR primers using the BigDye
by 10 min at 72°C. The amplified fragment is puri- Terminator v1.1 Cycle Sequencing Kit (Applied
fied by using a GENECLEAN kit (Bio 101) and Biosystems). Sequencing products are analyzed on
eluted into 1/10-strength TE. The purified fragment an automated sequencer ABI Prism 3100 genetic
is stored at −20°C until used in sequencing. analyzer. The BLAST analysis is used to compare
3. Sequencing is performed by using primers ITS1, the sequence with those at GenBank database.
ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′), ITS3
(5′-GCATCGATGAAGAACGCAGC-3′), ITS4 29.2.2.3 Sequencing Analysis of LSU rRNA Gene
(5′-TCCTCCGCTTATTGATATGC-3′), D1, D1R, Untereiner et al. [2] utilized primers WNS9 and LR5
D2, and D2R and Applied Biosystems DyeDeoxy (5′-ATCCTGAGGGAAACTTC-3′) to amplify a DNA fragment
sequencing kits. The sequencing reaction mixture is that extends from the 3′ end of the nuclear small subunit rRNA
prepared with 1.5 pmol of primer, 200–400 ng of the gene to approximately 1000 bp positions downstream from the
purified DNA fragment, and sequencing reagents. 5′ end of the nuclear LSU rRNA gene. The amplicon is purified
DNA sequences are analyzed on an ABI 373-auto- using a QIAquick PCR Purification Kit (Qiagen). Sequencing
mated DNA sequencer. reactions are performed using a Prism Dye Terminator Cycle
4. Alignment of overlapping and reverse complement Sequencing Ready Reaction Kit (Applied Biosystems) and
strands to form a consensus sequence for each iso- primers 5.8SR, LR1, WITS3, and WNS9. Excess dye termi-
late is accomplished with an ASCII text editor. nators are removed by centrifugation using Centrisep columns
Alignment of sequences from different isolates is (Princeton Separations) before analysis employing an Applied
performed visually also by using an ASCII text Biosystems 373A or 377 DNA sequencer.
editor. Phylogenetic relationships of isolates are Sequences are edited and assembled into larger consen-
determined by using PAUP 3.1.1. or programs from sus sequences using Sequencher 3.0 software (Gene Codes
the PHYLIP package. Corp.). Multiple alignments are produced using Clustal X
version 1.7. The final multiple alignments are adjusted manu-
29.2.2.2 Panfungal PCR and Sequencing Analysis ally after visual inspection and areas of sequence ambigu-
ity are eliminated. Phylogenetic relationships are inferred
Dot et al. [24] used panfungal primers His3 and His4
from aligned sequences using the maximum parsimony (MP)
for PCR amplification and sequencing identifica-
method found in PAUP (beta version 4.0b10). Gaps are treated
tion of Emmonsia and related genera. The primer His3
as missing in all analyses. Heuristic searches are performed
(5′-GTCGTAACAAGGTTTCCGTAG-3′) is located
employing tree bisection–reconstruction (TBR) branch swap-
at the end of 18S rRNA at positions 9–29, and His4
ping with the MulTrees and steepest descent options acti-
(5′-AGCGGGTATCCCTACCTGAT-3′) is located at the
vated, or using 1000 random addition sequence replicates.
beginning of 28S at positions 601–620 according to the
sequence of Histoplasma capsulatum U18363.
29.3 ConclusionS
Procedure
Genomic DNA is extracted from the BAL by using Members of the genus Emmonsia are saprophytic fungi
the High Pure PCR Template Preparation Kit (Roche that are commonly present in soil and small mammals (e.g.,
Diagnostics). rodents, otters, ground squirrels, goats, dogs, hedgehogs, rac-
coons, horses, and beavers). The teleomorphs of the genus
1. PCR mixture (50 μL) is composed of 2 U of FastStart Emmonsia genera with the Blastomyces and Histoplasma are
Taq DNA polymerase (Roche Diagnostics), 5 μL of found in the genus Ajellomyces.
10× PCR buffer-MgCl2 (20 mM), 20 pmol of each E. crescens and E. parva spores are capable of entering
primer, 200 μM of dATP, dGTP, dTTP, and dCTP, the alveoli of mammalian hosts through inhalation, which
and 5 μL of DNA. expand to become adiaspores (adiacanidia), and cause a
2. Amplification is conducted in an iCycler IQ Thermal granulomatous inflammatory reaction, leading to a pulmo-
Cycler (Bio-Rad) with initial denaturation at 94°C nary disease known as adiaspiromycosis in animals and
for 3 min; 30 cycles at 94°C for 1 min, 58°C for 40 s, occasionally in humans. Most human adiaspiromycosis cases
and 72°C for 1 min; and final extension at 72°C for are due to E. crescens. Although human adiaspiromycosis
5 min. often regresses spontaneously, sometimes the disease may
3. PCR product (5 μL) is electrophoresed in a 2% aga- persist, resulting in pneumonia or fatal respiratory failure
rose gel in the presence of ethidium bromide and [29,30].
visualized under UV light. The expected PCR prod- Because Emmonsia spp. are not easily cultured, diagnosis
uct size is 613 bp. If the band is faint, reamplifica- of adiaspiromycosis has relied on histological examination
tion of the product may be performed by using the of biopsies for large, round or oval, thick-walled adiaspores
same primers. using PAS or other stains. Use of molecular techniques,

Emmonsia 239
especially sequencing analysis of the D2 region of the LSU 16. England, D.M., Hochholzer, L., Adiaspiromycosis: An
rRNA gene and the ITS regions of rRNA biology, provides a unusual fungal infection of the lung. Report of 11 cases. Am J
valuable approach for improving the detection and diagnosis Surg Pathol. 1993;17:876–886.
17. Turner, D. et al., Pulmonary adiaspiromycosis in a patient with
of human adiaspiromycosis [31].
acquired immunodeficiency syndrome. Eur J Clin Microbiol
Infect Dis. 1999;18(12):893–895.
References 18. dos Santos, V.M. et al., Pulmonary adiaspiromycosis: Report
of two cases. Rev Soc Bras Med Trop. 2000;33(5):483–488.
1. The UniProt Consortium. Available at http://www.uniprot. 19. Wellinghausen, N. et al., Chronic granulomatous lung infec-
org/, accessed on August 1, 2010. tion caused by the dimorphic fungus Emmonsia sp. Int J Med
2. Untereiner, W.A. et al., The Ajellomycetaceae, a new fam- Microbiol. 2003;293(6):441–445.
ily of vertebrate-associated Onygenales. Mycologia. 2004; 20. Sun, Y. et al., Fine needle aspiration of pulmonary adiaspiro-
96:812–821. mycosis: A case report. Acta Cytol. 2007;51(2):217–221.
3. Sigler, L., Ajellomyces crescens sp. nov., taxonomy of 21. Denson, J.L. et al., Adiaspiromycosis mimicking widespread
Emmonsia spp., and relatedness with Blastomyces dermatiti- malignancy in a patient with pulmonary adenocarcinoma.
dis (teleomorph Ajellomyces dermatitidis). J Med Vet Mycol. J Clin Pathol. 2009;62(9):837–839.
1996;34(5):303–314. 22. de Almeida Barbosa, A. et al., Acute pulmonary adiaspi-
4. Peterson, S.W., Sigler, L., Molecular genetic variation in romycosis. Report of three cases and a review of 16 other
Emmonsia crescens and Emmonsia parva, etiologic agents cases collected from the literature. Rev Iberoam Micol.
of adiaspiromycosis, and their phylogenetic relationship 1997;14(4):177–180.
to Blastomyces dermatitidis (Ajellomyces dermatitidis) 23. Nuorva, K. et al., Pulmonary adiaspiromycosis in a two year
and other systemic fungal pathogens. J Clin Microbiol. old girl. J Clin Pathol. 1997;50:82–85.
1998;36(10):2918–2925. 24. Dot, J.M. et al., Molecular diagnosis of disseminated adi-
5. Krivanec, K., Otcenásek, M., Importance of free living aspiromycosis due to Emmonsia crescens. J Clin Microbiol.
mustelid carnivores in circulation of adiaspiromycosis. 2009;47(4):1269–1273.
Mycopathologia. 1977;60(3):139–144. 25. Mendes, M.O. et al., Acute conjunctivitis with episcleritis
6. Drouhet, E., Huerre, M., Yeast tissue phase of Emmonsia pas- and anterior uveitis linked to adiaspiromycosis and freshwa-
teuriana inoculated in golden hamster by intratesticular way. ter sponges, Amazon region, Brazil, 2005. Emerg Infect Dis.
Mycoses. 1999;42(Suppl 2):11–18. 2009;15(4):633–639.
7. Hubálek, Z., Emmonsiosis of wild rodents and insectivores in 26. Barbosa, A.d.A. et al., Acute pulmonary adiaspiromy-
Czechland. J Wildl Dis. 1999;35(2):243–249. cosis. Report of three cases and a review of 16 other
8. Chantrey, J.C. et al., Emmonsia crescens infection in a British cases collected from the literature. Rev Iberoam Micol.
water vole (Arvicola terrestris). Med Mycol. 2006;44:375–378. 1997;14:177–180.
9. Borman, A.M. et al., Adiaspiromycosis due to Emmonsia 27. Peterson, S.W., Kurtzman, C.P., Ribosomal RNA sequence
crescens is widespread in native British mammals. divergence among sibling species of yeasts. Syst Appl
Mycopathologia. 2009;168(4):153–163. Microbiol. 1991;14:124–129.
10. Cueva, J.A., Little MD. Emmonsia crescens infection (adi- 28. Borman, A.M. et al., An improved protocol for the preparation
aspiromycosis) in man in Honduras. Report of a case. Am J of total genomic DNA from isolates of yeast and mould using
Trop Med Hyg. 1971;20(2):282–287. Whatman FTA filter papers. Mycopathologia. 2010 February
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caused by Emmonsia crescens. Report of a unique case. Am J 29. Peres, L.C. et al., Fulminant disseminated pulmo-
Clin Pathol. 1971;56:394–399. nary adiaspiromycosis in humans. Am J Trop Med Hyg.
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human skin caused by Emmonsia crescens. Sabouraudia. 30. Echevarria, E., Cano, E.L., Restrepo, A., Disseminated adi-
1979;17(4):377–381. aspiromycosis in a patient with AIDS. J Med Vet Mycol.
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30 Epidermophyton
Dongyou Liu and Susan Coloe
Contents
30.1 Introduction...................................................................................................................................................................... 241
30.1.2 Clinical Features and Treatment........................................................................................................................... 242
30.2 Methods............................................................................................................................................................................ 243
30.2.2.1 PCR–RFLP Protocol............................................................................................................................. 244
30.2.2.2 Real-Time PCR Protocol....................................................................................................................... 244
30.3 Conclusion........................................................................................................................................................................ 244
References.................................................................................................................................................................................. 245
30.1 Introduction skin, nail, and hair of human and animal hosts, the genus
Epidermophyton consists of a single species: Epidermophyton
Dermatophytes are keratinophilic fungi belonging to the floccosum (obsolete synonyms: Acrothecium floccosum,
anamorphic (asexual or imperfect) genera Trichophyton, Epidermophyton clypeiforme, E. inguinale, E. plicarum,
Microsporum, and Epidermophyton. These organisms have Trichophyton cruris, T. floccosum, T. inguinale, T. intertrigi-
the capacity to invade keratinized tissues (skin, nails, and nis, and T. floccosum). A former Epidermophyton species,
hair), resulting in dermatophytosis (ringworm or tinea). that is, Epidermophyton stockdaleae is now considered as a
Unlike Trichophyton and Microsporum, which all encompass variant of Trichophyton ajelloi.
a large number of anthropophilic, zoophilic and geophilic E. floccosum colonies are generally slow growing, mus-
species, Epidermophton consists of a single anthropo- tard yellow to khaki green with a suede-like surface initially
philic species (E. floccosum). In addition, while members appearing flat but with age becoming raised and folded in
of Trichophyton and Microsporum infect skin, nails, and the center and having a submerged fringe. The reverse is a
hair, E. floccosum affects only skin and nails but not hair. yellowish tan color. E. floccosum rapidly becomes white,
In immunocompromised patients, dermatophyte infections downy, and sterile with age [2,3].
may assume insidious and severe forms such as disseminated Microscopically, septate, hyaline hyphae, macroconidia,
disease and dermatophytic granulomas. As clinical disease and chlamydospore-like cells are observed, but microco-
syndromes due to Epidermophton are indistinguishable from nidia are always absent. Occurring singly or in clusters (of
those caused by Trichophyton and Microsporum, accurate 2–3), macroconidia (measuring 20–40 μm × 6–12 μm, with
identification of these organisms is critical for the control and 1–9 septa) are smooth- and thin walled, clavate shaped with
prevention of dermatophytosis. rounded ends. Chlamydospore-like cells as well as arthroco-
nidia are common in older cultures [2,3].
30.1.1 Classification, Morphology, and Biology E. floccosum is differentiated from Trichophyton ajelloi
The genus Epidermophyton is a filamentous fungus belong- (obsolete synonym: E. stockdaleae) by the former’s inability
ing to the mitosporic (anamorphic) Arthrodermataceae to perforate hair, and by the latter’s tolerance to 7% NaCl.
group, family Arthrodermataceae, order Onygenales, In addition, macroconidia of E. floccosum are shorter than
class Eurotiomycetes, subphylum Pezizomycotina, phy- those T. ajelloi. On the other hand, E. floccosum is differenti-
lum Ascomycota, kingdom Fungi [1]. The mitosporic ated from Microsporum and other Trichophyton species by
Arthrodermataceae group consists of three genera: its absence of microconidia [4].
Epidermophyton, Microsporum, and Trichophyton, which E. floccosum is relatively sensitive to antifungal drugs
are collectively known as dermatophytes. While both the such as terbinafine, itraconazole, ketoconazole, voricon-
genera Trichophyton and Microsporum encompass a large azole, amorolfine, and naftifine, but somewhat resistant to
number of closely related species that are infective to the fluconazole.
241

30.1.2 Clinical Features and Treatment primarily colonizes and infects skin (tinea corporis, tinea
cruris, tinea manuum, and tinea pedis) and nails (tinea
Dermatophyte fungi Trichophyton, Microsporum, and unguium), and rarely cornea (keratitis) [10]. As the fungus
Epidermophyton have the capacity to invade keratinized tis- is incapable of penetrating the viable tissues of the immuno-
sues, such as hair, skin, or nails of humans and animals, and competent host, its infection is limited to the nonliving cor-
produce a variety of superficial diseases including tinea capi- nified layers of epidermis. However, invasive E. floccosum
tis, tinea corporis, tinea cruris, tinea manuum, tinea pedis, infection has been described in an immunocompromised
and tinea unguium, which are often referred to as dermato- patient with Behcet’s syndrome [11]. Typical of all forms of
phytosis (ringworm, or tinea) (Table 30.1) [5–7]. dermatophytosis, E. floccosum infections are communicable
Typically acquired directly from contact with infected and transmitted by contact, particularly in common showers
humans (anthropophilic organisms) or animals (zoo- and gym facilities.
philic organisms) or indirectly from exposure to contami- In a recent report, a 40-year-old man developed pain,
nated soil or fomites (geophilic organisms), Trichophyton, decreased vision, and multifocal stromal infiltrates in the left
Microsporum, and Epidermophyton are the only causes of eye 2 weeks after laser-assisted subepithelial keratectomy.
superficial mycoses in children [8,9]. The clinical presenta- The corneal infiltrate findings were suggestive of fungal
tions of dermatophytosis in immunocompromised hosts may keratitis, and corneal smears were positive for septate fungal
be atypical and variable. hyphae. Mycology culture and molecular analysis revealed
Epidermophyton floccosum is one of the common causes Epidermophyton floccosum as the infectious organism.
of dermatophytosis in otherwise healthy individuals. It
Table 30.1
Clinical Features, Common Causative Species, Differential Diagnosis, and Treatment of Human Dermatophytosis
Common Causative
Dermatophytosis Clinical Features Species Differential Diagnosis Treatment
Tinea capitis Superficial infection of the head and Trichophyton tonsurans, Alopecia areata, Terbinafine (Lamisil) for
invasion of hair shafts; scaling of the scalp, Microsporum trichotillomania, Trichophyton; griseofulvin
circumscribed alopecia with broken hair at audouinii, traction alopecia, and (Grifulvin) for
the scalp; adenopathy, pruritus Microsporum canis seborrheic dermatitis Trichophyton and
Microsporum
Tinea corporis Superficial infection of trunk, arms, and Trichophyton rubrum, Other annular skin Butenafine (Mentax) or
legs; annular patch or plaque with an Epidermophyton lesions terbinafine (Lamisil)
advancing, raised, scaling border, and floccosum
central clearing, diffuse erythema
Tinea cruris (jock Superficial infection of the groin Trichophyton rubrum, Candidal intertrigo (more Terbinafine (Lamisil) cream/
itch) predominantly in adolescent and young Trichophyton uniformly red with no spray; or butenafine
adults; active lesion border with pustules mentagrophytes, central clearing) and (Mentax) 1% cream
or vesicles; red to reddish brown Epidermophyton erythrasma (more
background rash; symmetric macule with floccosum uniformly brown with
well-demarcated borders; pruritus of slight scaling and no
burning quality active border)
Tinea manuum Superficial infection of the hand; Trichophyton rubrum, Contact and allergic Terbinafine (Lamisil) or
inflammatory rash with raised border and Trichophyton dermatitis; occasionally butenafine (Mentax) 1%
clearing in the middle; slowly extending mentagrophytes, atopic dermatitis
area of peeling, dryness, and mild itching Epidermophyton
on the palm of one hand floccosum
Tinea pedis Superficial infection of foot; common in Trichophyton rubrum, Contact and allergic Terbinafine (Lamisil) or
(athlete’s foot) adolescents; white macerated area between Trichophyton dermatitis; occasionally butenafine (Mentax) 1%
the toes; moccasin type with a more diffuse mentagrophytes, atopic dermatitis
dry scaling process often caused by T. Epidermophyton (dermatitis generally
rubrum; inflammatory vesiculobullous floccosum does not affect the
eruption on the soles of the feet; moderate intertriginous areas)
scaling and deep vesicular changes
Tinea unguium Infection of the finger- or toenails (a subset Trichophyton rubrum, Other acquired and Terbinafine for adolescents;
of onychomycosis); nail dystrophy Trichophyton congenital conditions griseofulvin for children;
mentagrophytes, itraconazole or fluconazole
Epidermophyton less effective but less costly
floccosum

Epidermophyton 243
After treatment with natamycin, the patient’s visual acuity polymerase chain reaction (PCR), restriction fragment
improved [10]. length polymorphism (RFLP), random amplification of
Most dermatophyte infections are treated with topical polymorphic DNA (RAPD), arbitrarily primed PCR, PCR
therapies, although tinea capitis, severe tinea pedis, and tinea fingerprinting, and Southern blotting [18–45]. In addition
unguium may require oral doses of griseofulvin, terbinafine, to random and repetitive genomic sequences, several con-
itraconazole, and fluconazole (Table 30.1) [12–15]. served gene regions have proven valuable for dermatophyte
identification and phylogenetic analysis, including small sub-
unit (SSU) 18S and large subunit (LSU) 28S rRNA genes,
30.1.3 Laboratory Diagnosis internal transcribed spacer (ITS) 1 and ITS2 regions, DNA
Considering that dermatophytosis caused by Trichophyton, topoisomerase II gene, and chitin synthase 1 (CHS1) gene
Microsporum, and Epidermophyton spp. is indistinguishable [25,46–56].
from each other and is easily confused with dermatitis and For example, De Baere et al. [56] utilized primers ITS86
other superficial infections caused by nondermatophyte fungi, (5′-GTGAATCATCGAATCTTTGAAC-3′) and ITS4
it is imperative that an etiologic agent involved in dermato- (5′-TCCTCCGCTTATTGATATGC-3′) to amplify the ITS2
phytosis is correctly identified to the species level for effective region followed by digestion with restriction enzyme BstUI
treatment. In addition, identification to the strain level is valu- (ITS2-RFLP). Subsequent electrophoretic examination of
able for epidemiological tracking of dermatophytes includ- digested PCR products facilitated the differentiation of 12
ing E. floccosum and for selection of tailored chemotherapy closely related species, including Arthroderma cajetani,
against drug-resistant dermatophyte strains [16]. Arthroderma grubyi, Arthroderma gypseum, Arthroderma
incurvatum, Arthroderma racemosum, Arthroderma
30.1.3.1 Conventional Techniques simii, Arthroderma uncinatum, Chrysosporium georgiae,
Traditionally, clinical diagnosis of dermatophytosis is Chrysosporium merdarium, Epidermophyton floccosum,
made with a focused history, physical examination. Wood’s Microsporum praecox, and Trichophyton balcaneum.
lamp examination, fluorescent microscopy, histological
tissue examination, and fungal culture further aid in the 30.2 Methods
diagnosis.
Wood’s lamp examination exploits the ability of cer- 30.2.1 Sample Preparation
tain dermatophyte species and other microbial agents to A portion of skin scrapings and nail clippings from patients
fluoresce under ultraviolet light. For example, zoophilic suspected of dermatophytosis is examined by microscopy
Microsporum canis and Microsporum audouinii causing upon treatment with 10% KOH. Another portion of the sam-
tinea capitis fluoresce blue-green; Malassezia furfur causing ples is inoculated onto Sabouraud dextrose agar (1% peptone,
tinea (pityriasis) versicolor fluoresces pale yellow to white; 1% glucose, and 1.5% agar) and incubated at 27°C for 5 days.
and Corynebacterium minutissimum causing erythrasma Further examination of micro- and macroscopic features is
fluoresces bright coral red. On the other hand, Trichophyton undertaken on the resultant fungal isolates.
and Epidermophyton causing tinea cruris and Candida spp. For DNA extraction, a small amount (50 mg) of myce-
do not fluoresce. Histological examination of nail clippings lium from Sabouraud dextrose agar is placed in lysis buf-
using a periodic acid-Schiff stain offers an accurate means to fer (200 mM Tris–HCl [pH 8.0], 0.5% [w/v] sodium dodecyl
confirm tinea unguium. sulfate [SDS], 250 mM NaCl, 25 mM EDTA) and crushed
Fungal culture is useful in cases that the infection is resis- with a conical grinder. The sample is then heated at 100°C
tant to standard topical therapy, the microscopic diagnosis is for 15 min and mixed with 150 μL of 3.0 M sodium acetate,
unclear, or there is a need for long-term oral therapy. Culture kept at −20°C for 10 min, and then centrifuged at 10,000 × g
can be carried out with skin scraping and hair shafts. In addi- for 5 min. The supernatant is extracted once with phenol–
tion, a clinical sample may be obtained with a toothbrush chloroform–isoamyl alcohol (25:24:1) and is subsequently
or cotton swab and then inoculated onto a fungal culture extracted once with chloroform. The DNA is precipitated
medium. The cotton-swab method is performed by moisten- with an equal volume of isopropanol at −20°C for 10 min,
ing a sterile cotton swab and vigorously rubbing it over the washed with 0.5 mL of 99% ethanol, dried, and suspended in
affected areas of the scalp [17,18]. 50 μL of ultrapure water. One microliter of solution is used as
Among the key features for the differentiation of E. the template for PCR. The total time required to prepare the
floccosum from Microsporum and Trichophyton are the DNA was 80 min [48].
former’s absence of microconidia and their inability to Alternatively, dermatophyte strains are cultured in 100 mL
invade hair. of Sabouraud liquid medium (Oxoid) and incubated with shak-
ing for up to 7 days at 27°C. Hyphal growth is harvested by fil-
30.1.3.2 Molecular Techniques tration and washed twice with 100 mL of sterile saline. Strains
Due to their relative simplicity and efficiency, molecular that are not processed immediately are frozen at −80°C prior to
techniques have been increasingly applied for the detec- extraction. Liquid nitrogen is added to 2–3 g of frozen hyphae
tion and identification of dermatophyte fungi. These include in a prechilled mortar, and the cells are ground finely with a

pestle. Approximately 200 mg of frozen, ground mycelium is to Trichophyton verrucosum, profile C corresponds to
placed in a 1.5 mL microcentrifuge tube, and 600 μL of lysis Trichophyton mentagrophytes var. interdigitale, profile D
buffer (400 mM Tris–HCl [pH 8.0], 60 mM EDTA, 150 mM corresponds to Epidermophyton floccosum, and profile E
NaCl, 1% SDS; 40 mg/mL proteinase K) is added. Samples corresponds to Microsporum canis [33] On the other hand,
are incubated for 1 h at 60°C with occasional mixing, and then MvnI digestion displays three genus-specific restriction pat-
100 μL of 5 M sodium perchlorate is added and incubated for terns I–III, with Epidermophyton floccosum showing three
a further 15 min at 60°C. Tubes are cooled on ice, and extrac- bands of approximately 150, 250, and 350 bp [25]. The iden-
tion is performed with 500 μL of ice-cold chloroform and tity of the dermatophytes of interest can be further confirmed
then with equal volumes of phenol–chloroform–isoamyl alco- by sequencing analysis of the PCR product.
hol (25:24:1 [pH 8.0]) and finally with chloroform. Purified
nucleic acids are precipitated with 2 volumes of ice-cold 95% 30.2.2.2 Real-Time PCR Protocol
ethanol, washed twice in 500 μL of 70% ethanol, air dried, Bergmans et al. [40] developed a single-tube
and resuspended in 150–200 μL of sterile water [25]. Lightcycler (LC) real-time PCR assay for rapid detec-
In addition, DNA can be prepared by using a genomic tion and identification of 11 clinically relevant
DNA extraction kit (Qiagen) according to the manufacturer’s Trichophyton, Microsporum, and Epidermophyton spe-
instructions, preceded by lyticase digestion. Lyticase digestion cies in nail, skin, and hair samples. For specific detec-
is carried out by transferring a loopful of mycelium to a 1.5 mL tion of Epidermophyton floccosum, dermatophyte ITS
tube containing 2.5 mg zymolase (Sigma–Aldrich) in 250 μL primer DERMF3 (5′-GGTTGCCTCGGCGGGCC-3′)
zymolase lysis buffer (10 mM Tris–HCl [pH 8.0], 1 mM and dermatophyte 5.8S rRNA primer B-DERMR2
EDTA) and incubating for 45 min at 37°C. Subsequently, (5′-CGGAATTCTGCAATTCACATTACT-3′) are com-
200 μL is transferred to another tube to carry out the DNA bined with two E. floccosum-specific ITS LC hybridiza-
extraction, with a final elution volume of 100 μL [56]. tion probes Efl3S (5′-LC705-CTACGAAATCTCCATAG
GTGGTTCAGTCT-3′) and Efl1A (5′-GCGCTCGCCGAAG
30.2.2 Detection Procedures GAGTGATTCTC-FL-3′) in a single tube.
30.2.2.1 PCR–RFLP Protocol Procedure
Universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-
3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) can be 1. PCR mixture (20 μL) is composed of 4 μL of LC
used for PCR amplification of the ITS1–5.8S rRNA–ITS2 Multiplex Master Hyb Probes (Roche), 400 nM
gene sequence. Subsequent digestion with restriction enzymes primer DERMF3 and 1300 nM primer DERMR2,
HinfI, HhaI, and MvnI generates distinct band patterns for 330 nM each of LC hybridization probes Efl3S and
dermatophytes including Epidermophyton floccosum [25,33]. Efl1A, 1.3 mM MgCl2, 0.5 μL of dimethylsulfoxide,
and 0.2 U of uracil DNA glycosylase (Roche), and
Procedure 5 μL of extracted DNA.
2. Amplification is conducted on a Lightcycler 2.0
1. PCR mixture (50 μL) is made up of 1 μL of 50 pmol (Roche) with initial denaturation for 10 min at 95°C,
of each primer, 25 μL of HotStarTaq Polymerase and 50 cycles of 20 s at 95°C, 20 s at 55°C, and 20 s
(Qiagen), 2 μL (10 ng) of extracted DNA, and 21 μL at 72°C. This is followed by a melting curve analysis
of distilled water. (1 min at 95°C, cooling for 1 min at 50°C, and ramp
2. The reaction mixture is subjected to an initial to 85°C, at a ramp rate of 0.1°C/s). Color compensa-
95°C for 6 min, 35 cycles of 95°C for 1 min, 55°C tion is performed according to the manufacturer’s
for 1 min, and 72°C for 1 min; and a final 72°C for instructions, and fluorescence signals are corrected
10 min. by the “back 530” signal.
3. A portion of PCR products (5 μL) is separated on 3. The presence of an amplification curve in the 705 nm
1% agarose gel, stained with ethidium bromide and channel of the Lightcycler, in conjunction with a
visualized by UV light. The expected sizes of PCR melting curve with the melting temperature (Tm) of
products are between 600 and 900 bp for all derma- 67.50°C–69.50°C and a Tm peak height of ≥0.01 is
tophyte strains. considered to be a positive result for E. floccosum.
4. Ten microliters of the PCR product is digested with
10 U of restriction enzymes HinfI, HhaI, or MvnI in Note. This single-tube Lightcycler real-time PCR assay
20 μL volume at 37°C for 2 h, electrophoresed on 8% enables sensitive and specific detection of E. floccosum in nail,
polyacrylamide gels, stained, and visualized as above. hair, and scales in 4 h (after overnight lysis of the samples).
Note: Restriction enzymes HinfI or HhaI digestion of the

30.3 Conclusion
PCR products from dermatophyte DNA yields five charac-
teristic, species-specific restriction patterns A–E. Profile A Dermatophytes classified in the genera Epidermophyton,
corresponds to Trichophyton rubrum, profile B corresponds Microsporum, and Trichophyton are keratinophilic fungi

Epidermophyton 245
with the capacity to invade the keratinized tissues (e.g., skin, 17. Karimzadegan-Nia, M. et al., 2007. Comparison of direct
nail, and hair) of humans and animals, resulting in a diverse smear, culture and histology for the diagnosis of onychomy-
range of superficial infections known as dermatophytosis, cosis. Aust J Dermatol. 48(1):18–21.
18. Uchida, T. et al., 2009. Comparative study of direct poly-
tinea, or ringworm. As the clinical manifestations of der-
merase chain reaction, microscopic examination and culture-
matophytosis caused by Trichophyton, Microsporum, and based morphological methods for detection and identification
Epidermophyton spp. are indistinguishable and dermato- of dermatophytes in nail and skin samples. J Dermatol.
phyte fungal spp. demonstrate varied sensitivity to commonly 36(4):202–208.
used antifungal drugs, correct identification of dermatophyte 19. Liu, D. et al., 1997. Molecular determination of dermatophyte
species involved is critical for the effective control of the dis- fungi using the arbitrarily primed polymerase chain reaction.
ease. Due to the slowness and impreciseness of conventional Br J Dermatol. 137:351–355.
diagnostic methods, molecular techniques have been increas- 20. Liu, D. et al., 2000 Jun. Application of PCR to the iden-
tification of dermatophyte fungi. J Med Microbiol.
ingly applied for identification and typing of dermatophyte 49(6):493–497.
fungi including Epidermophyton floccosum. This has not 21. Mochizuki, T., N. Sugie, and M. Uehara, 1997. Random
only led to a dramatic reduction in testing time from 3–4 amplification of polymorphic DNA is useful for the differ-
weeks to 3–4 days in most cases but also contributed to the entiation of several anthropophilic dermatophytes. Mycoses.
implementation of effective, tailor-made antifungal therapy 40:405–409.
against human dermatophytosis. 22. Baek, S.C. et al., 1998. Detection and differentiation of caus-
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31 Exophiala
Dongyou Liu and Shoo Peng Siah
Contents
31.1 Introduction...................................................................................................................................................................... 247
31.2 Methods............................................................................................................................................................................ 250
31.2.2.1 PCR Identification of Exophiala dermatitidis....................................................................................... 250
31.2.2.2 Pan-Fungal Real-Time PCR and Sequencing Analysis......................................................................... 250
31.2.2.3 Sequencing Analysis of the ITS1-5.8S-ITS2 Region............................................................................. 251
31.3 Conclusion........................................................................................................................................................................ 251
References.................................................................................................................................................................................. 252
31.1 Introduction The composition of the genus Exophiala has undergone

significant revisions following recent application of molecular
31.1.1 Classification, Morphology, and Biology techniques [4–6]. For example, E. jeanselmei is a well-known
The genus Exophiala is a dematiaceous (dark-walled) heterogeneous, cryptic species, which contains three varieties
fungus belonging to the mitosporic Herpotrichiellaceae (E. jeanselmei var. lecanii-corni, E. jeanselmei var. hetero-
group, family Herpotrichiellaceae, order Chaetothyriales, morpha, and E. jeanselmei) on the basis of its morphological
class Eurotiomycetes, subphylum Pezizomycotina, phy- features. Sequence analysis of E. jeanselmei rRNA internal
lum Ascomycota, and kingdom Fungi. The mitosporic transcribed spacer (ITS) and other gene regions revealed
Herpotrichiellaceae group encompasses 13 genera: that the former E. jeanselmei species can be separated into
Blastacervulus, Brycekendrickomyces, Cladophialophora, E. heteromorpha, E. lecanii-corni, E. oligosperma, and
Cladoriella, Cyphellophora, Exophiala, Heteroconium, E. xenobiotica plus E. jeanselmei [7]. Furthermore, molec-
Melanchlenus, Metulocladosporiella, Phaeococcomyces, ular examination of the Exophiala spinifera–E. jeanselmei
Rhinocladiella, Thysanorea, and Veronaea [1]. clade led to the differentiation of E. spinifera, E. jeanselmei,
In turn, the genus Exophiala consists of at least 29 species, E. attenuata, and E. (Phaeococcomyces) exophialae as well
the most notable of which are Exophiala castellanii (obso- as an unidentified Exophiala sp. (represented by strain CBS
lete synonyms: Aureobasidium mansonii, Cladosporium 725.88) [8,9].
mansonii, Dematium mansonii, E. jeanselmei var. castel- Morphologically, Exophiala colonies are slow growing
lanii, E. mansonii, Foxia mansonii, Malassezia mansonii, and change in texture and color with age. Initially, the culture
Microsporum mansonii, Rhinocladiella mansonii, Torula is yeast-like, moist (slimy), and brownish to greenish black in
mansonii, and Wangiella mansonii), Exophiala derma- color. As the colony ages, it becomes mold-like, suede-like
titidis (synonym: Wangiella dermatitis), Exophiala hetero- (velvety), or downy due to development of short, aerial gray-
morpha (synonym: Wangiella heteromorpha), Exophiala ish hyphae and varies from pale brown to black in color. The
jeanselmei var. heteromorpha, Exophiala jeanselmei var. colony is olivaceous-black on the front surface and black in
jeanselmei (synonym: E. jeanselmei; obsolete synonyms: the reverse surface in mature colonies [12].
Phialophora jeanselmei, Pullularia jeanselmei, and Torula The Exophiala conidiophores are hyaline to subhyaline
jeanselmei), Exophiala jeanselmei var. lecanii-corni and hyphae-like and these vary between the different spe-
(obsolete synonym: Pullularia fermentans and Torula cies. The conidium formation occurs on terminal or lateral
lecanii-corni), Exophiala moniliae, Exophiala pisciphila, conidiogenous cells (annellides), which are tubular and
Exophiala salmonis, Exophiala spinifera (obsolete syn- rocket shaped and typically taper to form a narrow elongated
onyms: Phialophora spinifera and Rhinocladiella spi- tip. The conidia, which ranged from 1–3 × 3–6 μm, are one
nifera) [2]. The teleomorphic state of the genus Exophiala celled, hyaline to pale brown, elongate, and accumulate in
is probably the genus Capronia [3]. balls at apices of annellides [10–12]. At the species level,
247

E. spinifera annellides arise on spine-like conidiophores Exophiala sp. 1 (1.6%), 3 E. attenuata (1.6%), 3 Phialophora
from the hyphae and have erect, multicellular, dark-brown europaea (1.3%), 1 E. heteromorpha (0.5%), and 1 Exophiala
stalks producing conidia from terminal and intercalary cells. sp. 2 (0.5%) strains [25].
Unlike E. spinifera, E. exophialae does not have well-differ-
entiated conidiophores. E. jeanselmei possesses non-spine-
like conidiophores and nonseptate, rocket-shaped, slightly
darkened conidiogenous cells, with cylindrical to lageni- As dematiaceous fungi, members of the genus Exophiala
form annellides. Like E. jeanselmei, the E. oligosperma often cause three types of mycotic infections in humans
conidiogenous cells are also nonseptated, rocket shaped, and and animals: phaeohyphomycosis, chromoblastomycosis,
slightly darkened. E. attenuate also has highly differenti- and mycetoma [26–29]. The infections due to Exophiala
ated conidiophores and is situated outside the E. spinifera spp. have been reported in both immunocompetent and
clade. E. moniliae has non-spine-like conidiophores and immunosuppressed patients [30,31]. Often the infection
moniliform annellides with long stalks [10–12]. Although takes hold following traumatic implantation of the fungus
Wangiella dermatitis is often considered as a synonym of through the skin or may occur as an opportunistic infec-
Exophiala dermatitis, both Wangiella and Phialophora dif- tion in patients with chronic or acute local or systemic
fers from Exophiala by forming phialides instead of annel- immunosuppression, such as organ transplantation [32–
lides. Wangiella phialides are brown and flask shaped to 37]. Phaeohyphomycosis usually presents as single cyst
cylindrical, without collarettes [13–18]. or abscess on exposed area while chromoblastomycosis
Besides microscopic features, growth at different tem- and mycetoma tend to display muriform cells or grains.
peratures (on potato dextrose agar [PDA] at 25°C, 37°C, and Clinical manifestations of Exophiala infections include
40°C for 2 weeks), hydrolysis of casein and tyrosine, abil- localized cutaneous infections, subcutaneous cysts, kera-
ity to grow in 15% NaCl, and KNO3 assimilation are use- titis, otitis, osteomyelitis, endocarditis, prosthetic valvular
ful for differentiation of Exophiala spp. as well as Hortaea vegetations, pneumonia, nosocomial outbreaks of funge-
werneckii (Table 31.1) [19,20]. Most Exophiala spp. are able mia, stomatitis, and other cerebral and disseminated infec-
to grow at 37°C, but Exophiala (Wangiella) dermatitis and tions [38–62].
Hortaea werneckii, in particular, are able to tolerate tem- A recent study surveyed 188 Exophiala strains isolated in
perature of 40°C. The teleomorphic genus Capronia species the United States and found that approximately 38%–40%
of E. psychrophila and E. mesophila from cold water and/ of the reported infections involved deep tissues. Specifically,
or fish are psychrophilic and do not grow at temperatures of 39.9% (73) of isolates came from deep infections involving
35°C or higher. In contrast, Capronia epimyces and Capronia the lung, pleural fluid, sputum, stomach, intestines, bile,
munkii, which are clustered in the E. dermatitidis clade, are heart, brain, spleen, bone marrow, blood, dialysis fluid, lymph
thermophilic and grow well at 37°C [3,21]. node, joint, breast, middle ear, throat, and intraocular tissues.
Exophiala spp. are saprophytes commonly distributed Notably, the lung, pleural fluid, and sputum accounted for
in environments including soil, plants, water, and decaying half of these cases. Another 38.3% (72) of the cases surveyed
wood material [22–24]. Of the 29 known species, 18 have were cutaneous infections involving the skin, mucous mem-
been isolated from humans and animals hosts, some (e.g., branes, nail, and corneal epithelium lesions. Subcutaneous
E. jeanselmei, E. moniliae, and E. spinifera) of which play infections including paranasal sinusitis, mycetoma, and sub-
a key role in human phaeohyphomycosis, mycetoma, and cutaneous cyst accounted for 12.0% (22) of the cases while
chromoblastomycosis [25]. A recent study reexamined 188 a remaining 0.5% (1) was isolated from superficial infec-
clinical Exophiala isolates from the United States. Using tions (including hair) [25]. Interestingly, the predominant
comparative sequencing of the ITS region in the rRNA gene, Exophiala spp. causing both the systemic, cutaneous, and
the frequency of Exophiala occurring in human hosts were as subcutaneous infections were E. dermatitidis and E. oligo-
follows: 55 E. dermatitidis (29.3%), 37 E. xenobiotica (19.7%), sperma. Specifically, the most frequently isolated Exophiala
35 E. oligosperma (18.6%), 13 E. lecanii-corni (6.9%), 12 E. spp. in systemic infections were (in decreasing order of fre-
phaeomuriformis (6.4%), 7 E. jeanselmei (3.7%), 7 E. bergeri quency): E. dermatitidis (36 of 73), E. oligosperma (16 of
(3.7%), 6 E. mesophila (3.2%), 5 E. spinifera (2.7%), 3 73), E. phaeomuriformis (9 of 73), E. xenobiotica (5 of 73),
TABLE 31.1
Physiological and Biochemical Characteristics of Some Exophiala spp.
Growth Growth Growth Hydrolysis Hydrolysis
Species at 25°C at 37°C at 40°C of Casein of Tyrosine
Exophiala jeanselmei + + − − +
Exophiala castellanii + + − − +
Exophiala (Wangiella) dermatitis + + + − +
Hortaea werneckii + + + + −

Exophiala 249
and E . lecanii-corni (4 of 73). In cutaneous and subcutane- infections by E. dermatitidis have been reported following
ous infections, the following species were reported: E. xeno- intravascular or intra-articular injection of contaminated
biotica (25 of 92), E. dermatitidis (16 of 92), E. oligosperma drugs. E. dermatitidis has been shown to cause pulmonary
(16 of 92), and E. jeanselmei (7 of 92) as well as E. bergeri, infections in humans, especially those suffering from cystic
E. spinifera, E. mesophila, and E. attenuate [25]. fibrosis (CF). Glucocorticoid treatment, antibiotic therapy,
As a localized, chronic infection of skin and subcutane- and malnutrition are known risk factors for the coloniza-
ous tissues (often following penetrating trauma on the foot), tion of the respiratory tract by E. dermatitidis in CF patients
mycetoma is characterized by tumefaction, abscess formation, [75–80].
painless slow-growing nodules or plaques, draining sinuses, Despite being a predominantly environmental agent and
and sclerotia (grains) within the abscesses and fistulae. Pain with only some Exophiala spp. being reported as human
and disfigurement may eventuate if the infection spreads to pathogens, colonization of medical equipment and con-
underlying bones. Mycetoma can be caused by either fila- sumables have been described and may be a risk factor for
mentous bacteria (actinomycetoma, 60%) or fungi (eumyce- opportunistic infections. There have been studies where
toma, 40%) [63] and as such should be properly investigated Exophiala spp. have been isolated from biofilms collected
as the treatment regime may differ. Over 30 fungal species from sterilization equipment and chemicals used, and indeed
are associated with eumycetoma in humans, among which some infections with the black yeast had resulted in fatal-
Madurella spp. are most common (causing so-called Madura ity [51]. For example, one study reported on the isolation of
foot, which was named after the district of India where it E. mesophila from chemical disinfectants used to reduce
was first described), while Exophiala jeanselmei has been bacterial load in dental unit waterlines, whereas another
reported as infrequent cause of eumycetoma [64–69]. study reported on the isolation of Phialophora spp. in the
Some case studies have been reported detailing the process biofilm collected from a water pipeline that supplied an auto-
of investigating mycetoma. In one case of Exophiala-related mated endoscope washer disinfectors [81,82]. The isolated
mycetoma, a 44-year-old man presented with a mass on his Phialophora strain was further shown to be resistant to UV
right foot that had multiple, recurrent sinuses that drained irradiation, peracetic acid, and hydrogen peroxide treatment
serosanguinous fluid within the area of tumefaction. When [82]. While the pathogenicity of these strains may not be
the patient had sustained minor trauma to the medial aspect known, it is possible that strains such as E. mesophila, which
of the right foot while outdoors about 20 years earlier, a pain- is unable to grow at temperatures of greater than 35°C, may
less mass occurred within weeks and slowly developed in size have cell walls that are capable of interfering with chemi-
over the years. Occasionally, minute dark granules (formed cal disinfectants and subsequently facilitate colonization by
by aggregates of fungal hyphae) were noticeable in the sinus bacteria [81].
drainage. A potassium hydroxide preparation of the lesion
material revealed hyphal elements and in vitro culture of tis-
sue biopsy grew Exophiala jeanselmei. Histopathological
examination showed a deeply seated abscess surrounded Apart from Exophiala spp., phaeohyphomycosis, chromo-
by an infiltrate of lymphocytes, plasma cells, and fibrosing blastomycosis, and mycetoma may be caused by other black
granulation tissue. Several grains of pigmented filamentous yeasts in the teleomorph family Herpotrichiellaceae and
fungi arranged radially at the periphery were stained within their filamentous relatives, anamorphs of members of the
the abscess using Gomori’s methenamine silver stain [70]. order Chaetothyriales. In addition, mycetoma may be due to
In another case of Exophiala infection, a 48-year-old man a number of actinobacteria. There is a need to identify these
developed a nodule on the right lower leg after trauma. The fungi for effective treatment and prevention purposes.
patient had undergone cardiac transplantation 8 months ear- Chaetothyrialean fungi including Exophiala often exhibit
lier with immunosuppressive therapy. Microscopic exami- a high degree of morphological variability and molecular
nation of the pus from lesion showed branched pale-brown diversity and encompass a wide array of clinically relevant
hyphae and rounded thick-walled vesicles. Histopathological species. As such, their identification and diagnosis by micro-
examination of tissue sections of a skin biopsy from the lesion scopic and histopathological examination of tissue speci-
displayed mild hyperplasia of the epidermis and suppurative mens and culture can be challenging and may take up to 12
granulomatous inflammation in dermis with budding yeasts weeks [25,83,84]. Cultures are frequently negative and when
and dematiaceous hyphae. On Sabouraud dextrose agar, positive, they may display poorly differentiated or delayed
dark, moist, olive to black yeast-like colony of E. jeanselmei sporulation. These fungi may also appear microscopically
was grown from the lesion material, which showed the long similar to phylogenetically remote species, thus leading to
mycelium, thick-walled septate conidiophores with a ball of misidentification [25].
conidia (annelophores) at the tip under the microscope. After In recent years, molecular approaches have been increas-
4 months of treatment with amphotericin B, the lesion showed ingly employed for improved identification of dematiaceous
marked reduction in the swelling and closure of sinuses and fungi. In particular, polymerase chain reaction (PCR) ampli-
residual scaring of the tissue [71]. fication and sequence analysis of the ITS1-5.8S-ITS2 gene
Besides mycetoma, Exophiala spp. have been implicated region with universal fungal primers provides a reliable
in other types of human infections [72–74]. Nosocomial tool for routine species distinction in the genus Exophiala

[85–87]. Furthermore, examination of the elongation factor centrifugation. The supernatant is transferred to a clean ster-
1-α and β-tubulin and mitochondrial cytochrome b gene ile Eppendorf tube and mixed with a 0.55 volume (~510 μL)
sequences has also proven valuable for confirmation of the of ice-cold isopropanol. After centrifugation for 7 min at
identity of some of the main clinically relevant species [88]. 14,000 rpm at 4°C (or room temperature), the supernatant is
The following section presents recent protocols for prepa- decanted and the pellet is washed twice with ice-cold 70%
ration of DNA from clinical samples and use of the ITS1- ethanol. The pellet is dried using a vacuum dryer, resus-
5.8S rRNA-ITS2 gene region to aid in the Exophiala spp. pended with 48.5 μL of Tris-EDTA buffer and 1.5 μL of
identification. RNase (10 mg/mL), incubated at 37°C for 15–30 min, and
stored at −20°C until use [25].
In addition, fungal strains can be cultured in 20 mL of
31.2 Methods
RPMI 1640 medium supplemented with l-glutamine but
31.2.1 Sample Preparation not sodium bicarbonate (Sigma-Aldrich). The RPMI 1640
medium is buffered to pH 7.0 with 0.165 M morpholinepro-
Clinical specimens suspected of dematiaceous fungi are panesulfonic acid (MOPS) (Sigma-Aldrich). After 3–14 days
treated with 10% KOH or stained with calcofluor white and of growth at 30°C with gentle agitation (100 rpm), the myce-
observed under the microscope for septate hyphal elements. lium is transferred into a tube and washed in 40 mL of sterile
Fungal isolation is carried out on inhibitory mold agar, distilled water. The mycelium is then stored at −20°C until
modified Sabouraud agar, or PDA at 25°C, 35°C, and 50°C. use. For DNA extraction, 100 mg of mycelium is homoge-
Preliminary identification is based on macro- and micro- nized for 1 min in a tube containing 1 mL of lysis buffer (2%
scopic features of the colonies. Halotolerance is tested in Triton X-100, 1% sodium dodecyl sulfate, 10 mM Tris–HCl
a liquid medium at 2.5%, 5%, and 10% (w/v) NaCl and pH 8, 100 mM NaCl, 1 mM EDTA pH 8), three 0.5 cm diam-
MgCl2 [89]. eter glass beads (Sigma), and approximately 500 mg of 425
The clinical specimen is grown on PDA slants for 1–7 days to 600 μm glass beads (Sigma). The homogenized mycelium
after which approximately 1 cm2 of mycelia is collected by is then snap-frozen in liquid nitrogen, thawed, and refrozen
scraping the slant with a sterile stick and emulsified in 1 mL once. DNA is then extracted with the DNeasy plant kit
of sterile H2O. The material is transferred to a 2 mL screw- (QIAGEN) [70].
cap tube, and the tube is centrifuged for 1 min at 6000 × g. If
the mycelia do not pellet, the material is concentrated with
a pediatric blood serum filter. After removal of superna- 31.2.2 Detection Procedures
tant, the material is resuspended in 200 μL of GenOhm IDI 31.2.2.1 PCR Identification of Exophiala dermatitidis
sample buffer (BD diagnostics) and transferred to the lysis
Nagano et al. [80] developed a novel species-specific PCR
tube containing glass beads. The lysis tube is vortexed on the
assay for E. dermatitidis using primers specific for the par-
highest setting for 5 min then boiled for 15 min. The sample
tial ITS1 region, the complete 5.8S rRNA region, and partial
is then centrifuged for 5 min at 16,000 × g and the superna-
ITS2 region. The primers ExdF (5′-CCG CCT ATT CAG
tant is stored at −20°C until PCR.
GTC C-3′) and ExdR (5′-TCT CTC CCA CTC CCG C-3′)
Alternatively, about 1 cm 2 of fungal material is trans-
amplify a 455 bp sequence from genomic DNA of E. derma-
ferred to a 2 mL Eppendorf tube containing a 2:1 (wt/wt)
titidis only. This method facilitates the investigation on the
mixture of silica gel and Celite (silica gel H, Merck 7736/
occurrence, etiology, and epidemiology of E. dermatitidis.
Kieselguhr Celite 545; Machery) and 300 μL of TES buffer
(2 g Tris (hydroxymethyl)-aminomethane, 0.38 g Na-EDTA 31.2.2.2 P an-Fungal Real-Time PCR and
[ethylenediaminetetraacetic acid], and 2 g sodium dodecyl Sequencing Analysis
sulfate prepared in 80 mL of ultrapure water [pH 8]). The
Pounder [89] described a real-time PCR assay utilizing
fungal material is grounded with a micropestle for 1–2 min
SYBR green DNA-binding dye and high-resolution melt-
then the volume is adjusted by adding 200 μL of TES buf-
ing temperature analysis for fungal detection. Amplicons
fer. The sample is shaken vigorously before 10 μL of pro-
were generated using pan-fungal primers ITS1 forward
teinase K (10 mg/mL) is added and the mixture incubated
at 65°C for 10 min. A measured quantity of 140 μL of 5 M
(5′-TCCTCCGCTTATTGATATGC-3′) [90]. The identity of
NaCl solution is added and the mixture is combined with
the fungi is then verified by subsequent sequence analysis.
0.1 volume (~65 μL) of CTAB (cetyltrimethylammonium
bromide) buffer 10%. The sample is then incubated for Procedure
another 30 min at 65°C. One volume (~700 μL) of chloro-
form-isoamyl alcohol (24:l vol/vol) is added and the sample 1. A PCR mixture is prepared comprising of
is mixed by inversion. After incubation for 30 min at 0°C 1 × Lightcycler FastStart DNA Master Hybridization
(on ice water) and centrifugation at 14,000 rpm at 4°C for Probes mixture (Roche Applied Science) (containing
10 min, the top layer is transferred to a clean Eppendorf deoxynucleoside triphosphates, FastStart Taq DNA
tube. Ammonium acetate (5 M, 225 μL) is added and the polymerase, and 1 mM MgCl2), 0.4 μM each of ITS1
sample returned to the ice bath for another 30 min before forward and ITS4 reverse primers, 1 × SYBR green

Exophiala 251
(Molecular Probes), and 3 μL template DNA. The 3. Amplicons are purified using a GFX column (GE
final MgCl2 concentration is adjusted to 4.6 mM. Healthcare) and sequenced separately with the V9G
2. The thermal cycling parameters used is 95°C for and LS266 primers. The sequencing reaction con-
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and sists of 1 μL of purified amplicon (1–10 ng), 3 μL
76°C for 30 s; and a final extension at 72°C for 2 min. of dilution buffer, 1 μL of BigDye v3.1, and 1 μL
3. The quality of the amplicon is determined using the of primer (4 pmol/μL) and sterile MilliQ water to
derivative of the melt analysis curve (55°C–99°C, a final volume of 10 μL. The sequencing reaction
45 s hold at 55°C, 5 s/°C) on the RotorGene 3000 cycling conditions consists of an initial 95°C for
(Corbett Robotics, Inc.). 1 min, 30 cycles at 95°C for 10 s, 50°C for 5 s, and
4. The amplified product is purified for bidirectional final extension of 60°C for 2 min. The sequencing
sequencing using ExoSAP-IT (USB Corp.). Five reactions are purified using Sephadex G-50 fine gel
microliters of BigDye Terminator Ready Reaction filtration medium (GE Healthcare Bio-Sciences)
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of and analyzed on an ABI Prism 3730xl DNA ana-
each primer (0.8 pmol/μL) and 3 μL of purified PCR lyzer (Applied Biosystems).
product. Cycle sequencing is performed with a 9700 4. The sequences are adjusted using the Lasergene’s
thermal cycler (ABI), using 25 cycles of 96°C for SeqMan Π software (DNASTAR, Inc.) and aligned
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing using Ward’s averaging in the Bionumerics package
reaction products are purified using a Sephadex v4.0 (Applied Maths). The nearest neighbors are
G-50 fine column to remove unincorporated dye identified by local BLAST searches. The distance
terminators then electrophoresed on an ABI Prism trees are based on a realigned file using the DCSE
3100 Genetic Analyzer with a 50 cm capillary array. program and calculated by the neighbor-joining
5. Sequences are analyzed with the SmartGene method of the Treecon package with Kimura-2 cor-
Integrated Database Network software version rection; only unambiguously aligned positions are
3.2.3. SmartGene is a web-based software and taken into account. A total of 100 bootstrap repli-
database system with reference sequences derived cates are used for analysis.
(NCBI) GenBank repository. Note: If the similarity of sequences at the ITS region is
greater than 99% between a studied strain and its nearest
Note: In case that real time PCR instrument is not available, neighbor and they are distributed in the same branch of the
standard PCR may be performed with primers ITS1 and phylogenetic tree, the strain is regarded as belonging to the
ITS4, and the resulting amplicon is sequenced with the same same species as its nearest neighbor.
primers. Sequence-based identifications are defined by per-
cent identity: An isolate is placed within a species if there is 31.3 Conclusion
greater than 99% sequence identity or within a genus if there
is 93%–99% identity, and classified as inconclusive when The genus Exophiala covers multiple species of dematia-
there is less than 93% sequence identity. ceous (dark-walled) fungi that are widely distributed in natu-
ral environments. Of the 29 recognized Exophiala species,
31.2.2.3 Sequencing Analysis of the 18 have been isolated from human and animal hosts, either
as colonizers or as disease-causing agents. Three types of
ITS1-5.8S-ITS2 Region
mycosis are attributable to Exophiala spp., namely, phaeo-
Zeng et al. [25] utilized primers V9G (5′-TTA CGT CCC hyphomycosis, chromoblastomycosis, and mycetoma, with
TGC CCT TTG TA-3′) and LS266 (5′-GCAT TCC CAA clinical manifestations ranging from localized cutaneous/
ACA ACT CGA CTC-3′) for PCR amplification and sequence subcutaneous infections, keratitis, otitis, endocarditis, pneu-
analysis of the ITS1-5.8S-ITS2 region [90]. monia, nosocomial outbreaks of fungemia, and disseminated
Procedure infections. Due to the fact that Exophiala spp. share mor-
phological and biochemical characteristics with each other
1. A 50 μL PCR reaction mixture comprising 10 mM and also with other related fungi that cause similar clinical
Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, diseases and that they frequently fail to produce character-
0.01% gelatin, 200 mM of each deoxynucleotide tri- istic diagnostic structures in culture, it is necessary to apply
phosphate, 25 pmol of each primer, and 0.5 U of Taq molecular techniques to supplement microscopic examina-
DNA polymerase (Sigma) is prepared. Between 10 tion and culture for their differentiation. To date, PCR ampli-
and 100 ng of rRNA is amplified. fication and sequencing analysis of the ITS1-5.8S-ITS2 gene,
2. Amplification is performed in a GenAmp PCR as well as the elongation factor 1-α and β-tubulin genes,
System 9700 thermocycler (Applied Biosystems) provide a useful way to identify these fungi. It is envisaged
with an initial 95°C for 4 min, 35 cycles of 94°C for that future identification of primers specific for individual
45 s, 52°C for 30 s, and 72°C for 2 min, followed by Exophiala spp. will further enhance the specificity of the
a final extension step of 72°C for 7 min. fungi classification and diagnosis.

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65. Hemashettar, B.M. et al. Mycetoma due to Exophiala jeansel- 87. Abliz, P. et al. Identification of pathogenic dematiaceous
mei (a case report with description of the fungus). Indian J. fungi and related taxa based on large subunit ribosomal DNA
Pathol. Microbiol., 29, 75, 1986. D1/D2 domain sequence analysis. FEMS Immunol. Med.
66. Neumeister, B. et al. Mycetoma due to Exophiala jeanselmei Microbiol., 40, 41, 2004.
and Mycobacterium chelonae in a 73-year-old man with idio- 88. Wang, L. et al. Identification, classification, and phylogeny
pathic CD4+ T lymphocytopenia. Mycoses, 38, 271, 1995. of the pathogenic species Exophiala jeanselmei and related
67. Brownell, I. et al. Eumycetoma. Dermatol. Online J., 11, 10, species by mitochondrial cytochrome b gene analysis. J. Clin.
2005. Microbiol., 39, 4462, 2001.
68. Capoor, M.R. et al., Eumycetoma pedis due to Exophiala 89. Pounder, J.I. Discovering potential pathogens among fungi
jeanselmei. Indian J. Med. Microbiol., 25, 155, 2007. identified as nonsporulating molds. J. Clin. Microbiol., 45,
69. Al-Tawfiq, J.A. et al. Madura leg due to Exophiala jeanselmei 568, 2007.
successfully treated with surgery and itraconazole therapy. 90. White, T.J. et al. Amplification and direct sequencing of fun-
Med. Mycol., 47, 648, 2009. gal ribosomal RNA genes for phylogenetics. In M.S. Innis
70. Desnos-Olliver, M. et al. Molecular identification of black- and D.H. Gelfand (eds.), PCR Protocols: A Guide to Methods
grain mycetoma agents. J. Clin. Microbiol., 44, 3517, 2006. and Applications, Academic Press, New York, 1990, p. 315.

32 Fonsecaea
Hideki Miyagi
Contents
32.1 Introduction...................................................................................................................................................................... 255
32.1.3 Diagnosis.............................................................................................................................................................. 256
32.2 Methods............................................................................................................................................................................ 260
References.................................................................................................................................................................................. 261
32.1 Introduction contains F. pedrosoi and F. monophora at present, and addi-

tional Fonsecaea species may exist as mentioned in litera-
The Fonsecaea infections such as chromoblastomycosis ture such as group C.7
and phaeohyphomycosis affect cutaneous and subcutane- Morphologically, F. monophora forms only slightly lon-
ous area and have been treated mainly in dermatological ger conidial chains and slightly shorter denticles than F.
field. Although these diseases occur chiefly in tropical and pedrosoi.2 Like other dematiaceous fungi, Fonsecaea sp. has
subtropical regions, they have also been reported in many melanin in its cell walls and this melanin seems to correlate
other parts of the world. Similar to other dematiaceous fungi, with pathogenicity. Colonies of Fonsecaea sp. look brown
Fonsecaea exists in environment as a saprophyte and is not with the naked eye. The slide culture of the Fonsecaea under
an anthropophile. Generally, Fonsecaea infection is not light microscopy also appears brown due to the presence
lethal. However, once it invades systemically, mortality rate of melanin. Three types of conidial formations are seen
is high. Following recent global climate changes, this infec- on micro-slide cultures of this polymorphic fungus, i.e.,
tion may extend from tropical and subtropical areas to the Cladosporium forms, denticulate forms, and phialides. The
temperate zone as well. With limited treatment options for conidiogenesis is important for morphological identification.
this fungal pathogen, it is important to identify it early and However, some strains with poor conidiogenesis exist and
correctly for control and prevention purposes. additionally some morphological mutants such as meriste-
matic mutant of F. monophora have been recovered. Xi et al.8
postulated that the order Chaetothyriales may have a plesio-
morphic potential to undertake extremotolerant growth.
The genus Fonsecaea consists of ascomycetous fungi that F. pedrosoi and F. monophora are nonanthropophilic but
are classified within order Chaetothyriales. Previously, two saprophytes in the environment, rotten plant, soil, wood, etc.
species, F. pedrosoi and F. compacta, are included in this F. monophora was sometimes recovered from the environ-
genus on the basis of morphological characteristics. Several ment, while F. pedrosoi was rarely isolated from the envi-
molecular investigations such as restriction fragment length ronment without mammal bait.2,7,9,10 There were reports on
polymorphism (RFLP) of mitochondrial DNA,1 ribosomal the isolation of F. pedrosoi from thorns of the plant Mimosa
RNA (rRNA) internal transcribed spacer (ITS) sequence,2 pudica,11 as well as from the shell of the babassu coconut
random amplified polymorphic DNA (RAPD),3 large sub- (Orbignya phalerata Martius) in the Amazon.12 However,
unit (LSU) rRNA D1/D2 domain sequence,4 and RFLP of these isolates were identified only morphologically but not
small subunit (SSU) rRNA and ITS regions5,6 have revealed yet molecularly. There is a need to evaluate whether clinical
that F. pedrosoi and F. compacta have few distinctions at isolates and environmental isolates of F. pedrosoi are exactly
the molecular level, and F. compacta is considered as a mor- same or not in terms of their pathogenicity. Molecular tech-
phological variant of F. pedrosoi now. A novel species F. niques have made it easier to examine environmental iso-
monophora was suggested from sequencing of rRNA region lates in detail and also previously recovered isolates. Indeed,
by de Hoog et al.2 in 2004. Thus, the genus Fonsecaea genetic diversity between pathogens of F. pedrosoi and
255

environmental encounters of F. pedrosoi has been noted. In with fibrosis. Pseudoepitheliomatous hyperplasia is often
addition, many morphologically Fonsecaea-like fungi from observed in the epidermis.
environment are verified as different species by molecular Chromoblastomycosis is recalcitrant and difficult to treat.
examinations. de Hoog et al.13 demonstrated that Fonsecaea- Indeed, some therapeutic approaches such as excision, che-
like strains are frequently found on rotten plant material and motherapy with antifungal drugs, cryotherapy, and topical
that fungal molecular diversity in the environment is higher thermotherapy have been applied to this disease but outcome
than that in the patient. This complicates the precise tracking is relatively variable. When the affected lesions are localized,
of the etiological agents of the disease and necessitates fur- excision is recommended. But once the lesions are expanded,
ther elucidation of the correlation between pathogenic agents chemotherapies with antifungal drugs including itraconazole
and environmental encounters. and terbinafine can be chosen. Chemotherapeutic effects
F. pedrosoi is usually found in tropical and subtropical depend on species of causative agent and the severity of the
regions, particularly in humid areas. F. pedrosoi causes disease. Each species has different sensitivity and F. pedro-
chromoblastomycosis as the principal agent, whereas F. soi is less sensitive than C. carrionii.
monophora causes not only chromoblastomycosis but also Phaeohyphomycosis is used to describe a broad group
phaeohyphomycosis, which may occasionally lead to possi- of infections ranging from localized cutaneous or subcuta-
ble invasive fungal infection. Indeed, there are a few reports neous disease to disseminated invasive disease17 and is an
of encephalitis cased by F. monophora.14 umbrella term for the fungal infections caused by a diversity
of dematiaceous fungi such as Cladophialophora bantiana,
Curvularia sp., Bipolaris sp., Exserohilum sp., Exophiala
jeanselmei, Scedosporium prolificans, Ochroconis gal-
Chromoblastomycosis is a cutaneous and subcutane- lopava, Coniothyrium fuckelii, Phialophora parasitica,
ous fungal infection caused by several fungi belong- P. repens, Wangiella dermatitidis, Lasiodiplodia theobro-
ing to the order Chaetothyriales such as F. pedrosoi, F. mae principally, although Fonsecaea sp. is one of the patho-
monophora, Cladophialophora carrionii, Phialophora ver- genic agents.
rucosa, Rhinocladiella aquaspersa, Exophiala dermatitidis, Phaeohyphomycosis is characterized pathologically by
Exophiala jeanselmei, and Exophiala spinifera. F. pedrosoi septate hyphae, toruloid hyphae, pseudohyphae, and no
is a leading causative agent of chromoblastomycosis among muriform cells. Subcutaneous phaeohyphomycosis presents
these fungi. However, each geographical area has a primary an asymptomatic mass or nodule mainly at the extremities
causative agent, e.g., in North America Phialophora verru- and head. It progresses chronically and the first-line regi-
cosa is the most common. In China, chromoblastomycosis men to localized lesion is surgical excision. Antifungal drugs
is primarily caused by C. carrionii in the north and by F. will be applied to widely spread lesion, but later relapse is
monophora and F. pedrosoi in the southern and eastern parts common. In systemic phaeohyphomycosis, not only brain
of the country.8,15 Fonsecaea sp. is not anthropophilic but but also joints, endocardium, and peritoneum through perito-
saprophytic and traumatic inoculation is needed to establish neal dialysis are affected. Disseminated phaeohyphomycosis
infection. These traumas are often microtrauma that patients is uncommon, but its incidence seems to increase gradually
are unaware of. Mainly immunocompetent rural workers are with the rising of immunocompromised individuals such as
infected with this disease but infection of immunosuppres- leukemia patients, hematopoietic stem cell transplant recipi-
sive patients exists. Exposed sites like the lower limbs are the ent, and solid organ transplant recipients. The treatment
most affected areas because traumatic inoculations are likely includes excision, antifungal or a combination of these, but
to take place in these areas, although the buttocks, trunk, and outcome is poor. The overall mortality rate amounts up to
face are affected as well. about 84% in immunocompromised patients. On the other
Clinical manifestations of chromoblastomycosis are wide- hand, especially cerebral phaeohyphomycosis is most com-
ranging and have been classified into several types includ- mon in immunocompetent individuals with no obvious risk
ing nodular type, tumorous type, verrucous type, cicatricial factors although cases have occurred in immunocompro-
type, and plaque type. Verrucous type is one of the most mised persons.17 The clinical outcome of cerebral phaeo-
manifested types. As yet it is unknown whether these types hyphomycosis is dismal too, with long-term survival being
are associated with specific etiologic agents or are depen- reported only when complete surgical resection of discrete
dent on host responses.16 The clinical course tends to be lesions is possible.17 The clinical potential of F. monophora
chronic and oligosymptomatic; occasionally it could last for differs from that of F. pedrosoi because F. monophora is
years. The detection of muriform cells (fumagoid, sclerotic, p redominantly neurotropic in the human host.18
Medlar body), which are spherical, thick walled and brown,
is needed for diagnosis of chromoblastomycosis in direct
32.1.3 Diagnosis
microscopic wet mount examination and histopathological
sections. Fungal elements can be detected on hematoxylin- Infectious diseases caused by the genus Fonsecaea such as
eosin-stained histopathological sections, and granulomatous chromoblastomycosis and phaeohyphomycosis are slow pro-
reactions consist of histiocytes, macrophages, and multinu- gressive and are refractory to treatment. If the diagnosis is
cleated giant cells. Lymphocytes may also be seen in dermis done while the lesions are still small, somewhat effective

Fonsecaea 257
choices of treatment including excision, antifungal, topical mentioned above: phialides (Figure 32.1, d1 and d2) observed
cryotherapy etc. are there. Once the lesions expand chroni- sporadically with collarettes of 5–6 μm. Cladosporium forms
cally, treatment effect will be limited and relapse is more (Figure 32.1, b1 and b2) observed regularly in the strains,
common. Therefore, the disease should be diagnosed as made of branched chains of blastospores showing little varia-
early as possible. Pathogenicity, virulence, and sensitivity tions between the strains. Denticulate (Figure 32.1, c1 and
of Chaetothyriales black yeasts may differ between closely c2) forms, acrotheca or Rhinocladiella form, have typically
related species.9 The causative agents have to be isolated and a sterile basal part and a distal fertile one which is more or
identified for precise diagnosis, treatment, and prevention. less developed, bearing conidia formed in a sympodial way.20
These efforts make an impact on prognosis of the disease. Exceptionally, F. compacta, which is not a species but a mor-
The cultures of causative agents are required for identifica- phological variant of F. pedrosoi today, shows that the conid-
tion although actually there are some attempts to identify ial head is elongated, and the chains of globose to subglobose
it directly from clinical samples by molecular procedures. conidia are held compactly together.21
Separation of F. pedrosoi and F. monophora is also sig- A delayed-type skin test using a prepared antigen has
nificant clinically because the spectrum of infection due to been used to diagnose and identify an isolate, but it seems
F. monophora has been shown to be more variable than that that specificity is limited yet.22,23 Some assimilation tests are
of F. pedrosoi15 and occasionally invasive. We have to know reported for identification.24,25
these differences for correct treatment.
32.1.3.1 Conventional Techniques Identification of pathogenic dematiaceous fungi including
The clinical pictures of the Fonsecaea infection are broad the genus Fonsecaea is typically done by morphological and
range. They are divided into several clinical types as men- physiological procedures. However, these procedures are
tioned above. The differential diagnosis includes sporotricho- time-consuming, require technical expertise, and are inef-
sis, protothecosis, leprosy, lupus erythematosus, sarcoidosis fective for identification of species with poor conidial pro-
and so on. If some kind of fungal infection is suspected, a duction. One of the problems for morphological identification
series of clinical and laboratory tests are started. The scales is morphological similarity among dematiaceous fungi and
are taken for direct wet mount preparation from especially polymorphic conidiogenesis of the Fonsecaea and some
“black dot,” which represents fungal elements during tran- other species. Recently, it has been recognized that molecu-
sepidermal elimination,19 and then are examined for the lar procedures (Figure 32.2) are useful for identification, phy-
presence of muriform cells and fungal elements. The skin logeny, taxonomy, typing, and epidemiology. The molecular
biopsy is also done for detection of muriform cells, fungal investigation is faster, less laborious, and highly reproducible
elements, granulomatous reaction, etc., in pathological sec- and could examine the strains with poor conidiogenesis and
tions. Clinical samples such scales, crusts, and biopsied skin morphological mutation. Moreover, it can detect geographi-
tissues will be submitted to a laboratory for culture isolation cal diversity, which is useful for epidemiology, and in the
and identification. Morphological, physiological, and bio- near future can evaluate pathogenicity of isolates, virulence,
chemical tests are available for conventional identification of and sensitivity to treatment from molecular diversity.
the Fonsecaea sp. Although evaluation and comparison of all genomic
On morphological identification, the causative agents are DNA will reveal many interesting details, this is practical.
cultured on Sabouraud’s dextrose agar and/or another appro- We have to target the proper DNA regions for classification
priate agar for observation by the naked eye, and microcul- and identification. It has been reported that the rRNA regions
ture is also done for observation under light microscope. (coding for rRNA) are suitable for the purpose of classifica-
Examination of microculture under light microscope is tion and identification of the Fonsecaea. rRNA is a compo-
sometimes more revealing than observation of plate with the nent of ribosome and plays a role in protein synthesis with
naked eye. Combining microscopic examination of micro- enzyme-like function. The rRNA is divided into several
culture with naked eye observation of the culture makes mor- parts such as SSU rRNA, 5.8S rRNA, LSU rRNA, ITS and
phological identification of an isolate easier. This is similar non-transcribed spacer (NTS) (Figure 32.3). A unit of rRNA
to a correlation between clinical pictures and pathological containing all these parts is arranged repeatedly and makes
sections in clinical practice. Pathological sections reveal a repeating structure in nucleus, although species with only
the details of the disease, but only sections without clinical one unit of rRNA in one cell such a Pneumocystis jirovecii
pictures make possible a poor diagnosis and bring us some exist. This repeating structure of rRNA in each cell means
confusion occasionally. Thus, using both types of obser- that enough template DNA is derived for investigation from
vations, one of clinical pictures at the naked eye level and relatively small amounts of sample, which then makes the
another of pathological sections at light-microscopic level, detection of nucleotide alignment easy.
makes more precise evaluation and diagnosis. Using molecular techniques, geographical genetic diver-
On culture, the colony of Fonsecaea grows relatively sity of F. pedrosoi was detected. Kawasaki et al.1 revealed
slowly and shows a velvety, dark colony on a slant agar and/ with RFLP of mtDNA that F. pedrosoi is divided into two
or on a petri dish agar (Figure 32.1a). The microculture groups and seven types correlating with geographical distri-
of the Fonsecaea shows three types of conidiogenesis as bution respectively and clarified that this molecular method is

(a)
BE FUJI 14 100 ACROS AACABE 133 15 100 ACP
0012 10 kv ×5.0 k 10 µm
(b) (c)
0012 8.0 kv ×3.0 k 10 µm
(d) (e)
0003 8.0 kv ×4.0 k 10 µm
(f) (g)
FIGURE 32.1 Plate of F. monophora (a) from chromoblastomycosis. The velvety, dark colony with relative slow growth is observed. The
three types of conidiogenesis are displayed. Cladosporium form (b, c), denticulate form (d, e) and phialides (f, g) as seen by microculture
technique (b, d, f) and scanning electron microscopy (c, e, g), respectively.

Fonsecaea 259
Clinical samples Culture Conventional tests
Morphological test
Physiological test
Analysis
Phylogeny
DNA extraction and purification classification
Identification
typing
Epidemiology, etc.
Molecular tests
Amplification of targeted DNA region RFLP

PCR
Specific PCR Sequence
FIGURE 32.2 Course of diagnostic investigation.
18s 5.8s 26s 18s 5.8s 26s 18s 5.8s 26s 18s 5.8s 26s
18s 5.8s 26s
FIGURE 32.3 Schematic view of rRNA region.
useful for identification. This geographical genetic diversity cytochrome b gene involved 33 clinical specimens that were
was confirmed with the ITS-RFLP analysis, which classi- identified as F. pedrosoi morphologically and other strains
fied 131 F. pedrosoi into five types (D1–D5) by Dde I, four of Fonsecaea sp. deposited already in the GenBank.26 It was
types (M1–M4) by Msp I, and a combined six rRNA types clear that Fonsecaea sp. is divided into two major clades,
corresponding to the six mtDNA types.5 Additionally, this groups A and B, besides subgroups A-1 and A-2 and B-1,
ITS-RFLP demonstrated that F. pedrosoi was discriminated B-2, and B-3, respectively. The groups A and B correspond
from 11 other dematiaceous fungi, making ITS-RFLP a use- to F. pedrosoi and F. monophora, respectively. The subgroup
ful tool for identification and epidemiological investigation.5 B-1 is equal to all the F. monophora strains described by de
In another report, the RFLP analysis of ribosomal gene Hoog et al.2 The subgroup B-2 consisted of all the strains
SSU rRNA and ITS regions of 12 isolates of dematiaceous from Japan identified as F. pedrosoi morphologically and one
agents showed inter- and intraspecific variability in ITS isolated from China. Intraspecies diversities corresponding
and conservation in SSU rRNA region.6 de Hoog et al.2 to geographic areas were detected as well. Moreover, 24 iso-
suggested the existence of the novel species F. monophora lated strains from China and some reference strains of the
from sequencing of ITS. Fonsecaea was classified into Fonsecaea were classified by ITS rRNA sequences into F.
group A and group B corresponding to F. pedrosoi and F. pedrosoi as genotypes A-1 and A-2 and F. monophora as
monophora, respectively, which is unrelated to F. compacta. genotypes B-1, B-2, and B-3.15 The correlation between geo-
Further investigation by RAPD procedure demonstrated that graphical distribution and genotypes was detected.
group A of F. pedrosoi divided further into RAPD groups I In addition, multi-locus sequence investigation on the
and III, and group B of F. monophora into RAPD groups II sequences of ITS, TUB1, and ACT1genes divided Fonsecaea
and IV. A subsequent phylogenetic study of ITS region and sp. into three clades as group A for F. pedrosoi, group B

for F. monophora, and group C for possible new species.7 sample, eliminating false negative results due to the presence
Not only ITS region but also LSU rRNA were investigated. of DNA polymerase inhibitors.
Analysis of the nucleotide sequences of the D1/D2 domains
of LSU ribosomal DNA of pathogenic dematiaceous fungi 32.2.2 Detection Procedures
and related taxa revealed that intraspecies sequence diversity
was extremely small, while interspecies diversity was higher. Sequencing, RFLP, and specific PCR targeting nuclear
Thus, the D1/D2 domain’s nucleotide alignments are valu- rRNA, mitochondrial DNA, and mitochondrial cytochrome
able for identification and phylogenetic analysis.4 Also, an b gene are useful for molecular investigation of Fonsecaea
oligonucleotide primer set targeting ITS region of the genus sp. (Figure 32.2), and to date most reports have focused on
Fonsecaea specifically was designed for more rapid identi- nuclear rRNA. The direct sequence of rRNA is one of the
fication in 200327 although the novel species F. monophora most practiced procedures in our lab.
was not yet suggested in 2003, and this valuable rapid iden- The universal primers targeting ITS region of broad fungi
tification method, specific PCR for Fonsecaea, may need include ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′; ITS2: 5′
reevaluation with the new species. Furthermore, the duplex -GCTGCGTTCTTCATCGATGC-3′; ITS3: 5′-GCATCGAT
PCR targeting the rRNA for rapid and specific identification GAAGAACGCAGC-3′; ITS4: 5′-TCCTCCGCTTATTGATA
was introduced.28 This duplex PCR amplifies one band with TGC-3′; ITS5: 5′-GGAAGTAAAAGTCGTAACAAGG-3′.29
universal primer pair ITS1 and ITS4 for broad species of The ITS region is amplified with these primers in PCR from
fungi and the other band with specific primer pair TSA1 and sample’s genomic DNA. The PCR mixture (20 μL) is com-
TSAS for detection of Fonsecaea simultaneously. The band posed of 10 μL of 2× Ampdirect Plus, 0.1 μL of Nova Taq™
generated by universal primer functions as a positive control, Hot Start DNA Polymerase (5 U/μL), 1 μL of 10 μM forward
making the reliable and correct identification of Fonsecaea primer, 1 μL of 10 μM reverse primer, 0.5–1 μL of template
possible. DNA, and 7.4 μL of distilled water.
Thus, these cutting-edge molecular techniques revealed Amplification is performed in Gene Amp PCR system
the existence of the new species F. monophora, which couldn’t 9700 with the following conditions: 1 cycle at 80°C for
be detected with conventional morphological techniques. 15 min and 94°C for 4 min followed by 45 cycles at 94°C for
Thanks to these molecular techniques, the phylogenetic 30 s, 55°C for 1 min, and 72°C for 1 min, with a final exten-
analysis can be performed in detail, and the identification of sion at 72°C for 7 min. The first cycle at 80°C for 15 min
not only the species level but also the strain level is possible. helps reduce activity of inhibitory substances present, And
Geographical diversity in each strain and its epidemiological the subsequent 45 cycles are neccessary because this PCR
characteristics can also be investigated. Evaluation of patho- has a relatively low amplification rate due to the existence of
genicity, virulence, and sensitivity of each strain represents inhibitory substances without purification.
another useful area for molecular techniques The amplified products are analyzed by 1% agarose gel
electrophoresis. On direct sequencing, at first the ampli-
fied products are recovered from electrophoresed agarose
32.2 Methods gel with QIAquick Gel Extraction Kit (Qiagen). The con-
centration of amplified product contained in this recovered
solution is measured using a spectrophotometer and by the
Clinical specimens containing causative fungus elements comparison of electrophoresed density between this recov-
such as scales, crusts, pus, biopsied and fragmented skin ered solution and a solution whose concentration of DNA
tissue, etc. are submitted for cultures and/or direct molec- already known e.g., size marker. Sometimes, the concentra-
ular tests. For fungus culture, the clinical specimens are tion measured by electrophoresed density is low in spite of
inoculated onto a slant agar such as Sabouraud’s dextrose the high concentration measured by the spectrophotometer in
agar and/or another proper agar containing antibiotics. the same sample. This is because the irradiation of UV light
Proliferated fungus elements from culture are offered to damages the structure of DNA when the amplified product is
molecular tests. A pick of fungus element with a loop is recovered. The exact amount of complete DNA is important
taken and immersed in 400 μL resolution buffer (20 mM for direct sequence reaction and the concentration value from
Tris–HCl pH 8.0, 5 mM ethylenediaminetetraacetic acid the electrophoresed density is more reliable.
(EDTA), 400 mM NaCl, 0.3% sodium dodecyl sulfate The sequencing reaction (10 μL) consists of 2 μL of premix
(SDS), and 200 μg/mL proteinase K) in 1.5 mL tube. Then, of BigDye™ Terminator Cycle Sequencing Ready Reaction
this mixture is incubated for several hours at 55°C and (Applied Biosystems), 2 μL of dilution buffer, 1.6 μL of
diluted with nine volumes of distilled water. This dilution primer (2 pmol/μL), 10–20 ng of template DNA, and up to
is done for reducing reaction of inhibitor to proper amounts 10 μL of distilled water. Sequencing reaction is performed in
for PCR. And then, this diluted solution becomes the tem- Gene Amp PCR system 9700 with the following conditions:
plate for molecular tests. 1 cycle at 96°C for 1 min followed by 25 cycles at 96°C for
Ampdirect• Plus kit (Analytical & Measuring Instruments 30 s, 48°C for 30 s, and 60°C for 2min, with a final exten-
Division, Shimadzu Corporation) may be used in PCR sion at 72°C for 7 min. Then this reaction solution is purified
m
ixture as it can neutralize the inhibiting materials in the with ethanol and applied to a sequencer of ABI PRISM 310

Fonsecaea 261
Genetic Analyzer. Acquired nucleotide alignment is com- References

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approaches, Med. Mycol., 38S, 243, 2000.
molecular techniques have led mycological diagnostics to
14. Takei, H., Goodman, J.C., and Powell, S.Z., Cerebral pha-
a new level of sensitivity, specificity, and speed. The novel eohyphomycosis caused by Cladophialophora bantiana
species F. monophora was discovered by molecular tech- and Fonsecaea monophora: Report of three cases, Clin.
niques, while traditional morphological and physiological Neuropathol., 26, 21, 2007.
techniques were unable to do so. Use of molecular tech- 15. Xi, L. et al., Molecular diversity of Fonsecaea
niques has also helped clarify intraspecies diversity, which (Chaetothyriales) causing chromoblastomycosis in southern
may underscore geographical diversity and will be instru- China, Med. Mycol., 47S, 27, 2009.
mental in the elucidation of Fonsecaea pathogenicity and 16. Najafzadeh, M.J. et al., Successful treatment of chromo-
blastomycosis of 36 years duration caused by Fonsecaea
virulence in future.
monophora, Med. Mycol., 48, 390, 2010.
Dematiaceous fungi can be divided into three ecologi- 17. Brandt, M.E. and Warnock, D.W., Epidemiology, clinical
cal groups: saprobes (that are not known as pathogens), low- manifestations, and therapy of infections caused by dematia-
virulent opportunists (that can directly be isolated from the ceous fungi, J. Chemother., 15S, 36, 2003.
environment), and highly specific pathogens (that cannot 18. Surash, S. et al., Cerebral phaeohyphomycosis caused by
be isolated from the environment directly but require liv- Fonsecaea monophora, Med. Mycol., 43, 465, 2005.
ing mammal bait).9 The correlation among the pathogen 19. Ameen, M. Chromoblastomycosis: Clinical presentation and
of the Fonsecaea, nonpathogen and/or low pathogen of the management, Clin. Exp. Dermatol., 34, 849, 2009.
20. Ibrahim-Granet, O. and de Bievre, C., Study of the conidial
Fonsecaea, and other dematiaceous fungi will be unraveled
development and cleistothecium-like structure of some strains
through further molecular analysis. Besides rRNA, other of Fonsecaea pedrosoi, Mycopathologia, 84, 181, 1984.
genetic regions may be investigated for identification purpose. 21. Wang, D.L., Li, R.Y., and Wang X.H., Observations of sporu-
Multi-locus genetic analysis, DNA microarray, and the DNA lation in Fonsecaea and Phialophora by scanning electron
chip will be also valuable for the study of Fonsecaea spp. microscopy, Mycopathologia, 98, 105, 1987.

22. Iwatsu, T. et al., Evaluation of skin test for chromoblasto- 27. Abliz, P. et al., Rapid identification of the genus Fonsecaea
mycosis using antigens prepared from culture filtrates of by PCR with specific oligonucleotide primers, J. Clin.
Fonsecaea pedrosoi, Phialophora werrucosa, Wangiella der- Microbiol., 41, 873, 2003.
matitidis and Exophiala jeanselmei, Mycopathologia, 77, 59, 28. Andrade, T.S. et al., Rapid identification of Fonsecaea by
1982. duplex polymerase chain reaction in isolate from patients with
23. Iwatsu, T. et al., Skin test-active substance prepared from cul- chromoblastomycosis, Diagn. Microbiol. Infect. Dis., 57, 267,
ture filtrate of Fonsecaea pedrosoi, Mycopathologia, 67, 101, 2007.
1979. 29. White, T.J. et al., Amplification and Direct Sequencing of
24. Espinel-ingroff, A. et al., Evaluation of the API 20C yeast Fungal Ribosomal RNA Genes for Phylogenetics, PCR: A
identification system for the differentiation of some dematia- Guide to Methods and Applications, Academic Press, Inc.,
ceous fungi, J. Clin. Microbiol., 27, 2565, 1989. New York, 1990, pp. 315–322.
25. Steadham, J.E., Geis, P.A., and Simmank, J.L., Use of carbo- 30. Miyagi, H. et al., Case of chromoblastomycosis appearing in
hydrate and nitrate assimilations in the identification of dema- an Okinawa patient with a medical history of Hansen’s dis-
tiaceous fungi, Diagn. Microbiol. Infect. Dis., 5, 71, 1986. ease, J. Dermatol., 35, 354, 2008.
26. Yaguchi, T. et al., Molecular phylogenetics of strains
morphologically identified as Fonsecaea pedrosoi from clini-
cal specimens, Mycoses, 50, 255, 2007.

33 Histoplasma
Rosely Maria Zancopé Oliveira, Allan Jefferson Guimarães,
Joshua D. Nosanchuk, Mauro de Medeiros Muniz,
Priscila Costa Albuquerque, and Rodrigo de Almeida Paes
Contents
33.1 Introduction...................................................................................................................................................................... 263
33.1.1.1 Classification.......................................................................................................................................... 263
33.1.1.2 Morphology............................................................................................................................................ 263
33.1.2 Biology and Epidemiology................................................................................................................................... 264
33.1.2.1 Biology................................................................................................................................................... 264
33.1.2.2 Epidemiology......................................................................................................................................... 264
33.1.3 Pathogenesis and Clinical Features...................................................................................................................... 265
33.1.3.1 Pathogenesis........................................................................................................................................... 265
33.1.4 Diagnosis.............................................................................................................................................................. 266
33.2 Methods............................................................................................................................................................................ 268
33.2.1.1 Growth of Histoplasma capsulatum...................................................................................................... 269
33.2.1.2 Extraction of Histoplasma capsulatum DNA from Culture.................................................................. 269
33.2.1.3 Extraction of Histoplasma capsulatum DNA on Blood Samples.......................................................... 269
33.2.2.1 PCR for Detection and Identification of Histoplasma capsulatum....................................................... 269
33.2.2.2 Nested PCR for Detection of Histoplasma capsulatum from Clinical Samples................................... 269
33.2.2.3 Identification of Histoplasma capsulatum by Sequencing.................................................................... 270
33.2.2.4 Molecular Typing of Histoplasma capsulatum..................................................................................... 270
References.................................................................................................................................................................................. 271
33.1 Introduction mules.5 However, based on molecular studies, Kasuga et al.6

suggest that H. capsulatum might be composed of six distinct
33.1.1 Classification and Morphology species, instead of three varieties. This chapter focuses on
33.1.1.1 Classification H. capsulatum var. capsulatum.
Histoplasma capsulatum is the anamorphic form of
Ajellomyces capsulatum. It belongs to the phylum 33.1.1.2 Morphology
Ascomycota, class Eurotiomycetes, order Onygenales, and H. capsulatum is a dimorphic fungal pathogen, since it dis-
family Onygenaceae and/or Ajellomycetaceae.1–3 A. cap- plays two distinct morphological forms, depending on the
sulatum is a heterothallic fungus with two distinct mating nutritional factors and temperature. The filamentous mor-
types: (+) and (−). They exist in a 1:1 ratio in soil; however, phological form occurs at temperatures below 35°C or in the
the (−) type is seven times more frequent in clinical samples.4 soil. This form is composed of hyaline septate hyphae that
There are three H. capsulatum varieties: H. capsulatum var. are 1–2.5 μm in diameter. Moreover, hyphae produce two dif-
capsulatum, the etiological agent of classic histoplasmosis; ferent hyaline asexual reproduction structures. Microconidia
H. capsultaum var. duboisii, the etiological agent of African are round to pear shaped, smooth, and 2–5 μm in diameter.
histoplasmosis; and H. capsulatum var. farciminosum, the Macroconidia are large, thick walled, round, and 7–15 μm
etiological agent of epizootic lymphangitis of horses and in diameter. The macroconidia are typically tuberculate,
263

knobby, or with short cylindrical projections, although they synthesize melanin in the absence of exogenous phenolic
may occasionally be smooth. These macroconidia are simi- substrate, it is probable that conidia are melanized in the
lar to those of Sepedonium; however, this genus is unable to environment. Thus, melanization may protect the conidia
produce microconidia.7 from environmental insults.13 Moreover, yeast melanization
In parasitism or when cultivated at 37°C in specific media, appears to contribute to virulence by reducing H. capsula-
H. capsulatum var. capsulatum are small yeast cells (2–4 μm tum susceptibility to host defense mechanisms and antifun-
in length) that are ovoid with a narrow base at the smaller gal drugs.15,16
end. The variety duboisii differs from the variety capsulatum Vesicular secretion of macromolecules has recently been
by producing larger yeast cells (8–15 μm in length). described in H. capsulatum. Macromolecules transported
The teleomorph state of H. capsulatum is A. capsulatum. via a trans-cell wall vesicular transport secretory mechanism
When (+) and (−) A. capsulatum strains are paired on alpha- by yeast cells are involved in diverse processes, including
cel-yeast extract agar they produce cleistothecia. They ini- metabolism, cell recycling, signaling, and virulence. The
tially appear globose or subglobose and then form irregular vesicles from the yeast form of H. capsulatum cells react with
stellates due to the extended growth of coils beyond the immune serum from patients with histoplasmosis, provid-
outer limit of the peridium. Asci are club to pear shaped and ing an association of the vesicular products with pathogen-
contain eight globose ascospores with a diameter of ~1.5 μm.4 esis and also represent a possible new target for antifungal
drugs.17
33.1.2 Biology and Epidemiology
33.1.2.1 Biology Numerous animal species can be infected by H. capsulatum.
H. capsulatum is a haploid ascomycete, although aneuploidy Contaminated soils are the infection source for both human
can occur in some strains, such as the reference Downs and other animals, such as dogs, cats, horses, cattle, pigs,
strain.8 It is closely related to other dimorphic fungi that also rodents, and marsupials.18 Usually the fungus is associated
cause pulmonary infections such as Blastomyces dermatiti- with soils enriched with organic nitrogen sources like ani-
dis and Coccidioides immitis or C. posadasii. H. capsulatum mal excrements, especially of bats and chickens.5,19 In fact,
has seven chromosomes and the genome size is approxi- H. capsulatum has been isolated from several protected
mately 25–30 Mb, although these values vary among differ- environments, such as caves, abandoned construction sites,
ent strains.9 and chicken coops. H. capsulatum does not cause disease in
The H. capsulatum cell wall is a rigid, largely polysac- birds, so their role in the environmental spread of H. capsu-
charide structure composed of four different glycans: soluble latum is probably due to fungal carriage on their feathers,
galactomannan, α-1,3-glucan, β-1,3-glucan, and a fibril- beaks, or claws. On the other hand, bats are fungal reservoirs,
lar chitin skeleton. The amount of each glycan depends on since they harbor the fungus in their intestinal mucosa and
the chemotype of the strain, although some fluctuations can release H. capsulatum in their feces.20 Also, histoplasmosis
occur in certain isolates from a single chemotype.10 The can occur in the bats, including lethal disease. Recently, Julg
α-1,3-glucan is considered relevant for H. capsulatum viru- et al. have detailed the acquisition of human histoplasmosis
lence, whereas the β-1,3-glucan, which is predominant in the at the entrances of bat caves, without exposure to deposits
fungal mycelial phase, is antigenic and participates in the of guano within the caves.21 Additionally, bat migration can
modulation of the host immune response.11 contribute for the generation of new H. capsulatum niches on
The vast majority of H. capsulatum biomass exists in the the environment.5
soil as a mold, since the parasitic stage is not necessary or H. capsulatum infection is typically acquired by micro-
advantageous for the survival of the fungus. Interestingly, conidia inhalation after disturbances of soil or excreta. For
the parasitic stage of H. capsulatum may have developed in this reason, histoplasmosis is not generally associated with
response to interactions between the fungus and other soil- person to person spread of disease. Rarely histoplasmosis can
living microorganisms, such as Acanthamoeba castella- be acquired by cutaneous inoculation of the fungus and, even
nii, since phagocytosis of the fungus can result in death of more unusually, vertical transmission can occur, given that
amoeba and H. capsulatum growth in the yeast form.12 there is one such case in the literature.4
In both saprophytic and parasitic stages, H. capsulatum Histoplasmosis is a cosmopolitan fungal infection with
must face an overabundance of diverse challenging envi- areas of high endemicity. However, patients are usually
ronmental conditions. In response, H. capsulatum produces unaware of their potential exposure.22 Endemic regions in
several biological molecules such as catalase to survive oxi- North America are located in Midwestern and Southeastern
dative stress conditions, siderophores to survive iron starva- regions of the United States of America, especially the
tion, and orotidine 5-monophosphate pyrophosphorylase to Mississippi, Ohio, and Missouri river valleys, locations
endure uracil limitation.9 This fungus is also able to produce where 80% of the resident population reacts to the histo-
melanin, on both conidia and yeast cells.13 Melanins are plasmin skin test.18 Data from the United States suggests
negatively charged, hydrophobic pigments of high molecular that half a million new human infections occur each year.9
weight, formed by the oxidative polymerization of phenolic In Latin America, the most prevalent areas of endemicity
and/or indolic compounds.14 Since H. capsulatum conidia are within Brazil, Venezuela, Ecuador, Paraguay, Uruguay,

Histoplasma 265
and Argentina. In Brazil, endemic areas are located in the fungus. Interestingly, vaccination with H. capsulatum HSP60
Midwestern and Southeastern portions of the country.19,23 induces protective host responses.45–46 The M and H antigens
Generally, the environmental conditions present in areas of are immunodominant antigens of H. capsulatum that have
high endemicity are a moderate climate with a relatively con- long been utilized as serological markers of histoplasmosis.19
stant humidity level. The M antigen, also known as catalase B, is a constitutively
Multiple typing methods have been developed to study expressed protein posited to play a role in counteracting the
the epidemiology of H. capsulatum. These methodologies oxidative defense mechanisms of host phagocytic cells.47–48
can be based on phenotypic characteristics such as anti- The H antigen is a secreted beta-glucosidase purportedly
genic profiles24 and multilocus enzyme electrophoresis25 involved in cell wall remodeling and nutrient acquisition.49
or on DNA-based analyses. Most recently, typing has been H. capsulatum secretes a calcium binding protein (CBP) dur-
accomplished by analysis of fatty acid profiles of H. capsu- ing yeast-phase growth that is essential for growth in cal-
latum.26 Molecular typing methods are generally considered cium limiting conditions, such as encountered in vivo, and
as having advantages over phenotypic methods in terms of is required for virulence during murine infection.50 Yeast-
the stability of genomic markers and providing greater lev- phase-specific protein 3 (YPS3) is a cell surface and secreted
els of typeability. Several genotype-based methods such as protein of uncertain function, but appears as a virulence
hybridization of target genes (probes),27–29 restriction frag- determinant, since silencing of the YPS3 gene significantly
ment length polymorphism (RFLP),30 karyotyping,31 random attenuates virulence in vitro and during murine infection.51
amplified polymorphic DNA (RAPD),32–36 and sequencing
have been described for H. capsulatum.6,37–39 33.1.3.2 Clinical Features
Histoplasmosis can affect any individual, independent of age
33.1.3 Pathogenesis and Clinical Features or sex. In general, the progress of the disease depends on the
amount of microconidia inhaled into the lungs. Contact with
33.1.3.1 Pathogenesis H. capsulatum is very common for persons living in endemic
After the inhalation of H. capsulatum conidia or mycelial areas; however, symptomatic infection is uncommon.52 Most
fragments by a host, the organism converts to its pathogenic primary infections in immunocompetent individuals result
yeast form that survives and replicates in the phagolysosomes in mild or asymptomatic respiratory disease. In such cases,
or modified phagosomes of host macrophages.40 H. capsu- the development of antigen-specific, T-lymphocyte-mediated
latum may be internalized after opsonization with antibody immunity and fungistatic activation of macrophages results
and/or complement. This process may be associated with in control of the infection and disease resolution within 1–2
host antimicrobial defense mechanisms, such as an oxidative weeks.9 However, children less than 1 year old and adults
burst mediated by nicotinamide adenine dinucleotide phos- older than 50 are at increased risk for more severe forms of the
phate (NADPH) oxidase, phagosome–lysosome fusion, vac- disease. Immunocompromised individuals, such as those with
uolar acidification, generation of nitric oxide, and exposure lymphomas, leukemia, AIDS, diabetes, and Hodgkin disease
to hydrolytic enzymes.9 The fungus can also bind directly to or patients on corticosteroid therapy are more likely to develop
CD18 integrins family on the surface of host mononuclear progressive opportunistic histoplasmosis, including fungal
cells, and this binding occurs through a specific ligand, a heat dissemination.5 Most recently, treatment with tumor necrosis
shock 60 kDa protein (HSP60),41 that interacts with CR3 on factor (TNF)-alpha inhibitors has been associated with a sig-
macrophages. The morphotypic transition from mold to yeast nificant increase in risk for histoplasmosis.53 However, histo-
in H. capsulatum is governed predominantly by temperature plasmosis even in immunocompetent people can frequently
and occurs after inhalation by a mammalian host. This inter- result in persistent, clinically inactive infection. This latent
action with the host involves a switch from infectious mold condition can erupt into active disease at a later time when
to pathogenic yeast that is well suited to intracellular parasit- the host–pathogen balance is disrupted following a decrease
ism of phagocytes and dissemination, either free in blood or in host immune function and/or due to uncharacterized con-
lymph, or more often carried within macrophages. tributions from the “quiescent” fungus. Many of these clinical
The dimorphic transition and the yeast morphotype are features of histoplasmosis resemble tuberculosis.52
required for the establishment of disease with H. capsula- There is a broad spectrum of clinical manifestations of
tum.9 This morphological change is the manifestation of histoplasmosis, ranging from a self-limited pulmonary infec-
several alterations in gene expression patterns that constitute tion, which subsides without treatment to chronic pulmonary
a switch to a pathogenic lifestyle. Chemical treatments of infection to more widespread disseminated disease. Acute
mycelia are able to prevent transition of Histoplasma to the pulmonary histoplasmosis is a usually a self-limited illness in
yeast form when grown at 37°C; although the treated myce- persons exposed to the organism for the first time, especially
lia remain viable at 37°C, they are avirulent.42 Therefore, after low inoculum exposure. In fact, over 90% of exposed
their virulence factors relate specifically to the yeast phase individuals are asymptomatic.54 However, symptomatic dis-
of the fungus.43 In addition to dimorphism, some virulence ease can occur after an incubation period of 1–3 weeks.55
determinants of H. capsulatum have been characterized and Symptoms include fever, malaise, headache, weakness, sub-
summarized.44 As noted, HSP60 is associated with initiation sternal chest pain, and dry cough.56 In some patients, chest
of phagocytosis, resulting in the intracellular phase of the radiographs are obtained only after the pulmonary infiltrate

has cleared, and hilar lymphadenopathy is the primary

finding. Acute self-limited pulmonary histoplasmosis is
accompanied by rheumatologic and/or dermatologic mani-
festations in a few patients. Disseminated histoplasmosis is
the most severe form of this disease. Clinical disseminated
disease most commonly occurs in individual patients receiv-
ing cytotoxic or steroid therapy, and in immunocompromised
persons, especially those with AIDS.53 Disseminated disease
is also more common in the very young or very old. The
manifestations of disseminated histoplasmosis are greatly
variable, commonly including fever, weight loss, hepato-
splenomegaly, generalized lymphadenopathy, lung infiltrates,
and haematological abnormalities.57–58
Figure 33.1 Histoplasmosis. Giemsa staining of a bone mar-
row smear showing intracellular H. capsulatum yeast phase, 40×.
33.1.4 Diagnosis (Courtesy of Bodo Wanke.)
Although the clinical manifestations of histoplasmosis are

well described, there is significant overlap of symptoms may be the most rapid method of establishing a definitive
with other diseases, and the diagnosis cannot be achieved diagnosis of disseminated disease (Figure 33.1). Skin scrap-
based on clinical information alone. The definitive diagnosis ings, exudates, and body fluids should be examined using
requires the isolation of the H. capsulatum on specific culture 10% KOH and Parker ink or calcofluor white mounts. Tissue
media or the visualization of the yeast form in direct exami- sections should be stained using PAS (periodic acid-Schiff)
nation of clinical specimens using specific fungal staining digest, Grocott’s methenamine silver (GMS), or Gram stain.61
techniques. The gold standard for diagnosis is the growth of H. capsulatum structures visualized microscopically can be
the organism; however, cultures typically take 2–4 weeks to confused with structures from other pathogens.19
grow and this technique lacks sensitivity. The likelihood of
a positive culture depends on the clinical form, ranging from 33.1.4.1.2 Culture
10% to 85%59 with the best yields in individuals with dis- Isolation of the fungus in its mycelial form can be achieved
seminated or chronic pulmonary disease. Direct microscopic on Sabouraud agar, following incubation at 25°C for 6–12
examination of tissues or body fluid can facilitate diagnosis, weeks. The fungal colonies are initially smooth, becom-
particularly of bone marrow in acute disseminated disease ing filamentous, cottony, and brownish with the age (Figure
where the yeast form can be identified in up to 50% of cases. 33.2A). Microscopically, they are composed of septated hya-
However, visual inspection can result in incorrect diagnosis. line hyphae that have tuberculated macroconidia and smooth-
Other techniques have been developed to supplement culture walled spherical, pyriform, or cigar-shaped microconidia in
and microscopic examination. Serological methods usually various developmental stages, ranging in size from 2 to 6μm
have a rapid turnaround time and detection of either antibod- in diameter4 (Figures 33.2B and C). The conversion from the
ies or antigens can provide information indicative of current
disease. The choice of each test to be applied for diagnosis
depends mainly on the clinical manifestation and host fac-
tors.56 Non-culture-based methods are used in conjunction
with culture to improve our ability to diagnosis H. capsu-
latum infection and can also guide therapy for histoplasmo-
sis. Recently, some molecular biology techniques have been
developed that may further improve the diagnosis of histo-
plasmosis, particularly for the detection of disease in an early (B)
stage and to improve the specificity of the diagnosis.
33.1.4.1 Conventional Techniques

33.1.4.1.1 Microscopy
Direct examination and histopathology are useful, especially
as they are the most important method for alerting laboratory
(A) (C)
personnel that they may be dealing with a potential fungal
pathogen.60 Histopathologic examinations of different clini- Figure 33.2 Histoplasma capsulatum. (A) Culture on Mycosel’s
cal specimens are frequently analyzed for the diagnosis of agar for 15 days at 25°C. (B) Macroconidia from mycelia form. The
histoplasmosis.53 Peripheral blood may show intracellular arrow indicates a microconidia, 40×. (C) Globose, tuberculated
organisms in white blood cells. However, bone marrow biopsy macroconidia, 100×.

Histoplasma 267
exposed to H. capsulatum.65 The presence of both the H and

M precipitins is considered to be conclusive for the diagno-
sis of histoplasmosis, though the status of disease requires
clinical assessment of the patient. Antibodies to H. capsu-
latum can also be detected by immunoenzymatic methods.
Western blot (WB) has been used successfully in order to
diagnose histoplasmosis and epidemiologically evaluate
the distribution of the disease66–69 and should be applied in
conjunction to ID in acute disease since the sensitivity can
be significantly improved.70 Several enzyme-linked immu-
nosorbent assays (ELISA) protocols have been described
for antibody detection using diverse antigenic preparations
showing sensitivity among 75%–100%.19,66 More recently, an
(A) (B) indirect ELISA for detecting antibodies against deglycosyl-
ated histoplasmin (HMIN) was found to have a sensitivity of
Figure 33.3 Histoplasma capsulatum. (A) Smooth yeast colony 92% and specificity of 96%.71 An advantage of this method
produced on YPD agar for 15 days at 37°C. (B) Globose and/or oval is that it can be readily applied in resource-limited labora-
yeast budding cells, 40×. tory settings.
Antigen detection methods are used when antibody
mold to the yeast phase is necessary for accurate diagnosis detection is unlikely, particularly in acute disease and for
of H. capsulatum and it is achieved using enriched media immunocompromised individuals, who frequently have the
such as Brain-heart infusion agar (BHI) supplemented with disseminated form of histoplasmosis.19,54 A radioimmunoas-
blood and cystein and incubated at 35°C–37°C. After conver- say for the detection of H. capsulatum antigen in urine and
sion, smooth white to brown colonies can be observed that serum specimens based on the detection of a polysaccharide
are ovoid thick-walled yeast cells by microscopic examina- antigen from H. capsulatum (HPA) has been particularly
tion (Figure 33.3). Although attempts to culture the organism effective, especially in patients with disseminated histoplas-
should be pursued for all forms of histoplasmosis, culture- mosis.72 This method has also proven especially useful for
based methods are most effective when the fungal burden is monitoring the effect of treatment.73 ELISAs for detection of
high, for instance, in blood samples of patients with chronic H. capsulatum antigens have been described and compared
or disseminated forms of histoplasmosis. Culture is insensi- with the solid-phase radioimmunoassay (Table 33.1).
tive in subacute and acute histoplasmosis. An inhibition ELISA has also been developed using a
murine monoclonal antibody that is specific for a 70 kDa H.
33.1.4.1.3 Serology capsulatum protein.75 The sensitivity varied from 57.1% to
Serologic diagnosis focuses on the identification of antibod- 88.9% depending on the clinical form of histoplasmosis, and
ies to the H62 and/or M antigens.47 The two routine antibody the specificity was 85.4% when serum samples of other myco-
detection methodologies are complement fixation (CF) sis were used. This methodology is also useful for monitor-
and immunodiffusion (ID), because of convenience, avail- ing histoplasmosis patients during treatment and following
ability, and accuracy of these assays. In the past, CF was a disease in non-AIDS patients with the acute or disseminated
popular test to diagnose histoplasmosis but ID was found to form of the disease.76 Recently, a capture ELISA for the detec-
be more specific than CF.19 The ID test qualitatively mea- tion of H. capsulatum using rabbit polyclonal antibodies as
sures precipitating antibodies and is highly specific for the capture and detection agents has been described.77 The assay
detection of antibodies to M and H antigens, ranging from had a sensitivity of 81% and specificity of 95% in the set-
70% to 100%.63 However, it has low sensitivity, principally ting of testing urine samples from Guatemalan patients with
in acute, disseminated, and opportunistic manifestation of AIDS who had culture-proven histoplasmosis. The authors
the disease. In general, ID is useful for detecting antibod- advertise this method for its simplicity and for its capacity to
ies 4–6 weeks after infection. The H band can be found in facilitate the rapid diagnosis of disseminated histoplasmosis
sera from patients during acute and/or progressive disease. in resource limited settings.
However, the H band is present in only 7% of serum from
patient with acute infection.64 Antibodies to the H antigen 33.1.4.2 Molecular Techniques
may be detected 1–2 years after the resolution of the disease, Molecular methods for the identification of fungal isolates
but usually disappear more quickly than the antibodies to can potentially reduce the time for diagnosis, while main-
the M antigen. The M band is more frequently detected than taining or improving the specificity, accuracy and sensitiv-
the H precipitin and appears soon after infection and can ity. Additionally, molecular methods can be safer as most
indicate prior infection, acute disease, or a chronic progres- methods do not require laboratory growth of the organism,
sive disease.19 The M precipitin can persist for up to 3 years limiting the possibility of laboratory infection.78 Polymerase
after disease resolution and can also be stimulated after chain reaction (PCR) based on the amplification of fungal
skin testing with histoplasmin in people who have not been gene sequences is a powerful tool for identifying invasive

Table 33.1
Parameters of Antigen Detection Techniques for Histoplasma capsulatum
Sensitivity (%)
Antigen Detection Test Disseminateda Non-Disseminatedb Specificity (%) References
RIA 95 48 96 Wheat et al.72
Sandwich ELISA APc 89 25 92 Durkin et al.74
HRPd 89 16 96 Durkin et al.74
Inhibition-ELISA 68 75 85 Gomez et al.75
a Disseminated includes disseminated non AIDS and opportunistic infection.

b Non disseminated includes acute, chronic, mediastinal and self-limited infections.
c Alkaline phosphatase.
d Horseradish peroxidase.
mycoses.52 Several PCR-based methods have been described and paraffin-embedded tissue has been described using
for H. capsulatum diagnosis. nested PCR assays. Using the sequence of the H antigen gene,
A PCR was developed for rapid identification of primers were selected and a semi-nested PCR developed. A
H. capsulatum isolates in culture based on the sequences of comparison of a semi-nested PCR method targeting the H
the M gene.79 The M antigen gene was amplified successfully antigen gene to biopsy, culture, and skin scraping with May–
from 31 H. capsulatum strains, showing 100% sensitivity. Grunwald–Giemsa staining revealed similar results between
Therefore, this alternative methodology appears to offer the the approaches, except that the PCR method detected
potential for improving the current methods for identification H. capsulatum DNA in two culture-negative blood samples.87
of this pathogenic mold, though it has mainly been applied to A colorimetric microtiter plate PCR–enzyme immunoas-
atypical isolates. Chemiluminescence-labeled DNA probes say (PCR-EIA) for the detection of H. capsulatum in urine
have been developed for the detection of specific sequences of showed positivity in 80% of urine specimens that were posi-
rRNA that have been able to detect and confirm all of the 41 tive by culture and no correlation was observed with anti-
H. capsulatum clinical isolates tested.80–81 Also, these probes genuria, limiting its use in the diagnosis of disseminated
could detect H. capsulatum directly on paraffin embedded histoplasmosis.88 Recently, real-time PCR has also shown a
tissue sections,81 such as excised heart valves from patients potential tool for the diagnosis of histoplasmosis.89–90
with Histoplasma endocarditis.80
A 100 kDa H. capsulatum protein (HcP100) has shown
promise as a diagnostic target using specific primers target- 33.2 Methods
ing the gene coding for HcP100. The method successfully
amplified the gene in 20 of 29 histopathologically positive
biopsy specimens, but, had no false-positive results.82 Using The application of molecular biology techniques to the anal-
whole blood, a nested PCR targeting a 210 bp specific seg- ysis of complex genomes, such as eukaryotic organisms,
ment of the HcP100 gene showed 89% sensitivity and 98% depends on the ability to prepare pure DNA. One of the most
specificity.83–84 A nested PCR based on the small-subunit essential steps is the extraction of adequately pure and suit-
(18S) rRNA gene of H. capsulatum has been tested in murine ably sized nucleic acids. Several in-house methods have been
models of histoplasmosis and compared to quantitative cul- described for DNA extraction from filamentous fungi and
tures.85 The nested PCR efficiently detected H. capsulatum yeast,91–94 and most of them involve disruption of the cells,
DNA in tissue and blood samples from infected animals, but and digestion with proteinase K followed by phenol and phe-
the assay also detected DNA of B. dermatitidis and P. brasil- nol/chlorophorm extractions.
iensis. Using the same biopsy specimens described above, the In this chapter, we describe conventional microbiologi-
nested PCR targeting the 18S rRNA gene was positive in 26 cal approaches and selected methods of DNA preparation
or 29 H. capsulatum infected tissues, but sequencing of the from H. capsulatum yeast phase that we have adapted from
PCR products revealed that only half were identical to the H. diverse techniques previously described in the specialized
capsulatum gene sequence and the assay was also positive in literature.93 For atypical strains, which are not able to convert
18 of negative control samples82 and nonspecific amplifica- to the yeast phase, the DNA should be extracted from myce-
tion of DNA from nonpathogenic fungal species had to be lia and/or spores by previously described methodologies.95
confirmed by sequencing. The detection of H. capsulatum All the work should be performed in a biosafety level two
DNA in choroidal lesions in histopathological sections from or three biological safety cabinet until cells are lysed. The
a patient with a chronic ocular histoplasmosis syndrome was methods described represent the state of the art at the present
also accomplished using another set of primers to the rRNA time in our laboratory and certainly will be changed as inno-
gene.86 Detection of fungal DNA in human formalin-fixed vations occur and newer procedures are developed.

Histoplasma 269
33.2.1.1 Growth of Histoplasma capsulatum proteinase K [0.5 mg/mL]) for 2 h at 55°C, and 16 h
(i) Grow H. capsulatum yeast phase in HAM’s F12 or BHI at 37°C.
agar plate, and incubate at 37°C until colonies appear in 5. Adjust 1.4 M NaCl with stock solution (140 μL of
order to isolate a single colony. (ii) Transfer a single colony 5M NaCl stock solution), and 1% hexadecyltrimeth-
to HAM’s F12 or YPD (1% yeast extract, 2% peptone, 1% ylammonium brominde (CTAB) (65 μL of 10%
dextrose) broth, and incubate at 37°C for 72–96 h. CTAB). Incubate 10 min at 65°C.
33.2.1.2 Extraction of Histoplasma 33.2.2 Detection Procedures

capsulatum DNA from Culture 33.2.2.1 P
CR for Detection and Identification
of Histoplasma capsulatum
1. Harvest 5 mL H. capsulatum yeast culture.
2. Centrifuge at least 2 mL of the culture 10 min at 1. Prepare a PCR mixture (25 μL) containing 100 ng
900 × g (3000 rpm) and discard supernatant. DNA, PCR buffer (10 mM Tris–HCl, 50 mM KCl),
3. Suspend cells in 500 μL TES buffer (100 mM Tris, 1.5 mM MgCl2, 200 μM deoxynucleoside triphos-
pH 8.0; 50 mM EDTA, 1% sodium dodecil sulphate phates, 2.5 U of Taq DNA polymerase, and 20 pmol
[SDS]). of each primer Msp1F (5′-ACA AGA GAC GAC
4. Add equal volume of glass or zirconium beads GGT AGC TTC ACG-3′) and Msp1R (5′-GCG TTG
0.5 mm. Vortex 2 min. GGG ATC AAG CGA TGA GCC-3′).79
5. Add 275 μL of 7.0 M ammonium acetate. 2. Carry out the PCR amplification in a thermocycler
6. Incubate samples in tightly capped tubes 5 min at with a cycling program consisting of a denaturation
65°C, and 5 min on ice. step at 95°C for 5 min 35 cycles of 1 min at 95°C,
7. Extract samples with 500 μL of 25:24:1 phenol/ 1 min at 70°C and 1 min at 72°C, followed by a sin-
chloroform/isoamyl alcohol. Vortex for 30 s. gle terminal extension at 72°C for 5 min.
Centrifuge 5 min at full speed (20,000 × g; 3. Include an internal PCR control to verify the effi-
14,000 rpm). Transfer top layer (aqueous phase) to a ciency of the test and to ensure that PCR inhibi-
new tube. tion is absent. The universal fungal primers ITS1
8. Add 1mL of isoprapanol. Incubate 5 min at room and ITS4, derived from highly conserved regions
temperature (RT). Centrifuge 10 min at full speed of the fungal rRNA gene, should be used for this
(20,000 × g; 14,000 rpm). purpose.96
9. Wash the pellet with 70% ethanol. Mix gen- 4. Separate the PCR product on a 1% agarose gel, and
tly several times. Centrifuge 5 min at full speed visualize with a UV transilluminator after ethidium
(20,000 × g; 14,000 rpm), air dry at RT, and suspend bromide (0.5 μg/mL) staining.
in 50 μL dH2O or TE buffer.
33.2.2.2 Nested PCR for Detection of Histoplasma
33.2.1.3 Extraction of Histoplasma capsulatum capsulatum from Clinical Samples
DNA on Blood Samples Bialek et al.82 developed a nested PCR assay targeting the gene
In order to perform a sensitive, specific, and reliable nucleic coding a 100 kDa-like protein described as being essential
acid-based diagnostic test, availability of pure DNA-lacking for the survival of H. capsulatum in human cells.97 The outer
inhibitors as well a rapid and easy-to-perform DNA extrac- primer set is Hc I (5′-GCG TTC CGA GCC TTC CAC CTC
tion protocol is essential. Protocols for extraction of DNA AAC-3′) and Hc II (5′-ATG TCC CAT CGG GCG CCG TGT
of fungal cells either are time consuming or show poor AGT-3′) and delimits a 391 bp sequence. The inner primer set
release of fungal DNA.9 In this procedure, prior to the DNA is Hc III (5′-GAG ATC TAG TCG CGG CCA GGT TCA-3′)
extraction with 25:24:1 phenol/chloroform/isoamyl alcohol, and Hc IV (5′-AGG AGA GAA CTG TAT CGG TGG CTT
the whole blood sample should be prepared as follow84: G-3′) and the nested PCR product is 210 bp long.82,84
1. Centrifuge 2.5–4 mL of fresh blood for 10 min at 1. For the first amplification prepare a PCR mixture
2.500 × g, 4°C. Do not freeze the blood samples. (25 μL) containing 2 μL of DNA, PCR buffer (10 mM
Better to use after 7 days at 4°C. Tris–HCl, 50 mM KCl), 2 mM MgCl2, 200 μM
2. Wash the cells pellet with TE buffer. Repeat this deoxynucleoside triphosphates, 1 U of Taq DNA
step. polymerase, 0.1% BSA Fraction V and 1 μM of each
3. Suspend cells in lysing solution I (6.5 μL lyticase; primer Hc I and Hc II. Perform the first PCR ampli-
83 μL lysing enzyme from Trichoderma harzianum fication using a cycling program consisting of 94°C
[6 μg/μL] in 230 μL sorbitol buffer). Incubate 2 h at for 5 min; 35 times at 94°C for 30 s, 65°C for 30 s, and
25°C. 72°C for 1 min; and then once at 72°C for 5 min.
4. Complete spheroplasting with lysing solution II 2. For the nested PCR the reaction mixture is identi-
(500 mM EDTA; 1% [w/v] N-laurylsarcosine; cal, except that 2 μL of the first PCR product, 1 mM

MgCl2, and 1 μM of each primer Hc III and Hc IV is 1. Prepare the master mixture (50 μL total volume)
used. The reaction mixture is thermally cycled once solution containing 1 × PCR buffer (10 mM Tris–
at 94°C for 5 min, 30 cycles of 30 s at 94°C, 30 s at HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM
67°C and 1 min at 72°C, and then once at 72°C for each of the dATP, dCTP, dGTP and dTTP (Roche
5 min. Diagnostics), 3 mM magnesium acetate, 30 ng
3. Separate the PCR product on a 2% agarose gel, and of primer, and 2.5 U Amplitaq DNA polymerase
visualize with a UV transilluminator after ethidium (Applied Biosystems). Add 25 ng of genomic DNA.
bromide (0.5 μg/mL) staining. 2. PCR is performed for 35 cycles in a thermalcycler
with 20 s of denaturation at 94°C, 1 min annealing at
33.2.2.3 Identification of Histoplasma 50°C, and 20 s extension at 72°C, followed by a final
capsulatum by Sequencing extension cycle for 6 min at 72°C.
The sequencing of internal transcribed spacer (ITS) regions 3. Amplification products were removed, concentrated
of rDNA with different primers have been chosen for iden- to approximately 20 μL and separated by electro-
tifying more precisely this species.6,37–38,98–99 The primers phoresis on 1.4% agarose gels (stained with ethid-
ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 ium bromide, 0.5 μg/mL final concentration) in 1×
(5′-TCCTCCGCTTATTGATATGC-3′) are the most used in Tris-borate-EDTA (TBE) buffer at 60 V for 14 cm,
our laboratory. and visualized under UV light.
4. All visualized bands on the gel were counted, inde-
1. Prepare a PCR mixture consisting of 10 mM pendent of their intensity, and data were scored
Tris–HCl buffer containing 50 mM KCl, pH 8.0, for the presence or absence of the amplified DNA
1.5 mM MgCl2, 0.2 mM deoxynucleoside triphos- bands.
phate (Invitrogen), and 1.25 U of Taq polymerase
(Invitrogen). Add primers ITS1 and ITS4 (final con- 33.2.2.4.2 PCR-RFLP (rRNA Gene)
centration of 0.2 mM each), and template DNA at a PCR-RFLP analysis of the ITS region of the rDNA gene
final concentration of 20 ng per 100 μL of the reac- reveals species-specific variations of restriction enzymes
tion mixture. (RE) cut sites.
2. Carry out an initial denaturation of template DNA
at 95°C for 5 min followed by 30 cycles of 30 s at 1. PCR mixture (50 μL) is composed of 50 ng of
95°C, 30 s at 58°C, and 1 min at 72°C. A final exten- DNA, 1 × PCR buffer (10 mM Tris–HCl pH 8.3,
sion step is conducted for 10 min at 72°C. 50 mM KCl, 1.5 mM MgCl2)(Applied Biosystems),
3. Separate the PCR product on a 1% agarose gel, and 0.2 mM each of dATP, dCTP, dGTP, and dTTP
visualize with a UV transilluminator after ethidium (Roche Diagnostics), 3 mM magnesium ace-
bromide staining. tate, 1.5 U AmpliTaq DNA polymerase (Applied
4. Purify PCR product using QIAquick PCR Biosystems), and 50 ng of each primer SR6R
Purification Kit (Qiagen). (5′-AAGTARAAGTCGTAACAAGG-3′) and LR1
5. Prepare a sequencing mixture (10 μL) containing (5′-GGTTGGTTTCTTTTCCT-3′).
premix terminator (Applied Biosystems), 100 ng 2. PCR is performed in a thermalcycler with 1 cycle
purified PCR product, and 3.2 pM of primers ITS1 of 94°C for 2 min, 35 cycles of 45 s at 94°C, 1 min at
and ITS4. 61°C, and 2 min at 72°C followed by a final exten-
6. Direct sequencing of the PCR product is performed sion cycle of 10 min at 72°C.
with a sequencing kit using the ABI PRISM 3100 3. PCR products (20 μL) are incubated with Sau96I
sequencer (Applied Biosystems). (10 U/μL) and HhaI (20 U/μL) at 37°C overnight
7. Edit and format the DNA sequences and align, by and separated by 3% agarose gel electrophoresis at
sequencing alignment programs, with those from 100 V for 5 h after mixing with one fifth volume of
other medically important dimorphic fungi that are loading buffer.
available at the GenBank.
33.2.2.4.3 Sequencing
33.2.2.4 Molecular Typing of Histoplasma The population structure of H. capsulatum by DNA
capsulatum sequences of partial gene coding proteins arf, H, ole, tub,
Muniz developed a typing system composed of three meth-
100 and ITS regions of rDNA has been used for phylogenetic
odologies, M13 DNA fingerprinting, PCR-RFLP, and sequenc- analyses.6,38
ing of four partial gene coding protein described below.
1. PCR is performed with 100 ng genomic DNA
33.2.2.4.1 M13 PCR Fingerprinting template in 50 μL reaction mixture, consisting of
The minisatellite-specific core sequence of the wild-type 0.45 μM of each primer, 1.0 U of AmpliTaq DNA
phage M13 (5′-GAGGGTGGCGGTTCT-3′)101 must be used polymerase (Perkin-Elmer), 10 mM Tris–HCl (pH
as a single primer. 8.3), 1.5 mM MgCl2, 50 mM KCl, and 0.2 mM

Histoplasma 271
deoxynucleotidetriphosphates. The primers (5′-3′) are the gold standard in diagnosis continues to be culture,
as follows: arf1 (AGAATATGG GGCAAAAAGGA′) although results are usually available after presumptive ther-
and arf2 (CGCAATTCATCTTCGTTGAG); apy has been initiated.
H - a n t i 3(C G CAGT CAC C T C CATAC TAT C )
and H-anti4 (GCGCCGACATTAACCC); ole3 References
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34 Lacazia
Dongyou Liu and Yi-Wei Tang
Contents
34.1 Introduction...................................................................................................................................................................... 275
34.1.3 Diagnosis.............................................................................................................................................................. 276
34.2 Methods............................................................................................................................................................................ 277
34.2.2.1 PCR Amplification of gp43 Gene.......................................................................................................... 277
34.2.2.2 Sequencing Analysis of L. loboi 18S SSU rRNA Gene........................................................................ 277
34.3 Conclusion........................................................................................................................................................................ 277
References.................................................................................................................................................................................. 278
34.1 Introduction morphology in infected tissues and its sequence homology

in 18S small-subunit (SSU) ribosomal RNA (rRNA) gene,
34.1.1 Classification, Morphology, and Biology chitin synthase 2, chitin synthase 4, ADP-ribosylation factor,
The genus Lacazia belongs to the mitosporic Onygenales and gp43 genes, L. loboi is considered as the phylogenetic
group (family), order Onygenales, class Eurotiomycetes, sister group to, but independent from, Paraccidioides brasil-
subphylum Pezizomycotina, phylum Ascomycota, and king- iensis, and is linked to the other dimorphic members of the
dom Fungi. Apart from Locazia, the mitosporic Onygenales mitosporic Onygenales group.5–7
group also includes the following genera: Blastomyces, Morphologically, Lacazia loboi in vivo phenotype con-
Chrysosporium, Coccidioides, Emmonsia, Geomyces, sists of unicellular, thick-walled yeast-like globose to sub-
Malbranchea, Myriodontium, and Paracoccidioides. The globose cells of 7.6–7.9 μm (range 5–12 μm) in diameter that
genus Lacazia consists of a single species Lacazia loboi occur singly as well as in branched and unbranched chains of
(formerly, Loboa loboi, Paracoccidioides loboi) that is three or more cells connected by short tubules. Constitutive
responsible for lacaziosis. melanin is present in yeast cell walls, which can be detected
Lacaziosis (also known as lobomycosis, Lobo’s disease, by the Fontana-Masson histologic stain. Old cells show thick-
keloidal blastomycosis, Amazonian blastomycosis, and blas- ened cell wall, leading to the separation of adjacent cells.8–10
tomycoid granuloma), which was first described by Jorge Lacazia loboi is an uncultivated fungal pathogen that
Lobo in 1930,1 is a chronic, granulomatous infection of the causes chronic, localized, subepidermal infection called
skin and subcutaneous tissues of humans and members of lacaziosis (lobomycosis) in humans and dolphins, occasion-
the family Delphinidae (e.g., bottle-nosed dolphins).2,3 A ally in the tropical areas of the Americas.11,12 This disease is
fungus related to Paracoccidioides brasiliensis was isolated characterized by the presence of keloidal, verrucoid, nodular
using Sabouraud glucose agar, which consisted of solitary, lesions (containing masses of spheroidal, yeast-like organ-
budding yeast cells and pseudohyphae in tissue. This etio- isms) or sometimes by vegetating crusty plaques and tumors,
logic agent was referred to as Blastomyces loboi in 1952 and on the face, ears, or extremities. First reported in Brazil in
as Loboa loboi (which is now a synonym of P. brasilien- 1930, lacaziosis appears to be restricted to the geographic
sis) in 1956. Another generic name Lobomyces was used to area of the Amazon (Brazil, Ecuador, Venezuela, Guyana,
describe the fungus in the tissue in 1968. Then, the species Suriname, Bolivia, Peru, and Colombia) as well as other
name Paracoccidioides loboi was proposed in 1971 for the South and Central American countries.13–16 Although several
etiologic agent of lobomycosis. Finally, the etiologic agent recent studies indicated its occurrence in Europe, Canada,
of lobomycosis was renamed as Lacazia loboi in 1999, with the United States, and South Africa, most patients had vis-
the generic name Lacazia acknowledging the major contri- ited areas of endemicity in Central or South America or had
butions to our understanding of lobomycosis by Carlos da handled infected dolphins.17,18
Silva Lacaz and the species name loboi honoring the origi- Lacaziosis has been documented in bottlenose dol-
nal discoverer Jorge Lobo.4 On the basis of its yeast-like phins (Tursiops truncatus) and Guiana dolphins (Sotalia
275

guianensis) along the Florida and Texas coasts, the South In another report, a 41-year-old female veterinarian from
Brazilian coast, the Suriname river estuary, and the Spanish– Brazil complained of a slowly growing, painless subcutane-
French coast since the 1970s.4,19–24 The infected dolphins ous nodule of 2.0 × 1.5 cm in diameter on the inner side of
show white to pink, verrucous lesions on the dorsal fin, head, her left-hand middle finger, which had appeared 10 months
flukes, and peduncle, which may ulcerate and form large earlier as a small hard cutaneous swelling. The patient had
plaques.25 worked extensively with the fungus L. loboi in experimen-
Being phylogenetically linked to other dimorphic fungal tally infected mice for 10 years, and her left-hand middle
pathogens in the mitosporic Onygenales group, L. loboi may finger had been extensively used to manipulate biopsied
have a mycelial form in nature with high tropism for soil of tissues and to inoculated mice with live yeast-like cells.
damp wooded areas. Humans may become infected with the Histopathological examination of hematoxylin–eosin-
fungus through skin trauma (minor scratches or insect bites) stained sections of the excised nodule displayed a granulo-
that facilitates fungal establishment from environmental matous infiltrate of histiocytes and giant cells together with
propagules or through contact with propagules from diseased numerous uniform thick-walled yeast-like cells, either singly
hosts (humans, dolphins, and experimentally infected mice). or in chains, characteristic of L. loboi.29
Transmission of lobomycosis among Delphinidae may occur While lacaziosis in humans is associated with a partial
by direct contact including transmission from mother to calf. deficit of cell-mediated immunity that is responsible for the
lack of containment of the pathogen and no alterations of
humoral immunity, the disease in dolphins shows a substan-
tial decrease in CD4+ helper T lymphocytes and CD19+ and
Lacaziosis is a chronic cutaneous and subcutaneous disease CD21+ B cells.30,31
of humans and dolphins, such as marine dolphins (Tursiops
truncatus) and marine-freshwater dolphins (Sotalia fluviati-
34.1.3 Diagnosis
lis), that is characterized by the development of nodular para-
keloidal lesions (solitary or multiple) on the ears, face, arms, As Lacazia loboi has been uncultivable, laboratory diagnosis
and legs in humans and head, back, dorsal fin, flanks, caudal of locaziosis (Lobo’s disease) relies on microscopic examina-
peduncle, and tail in dolphins. Being well defined, smooth, tion of the infected cutaneous and subcutaneous tissue biop-
shiny, and painless, developing lesions may be easily moved sies to show yeast-like cells of 5–12 μm in diameter, often
around as they lie free over the deeper tissues. Older lesions forming long chains of spherical cells interconnected by
may be macular, gummatous, verrucoid, or ulcerative with tubules. Differentiation diagnosis for Lacazia loboi includes
satellite lesions resulting from autoinoculation. Paracoccidiodes brasiliensis or Blastomyces dermatitidis
Lacaziosis predominantly affects males in close contact that shares similar morphological features.
with vegetation and aquatic environments. Many patients Microscopically, Lacazia loboi nodules are located
with lacaziosis recalled accidental trauma with plant thorns between the skin and subcutaneous tissue and consist of
or insect bites prior to onset of the lesions. Although lacazio- subepidermal histiocytic granulomas, with fibrous tissue dis-
sis is generally regarded as a slowly evolving disease, some persing between large numbers of multinucleated giant cells
variations in its incubation period have been observed. For (of 40–80 μm in diameter) and histiocytes. Special fungal
example, after handling an L. loboi-infected dolphin, an stains such as Grocott’s methenamine silver (GMS)32 and
aquarium caretaker developed the disease 3 months later, periodic acid-Schiff (PAS) are used for the histopathological
while man acquired the infection 2½ years after his trip to the diagnosis. Under GMS staining, L. lobio yeast-like cells are
endemic area. In experimental animal model, BALB/c mice dark or have the appearance of “empty” cells. The yeast-like
showed extensive granulomatous infiltrate and macroscopic cells are in chains of two or more cells and from branches,
lesions 7–8 months after inoculation at hind foot pads with similar to those yeast cells observed in the infected tissue of
L. loboi cells obtained from patients with the disease. On patients with paracoccidioidomycosis. The cells are typically
continuous passages from mice to mice, lesions developed connected with small tubules, a feature also observed using
within 4 months of L. loboi inoculation.26,27 other fungal stains including PAS. Alternatively, cutaneous
In a recent case of human locaziosis, a 62-year-old fisher- and subcutaneous tissue is mounted in 10% KOH and exam-
man from Venezuela presented extensive lesions on the left ined for the presence of chains of globose cells.33
ear. The patient recalled that his illness began after he acci- Compared to Paracoccidioides brasiliensis, which dis-
dentally injured the posterior portion of the helix of that ear plays expanding globose to subglobose solitary yeast cells
with a fishhook 10 years earlier. Initially, a small, solitary, with multiple daughter blastoconidia, L. loboi forms yeast
and hard nodule appeared, which was followed by similar cells of consistent size, generating branching chains of blas-
satellite lesions with confluent and harder nodules with flat toconidia. In addition, while P. brasiliensis is a geophilic
and shiny surfaces, causing occasional pruritus. Silver- dimorphic fungus that is associated with tegumentary sur-
stained sections of the parakeloidal lesions revealed single, faces, acquired through inhalation and disseminated from
uniform yeast-like cells as well as branched and unbranched lungs to other organs by hematogenous means, L. loboi only
chains of three or more cells connected by short tubules, infects cutaneous–subcutaneous tissue, especially the skin of
typical of L. loboi organism.28 ears, elbows, the anterior aspect of legs, and ankles, with a

Lacazia 277
lower body temperature, due to its apparent intolerance of Lacazia loboi in case of concomitant lobomycosis and para-
37°C temperature (refer to Chapter 40 for further details). coccidioidomycosis.5 For in vivo propagation, several BALB/c
Similarly, Blastomyces dermatitidis frequently infects the mice are inoculated in the footpads with L. loboi suspension.32
lungs as well as skin and genitourinary tract. In calcofluor For DNA extraction, L. loboi-infected skin tissue is ground
white or KOH-stained sputum, exudates or tissue, B. derma- under liquid nitrogen. The DNA from the ground tissues is
titidis shows large, spherical, and thick-walled yeast cells of then transferred to microcentrifuge tube, treated with sodium
8–15 μm in diameter, which bud singly and have wide base dodecyl sulfate, subjected to proteinase K digestion, and
attachment between the buds and parent cells (see Chapter 24 extracted with phenol and chloroform. The extracted L. loboi
for details). In addition, as both B. dermatitidis and P. brasil- genomic DNA is stored at −80°C until use.46
iensis grow in vitro and L. loboi does not, a useful way to
distinguish B. dermatitidis and P. brasiliensis from L. loboi
is to inoculate them on Sabouraud agar.5 34.2.2 Detection Procedures
L. loboi yeast cells can be extracted from biopsies of lobo- 34.2.2.1 PCR Amplification of gp43 Gene
mycosis skin lesions with the help of proteolytic enzyme dis-
Vilela et al. designed four primers from three of the highly con-
pase, which is known for its action against fibronectin and
served regions of the gp43 gene sequence of Paracoccidioides
collagen type IV.34 A number of studies have examined the
brasiliensis for detection and differentiation of Lacazia loboi
potential of immunological methods for diagnosis of loca-
and P. brasiliensis.46 The sequences of these primers are primer
ziosis.30,31,35–40 Using Western blot, an immunodominant
NL1 (5′-TGC TGG AGC CAT GGA TC-3′), primer NL2 (5′-
193 kDa Lacazia loboi antigen was detected in dolphin,
AAC GGC TTC GAC AAC AGC-3′), primer NL3 (5′-GCT
human, and mouse sera infected with lacaziosis.41 Because
GTT GTC GAA GCC GTT-3′), and primer NL4 (5′-TAG ATA
L. loboi does not grow in vitro, BALB/c and other rodent
CAT GGC GCA GTC-3′). While primers NL1–NL4 amplify
strains have been used for its maintenance.27,42–44 The viabil-
a 917 bp fragment within the 1329 bp gp43 molecule of P.
ity of Lacazia loboi may be assessed by means of fluorescein
brasiliensis, primers NL1–NL3 and NL2–NL4 amplify the
diacetate–ethidium bromide (FD-EB) staining.32
917 bp amplicon in two fragments of 431 and 486 bp, respec-
Molecular techniques have proven instrumental in clari-
tively. However, primers NL1–NL4 and NL1–NL3 do not
fying the taxonomic status of L. loboi.7,9,45,46 Sequencing
amplify the predicted 917 and 431 bp fragments, respectively,
analysis of L. loboi 18S SSU ribosomal RNA (SSU rRNA)
from L. loboi DNA, and only primers NL2 and NL4 generate
and partial chitin synthase-2 gene indicated that L. loboi
a 483 bp fragment from L. loboi DNA. This allows differentia-
is the sister taxon of the human dimorphic fungal patho-
tion of L. loboi from P. brasiliensis. The PCR protocol con-
gen Paracoccidioides brasiliensis within the mitosporic
sisted of an initial activation at 95°C for 10 min (for activation
Onygenales group in the order Onygenales.5,45 Further inves-
of Taq Gold polymerase) and 40 cycles of 1 min at 94°C, 2 min
tigation on rRNA internal transcribed spacer (ITS) and chitin
at 50°C, and 3 min at 70°C, followed by an extension at 72°C
synthase 4, ADP-ribosylation factor, and gp43 genes con-
for 7 min. The amplicons are separated on 1% agarose gels,
firmed that L. loboi is a well-supported, monophyletic group
stained with EB and visualized under UV light.
within the Paracoccidioides clade, but clearly distinct from
Paracoccidioides.7 Through comparative assessment of the
34.2.2.2 Sequencing Analysis of L. loboi
gene encoding the gp43 homologous protein in P. brasiliensis
and L. loboi, three sets of primers were developed for specific 18S SSU rRNA Gene
differentiation of these two closely related fungi.7,47 Current Herr et al. utilized forward primer NS1 (5′-GTA GTC ATA
MicroSeq D2 sequence database (Applied Biosystems) does TGC TTG TCT C-3′) and reverse primer NS8 (5′-TCC GCA
not cover L. loboi. GGT TCA CCW ACG GA-3′, W = A or T) to amplify the
18S SSU rRNA gene from L. loboi for phylogenetic analy-
sis.5 The resulting PCR amplicon (1768 bp) is purified and
34.2 Methods sequenced; and the sequence is analyzed using Parsimony
(version 4.0b.4a; D. L. Swofford, Illinois Natural History
34.2.1 Sample Preparation Survey, Champlain). Neighbor-joining analysis of the 18S
The clinical specimens collected in cases of lacaziosis are SSU rDNA sequences uses a maximum-likelihood multiple-
mostly biopsy tissues from the infected sites. Fine-needle hit correction with an empirical transition/transversion ratio,
aspiration has been shown recently as an effective alterna- empirical base frequencies, a gamma distribution of 0.5, and
tive to skin biopsy for collection of material for experimen- four categories of variation. Thousand bootstrap-resampled
tal infection of mice with unculturable pathogens such as data sets analyzed by both neighbor-joining and parsimony
Mycobacterium leprae and L. lobio.48 A portion of biopsied methods (heuristic) are used to assess branch support.
tissues with Lacazia loboi yeast cells is fixed in formalin, sec-
tioned and stained by hematoxylin and eosin, and GMSs for
34.3 Conclusion
microscopic examination. The remainder is used for culture, in
vivo propagation, and DNA extraction. Growth in Sabouraud Lacazia loboi (formerly Loboa loboi, Paracoccidioides loboi)
agar helps differentiate Paracoccidioides brasiliensis from is a fungal pathogen that causes a chronic, granulomatous

disease of the skin and subcutaneous tissues of humans and 12. Rodriguez-Toro, G. Lobomycosis. Int J Dermatol 32, 324–
dolphins, mainly in Central and South America. Due to its 332 (1993).
inability to grow in culture media and its morphological 13. Jaramillo, D., Cortes, A., Restrepo, A., Builes, M., and
Robledo, M. Lobomycosis. Report of the eighth Colombian
resemblance to Paracoccidioides and Blastomyces spp., the
case and review of the literature. J Cutan Pathol 3, 180–189
taxonomic status of this organism remained unsolved until (1976).
recently. Following the examination of its 18S SSU rRNA, 14. Paniz-Mondolfi, A.E., Reyes Jaimes, O., and Davila Jones,
chitin synthase 2, chitin synthase 4, ADP-ribosylation factor, L. Lobomycosis in Venezuela. Int J Dermatol 46, 180–185
and gp43 gene sequences, it is apparent that Lacazia loboi (2007).
is phylogenetically related to Paraccidioides brasiliensis 15. Ramos, E.S.M., Aguiar-Santos-Vilela, F., Cardoso-de-Brito,
but contains sufficient genetic difference to warrant its inde- A., and Coelho-Carneiro, S. Lobomycosis. Literature review
pendent species identity within the mitosporic Onygenales and future perspectives. Actas Dermosifiliogr 100(Suppl 1),
92–100 (2009).
group. This implies that as a dimorphic fungus, L. loboi may 16. Rodriguez-Toro, G. and Tellez, N. Lobomycosis in Colombian
go through a mycelial stage in nature as well as a yeast-like Amer Indian patients. Mycopathologia 120, 5–9 (1992).
stage in the host. In the mammalian hosts, L. loboi typically 17. Al-Daraji, W.I., Husain, E., and Robson, A. Lobomycosis in
produces parakeloidal lesions containing uniform yeast-like African patients. Br J Dermatol 159, 234–236 (2008).
cells, singly or in chains of three or more cells connected by 18. Burns, R.A., Roy, J.S., Woods, C., Padhye, A.A., and
short tubules. While the mycelial stage of L. loboi has yet Warnock, D.W. Report of the first human case of lobomycosis
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tool for lobomycosis. Mycoses 52, 187–189 (2009). lacaziosis and sera from experimentally infected mice for
34. Salgado, C.G. et al. Enzymatic isolation of Lacazia loboi cells Western Blot analyses of Lacazia loboi antigens. Clin Vaccine
from skin lesions of lobomycosis. Med Mycol 47, 119–123 Immunol 15, 164–167 (2008).
(2009). 42. Madeira, S., Opromolla, D.V., and Belone, A. Inoculation
35. Camargo, Z.P., Baruzzi, R.G., Maeda, S.M., and Floriano, of BALB/c mice with Lacazia loboi. Rev Inst Med Trop Sao
M.C. Antigenic relationship between Loboa loboi and Paulo 42, 239–243 (2000).
Paracoccidioides brasiliensis as shown by serological meth- 43. Opromolla, D.V., Madeira, S., Belone, A.F., and Vilani-Moreno,
ods. Med Mycol 36, 413–417 (1998). F.R. Jorge Lobo’s disease: Experimental inoculation in Swiss
36. Landman, G., Velludo, M.A., Lopes, J.A., and Mendes, E. mice. Rev Inst Med Trop Sao Paulo 41, 359–364 (1999).
Crossed-antigenicity between the etiologic agents of lobomy- 44. Opromolla, D.V. and Nogueira, M.E. Inoculation of Lacazia
cosis and paraccocidioidomycosis evidenced by an immuno- loboi into the subcutaneous tissue of the hamster cheek pouch.
enzymatic method (PAP). Allergol Immunopathol (Madr) 16, Rev Inst Med Trop Sao Paulo 42, 119–123 (2000).
215–218 (1988). 45. Vilela, R., Martins, J.E., Pereira, C.N., Melo, N., and
37. Taborda, C.P. and Camargo, Z.P. Diagnosis of paracoccidi- Mendoza, L. Molecular study of archival fungal strains iso-
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using a purified and specific antigen-gp43. J Med Vet Mycol 50, 470–474 (2007).
31, 155–160 (1993). 46. Vilela, R. et al. Molecular model for studying the uncultivated
38. Taborda, C.P. and Camargo, Z.P. Diagnosis of paracoc- fungal pathogen Lacazia loboi. J Clin Microbiol 43, 3657–
cidioidomycosis by dot immunobinding assay for antibody 3661 (2005).
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Microbiol 32, 554–556 (1994). Paracoccidioides brasiliensis: Immunochemical reactions with
39. Taborda, C.P., Juliano, M.A., Puccia, R., Franco, M., sera from patients with paracoccidioidomycosis, histoplasmosis,
and Travassos, L.R. Mapping of the T-cell epitope in the or Jorge Lobo’s disease. J Clin Microbiol 29, 1610–1615 (1991).
major 43-kilodalton glycoprotein of Paracoccidioides brasil- 48. Rosa, P.S., Belone, A.D., Lauris, J.R., and Soares, C.T. Fine-
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(1998). leprae and Lacazia loboi. Int J Infect Dis 14, e49–e53 (2010).

35 Lecythophora
Dongyou Liu
Contents
35.1 Introduction...................................................................................................................................................................... 281
35.1.2.1 Lecythophora hoffmannii (Phialophora hoffmannii)........................................................................... 281
35.1.2.2 Lecythophora mutabilis (Phialophora mutabilis)................................................................................. 282
35.1.3 Diagnosis.............................................................................................................................................................. 282
35.2 Methods............................................................................................................................................................................ 282
35.2.2.1 Real-Time PCR and Sequencing Analysis of ITS Regions................................................................... 282
35.2.2.2 Sequencing Analysis of D1/D2 Domains of LSU rRNA Gene............................................................. 283
35.2.2.3 PCR–Restriction Fragment Length Polymorphism............................................................................... 283
35.3 Conclusion........................................................................................................................................................................ 284
References.................................................................................................................................................................................. 284
35.1 Introduction 35.1.2 Clinical Features

35.1.1 Classification and Morphology Of the six recognized dematiaceous fungal species in the
genus Lecythophora, two (Lecythophora hoffmannii and
The genus Lecythophora is a dematiaceous (dark-walled) Lecythophora mutabilis) have been implicated in human
fungus belonging to the mitosporic Coniochaetaceae phaeohyphomycosis, leading to abscess, sinusitis, mastoid-
group, family Coniochaetaceae, order Coniochaetales, itis, endocarditis, endophthalmitis, peritonitis, and invasive
class Sordariomycetes, subphylum Pezizomycotina, phy- disease [3–6].
lum Ascomycota, and kingdom Fungi. As the only mem-
ber of the mitosporic Coniochaetaceae group, the genus
Lecythophora currently consists of six recognized species, 35.1.2.1 L ecythophora hoffmannii
that is, Lecythophora decumbens, Lecythophora fascicu- (Phialophora hoffmannii)
lata, Lecythophora hoffmannii (teleomorph: Coniochaeta Rinaldi et al. [7] reported a case of L. hoffmannii (P. hoffman-
ligniaria; obsolete synonyms: Margarinomyces hoffmannii) infection causing a gluteal abscess after multiple intra-
nii and Phialophora hoffmannii), Lecythophora lignicola, muscular injections of antimicrobial agents. Treatment of the
Lecythophora luteoviridis, and Lecythophora mutabilis (obso- purulent exudate and paraffin block sections of the exudate
lete synonyms: Margarinomyces mutabilis and Phialophora with an N-acetyl cysteine–NaOH digestion–decontamina-
mutabilis), in addition to 25 unassigned species [1]. Of these, tion procedure demonstrated hyphal elements. Culture of the
Lecythophora hoffmannii and Lecythophora mutabilis have abscess material yielded a pure growth of the mold L. hoff-
been associated with human phaeohyphomycosis. mannii (P. hoffmannii). Marriott et al. [8] described a rare
Lecythophora hoffmannii colonies are flat, smooth, moist, case of simultaneous infections with Lecythophora hoffman-
and pink to orange, with regular and sharp margin; reverse is nii (causing chronic sinusitis) and Scytalidium dimidiatum
pink. Hyphae are narrow and hyaline, producing conidia lat- (causing skin lesions, lymphangitis, and lymphadenitis) in a
erally from small collarettes directly on the hyphae or from 44-year-old human immunodeficiency virus-infected man.
lateral cells sometimes arranged in dense groups; lateral cells Slide cultures of sinus aspirates yielded pink colonies with a
are flask shaped or nearly cylindrical. Collarettes (1.5 μm tan reverse on Sabouraud’s dextrose agar. On potato dextrose
wide) are unpigmented. Conidia (3.0–3.5 × 1.5–2.5 μm) are agar, colonies became darker olivaceous in color with age.
hyaline, smooth, thin walled, slightly curved, and broadly Microscopy showed packed strands of hyphae with simple,
ellipsoidal to cylindrical or allantoid and tend to aggregate smooth, hyaline conidiophores. Phialides were flask shaped to
in clusters at the tip of the phialides. Lecythophora mutabilis cylindrical, single, and without collarettes. Conidia were hya-
conidia form from intercalary cells along the hyphae [2]. line, smooth, and slightly curved (0.6–2.3 μm × 2.2–6.0 mm)
281

and tended to aggregate in clusters at the tip of the phialides. targets include small-subunit (SSU) and large-subunit (LSU)
The fungus was identified as L. hoffmannii. Chang et al. [9] rRNA genes, internal transcribed spacer (ITS) regions, and
also documented an extremely rare fungal mastoiditis due other genes [15–19].
to Lecythophora hoffmannii in an immunocompetent host.
Successful treatment involved a combination of radical sur-
gical removal of all apparent infected tissue along with local 35.2 Methods
treatments with polyhexamethylene biguanide, a common 35.2.1 Sample Preparation
swimming pool biocide agent.
Clinical specimens (including smears and tissue sections) are
35.1.2.2 L ecythophora mutabilis (Phialophora stained by hematoxylin and eosin, methenamine silver stain,
mutabilis) or other fungal stains for microscopic examination. Portions
of samples are inoculated on Sabouraud’s dextrose agar and
Slifkin and Bowers [10] described a case of fungal endo-
other mycological media. Slide cultures of resulting isolates
carditis due to vegetative growth of L. mutabilis (P. muta-
are prepared on potato dextrose agar, cornmeal agar, and 2%
bilis) on a prosthetic mitral valve in a patient with rheumatic
agar for improved identification to the species level [8].
heart disease. The patient developed congestive failure, and
Fungal strains are cultured in 20 mL of RPMI 1640
a large matted obstructive fungal vegetation was found on
medium with l-glutamine but without sodium bicarbonate
the prosthesis. Culture of this vegetation grew L. mutabilis
(Sigma-Aldrich) buffered to pH 7.0 with 0.165 M morpho-
(P. mutabilis). Conidia form from intercalary cells along the
linepropanesulfonic acid (MOPS) (Sigma-Aldrich). After
hyphae. In a separate study, Drees et al. [11] described a case
3–14 days of growth at 30°C under agitation (100 rpm), the
of Lecythophora mutabilis prosthetic valve endocarditis in a
mycelium is transferred into a tube and washed in 40 mL
diabetic patient, who was treated surgically and with ampho-
of sterile distilled water. Mycelium is then stored at −20°C
tericin B lipid complex and voriconazole.
until use. Hundred milligram of mycelium is homogenized
Scott et al. [12] reported delayed-onset, bleb-associated
for 1 min in a tube containing 1 mL of lysis buffer (2%
endophthalmitis caused by Lecythophora mutabilis in a
Triton X-100, 1% sodium dodecyl sulfate, 10 mM Tris–HCl
57-year-old woman who underwent trabeculectomy with
pH 8, 100 mM NaCl, and 1 mM EDTA pH 8), three 0.5-cm-
Mitomycin C in her left eye. The patient presented with a
diameter glass beads (Sigma), and approximately 500 mg of
10 day history of pain and decreased vision in her left eye.
425–600 μm glass beads (Sigma). The homogenized mycelia
Cultures of scraping sample yielded L. mutabilis. After treat-
are then snap-frozen in liquid nitrogen, thawed, and refrozen
ment with intraocular and systemic antifungal agents, three
once. DNA extraction is then performed with the DNeasy
pars plana vitrectomies, partial iridectomy, and cryopexy to
plant kit (QIAGEN) [18].
the bleb and angle structures, her left eye showed no residual
Alternatively, 50 mg of fungal elements is suspended in
infection, and vision corrected to 20/80.
600 μL extraction buffer (200 mM Tris–HCl, pH 7.5, 25 mM
Taniguchi et al. [13] documented an invasive fungal infec-
EDTA, 0.5% w/v sodium dodecyl sulfate, and 250 mM NaCl).
tion (IFI) due to Lecythophora mutabilis in an 18-year-old
The mixture is vortexed for 15 s, incubated at 100°C for 15 min,
man with mitochondrial encephalomyopathy accompanied
kept on ice for 60 min, and then centrifuged at 14,000 × g for
with refractory anemia and chronic renal failure. The patient
15 min. Supernatants are transferred to new tubes and extracted
developed septic shock, and L. mutabilis was detected from a
with phenol–chloroform–isoamyl alcohol (25:24:1 v/v). Each
blood culture and was identified morphologically and geneti-
sample DNA is precipitated with cold isopropanol (−20°C),
cally. Despite treatment with micafungin and liposomal
dried, and resuspended in 100 μL distilled water [16].
amphotericin B, the patient succumbed to the infection.

35.1.3 Diagnosis
35.2.2.1 Real-Time PCR and Sequencing
Lecythophora is one of the 70 dematiaceous (melanized) Analysis of ITS Regions
fungal genera that are opportunistic human pathogens caus-
ing phaeohyphomycosis, chromoblastomycosis, and eumyce-
DNA-binding dye and amplicon-melting temperature anal-
toma in humans. Because of the morphological, ecological,
ysis for fungal detection using pan-fungal primers ITS1
and biochemical diversity of the dark-walled fungi, effective
treatment and control of these organisms are reliant on their
accurate identification and speciation.
of the fungi is verified by subsequent sequencing analysis.
Given the lengthy periods it takes to grow dematiaceous
fungi and the technical expertise it demands to speciate them Procedure
based on macroscopic and microscopic characteristics [14],
molecular methods such as PCR and sequencing have been 1. PCR mixture is composed of 1 × Lightcycler
developed and applied for their rapid, accurate, and sensitive FastStart DNA Master Hybridization Probes
detection and identification. The most common molecular mixture (Roche Applied Science) containing

Lecythophora 283
deoxynucleoside triphosphates, FastStart Taq DNA 3. The amplified products are purified and sub-
polymerase, and 1 mM MgCl2 (additional MgCl2 is jected to direct sequencing with an ABI Prism
added to a final concentration of 4.6 mM), 0.4 μM 3100 sequencer after labeling with BigDye™
each of ITS1 forward and ITS4 reverse primers, Terminator Cycle Sequencing Ready Reaction
1 × SYBR green (Molecular Probes), and 3 μL tem- (Applied Biosystems). The external primers, NL-1
plate DNA. and NL-4m, and the internal primers, NL-2m,
2. Thermal cycling parameters include 95°C for 5′-CTTGTGCGCTATCGGTCTC-3′, and NL-3m,
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and 5′-GAGACCGATAGCGCACAAG-3′, are used to
76°C for 30 s; and a final extension at 72°C for 2 min. sequence each DNA sample.
3. The quality of the amplicon is determined using the 4. The sequence data are aligned with CLUSTAL W
derivative of the melt analysis curve (55°C–99°C, (version 1.6). Phylogenetic trees are constructed
45 s hold at 55°C, and 5 s/°C) using the RotorGene with the neighbor-joining (NJ) method.
3000 (Corbett Robotics, Inc).
4. The amplified product is purified for bidirectional Note. The sequences of D1/D2 domains of the medically
sequencing using ExoSAP-IT (USB Corp). Five important species are highly conserved. For the spe-
microliter of Big Dye Terminator Ready Reaction cies in which the number of nucleotide differences is less
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of than three, the sequences of D1/D2 domains are consid-
each primer (0.8 pmol/μL) and 3 μL of purified PCR ered to be insufficient criteria to identify confidently each
product. Cycle sequencing is performed with a 9700 species.
reaction products are passed through a Sephadex 35.2.2.3 P CR–Restriction Fragment
G-50 fine column to remove unincorporated dye ter- Length Polymorphism
minators. Purified sequencing reaction products are Destino et al. [18] described the use of universal fungal prim-
run on an ABI Prism 3100 Genetic Analyzer with a ers V9D (5′-TTAAGTCCCTGCCCTTTGTA-3′) and LS266
50 cm capillary array. (5′-GTAGTCATATGCTTGTCTC-3′) for PCR amplification
5. Sequences are analyzed with the SmartGene and restriction fragment length polymorphism analysis for
Integrated Database Network software version identification of fungal organisms. In the case of negative
3.2.3 vr. SmartGene is a web-based software and amplification with V9D and LS266, other fungal universal
database system with reference sequences derived primers (e.g., ITS4/ITS5) may be employed.
(NCBI) GenBank repository. Procedure
PCR mixture (20 μL) is composed of 1 μL of genomic DNA,
Note. If real time PCR instrument is not available, standard
1.25 U of AmpliTaq gold (Roche), 2 μL of 10 × PCR buffer
PCR may be performed with primers ITS1 and ITS4, and
(Roche), 2 μL of 25 mM MgCl2, 2 μL of 2.5 mM deoxynu-
the resulting amplicon is sequenced with the same prim-
cleoside triphosphate, and 1 μL of each 10 μM concentrated
ers. Sequence-based identifications are defined by percent
primers.
identity: species, ≥99%; genus, 93%–99%; and inconclusive,
Amplification is conducted with a first cycle of 95°C for
≤93%.
10 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s,
and 72°C for 30 s, with a final extension of 72°C for 10 min.
35.2.2.2 Sequencing Analysis of D1/D2 PCR products are digested with endonuclease SmaI for 1 h
Domains of LSU rRNA Gene at 27°C. Restriction fragments are visualized on a 3% aga-
Abliz et al. [16] utilized primers NL-1, 5′-GCATATCAATA rose gel after ethidium bromide staining.
AGCGGAGGAAAAG-3′, and NL-4m, 5′-GGTCCGTGTTTC
Note. PCR products may be also purified and sequenced
AAGACG-3′, for amplification of the D1/D2 domains of the
by using the BigDye Terminator Cycle Sequencing Ready
LSU rRNA gene followed by sequencing for identification of
Reaction kit, version 3.1 (Applied Biosystems), with the
melanized fungi.
primers V9D and LS266. Reaction products are ana-
Procedure lyzed using an ABI Prism 3700 automated DNA analyzer
(Applied Biosystems). Sequences are then edited and manu-
1. PCR (50 μL) is made up of 5 μL of template DNA, ally corrected with Chromas, version 2.24 (Technelysium,
5 μL (2 pmol) each primer, 4 μL (2.5 mM) dNTP Helensvale, Queensland, Australia). Multiple-sequence
mixture, 0.25 μL (5 U/μL) Taq polymerase, and 5 μL alignment was carried out using ClustalW 1.8. Phylogenetic
10× reaction buffer. trees are constructed by the NJ method using the Phylip
2. Amplification is performed with 1 cycle of 95°C for package (http://www.infobiogen.fr) and visualized using
4 min; 30 cycles of 94°C for 1 min, 55°C for 2.5 min, Treeview. Pseudallescheria boydii is selected as the out-
and 72°C for 2.5 min; and 1 cycle of 72°C for 10 min. group (GenBank accession number AY228119).

35.3 Conclusion 9. Chang CY et al., Novel use of a swimming pool biocide in

the treatment of a rare fungal mastoiditis. Laryngoscope.
The genus Lecythophora contains six recognized dematia- 2005;115(6):1065–1069.
ceous fungal species, two of which (Lecythophora hoffman- 10. Slifkin M, Bowers HM Jr., Phialophora mutabilis endocardi-
nii and Lecythophora mutabilis) have been implicated in tis. Am J Clin Pathol. 1975;63(1):120–130.
human phaeohyphomycosis, resulting in a range of symp- 11. Drees M et al., Lecythophora mutabilis prosthetic
valve endocarditis in a diabetic patient. Med Mycol.
toms (e.g., abscess, sinusitis, mastoiditis, endocarditis, endo-
2007;45(5):463–467.
phthalmitis, peritonitis, and invasive disease). Considering 12. Scott IU et al., Delayed-onset, bleb-associated endophthal-
the time-consuming nature and difficulty of using conven- mitis caused by Lecythophora mutabilis. Am J Ophthalmol.
tional laboratory procedures for correct identification of 2004;137(3):583–585.
Lecythophora and other melanized fungi, it is critical that 13. Taniguchi Y et al., Septic shock induced by Lecythophora
molecular methods (e.g., PCR and sequencing) are adopted for mutabilis in a patient with mitochondrial encephalomyopathy.
their rapid, accurate, and sensitive detection and identification. J Med Microbiol. 2009;58:1255–1258.
14. Espinel-Ingroff A et al., Evaluation of the API 20C yeast iden-
tification system for the differentiation of some dematiaceous
References fungi. J Clin Microbiol. 1989;27(11):2565–2569.
1. The UniProt Consortium. Available at http://www.uniprot.org/, 15. Fell JW et al., Biodiversity and systematics of basidiomy-
accessed on August 1, 2010. cetous yeasts as determined by large-subunit rDNA D1/
2. Rippon JW, Medical Mycology, 3rd edn. W.B. Saunders Co., D2 domain sequence analysis. Int J Syst Evol Microbiol.
Philadelphia, PA, 1988. 2000;50:1351–1371.
3. Pierach CA et al., Phialophora mutabilis endocarditis. Ann 16. Abliz P et al. Identification of pathogenic dematiaceous fungi
Intern Med. 1973;79(6):900–901. and related taxa based on large subunit ribosomal DNA D1/D2
4. Ahmad S et al., Fungal peritonitis caused by Lecythophora domain sequence analysis. FEMS Immunol Med Microbiol.
mutabilis. J Clin Microbiol. 1985;22(2):182–186. 2004;40(1):41–49.
5. Marcus DM et al., Lecythophora mutabilis endophthal- 17. Bagyalakshmi R et al., Newer emerging pathogens of ocular
mitis after long-term corneal cyanoacrylate. Retina. non-sporulating molds (NSM) identified by polymerase chain
1999;19(4):351–353. reaction (PCR)-based DNA sequencing technique target-
6. Sakaeyama S et al., Lecythophora hoffmannii isolated ing internal transcribed spacer (ITS) region. Curr Eye Res.
from a case of canine osteomyelitis in Japan. Med Mycol. 2008;33(2):139–147.
2007;45(3):267–272. 18. Destino L et al., Severe osteomyelitis caused by
7. Rinaldi MG, McCoy EL, Winn DF. Gluteal abscess caused by Myceliophthora thermophila after a pitchfork injury. Ann Clin
Phialophora hoffmannii and review of the role of this organ- Microbiol Antimicrob. 2006;5:21.
ism in human mycoses. J Clin Microbiol. 1982;16:181–185. 19. Pounder JI, Discovering potential pathogens among fungi
8. Marriott DJ et al., Scytalidium dimidiatum and Lecythophora identified as nonsporulating molds. J Clin Microbiol.
hoffmannii: Unusual causes of fungal infections in a patient 2007;45(2):568–571.
with AIDS. J Clin Microbiol. 1997;35(11):2949–2952.

36 Microsporum*
Rahul Sharma and Yvonne Gräser
Contents
36.1 Introduction...................................................................................................................................................................... 285
36.1.1 Classification, Morphology, Biology, and Epidemiology..................................................................................... 287
36.1.1.1 Classification.......................................................................................................................................... 287
36.1.1.2 Morphology............................................................................................................................................ 288
36.1.1.3 Biology and Epidemiology..................................................................................................................... 289
36.1.2.2 Pathogenesis........................................................................................................................................... 290
36.1.3 Diagnosis.............................................................................................................................................................. 291
36.2 Methods............................................................................................................................................................................ 292
36.2.1.1 DNA Extraction from Fungal Colony.................................................................................................... 292
36.2.1.2 DNA Extraction from Clinical Specimen.............................................................................................. 293
36.2.2.1 Species Recognition............................................................................................................................... 293
36.2.2.2 Strain Typing.......................................................................................................................................... 293
Acknowledgments...................................................................................................................................................................... 295
References.................................................................................................................................................................................. 295
36.1 Introduction phytes—plants); the other two are Trichophyton Malmsten5

and Epidermophyton Sabouraud6 first described in 1845 and
More has been written about ringworm than any other
mycotic disease of animals and man and for no other myco-
1910, respectively. The name Microsporum refers to numer-
sis is the older literature more confused. ous micro-arthrospores covering formed during its (ectothrix
type) growth on hair surface unlike Trichophyton that grows
G.C. Ainsworth and P.K.C. Austwick, 19731 inside hair shaft.
The similarity among these dermatophytic genera extends
The above quote was written when polymerase chain reac- to their nutritional requirement, which is mainly keratin7 that
tion (PCR)-based molecular methods have not yet arrived forms exoskeleton covering in three groups of higher verte-
at the fungal taxonomic scene and especially for mycolo- brates, i.e., birds, reptiles, and mammals. Their keratinolytic
gists’ studying dermatophytes. The PCR technology as we nature itself suggests that they originated when life came to
know now was only available when Kary B. Mullis made land from water because aquatic vertebrates (e.g., whales)
a breakthrough of amplifying DNA in vitro,2 and 2 years lack exoskeleton adnexes like hair, feather, hooves, which are
later the use of thermostable polymerase3 made the whole primarily made of keratin and we don’t have Microsporum
process automated, which revolutionized biological inves- (or other dermatophyte), which is aquatic (marine) in nature
tigations. This single method has undoubtedly had more because it has been shown that growth of human pathogenic
application in biology today than any other technique in fungi (including dermatophyte M. gypseum) is inhibited by
whole of biological sciences including diagnosing human high salt concentrations.8 Also, rhexolytic dehiscence mech-
fungal infections. anism is found among fungi that have dry spores as in all
The genus Microsporum established by Gruby in 18434 Microsporum species (and not in mucilaginous mass as in
was the first genus to be described among the three genera Fusarium spp., which form blastospores). Among the three
commonly referred to as dermatophytes (dermal—skin; dermatophytic genera, Microsporum is well adapted to soil
* The authors dedicate this chapter to Prof. Libero Ajello, the man who knew so well the natural history of dermatophytes.
285

as (except a few that have newly evolved as anthropophilic dermatophytes. The gene(s) responsible for triggering or
species) its entire species produce macroconidia, which have inhibiting the production of macroconidia and the extent of
rough surfaces unlike their Trichophyton counterparts. this inactivation in anthropophiles is yet to be investigated.
The member of the genus can be grouped on the basis It is seen in agar or hair cultures of dermatophytes that the
of ecological preference as geophilic (saprophytes in soil), fungus forms spores once the nutrients are depleted (this is
zoophilic (adapted to lower animals), or anthropophilic true for the formation of sexual stages, i.e., sexual repro-
(adapted to human).9 These ecological groupings of various duction and formation of ascomata as in Takashio dilute
species are reflected in the phylogenetic clusters obtained by Sabouraud agar 20) and the phenomenon is not uncommon in
mtDNA10 and rDNA studies.11 This suggests that they all were wide range of fungi. Once a fungus gets human skin (which
previously soil dwellers and used to thrive on shed keratin. is still attached to human body), it can ideally grow over
Close or direct contact with live animals and later humans its whole body, but due to variable microenvironment and
made them well adapted to their zoophilic and anthropo- medical arrangements, the fungus is prevented from spread-
philic lifestyle, respectively. Also, the anthropophilic mem- ing and mostly controlled; however, severe cases are often
bers (M. ferrugineum and M. audouinii) must have evolved reported when no medical treatment is done or due to altered
along with Homo sapiens (modern human), which have microclimate (caused by drugs or reduced inherent immu-
sparse hairs on their body unlike its earlier hominid viz. nity). Human skin is like a continuous culture with regular
Australopithecus afarensis, Homo habilis or related primate supply of fresh keratin from beneath. The fungus anchors its
like ancestors. This hypothesis needs further investigation mycelia with the fresh integument by spreading radial in a
with right molecular marker and algorithm,12,13 which could ring-like fashion (hence, the popular term ringworm coined
precisely date and calibrate the divergence of M. canis into by Anglo-Saxon ancestors in sixteenth century),9 and thus
sibling species M. auduoinii in Africa and M. ferrugineum ensuring its survival on host.
in East Asia to superimpose precisely with the divergence Africa is thought to be the center of origin of Homo sapi-
of Homo sapiens from Africa to East Asia. One theory sug- ens based on the available evidence.21 Ancestral dermato-
gests that modern Homo sapiens developed relatively hair- phytes including young anthropophiles also might have their
less bodies to evade parasites14 which certainly would have origin in Africa.22 If this is true, then the separation of two
included dermatophytic fungi. For adaptation to this altered lineage to become distinct species of the M. canis clade after
trait of nakedness15 in humans, the evolving dermatophytes early humans moved “out of Africa” to Southeast Asia23,24
(geophiles > zoophiles > anthropophiles) that produce mac- is probable, thereby causing allopatric speciation25 resulting
roconidia for transmission, slowly lost their ability by natural into two closely related anthropophilic species M. auduoi-
selection since transmission became mainly through direct nii and M. ferrugineum. A recent trend of cryptic lineages26
contact and propagules like arthroconidia. Macroconidia is witnessed in the distribution of minus (−) mating type,
cling to hairs of animals for dispersal and host can be symp- which tend to be well adapted to human but a lineage origi-
tomless carriers like the cats,16 which is an indicator that nally occurring on animals.27 This assumption gains support
cat dwelling areas are survival grounds of M. canis. Cats’ from the study establishing first domestication of middle
loose hairs are shed in large numbers (an attribute familiar eastern wild cat by people of Israel 9500 years ago based
to cat owners), enabling the host to dislodge clinging mac- on archeological and historical records.28 Also, examination
roconidia and avert infection; however, the macroconidia of of DNA from 1000 wild cats and domestic cats across old
M. canis in particular is well adapted to zoophilic nature world showed all modern domestic cats descended from the
as its curved and roughened apex help attachment to ani- middle eastern wild cat Felis silvestris lytica.29 Could this
mal skin, and the overall shape resemble an arrow head be the time (10,000 years ago) when M. canis first seriously
that penetrate through the dense hairs to reach skin apart interacted with humans?
from being anti-grazing devise postulated by Summerbell.17 The cosmopolitan distribution of certain species of der-
Macroconidia are rare or absent in present anthropophiles matophytic fungi (not associated with birds) was greatly
because the transmission also is hardly a problem for the facilitated by humans and the animals he transported (wild
fungus when the host are in plenty (considerable rise in and domestic) in recent past, i.e., within 5000 year BP. The
human population), and chances of not finding a suitable massive movement of human population during wars in more
host are rare because urban population recently reached 3.2 recent time (1–2k years) dispersed dermatophytic fungi,
billion (crossing the world rural population of 3.1 billion),18 which otherwise were restricted continently. Unlike certain
where more humans live per unit area, a condition favorable rust fungi, which infect huge number of host plants at a time
for anthropophilic adaptation and evolution. Therefore, the that could generate unimaginable spore inoculums and due to
need for dispersal spores adapted to transfer through furred small size and lighter spores have intercontinental dispersal
animals was gradually lost or greatly reduced, leaving the through air current,30 the dermatophytes have comparatively
production of resting spores resistant to adverse condi- large and heavy spores (due to which it can hardly reach the
tions like arthrospores (chlamydospores). These contagious turbulence layer)31 and are never able to develop such a huge
propagules are produced in large numbers and have shown spore inoculum since they infect meager number of hosts
successful adhesion to stratum corneum under experimental at a time to have intercontinental dispersal by themselves
conditions,19 thereby effectively transmitting anthropophilic and need animal or other aid for long-distance travel. Due

Microsporum 287
to preening habit and flight, dispersal by birds is limited to tool(s). Also, we incorporate a portion of morphological detail
fungal species that have specialized clinging structures like in the conventional method section to help non-taxonomist,
Ctenomyces serratus (that forms ctenoid appendages)32 whose parataxonomist, or even pure molecular taxonomist to appre-
intercontinental dispersal through birds might have enabled ciate the elaborate morphology that Microsporum species
regular gene flow among its populations across continents represent through their millions of years of evolutionary his-
and prevented allopatric speciation (the species is monotypic) tory, something Perkins felt for Neurospora.40 The principle
as noted for some of the M. fulvum strains infrequently iso- aim of our article is to help user accurately identify known
lated in Central Europe from sand-martin (Riparia riparia Microsporum species and able to recognize newer ones in
L.) whose winter migration is in East Africa (with 8000 km their future encounters with the genus. The article also dis-
migratory route).33 The species of dermatophytes and kerati- cusses the recent developments in the dermatophyte identifi-
nophilic fungi reported by Ajello and Padhye in 197434 from cation like the use of alternate locus to the ribosomal DNA
Galapagos Islands (Darwin fame) mostly are bird associated like the chitin synthase gene for rapid detection,41 use of mul-
and no Microsporum species was recorded. The lack of zoo- tiplex RT-PCR,42,43 oligonucleotide array44 along with the sta-
philic or geophilic dermatophyte species (either Microsporum tus of genome sequencing in two of the Microsporum species
or Trichophyton) pathogenic to humans is understandable viz. M. canis (a zoophile) and M. gypseum (a geophile) pre-
in terms of the relative lack of past human disturbance (or viously prioritized among five dermatophytes (include three
migration) in the Galapagos before Darwin arrived. Trichophytons—T. rubrum, T. tonsurans, and T. equinum) by
Since the inception of molecular methods in disease diag- the Dermatophyte Genome Steering Committee.45,46
nostics, dermatophyte detection, and species identification
of clinically relevant (almost all) Microsporum species has
36.1.1 C
lassification, Morphology,
become more accurate and precise in comparison to the less
consistent morphology-based methods. However, even today Biology, and Epidemiology
morphology is still important in providing preliminary data 36.1.1.1 Classification
for starting the molecular analysis for species identification. The genus Microsporum is a mitosporic fungus that multi-
In the absence of any supporting morphological details viz. plies by producing mitospores: macro- and microconidia
clinical symptoms, source of infection or even preliminary whose sexual state lies in ascomycete genus Arthroderma,
slides prepared from clinical specimen, molecular identifi- some species of which produces Chrysosporium and
cation becomes blind and sometimes time consuming; how- Trichophyton asexual stages.11,47,48 Table 36.1 lists all known
ever, the above morphological attributes, if checked, greatly teleomorphs, i.e., fungi having Microsporum asexual states
help apply molecular methods precisely. including those that lack sexual stage in their life cycle, along
For molecular identification of Microsporum species, sev- with their natural habitat and distribution, which has been
eral approaches are now available, which initially started with greatly altered due to human migration in the last century.
the use of DNA homology studies35 or mitochondrial DNA The taxonomic positioning of Microsporum as per current
analysis.10 Now, we have better approaches or loci available scheme of fungal classification49,50 and the reason thereof is
that can utilize even fraction of the genomic DNA to cor- given below, with example of Microsporum gypseum, the
rectly diagnose a Microsporum species. The present chapter first Microsporum species (in fact the first dermatophyte) to
provides account of the preferred method, which is conve- have complete genome sequenced.
nient in application by even small laboratories and diagnostic
facilities. Although, in recent times, excellent reviews have Superkingdom Eukarya True nucleus
been published36–38 that gives an overall view in itself of the Kingdom Eumycota True fungi
dermatophyte species definition, strain differentiation or the Subkingdom Dikarya Dikaryotic hyphae
molecular methods involved in their identification and men- Phylum Ascomycota Ascus as diagnostic character
tioning them all again will be repetition of the already avail- Subphylum Pezizomycotina Asci within fruit bodies enclosed
able text. However, we do discuss in some detail a method partially or completely
that is widely applied in population-based investigations, Class Eurotiomycetes Mostly heterogenous assemblage
i.e., microsatellite markers for strain typing.37 Microsatellite of taxa
markers are useful in identifying origin of infection (e.g., a Subclass Eurotiomycetidae Stroma absent, ascomata mostly
pet, cattle, or environment), endemic strain introduced into cleistothecial, often brightly
a new area, multiple strain infection, or even relapse or rein- colored, evanescent asci
Order Onygenales Ascomata from coiled initials,
fection, all of which can be detected if a good polymorphic
peridium loosely interwoven
marker is available. Recently developed microsatellite mark-
thick-walled hyphae
ers for two Microsporum species were able to detect poly-
Family Arthrodermataceae Rough-walled, dumbbell-shaped
morphisms first in an otherwise known clonally reproducing ossiform peridial hyphal cells,
species (M. canis) and in the second (M. persicolor), to be a ascospores-minute smooth-
common old species in India and not introduced recently.27,39 walled discoid, anamorph either
Here we present the practical approach of “how to” iden- Chrysosporium, Microsporum,
tify or detect Microsporum species using modern molecular or Trichophyton

Genus Microsporum Forms echinulate holothallic two macroconidia in Malt agar or Sabouraud agar supplemented
to multiseptate macroconidia with horse or human hair except in species in which they are
Species Microsporum Macroconidia in large clusters, seldom formed.
gypseum rather thin-walled, regularly There exist gross similarities among these fungal forms
verrucose, fusiform, 3–6(−8) in their macrospore morphology in that they are multi-
celled, 25–60 × 8.5–15 μm septate and have rhexolytic dehiscence mechanism of dis-
charge.51 The similarity in their macrospore morphology is
probably a result of adaptive evolutionary pressures for the
36.1.1.2 Morphology dermatophytic lifestyle, which is supported by their phylo-
Characteristic morphology of Microsporum is the holothallic genetic and morphological closeness to keratin degrading
macroconidia the species produce, which is mainly two to Chrysosporium spp. whose aleurioconidia are indistinguish-
multi-septate transmission propagules. The number of cells able from microspore morphology of Microsporum spp and
in a single macroconidium represents the same number of Trichophyton spp suggesting Chrysosporium to be ances-
sub-propagules, all of which can germinate to form indi- tral to both (macroconidia forming) Microsporum and
vidual thalli. Microsporum spp. mostly produces abundant Trichophyton.52
Table 36.1
Known Sexual States of Microsporum Species and Their Habitat/Host Preference
Teleomorph Anamorph Natural Prevalent Biosafety Reference Strain Sequence
S. No. (Sexual State) (Asexual State) Reservoir Host(s) Distribution Levela Number and Statusb Accession
1 Arthroderma Microsporum Soil — Brazil/Africa BSL1 CBS 967.68, ST AJ877220
borelli amazonicum
2 Arthroderma Microsporum Soil — Global BSL1 CBS228.58, AUT AJ970145
cajetanum cookei
3 Arthroderma Microsporum Soil — Global (seems — CBS101.83, HT, MT− AM000035
cookiellum anamorph to be)
4 Arthroderma Microsporum Soil — Africa (rare) BSL1 CBS364.81, HT, MT+ AJ970143
corniculatum anamorph
5 Arthroderma Microsporum Soil — Global BSL1 CBS287.55, T AJ000627
fulvum fulvum
6 Arthroderma Microsporum Soil/animal Fowl Global BSL2 CBS243.66, T, MT+ AJ000612
grubyi gallinae
7 Arthroderma Microsporum Soil — Global BSL1 CBS258.61, NT AJ970141
gypseum gypseum
8 Arthroderma Microsporum Soil — Global BSL1 CBS174.64, T AJ970153
incurvatum gypseum
9 Arthroderma Microsporum Animal Pig Global BSL2 CBS322.61, T, MT− AJ970149
obtusum nannum
10 Arthroderma otae Microsporum Animal Cat, dog, horse, Global BSL2 CBS496.86, ET, MT− AJ000619
canis human
11 Arthroderma Microsporum Soil — Global BSL1 CBS424.74,T,MT− AJ970146
racemosum racemosum
12 Arthroderma Microsporum Soil Vole, rat Global BSL2 CBS468.74, MT− AJ000615
persicolor persicolor
13 — Microsporum Human Human Africa BSL2 CBS545.93, −NT AJ000623
audouinii
14 — Microsporum Human Human Asia, Africa BSL2 CBS497.48, AUT AJ252336
ferrugenium
15 — Microsporum Soil — France and BSL2 CBS288.55, AUT AJ970148
praecox USA (rare)
Source: de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn., Centraalbureau voor Schimmelculturales, Utrecht, the Netherlands, 2000.
a BSL1, saprobes occupying non-vertebrate ecological niches; BSL2, species principally occupying non-vertebrate ecological niches, but with a relatively
pronounced ability to survive in vertebrate tissue.

b AUT, authentic strain; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, HT, holotype strain; MT, mating type; NT, neotype strain;
ST, syntype strain; T, type strain.

Microsporum 289
36.1.1.3 Biology and Epidemiology depends largely on hosts’ immunity, pathogen availability,
Microsporum is an asexual stage of the sexually reproducing and virulence along with favorable microenvironmental
ascomycete genus Arthroderma.47 The fungus is mostly het- conditions (e.g., traumatized skin—wet for long time as in
erothallic with two mating types plus (+) and minus (−) that swimming pools, sports person or soldiers during training
mates under favorable conditions in soil to produce ascomata with shoes on for long hours, or farmers or farm workers in
as in Arthroderma otae.53 as the phenomenon has been dem- developing world working barefoot) and most importantly
onstrated in laboratory on Takashio’s dilute Sabouraud agar reduced hygiene that brings all these conditions together.
and soil-keratin culture where compatible mating types fuse This is true for animals also, e.g., cats licking habit (at least
to form fertile ascomata, which bear innumerable asci and once a day) and avoidance of water allows it to avert derma-
ascospores (the product of meiosis),54 also referred as meio- tophyte spores to reach its skin surface that remain clinging
spores. Sexual reproduction is essential for genetic fitness in to its dense but loose body hairs and hence are mostly non-
an organism as it involves recombination and production of symptomatic carriers; inability to lick their face might be
newer genetic traits for better adaptability to the continuously the reason why cats have inconspicuous lesions around nose,
changing environment. Figure 36.1 illustrates a life cycle of eyes, and ears caused by M. canis.57 However, the habit is
sexual Microsporum species (Arthroderma sp.); only the uncommon in dogs, which have low rates of infection in pet
anamorphic stage is clinically active and acquires greater dogs58; however, this does not hold true in stray dogs (with
virulence27 to be able to infect humans. little hygiene and medical care) that have much higher rates
Infections by Microsporum species (and other dermato- of dermatophyte infection (personal observation of RS in
phytes) affect a considerable part of human body55 and popu- India), thus helping the fungus maintain itself in nature simi-
lation worldwide.56 The incidence of dermatophyte infection lar to the mechanism known for obligate plant pathogens like
1
Sexual Arthroderma
state (in soil)
7
2
T cap
T corp
6 3
T ped
4.5
+ –
4
5
(a)
(g) (f) (b)

Asexual Microsporum state (d)
(e)
(on host and in soil)
(c)
Figure 36.1 Life cycle of Arthroderma species (clockwise, not to scale) having Microsporum anamorph, which cause skin infections
in humans. The line separating the two states are crossed when keratin is in plenty or scarcity. 1: Ascoma (peridium) bearing thick-walled
dumble-shaped ascomatal hyphae with coiled appendages; 2: Evanescent asci and ascosores shed freely from ascoma after maturity; 3:
Ascospores germinating and colonizing hair in soil; 4a: Fungal mycelium with conidiophores bearing young (nonseptate) and mature (sep-
tate) macroconidia, b: Clavate sessile microconidia, c: Thick-walled arthroconidia, d: Typical racquet hypha, e: Enlarged single five-celled
macroconidium (four septate), f: Germinating macroconidium, g: Liberated microconidium; 4.5: Entry of Microsporum into human niche
directly or indirectly through contact with infecting (mitotic) propagule which cause three main clinical manifestations: Tinea capitis, Tinea
corporis, or Tinea pedis (first two due to adaptation to active dispersal of macroconidia by flight or through animal contact and third due to
passive dispersal by arthroconidia through direct ground contact of humans); 5: Approaching receptive hyphae of opposite mating type (+)
and (−); 6: Coiling of ascomatal initials; 7: Nascent asci formed after genetic recombination and meiosis.

the physiological races of wheat rusts (having alternate and time like wet humid skin. Humans get easily infected by zoo-
collateral hosts systems)59 for survival in absence of favored philic dermatophyte through contact with infected or nonin-
host, i.e., wheat crop. Birds are another example of hygiene fected symptomatic carrier animals57 that may cause severe
maintenance as their preening habit meant for removing symptoms in humans because naked human skin has fewer
parasites or unwanted objects along with application of oil barriers compared to animals skin (which is densely covered
coating on feathers60 prevents them from potential infections. with hair) and, in general, have high levels of immunity.
Also, their tough covering on feet makes it difficult for a der-
matophyte to colonize and digest, similar to tail hair of lions, 36.1.2 Clinical Features and Pathogenesis
which is also relatively unaffected compared to hairs of other
animals including human when subjected to Microsporum 36.1.2.1 Clinical Features
gypseum treatment.61 Birds also get rid of occasional clinging Clinical features caused by Microsporum apparently visible
of propagules onto their feather by flight strokes; however, on human host are mostly characteristic for a dermatophytic
weak or ill ones are prone to dermatophytic infections (since infection but not always as the name suggests in the form of
they can’t fly). rings. And thus, are commonly but inappropriately referred
Tinea capitis in pre-independent India was mostly found in to as ringworm (as no worm is involved) because of a ring-
Europeans, Jews, and Anglo-Indians as reported in an exten- like appearance of the advancing infection, which is due
sive (50,000 skin samples during 5 years) study of fungal to the radial growth of fungus on the skin (similar to their
infections in Calcutta62 possibly due to application of wide growth on artificial medium viz. Sabouraud Dextrose Agar
variety of hair oils viz. amla, almond, coconut, and mustard [SDA]).
by natives, a custom followed from ages by both males and Principally, clinical features of dermatophytes are recog-
females (a similar story in nature can be found among birds nized and named after the part of the human body infected
that have natural coating). Garg63 using a combination of bio- along with the word “tinea” that is being used since the time
chemical assay and transmission electron microscopy showed of first recorded reference of dermatophyte infection (around
the toxicity of certain hair oils used by Indians against four 30 AD).69 It was used as a generic name for the cloth moth
dermatophytes including M. canis and M. gypseum in which (Tinea sp.) that feeds on keratin of woolen garments, thus
Amla (Emblica officinalis) oil was most effective. making characteristic holes in them, whose resemblance to
Infections are more prevalent in persons with lower ring-like lesion caused by fungal infection on smooth skin
socioeconomic conditions but such cases are greatly under made Anglo-Saxon people to coin the term ringworm for
reported and unrecorded (reason for this is dermatophytes such fungal infection in sixteenth century.9 The term is now
are never life threatening and food is a priority for survival more popular for designating fungal infection types than the
than a mere skin infection), unlike in persons with good insect whose generic name is Tinea. The various types of der-
socioeconomic conditions and better hygiene (in such cases, matophyte infection or the clinical manifestations in which
90%–95% cases are recorded). On a community scale, this Microsporum spp. are involved (including other dermato-
holds good for developed and underdeveloped locations, cit- phytes) are Tinea capitis-infection of head, scalp, eyebrows,
ies, or even countries. Lack of basic microbiological knowl- and eyelashes (all Microsporum spp.); Tinea favosa infection
edge among common people is one of the foremost causes of of hair called crusty hair (M. gypseum, rare), Tinea barbae
increased incidence of dermatophytic infections worldwide. infection of beard (M. canis), and Tinea corporis infection of
This includes knowledge that environment is the potential body or smooth skin (M. audouinii, M. canis, or any derma-
reservoir of dermatophytes64; stray animals like cats and tophyte).70 All Microsporum species grow as ectothrix, i.e.,
dogs (with or without visible symptoms); garbage (which is they grow outside the hair shaft and form numerous arthro-
dumped indiscriminately in landfills areas, sewage (a big conidia surrounding it while destroying the cuticle.
source of shed keratin),65 etc. Also, a fact that a dermatophyte
takes a day or two to cause primary athlete’s foot infection in 36.1.2.2 Pathogenesis
a human being after adherence of infectious propagules (viz. Pathogenesis of keratinophilic fungi including dermato-
microconidia, macroconidia, chlamydospores) on human phytes is due to their ability (they possess from their early
skin or its adnexes,66,67 infections in other body parts may origin) to break down keratin,71 the second most abundant
take longer due to not so congenial microenvironment like polymeric molecule on earth after cellulose, by releasing
feet; not washing oneself for a day or two renders a person extraordinary amounts of enzymes often referred to as kera-
susceptible for infection if one is in contact with infectious tinases, which are in true sense proteases.72 As studies in
propagule(s).66 This condition can be with soldiers, hik- M. gypseum using radioactive tracer technique (35S cystine)
ers, mountaineers, slum dwellers, or all those persons liv- showed the breakdown of native keratin is by the process
ing in water scarcity areas (a considerable portion of human of sulphitolysis,71,73 and production of keratin digesting
population does not have enough water).68 Human infec- enzymes as noted in M. canis.74,75 The fungus mainly attack
tions are generally caused when they come in contact with keratinous material by surface erosion and radial penetra-
propagules(s), which are the overwintering structures formed tion by formation of boring hyphae (similar to appressoria
in the life cycle (Figure 36.1) of a dermatophyte and when or haustoria formed by plant pathogenic fungi to absorb
favorable environmental conditions are prolonged for some nutrients from host plants59) where most of the secreted

Microsporum 291
proteolytic enzymes are concentrated.76 Once the mycelia fragments by the brush) and can be brought to the labora-
formed from freshly germinated arthroconidia have pene- tory. The brush can then be pressed onto the SDA plate with
trated keratin substrate and had established infection, exten- cycloheximide.
sive radial colonization under skin follows, which is seen as Microscopic observation: KOH or NaOH mounts (10%–
various clinical manifestations like the characteristic rings 30%) plus 5% glycerol (to emulgate lipids) can be prepared
on smooth skin. directly from the clinical specimen, gently heated to observe
for any hyphae or propagules, the preparation can be appro-
priately stained with cotton blue to visualize fungal mate-
36.1.3 Diagnosis
rial. Fluorescence stain with Calcoflour,84 Kongo red or
Diagnosis is the key for all successful medical treatment Mycetfluo®79 can improve the visualization.
for all types of diseases including dermatophytic infection. Culture preparation on Sabouraud’s dextrose agar or
If diagnosis is correct, right medication can prevent fur- mycotic agar: Once microscopy has revealed the presence of
ther spread and agony to the patient. A dermatophytic fun- fungal material, a second half of the material can be planted
gal diagnosis is primarily important to successfully render aseptically onto SDA or Mycotic agar containing antibac-
appropriate therapy and eventually control of the disease. terials (usually gentamicine and/or chloramphenicol plus
Diagnosing in early days of infection allows for immediate cycloheximide).
antifungal treatment and possible control; however, in the Hair perforation tests: Using human or horse hair (ster-
absence of correct diagnosis, the treatment could be false, ilized by tindalization), hair perforation test85 can be per-
which might help the fungus to further establish infection formed, which involves placing the hair on glass beads
like in nail infections, which later becomes difficult to treat. kept inside a presterilized Petri dish containing little distil
Once infection is deep, the treatment takes long and relapses water. The fungus cultured in pure form can then be inocu-
can frequently occur. lated aseptically onto the hair and incubated for 1–2 weeks.
Dermatophytes can establish infection faster than we The fungus causing active degradation by means of boring
can imagine, i.e., even half day (T. tonsurans),67 which hyphae can be visualized by cotton blue mounts of the inocu-
prompt us to find faster and accurate diagnostic procedures. lated hair. The test is particularly useful in differentiating M.
Conventional culture-based techniques are time taking and canis (positive) from M. audouinii (negative).86 However, the
often do not always give positive results even if sample is horse variant of M. canis cannot be distinguished from M.
positive for dermatophytic infection.77 Currently, laboratory audouinii due to being hair-perforation negative for which
testing procedures are integrating the two techniques for bet- molecular methods are useful.27
ter diagnoses and understanding of dermatophytes diversity,
since culturing a new dermatophyte to reveal its morphology 36.1.3.2 Molecular Techniques
is essential in describing a novel species78 and future drug With molecular techniques available, a diagnosis of infec-
testing for control therapy. Further section describes briefly tions caused by Microsporum from a clinical sample or
the conventional techniques used in dermatophyte diagnoses material has become relatively rapid, exact, and authentic.
and molecular techniques in more detail. In a recent study published comparing the conventional
(microscopic examination and culture-based method) and
36.1.3.1 Conventional Techniques molecular methods to detect dermatophytes in nail and skin
Recently, an excellent review by Robert and Pihet79 has samples clearly showed the contrast in positive rates of PCR
appeared on conventional methods for diagnoses of dermato- versus culture (95%–99%—PCR versus culture—67% [skin]
phytoses apart from a more comprehensive earlier treatment and 33% [nail]) clearly in favor of PCR method based on
by Kane et al.80 that greatly complement the rapid molecular nuclear ribosomal internal transcribed spacer (ITS1) DNA
techniques. Some of the conventional techniques mentioned sequences.77
below can be applied as preliminary tests prior to performing However, morphological features are still useful like
molecular analysis, all of which starts with the right method clinical symptoms on host or colony appearance on artificial
of collecting clinical specimen, i.e., infected material from media (if culture is successful, which is when the samples are
an infected host.81 collected from growing portion of the lesion) or the presence
Wood’s lamp: It is used to visualize dermatophytic infec- of racquet hyphae in culture; all of which even makes the
tion (caused by M. canis and M. audouinii), seen as yellowish molecular identification more focused by excluding most of
green fluorescence under dark, however not all fluoresce. the non-dermatophytes from picture and suggesting the use
Hair brush technique: Although the method has been of species-specific primers (for M. canis87 and M. audoui-
devised for animals,82 it can be used for humans as well.83 nii88) or probes (for T. rubrum).89 If culture is successful, and
It includes use of a presterilized hair brush (having dense macroconidia are formed, then their surface feature indicates
arrangements of hair bristles) to brush the infected portion whether it is Microsporum (rough walled) or Trichophyton
of the hairy body (head of human or body of pet). The brush species (smooth walled).
can be placed in sterile, preferably paper bags (since poly- Currently, the technique(s) most commonly applied in rec-
thene bags tend to possess static charge, which attracts loose ognizing Microsporum species due to its simplicity (as it can
particles and hence can prevent retention of spores or skin be performed even by nonspecialists) and cost effectiveness

(can be routinely applied by a small diagnostic laboratory) can be collected in fresh paper envelops or sealed plastic bags
is—ITS-PCR-RFLP. ITS-PCR-sequencing is able to differ- and brought to the laboratory for processing. Material (skin
entiate also close-related species but a method that is not so or hair) can be collected from growing peripheral portion of
easy to perform and more expensive. Both techniques involve the lesions.
the targeted in vitro amplification of fungal pathogen DNA Most samples can be directly subjected to DNA extraction
(Microsporum species) in thermal cycler to enhance its vis- (preferably using a DNA extraction kit if the sample is less
ibility (on a gel matrix). The amplified DNA usually from a than 5–10 mg) but a part of the sample can be sent for culture
selected site of the genome (viz. best studied is the ITS region (and slide preparation) in appropriate laboratory (if host labo-
of the ribosomal DNA) can be cut with restriction enzyme(s) ratory lacks culturing facility) so that bulk DNA extraction
to reveal a not always species-specific profile using com- can be made, which could be stored for decades for future
parison with the reference strains. The drawback of RFLP is reference studies. Several commercial kits are available for
that close-related species like M. canis and M. ferrugineum extracting DNA from clinical sample like infected nail, hair,
are not differentiated and a large amount of PCR product is or skin scrapings; however, it depends on the user for prefer-
essential. A better alternative is sequencing of the ITS region. ring a particular kit in terms of performance and cost (since
Comparison with the reference sequences in the nucleotide kits tend to increase the cost of analysis).
databases (e.g., NCBI); better and recommended is the vali-
dated CBS database (http://www.cbs.knaw.nl) that helps in 36.2.1.1 DNA Extraction from Fungal Colony
identifying the causative fungal species. Other methods, The same kits used for clinical material can be applied when
which have now become obsolete, are the mtDNA and DNA– extracting DNA from a grown colony. In this case, colonies
DNA hybridization. However, many so-called “in house” not older than 1–2 weeks should be used for the DNA prepa-
PCR assays have been developed already, which mainly ration because pigmentation of the culture can cause problem
use nested PCR to increase the sensitivity in addition to a and inhibit the following PCR reaction. The cetyl trimethyl
species-specific gene probe, which hybridizes in liquid (PCR ammonium bromide (CTAB) method described briefly, is,
ELISA) or firm (blot) manner to the amplified and univer- however, not so expensive and yields a larger amount of
sal target gene.90–92 Although a few assays using real-time DNA. Mycelia were scraped from the surface of agar col-
PCR have been developed preventing contamination of the ony; also portions of colony are cut and placed inverted on
sample during the identification process, two out of three are the plain agar. The agar is then scraped off from the surface
only able to identify species groups.41,92–94 All of these assays till only the mycelial matt remained. Three to four of these
involve extra cost but were developed for use in routine diag- mycelial mats is taken in sterile pestle and grinded in liquid
nostic laboratories. Here we describe the method that is able nitrogen using a mortar. The finely grinded mycelial powder
to recognize almost all currently known Microsporum spe- is then added to 2 mL Eppendorf tube containing 1 mL of
cies (except anthropophilic ones that can be recognized by CTAB extraction buffer (100 mM Tris–HCl pH 8.0, 1.4 M
sequencing) based on the new species concept.36 The meth- NaCl, 20 mM EDTA, 2% cetylmethylammonium bromide,
ods described can be applied by most laboratories and espe- 2% β-mercaptoethanol). The suspension is then subjected to
cially sequencing is a tool to identify the causative agent of three rounds of freezing and thawing. Then, proteinase K
any fungal infection. is added to give a final concentration of 50 μg/mL and mix-
ture is incubated for 1.5 h at 60°C. After centrifugation at
13,000 × g for 5 min, one volume of phenol/chloroform/isol-
36.2 Methods amyl alcohol (25:24:1) is added to the supernatant and tube
is vortexed and centrifuged again for 15 min at 13,000 × g.
The upper aqueous layer is taken in a fresh tube and the
Direct molecular analysis of clinical sample(s) viz. hair, procedure repeated four to five times until the middle layer
skin, and nail for diagnosis of fungal infectious agents is clear and shows no further protein precipitation. Finally,
makes the detection of dermatophytic species rapid in con- one volume of chloroform was added to the supernatant
trast to culture-dependent approach, which at least takes from previous step, mixed and centrifuged at 13,000 × g for
a couple of weeks time. In direct approach, total genomic 15 min. Finally, DNA was precipitated with 1/10 volume of
DNA can be isolated from clinical sample using DNA sodium acetate (4.0 M, pH 5.2) and 1 mL of cold isopropanol.
extraction kits (e.g., Illustra Tissue & Cells GenomicPrep The content of tube is mixed by gentle inversion and incu-
Mini Spin Kit, GE Healthcare Life Sciences; QIAamp DNA bating overnight at −20°C. Next day, the precipitated DNA
isolation Kit, Quiagen); High Pure-PCR-Template prepa- is centrifuged at 10,000 × g for 10 min and pellet is washed
ration Kit, Roche), and pegGOLD Fungal DNA Mini kit, with 70% ethanol and air-dried. DNA is dissolved in 1× TE
Peqlab) and KOH. buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0) and treated
Preparation of sample(s) for molecular detection of with RNase with final concentration 100 μg/mL for 15 min
Microsporum does not require special care unlike systemic at 37°C. RNase was inactivated by 5 min incubation at 65°C
fungi; however, care must be taken while handling since cer- and the pure DNA was stored at 4°C. DNA concentration
tain animal origin strains are more virulent. Samples that was estimated by spectrophotometer reading at 280 and
include hair, nails, skin scrapings, and even floor dust or soil 260 nm, whose ratio gives the purity of DNA extracted, and

Microsporum 293
also physically checked by running genomic DNA on 0.8% 55°C for 1 min, 72°C for 1 min; a final extension 72°C for
agarose gel along with known concentration of λ DNA. 10 min. Five microliter PCR product is then checked on 1.2%
agarose gel for successful amplification.
36.2.1.2 DNA Extraction from Clinical Specimen
For extraction of DNA from clinical sample—hair, nail, or 36.2.2.1.2 Restriction Digestion (ITS-PCR-RFLP)
skin, 0.5–3 mg of material previously collected is cut into The resulting amplicon can be digested by MvaI restriction
small pieces (2 mm) and subjected to commercially available enzyme to give species-specific profile (Figure 36.2). For
kit extraction as per manufacturer’s instruction. restriction digestion, 20 μL of fresh PCR product (showing
Alternatively, homogenization of tissue for DNA extrac- single clear band on 1.2% agarose) is first transferred to
tion, clinical specimen can be placed in a 2 mL screw cap an Eppendorf tube. The DNA is precipitated by 1/10 vol-
tube containing CTAB buffer11 (100 mM Tris–HCl pH 8.0, ume of sodium acetate (3 M, pH 4.8–5.2) and 2.5 volume of
1.4 M NaCl, 20 mM EDTA, 2% cetyl methylammonium ethanol (95%) and incubated overnight at 4°C. Precipitated
bromide, 2% β-mercaptoethanol), sterile sand and a 5 mm DNA is centrifuged at 13,000 × g for 15 min and pellet
solid ceramic bead in an MPBio Fast Prep 24 homogenizer (mostly not visible) washed with ethanol (70%) and again
(Biomedicals GmbH, Germany). Further steps remain same centrifuged at 13,000 × g for 15 min. The pellet is dried
as in the previous section on extraction from colony. A very in vacuum centrifuge for 10 min at 30°C. The dried pellet
rapid method (1 h) with slight optimization can be readily is dissolved in 18 μL of PCR grade water with the help of
applied to bulk samples obtained by small laboratories or micropipette. 2 μL of 10× RE buffer (10 mM Tris–HCl pH
private clinics94 or the one by Brillowska-Dobrowska et al.,95 8.5, 10 mM MgCl2, 100 mM KCl, and 0.1 mg/mL BSA) is
which is mainly for nail but can be applied to other clinical added to the cleaned PCR product along with 5–10 U of
specimens as well. MvaI RE (Fermentas) before incubating for 4 h at 37°C.
The digested product is separated on 2% Metaphor agarose
and stained with ethidium bromide and photographed for
36.2.2 Detection Procedures documentation.
The results of characteristic species-specific pattern con-
36.2.2.1 Species Recognition
siderably depend on the success of digestion reaction. Partial
Identification of species of Microsporum using molecu- or incomplete digestion or even mutation (which is rare) at
lar means involves primarily the comparison of rDNA restriction site may give altered pattern. The method is most
sequences, some regions of which are highly conserved on suitable for routine bulk samples. Figure 36.2A shows the
one hand and variable on the other to reveal interspecies dif- MvaI restriction profile of selected species of Microsporum
ferences. To visualize the interspecific variations for diag- generated from nearly 1000 bp amplicon covering ITS1, 2
nosis, rDNA regions being multi-copy are first amplified by flanking the 5.8S region of rDNA.
PCR technique and then analyzed in one or the other ways
for species identification.
36.2.2.1.3 Sequencing of PCR Products
36.2.2.1.1 PCR Amplification Direct sequencing of PCR product is done by cycling
Once the genomic DNA is extracted in sufficient amount and sequencing kits using an automated sequencer (gel or
quantified, PCR can then be performed with several universal capillary based) or first cloning them in appropriate vec-
fungal primers, e.g., ITS4 (5′TCC TCC GCT TAT TGA TAT tor and then performing cycling sequencing reaction using
GC3′-LSU 69-50 of Saccharomyces cerevisiae) and ITS5 M13 universal primer. Direct sequencing of PCR product
(5′GGA AGT AAA AGT CGT AAC AA3′),96 or LSU26697 (obtained by primer pair V9D and LSU266) is done by per-
and V9D,98 which were used for Microsporum species to forming sequencing PCR with internal primers ITS4 and
amplify the ITS1, 5.8S, and ITS2 regions of rDNA giving ITS5.96
an amplicon of nearly 1000 bp. PCR conditions-50 μL reac-
36.2.2.1.4 Sequence Alignment and Identification
tion mixture contained 10 mM Tris–HCl, 50 mM KCl, 3 mM
MgCl2, 21 pmol each primer (V9D and LSU266), 50 μM Sequence obtained in the form of chromatogram from auto-
concentration of each deoxynucleotide triphosphate, 2.5 U mated sequencer has to be manually checked for inconsis-
of Taq polymerase, and 50 ng of template DNA. Appropriate tency and accordingly edited for accuracy. Edited sequences
controls viz. positive (containing template known to amplify can then be aligned with known (reference) sequences in the
in given conditions), negative (reaction mixture lacking any database using CBS database or NCBI-BLAST search to
template), and inhibition (reaction mixture containing 25 ng finally give identification results.
test template and 25 ng template previously known to amplify
under given conditions) are required. In case of non-amplifi- 36.2.2.2 Strain Typing
cation, even when there is no inhibition, 10% DMSO can be For epidemiological study including tracing the source of
added.99 The reaction mixture is finally overlaid with light infection (like pet) intra-specific markers are needed like
mineral oil. Amplification conditions in a thermo cycler con- microsatellites or randomly amplified polymorphic DNA
sist of 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 1 min, (RAPD). The choice of the former depends on the availability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1636 bp
1018 bp
506 bp
396 bp
344 bp
298 bp
220 bp
201 bp
154 bp
134 bp
75 bp
(A)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
M. Ferrugineum
M. Canis
M. Audouinii
140 bp
108 bp
G A A B C D E E E E E E E F
(B)
Figure 36.2 (A) Restriction digestion profile (MvaI) of reference strains (of seven Microsporum sp.) Microsporum gypseum var.
gypseum (CBS161.69) (lane 2), same strain as previous (lane 3), Arthroderma incurvatum (M. gypseum) (CBS173.64, MT+) (lane 4),
A. incurvatum (CBS 174.64, T) (lane 5), A. gypsea (CBS 258.61, NT) (lane 6), A. fulvum (CBS 167.64, MT+) (lane 7), M. fulvum (CBS
287.55, T) (lane 8), A. persicolor (CBS 468.74, MT–) (lane 9), A. persicolor (CBS 469.74, MT+) (lane 10), M. canis (CBS 132.88) (lane
15), A. borelli (M. amazonicum) (CBS 967.68, ST) (lane 17), A. cookiella (CBS 102.83) (lane 18), Epidermophyton floccosum (CBS
358.93) (lane 19). Test Microsporum species profile (lanes 11–13) corresponds to reference profile of M. persicolor, M. fulvum, and
M. gypseum (lane 10, 8, and 6 respectively). Abbreviations: CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands,
MT, mating type; NT, neotype strain; ST, syntype strain; T, type strain. (Reproduced from Sharma, R. et al., Med. Mycol., 46, 67, 2008.
With permission.) (B) Polyacrylamide gel (12%) showing seven different alleles among strains of M. canis (G-10, A-12, B-13, and C-17
repeats), M. ferrugenium (D-25 repeats), and M. audouinii (F-7 and E-9 repeats) analyzed by marker Mc(CA)13.
of already developed markers (e.g., M. canis and M. persi- marker (i.e., markers 300_27ga [For: 5′-GGCTTGAGTG
color),27,39 otherwise they have to be generated afresh in the GCGTCTTC-3′ Rev: 5′-AGCAAACGAACCGCTGAG-3′]
species under investigation by one of the several methods 196_23ca [For: 5′-TCGGCCTCCTCATCCTTC-3′ Rev:
available.100,101 With the availability of the genome project of 5′-TCGGGATGTAAGTAAAGG-3′] and 300_17ct
M. canis and M. gypseum, the design of such markers at least [For: 5′-GGGCAATTCTATGGGCAAG-3′3′ Rev:
for these two species became easy. 5′-CTTCTTCCAAGCTCTGCCTG-3′]) had maximum
38, 26, and 18 dinucleotide repeats, respectively. The high
36.2.2.2.1 Microsatellites number of multilocus genotypes (26) obtained in small
Microsatellites or simple sequence repeats (SSRs) are short population size (56 strains) suggested a fairly high level
stretches of di-, tri-, tetra-, penta-, or hexa-sequences repeated of recombination in the population and its presence in the
more than five times. The more the number of repetitions are region to be much older than its first isolation from India.39
present, the better the marker. The flanking regions of the mic- Misidentification of this species in earlier works on Indian
rosatellite repeats are conserved (where primer binds), while soils is probable because they all were morphology based
variations in the number of repeat results in polymorphisms and M. persicolor is known to be misinterpreted by morpho-
among individual of a population, which can be visually logical features.102,103 In zoophilic species M. canis, 11 mul-
observed on PAGE gel or fragment analyzer. In the geophilic tilocus genotypes were obtained with 2 microsatellite loci
species M. persicolor, Sharma et al.27 detected 26 genotypes Mc(GT)13 (forward: 5′-GATCGGAGCATGCCATACAG3′;
among 56 nonclinical strains analyzed, which were isolated reverse: 5′-TCTTCCCACCCTTCTCAATG-3′) and Mc(GT)17
from various soil locations in Central India viz. public places, (forward: 5′-GCTCTGGGATAAGGTGTTTG-3′; reverse:
hair salon, burrow, cattle shed, garbage, cave, and rat carcass. 5′-GTAGCAGTAAAGCCAAGAGGG-3′) among 101 global
The number of genotypes obtained with each of the three mic- strains analyzed.27 The markers were also able to differenti-
rosatellite marker (with 13, 9, and 5 genotypes) corresponded ate M. canis from close-related anthropophiles M. audoui-
with the increase in repeat length of the microsatellite nii and M. ferrugenium, which represented three additional

Microsporum 295
genotypes and thus can be differentiated by gel electrophore- to a highly specialized life style (like rusts in plants) that will
sis (Figure 36.2B). lead them to become future obligate parasites and their (evo-
lutionary) survival will last until its host species.
36.2.2.2.2 RAPD Markers, AP-PCR, and Apart from detecting fungal pathogens in clinical speci-
PCR-Fingerprinting mens, molecular methods have proved useful in more than
A number of techniques are available like AP-PCR (arbi- three ways for the study of dermatophytes, thus having direct
trary primed-PCR), PCR-fingerprinting, and RAPD, which implication on the clinicians’ knowledge of dermatophytes.
employ a single short primer to amplify fragments scattered First, in detecting species in location where they are unre-
on the chromosomes for strain typing and population genetic corded,39 second validating the taxonomic status of a closely
studies. DNA polymorphisms, in terms of different band pro- related or cryptic species114 or atypical isolates,115 and third
file, are obtained (presence or absence of band(s) that may in providing phylogenetic positioning of newly found spe-
be of variable intensities). The reliability of these methods cies78,116 for nomenclatural and taxonomic considerations. We
greatly depends on standard conditions used (including make now have numerous markers and methods with us like never
of thermal cycler); however, reproducibility is a problem104 before; non-detection of dermatophytes in clinical specimen
among laboratories. Although the methods are for assessing or the environmental samples becomes relatively rare, the
intraspecific variability, they are of little use in case of der- only impetus that remains is how faster we can diagnose their
matophytes that lack variability due to their recent adaptive presence for appropriate therapy. Although dermatophytes
radiation; and hence reveal only interspecific differences105,106 are not life threatening, they can influence the quality of life
unlike seen in other older fungal lineages. drastically.
36.3 C
onclusions and Acknowledgments
Future Perspectives
Drs. G.S. de Hoog and N.D. Sharma are greatly acknowl-
Diagnosing fungal pathogen is the first step in treatment of a edged for helpful discussions on dermatophytes and fungi
dermatophytic infection, which is one of the most common during several years of their association with Y.G. and R.S.
fungal infections worldwide infecting almost 20%–25% of respectively and Dr. S.N. Kulkarni, ARI Library, for access
population.107 Molecular methods have proved greatly ben- to current literature. R.S. thanks Department of Science and
eficial and have revolutionized detection of pathogens due Technology (DST), Govt. of India, for Fast Track grant SR/
to sensitivity of techniques involved (even in cases where FT/L-36/2005 supporting the work on keratinophilic fungi
symptoms are unapparent) and the rapidity in relation to the of national parks of Central India, which led to many con-
conventional techniques like hair perforation, mating com- cepts mentioned in this chapter and Deutscher Akademischer
petency tests, and even nutritional tests. Apart from the cur- Austausch Dienst (DAAD), Bonn, for research fellowship
rently available molecular methods, newer improved ones are that enabled collaboration between the two authors to study
still being regularly developed like oligonucleotide array44,108 and generate new information on Microsporum.
with increased rapidity95,109 mainly due to the reduced time
required for DNA extraction.95
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37 Myriodontium
Dongyou Liu
Contents
37.1 Introduction...................................................................................................................................................................... 299
37.1.3 Diagnosis.............................................................................................................................................................. 300
37.2 Methods............................................................................................................................................................................ 300
37.3 Conclusion........................................................................................................................................................................ 300
References.................................................................................................................................................................................. 301
37.1 Introduction 37.1.2 Clinical Features

37.1.1 Classification and Morphology Maran et al. [3] reported the first case of Myriodontium kera-
tinophilum infection in 53-year-old Nigerian man, who was
The genera Myriodontium is a mold belonging to the not immunosuppressed nor diabetic. The patient complained
mitosporic Onygenales group, order Onygenales, sub- of facial pain and underwent nasal polypectomy after diag-
class Eurotiomycetidae, class Eurotiomycetes, subphylum nosis of sinusitis secondary to nasal polyps. Polypectomy
Pezizomycotina, phylum Ascomycota, and kingdom Fungi. was repeated three times during the subsequent months, with
The mitosporic Onygenales group encompasses nine gen- the polyps recurring often and at short intervals. Four months
era: Blastomyces, Chrysosporium, Coccidioides, Emmonsia, after the last operation, the patient developed a swelling on
Geomyces, Locazia, Malbranchea, Myriodontium, and his forehead, which gradually enlarged and was accompa-
Paracoccidioides. Currently, the genus Myriodontium con- nied by left proptosis. Upon further examination, brown
sists of a recognized species Myriodontium keratinophilum necrotic-like material was found in the sinus, leading to the
and an unassigned species Myriodontium sp. SW150 [1]. erosion of the roof of the left orbit and both ethmoid bones
First described in 1978 as a new species [2], Myriodontium and blocking of both sides of the nose by polypoidal mucosa.
keratinophilum is widespread in nature, especially where The posterior wall of the frontal sinus was also thinned
keratinous substrates are present. The fungus has been and eroded. The tissue lining the sinus consisted of floridly
isolated from soil in Italy and California, from the hair of inflamed granulation tissue containing large numbers of
shrews and cats in the United Kingdom, from the penis of plasma cells and eosinophils; most of the mucosal lining was
a bull in Germany, and also from an unknown source in ulcerated. There was no evidence that the tissue had been
Nigeria [3]. invaded by the fungus in the lumen. The contents of the sinus
M. keratinophilum colonies are slow growing, with the were mainly cellular debris and some septate hyphae, which
appearance of a white mold. Hyphae are septate, branch nonbranched non-dichotomously and appeared ribbon like and
dichotomously, and appear ribbon-like and folded. Conidia folded. These features are characteristic of mycetoma of the
are single celled, appearing on long denticles along the paranasal sinus seen in the Sudani and of allergic aspergillo-
sides of the fertile hyphae [3]. The teleomorph of M. kera- sis of the paranasal sinus [5,6]. Growth of the hyphae yielded
tinophilum is found in the genus Neoarachnotheca, order a slow-growing white mold that produced a few single-celled
Onygenales. The genus Neoarachnotheca is characterized by conidia on long denticles along the sides of the fertile hyphae.
its white, spherical ascomata with a wall formed by a network This isolate was identified as M. keratinophilum. An anti-
of hyphae and spherical, subhyaline ascospores with an irreg- genic extract prepared from the isolate produced a single dif-
ular sheath. The type species of the genus Neoarachnotheca, fuse precipitin line when tested against the patient serum by
Nt. keratinophila, displays wavy peridial hyphae and has double diffusion. No reaction was observed between com-
been isolated from marine and river sediments [4]. parable extracts of A. flavus, A. fumigatus, and A. versicolor
299

and patient serum. The patient was treated with ketoconazole deoxynucleoside triphosphates, FastStart Taq DNA
200 mg daily for 3 weeks, recovered with no recurrence. polymerase, and 1 mM MgCl2 (additional MgCl2 is
added to a final concentration of 4.6 mM), 0.4 μM
each of ITS1 forward and ITS4 reverse primers, 1×
37.1.3 Diagnosis
SYBR green (Molecular Probes), and 3 μL template
Molds such as Myriodontium keratinophilum are environ- DNA.
mental organisms that often transiently colonize the respi- 2. Thermal cycling parameters include 95°C for
ratory, integument, and gastrointestinal systems of human 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and
hosts. Traditionally, molds are identified by direct micros- 76°C for 30 s; and a final extension at 72°C for 2 min.
copy in combination with culture using a variety of myco- 3. The quality of the amplicon is determined using the
logical media. To augment specific identification, in vitro derivative of the melt analysis curve (55°C–99°C,
sporulation using cultured isolate may also be attempted, 45 s hold at 55°C, and 5 s/°C) using the RotorGene
which may take up to 3 weeks of incubation and for which 3000 (Corbett Robotics, Inc).
success is not guaranteed, however. 4. The amplified product is purified for bidirectional
For more rapid and more accurate identification of molds sequencing using ExoSAP-IT (USB Corp). Five
including M. keratinophilum, PCR amplification, and microliters of Big Dye Terminator Ready Reaction
sequencing analysis of ribosomal RNA (rRNA) genes, inter- Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
nal transcribed spacer (ITS) regions as well as other genes each primer (0.8 pmol/μL) and 3 μL of purified PCR
(e.g., EF1α) can be utilized [7,8]. product. Cycle sequencing is performed with a 9700
37.2 Methods
37.2.1 Sample Preparation G-50 fine column to remove unincorporated dye ter-
minators. Purified sequencing reaction products are
Molds from clinical specimens are grown on inhibitory mold run on an ABI Prism 3100 Genetic Analyzer with a
agar, modified Sabouraud agars, or potato dextrose agar. 50 cm capillary array.
Microscopic structures are observed on tease or tape prepa- 5. Sequences are analyzed with the SmartGene
rations and slide cultures for up to 21 days. Integrated Database Network software version
After growing for 1–7 days on potato dextrose agar slants, 3.2.3 vr. SmartGene is a web-based software and
approximately 1 cm2 of mycelia are collected by scraping the database system with reference sequences derived
slant with a sterile stick in 1 mL of sterile, molecular-grade from the National Center for Biological Information
H2O. The material is transferred to a 2 mL screw-cap tube. (NCBI) GenBank repository.
The tubes are centrifuged for 1 min at 6000 × g. If the mycelia
do not pellet, the material is contained with a pediatric blood Note. If preferred or real time PCR instrument is not avail-
serum filter (Porex Corp., Fairburn, GA). After removal of able, standard PCR may be performed with primers ITS1 and
supernatant, the material is resuspended in 200 μL of IDI ITS4, and the resulting amplicon is sequenced with the same
sample buffer (IDI lysis kits, GeneOhm Sciences) and trans- primers. Sequence-based identifications are defined by per-
ferred to the lysis tubes containing glass beads. Lysis tubes cent identity: species, ≥99%; genus, 93%–99%; and incon-
are vortexed on the highest setting for 5 min. The tubes are clusive, ≤93%.
placed in a boiling water bath for 15 min. Tubes are centri-
fuged for 5 min at 16,000 × g. The supernatant was stored at
−20°C until amplification [7]. 37.3 Conclusion
Myriodontium keratinophilum (teleomorph Neoara
37.2.2 Detection Procedures chnotheca keratinophila) is a mold classified in the genus
Myriodontium, the mitosporic Onygenales group. Similar to
Pounder et al. [7] described a real-time PCR with SYBR other mold organisms, M. keratinophilum is commonly pres-
green DNA-binding dye and amplicon-melting temperature ent in the environment and has been considered as a labora-
analysis for fungal detection using pan-fungal primers ITS1 tory contaminant. After the description of the first case of
forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 M. keratinophilum-associated sinusitis in an immunocompe-
reverse (5′-TCCTCCGCTTATTGATATGC-3′) [9]. The tent man in 1985 [3], no new cases due to this organism have
identity of the fungi is verified by subsequent sequencing bee reported.
analysis. Considering the continuing ageing of human populations
Procedure and the increasing occurrence of suppressed immune func-
tions in individuals with underlying diseases or undergoing
1. PCR mixture is composed of 1 × Lightcycler chemotherapies, it is likely that uncommon infections due to
FastStart DNA Master Hybridization Probes opportunistic mold pathogens will appear with heightened
mixture (Roche Applied Science) containing frequency in future. The availability of rapid, sensitive, and

Myriodontium 301
specific laboratory techniques such as those based on PCR 5. Milosev B et al. Primary aspergilloma of the paranasal sinuses
and sequencing is critical for correct determination and dif- in the Sudan. Br J Surg. 1969;56:132–137.
ferentiation of molds that are difficult to identify on the basis 6. Miller JW, Johnson A, Lamb D. Allergic aspergillosis of the
maxillary sinuses. Thorax. 1981;36:710.
of macroscopic and microscopic features alone.
7. Pounder JI et al., Discovering potential pathogens among
fungi identified as nonsporulating molds. J Clin Microbiol.
References 2007;45:568–571.
8. Bagyalakshmi R et al., Newer emerging pathogens of ocular
1. The UniProt Consortium. Available at http://www.uniprot. non-sporulating molds (NSM) identified by polymerase chain
org/, accessed on August 1, 2010. reaction (PCR)-based DNA sequencing technique target-
2. Samson RA, Polonelli L. Myriodontium keratinophilum. gen. ing internal transcribed spacer (ITS) region. Curr Eye Res.
et sp. nov. Persoonia. 1978;9:505–509. 2008;33(2):139–147.
3. Maran AG et al., Frontal sinusitis caused by Myriodontium 9. White, TJ et al., 1990. Amplification and direct sequenc-
keratinophilum. Br Med J. 1985;290:207. ing of fungal ribosomal RNA genes for phylogenetics.
4. Cano J et al., Studies on keratinophilic fungi. IX: Neoara In M. A. Innis et al. (eds.), PCR Protocols: A Guide to
chnotheca gen. nov. and a new species of Nannizziopsis. Methods and Applications. Academic Press, San Diego,
Anton Van Leeuwen. 1997;72(2):149–158. CA, pp. 315–322.

38 Onychocola
Dongyou Liu
Contents
38.1 Introduction...................................................................................................................................................................... 303
38.1.2.1 Onychocola canadensis......................................................................................................................... 304
38.1.2.2 Onychocola kanei.................................................................................................................................. 304
38.1.3 Diagnosis.............................................................................................................................................................. 304
38.2 Methods............................................................................................................................................................................ 305
38.2.2.1 Standard PCR and Sequencing.............................................................................................................. 306
38.2.2.2 Real-Time PCR and Sequencing............................................................................................................ 306
38.3 Conclusion........................................................................................................................................................................ 306
References.................................................................................................................................................................................. 307
38.1 Introduction grayish yellow later. Growth at 37°C is restricted, reaching a

diameter of 6–13 mm in 21 days. On oatmeal agar at 25°C,
38.1.1 Classification and Morphology colonies are white to grayish white, flat, and initially glabrous
The genus Onychocola is the only member of the mitosporic and then turn downy tufts by 21 days. Ascomata (cleistothe-
Arachnomycetaceae group, family Arachnomycetaceae, cia) formed in paired isolates are superficial or submerged in
order Onygenales, class Eurotiomycetes, subphylum mycelia, reddish brown, nonostiolate, and subglobose to glo-
Pezizomycotina, phylum Ascomycota, and kingdom Fungi. bose with a diameter of 175–300 μm and bear 5–8 appendages
Currently, the genus Onychocola consists of four species: (setae). Peridial wall of the ascoma is membranous of the tex-
Onychocola canadensis (teleomorph: Arachnomyces nodo- tura angularis type. Seta (115–125 μm × 4–10 μm) is smooth
setosus), Onychocola kanei (teleomorph: Arachnomyces to intermittently slightly nodose, sparsely septate, and unci-
kanei), Onychocola glareosa (teleomorph: Arachnomyces nate to loosely coiled at the tip. Asci (7.5 μm × 6.5 μm) are
glareosus), and Onychocola sclerotica (teleomorph: evanescent, eight spored, and subglobose. Ascospores (3.5–
Malbranchea sclerotica), of which Onychocola canadensis 4.5 μm × 2.5–3 μm) are smooth, oblate with a polar boss at
and possibly Onychocola kanei are implicated in subungual the center, and pale to light brown. Conidia (aleurioconidia)
or superficial onychomycosis in humans [1–3]. (3.5–7 μm × 3–4.5 μm) are sessile or borne on stalks and are
Onychocola canadensis (teleomorph: Arachnomyces smooth to finely asperulate and clavate to pyriform or are
nodosetosus) colonies grow slowly, with velvety to lanose intercalary and alternate and cylindrical or irregular with one
texture and yellow to pale sandy brown color; reverse is deep or both sides swollen. Conidia detach by lytic dehiscence [2].
brown-gray. Arthroconidia (4–16 × 2–5 μm in size) appear A. kanei is distinguished from other related species by
after 14–21 days; are broadly ellipsoidal to nearly spherical, its setal and anamorph morphology, and colony color. A.
smooth, usually single-celled (but occasionally two-celled), kanei produces cylindrical to irregularly swollen alternate
and hyaline to subhyaline; and often form long, more or less, arthroconidia but differs in producing solitary aleurioco-
upright chains that do not readily fragment into separate nidia. A. nodosetosus differs in producing swollen, one or
conidia. Old cultures may form distinctive broad, brown, two-celled arthroconidia in chains that do not easily break
thick- or rough-walled, nodose hyphae resembling peridial apart. Dehiscence occurs by rhexolysis of thin-walled cells
appendages of the Arachnomyces sexual state [4,5]. adjacent to, or between, chains of conidia and sometimes
Onychocola kanei (teleomorph Arachnomyces kanei) by schizolysis of adjacent conidia. Arachnomyces minimus
(kanei is named after Canadian mycologist Julius Kane) colo- has setae that are more nodose than those of A. kanei and it
nies reach a diameter of 15–21 mm in 21 days on potato dex- lacks an anamorph. Arachnomyces gracilis differs in having
trose agar (PDA) at 25°C are downy, raised, rugose, umbonate setae that are straight at the tip and producing nonswollen or
to crateriform, initially white and often becoming yellow to cylindrical, alternate arthroconidia of the Malbranchea type,
which secede by rhexolytic dehiscence [2].
303

Onychocola glareosa (teleomorph: Arachnomyces Direct microscopy revealed hyaline, round-to-barrel-shaped

glareosus) colonies attain a diameter of 18–25 mm in 21 days arthroconidia, hyaline hyphae of varying width, and broad,
on PDA at 30°C, are initially white, later grayish brown cen- thick-walled brownish hyphae. The fungus is identified as
trally, downy, and raised to crateriform and produce abundant Onychocola canadensis Sigler gen. et sp. nov. Subsequently,
sclerotia giving the colony a grainy appearance. Droplets of Sigler et al. [18] described six additional cases of toenail
dark reddish brown exudate occur in areas where sclerotia infection in elderly individuals and one case of glabrous skin
are produced. A pale brown diffusing pigment first appears infection due to Onychocola canadensis (Arachnomyces
after 7 days and turns medium dark reddish brown within nodosetosus), originating from New Zealand and Canada.
21 days. Growth at 35°C is restricted, reaching 4–5 mm The development in culture of broad, brown, nodose, thick-
diameter in 21 days. Ascomata (cleistothecia) (185–310 μm walled hyphae suggested an affinity to the ascomycete genus
in diameter) is reddish brown, subglobose to globose, and Arachnomyces. Ascocarps were produced in six mated pairs
non-ostiolate, bearing 1–5 setae. Peridial wall is membra- on sterilized rice grains or rice extract agar after 7–12 month
nous of textura angularis. Setae are smooth to intermittently incubation. Furthermore, Gupta et al. [19] documented 10
nodose, regularly septate, and uncinate to loosely coiled or previously unreported cases of O. canadensis onychomy-
straight at the tip. Asci (6 μm × 5 μm) are evanescent, eight cosis in Canada, which were diagnosed on the basis of (i)
spored, and subglobose. Ascospores (3–3.5 μm × 1.5–2 μm) the finding of compatible filaments on direct microscopy of
are smooth, oblate with polar boss, and light brown. Fertile nail and (ii) consistent culture from repeated specimens. Of
hyphae are arcuate, bearing intercalary alternate arthroco- these 10 cases, O. canadensis was associated with DLSO (six
nidia. Arthroconidia (2.5–4.5 μm × 1.5–2 μm) are cylindri- patients), WSO (one patient), and as an insignificant contami-
cal, sometimes slightly curved, or irregular with one or both nant in the nails (three patients).
sides swollen, detaching by lytic dehiscence. Racquet hyphae Erbagci et al. [20] described the first Turkish case of
are present. Sclerotia (45–60 μm in diameter) develop from skin and nail infection due to Onychocola canadensis in a
swollen, thick-walled, and pale brown cells of the vegetative farmer who frequently worked barefoot on soil. The patient
hyphae, enlarging and aggregating to form a solid tissue with presented with scaly and hyperkeratotic lesions resembling
a membranous rind of textura angularis. Sclerotia are pale tinea pedis, erythematous plaques, and Majocchi’s granu-
brown and subglobose to globose in shape [3]. loma-like dermal papulonodules. Cultures of nail plates,
skin scrapings, and needle aspiration materials from papules
or nodules on Sabouroud dextrose media with and without
cycloheximide, trichophyton agar, and PDA at 26°C all grew
Human onychomycosis can be separated into five types: distal a mold, which was identified as Onychocola canadensis on
and lateral subungual onychomycosis (DLSO), white superfi- the basis of its colonial and microscopic morphology. While
cial onychomycosis (WSO), proximal subungual onychomy- skin lesions responded to daily itraconazole in a dose of
cosis (PSO), Candida onychomycosis, and total dystrophic 200 mg for 3 months, the onychomycosis was resistant to
onychomycosis (TDO) (primary or secondary type). The most therapy.
common causal organisms for onychomycosis in humans
are Trichophyton mentagrophytes, T. rubrum, Acremonium 38.1.2.2 Onychocola kanei
spp., Alternaria spp., Aspergillus spp., Fusarium oxysporum, Gibas et al. [2] reported the identification of Onychocola
Scopulariopsis brevicaulis, Scytalidium, and Onychocola kanei from two patients. The first patient was a 78-year-old
canadensis [5–12]. man from Toronto, Ontario, who had a 3–4 year history of
an abnormally appearing left great toenail, typical of WSO.
38.1.2.1 Onychocola canadensis Direct examination of the nail revealed irregular filaments,
Onychocola canadensis is a nondermatophyte first isolated and cultures yielded a slow-growing yellow fungus, which
from chronic infection of the great toenail in Canada [17]. was identified as Onychocola kanei. The second patient was
The slow growth rate of O. canadensis and lack of resem- an elderly female with an abnormal left and right great toe-
blance to any other known nail-infecting fungus may have nail, which was thickened with whitish plaques on the upper
contributed to its delayed discovery. The fungus has since surface, suggestive of WSO. Direct microscopy of nail scrap-
been reported in New Zealand, France, Britain, Belgium, ings revealed irregular filaments, and cultures of nail scrap-
Spain, Italy, Turkey, and other parts of the world [13–16]. As ings grew a yellow, slow-growing fungus consistent with
an uncommon cause of DLSO or WSO, O. canadensis makes Onychocola kanei as well as Aspergillus sydowii.
the nail white or yellow in color and often hyperkeratotic and
friable. O. canadensis onychomycosis mostly affects gar-
38.1.3 Diagnosis
deners or farmers, suggesting its soil origin. Sometimes, O.
canadensis may be present in an abnormal-appearing nail as Onychomycosis may result from infections with dermato-
an insignificant finding, not as a pathogen. phytes (e.g., Trichophyton mentagrophytes and T. rubrum,
Sigler and Congly [17] reported three cases of great toenail which invade the nail plate, resulting in tinea unguium), nonder-
infection in Canada, in which a slow-growing arthroconidial matophytic filamentous fungi (e.g., Acremonium, Alternaria,
hyphomycete was isolated repeatedly and in pure culture. Aspergillus, Fusarium, Scopulariopsis, Scytalidium,

Onychocola 305
Onychocola, Pyrenochaeta, and Botryodiplodia), and medium, and inhibitory mold agar without cycloheximide) to
Candida yeast. Although not life threatening, onychomyco- facilitate such growth. A dermatophyte tends to grow on both
sis can impact negatively on patients’ emotional, social, and types of media. As nails are nonsterile, fungal and bacterial
occupational functioning as well as health budget. Correct contaminants may obscure the nail pathogen. In addition,
identification of the causative agents for onychomycosis is approximately 40% of nail specimens may not yield fungal
vital for the implementation of effective treatment and con- growth.
trol measures. Because culture-based diagnosis of onychomycosis is
Because nondermatophytes are opportunistic fungal time-consuming and difficult, molecular techniques espe-
pathogens with widespread distribution, isolation of these cially PCR and sequencing of rRNA genes and internal tran-
fungi from a clinical specimen alone does not prove con- scribed spacer (ITS) regions have been developed for rapid
clusively their direct involvement in the disease process. For and accurate identification of causal fungal agents directly
reliable diagnosis of opportunistic onychomycosis, direct from clinical specimens and cultured isolates [2,3,21].
positive microscopy and repeat isolations of the proposed
organism are necessary.
To ensure that the samples are free of contaminating bac- 38.2 Methods
teria, thorough cleansing of the nail area with alcohol rep-
resents the first step of the sample collection. (i) For distal
subungual onychomycosis (DSO) that occurs in the nail bed, Nail specimens are divided into three portions. The first por-
the specimen is obtained from the nail bed, which contains tion is mounted on a slide with 20% KOH with 40% dimethyl
the greatest concentration of viable fungi. The nail is clipped sulfoxide. The glass slide is heated for 1 min under a cover
short with nail clippers, and the specimen is taken from the glass and then examined microscopically for the presence
nail bed as proximally to the cuticle as possible with a small of fungal elements: spores and hyphae. The second portion
curet or a no. 15 scalpel blade. Material is also obtained from is cut into small pieces by scalpel, cultured on Sabouraud’s
the underside of the nail plate, with emphasis placed on sam- dextrose agar containing chloramphenicol (0.05%) with and
pling from the advancing infected edge most proximal to the without cycloheximide (0.5%), and incubated at 25°C for 4–6
cuticle. This helps reduce contaminants. (ii) For PSO that weeks. Clinical isolates are identified on the basis of pheno-
takes place under the cuticle before settling in the proximal typic characteristics of the colonies (e.g., growth morphol-
nail bed, the healthy nail plate is gently pared away with a no. ogy, colonial appearance, surface texture, shape, size, color,
15 scalpel blade. A sharp curet is then used to remove mate- rate of growth, undersurface, and edge), microscopic exami-
rial from the infected proximal nail bed as close to the lunula nation of lactophenol cotton blue wet mounts, and physiologi-
as possible. (iii) For WSO that affects the nail plate surface, a cal tests such as urease production, in vitro hair perforation,
no. 15 scalpel blade or sharp curet is used to scrape the white and nutritional requirement tests. Characteristics of conidia
area and remove the infected debris. (iv) For Candida ony- are examined in slide culture preparations on Pablum cereal
chomycosis, material is taken from the proximal and lateral agar (CER). The third portion is used for DNA extraction
nail edges; the lifted nail bed is scraped; scrapings are taken after crushing in liquid nitrogen. The crushed scrapings are
from the undersurface of the nail if insufficient debris is pres- suspended in 200 μL of Tris–EDTA buffer and subjected to
ent in the nail bed. repeated freezing and thawing. Then, 300 μL of 0.1% Triton
The specimen is divided into two portions for direct X-100 (pH 8) and 2 μL of proteinase K solution (20 mg/mL)
microscopy and culture. As nail debris is thick and coarse were added and incubated for 2 h at 65°C. The extracted
and hyphae are usually only sparsely present, direct micros- DNA is purified by the phenol–chloroform–isoamyl alcohol
copy serves only as a screening test for the presence or (25:24:1) method and resuspended in 30 μL of Tris–EDTA
absence of fungi, and its false-negative rates may range buffer [22].
from 5% to 15%. The specimen is mounted in a solution of Alternatively, DNA is extracted from cultured isolate.
20%–25% KOH (or NaOH mixed with 5% glycerol or 20% Approximately 100 mg of fresh mycelium is scraped from the
KOH and 36% dimethyl sulfoxide), heated to emulsify lipids agar surface and placed in a precooled sterile porcelain mor-
(1 h at 51°C–54°C), and observed under ×40 magnification. tar containing a small amount of acid-sterilized sand. Liquid
The specimen may be counterstained with chitin-specific nitrogen is added and the frozen mycelium is ground to a
Chlorazol black E to accentuate hyphae and prevent false- powder. 750 μL of extraction buffer (1% w/v cetyl–trimethyl
positive results. Direct microscopy helps determine if the ammonium bromide, 1 M NaCl, 100 mM Tris, 20 mM EDTA,
hyphae are typical of dermatophyte fungi, nondermatophyte and 1% w/v polyvinyl polypyrolidone) is added to the ground
molds, or yeasts. material. The mixture is transferred into a sterile 2 mL screw-
Culture provides an ultimate way to identify the causative capped microcentrifuge tube and incubated for 30 min at
microorganism of onychomycosis. Specimens are often plated 65°C. An equal volume of chloroform:isoamyl alcohol (24:1
on two different media: a primary medium (e.g., Sabouraud v/v) was added. The resulting complex is mixed by invert-
peptone–glucose agar with cycloheximide) to select against ing the tube about 20 times and centrifuged for 15 min at
most nondermatophytic molds and bacteria and a secondary 10 000 × g at room temperature. The upper layer, which con-
medium (e.g., Sabouraud’s glucose agar, Littman’s oxgall tained crude DNA material, is collected and purified using

the QIAquick DNA purification kit (QIAgen) and the puri- microliters of Big Dye Terminator Ready Reaction
fied DNA is stored at −20°C [2]. Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
each primer (0.8 pmol/μL) and 3 μL of purified PCR
38.2.2 Detection Procedures thermal cycler (ABI), using 25 cycles of 96°C for
38.2.2.1 Standard PCR and Sequencing
Gibas et al. [2] utilized primers ITS3 and ITS4 to amplify G-50 fine column to remove unincorporated dye ter-
a fragment of 271–302 bp including a portion of the 5.8S minators. Purified sequencing reaction products are
gene, the complete ITS2 region and a portion of the 28S run on an ABI Prism 3100 Genetic Analyzer with a
gene followed by sequencing analysis for identification of 50 cm capillary array.
Onychocola/Arachnomyces. 5. Sequences are analyzed with the SmartGene
PCR amplification is performed with initial denaturation Integrated Database Network software version 3.2.3
at 94°C for 2 min; 30 cycles of 94°C for 1 min, 55°C for 1 min, vr. SmartGene is a web-based software and database
and 72°C for 2 min; and final extension 72°C for 7 min. system with reference sequences derived from the
Sequencing of the amplicons is conducted with the National Center for Biological Information (NCBI)
DYEnamic™ ET terminator kit (Amersham Pharmacia GenBank repository.
Biotech) and run on an ABI 377 Automated sequencer
(Amersham). Consensus sequences are determined using
Note. If real time PCR instrument is not available, standard
the Sequencher™ for Windows 4.0.2 (Gene Codes Corp.)
and alignment is done manually using Se-Al v1.0a1 Fat, a
the resulting amplicon is sequenced with the same primers.
sequence alignment program. Phylogenetic analysis is done
Sequence-based identifications are defined by percent iden-
using PAUP (Phylogenetic Analysis Using Parsimony) v.
tity: species, ≥99%; genus, 93%–99%; and inconclusive,
4.0b8, and the robustness of the resultant phylogenetic tree
≤93%.
or inferred clades is tested using bootstrap analysis of 1000
resamplings. Ajellomyces capsulatus (GenBank AF038354)
is used as the outgroup taxon.
38.3 Conclusion
38.2.2.2 Real-Time PCR and Sequencing The genus Onychocola consists of four nondematophyte fungal
species: Onychocola canadensis (teleomorph: Arachnomyces
nodosetosus), Onychocola kanei (teleomorph: Arachnomyces
green DNA-binding dye and amplicon-melting temperature
kanei), Onychocola glareosa (teleomorph: Arachnomy
ces glareosus), and Onychocola sclerotica (teleomorph:
Malbranchea sclerotica), which are commonly present in the
environment. Of these, Onychocola canadensis and possibly
Onychocola kanei are implicated in subungual or superficial
Procedure onychomycosis in humans.
Besides Onychocola spp., a number of dermatophytes (e.g.,
1. PCR mixture is composed of 1× Lightcycler FastStart Trichophyton mentagrophytes and T. rubrum), nondermatopy-
DNA Master Hybridization Probes mixture (Roche tic fungi (e.g., Acremonium, Alternaria, Aspergillus, Fusarium,
Applied Science) containing deoxynucleoside tri- Scopulariopsis, Scytalidium, Onychocola, Pyrenochaeta, and
phosphates, FastStart Taq DNA polymerase, and Botryodiplodia), and Candida yeast may also be responsible
1 mM MgCl2 (additional MgCl2 is added to a final for onychomycosis. In order to implement effective treatment
concentration of 4.6 mM), 0.4 μM each of ITS1 for- and control measures, it is important that the causative agents
ward and ITS4 reverse primers, 1× SYBR green for onychomycosis be identified to species level.
(Molecular Probes), and 3 μL template DNA. Given their time-consuming nature and requirement
2. Thermal cycling parameters include 95°C for of specialized skills, direct microscopy and culture for the
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, identification of causal agents for onychomycosis are far
and 76°C for 30 s; and a final extension at 72°C for from being satisfactory. To speed up the diagnostic process
2 min. and to reduce misidentification, molecular techniques based
3. The quality of the amplicon is determined using the on PCR amplification and sequencing analysis have been
derivative of the melt analysis curve (55°C–99°C, applied for improved determination of Onychocola and other
45 s hold at 55°C, and 5 s/°C) using the RotorGene fungi and Candida yeast. It is envisaged that future devel-
3000 (Corbett Robotics, Inc). opment of Onychocola-species-specific primers will further
4. The amplified product is purified for bidirectional streamline the direct detection and identification of this fun-
sequencing using ExoSAP-IT (USB Corp). Five gus from clinical specimens.

Onychocola 307
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sis: Report of the first two Spanish cases. Med Mycol.
1. The UniProt Consortium. Available at http://www.uniprot.org/ 2002;40(2):209–212.
taxonomy/, accessed on August 1, 2010. 15. Fanti F et al. First Italian report of onychomycosis caused by
2. Gibas CF et al. Arachnomyces kanei (anamorph Onychocola canadensis. Med Mycol. 2003;41(5):447–450.
Onychocola kanei) sp. nov., from human nails. Med Mycol. 16. Van Esbroeck M et al. Isolation of Onychocola canadensis
2002;40(6):573–580. from four cases of onychomycosis in Belgium. Acta Clin
3. Gibas CF et al. Mating patterns and ITS sequences distinguish Belg. 2003;58(3):190–192.
the sclerotial species Arachnomyces glareosus sp. nov. and 17. Sigler L, Congly H. Toenail infection caused by
Onychocola sclerotica. Stud Mycol. 2004;50:525. Onychocola canadensis gen. et sp. nov. J Med Vet Mycol.
4. de Hoog GS et al. (eds.). Atlas of Clinical Fungi, 2nd edn., 1990;28(5):405–417.
p. 325. Centraalbureau voor Schimmelcultures, Utrecht, the 18. Sigler L, Abbott SP, Woodgyer AJ. New records of nail and
Netherlands, 2004. skin infection due to Onychocola canadensis and description
5. Gupta AK. Non-dermatophyte onychomycosis. Dermatol of its teleomorph Arachnomyces nodosetosus sp. nov. J Med
Clin. 2003;21(2):257–268. Vet Mycol. 1994;32(4):275–285.
6. Vélez H, Díaz F. Onychomycosis due to saprophytic fungi. 19. Gupta AK, Horgan-Bell CB, Summerbell RC. Onychomycosis
Report of 25 cases. Mycopathologia. 1985;91(2):87–92. associated with Onychocola canadensis: Ten case reports
7. Elewski BE. Onychomycosis: Pathogenesis, diagnosis, and and a review of the literature. J Am Acad Dermatol. 1998;39
management. Clin Microbiol Rev. 1998;11:415. (3):410–417.
8. Gupta AK, Summerbell RC. Combined distal and lateral sub- 20. Erbagci Z et al. Cutaneous hyalohyphomycosis and onycho-
ungual and white superficial onychomycosis in the toenails. mycosis caused by Onychocola canadensis: Report of the first
J Am Acad Dermatol. 1999;41(6):938–944. case from Turkey. J Dermatol. 2002;29(8):522–528.
10. Gupta AK et al. Itraconazole and terbinafine treatment of some 21. Walberg M et al. 18S rDNA polymerase chain reaction
nondermatophyte molds causing onychomycosis of the toes and sequencing in onychomycosis diagnostics. Acta Derm
and a review of the literature. J Cutan Med Surg. 2001;5:206. Venereol. 2006;86(3):223–226.
11. O’Donoghue NB, Moore MK, Creamer D. Onychomycosis 22. Garg J et al. Evaluation of pan-dermatophyte nested
due to Onychocola canadensis. Clin Exp Dermatol. PCR in diagnosis of onychomycosis. J Clin Microbiol.
2003;28(3):283–284. 2007;45(10):3443–3445.
12. Piraccini BM, Tosti A. White superficial onychomyco- 23. Pounder JI et al. Discovering potential pathogens among
sis: Epidemiological, clinical, and pathological study of 79 fungi identified as nonsporulating molds. J Clin Microbiol.
patients. Arch Dermatol. 2004;140(6):696–701. 2007;45(2):568–571.
13. Campbell CK, Johnson EM, Warnock DW. Nail infection
caused by Onychocola canadensis: Report of the first four
British cases. J Med Vet Mycol. 1997;35(6):423–425.

39 Paecilomyces
Ana Alastruey-Izquierdo, Maria Victoria Castelli,
Leticia Bernal-Martinez, and Manuel Cuenca-Estrella
Contents
39.1 Introduction...................................................................................................................................................................... 309
39.1.1 Classification, Morphology, and Epidemiology.................................................................................................... 309
39.1.1.1 Classification.......................................................................................................................................... 309
39.1.1.2 Morphology............................................................................................................................................ 309
39.1.1.3 Epidemiology and Susceptibility Profile................................................................................................310
39.1.2 Clinical Features....................................................................................................................................................310
39.1.2.1 Paecilomyces lilacinus Infections..........................................................................................................310
39.1.2.2 Paecilomyces variotii Infections............................................................................................................311
39.1.3 Treatment of Paecilomyces Infections...................................................................................................................311
39.1.4 Diagnosis...............................................................................................................................................................311
39.2 Methods.............................................................................................................................................................................313
39.2.1.1 Clinical Specimens.................................................................................................................................313
39.2.1.2 DNA Extraction from Cultured Strains..................................................................................................313
39.2.1.3 DNA Extraction from Clinical Samples.................................................................................................313
39.2.2.1 PCR Identification...................................................................................................................................313
39.2.2.2 Quantitative PCR Detection of Paecilomyces Species...........................................................................314
39.3 Conclusions and Future Perspectives................................................................................................................................314
References...................................................................................................................................................................................315
39.1 Introduction (MIC) of amphotericin B, itraconazole, and of new azoles

but in vitro resistance to voriconazole has been reported [7].
Paecilomyces species are widespread soil saprophytes [1]. In addition, the echinocandins show good in vitro activity
Fungal infections caused by these species are not common against P. variotii, while P. lilacinus is not susceptible to this
despite their ubiquity in the human’s environment [2,3]. family of antifungal compounds [7–9].
These species have been mainly described as the cause of
hyalohyphomycosis associated with traumatic inoculation
and contamination of human skin or prosthetic devices. In 39.1.1 Classification, Morphology,
the last few years, Paecilomyces have been reported in occa- and Epidemiology
sional cases of sinusitis or pulmonary infection afflicting
immunosuppressant patients after inhalation of their conidia 39.1.1.1 Classification
[3,4] and even as the cause of disseminated mycoses in some Paecilomyces are hyaline species of filamentous fungi
patients with predisposing factors [5,6]. Paecilomyces lilaci- whose sexual telemorphs belong to the division Ascomycota
nus and Paecilomyces variotii are the species most frequently (Euascomycetes, Eurotiales: Trichocomaceae) of kingdom
involved in human infection. The differentiation between Eumycota and are classified in the genera Byssochlamys,
these two species is clinically important, since P. lilacinus Chromocleista, Talaromyces, and Thermoascus [10].
and P. variotii seem to present marked differences in their
in vitro susceptibility to the antifungal agents. P. lilaci- 39.1.1.2 Morphology
nus is resistant to amphotericin B and itraconazole, while Colonies of organisms belonging to these species are fast
other compounds such as posaconazole and voriconazole growing. Some species are termophilic; thus, Paecilomyces
have good in vitro activity against this fungus. In contrast, crustaceus and P. variotii can grow well at temperatures
P. variotii exhibits lower minimal inhibitory concentrations above 50°C [10]. Colonies are flat spreading, powdery or
309

The identification of Paecilomyces spp. has clinical

significance as differences in their susceptibility profile
have been described. Table 39.1 displays the data of sus-
(a) ceptibility in vitro of P. variotii and P. lilacinus, including
(c) data of 69 clinical strains tested in the Spanish Mycology
Reference Laboratory following the EUCAST (European
(b) Committee on Antimicrobial Susceptibility Testing) ref-
erence procedure for susceptibility testing of filamentous
fungi.

Figure 39.1 Microscopy of Paecilomyces spp. Conidiogenous
cells phialidic, swollen at the base (a) and gradually narrowed into
Despite its ubiquity in the human’s environment, fungal
a long beak (b). Conidiophores occur as verticils (c). infections caused by Paecilomyces species are uncommon.
They can cause the same clinical entities that are described
for other hyaline moulds such as Aspergillus, Fusarium, and
velvety, and are usually white, brownish, or in bright col- Penicillium. Most cases occur in patients with impaired host
ors, reverse off-white to brown. The most common clinical defences or following trauma or surgical procedures [11], but
species are P. variotii with a yellowish brownish greenish allergic bronchopulmonary diseases and pulmonary fungus
colony and P. lilacinus with characteristic vinaceous to violet ball have also been reported [3,13].
colonies. Invasion of tissues by these species causes a hyalohypho-
Conidiophores occur solitarily, in pairs, as verticils, and mycosis in which the invasive form of the etiologic agent is
in penicillate heads. Conidiogenous cells are phialidic, swol- septate hyphae with no pigment in the wall. Histopathology
len at the base, and gradually narrowed into a long beak; they is very similar to that observed in infections by other hya-
bend away from the axis of the conidiophore. Microscopically line fungus such as Aspergillus spp., with infected foci that
resembles Penicillium spp. but the phialides are more elon- contain a central cluster of radially arranged hyphae and a
gated (see Figure 39.1) [10]. margin of necrosis, with or without hemorrhagic borders.
Ischemia and edema result in lesions that are grossly firm
39.1.1.3 Epidemiology and Susceptibility Profile and pale. The inflammatory response is minimal in immuno-
Paecilomyces species are saprophytic hyalohyphomycetes suppressed patients [20,21].
filamentous fungi that are found worldwide in soil, decaying
vegetable matter, and as air and water contaminants causing 39.1.2.1 Paecilomyces lilacinus Infections
deterioration of grain, food, and paper. Moreover, they can In 2006, Pastor and Guarro published the most recent review
contaminate creams and lotions of clinical use and colonize so far on clinical importance of P. lilacinus. The review iden-
catheters and plastic implants [11,12]. tified 119 reported cases of human infection by P. lilacinus
As stated above, P. lilacinus and P. variotii are the between 1964 and 2004. Most were cases of oculomycosis
most ubiquitous species of the genus and most frequently (51.3%), followed by cutaneous and subcutaneous infections
involved in human infection. Other species of the genus (35.3%), and a smaller group of miscellaneous infections
such as Paecilomyces crustaceus, Paecilomyces marqui- (13.4%) [12].
andii, Paecilomyces viridis, Paecilomyces javanicus, and As stated above, this species has most often associated
Paecilomyces puntonii [13–18] have also been reported occa- with ocular mycosis and intraocular lens implantation has
sionally from human infections. Some of these species have been reported as the most frequent predisposing factor for
also been described as etiology of animal diseases [19]. that. Other risk factors such as the use of extended-wear
Table 39.1
Summary of Susceptibility Results In Vitro of Antifungal Agents Tested against Paecilomyces speciesa
AMB ITC VRC POS CPF ANF MCF
Species N MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
P. variotii 37 0.03 0.50 0.06 0.25 1.0 8.0 0.03 0.25 0.50 4.0 0.03 0.03 0.03 0.03
P. lilacinus 32 >16.0 >16.0 >8.0 >8.0 0.50 4.0 0.25 0.50 >16.0 >16.0 >16.0 >16.0 >16.0 >16.0
MIC50, MIC causing inhibition of 50% of isolates; MIC90, MIC causing inhibition of 90% of isolates; AMB, Amphotericin B; ITC, itraconazole;
VRC, Voriconazole; POS, Posaconazole; CPF, Caspofungin; ANF, Anidulafungin; MCF, Micafungin.
a Table 39.1 displays MIC and MIC a in μg/mL of antifungal agents tested.
50 90

Paecilomyces 311
contact lenses and trauma have also been described. It can mellitus [43], and complicating hairy cell leukemia in a mid-
cause endophthalmitis, keratitis, corneal ulceration, infection dle-aged woman [44]. Cellulitis, subcutaneous infections,
of the lacrimal sac, and orbital granuloma. The first reported and multifocal osteomyelitis have been described in renal
case of endophthalmitis due to P. lilacinus resulted from transplant recipients and in a patient with chronic granu-
surgical procedure to treat glaucoma in Argentina [11]. An lomatous diseases [11,45]. Finally, a case of disseminated
endophthalmitis epidemic caused by P. lilacinus occurred in infection in a neutropenic patient with leukemia was recently
the United States in 1975. A total of 13 cases were collected reported [7].
and it was proved that the infection resulted from the inser- In addition, P. variotii has been isolated from deep sites
tion of an intraocular lens that had been sterilized in sodium after surgical procedures. It was collected from the airways
hydroxide and neutralized in contaminated sodium bicarbon- of a pediatric patient with cystic fibrosis who underwent
ate solution [22,23]. bilateral living-donor lobar lung transplantation [46]. This
P. lilacinus has been involved in a number of cutane- microorganism was found to colonize a cerebrospinal fluid
ous and subcutaneous infections. These mycoses occur shunt of a woman who underwent ventricle-peritoneal shunt
mainly in solid organ and bone marrow transplant recipi- placement for noncommunicating hydrocephalus [47]. It
ents, although surgery and primary or acquired immuno- also was isolated from the renal pelvis at ureterolithotomy
deficiency are also relevant predisposing factors [2,24–26]. of a patient who had a long history of nephrolithiasis [48].
There are also cases of histopathologically proven cuta- In these cases, infection was noninvasive but the isolation of
neous infections due to P. lilacinus in immunocompetent that fungus further complicated the approach to postsurgical
patients [27,28], even one in a healthy patient without any management and antimicrobial therapy.
apparent portal of entry [29].
Other entities have also been associated with P. lilaci- 39.1.3 Treatment of Paecilomyces Infections
nus. Several cases of sinusitis have been reported in patients
with predisposing causes. Diabetes mellitus and neutropenia There are no guidelines to treat these infections. But accord-
have been reported as risk factors of these infections [4,30]. ing to expert recommendations, the therapeutic strategy
A chronic maxillary sinusitis was described in an immu- should be based on three points, that is, surgery, antifun-
nocompetent patient [31], and one more infection of the gal treatment, and adjuvant therapy to control predispos-
paranasal sinuses was reported in a pediatric patient with- ing causes. When possible, extensive surgical debridement
out predisposing causes [32]. Nasal obstruction, discharge, of necrotic and infected tissue and replacement of infected
telecanthus, diplopia, and epiphora were the symptoms prosthesis should be done. Antifungal treatment should be
observed. administered taking into account the susceptibility profile
P. lilacinus has also been implicated in catheter-related of those species. Infections by P. lilacinus should be treated
fungemia in an immunocompromised pediatric patient [6], with voriconazole or posaconazole as it seems to be resis-
in endocarditis and subaortic aneurysm after prosthesis tant to amphotericin B and echinocandins. P. variotii unlike
implantation [33], in lung abscess in a 57-year-old man with- P. lilacinus is in vitro resistant to voriconazole and suscep-
out immunosuppression [34], in a report of pleural effusion tible to the other licensed antifungal compounds [9].
[35], an in a case of proven vaginitis in an immunocompetent Disseminated infections are difficult to treat and surgery
woman [36]. cannot be performed in most cases. The granulocyte colony
stimulating factor and granulocyte transfusion could play an
39.1.2.2 Paecilomyces variotii Infections important role in the management of these infections since
P. variotii has been mainly described as cause of endocarditis they are showing some usefulness in the therapy of other life-
and peritonitis, although other rare infections have also been threatening mycoses such as zygomycosis [49]. Concomitant
reported. Endocarditis by P. variotii has been observed in combination antifungal therapy could be another option to
prosthetic heart valves [11,37] and one complicating an aortic treat disseminated infections. No clinical data are available
valve allograft [38]. Other species of Paecilomyces have been on it, but results in vitro indicate that combination between
hardly ever described as etiology of endocarditis. An infec- triazoles and terbinafine could have synergy against both
tion afflicted a diabetic woman who developed endocarditis species. Other antifungal combinations have shown indiffer-
of the aortic valve 6 years after insertion of a porcine mitral ence [50].
valve heterograft. Infection was caused by P. javanicus in
this case [14]. 39.1.4 Diagnosis
Peritonitis by P. variotii has been normally associated with
continuous ambulatory peritoneal dialysis. Approximately In general, the fungal diagnostic methods include the
half of the patients had received multiple antibiotics before identification of disease symptoms as well as isolation
the onset of the fungal peritonitis because of either bacterial and laboratory identification of the fungus [51]. However,
peritonitis or exit site infection. No other predisposing factors in recent years, important advances in polymerase chain
seem to be related to this Paecilomyces infection [39–42]. reaction (PCR)-based methods and other molecular tools
Other rare mycoses by P. variotii have been reported. have increased their importance in the diagnosis of fungal
Pneumonia has been described in a patient with diabetes infections.

39.1.4.1 Conventional Techniques these species can trigger the immunological response as sev-
Conventional techniques for the diagnosis of this mycosis eral reports have shown [13,52–54] and subsequently, spe-
rely on the isolation and culturing of the fungus and its iden- cific antibodies could be detected.
tification including macro and micro-morphological identifi- Regarding antigen quantification, Paecilomyces produce
cation. However, the two most common species, P. variotii beta-d-glucan [55,56], an antigen that is used for the diag-
and P. lilacinus, are frequently encountered as laboratory nosis of invasive fungal infections and that is a criterion
contaminants or as saprophytic flora, and cannot be taken as of probable invasive mycoses by EORTC/MSG (European
clinically significant organisms unless the presence of fungal Organization for Research and Treatment of Cancer/Mycoses
filaments are histologically confirmed [30]. Study Group) [57]. No data are available about the clinical
Although the morphological techniques are often enough utility of that technique to detect Paecilomyces infections
to identify the clinically relevant species of these genera, since the prevalence of these mycoses is low and conclu-
they require high level of expertise from the laboratory staff. sions can not be drawn from studies published so far [58].
Another disadvantage is that these techniques are time con- Paecilomyces species have been reported as having galacto-
suming, and can often delay treatment. mannan as well, an antigen that is specific for the diagnosis
of aspergillosis. That cross-reactivity could allow the detec-
39.1.4.1.1 Direct Microscopy tion of Paecilomyces by the galactomannan quantification in
patients suffering from invasive fungal infection [59].
Material from specimens should be analyzed using 10% potas-
sium hydroxide (KOH) or optical brighteners such as calco- 39.1.4.2 Molecular Techniques
fluor. Septate hyaline hyphae may be observed in the direct
An increasing number of molecular techniques for the diag-
examination of the biological samples. However, as stated
nosis of fungal infections have been developed in the last
above, the presence of fungal filaments in tissues is compul-
few years, due to the growing prevalence of mycoses and the
sory to confirm the fungal infection. Stains of fixed tissues
length of time required for diagnosis when classical micro-
with hematoxylin and eosin, methenamine-silver or periodic
biological methods are used.
acid-Schiff show hyaline hyphae only. This technique is not
With the PCR, it is possible to multiply a given DNA
specific enough to reach species identification since other gen-
segment quickly and identify the fungus. This method has
era as Aspergillus, Penicillium or Fusarium, produce similar
provided a rapid and powerful tool to identify organisms by
structures. Lesions in tissues are caused by hyaline septate
amplifying specific DNA regions. PCR-based assays for the
filaments with acute-angle (< 45°) branching within blood
diagnosis of these species (and all mycosis) include three
vessels, causing infarction, edema, and hemorrhage [11].
common steps: (i) the selection of a specific target region of
Therefore morphological characteristics of the fungus are
DNA to identify the fungus, (ii) DNA extraction method, and
often overlooked by pathologists leading to a misidentification
(iii) a method to identify the presence of the target region.
and a delay in the prescription of the correct treatment [30,51].
Conventional PCR assays have been developed to
detect DNA from tissues or cultured strains and are tar-
39.1.4.1.2 Isolation geted mostly toward the internal transcribed spacer (ITS)
These fungi grow well on conventional culture media. region of the ribosomal DNA gene cluster. Once ampli-
However, fungi are difficult to recover from some samples, fication of the target region has been performed, PCR-
particularly from biopsies of tissues. Specimens from sterile amplified segments must be identified. This region can also
sites can be directly inoculated. Contaminated specimens be used to detect Paecilomyces spp. from clinical samples
should be treated with antibacterial agents before inocula- or to identify theses species from cultures, but no reliable
tion or be inoculated on assay media including antimicrobial data are available so far [60]. DNA segments comprising
agents in their formulation. Clinical material can be inocu- the ITS1 and ITS2 regions can be amplified with prim-
lated onto Sabouraud dextrose agar, blood agar, and choco- ers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
late agar and incubated at 28°C–30°C and 37°C. Alternative (5′-TCCTCCGCTTATTGATATGC-3′) [61].
media such as malt extract or broths such as brain heart infu- Regarding quantitative PCR-based techniques, Atkins
sion may be used for biopsies. Recovery of the fungus from et al. [51] developed a real time PCR using TaqMan probes
cultures allows for the identification of the organism at species and species-specific primers targeting the ITS region of the
level according to morphological characteristics stated earlier. rDNA to identify and detect P. lilacinus from nematode, root
Although Paecilomyces are saprophytes and laboratory soil, and clinical isolates. The test showed a high specificity
contaminants, the isolation of these organisms in specimens and authors speculated its usefulness in clinical samples.
from patients with severe immunosuppression or other pre- Haugland et al. developed quantitative PCR assays of
disposing factor should not be disregarded as contamination selected Aspergillus, Penicillium, and Paecilomyces species
and patients should be carefully evaluated. to analyze dust samples from different sources around the
United States. The assay incorporated fluorigenic 5′ nuclease
39.1.4.1.3 Serology and Antigen Detection (TaqMan) chemistry and was directed at the nuclear ribo-
There are no specific serological techniques developed for somal RNA operon, internal transcribed spacer regions (ITS1
the detection of Paecilomyces in clinical samples. However, or ITS2). The assay showed to be reliable for the detection of

Paecilomyces 313
Paecilomyces species, but nothing is known on its applica- 11. Transfer supernatant to a new tube and add an
tion in clinical samples [62]. equal volume of phenol:chloroform:isoamyl alcohol
(25:24:1) and vortex for 30 s. Centrifuge 15 min a
12,000 rpm at 4°C–8°C.
39.2 Methods 12. Remove supernatant and extract with an equal vol-
No particular procedures exist to be used in the molecular ume of chloroform:isoamilic alcohol (24:1).
diagnosis of Paecilomyces. DNA extraction, amplification, 13. Vortex for 30 s and centrifuge for 5 min at 12,000 rpm
sequencing, and classification follow the general principles at 4°C–8°C.
of mycological taxonomy. 14. Take supernatant and precipitate DNA with isopro-
panol. Centrifuge for 15 min at 12,000 rpm at room
temperature.
39.2.1 Sample Preparation 15. Aspirate supernatant and wash the pellet with 70%
ethanol. Centrifuge at 12,000 rpm for 10 min at
39.2.1.1 Clinical Specimens 4°C–8°C.
Adequate specimens for detection of species of Paecilomyces 16. Aspirate supernatant and suspend the pellet again in
are similar to those for other fungal species. Ocular exudates 25–50 μL RNAase (1 mg/mL). Incubate at 37°C for
from oculomycosis, skin scrapings or biopsies from cutane- 1 h.
ous infection, aspirates from sinuses in patients with sinusitis,
bronchoalveolar lavages from pulmonary lesions, and biop-
39.2.1.3 DNA Extraction from Clinical Samples
sies of tissues in disseminated infections have been shown
useful for the detection of these species. Blood cultures Different DNA extraction kits can be employed depending on
were positive in case of endocarditis. No data are available the clinical sample. In-house methods can be used as well.
on reliability of that specimen in other sort of Paecilomyces There are some examples of commercial kits available to
infection. Sample transportation should follow the regular extract DNA such as Wizard (Promega) and QiAmpTissue
recommendations on biosafety, delivery, and storage. (Qiagen), which can be used to extract DNA from tissues, and
NucleoSpinBlood (Macherey-Nagel) ) and QiAmp (Qiagen)
39.2.1.2 DNA Extraction from Cultured Strains to be used with blood and other fluids (serum, bone marrow
aspirate, bronchoalveolar lavage, etc.). Manufacturer’s rec-
Fungal DNA extraction is the first step in the molecular diag-
ommendations must be strictly followed if these kits are used
nosis. There are several commercial and in-house methods
in the clinical laboratory.
to perform this action. The extraction method described by
Maresca et al. [63] is included below:
1. Subculture the isolated fungus in malt extract agar
(2% malt extract) or potato dextrose agar until vis- 39.2.2.1 PCR Identification
ible growth at 30°C ± 2°C. A description of PCR procedures used in the Spanish
2. Add distilled water to the subculture and scrape Mycology Reference Laboratory to detect Paecilomyces
with a swab. DNA is included.
3. Inoculate plastic Petri dish containing 3 mL GYEP The Spanish Reference Laboratory used a panfungal
(0.3% yeast extract, 1% peptone, 2% glucose) with PCR-based technique to detect DNA of these species by ITS
50–100 μL of the spore suspension obtained. fragment amplification. There are several universal primers
4. Incubate until growth at 30°C ± 2°C. to amplify the ITS regions. Table 39.2 displays the most com-
5. Harvest mycelium with a plastic tip and put on a monly used.
Whatman paper. It is important to dry mycelium Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)
completely. and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) can be
6. Transfer dry mycelium to a 50 mL propylene tube used to amplify the complete region ITS1–5.8s–ITS2 of
with screw cap and add six glass beads (4 mm Paecilomyces species. The methods used are as follows:
diameter).
7. Introduce the tube into liquid N2 for 30 s and vortex 1. The reaction mixtures contain 0.5 μM of each primer,
for 30 s. 0.2 mM of each deoxynucleoside triphosphate, 1.5 mM
8. Repeat operation until all mycelial powder is MgCl2, 2.5 U Taq DNA polymerase, and 25 ng of
obtained. DNA in a final volume of 50 μL of PCR buffer.
9. Add 0.8 mL of extraction buffer (0.2 M Tris–HCl, 2. Cycling conditions include 1 initial cycle at 94°C for
0.5 M NaCl, 10 mM EDTA, sodium dodecyl sul- 2 min, followed by 35 cycles of 30 s at 94°C, 45 s at
fate [SDS] 1%) and 0.8 mL of phenol:chloroform: 56°C, and 2 min at 72°C, with 1 final cycle of 5 min
isoamilic alcohol (25:24:1) and shake gently. at 72°C.
10. Transfer to an eppendorf and centrifuge at 3. The reactions products must be analyzed in a 0.8%
12,000 rpm for 15 min at 4°C–8°C. agarose gel.

7700. The reaction was made up to 20 μL using

Table 39.2 ultrapure dH2O. Universal thermal cycle protocol
Sequences of Primers Used to Amplify ITS Regions was used for PCR amplification: Stage 1, 50°C for
ITS-1-5,8s-ITS2 2 min; stage 2, 95°C for 10 min (activation of Taq
DNA polymerase); and stage 3, 50 cycles of 95°C
Primer Sequence (5′–3′) Size (bp)
for 15 s and 60°C for 1 min. The amplicon generated
ITS1 TCCGTAGGTGAACCTGCGG 600–630 was 130 bp for P. lilacinus.
ITS4 TCCTCCGCTTATTGATATGC
ITS3 GCATCGATGAAGAACGCAGC ITS3 + 4 = 330
ITS2 GCTGCGTTCTTCATCGATGC ITS1 + 2 = 290 39.3 C
onclusions and
ITS5 GGAAGTAAAAGTCGTAACAAGG ITS5 + 4 = 645
Future Perspectives
Source: White, T.J. et al., PCR Protocols: A Guide to Methods and According to latest studies Candida, Aspergillus and, in
Applications, Innis, M. (Ed.), Academic Press, San Diego, CA,
some geographical areas, the Zygomycetes are the most com-
1990, pp. 315–322.
mon causes of opportunistic fungal infections. However,
severe infections due to other rare and uncommon fungal
species are increasingly reported. It is hard to know the real
The above conditions are examples, and each PCR-based prevalence of emerging pathogens such as Paecilomyces but
assay should be optimized in each laboratory. it seems that their frequency could be growing.
The identity of Paecilomyces can be further verified Several points have been signaled as causes of the rise
by sequencing analysis of the amplified product. Prior to of opportunistic infections and the emergence of rare molds.
sequencing, the PCR product is purified. There are several Environmental changes, antimicrobial pressure, and an
commercial kits available to perform this purification and expanding population of immunocompromised hosts and of
are well-known for experts working in molecular procedures. patients with predisposing factors have been argued in many
For sequencing reactions, Spanish Reference Laboratory uses publications. In addition, an efficient control of more preva-
BigDye Terminator Cycle Sequencing Ready Reaction by lent species could be extending the niche of rare and may be
Applied Biosystems Inc, typically with 1 μM of the primer more resistant species. The use of voriconazole for the treat-
and 3 μL of the purified PCR product in a final volume of ment of aspergillosis and of echinocandins as empirical treat-
10 μL. ment of invasive fungal infections have been claimed to be
responsible for the increase of mycoses due to multi-resistant
39.2.2.2 Q uantitative PCR Detection emerging pathogens such as zygomycosis or of infections by
of Paecilomyces Species Scedosporium or Fusarium [64].
Two real-time PCR-based assays to specifically detect In addition, there has been an increase in number of
Paecilmyces have been developed [51,62]. Atkins et al. devel- experts in Medical Mycology with capabilities to identify
oped an assay for the identification and detection of P. lilacinus rare fungi such as Paecilomyces at species level. Moreover,
from nematode, root soil, and clinical strains. This technique there is a novel interest in identifying pathogens at species
could be applied in clinical laboratory as it showed good rates level since distinct alternatives with specific spectrum of
of reproducibility and specificity, and a low limit of detection. action are available to treat patients. From our point of view
A summary of this assay is attached below: emergent fungal infections will continue to increase in those
clinical settings where experts such as microbiologists, infec-
1. Real-time PCR reactions were performed in tious diseases experts, hematologists, and pediatricians are
Bioplastics (EU) 96 × 0.2 mL PCR plates capped interested in fungal identification at species level.
with Bioplastics EU Optical thin wall eight-cap The differentiation between these Paecilomyces species is
strip low background (BIOplastics BV, Landgraaf, clinically important, since P. lilacinus and P. variotii seem
the Netherlands). to present marked differences in their in vitro susceptibility
2. Stratagene Brilliant QPCR Master Mix TaqMan to the antifungal agents. P. lilacinus is resistant to amphoteri-
Kit was used for real-time reactions (Stratagene, cin B, itraconazole, and echinocandins, while P. variotii is in
Amsterdam, the Netherlands). vitro resistant to voriconazole only.
3. Primers PLrtF and PLrtR were included at a final Molecular methods of identification and classification of
concentration of 900 nM each and the TaqMan these species can be more useful from a clinical point of
probe (PLrtP) was used at 225 nM. view than conventional techniques. A panfungal PCR-based
4. Additional MgCl2 was added to increase the con- technique is able to identify Paecilomyces spp. from cul-
centration to 3.5 mM. tures by ITS fragment amplification. Quantitative specific
5. The automated ABI Prism 7700 sequence detector PCR methods are useful to detect these species in envi-
(Applied Biosystems) was used for reactions. ronmental and animal samples and even to identify them
6. Reference dye was added in accordance to the man- from cultures, but no data are available yet about useful-
ufacture’s recommendations for use with the ABI ness of these molecular methods in clinical samples. Direct

Paecilomyces 315
detection of fungal DNA from clinical samples could be 19. Quance-Fitch, F.J., Schachter, S., and Christopher, M.M.,
soon the reference procedure for the diagnosis of invasive Pleural effusion in a dog with discospondylitis, Vet. Clin.
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20. Naggie, S. and Perfect, J.R., Molds: Hyalohyphomycosis,
phaeohyphomycosis, and zygomycosis, Clin. Chest. Med., 30,
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40 Paracoccidioides
Eduardo Bagagli, Sandra de Moraes Gimenes Bosco,
Virgínia Bodelão Richini-Pereira, Raquel Cordeiro Theodoro,
and Sílvio Alencar Marques
Contents
40.1 Introduction.......................................................................................................................................................................317
40.1.1 Paracoccidioidomycosis (PCM): PCM Infection and PCM Disease....................................................................317
40.1.2 Mycological and Ecological Features of Paracoccidioides brasiliensis...............................................................317
40.1.3 P. brasiliensis Detection: Clinical and Environmental Sources...........................................................................318
40.2 Methods............................................................................................................................................................................ 320
40.2.1.1 DNA Extraction..................................................................................................................................... 321
40.2.1.2 PCR Protocols........................................................................................................................................ 322
40.2.1.3 Molecular Strategies to Differentiate Cryptic Species of P. brasiliensis Complex............................... 322
References.................................................................................................................................................................................. 326
40.1 Introduction The chronic form of PCM disease corresponds to 90% of

the cases and is often observed in men between 40 and 60
40.1.1 P
aracoccidioidomycosis (PCM): years old, whose main occupation is agricultural work [1,5].
PCM Infection and PCM Disease These patients in general present some predisposing factors,
such as smoking, alcoholism, and innutrition, and the main
Paracoccidioidomycosis is the most important and preva-
clinical manifestation is the involvement of the lungs. The
lent systemic mycosis in Latin America, mainly in Brazil,
pathogen can also present hematogenic dessemination to
Colombia, and Venezuela. In these endemic areas, while
other sites such as brain [7,8], spinal cord [9], bone [10], bone
PCM infection seems to occur at high frequencies, with
marrow [11], genital tract [12,13], adrenal glands [14], oral
no clinical symptoms, PCM disease occurs less frequently
cavity [15,16], and cutaneous tissues [17].
(0.1–3 cases per 105 individuals, approximately), affecting
Autochthonous cases of PCM are observed exclusively
both immunocompromised and immunocompetent indi-
in regions of Latin America; however, imported PCM cases
viduals [1]. In Brazil, the disease represents the eighth most
have also been observed worldwide, mainly resulting from
common cause of death from chronic or recurrent types of
the increase in immigration of individuals from Central and
infectious and parasitic diseases, with a mean annual mortal-
South America to other regions [18–26]. In such imported
ity rate of 1.45 per million inhabitants [2].
cases, the diagnosis is often delayed because PCM disease fre-
Two distinct clinical forms of PCM disease have been
quently imitates other well-known diseases like tuberculosis
observed: the acute–subacute form, which affects mainly
or sarcoidosis [27]. It is important to call attention to the long
young patients and evolves quickly as soon as the infection
period between the infection and the disease development in
takes place, and the chronic form, which usually affects
these imported cases, which in three well-documented ones
adults and has a long period of latency [3]. The acute–sub-
were, respectively, 25, 38, and 50 years, causing many cases
acute form of the disease, also called juvenile, may be classi-
to remain undiagnosed without suspicion of PCM [22].
fied into moderate and severe, according to the dissemination
level. It occurs in young adults under 30 years old of both
sexes and represents around 5%–10% of the total casuistry of 40.1.2 Mycological and Ecological Features
the disease. The main clinical manifestation is the involve-
of Paracoccidioides brasiliensis
ment of the monophagocytic system showing fever, weight
loss, and diffuse superficial and intraabdominal adenopa- Paracoccidioides brasiliensis is a thermally dimorphic fun-
thy with or without hepatosplenomegaly. Mucocutaneous gus that grows as yeast in the host tissues or when cultured at
involvement may be associated, however, in no more than 35°C–37°C and as mycelia under saprobic conditions or when
5% of the cases [3–6]. cultured at room temperature, 18°C–23°C. The teleomorphic
317

18°C–23°C 35°C–37°C (MLST) [55–57], microsatellite analysis [58], and prp8 intein
sequencing [59]. One of these four cryptic species, commonly
denominated Pb01-like, differs substantially from the others
(S1, PS2, and PS3), and the divergence time between it and
the remaining ones was estimated as 30 mya. This phyloge-
netic species represented by the Pb01-like genotype has been
described as a new taxon, Paracoccidioides lutzii, in honor
of Adolpho Lutz, a physician who first recognized PCM and
its agent in 1908 [57,60].
Although the occurrence of distinct genotypes in P. brasil-
iensis appears to interfere directly in the serological diag-
nosis, its implications for clinical and treatment responses
Mycelia Yeast have not yet been properly evaluated. For example, patients
Figure 40.1 Thermal dimorphism of P. brasiliensis species.
infected with Pb01-like genotype present negative serologi-
The arrow indicates the conidia produced during the mycelia phase. cal results, or very low positivity, by the immunodiffusion
test when its serum is challenged against antigens produced
by the other genotypes of P. brasiliensis [61,62]. The dis-
or sexual phase is still unknown [28]. The exact places where covery of cryptic species in P. brasiliensis has increased the
the fungus occurs in nature have not been completely deter- necessity of comparative studies aiming to detect relevant
mined, while several pieces of evidence indicate that its phenotypic differences and molecular markers among the
saprobic form may occur in some restricted soil conditions, species in order to provide the correct diagnosis, treatment,
producing the infective propagula in the form of arthoco- and prophylactic measures.
nidia and aleuroconidia that cause primary infection in the
lung by the airborne route [29,30] (Figure 40.1). Natural 40.1.3 P. brasiliensis Detection: Clinical
infection in several wild and domestic animals has been
and Environmental Sources
observed by intradermal, serological, and molecular tests
[31–34]. The fungus has been repeatedly isolated from arma- PCM diagnosis is usually made by microscopic detection
dillos, in which scarce granulomas with fungal elements have of multi-budding yeast cells in direct mycological exami-
been observed, suggesting that PCM disease may also occur nation and/or fungal cultures from clinical specimens,
in this wild mammals [35–39]. Naturally, PCM disease was histopathology of biopsied tissue, and also by serological
also reported in dogs with generalized adenomegaly [40,41]. tests, mainly with gp43, the immune dominant antigen of
Morphological and molecular findings have confirmed P. brasiliensis [28].
that P. brasiliensis and the main pathogenic fungi that cause Although a definitive diagnosis can be made through fun-
systemic mycosis are phylogenetically related, thus support- gal culture, this procedure involves high contamination risk,
ing its classification into Ajellomycetaceae, a new family of long incubation period, and low sensitivity [63,64]. In histo-
ascomycetes from the Onygenales order [42,43]. All members logical sections, the etiological agent might be undetected in
of this family, which also includes the species Blastomyces some pauci-cellular samples or confused with other dimor-
dermatitidis, Histoplasma capsulatum, Emmonsia parva, phic fungi [65]. Several serological techniques using differ-
E. crescens, and Lacazia loboi, might have evolved in close ent P. brasiliensis antigen types have been employed for both
association with vertebrate hosts [42,44,45]. The knowledge diagnosis and monitoring therapy of patients; however, some
of the correct phylogenetic and taxonomic position of P. problems such as anergy, poor production of antibodies, cross
brasiliensis has contributed to a better comprehension on the reactivity, and the sensitivity and specificity of the technique
fungus’s lifestyle and/or strategies that the pathogen presents must be considered [66]. Besides that, patients infected with
both to survive in nature and to interact with the hosts as well, different P. brasiliensis genotypes can respond differently to
including humans. For example, some clinical aspects of serological assays, as already mentioned in this chapter.
PCM disease, including its characteristic long latency period Molecular approaches for detecting P. brasiliensis, both
and its tendency to develop into a chronic form and to pres- in several clinical specimens and in environmental sources,
ent relapses, might be greatly shaped by the long evolution of have been increasingly used, exploring different genomic
the pathogen in close association with vertebrate hosts, rather regions as targets to develop primers and/or probes. Table
than into a free-living decomposer fungus [46,47]. 40.1 summarizes the primer/probe sequences and their appli-
Like all living organisms, P. brasiliensis presents a signif- cations for fungal detection.
icant variability, which has been well documented by differ- Goldani et al. [67] designed specific primers, whose target
ent mycological, antigenic, and virulence studies, as well as is based on a specific sequence of β-actin gene from P. brasil-
by molecular techniques [48–54]. Instead of being a unique iensis, and, therefore, they are not detected in other fungi such
species, P. brasiliensis appears to contain at least four dif- as H. capsulatum, B. dermatitidis, Cryptococcus neoformans,
ferent groups of genotypes (cryptic species), as observed by Candida albicans, Aspergillus fumigatus, Saccharomyces
the phylogenetic studies through multi-locus sequence type cerevisiae, or Pneumocystis carinii. Later, Goldani and Sugar

Paracoccidioides 319
TABLE 40.1
Primers and Probes for the Molecular Detection of P. brasiliensis
Primers/ Amplicon
Probes Gene Primer Sequences (5′–3′) Size (bp) Evaluated Samples References
Primer 1 β-actin TCGTTATCCTCATCGAA 62 Fungal culture, serum (inoculated mice) [67,68]
Primer 2 AAGAGTCTTCCCTCGC
PC1 gp43 TCATCTCACGTCGCATCTCACATT 1030 Clinical (sputum) [69]
PC5 AGCGCCAGATGGTTTGCCCGCTAGGAACGAA
PC2 ATAGAGGGAGAGCCATATGTACAAGGT 600
PC6 GGCTCCTCAAAGTCTGCCATGAGGAAG
para I gp43 AAC TAG AAT ATC TCA CTC CCA GTC C 355 Fungal culture, tissue (mice inoculated [65,70]
para II TGTAGACGTTCTTGTATGTCTTGGG and paraffin embedded)
para III GATCGCCATCCATACTCTCGCAATC 196
para IV GGGCAGAGAAGCATCCGAAATTGCG
LO p27 CAACTCTTGGCTTTGGTTGAAG 536 Tissue (armadillos, inoculated mice), soil [71,72]
UP CTGTTGTTTCCGTCCTTGCGC artificially contaminated
MG2(1)F Genomic GGGATTCCCTAGGCAAACACTTGTGTGA 285 Fungal culture, clinical (sputum, CSF) [73]
MG2(1)R DNA GTGCAGTTATCCACAAGCCATATATTC
MG2(2)F GGAGATGATCTGACGTTAGTACGTGATG 288
MG2(2)R ATGCTAATTTATGTCATTCCGCGTCTG
OL3 rRNA CTCAGCGGGCACTT 203 Fungal culture [74]
UNI-R GGTCCGTGTTTCAAGACG
PbITS1sa rRNA CCGCCGGGGACACCGTTG 418 Fungal culture [75]
PbITS3aa AAGGGTGTCGATCGAGAG
Pb-ITS-Ea rRNA GAGCTTTGACGTCTGAGACC 387 Fungal culture, tissue (armadillos and [33,76,77]
Pb-ITS-Ra AAGGGTGTCGATCGAGAGAG road-killed wild animals), soil
OliPbMB1 rRNA ACCCTTGTCTATTCTACC 144 Fungal culture, clinical (blood, sputum, [78]
OliPbMB2 TTACTGATTATGATAGGTCTC and skin biopsy)
PbMB1b rRNA cgcgatCGCCGGGGACACCGTTGAAatcgcg —
Source: Adapted from Richini-Pereira, V.B. et al., Mem. Inst. Oswaldo Cruz, 104, 636, 2009.
a Inner primers used in nested PCR, after the first PCR with panfungal sets of primers such as ITS1-ITS4 or ITS4-ITS5 [79].
b Probe used in the real-time PCR to detect the OliPbMB1/2 amplicon.
[68] used the same set of primers and obtained positivity in Since gp43 was shown to be one of the most polymor-
sera from five experimentally infected mice. phic genes that clearly separates the four cryptic species of
Polymerase chain reaction (PCR) assays targeting gp43 P. brasiliensis [55–57], it is now indicated for diagnostic pur-
gene are widely used for molecular detection of P. brasiliensis poses and genotyping, by PCR, of clinical samples. By using
DNA in several samples. Cisalpino et al. [80] cloned and char- this strategy, Ricci et al. [81] detected the pathogen and dif-
acterized the entire coding region of the gp43 gene, which is ferentiated it between S1 and PS2 genotypes from paraffin-
constituted by two exons interrupted by a 78 bp-intron. After embedded tissue.
this study, Gomes et al. [69] combined five primer pairs and As new genomic regions of the fungus became known, addi-
suggested a PCR using the PC2–PC6 primer pair for direct tional primers for molecular detection have been described.
amplification from clinical material (sputum) or nested PCR For example, the gene that codes for a 27 kDa antigen protein
following the PC1–PC5 primer set amplification. In an experi- of P. brasiliensis was also cloned and sequenced [82], per-
mental infection with H. capsulatum and P. brasiliensis mitting the design of specific primers (LO and UP) that were
yeasts cells, Bialek et al. [65] employed a nested PCR, also used in ecological studies in tissue samples of armadillos,
based on gp43 gene, with high sensitivity and specificity for experimental infection, and contaminated soil [71,72]. After
the sets of primers para I/II and para III/IV. Using these same observing the common presence of a 0.72 kb fragment, gener-
sets of primers, Ricci et al. [70] demonstrated 30% positivity ated by random amplification of polymorphic DNA (RAPD)
in pathogen detection in biopsies from PCM patients. Those with the arbitrary primer OPG18 (Operon Biotechnology), in
authors emphasized that this low positivity might be due to the a large number of different P. brasiliensis isolates, from dis-
procedures used for fixation, paraffin embedding, and stor- tinct geographic origins [49], San-Blas et al. designed specific
age of the material, which favored DNA degradation, and the primers (MG2(1)F/MG2(1)R and MG2(2)F/MG2(2)R) based
use of primers from gp43 regions that have been proven to be on this region, which were specific and highly sensitive for P.
polymorphic. brasiliensis. These primers were capable of identifying the

PbITSE
PbITS1s
OliPbMB1 OL3
18S ITS1 5.8S ITS2 28S
PbMB1-probe UNI-R
OliPbMB2 PbITSR
PbITS3a
Sequence Similarity to P. brasiliensis Cryptic Species

Primer/Probe
S1 PS2 PS3 Pb01-Like
UNI-R + + + +
OL3 – – – +
PbITS1s + + + +
PbITS3a + + + –
Pb-ITS-E + + + +
Pb-ITS-R + + + –
OliPbMB1 + + + –
OliPbMB2 + + + –
PbMB1 + + + +
Note: +: Complete similarity; –: Incomplete similarity.
Figure 40.2 Scheme of primers and/or probe annealing in rRNA region and their similarity to the four species of P. brasiliensis complex.
pathogen in sputum and in cerebrospinal fluid samples from is based on just one-step PCR, it minimizes the contamination
patients with different clinical forms of the disease [73]. risks [78].
The rRNA region, compounded by 18S, internal tran- Since P. brasiliensis actually consists of a species complex
scribed spacer (ITS) 1, 5.8S, ITS2, and 28S, has been widely with distinct genetic groups, and given that new still undiscov-
employed for molecular detection and distinction of different ered genotypes will probably be detected in the near future,
fungal species, including P. brasiliensis (Figure 40.2). The molecular protocols for P. brasiliensis detection should be
ribosomal subunit regions (28S, 5.8S, and 18S) are known routinely reevaluated, both in silico and experimentally, in
to accumulate mutations at a lower rate than the ITS regions order to confirm their homology to the target sequences and
over time. The first regions are used to establish phylogenetic their potential to discriminate among the several genotypes
relationships among taxonomic levels above genus, and the that occur in the endemic areas. For example, most of the
second ones are useful for the separation between genera and rRNA primers designed until now that are supposed to be
species, being considered the main candidate for the barcod- species specific for P. brasiliensis do not have complete simi-
ing system of fungal identification [83]. Furthermore, this larity to all P. brasiliensis species (Figure 40.2).
target provides higher sensitivity in PCR due to its several
copies per genome (more than 100 copies) [79,84].
Using panfungal oligonucleotides, as outer primers, 40.2 Methods
which anneal in 18S and 28S [79], Imai et al. [75] as well as
Theodoro et al. [76] designed species-specific inner primers
derived from ITS-5.8S rDNA (PbITS1s/PbITS3a and PbITSE/ Despite the fact that P. brasiliensis presents hematogenic dis-
PbITSR, respectively) for P. brasiliensis detection in nested semination to other tissues, blood and serum from patients
PCR. This approach proved to be useful for fungal detection with chronic PCM do not represent the samples of choice for
in soil [76,77], road-killed wild animals [33], and also in clini- molecular diagnosis, neither for PCR nor real-time PCR, even
cal samples, such as skin biopsies and cerebrospinal fluid (data when carried out with primers derived from the rRNA, a step
not published). Motoyama et al. [74] proposed the OL3 and that substantially increases the detection sensitivity [73,78].
UNI-R primers for PCR, also from the rRNA region (ITS2 The main clinical samples employed for molecular detec-
and 28S), and obtained a 203 bp fragment when P. brasiliensis tion of P. brasiliensis are sputum, skin biopsies, ganglionary
(Pb01 genotype) DNA was used as the template. These prim- secretion, cerebrospinal fluid, and paraffin-embedded tissue.
ers were capable of discriminating between Pb01-like isolates Special attention is given to the collection of skin biopsies, in
and H. capsulatum. Based on the same region, a real-time which the tissue fragment must be collected by puncturing
PCR, using a beacon probe, was successfully applied for P. the most infiltrated skin lesions or on the border of ulcerated
brasiliensis detection in clinical samples. This approach is lesions. Since P. brasiliensis is a fungal pathogen of biosafety
considered even more sensitive than nested PCR and since it level 2, it is recommended that the procedures, which involve

handling the fungus and clinical specimens, should be per- 6. The aqueous phase must be re-extracted with PHE/
formed within a class II safety cabinet. CHL/IAA (25:24:1) twice and with CHL/IAA (24:1)
once. After each procedure, centrifuge as already
40.2.1.1 DNA Extraction described.
40.2.1.1.1 Fungal culture 7. Add 1000 μL of cold isopropanol and 10 μL of
sodium acetate 3M and incubate for 20–60 min at
1. Transfer yeast cells or fragment mycelia of P. brasil-
−20°C or overnight at 4°C.
iensis to a 2.0 mL microcentrifuge tube (Safe-Lock
8. Centrifuge at 14,000 rpm for 15 min at 4°C and dis-
type) containing 500 mg of acid-washed glass beads
card the alcohol.
(0.4–0.6 mm diameter, Sigma, St. Louis, MO) and
9. Wash the pellet twice with 500 μL of cold 70%
500 μL of 125 mM EDTA/1 M sorbitol solution.
alcohol.
2. Homogenate in a Precellys (Precellys 24, Bertin
10. Dry the pellet and then resuspend with 50 μL of
Technologies) in three cycles of 40 s at 20 s inter-
ultrapure water.
vals. Alternatively, vortex at the highest setting for
11. The purity and concentration of DNA can be deter-
5 min, in a common laboratory shaker. Precautions:
mined in a drop-and-measure spectrophotometer or by
Tubes must be appropriate to avoid disruption.
2% agarose gel electrophoresis, using a known DNA
3. Centrifuge at 13,000 rpm for 10 min at room tem-
marker as standard (Low Mass Ladder, Invitrogen).
perature (around 22°C) and remove the supernatant.
12. Store at −20°C.
4. Add 500 μL of extraction buffer (100 mM Tris–HCl,
20 mM EDTA, and 1% SDS) and 20 μL of protein- 40.2.1.1.3 Tissue samples (Procedure
ase K (20 mg/mL) to the pellet of cells and beads. II—without phenol)
Incubate overnight in a water bath at 54°C.
1. Grind 100 mg of frozen tissue with liquid nitrogen,
5. Add 500 μL of sodium acetate 3 M and keep the
using mortar and pestle, until the tissue becomes
microtube on ice for 1 h.
powdered.
6. Centrifuge at 13,000 rpm for 10 min at 4°C. Transfer
2. Transfer the powdered tissue to a 1.5 mL microtube
the supernatant to a new microtube containing
and add 600 μL of extraction buffer (100 mM Tris–
1000 μL of cold absolute ethanol and mix gently by
HCl, pH 7.2, 20 mM EDTA, 1% SDS, and 0.02%
inversion.
2-mercaptoethanol) and 20 μL of proteinase K
7. Centrifuge at 13,000 rpm for 10 min at 4°C and dis-
(20 mg/mL).
card the supernatant. Wash the pellet twice with
3. Incubate in water bath at 65°C for 2 h or at 56°C
500 μL of cold ethanol 70%. Centrifuge as men-
overnight.
tioned above.
4. Add 100 μL of 5 M sodium chloride solution.
8. Dry the pellet and then resuspend with 100 μL of
5. Preheat the solution of cetyltrimethylammonium
ultrapure water, homogenizing carefully.
bromide (CTAB) and sodium chloride (12.5%
9. The purity and concentration of DNA can be deter-
CTAB and 5.125% NaCl) at 65°C for 10 min.
mined in a drop-and-measure spectrophotometer or
6. Add 100 μL of preheated CTAB/NaCl solution and
by 2% agarose gel electrophoresis, using a known
gently homogenize the microtubes.
DNA marker as standard (Low Mass Ladder,
7. Add 750 μL of chloroform/isoamyl alcohol (24:1)
Invitrogen™).
and vortex for 10 s.
10. Store at −20°C.
8. Centrifuge at 14,000 rpm for 5 min at room
temperature.
40.2.1.1.2 Tissue samples (Procedure I) 9. Transfer the supernatant to a fresh tube, taking care
1. Grind 100 mg of frozen tissue with liquid nitrogen, to avoid contact with the sediment.
using mortar and pestle, until the tissue becomes 10. Add 450 μL of absolute ethanol or isopropanol kept
powdered. at −20°C.
2. Transfer the powdered tissue to a 1.5 mL microtube 11. Incubate at −20°C for 10 min and then centrifuge at
and add 600 μL of extraction buffer (100 mM Tris– 14,000 rpm for 20 min at 4°C.
HCl, pH 7.2, 20 mM EDTA, 1% SDS, and 0.02% 12. Discard the supernatant and wash the pellet twice
2-mercaptoethanol) and 20 μL of proteinase K with 500 μL of 70% ethanol.
(20 mg/mL). 13. Dry the pellet and resuspend with 50 μL of ultrapure
3. Incubate in a water bath at 65°C for 2 h or at 56°C water.
overnight. 14. The purity and concentration of DNA can be deter-
4. Add 500 μL of phenol/chloroform/isoamyl alcohol mined in a drop-and-measure spectrophotometer or
(PHE/CHL/IAA—25:24:1) and gently homogenize by 2% agarose gel electrophoresis, using a known
the microtubes). DNA marker as standard (Low Mass Ladder,
5. Centrifuge at 14,000 rpm for 15 min at room tem- Invitrogen).
perature (around 22°C). 15. Store at −20°C.

40.2.1.1.4 Paraffin-embedded tissues (ITS1 and ITS4 or ITS4 and ITS5; see Table 40.1).
DNA extraction from paraffin-embedded tissue may also be 2. Include a positive control (DNA of the pathogen)
performed by using commercial kits and with an in-house and a negative control (ultrapure water as template).
protocol. 3. Cycling conditions: initial denaturation at 94°C for
5 min, 25 cycles at 94°C for 1 min, 60°C for 2 min,
1. Cut two or three sections of 5 μm thickness (or one and 72°C for 2 min, and a final extension at 72°C for
of 20 μm thickness) and place into a 1.5 mL micro- 7 min.
tube. The sections must be cut in triplicate and with 4. The size of the ITS-5.8S amplicon of P. brasilien-
different blades. sis is around 634 bp and must be visualized in 1.5%
2. Add 1000 μL of xylol and submit the microtube to agarose gel.
a continuous vortexing for 30 min at room tempera-
ture (around 22°C). Protocol step for second amplification:
3. Centrifuge at 13,000 rpm for 5 min at room
temperature. 1. The PCR reaction is carried out in 25 μL of reaction
4. Remove the xylol while taking care to avoid contact mixture (1 × PCR buffer) (50 mM KCl and 10 mM
with the sediment. Tris–HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM dNTP,
5. Repeat steps 2–4 twice. and 1 unit of Taq polymerase—GE Healthcare) and
6. Add 500 μL of 100% ethanol and mix by inverting 10 ρM of each primer (PbITSE and PbITSR; see
the microtubes for 5 min at room temperature. Table 40.1).
7. Centrifuge at 13,000 rpm for 3 min at room temper- 2. Add 2.0 μL of amplified product of PCR reaction
ature. Remove the ethanol. (first amplification) as DNA template. Caution: this
8. Repeat steps 6 and 7 once. step must be carried out in a different room from
9. Dry the samples to evaporate the ethanol. the one in which the reaction mixture is prepared to
10. Proceed to the DNA extraction for tissue as avoid contamination.
described above (protocol I or II) or follow the 3. Include positive and negative controls from the first
instructions for an appropriate commercial kit. amplification and also include a new negative con-
trol for the nested-PCR mixture reaction (ultrapure
water as template).
40.2.1.2 PCR Protocols
4. Cycling conditions: initial denaturation at 94°C for
The majority of the several PCR protocols proposed to detect 5 min, 25 cycles at 94°C for 1 min, 62°C for 2 min,
P. brasiliensis are based on the amplification of the genes and 72°C for 2 min, and a final extension at 72°C for
gp43 and ribosomal (Table 40.1). While the gp43 gene is 10 min.
restricted to the P. brasiliensis complex, it is highly poly- 5. Amplicon of nested PCR presents a size of 387 bp
morphic and occurs as a single copy or small number of and must be visualized in 1.5% agarose gel.
copies per genome. However, the ribosomal genes occur as
multicopies per genome and with conserved and moderately
polymorphic regions that permit the design of primers, both 40.2.1.3 Molecular Strategies to Differentiate
generic and species specific. Cryptic Species of P. brasiliensis Complex
40.2.1.3.1 Using PRP8 Intein
40.2.1.2.1 Nested PCR with Ribosomal Primers The discovery of cryptic speciation in P. brasiliensis has high-
This protocol has proved to be highly sensitive for clinical lighted the need to discriminate among these species when
and environmental sampling and for detection of the three diagnosing PCM. To that end, Matute et al. [55] and Teixeira et
principal genotypes of P. brasiliensis (S1, PS2, and PS3). al. [57] accomplished multilocus sequencing typing (MLST)
However, this protocol might not work as expected for the with eight nuclear coding regions. The primers, PCR, and phy-
Pb01-like genotype, which is genetically very distant from logenetic analysis conditions for these experiments are detailed
the others and has been proposed as a new species (P. lutzii). in their articles. However, sequencing many regions for each
Therefore, a new combination of primers should be designed new isolate is too much laborious for a lab that performs rou-
and properly evaluated. tine clinical diagnosis. The challenge now is to find molecular
markers that easily differentiate among the four species from
Protocol step for first amplification: P. brasiliensis complex. For that, Theodoro et al. [59] propose
to sequence the PRP8 intein, a parasitic genetic element in the
1. The PCR reaction is carried out in 25 μL of reaction prp8 gene. An intein is transcribed and translated together
mixture (1 × PCR buffer) (50 mM KCl and 10 mM with the host gene and then it performs an autocatalytic pro-
Tris–HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM dNTP, tein splicing. It has a splicing domain in its N- and C-terminus
and 1 unit of Taq polymerase—GE Healthcare), ends responsible for the protein splicing and also a homing
10 ng of genomic DNA, and 10 ρM of each primer endonuclease (in its core) that accounts for its mobility.

Protocol steps: while the second relies on only one PCR step with specific
primers for P. lutzii isolates [57].
1. The PCR for the entire PRP8 intein is carried out in Protocol steps:
25 μL of reaction mixture containing 20 ng of genomic
DNA, 50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 1. The PCR reaction is carried out in 25 μL of reaction
0.2 mM dNTP, and 0.4 μM of each primer: RCTfwd2 mixture (10 ng of genomic DNA, 1 × PCR buffer
(5′ GCCTATCGAACCGATGTTATCC 3′) and [50 mM KCl and 10 mM Tris–HCl], 1.5 mM MgCl2,
RCTrev2 (5′ TGAACCTGGAAGCCAACGTAG 3′) 0.2 mM dNTP, 0.4 μM of each primer, and 1 unit
and 1 unit of Taq polymerase (GE Healthcare) in a of Taq polymerase—Amersham Biosciences). The
thermalcycler. The amplified fragment is ~1.9 kb. primers used in this reaction, HSPMMT1 (forward: 5′
2. The thermal cycling conditions are 94°C for 4 min fol- AACCAACCCCCTCTGTCTTG 3′) and PLMMT1
lowed by 40 cycles at 94°C for 1 min, 55°C for 1 min, (reverse: 5′ GAAATGGGTGGCAGTATGGG
72°C for 2 min and a final cycle at 72°C for 5 min. 3′) [57], anneal to an indel region exclusive to the
3. The same procedure is carried out with the primers P. lutzii species and amplify ~500 pb.
FwdIn3 (5′ CATCGCTGACCATGCTGC 3′) and 2. The reactions are performed in a thermocycler at
RevIn2 (5′ TGAAGCCGAACGTGTTTTCTG 3′) 94°C for 4 min followed by 35 cycles at 94°C for
for core amplification of the PRP8 intein (~860 kb). 1 min, 55°C for 1 min, and 72°C for 2 min and a final
4. The PCR products are identified by 1.5% agarose cycle at 72°C for 5 min. The PCR products are iden-
gel electrophoresis stained with ethidium bromide. tified by 0.8% agarose gel electrophoresis stained
5. The PCR amplicons are purified by the com- with ethidium bromide.
mercial kit GFX PCR DNA and Gel Band (GE
Healthcare), whereas the sequencing reactions are
carried out on both strands, using the DYEnamic 40.2.1.3.3 Using SNP
ET Dye Terminator Kit, code US81090 (GE Once the MLST studies have revealed the main polymor-
Healthcare). The MegaBACE 1000 DNA Analysis phisms that are conserved among isolates from the differ-
System is used for capillary electrophoresis, and ent cryptic species, we can use this information to detect
the chromatogram is visualized by the Chromas single nucleotide polymorphisms (SNP) that can be useful
program. for species identification. Among the many options available
6. The sequences are sent to blastn for comparison for the study of SNP, we have been testing the ability of the
with the NCBI database (http://www.ncbi.nlm.nih. SNaPshot® Multiplex, from Applied Biosystems, to differ-
gov/BLAST) and to blastn of the Broad Institute entiate isolates from the four cryptic species. This is a very
Web site for comparison with the sequences from simple technique in which we use a PCR product to perform
the isolates Pb18 (S1 species), Pb01 (P. lutzii), and a mini-sequencing reaction with a primer that is designed
Pb03 (PS2 species), available in the P. brasiliensis right in the upstream of the SNP, so that the first nucleotide
genome database (http://www.broad.mit.edu/anno- to be incorporated by the sequencing reaction will be the one
tation/genome/paracoccidioides_brasiliensis). Table corresponding to the SNP because all the dNTPs used in this
40.2 [59] can be useful for species identification reaction are dideoxynucleotides labeled with fluorescence
because it presents all the polymorphic positions (different for each nucleotide). Basically, we have been using
in the intein PRP8 for the four cryptic species of three SNP for species identification in P. brasiliensis com-
P. brasiliensis complex. plex, one from gp43, another from chs2, and the other from
arf. The primers used for the SNaPshot reaction and SNP
40.2.1.3.2 Using HSP70 positions for each species are listed in Table 40.3.
Teixeira et al. [57] revealed, by MLST, that the species Protocol steps:
P. lutzii is highly divergent when compared to the remain-
ing species. Furthermore, subsequent works have shown that 1. PCR of the genes gp43, chs2, and arf: The prim-
patients infected with this genotype present negative serolog- ers Gp43-E2-sense2 (5′ CTAGAATATCT
ical results, or very low positivity, by the immunodiffusion CACTCCCAG 3′) (not published yet) and Gp43-E2-
test when its serum is challenged with antigens produced by antisense (5′ GCCCCCTCCGTCTTCCATGTCC
the other genotypes of P. brasiliensis [61,62]. In this sense, 3′) [55] anneal in exon 2 of gp43 gene and
it is very important to distinguish between P. lutzii and the amplify ~700 pb. The primers CHS2 E2-4 Fwd (5′
other species. To that end, two nuclear coding regions have CTTAACGGTGCCTTCTTTGCGG 3′) and CHS2
been used: ITS1-5.8S-ITS2 and HSP70. The first one clearly E2-4 Rev (5′ GTGAAAGTATTGTTGCCCAGCG
differentiates P. lutzii from the remaining species by PCR 3′) [55] are used for chs2 amplification of a 549 pb
with ITS4 and ITS5 primers, followed by sequencing and fragment, while the primers Arf–fwd (5′ TCTCATG
blast comparisons (protocol described in Section 40.2.1.2), GTTGGCCTCGATGCTGCC 3′) and Arf–rev

Table 40.2
Polymorphism in the PRP8 Intein of P. brasiliensis Isolates Belonging to the Four Cryptic Species, according to
Theodoro et al.
Isolate or Block/Site
Groupa
A51 A78 A90 A99 A135 A159 B183 B219 B245 B246 B247 B248 B249 B250 B251 B252 B253 B254 B257 B266 B267 B279 B304
Con A G C C T G T G C C C G T C C A T T C T A T T
T1F1-like . . . . . . . . . . . . . . . . . . . . . . .
T10B1-like . . . . . . . . . . . . . . . . . . . . . . .
Bt84-like . . . . . . . . . . . . . . . . A . . . . . .
Pb262 . . . . . . . . . . . . . . . . A . . . . . .
Epm77-like . . . . . . . . . . . . . . . . . . . . . . .
Pb01 G T T G C C C A — — — — — — — — — A T C G C C
C766 C776 C825 C837 C859 C864 C879 C887 C902 C925 C937 C955 C963 C966 C970 C975 C980 C999 C1077 C1146 C1259 D1297 D1309
Con C A A G G G G T T A T A A C A G C C A T A A A
T1F1-like . . . . . . . . . . . . . . . . . . . . . . .
T10B1-like . G . A . A . . . . . . . . . . . . . . . . C
Bt84-like . G . A . A . . . . . . . . . . . . . . . . C
Pb262 . G . A . A . . . . . . . . . . . . . . . C C
Epm77-like . . . . . . . . . . . . . . . . . . . . . . C
Pb01 T . T . T A C C A T C G C T C A T T T C G . C
Source: Theodoro, R.C. et al., Fung. Genet. Biol., 45, 1284, 2008.
Blocks A, B, and F correspond to the splicing domain and the blocks C, D, E, and H to the homing endonuclease domain. Con, consensus sequence.
a T1F1-like (S1 genotype) T10B1-like; Bt84-like and Pb262 (PS2 genotype); EPM77-like (PS3 genotype) and Pb01 (P. lutzii genotype).

B310 B326 B337 B357 B405 B433 B441 C471 C484 C501 C577 C583 C625 C630 C638 C653 C672 C699 C713 C731 C738 C740
A A A T T C C G C T T T T T A C C G C A T T
. . . . . . . . . . . . . . . . . . . . . .
. . . . . . T . . . . . . . . . . . T . . .
. . . . . . T . . . . . . . . . . . T . . .
. . . . . . T . . . . . . . . . . . T . . .
. . . . . . . . . . . . . . . . . . . . . .
G T G G A G . A G A C G C G G T T A . C A C
D1312 E1338 E1351 E1371 E1378 E1413 H1443 H1470 H1486 H1491 H1530 H1548 H1561 H1606 H1609 H1632 H1645 F1692
G A G A A A G A C A G C C C A G G A
. . . . . . . . . . . . . . . . . .
. . . . . . A . . . . . . . . . . .
. . . . . . A . . . . . . . . . . .
. . . . . . A . . . . . . . . . . .
. . . . . . . . . . . . . . . . . .
C G A G G C . G G T T A A G G A A G

Table 40.3
SNP Primers Used for Mini-Sequencing Reactions to Differentiate the Four Species of
P. brasiliensis Complex
SNPs/Species
SNP
Primer Sequence 5′–3′ a Gene Positionb S1 PS2 PS3 P. lutzii
SNP3 4(C) GTATCTAAATGGGCGTGGCA GP43-exon 2 344 T T T G
SNP9 21(C) CATCCAATTTACCGTGTGGGA ARF 298 C T C C
SNP13 32(C) CTCCAGCAAAATACTCCCAGAT CHS2 (exon 2–4) 185 C C T NA
a The cytosines in the 5′ end of the primers were added in order to distinguish the different mini-sequencing products accord-
ing to their size, in case the three reactions are performed together, in the same tube.
b According to the sequences deposited by Matute et al. [55] in GenBank. NA means “no annealing.”
(3′ GAGCCTCGACGACACGGTCACGATC 5′) [55] 40.3 C

onclusions and
are used for arf amplification of a 407 pb fragment. Future Perspectives
PCR reactions are carried out in 25 μL of reac-
tion mixture (10 ng of genomic DNA, 1 × PCR buf- The dimorphic pathogenic fungus Paracoccidioides brasil-
fer [50 mM KCl and 10 mM Tris–HCl], 1.5 mM iensis occurs in large areas of Latin America and provokes
MgCl2, 0.2 mM dNTP, 0.4 μM of each primer, and PCM, a life-threatening systemic infection that still presents
1 unit of Taq polymerase—Amersham Biosciences) serious challenges for its treatment and control. The exact
The reactions are performed in a thermocycler place where the fungus occurs in nature, producing the infec-
at 94°C for 4 min followed by 40 cycles of 94°C for tive propagula, is not completely known, which hinders the
1 min (annealing temperature 55°C for gp43 and adoption of preventive measures to avoid new infections.
60.4°C for chs2 and arf ) and 72°C for 1 min and Molecular techniques have greatly improved our capacity to
a final cycle of 72°C for 5 min. The PCR products address important aspects of the pathogen and the disease.
are identified by 1.5% agarose gel electrophoresis Besides providing a better comprehension of the fungus taxon-
stained with ethidium bromide. omy, phylogeny, and ecology, the molecular approaches have
2. Purification of PCR products: 10 μL of each PCR also substantially expanded the disease diagnosis. This chap-
product is purified with 4 μL of ExoSAP-IT (GE ter presents some recent findings on P. brasiliensis molecular
Healthcare) for 1 h at 37°C, followed by 20 min at taxonomy and the state of the art in molecular detection of the
80°C to inactivate enzymes. pathogen, in both environmental and clinical materials.
3. Mini-sequencing reaction: 6 μL of ultrapure water, The discovery of cryptic speciation in Paracoccidoides
1 μL of 1/2TERM Buffer (200 mM Tris–HCl, genus has added new challenges for PCM researchers in
5 mM MgCl2, and pH9.0), 1 μL of SNaPmix (GE relation to the clinical aspects, treatment, and, especially,
Healthcare), 1 μL of the 1 ρmol/μL SNP primer the correct diagnosis. The structural genomes of the main
(listed in Table 40.3), and 1 μL of the purified PCR genetic groups or cryptic species of P. brasiliensis have
product were used for each mini-sequencing reac- been established, and several molecular identification pro-
tion. The dideoxynucleotides, which are left from tocols have already been proposed, with promising results.
the mini-sequencing reaction, are removed by treat- Additional studies that focus on population are still neces-
ing the samples with 1 μl of SAP (Shrimp Alcaline sary in order to provide a clearer picture of the pathogen
Phosphatase, GE Healthcare) for 1 h at 37°C fol- biodiversity. Therefore, the use of rapid, contamination-free,
lowed by 20 min at 80°C. and specific methodologies such as real-time PCR together
4. Electrophoresis: The reactions are diluted to 1:1 with primers and/or probes specific for all species from the
with loading buffer containing Hi-Di™ formamide Paracoccidioides genus should be implemented as soon as
(Applied Biosystems). Before loading the samples possible for routine diagnosis of PCM.
on the gel, they are heated at 95°C for 3 min and
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41 Penicillium: Mycoses and Mycotoxinoses
R.R.M. Paterson and N. Lima
Contents
41.1 Introduction...................................................................................................................................................................... 329
41.1.1 Mycosis................................................................................................................................................................. 330
41.1.1.1 Classification and Biology..................................................................................................................... 330
41.1.1.2 Clinical Features and Pathogenesis....................................................................................................... 332
41.1.1.3 Phenotypic Identification....................................................................................................................... 332
41.1.2 Mycotoxinosis....................................................................................................................................................... 332
41.1.2.1 Human Disease Etiology....................................................................................................................... 333
41.1.2.2 Animal Models of Human Disease........................................................................................................ 333
41.1.2.3 Mycotoxin Toxigenic Mechanisms........................................................................................................ 334
41.1.2.4 Taxonomy of Mycotoxin Fungi.............................................................................................................. 334
41.1.3 Molecular Diagnosis of Mycosis and Mycotoxinosis........................................................................................... 335
41.1.3.1 Molecular Identification of P. marneffei................................................................................................ 335
41.1.3.2 Molecular Studies of Mycotoxin/Occasional Mycosis Penicillia.......................................................... 336
41.2 Methods............................................................................................................................................................................ 337
41.2.1 Sample Preparation of P. marneffei...................................................................................................................... 337
41.2.2.1 Basic PCR for Fungi.............................................................................................................................. 337
41.2.2.2 PCR Identification of Mycotoxin/Occasional Mycosis Penicillia......................................................... 338
References.................................................................................................................................................................................. 340
41.1 Introduction Identifications are time-consuming, and decisions regard-

ing what represents a species tend to be subjective. The
Fungal species are difficult to identify. The organisms are standard characters for identifying and classifying filamen-
complex from a biological perspective, which contributes to tous fungi remain morphology, and the literature provides
the identification problem. Investigations involving human extensive examples of problems. Unreliable morphological
mycoses and mycotoxinoses require an improved compre- minutia to describe new species and variability within the
hension of the structure and behavior of fungal populations. morphological characters of accepted species are constant
(We have employed the term mycotoxinoses as the fungal issues. Penicillium is difficult in these respects and the situ-
equivalent of bacterial toxinoses.) However, an understand- ation in the terverticillate penicillia (heavily responsible for
ing of the speciation, population biology, phylogeny, and evo- toxin production) is particularly problematic [2,3]. In general,
lution of fungi is not so developed as for other organisms [1]. it is accepted that a “polyphasic” approach is optimal where
Classification undergoes almost continual change, par- morphological, physiological, biochemical, and molecular
ticularly since the introduction recently of nucleic acid-based biological characters are employed to characterize fungi.
methodology where, for example, “cryptic species” are Penicillium Link is the most common of the ubiqui-
revealed. These are species determined by “molecular biol- tous fungi. It is related to other genera that produce peni-
ogy” to have (significantly) different nucleic acid structures cilli (i.e., Scopulariopsis, Geosmithia, and Paecilomyces)
to other strains that are indistinguishable morphologically, to which it could be confused. The teleomorphic genera
although distinguishing morphology can often be found with anamorphs which produce penicilli are Byssochlamys,
when the genetic differences are known. Additional com- Eupenicillium, and Talaromyces. Furthermore, the genus
plexity arises as systematic mycologists must work within the can usefully be separated into subgenera where the numbers
botanical code of nomenclature that emphasizes the naming of branch points in conidiophores determine to which sub-
of sexual states (teleomorphs) over asexual vegetative growth genus strains belong. In Aspergilloides, there is one branch
forms (anamorphs). point and so are monoverticillate. The biverticillate subgenus
329

has two branch points within which there are described two 41.1.1.1 Classification and Biology
subgenera: Furcatum and Biverticillium. The most complex
41.1.1.1.1 Classification
form of the penicillus has characteristically three branch
points; these are classified as terverticillate, that is, subgenus P. marneffei was first classified into section Asymmetrica
Penicillium. The genus contains numerous similar species and subsection Divaricata (Raper and Thom) in 1959 [11],
that are difficult to distinguish and therein lie the problems which is equivalent to Pitt’s subgenus Furcatum. Frisvad and
for identifying species with human pathogenic properties [4]. Filtenborg [12] placed the species in subgenus Biverticillium
There are two related areas of concern in the present confirming Pitt’s assignation to the subgenus. A phyloge-
chapter: (i) mycosis and (ii) “myco”toxinosis. The molecular netic analysis established that the fungus is related closely to
biology of the fungi is presented in a separate section as it Penicillium subgenus Biverticillium and sexual Talaromyces
has assumed such importance and is, nevertheless, problem- species with asexual biverticillate Penicillium states [13]. It
atic [1]. is of relevance that 18S rRNA indicated that the fungus was
related closely to a Talaromyces flavus strain (and so is the
teleomorph?), second only to a near identical similarity to
41.1.1 Mycosis one of Penicillium verruculosum [14].
The most important Penicillium species causing mycosis is The first naturally occurring human case of “penicillio-
the surely now-emerged fatal systemic pathogen, Penicillium sis marneffei” was reported in 1973, of a North American
marneffei (Figure 41.1). However, other penicillia are impli- minister who lived in Southeast Asia. The second (1984)
cated in human mycoses (e.g., Penicillium spp., P. bertai, P. was a compatriot who travelled in the Far East. Tissue sec-
bicolor, P. brevicompactum, P. chrysogenum, P. citrinum, tions demonstrated yeast-like cells of P. marneffei that were
P. commune, P. crustaceum, P. expansum, P. glaucum, P. confirmed in culture. Five more cases were reported from
purpurogenum, and P. spinulosum) [5,6]. Clearly, a thorough Bangkok in the same year. Furthermore, eight cases of infec-
knowledge of Penicillium is required even for those inter- tion were reported from China in 1985 observed between
ested predominately in P. marneffei. 1964 and 1983, and 20 and 6 cases from the Guangxi region
Patients with human immunodeficiency virus are suscep- and Hong Kong, respectively, were reported shortly after
tible particularly to P. marneffei. The disease is endemic in [7]. However, from 1988 penicilliosis marneffei increased in
Asian countries such as Vietnam, Thailand, China, India, and Southeast Asia in patients with advanced HIV infection. The
Taiwan [7]. P. marneffei was first isolated from the hepatic first cases were foreign AIDS patients who had visited regions
lesions of a bamboo rat (Rhizomys sinensis) that was in cap- of endemicity [15] and HIV patients who were native to such
tivity in 1956 [8]. The importance for humans was estab- regions within Thailand [16]. However, the fungal infections
lished with the HIV pandemic in Asia: infection increased were also observed in Cambodia, China, India, Malaysia,
in local and visitor populations from non-endemic countries Taiwan, and Vietnam. External cases were detected in HIV-
[7] and only cryptococcosis and tuberculosis are of greater infected patients from Australia, Belgium, France, Germany,
significance in the relevant countries [9]. Frequent manifesta- Japan, Sweden, Switzerland, the Netherlands, the United
tions are respiratory signs, anemia, fever, weight loss, lymph- Kingdom, and the United States [7]. Finally, the infection is
adenopathy, hepatosplenomegaly, and skin lesions. Primary decreasing in areas where HIV infection is contracting (and
treatment with amphotericin B and secondary prophylaxis vice versa, e.g., Vietnam): The welcome recent reports of a
with itraconazole are effective [10]—otherwise there is poor vaccine for HIV in Thailand (U.S. Military HIV research
prognosis; infected patients who are HIV positive need sup- program [www01.hjf.org/apps/internet/hivnewscenter.nsf/
pressive therapy to prevent relapse [7]. These treatments are phase3pressrelease]) and, for example, the U.K. newspa-
fairly standard for mycoses in general. per The Guardian (http://www.guardian.co.uk/world/2009/
sep/24/hiv-infection-vaccine-aids-breakthrough, accessed
on March 24, 2011) may result ultimately in a decrease in
the fungal infection, although publication in a non-scientific
journal is somewhat worrying.*
41.1.1.1.2 Physiology
P. marneffei is the only Penicillium species where tempera-
ture-dependent dimorphic growth is reported. Dimorphism
occurs in P. chrysogenum [17] in relation to carbon starvation
and may be more widespread amongst fungi. It is well known
that temperature affects, amongst other things, atmospheric
CO2 concentrations, and hence, the temperature effect may in
reality be from other physical factors [18].
Conventional knowledge states that the fungus grows as (i)
FIGURE 41.1 Hyphae and conidiophores of Penicillium mycelia at temperatures below 37°C and (ii) yeast at 37°C [7].
marneffei. However, the yeast transition occurs at 32°C and possibly above

Penicillium: Mycoses and Mycotoxinoses 331
30°C. One strain of those tested (9%) became yeast only at 39°C of P. marneffei possessed the urease enzyme, which may be
[19], again with significant ramifications for identifications. of use in biotyping. Some heterogeneity between isolates was
Furthermore, P. marneffei produces a red pigment and observed in biochemical profiles leading to 17 biotypes being
a wide range of penicillia produce the same color [3,20]. recognized. As mentioned, more work is emerging on sec-
Mapari et al. [21] report that the P. marneffei pigments are ondary metabolite production [21,23], which will have diag-
mitorubrinol (orange-red), monascorubramine (purple-red), nostic utility.
purpactin, rubropunctatin (orange), and secalonic acid (yel-
low). Secalonic acid is a mycotoxin produced by, inter alia, 41.1.1.1.3 Ecology and Epidemiology
P. chrysogenum: The compound is also a mutagen [22] with P. marneffei was isolated from (a) rodents R. sinensis, R. pru-
implications for nucleic acid analysis of producing fungi (see inosus, R. sumatrensis, and Cannomys badius and (b) soil
[1] and below). Mitorubrinol is also produced by P. purpu- samples from bamboo rat burrows [26,27]. Typing, including
rogenum (Table 41.1) with consequences for the use of “red population genetic analyses, showed that genetically identi-
pigment” as a character used in diagnosis. A red pigment was cal strains were isolated from humans and bamboo rats [7].
characterized as being (remotely) similar to herquinone by Furthermore, genes involved in asexual development, mor-
Bhardwaj et al. [23]. Color in particular is a subjective char- phogenesis, and host immune response were reported [28].
acter about which an individual’s perception can vary mark- The human disease may occur from zoonotic (animal) or
edly. Obviously, great care is required when using these traits sapronotic (environmental) transmission. The prevalence of
as characters for identification although chemical analysis infection in rats varies widely across Southeast Asia, which
will improve objectivity. A list of red or similarly colored suggests that there are regional variations in the endemicity
pigments produced by penicillia is available in Table 41.1. or that there are geographical variations in the predisposition
The enzymatic activities of P. marneffei have been stud- to infection within different species of bamboo rats. Gugnani
ied [24]. However, characterization of the effects are required et al. [26] surveyed six species of sympatric rodents in north
to determine whether secreted enzymes are linked to viru- eastern India and found that only C. badius harbored infec-
lence and similarly for the pigments and mycotoxin dis- tion; hence, host-specific factors may govern infection. All
cussed above. Wong et al. [25] demonstrated that 32 isolates 51 animals of the grayish-black subspecies of C. badius were
negative for P. marneffei, while 3 of the 10 rats in the red-
dish-brown group were positive [29]. These studies indicate
TABLE 41.1 host and geographical components.
An accurate description of the factors governing the dis-
List of Red or Similarly Colored Pigments from
tribution of P. marneffei infection in bamboo rats is neces-
Penicillium (i.e., Excluding Yellow) sary to understand the epidemiology in human populations.
Taxon Name The endemic geographic range of P. marneffei is wide [7].
Ranges of the lesser and greater bamboo rats (Cannomys spp.
Subgenus Biverticillium
and Rhizomys spp.) follow broadly the known distribution of
P. marneffei Monascorubramine Purple red
P. marneffei. This may indicate that bamboo rats are an obli-
Rubropunctatin Orange
gate stage in the life cycle of the fungus or that the ecotype
Mitorubrinol Orange red
favored is shared with P. marneffei and they are examples
Some similarity to Red
herqueinone (see below)
of sylvatic sapronotic infections. A case-control study of
P. purpurogenum Mitorubrinol Orange red HIV-positive patients [30] did not implicate bamboo rats
PP-R Purple red as a reservoir of infection for humans. Age and an agricul-
Purpurogenome Yellow orange tural occupation were factors associated with increased risk
P. islandicum Erythroskyrin Orange red of infection. Temporal analyses of the incidence [7] showed
Skyrin Orange extensive seasonal variation in infection rates and increased
disease was associated with the rainy season. The conclusion
Subgenus Penicillium
was that soil exposure is a critical risk factor associated with
P. atramentosum Uncharacterized Dark brown
infection by P. marneffei: The fungus must have a reservoir
P. atrosanguineum Phoenicin Red
in the environment if it is a sapronosis.
Uncharacterized Red
As mentioned, a general assumption that penicillia are
P. atrovenetum Norherqueinone Red
P. cyclopium, P. freii Viomellein Red brown
associated with soil has been extrapolated to P. marneffei.
Xanthomegnin Orange
However, in Talaromyces and subgenus Biverticillium, the
P. herquei Herqueinones Red primary habitats are wood and its products, textiles, and
P. oxalicum “Arpink red” Red “bird feathers” [31]. Hence, soil may not be the principal hab-
Anthraquinone derivative Red itat: Vanittanakom et al. [32] demonstrated only 6% recovery
P. paneum Uncharacterized Red from seeded non-sterilized soil, and P. marneffei can survive
P. persicinum Uncharacterized Cherry red only for a few days in non-sterilized soil [7]. Furthermore,
P. viridicatum Viomellein Red brown P. marneffei was isolated from three soil samples collected
from R. pruinosus burrows and one out of 28 soil samples

from that of R. sumatrensis. Definitive proof of an environ- conidial [7]. Youngchim et al. [24] found an expression of
mental reservoir is lacking. Many penicillia are linked to acid phosphatase activity by P. marneffei, which is one of the
excretion, so it is perhaps surprising that examination of rat virulence factors for other intracellular pathogens. Finally an
feces has not been reported. antigenic catalase-peroxidase protein-encoding gene (cpeA)
was isolated recently from P. marneffei, by antibody screen-
41.1.1.2 Clinical Features and Pathogenesis ing of a cDNA yeast-phase library of this fungus [28].
Approximately 8552 cases of P. marneffei infection were
reported in HIV-infected patients at Chiang Mai University 41.1.1.3 Phenotypic Identification
Hospital and Thailand from 1984 to 2004. The symptoms 41.1.1.3.1 Microscopy and Culture
of 74 patients were weight loss, skin lesions, fever, hepato- Traditionally, identifications in clinical specimens are by
megaly, and generalized lymphadenopathy respiratory signs. microscopy and culture [9,33]. Presumptive diagnosis is
Observed lesions in 54 patients were papules with central made by microscopic examination of bone marrow aspirates
necrosis, and the rest were papules or maculopapules. All and/or smears of skin or lymph node biopsy specimens [36].
patients acquired lesions on the face, neck, and other sites. P. marneffei in histopathological sections can be stained var-
Six patients had papular lesions on the palate and the aver- iously and appears as fission arthroconidia or roundish cells
age number of CD4+ T lymphocytes in these patients was [7]. Definite diagnosis is from culture where bone marrow,
64 cells/mm3. Patients (76%) were anemic, with a hemoglo- blood, and skin biopsy give positive results in particular [9].
bin level of 10 g/dL or less. Importantly, some patients had Identification of P. marneffei involves the morphology of the
hepatic penicilliosis without any skin lesions. Osteoarticular colony, mold-to-yeast conversion, and microscopic morphol-
lesions were seen in multiple sites and arthritis was wide- ogy (also including production of the pigment).
spread. Multiple lytic bone lesions involving flat bones of the
skull, long bones, and small bones of the fingers were also 41.1.1.3.2 Immunological Tests
seen. However, attributing these signs to P. marneffei infec- Experimental methods include identification from
tion alone was equivocal [7]. infected guinea pigs by monoclonal antibodies [39,40].
P. marneffei is a primary pulmonary pathogen that dis- Immunoperoxidase staining was developed [41]: cells in
seminates to other organs by hematogenous spread. The skin biopsies were intracellular yeasts. Also, a specific indi-
onset of symptoms was rapid and severe in the absence of rect fluorescent-antibody reagent in histologic sections was
early treatment [33]. Necrotizing reactions with macro- devised [42]. Relevant murine immunoglobulin (IgM) mono-
phage and histiocyte infiltrations are seen in immunocom- clonal antibodies reacted in immunofluorescent staining of
promised patients. P. marneffei yeasts were observed with the yeast in tissue biopsies of patients [43]. Fine-needle aspi-
macrophages and histiocytes being oval or spherical cells ration cytology [44,45] was useful “potentially” to patients
of 2–3 μm diameter, with elongated cells of 13 μm observed in whom lymphadenopathy was confined to intra-abdominal
extracellularly. Granulomatous and suppurative reactions nodes. Interestingly, an exoantigen immunodiffusion test
were seen in tissues [7]. was described [46] differentiating P. marneffei from other
The mechanisms of infection are understood poorly: P. Penicillium species, such as P. citrinum and P. commune
marneffei conidia can recognize fibronectin and bind to lam- [47], implying these could be problematic, although Ref. [47]
inin [34,35], which are involved possibly in the attachment does not mention other fungi and so from where does this
of conidia to bronchoalveolar epithelia. Monocyte-derived information derive?
macrophages phagocytose P. marneffei. The fungus can Specific antibodies against antigens of P. marneffei in
be cleared within 2–3 weeks in healthy hosts, whereas the clinical specimens [48] have low sensitivity [49]. A fluo-
infection is fatal in nude or in T-cell-depleted mice [7]. These rescent-antibody test [50], an immunoblot assay reactive
results demonstrated that T cells are necessary for clearing with serum from patients [51], and a diagnostic antigen of
infection in mice [36]. The host immunological response to 38 kDa [52] have been reported. Antigens from P. marnef-
P. marneffei probably is activation of macrophages by T-cell- fei confirmed a humoral response [53] and an enzyme-linked
derived cytokines. Human and mouse macrophages control P. immunosorbent assay was developed [54]. A latex agglutina-
marneffei growth and kill intracellular yeast when activated tion (LA) test using a monoclonal antibody [55] and a spe-
in vitro by T-cell-derived cytokines. It was demonstrated cific LA for the detection of P. marneffei antigens were also
that protective immunity follows a Th1 response, playing devised [49]. An enzyme immunoassay for the quantification
a crucial role in host resistance to intracellular pathogens of P. marneffei [56] was used in a dot blot ELISA and an LA
[37]. Phosphoprotein osteopontin OPN, secreted from mono- test [57]. All tests were highly sensitive and specific. Finally,
cytes, is involved in the production of IL-12 from peripheral a monoclonal antibody-based sandwich ELISA was investi-
blood mononuclear cells after stimulation with P. marneffei gated [43,58], which requires further testing.
[38]. Rabbit pulmonary alveolar macrophages and circulat-
ing human mononuclear phagocytes manifested antifungal
41.1.2 Mycotoxinosis
activity against the fungus. Granulocyte-macrophage colony-
stimulating factor enhanced the antifungal activity of human This section deals with chemical substances that cause dis-
neutrophils against the yeast form of P. marneffei but not the ease rather than biological entities. Life expectancy has

increased dramatically in the developed world at least indi- Japanese government decreased the availability of moldy rice
cating that acute poisonings from toxins are not major prob- in the markets and resulted in rapid decline of the disease.
lems. However, this has not occurred in less technologically Improved diet and inspection have made cardiac beriberi of
advanced countries. In either case, there is a vast amount of little importance in modern times [62] but provide a useful
general ill health in the populations and it is required to be historical example of mycotoxin-related pathogenicity and
known to what extent mycotoxins play roles. (It has been esti- how it can be ameliorated.
mated that 20,000 deaths/year occur in Indonesia from afla- Nevertheless, the Penicillium mycotoxins of prime impor-
toxin [an Aspergillus toxin]-induced liver cancer [59].) tance are ochratoxin A and patulin, which have statutory
Mycotoxins are fungal metabolites, which cause sickness limits imposed on them in foodstuff in the European Union.
or death in people when ingested, inhaled, and/or absorbed Table 41.2 indicates from which food penicillia are found
[60]. They occur and co-occur naturally in food allowing and the associated mycotoxins. When mycotoxins are fed to
synergistic effects. Furthermore, Penicillium species are animals in combination, interactive effects can be classified
ubiquitous in nature and unavoidable, although they can as additive, less than additive, synergistic, potentiative, or
be controlled: those that produce mycotoxins are common even antagonistic [60]. Hence, in effect, many of the studies
and are associated predominantly with the terverticillate described below provide only a partial view.
penicillia, which are difficult taxonomically. The chemical There is growing concern within medicine about myco-
structures of a few are provided in Figure 41.2. Mycotoxins toxin involvement in human diseases [60]. More occult dis-
may also be considered as weapons [61] although those from ease may occur when the mycotoxin interferes with immune
Penicillium are not important in this regard at present. processes, rendering the patient more susceptible to infec-
tious diseases (cf. P. marneffei). This may explain why few
41.1.2.1 Human Disease Etiology data are available on mycotoxins being implicated in immu-
Two “named” human diseases are considered somewhat nosuppressive illnesses as the presenting diseases are more
inconclusively to be caused by Penicillium mycotoxins [62]. obvious. The morphological effects of ochratoxin A on lym-
Balkan endemic nephropathy (BEN) is presumed to be from phocytes and neutrophils from healthy subjects and patients
cereals containing ochratoxin A, and associated urinary tract with oesophageal and breast carcinomas indicated that leu-
tumors are linked to P. verrucosum contamination. Evidence kocyte death was evident from the decrease in cell survival of
for a role of ochratoxin A in BEN has accumulated, but there those exposed. The study supported that homeostasis of the
is no direct epidemiological proof at the level of individual immune system is compromised in cancer patients exposed to
subjects. ochratoxin A, including those with breast cancer. Gliotoxin
Cardiac beriberi or “shoshin kakke” is associated with may be involved in the mycosis referred to as aspergillosis
rice containing citreoviridin produced by P. citreoviride, a and Penicillium bilaii can produce this mycotoxin.
disease that occurred for centuries, including the early twen-
tieth century in Japan and other Asian countries. The dis- 41.1.2.2 Animal Models of Human Disease
ease was characterized by palpitations, nausea, vomiting, The effects of mycotoxins are usually more obvious in domes-
rapid and difficult breathing, rapid pulse, abnormal heart ticated animals and extrapolations to humans are often based
sounds, low blood pressure, restlessness, and violent ania on these observations and supported by laboratory animal
leading to respiratory failure and death. The causative agent studies [60]. Ochratoxin A in animals (i) damages kidneys,
was regarded as infection or avitaminosis, until a fungus was (ii) causes intestinal necrosis and hemorrhage, (iii) sup-
isolated and identified. An extract component was identified, presses immunity, and (iv) is carcinogenic. Dogs, rats, and
its structure elucidated, and named as citreoviridin. The neu- swine are affected with kidney problems from ochratoxin A.
rologic syndrome and respiratory failure were reproduced The impaired renal function results in glucosuria and protein-
in laboratory animals. The Rice Act of 1921 passed by the uria, with casts evident in the urine, and absorption occurs in
H3C O CH3
OH Patulin
HOOC O H3C N O
O
O H
CH3 H OH
O H
CH3 CH3
Citrinin Cyclopiazonic acid
O OH NH
CO2H O OH O
N O
H
CH3
Ochratoxin A (OTA) CI
FIGURE 41.2 Chemical structure of selected mycotoxins.

TABLE 41.2
Penicillia Commonly Isolated from Food and Associated Mycotoxins
Food, Commodity Fungi Mycotoxins
Cereals P. verrucosum Ochratoxin A, citrinin
Dried meat products P. nordicum Ochratoxin A
Pomaceous fruit (e.g., apples) P. expansum Patulin, citrinin
Tap root plants P. radicicola Citrinin, penicillic acid
Beer, wine, rye bread, dry meat P. carneum Patulin, penicillic acid
Cereals, dry foods, indoor air P. griseofulvum Patulin, cyclopiazonic acid, secalonic
acid D, F
Rye bread P. paneum Patulin
Nuts P. expansum Patulin
Yams P. sclerotigenum Patulin
Cereals P. aurantiogriseum, P. cyclopium, P. freii, Penicillic acid
P. melanoconidium, P. neoechinulatum,
P. tulipae, P. viridicatum
Cereals, dried meat products P. polonicum Penicillic acid
Cheese P. camemberti, P. commune, P. dipodomyicola, Cyclopiazonic acid
P. palitans, P. griseofulvum
the proximal and distal tubules of the kidney. Changes in the Mutagenic effects involving nucleic acids can be direct
renal function of pigs include impairment of proximal tubu- (e.g., changing bases) and/or indirect (e.g., inhibiting
lar function, altered urine excretion, and increased excretion enzymes) [65]. Ochratoxin A, citrinin, patulin, penicillic
of urine glucose. Carcinogenicity was evident in rats/mice. acid, and penitrem have carcinogenic effects and are DNA
Ochratoxin A may have an immunological effect via immu- damaging. Ochratoxin A is a potent carcinogen in rats and is
noglobulins and phagocytic cells and can cause liver damage classified as a possible human carcinogen by the International
particularly at higher doses. It is reported to affect embry- Agency for Research on Cancer [66]. DNA–DNA cross-links
onic survival. Tumorigenesis/carcinogenesis was reported are induced by patulin: Mutations of cells might be from an
although mutagenicity was not established. indirect mutagenic mechanism (e.g., inhibition of enzymes)
Cyclopiazonic acid intoxication includes weight loss, and direct reactivity to DNA has been demonstrated [67].
anorexia, diarrhea, dehydration, pyrexia, ataxia, immobility, The mode of action of penitrem A is by the release of
and extensor spasm at death in dogs, rats, pigs, laying hens, neurotransmitters from synaptosomes in the CNS and
sheep, and chickens. Patulin has an immunology effect and in peripheral nerves at the neuromuscular junction [68].
inhibits multiple aspects of macrophage function in vitro: neu- Mycelianamide and gliotoxin function as enzyme inhibitors:
trophilia in rats was attributed to GI inflammation. Gliotoxin Low concentrations of gliotoxin inhibited specifically the
has antimicrobial and immunosuppressive capabilities. The activation of NF-κB. Hundreds of mycotoxins are known and
compound is produced in infected animal tissue strengthen- detected frequently in plant-derived products. Various myco-
ing its involvement in pathogenesis. Rubratoxin has been sus- toxins may occur simultaneously, depending on, amongst
pected as the cause of hepatotoxic, hemorrhagic disease of other things, the environmental and substrate conditions [60].
cattle and pigs. Ochratoxin A and rubratoxin B (from P. pur-
purogenum) are teratogenic in mammalian species. Finally, 41.1.2.4 Taxonomy of Mycotoxin Fungi
penitrem A elicits a tremogenic response in animals [60,63]. The penicillia can be divided into the monoverticillate,
biverticillate, and terverticillate, which refer to the number
41.1.2.3 Mycotoxin Toxigenic Mechanisms of branches in the conidiophores as mentioned. The predomi-
Mycotoxins as a unit are unclassified according to mecha- nant mycotoxin producers belong to the terverticillates (cf.
nisms of action because of the high diversity of chemical subgenus Penicillium) [3]. P. expansum produces smooth-
structures. The potential for complex toxin interactions is walled penicilli, synnemata, and rots apples: It is the predom-
great given the large number and diversity of the action mech- inant fungus associated with patulin production. The fungus
anisms. Mycotoxins with similar modes of action would be typifies the genus although the classic features are variable.
expected to have at least additive effects. Conversely, some This plasticity in characters is a general feature in terverticil-
interactions could have subtractive effects. For example, late fungi making the taxonomy unstable and requiring other
cyclopiazonic acid prevents the lipid peroxidation induced characters to stabilize the taxonomy, hence assisting in spe-
by patulin [64]. The actual combined health risk from myco- cies recognition. DNA-sequencing data have contributed, but
toxin exposure is unknown because many mycotoxins are are not suited particularly for classification and identification,
undiscovered. although defining phylogenetics. However, Paterson et al.

have questioned the validity of such data because of “self” [2,76] mentioned above were valid. When fungi are grown,
mutagen and inhibitor production as described in a series of they may produce (i) inhibitors of enzymes and (ii) muta-
papers (see [65]). gens of nucleic acid. Hence, the DNA polymerase of PCRs
Pitt [69] reintroduced some novel characters and made could be inhibited. Most diagnostic PCR methods lack an
rearrangements; however, overall, the concepts were similar internal amplification control (IAC) leading to potential
to those in Frisvad et al. [31] and the sequence-based ribo- false-negative results from inhibitors produced in media
somal DNA phylogeny of Peterson [70]. Bridge et al. [71] [65]. Also, IACs are required to account for faulty thermal
attempted probably the first polyphasic approach and also cyclers and reagents. This permits the possibility of false-
employed numerical taxonomy, which indicated a general negative results—the worst possible outcome for diagnostic
similarity in most of the strains tested; some species con- microbiology, in that samples containing a pathogen may be
cepts were confirmed or new groupings were made in some considered as safe.
other cases. Indeed, few differences were found between Furthermore, the nucleic acid of interest could be mutated
terverticillate penicillia in the ribosomal DNA gene [70]. and/or the stabilizing enzymes could be inhibited, allow-
Nevertheless, Frisvad and Samson [3] describe, illustrate, ing for further mutations [65]—an undesirable situation for
and key out 58 species. Seifert and Lous-Seize [72] indicated nucleic acid analysis. Factors such as these had not been con-
that β-tubulin was more useful in delineating species, which sidered previously by other authors. In pure (batch) liquid
was confirmed by Samson et al. [73]. culture or agar (which is how fungi are grown in molecular
biology), these mutagens and inhibitors accumulate normally
41.1.2.5 Phenotypic Identification to a high level and will tend to overwhelm natural mecha-
Conventional methods to identify these fungi involve isola- nisms to ameliorate their effects.
tion from a substrate and microscopic observation of mor- Penicillium mycotoxins, which are DNA damaging,
phology, growth rates, and colors/morphology on growth include ochratoxin A, citrinin, patulin, penicillic acid,
media [3,4]. The use of secondary metabolites as characters and secalonic acid produced by a wide range of penicillia.
in the group has been advocated by Frisvad and Samson [3]: Interestingly, secalonic acid is produced by P. marneffei (see
Paterson found that the profiles in [3] gave relatedness to spe- above). Many other secondary metabolites are likely to be
cies concepts only in some cases, and there is a sense of com- mutagenic and inhibitory, which may not be mycotoxins, and
ing full circle as the species concepts were closer to Raper so avoidance needs to be universal when analyzing fungi.
and Thom in 1948 than Pitt in 1980 (see [74]). Extracellular However, this has not been done with potential negative con-
enzymes were shown to be variable in most species by sequences for the molecular biology of fungi. None of the
Paterson et al. [75] and have not been used subsequently with research described below has addressed these issues and the
significance, although P. brevicompactum did appear differ- onus is on researchers to prove these effects do not influence
ent. A solution to identifying these penicillia was offered by their results.
Paterson et al. [2,76], which may be useful particularly in
determining those strains which affect humans. This was to 41.1.3.1 Molecular Identification of P. marneffei
identify the conidiophores and analyze for the mycotoxin of Molecular diagnosis of P. marneffei is based on standard
interest. For patulin producing strains, an identification of specific oligonucleotide primers designed from the internally
subgenus Penicillium, patulin positive could be obtained. transcribed spacer and 5.8S rRNA gene (ITS1-5.8S-ITS2) of
Finally, some of the constraints have led to an interest in P. marneffei [13]. First, fungal DNA was amplified with the
matrix-assisted laser desorption/ionization time-of-flight primer pair ITS5 and ITS4 [78]. Second, nested PCR was
intact cell mass spectrometry (MALDI-TOF ICMS) for rapid performed successfully with primer pairs “PM2 and PM4”.
and reliable identifications [1]: much more work is required A 347 bp PCR product was obtained, which was used to iden-
before its use is routine. tify P. marneffei from skin [79].
Furthermore, P. marneffei was identified using a specific
41.1.3 Molecular Diagnosis of Mycosis PCR-enzyme immunoassay method [80]: Applicability with
clinical samples required further evaluation. An 18S rRNA
and Mycotoxinosis
gene probe was specific in a PCR-hybridization reaction
Nucleic acid techniques are extremely sensitive and have from human and environmental isolates [81]. However, the
been employed apparently for the successful specific detec- hybridization technique is labor- and skill-intensive; hence,
tion of fungi. However, problems with the methods have been single and nested PCR for the rapid identification of P.
described recently by the authors of the present chapter in marneffei were developed using specific primers, also based
particular. Dupont [77] suggests that “molecular methods on the 18S rRNA gene sequence [82]. Successful discrimi-
offer a big advantage over conventional phenotypic methods nation of a young culture of P. marneffei was performed.
for species diagnosis in that they measure “stable” genotypic Finally, a sensitive one-tube seminested PCR assay based on
characteristics and do not rely on culture and operator inter- the 18S rRNA sequences [83] was used in a cultural-depen-
pretation.” This may not be the case in what is an excellent dent PCR (CDP) and clinical samples (or cultural-indepen-
review of molecular detection of food-borne penicillia. The dent PCR [CIP]). The methods described in [82,83] need to
author also considered that the methods of Paterson et al. be studied further to prove their utility. In none of the above

studies were IACs employed allowing for the potential of the realized that these can become involved in mycosis as dis-
worst possible outcome of diagnostic PCR—false-negative cussed briefly above. For example, P. brevicompactum, P.
results. chrysogenum, and P. expansum can fall into both categories
In addition to a draft sequencing of the genome [14], ran- (cf. P. marneffei producing the mycotoxin secalonic acid).
dom sampling of genetic variation within the P. marneffei As mentioned, the methods have been reviewed previ-
genome has been undertaken. Human isolates had type I ously by the current authors, which can be consulted for
(73%) and type II (27%) DNAs whereas bamboo rat isolates more detail (see [65]). Also, the methods in [77] can be con-
were type I from R. sumatrensis and type II from C. badius sulted. A novel aspect of the molecular biology of myco-
for 20 and 3 strains, respectively [84]. Twenty P. marneffei toxin-producing fungi is the possibility to analyze for the
isolates from patients [85] had a similar pattern although ran- genes of the metabolic pathway that creates the mycotoxin
domly amplified polymorphic DNA (RAPD) assays yielded of interest, in addition to genes specific to particular taxa
eight different patterns. Four genotypes were found that var- [93]. These have been reported for patulin, ochratoxin A,
ied in frequency between northern and southern Thailand and PR-toxin.
[86]. Isolates from Thailand [87] grouped into nine subpro- Marek et al. [94] report their assay for Penicillium expan-
files yielding 54 genotypes. However, no correlation with sum on fruits using the polygalacturonase gene although
geographic region or specimen source was observed [7]. In without using an IAC. Calderon et al. [95] describe a method
addition, sequence-specific assays of genetic variation in the for determining spores of Penicillium roqueforti from
P. marneffei genome have been investigated [88]. Some of an air sampler using PCR. There is no mention of an IAC
the sequences resulted in 30 dinucleotide, 14 trinucleotide, although the possibility of inhibition of the PCR is discussed.
and 5 tetranucleotide repeats [89]. Only three microsatellites Colombo et al. [96] provided a method that claimed to dis-
were uncovered subsequently [90] and why this discrepancy tinguish Penicillium aurantiogriseum from foods. Sholberg
between the two studies exists remains unclear. Of the (i) et al. [97] did not use an IAC for Penicillium blue rot molds
49 repeats in [89], 24 were amplified to type 29 clinical and although a negative control of “no DNA” in the reaction mix-
bamboo rat isolates [26,89,91], and (ii) 24 loci, 23 were ampli- ture was included. Again, IAC were not employed in Dupont
fiable and 21 were polymorphic with between 2 and 14 alleles et al. [98], and the RFLP patterns could represent inhibition
present at each locus, comprising 19 unique microsatellite rather than true differences in DNA between Penicillium
type (MT)s. Clustering showed that isolates occurred within camemberti and Penicillium nalgiovense. The conclusion
one of two separated clades geographically that accounted that IACs are required in RFLP cannot be avoided, as the
for 26% of the total observed genetic diversity. The “east- method is also enzyme based, although IAC will be more
ern” clade contained isolates from mainland China, Hong difficult to employ. Pedersen et al. [93] investigated subgenus
Kong, Indonesia, and Vietnam, while the “western” clade Penicillium with two sets of primers and again an IAC was
contained isolates from Thailand and India, showing that P. not used.
marneffei has a geographic component to its genetic struc- Dao et al. [99] describe a method for detecting ochratoxin
ture. Extensive linkage disequilibria existed between loci [7], A and citrinin producing fungi (see Paterson [100]). Geisen
suggesting that there were either genetically differentiated et al. [101] did not use an IAC and so the negative strains
subpopulations or extensive clonal reproduction occurring. tested with the primers may have been inhibited and not spe-
A study over a smaller geographical scale in India showed cific for Penicillium nordicum in terms of the nucleic acid
that the MTs of isolates were dissimilar between plantations involved. Bogs et al. [102] propose a method for differenti-
[26]. The population genetic structure of P. marneffei may ating the ochratoxin A producing species P. nordicum and
be partitioned over local and geographical scales. The type Penicillium verrucosum. As there was no IAC, the usual pro-
isolate of P. marneffei [7] was characterized by a unique MT visos apply concerning negative results and nonappearance
and was therefore dissimilar to human isolates [26]; a simi- of bands.
lar result was found by comparing two bamboo rat isolates An IAC was not employed in the reports on patulin pro-
with 32 clinical isolates [90]. However, 10 bamboo rat iso- ducing penicillia [100,103,104]. Nevertheless, the possi-
lates collected in India demonstrated that one was identical bility of inhibition at least was recognized. Unpredictable
to all 21 microsatellite loci of the human isolate CBS 101038 results occurred on some occasions with idh, such as (i)
[92]. This is the first molecular epidemiological evidence that presence of the gene in Penicillium brevicompactum and
humans and bamboo rats share identical strains of P. marnef- (ii) P. expansum lacking the gene very occasionally. One
fei and that host-to-host transmission may occur. However, explanation for not obtaining a positive result from all
it is possible that coinfection had occurred from a common members of a species was the occurrence of false-negative
environmental source. results from inhibitors [65]. In all cases bar one, the vari-
ous media used by authors were not designed to inhibit sec-
41.1.3.2 Molecular Studies of Mycotoxin/ ondary metabolites. Furthermore, Dombrink-Kurtzman
Occasional Mycosis Penicillia [105] investigated the differences between the sequences
This work relates normally to fungi that have grown on of the idh of P. griseofulvum and P. expansum. Both spe-
foods, produced toxins that are then ingested by humans cies produced patulin in culture with P. griseofulvum
(or other animals), and caused disease. However, it has to be manufacturing much more under the conditions employed.

The analysis of the sequences revealed various differences Sabouraud glucose agar at 28°C [82]. The DNA was the same
between P. griseofulvum strains. The three P. expansum in each case that was extracted from fungal cells or from fun-
strains were identical for introns “1 and 2,” but there were gal spheroplasts. Fungal cells from the surface of the agar
nine additional bases in intron 1 compared to the P. griseo- medium were suspended in lysis buffer, boiled, and vortexed
fulvum idh gene. P. expansum had more nucleotide differ- in the case of the direct method. Phenol–chloroform–isoamyl
ences in both introns than did strains of P. griseofulvum. alcohol (25:24:1 ratio) was added and re-vortexed to extract
One P. griseofulvum had nine bp differences and another the DNA. The upper phase was removed and DNA precipita-
five differences compared to the standard. Dombrink- tion was carried out with cold acetone. The DNA pellet was
Kurtzman [106] extended the work to many other patulin- air dried and resuspended in water.
producing species and demonstrated good correlation with Spheroplasts were prepared by suspending fungal cells in
species concepts, although P. griseofulvum “3523” was dif- filter-sterilized osmotic medium, placing on ice and incubat-
ferent considerably from other strains of the species. A rea- ing with NovoZym 234 in the presence of bovine serum albu-
son may have been mutations of idh because of mutagens min at 37°C. The spheroplasts were lysed with buffer and
in the growth medium as discussed herein. Furthermore, a deproteinized. The nucleic acids were collected by 2-propa-
discussion of various PCR methods for mycotoxigenic fungi nol precipitation, resuspended in buffer, extracted by phenol-
is provided in Paterson [100] including details of the various chloroform–isoamyl alcohol, precipitated with ethanol, and
growth media employed. It can be assumed that mutagenic resuspended in buffer.
compounds will be produced in these media—an unsatis- PCR to identify the organism in skin specimen was
factory position for nucleic acid analysis [65]. attempted when culturing failed [79]. DNA was extracted
“Bar coding” to identify fungi is being considered actively from the skin specimen fixed with formaldehyde and embed-
[107], in part because of problems with existing characters. ded in paraffin. (The use of formaldehyde warrants some
The authors are also very familiar with secondary metabo- concern as it is a mutagen.) The control was the unaffected
lites production from these penicillia. Results of a maximum part of the skin in a fixed specimen of a benign subcutaneous
parsimony analyses indicated that P. carneum and P. paneum tumor.
were closely related: They had a 12 nucleotide difference in
the ITS regions [108]. Peterson [70] found few differences
between terverticillate penicillia in the ribosomal DNA 41.2.2 Detection Procedures
gene. Seifert and Lous-Seize [72] indicated that β-tubulin
41.2.2.1 Basic PCR for Fungi [13]
was more useful in delineating species, which was con-
firmed by Samson et al. [73] who investigated the β-tubulin 41.2.2.1.1 Reagents
sequences for the phylogenetic analysis of Penicillium subge- 10× amplification buffer
nus Penicillium in general.
15 mM MgCl2
500 mM KCl
41.2 Methods 100 mM Tris–HCl (pH 8.3) at 23°C
0.1% gelatine
41.2.1 Sample Preparation of P. marneffei
As a general observation, many anatomical locations within 10× dNTP stock solution
patients have been demonstrated to contain the fungus, and
so CIP may be possible from these sites [7,44,45]. Details 2 mM each of dATP, dGTP, dTTP, and dCTP
of how the organism was isolated were not provided in [13]
although these need to be known for comparative purposes. 41.2.2.1.2 Procedure
Growth of P. marneffei mycelium for DNA isolation was on 1. Prepare a working reaction solution sufficient for 10
malt extract agar at 25°C, and DNA extraction methods were amplifications using positive displacement pipettes
described in [109]. In Vanittanakom et al. [81], filamentous- for steps 1–4, which consists of
phase cultures were obtained in Sabouraud dextrose broth at 275μL sterile distilled water
25°C. Only details were provided of the culture collection 100 μL 10× amplification buffer
and from whom the cultures were obtained and not of how 100 μL dNTP stock mixture (2 mM each dNTP)
the strains were actually isolated nor from which substrate. 10 μL excess primer (50 μM stock)
DNA was extracted by grinding the cellular mats with a mor- 10 μL limiting primer (5 μM stock)
tar and pestle in the presence of liquid nitrogen. The fungal 5 μL Taq polymerase (5 units/μL)
mat was ground into a fine powder with a sterile pestle. DNA 2. Add 50 μL of the working solution to each tube.
was then extracted and purified using serial applications of 3. Dilute the DNA samples in TE (10 mM Tris–HCl
proteinase K and RNase. [pH 8.0] and 0.1 mM EDTA) to 0.1–10 ng 50 μL−1.
How the human isolates were obtained was not described 4. Add 50 μL of the diluted sample DNA to each tube
in [81,82]. However, the yeast form was maintained in followed by two drops of mineral oil.
brain heart infusion agar at 37°C and the hyphal stages on 5. Spin briefly in a microcentrifuge

6. Cycling parameters 41.2.2.1.4 Specific PCR of P. marneffei Protocol

1. Amplify isolates with primers designed
Initial denaturation 2–3 min at 95°C as general fungal primers, namely, ITS5
Annealing 30 s at 50°–60°C (GGAAGTAAAAGTCGTAACAAGG) and ITS4
Extension 0.5–2 min (depending on prod- (TCCTCCGCTTATTGATATGC), with an anneal-
uct size) at 72°C ing temperature of 48°C.
Denaturation 30 s at 95°C 2. Purify the ITS1-5.8S-ITS2 rDNA PCR products to
Final extension 10 min at 72°C eliminate primers.
3. Dilute PCR products × 1000 and then amplify with
7. Cycle numbers: Use (i) 25 for double-stranded prod- two primer combinations, PM1–PM4 and PM2–
uct with 50 pM of each primer and (ii) 35 for sin- PM4, at annealing temperatures of 60°C or 65°C.
gle-stranded product using a primer ratio of 50:1 or 4. Test PCR amplifications from P. marneffei mini-
50:2.5 pM. prep DNA diluted 1000-fold with PM1–PM4 and
8. Briefly centrifuge the tubes when the amplification PM2–PM4 to ensure that the primers amplified
is completed and take 5 μL samples for analysis by genomic DNA.
minigels. Specific primers
9. Remove the oil by extraction in chloroform and
remove the unincorporated nucleotides while con- Primer Primer Locationa Nucleotide Sequence (59–39) bp
centrating the DNA by centrifugal filtration before PM1 (59) ITS1 (bp 125–142) TCGCCGGGGGACGTTTGT
sequencing the single-stranded PCR. PM2 (59) ITS1 (bp 187–205) GATGGACTGTCTGAGTACC
10. Routinely, 7 μL of the retentate is used for sequenc- PM4 (39) ITS2 (bp 498–519) ATGGTGGTGACCAACCCCCGCA
ing reactions. Concentrate in a Speed Vac if the a Primer location is based on P. marneffei ITS sequence, GenBank
DNA is too dilute. Use 1 pM of the limiting primer accession no. L37406.
(or an internal primer) as the sequencing primer.
Anneal at 65°C, not 90°C–95°C, to avoid denatur-
ing the double-stranded product prior to primer 41.2.2.2 P CR Identification of Mycotoxin/
annealing. Occasional Mycosis Penicillia
11. Primers NS1–NS8 and ITS1–ITS4 [78] have been 1. Marek et al. [94] report their assay for Penicillium
used with lower dNTP concentrations (32 μM each expansum on fruits using the polygalacturonase
dNTP, instead of 200 μM in the reaction). gene although without using an IAC.
12. The above conditions were not optimized for 2. Calderon et al. [95] describe a method for deter-
enzyme and magnesium ion concentrations. mining spores of Penicillium roqueforti from an air
sampler using PCR. There is no mention of an IAC
Primer sequences for fungal ribosomal RNA genes are pro- although the possibility of inhibition of the PCR is
vided in [78]. discussed.
3. Colombo et al. [96] provided a method that claimed
41.2.2.1.3 PCR Identification of P. marneffei to distinguish Penicillium aurantiogriseum from
The PCR conditions in [13] were described in [91] and as foods.
modified in [109]. Primers were MS1 and MS2 for the 4. Dupont et al. [98] detected the RFLP patterns
mtSrDNA and ITS2, ITS3, ITS4, and ITS5 for the ITS1- in DNA between Penicillium camemberti and
5.8S-ITS2 rDNA region [109]. Nucleotide similarity among Penicillium nalgiovense.
six isolates of P. marneffei was analyzed initially by restric- 5. Pedersen et al. [93] investigated subgenus
tion fragment length polymorphisms of the ITS1-5.8S-ITS2 Penicillium with two sets of primers.
rDNA PCR products. The ITS1-5.8S-ITS2 rDNA PCR prod- 6. Dao et al. [99] describe a method for detecting
ucts were digested singly with 1–3 U of the four-base-cutter ochratoxin A and citrinin, producing fungi (see
restriction enzymes AvaI, AvaII, HhaI, MboI, and TaqI. The Paterson [100]).
mtSrDNA and ITS1-5.8S-ITS2 rDNA regions of P. marnef- 7. Bogs et al. [102] propose a method for differentiat-
fei were sequenced from single-stranded template generated ing the ochratoxin A producing species P. nordicum
from asymmetric PCR amplifications. Nucleotide sequences and Penicillium verrucosum.
from other species used in phylogenetic analyses were deter-
mined from previous studies [109]. DNA sequences were 41.2.2.2.1 Protocol for Penicillium Subgenus Penicillium
initially aligned by the use of the Genalign option in the In the first instance, PCR conditions can be optimized by
Intelligenetics software package and then by visual optimi- varying the concentrations of KCl, MgCl, and Tris–HCI pH
zation. Only regions without ambiguity were included in the 8.3 using purified Penicillium subgenus Penicillium DNA as
analysis. a template.

41.2.2.2.2 Protocol for Penicillium Subgenus buffer (both from HT Biotechnology, Cambridge,

Penicillium as a Whole United Kingdom), 0.8 μM of primer IDH1 and
1. Wash the previously obtained DNA pellet with 80% IDH2, and template DNA.
ethanol and dry. 2. Undertake an initial denaturing step of 94°C for
2. Dissolve in TE buffer (10 mM Tris–HCl pH 8.0 and 3 min. Amplify the idh gene in a thermal cycler
1 mM EDTA pH 8.0; 50 μL). (e.g., Hybaid, Omn-E) under the following condi-
3. Use Penicillium subgenus Penicillium specific tions: 94°C for 1 min, 52°C for 1 min, 72°C for 1 min
primers: ITS 212d (5′-AAA TAT AAA TTA TTT for 30 cycles and end the programme with an exten-
AAA ACT TTC-3′) and ITS 549 (5′-CTG GAT sion step of 72°C for 5 min.
AAA AAT TTG GGT TG-3′). 3. Separate the amplified products by electrophoresis
4. Add 1–5 μL DNA (10 ng) to a reaction mixture con- in 1.5% agarose gel (SeaKem LE, FMC Bioproducts)
taining 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1 mM in Tris-acetate-EDTA buffer at a constant voltage of
MgCl2, 200 μM, dNTPs, 1, μM ITS 212d, and 1 μM 5 V/cm.
ITS 549 and 2.5 U (Ampli)Taq (Perkin Elmer Cetus) 4. Stain the gels with ethidium bromide and view
polymerase in a final volume of 100 μL. under UV light.
5. Amplify using the conditions of 45 cycles at
94°C for 30 s, 48°C for 30 s, 72°C for 30 s, and 41.3 C
onclusions and
finally 72°C for 10 min using a Perkin Elmer 9600 Future Perspectives
Thermocycler.
There is not an absolute distinction between fungi that cause
6. Alternatively, use a Perkin Elmer 480 thermocycler
mycosis and those that produce mycotoxins as discussed
for 40 cycles at 94°C for 30 s, 48°C for 60 s, and
above. Furthermore, standard treatments for mycosis should
72°C for 60 s followed by 72°C for 10 min.
be considered immediately for patients with P. marneffei
7. Add 10 μL aliquots to a 2.5% agarose gel in TAE
infections without waiting for a definitive identification: The
buffer and electrophorese.
rational being that treatment is not specific to any marked
8. Staining in 1 μg/mL ethidium bromide to observe
degree, although this needs to be confirmed by medical
PCR products.
practitioners (see [110] for general treatments of Penicillium
infections). Finally, analyzing samples from patients for a
41.2.2.2.3 Protocol Adaption for Penicillium general fungal test such as the fungal lipid ergosterol (see
Subgenus Penicillium Species P. [111]) may be worthwhile to facilitate immediate treatment.
roqueforti and P. carneum It appears to be assumed that the natural habitat for P.
1. Use ITS 183 (5′-CTG TCT GAA GAA TGC AGT marneffei is soil because “penicillia are associated with
CTG AGA AC-3′) and ITS 401 (5′-CCA TAC GCT soil”. However, Talaromycetes and Biverticillate penicillia
CGA GGA CCG GAC-3′). are found more in wood and its products, textiles, and bird
2. Add 5 μL template DNA (10 ng) to a 50 μL reaction feathers. In addition, Subgenus Penicillium is associated with
mixture containing 10 mM Tris–HCl pH 8.3, 50 mM animal excretion, and the feces of rats require to be investi-
KCl, 2.5 mM MgCl2, 0.01% gelatine, 200 μM gated (despite the fungus being biverticillate). After all, it is
dNTPs, 0.25% Tween 20, 10% DMSO, 0.3 μM of possible that infection of humans is via rat feces in contami-
each of ITS 183 and ITS 401, and 2.5 U AmpliTaq nated food.
polymerase. The pigments produced by P. marneffei could be ana-
3. Amplify at 45 cycles for 30 s at 94°C, 1 min at 60°C lyzed as a means to identification. Hence, adapting Paterson
and 1 min at 72°C and finally 72°C for 10 min (the et al. [2], the distinctive conidiophore could be identified by
Perkin Elmer 9600 Thermocycler is adequate for microscopy and then chromatography undertaken to deter-
this purpose). mine the pigments. MALDI-TOF needs to be employed to
investigate the yeast/hyphal transition in P. marneffei [1] and
to compare this with other penicillia.
41.2.2.2.4 PCR for a Mycotoxin Metabolic Pathway The issue of climate change is relevant to fungal diseases
Using Patulin as an Example [112]: P. marneffei may become less prevalent in tropical
Use the following idh primers that were designed to amplify climates as the temperature increases beyond tolerable lev-
the 600 base pairs of the idh gene which had the following els for the fungus. However, this may allow increased dis-
structure: 5′CAA TGT GTC GTA CTG TGC CC and 5′ACC ease in currently temperate countries (e.g., parts of Europe
TTC AGT CGC TGT TCC TC (GIBO BRL, Paisley, Scotland). and United States) as the temperatures increase. Equivalent
speculation can be made with Penicillium mycotoxins, with
1. Prepare the PCR reaction mixture consisting of currently colder countries becoming more susceptible to och-
200 μM of dNTPs (Pharmacia, Herts, United ratoxin A and patulin, for example. Certainly the situation
Kingdom), 1.25 units of Tth polymerase enzyme, Tth merits careful monitoring.

Growing fungi in media that do not stimulate secondary 9. Supparatpinyo, K. et al., Disseminated Penicillium marneffei
metabolites production need to be investigated, and these infection in Southeast Asia, Lancet, 344, 110, 1994.
should be employed for nucleic acid analysis. Secondary 10. Supparatpinyo, K. et al., A controlled trial of itraconazole to
prevent relapse of Penicillium marneffei infection in patients
metabolites can be controlled by harvesting the fungus in tro-
infected with the human immunodeficiency virus, N. Engl. J.
phophase (i.e., growth) rather than idiophase (i.e., secondary Med., 339, 1739, 1998.
metabolites production). In this way, secondary metabolites 11. Segretain, G., Description d’une nouvelle espèce de penicil-
can be reduced. Growing fungi for the minimum amount of lium: Penicillium marneffei n. sp., Bull. Soc. Mycol. Fr., 75,
time may be useful as this will limit the contact of secondary 412, 1959.
metabolites with nucleic acids. Finally, preserving the fungi 12. Frisvad, R.A. and Filtenborg, O., Revision of Penicillium
in the original substrate could be undertaken. A reservoir of subgenus Furcatum based on secondary metabolites and
substrates from which a particular fungus was isolated could conventional characters, in Modern Concepts in Penicillium
and Aspergillus classification, Samson, R.A., Pitt, J.I., Eds.,
be maintained in freezers, freeze dried ampoules, and liquid Plenum Press, New York, 1990, p. 159.
nitrogen specifically for nucleic acid analysis. If these proce- 13. LoBuglio, K.F. and Taylor, J., Phylogeny and PCR identifica-
dures appear laborious, then this has to be balanced with (i) tion of the human pathogenic fungus Penicillium marneffei, J.
the waste of financial, intellectual, and time resources, and Clin. Microbiol., 33, 85, 1995.
(ii) the consequences of false diagnostic results, if the nucleic 14. Cai, J.J., Understanding the pathogenic fungus Penicillium
acid does not represent the taxa under analyses. There are marneffei: A computational genomics perspective, PhD the-
various areas that require clarification as described above in sis, The University of Hong Kong, 2006.
15. Piehl, M.R., Kaplan, R.L., and Haber, M.H., Disseminated
the field of human pathogenicity by Penicillium and which
penicilliosis in a patient with acquired immunodeficiency
may also apply to other fungi. Finally, it is to be hoped that syndrome, Arch. Pathol. Lab. Med., 112, 1262, 1988.
recent success with a vaccine for HIV is confirmed (see 16. Sathapatayavongs, B. et al., Disseminated penicilliosis associ-
above) and will result in a concomitant reduction in the ated with HIV infection, J. Infect., 19, 84, 1989.
incidence of P. marneffei infection.* 17. Pócsi, Z. et al., Yeast-like cell formation and glutathione
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Acknowledgments 18. Devlieghere, F., Debevere, J., and Van Impe, J., Concentration
of carbon dioxide in the water-phase as a parameter to model
RRMP is employed by the FCT framework, Commitment to
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42 Phialophora
Contents
42.1 Introduction...................................................................................................................................................................... 345
42.1.2.1 Phialophora verrucosa.......................................................................................................................... 346
42.1.2.2 Phialophora richardsiae....................................................................................................................... 347
42.2 Methods............................................................................................................................................................................ 347
42.2.2.2 Sequencing Analysis of the ITS1-5.8S-ITS2 Region............................................................................. 348
42.2.2.3 Sequencing Analysis of the D1/ D2 Domains of 28S rRNA Gene........................................................ 349
42.3 Conclusions....................................................................................................................................................................... 349
References.................................................................................................................................................................................. 349
42.1 Introduction parasiticum), Phialophora pedrosoi (synonym: Fonsecaea

pedrosoi), and Phialophora spinifera (synonym: Exophiala
42.1.1 Classification, Morphology, and Biology spinifera).
The genus Phialophora is a dematiaceous (dark-walled) fila- Phialophora is a ubiquitous organism that inhabits the
mentous fungus belonging to the mitosporic Magnaporthaceae soil, plants, and decaying food [2,3]. Several Phialophora
group, family Magnaporthaceae, order Magnaporthales, species are the causative agents of human infections such
class Sordariomycetes, subphylum Pezizomycotina, phylum as chromoblastomycosis, phaeohyphomycosis, and eumy-
Ascomycota. The obsolete synonyms of the genus include cetoma. P. verrucosa and P. richardsiae are often involved
Cadophora and Margarinomyces, and teleomorphs of this in phaeohyphomycosis, with clinical presentations rang-
genus are found in Chaetosphaeria, Gaeumannomyces, and ing from cutaneous infections, subcutaneous cysts, kerati-
Mollisia. tis, endocarditis, arthritis, osteomyelitis, cerebral infection,
Currently, the genus Phialophora contains 16 recognized fatal hemorrhage, and disseminated infection. Phialophora
and over 69 unassigned species, the most notable of which are verrucosa is the principal causative agent of chromoblas-
Phialophora bubakii, Phialophora europaea, Phialophora tomycosis in tropical and subtropical areas, particularly
gregata, Phialophora repens (obsolete synonym Cadophora in Japan and South America. Phialophora europaea has
repens), Phialophora reptans, Phialophora richardsiae been isolated from cutaneous and nail infections in North-
(obsolete synonyms: Cadophora brunnescens, Cadophora Western Europe.
richardsiae, P. brunnescens, and P. caliciformis), and Phialophora has short conidiophores (sometimes reduced
Phialophora verrucosa (synonym: P. americana; obsolete to phialides) and unicellular conidia, although conidia are
synonym: Cadophora americana; and teleomorph: Capronia sometimes absent. Phialophora colonies grow moderately
semiimmersa) [1]. slowly and reach a diameter of 2–3 cm after 7 days at 25°C.
A number of former Phialophora species have been reclas- Colonies are gray to olivaceous black and often become dark
sified: Phialophora compactum (synonym: Fonsecaea com- gray-green, brown, or black with age. From the reverse, colo-
pacta), Phialophora cyanescens (synonym: Cylindrocarpon nies appear iron-gray to black; colony texture is cottony to
cyanescens), Phialophora dermatitidis (synonyms: Exophiala velvety and may be heaped and granular.
dermatitidis and Wangiella dermatitidis), Phialophora hoff- Phialophora hyphae (up to 5 μm wide) are branched and
mannii (synonym: Lecythophora hoffmannii), Phialophora hyaline to brown. If present, condiophores are short and pale
jeanselmei (synonym: Exophiala jeanselmei var. jeansel- brown; conidiogenous cells (phialides) have characteristic
mei), Phialophora mutabilis (synonym: Lecythophora muta- flask-shaped or cylindrical phialides with distinctive collar-
bilis), Phialophora parasitica (synonym: Phaeoacremonium ettes, which are terminally or laterally located on the hyphae;
345

conidia are one celled, hyaline to olivaceous brown, smooth osteomyelitis, and prosthetic valve endocarditis as well
walled, ovoid to cylindrical or allantoid, and often aggregate as disseminated infections [7–13], with P. verrucosa and
in slimy heads at the apices of the phialides, giving the appear- P. richardsiae being most commonly reported [9,14–17].
ance of a vase of flowers; phialides may be solitary or in a
brush-like arrangement. In tissues infected with Phialophora 42.1.2.1 Phialophora verrucosa
species, spherical or polyhedral, dark brown, thick-walled A case of fatal hemorrhage due to Phialophora verrucosa
sclerotic bodies (muriform cells) and phaeoid hyphae are occurred in a patient with prolonged neutropenia undergoing
often present, although absence of muriform cells is noted in autologous bone marrow transplant for acute myelogenous
cases involving immunosuppressed or debilitated patients. leukemia. Autopsy uncovered granulation tissue on the tra-
At the species level, Phialophora ripens produces inter- cheal wall, which showed fungal hyphae by histopathological
calary phialides without basal septa or cylindrical to lageni- examination and grew P. verrucosa in culture [18].
form phialides (<20 μm in length) with narrow collarettes; In a case of Phialophora verrucosa-related multifocal
Phialophora richardsiae conidia are globose to cylindrical subcutaneous phaeohyphomycosis, an 85-year-old woman
and phialides with saucer- or vase-shaped collarettes; and presented with purulent multifocal subcutaneous nodules
Phialophora verrucosa produces flask-shaped phialides with on the right forearm and hand. The patient reported a his-
vase-shaped collarettes; Phialophora europaea—a member tory of gardening and recalled the occurrence of a painless,
of the P. verrucosa complex—shows reduced, flaring phi- soybean-sized nodule on the dorsal site of the right forearm
alidic collarettes [4]. 3 years earlier, which gradually extended peripherally to the
dorsum of the right hand. The pustules and nodules (rang-
ing from 5 × 5 mm to 20 × 40 mm in size) showed a yellow
crust on the surface and were surrounded by erythema and
The term dematiaceous (dark-pigmented) fungi refers to a discharged a purulent fluid upon pressure. Examination of
large and heterogeneous group of molds belonging to sev- the biopsied specimen revealed a subcutaneous granuloma
eral different genera. Some are capable of causing a diverse (containing faint brown septate hyphae with rare branch-
range of clinical diseases such as phaeohyphomycosis, ing) with central abscess and necrosis, consistent with a
chromoblastomycosis, and eumycetoma. The most notable phaeomycotic cyst. Culture of the discharged purulent fluid
human pathogens in the dematiaceous fungal group include on potato dextrose agar (PDA) at room temperature grew a
Alternaria, Bipolaris, Cladophialophora, Curvularia, nearly black fungus, attaining a diameter of 20 mm in 14 days
Exophiala (Wangiella), Fonsecaea, Madurella, Phialophora, and 50 mm in 35 days. Under light microscope, flask-shaped
Rhinocladiella, Scedosporium, and Scytalidium. Being phialides (conidiogenous cells) with cup-shaped collarettes
widely distributed in the environment including soil, wood, and a bunch of elliptical conidia at the apices of the collar-
and decomposing plant debris, these organisms have the ettes were noted, along with brown to light-brown hyphae.
capacity to take advantage of weakened defense mecha- These morphologic features confirmed the fungus as P. ver-
nism of human hosts, inducing cutaneous, subcutaneous, rucosa, which responded to oral itraconazole treatment [19].
and corneal infections, invasive and allergic sinusitis, arthri- A cutaneous fungal infection due to P. verrucosa was
tis, endocarditis, and systemic and disseminated infections. reported in a 53-year-old woman who had a history of sys-
Traumatic implantation, injury, and inhalation represent the temic corticosteroid treatment for Evans’ syndrome. The
main routes of entry, and host immune deficiency (suppres- patient presented asymptomatic multiple nodules on her glu-
sion) plays a critical role in the development of systemic and teal region. A smear from the purulent exudate of the nodules
disseminated infections [5,6]. showed brown-colored hyphae, spores, and large dark-brown
Pheohyphomycosis affects the skin and subcutis, parana- cells; and a dark-brown-colored colony grew on Sabouraud
sal sinuses, or central nervous system and is often catego- glucose agar, showing Phialophora-type conidia formation.
rized into four types by the region of the body in which the The fungus was diagnosed as Phialophora verrucosa [20].
infection occurs: superficial, cutaneous, subcutaneous, and Phialophora verrucosa was also shown to be respon-
systemic [2]. Chromoblastomycosis is a cutaneous and subcu- sible for a severe, verrucous facial mycosis and sinusitis
taneous mycotic disease (mostly of the feet and legs) with the in a 12-year-old girl. The disease began with the develop-
appearance of pigmented sclerotic bodies, commonly called ment of verrucous, hyperkeratotic plaques, and subcutane-
“copper pennies” [5]. Eumycetoma is a subcutaneous fungal ous violet nodules on the face and upper extremities and
infection (mostly of the feet and legs) showing a mass with later evolved into severely progressive tumorous cutaneous
multiple sinuses draining pus and “grains.” The grains (0.5– and nasal lesions. Microscopic examination of scale samples
1.5 mm in diameter) in eumycetoma may appear white (due from the upper extremities and the face showed brown, thick-
to infection with Acremonium, Cylindrocarpon, Fusarium, walled fungal elements, which were subsequently identified
Neotestudina, or Pseudallescheria) or black (due to infection as Phialophora verrucosa. Oral treatment with itraconazole
with Exophiala, Madurella, Leptosphaeria, Pyrenochaeta, and fluorocytosin resulted in significant improvement of the
or Phlenodomus). lesions [17].
Phialophora spp. have been shown to cause chromo- A case of polymicrobial keratitis caused by Phialophora
blastomycosis, mycotic keratitis, cutaneous infections, verrucosa, Candida tropicalis, and Propionibacterium acnes

Phialophora 347
has also been described. Histopathologic evaluation revealed 50°C, respectively. Preliminary identification of the colonies
penetration of the crystalline lens by fungus at the site of is based on macroscopic and microscopic features [25].
synechiae between the intact lens capsule and iris [13]. After the growth for 1–7 days on PDA slants, approximately
1 cm2 of mycelium is collected by scraping the slant with a
42.1.2.2 Phialophora richardsiae sterile stick that is placed in 1 mL of sterile H2O. The material
A case of subcutaneous phaeohyphomycosis due to is transferred to a 2 mL screw-cap tube, and the tube is centri-
Phialophora richardsiae was described in a renal transplant fuged for 1 min at 6000 × g. If the mycelium does not pellet,
recipient. The patient developed dark-colored nodules that the material is passed through a pediatric blood serum filter.
subsequently ulcerated after transplantation. Histopathologic After removal of the supernatant, the material is resuspended
examination showed dematiaceous fungal hyphae with a sur- in 200 μL of IDI sample buffer (IDI lysis kit, BD) and trans-
rounding granulomatous reaction, and the fungus was identi- ferred to the lysis tube containing glass beads. The lysis tube is
fied as P. richardsiae [21]. vortexed on the highest setting for 5 min, and the tube is placed
Diseases due to dematiaceous fungi require antifun- in a boiling water bath for 15 min. Following centrifugation
gal and surgical treatments. Current antifungal therapy for for 5 min at 16,000 × g, the supernatant is stored at −20°C until
chromoblastomycosis and subcutaneous phaeohyphomycosis polymerase chain reaction (PCR) is undertaken [25].
relies on itraconazole, and for eumycetoma it is ketoconazole. Alternatively, about 1 cm2 of fungal material is transferred
Surgical debridement in combination with amphotericin B to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) mixture
treatment is useful for chronic invasive sinusitis [6,9]. of silica gel and Celite (silica gel H, Merck 7736/Kieselguhr
Celite 545; Machery) and 300 μL of TES buffer (2 g Tris
[hydroxymethyl]-aminomethane, 0.38 g Na-EDTA, and 2 g
42.1.3 Laboratory Diagnosis sodium dodecyl sulfate in 80 mL of ultrapure water pH 8).
Phialophora represents one of the dematiaceous fungi The fungal material is ground with a micropestle for 1–2 min.
responsible for causing phaeohyphomycosis and chromoblas- The volume is adjusted by adding 200 μL of TES buffer.
tomycosis in humans. There is a need to differentiate among After vigorous shaking and the addition of 10 μL of a 10 mg/
the other dematiaceous fungi in order to implement appropri- mL concentration of proteinase K to the tube, the mixture is
ate treatment regimens as these organisms demonstrate var- incubated at 65°C for 10 min. With the addition of 140 μL of
ied sensitivity to the currently available antifungal drugs [6]. 5 M NaCl solution, the mixture is combined with 1/10 vol-
Dematiaceous fungi including Phialophora spp. have ume (∼65 μL) of cetyltrimethylammonium bromide (CTAB)
been traditionally identified by macroscopic and microscopic buffer 10%, followed by incubation for another 30 min at
characteristics as well as biochemical properties. For exam- 65°C. One volume (∼700 μL) of chloroform–isoamyl alcohol
ple, phospholipase production, induced by egg yolk substrate, (vol/vol = 24/l) is added and mixed by inversion. After incu-
offers a useful means for the differentiation of the taxonomi- bation for 30 min at 0°C (on ice water) and centrifugation at
cally related dematiaceous fungal species on the basis of their 14,000 rpm at 4°C for 10 min, the top layer is transferred to
distinct enzymatic profiles [22]. Nevertheless, these methods a clean Eppendorf tube. The sample is added with 225 μL
are laborious and sometimes incapable of distinguishing spe- of 5 M NH4 –acetate and incubated for at least 30 min (on
cies with polymorphic conidiogeneses or lacking conidia ice water) and centrifuged. The supernatant is transferred to
formation. a clean sterile Eppendorf tube and mixed with a 0.55 vol-
For these reasons, molecular techniques such as random ume (∼510 μL) of ice-cold isopropanol. After centrifugation
amplified polymorphic DNA (RAPD), restriction fragment for 7 min at 14,000 rpm and 4°C (or room temperature), the
length polymorphism (RFLP), and DNA sequencing have supernatant is decanted. The pellet is washed with ice-cold
been adopted increasingly for identification [23]. In particu- ethanol 70% twice and air dried. The powder is resuspended
lar, sequencing analysis of small subunit (SSU) 18S and large in 48.5 μL of Tris–EDTA buffer with 1.5 μL of 10 mg of
subunit (LSU) 28S rRNA genes and internal transcribed RNase/mL, incubated at 37°C for 15–30 min, and stored at
spacer regions (ITS) has proven valuable. Indeed, many −20°C until use [26].
Phialophora species can be correctly identified by examin- In addition, fungal strains can be cultured in 20 mL of
ing the nucleotide sequence of the D1/D2 domains in the 28S RPMI 1640 medium with l-glutamine but without sodium
rRNA gene [24]. bicarbonate (Sigma-Aldrich) buffered to pH 7.0 with 0.165 M
morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich).
After 3–14 days of growth with agitation (100 rpm) at 30°C,
42.2 Methods the mycelium is transferred into a tube and washed in 40 mL
of sterile distilled water. Mycelium is then stored at −20°C
until use. For DNA extraction, 100 mg of mycelium is homog-
Clinical specimens suspected of containing dematiaceous enized for 1 min in a tube containing 1 mL of lysis buffer (2%
fungi are treated with 10% KOH or stained with calcofluor Triton X-100, 1% sodium dodecyl sulfate, 10 mM Tris–HCl
white and observed by microscopy for septate hyphal ele- pH 8, 100 mM NaCl, and 1 mM EDTA pH 8), three 0.5 cm
ments. Samples are then inoculated onto inhibitory mold diameter glass beads (Sigma), and approximately 500 mg
agar, modified Sabouraud agar, or PDA at 25°C, 35°C, and of 425–600 μm glass beads (Sigma). The homogenized

mycelium is snap-frozen in liquid nitrogen, thawed, and 3.2.3 vr. SmartGene is a web-based software and
refrozen once. DNA is then extracted using a DNeasy plant database system with reference sequences derived
kit (Qiagen) [27]. from the National Center for Biological Information
Also, fungal DNA may be prepared by suspending 50 mg (NCBI) GenBank repository.
of mycelium in 600 μL extraction buffer (200 mM Tris–HCl,
pH 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate, and Note. Sequence-based identifications are defined by percent
250 mM NaCl). The mixture is vortexed for 15 s, incubated identity: species, ≥99%; genus, 93%–99%; and inconclusive,
at 100°C for 15 min, kept on ice for 60 min, and then centri- ≤93%.
fuged at 14,000 × g for 15 min. Supernatants are transferred
to new tubes and extracted with phenol–chloroform–isoamyl 42.2.2.2 Sequencing Analysis of the
alcohol (25:24:1 v/v). Each sample DNA is precipitated with ITS1-5.8S-ITS2 Region
cold isopropanol (−20°C), dried, and resuspended in 100 μL Zeng et al. [26] utilized primers V9G (5′-TTA CGT CCC
distilled water [24]. TGC CCT TTG TA-3′) and LS266 (5′-GCAT TCC CAA
ACA ACT CGA CTC-3′) for PCR amplification and sequence
42.2.2 Detection Procedures analysis of the ITS-5.8S-ITS2 region.
42.2.2.1 P an-Fungal Real-Time PCR and Procedure

Sequencing Analysis
1. PCR mixture (50 μL) is composed of 10 mM Tris–
Pounder [25] reported a real-time PCR with SYBR green HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl · 6H2O,
DNA-binding dye and amplicon-melting temperature anal- 0.01% gelatin, 200 mM of each dNTP, 25 pmol of
ysis for fungal detection using pan-fungal primers ITS1 each primer, 0.5 U of Taq DNA polymerase (Sigma),
forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 and 10–100 ng of extracted DNA.
reverse (5′-TCCTCCGCTTATTGATATGC-3′) [28]. The 2. The mixture is subjected to an initial 95°C for
identity of the fungus is confirmed by sequencing. 4 min, 35 cycles of 94°C for 45 s, 52°C for 30 s,
Procedure and 72°C for 2 min, and a final 72°C for 7 min in a
GenAmp PCR System 9700 thermocycler (Applied
1. PCR mixture consists of 1 × Lightcycler FastStart Biosystems).
DNA Master Hybridization Probes mixture (Roche 3. Amplicons are purified with GFX columns (GE
Applied Science) containing deoxynucleoside tri- Healthcare), and both strands are sequenced with
phosphates, FastStart Taq DNA polymerase, and each of the two primers separately. The sequenc-
1 mM MgCl2 (additional MgCl2 is added to a final ing PCR mixture consists of 1 μL of template DNA
concentration of 4.6 mM), 0.4 μM each of ITS1 for- (1–10 ng), 3 μL of dilution buffer, 1 μL of BigDye
ward and ITS4 reverse primers, 1 × SYBR green v3.1, and 1 μL of 4 pmol primer filled with 4 μL of
(Molecular Probes), and 3 μL template DNA. MilliQ water to a final volume of 10 μL. The ampli-
2. Thermal cycling parameters include 95°C for fication is performed with initial 95°C for 1 min, 30
10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and cycles of 95°C for 10 s, 50°C for 5 s, and 60°C for
72°C for 30 s; and a final 72°C for 2 min. 2 min. Reaction products are cleaned with Sephadex
3. The quality of the amplicon is determined using the G-50 Fine (GE Healthcare Bio-Sciences) and ana-
derivative of the melt analysis curve (55°C–99°C, lyzed by using an ABI Prism 3730xl DNA analyzer
45 s hold at 55°C, and 5 s/°C) using the RotorGene (Applied Biosystems).
3000 (Corbett Robotics, Inc). 4. The sequences are adjusted by using the Lasergene
4. The amplified product is purified for bidirectional software program SeqMan Π (DNASTAR, Inc.)
sequencing using ExoSAP-IT (USB Corp). Five and aligned iteratively using Ward’s averaging in
microliters of Big Dye Terminator Ready Reaction the Bionumerics package v. 4.0 (Applied Maths).
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of Nearest neighbors are identified by local BLAST
each primer (0.8 pmol/μL) and 3 μL of purified PCR searches. The distance trees are based on a realigned
product. Cycle sequencing is performed with a 9700 file using the DCSE program and calculated by the
thermal cycler (ABI), using 25 cycles of 96°C for neighbor-joining (NJ) method of the Treecon pack-
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing age with Kimura-2 correction; only unambiguously
reaction products are passed through a Sephadex aligned positions are taken into account. A total
G-50 fine column to remove unincorporated dye ter- of 100 bootstrap replicates are used for analysis.
minators. Purified sequencing reaction products are Bootstrap values of >90 from 100 resampled datas-
run on an ABI Prism 3100 Genetic Analyzer with a ets are shown.
50 cm capillary array.
5. Sequences are analyzed with the SmartGene Note. If the similarity of sequences of the ITS region is more
Integrated Database Network software version than 99% between a studied strain and its nearest neighbor

Phialophora 349
and they are distributed in the same branch of the phyloge- lacking conidia. Exploiting the nucleotide variations within
netic tree, the strain is regarded as belonging to the same the ITS by PCR followed by sequencing, offers a valuable
species as its nearest neighbor. tool for precise determination of Phialophora species.
42.2.2.3 Sequencing Analysis of the D1/ D2 References

Domains of 28S rRNA Gene
Abliz et al. [24] utilized primers NL-1 (5′-GCATATCAAT org/, accessed on August 1, 2010.
AAGCGGAGGAAAAG-3′) and NL-4m (5′-GGTCC 2. Revankar SG et al., Disseminated phaeohyphomyco-
GTGTTTCAAGACG-3) to amplify the conserved D1/ D2 sis: Review of an emerging mycosis. Clin Infect Dis.
domains of the 28S rRNA from Phialophora and other 2002;34:467–476.
dematiaceous fungi. Subsequent analysis of the ampli- 3. Vicente VA et al., Environmental isolation of black yeast-
con with primers NL-1 and NL-4m and internal primers like fungi involved in human infection. Stud Mycol.
2008;61:137–144.
NL-2m (5′-CTTGTGCGCTATCGGTCTC-3′) and NL-3m
4. de Hoog GS et al., A new species, Phialophora euro-
(5′-GAGACCGATAGCGCACAAG-3′) permitted the correct paea, causing superficial infections in humans. Mycoses
identification of Phialophora spp. 2000;43:409–416.
5. McGinnis MR. Chromoblastomycosis and phaeohyphomy-
Procedure cosis: New concepts, diagnosis, and mycology. J Am Acad
Dermatol. 1983;8:1–16.
1. PCR mixture (50 μL) is made up of 5 μL of tem- 6. Brandt ME, Warnock DW. Epidemiology, clinical manifesta-
plate DNA, 5 μL (2 pmol) each of primers NL-1 tions, and therapy of infections caused by dematiaceous fungi.
and NL-4m, 4 μL (2.5 mM) dNTP mixture, 0.25 μL J Chemother. 2003;15(Suppl. 2):36–47.
(5 U/μL) Taq polymerase, and 5 μL 10 × reaction 7. Duggan JM, Wolf MD, Kauffman CA. Phialophora infection
buffer. in an AIDS patient. Mycoses 1995;38:215–218.
2. Amplification is conducted with an initial 95°C for 8. Hirst LW, et al., Phialophora corneal ulcer. Aust N Z J
Ophthalmol. 1995;23(3):223–225.
4 min; 30 cycles of 94°C for 1 min, 55°C for 2.5 min,
9. Guerriero C, De Simone C, Tulli A. A case of chromoblasto-
and 72°C for 2.5 min; and a final 72°C for 10 min. mycosis due to Phialophora verrucosa responding to treat-
3. The amplified products are first checked on 1.5% ment with fluconazole. Eur J Dermatol. 1998;8(3):167–168.
agarose gel, purified with SUPREC™-02 (TaKaRa) 10. Sharma NL et al., Subcutaneous phaeohyphomycosis in India:
and sequenced with an ABI Prism 3100 sequencer A case report and review. Int J Dermatol. 2002;41:16–20.
after labeling with BigDye™ Terminator Cycle 11. Cornia PB, Raugi GJ, Miller RA. Phialophora richardsiae
Sequencing Ready Reaction (Applied Biosystems) bursitis treated medically. Am J Med. 2003;115(1):77–79.
12. Eckhard M et al., Fungal foot infections in patients with dia-
using external primers, NL-1 and NL-4m, and the
betes mellitus—Results of two independent investigations.
internal primers, NL-2m and NL-3m. Mycoses 2007;50(Suppl. 2):14–19.
4. The sequence data are aligned with CLUSTAL W 13. Banitt M et al., A case of polymicrobial keratitis violating an
(version 1.6). Phylogenetic trees are constructed intact lens capsule. Cornea 2008;27(9):1057–1061.
with the NJ method. 14. Iwatsu T, Miyaji, M. Subcutaneous cystic granuloma caused by
Phialophora verrucosa. Mycopathologia 1978;64:165–168.
15. Schnadig VJ et al., Phialophora-verrucosa induced subcuta-
42.3 Conclusions neous phaeohyphomycosis. Acta Cytol. 1986;30:425–429.
16. Lieb DF et al., Case report: Fungal endophthalmitis caused by
The genus Phialophora is a dematiaceous fungus that rep- Phialophora richardsiae. Retina 2003;23(3):406–407.
resents one of the causes for human phaeohyphomycosis 17. Hofmann H et al., Invasive chromoblastomycosis and sinus-
and chromoblastomycosis. The most common Phialophora itis due to Phialophora verrucosa in a child from northern
spp. identified in human infections are P. verrucosa and Africa. Mycoses 2005;48(6):456–461.
18. Lundstrom TS et al., Phialophora verrucosa infection in a
P. richardsiae, with clinical symptoms ranging from cutane-
BMT patient. Bone Marrow Transplant. 1997;20:789–791.
ous and subcutaneous infections, mycotic keratitis, osteomy- 19. Kimura M et al., Multifocal subcutaneous phaeohyphomyco-
elitis, endocarditis, and disseminated infections. sis caused by Phialophora verrucosa. Arch Pathol Lab Med.
Because different dematiaceous fungal taxa demonstrate 2003;127:91–93.
varied sensitivity and resistance to antifungal reagents, it is 20. Ohira S et al., Case report: Phaeohyphomycosis caused
important to determine their species identity with the aim by Phialophora verrucosa developed in a patient with
of implementing the most effective treatment measures for non-HIV acquired immunodeficiency syndrome. Mycoses
these diseases [6]. While traditional macroscopic, micro- 2002;45:50–54.
21. Yehia M et al., Subcutaneous black fungus (phaeohyphomy-
scopic, and biochemical methods provide a useful approach
cosis) infection in renal transplant recipients: Three cases.
for the differentiation of the taxonomically related dema- Transplantation 2004;77(1):140–142.
tiaceous Phialophora spp., they suffer from the shortcom- 22. Souza TF et al., Secretion of five extracellular enzymes by
ings of being time consuming, laborious, and incapable of strains of chromoblastomycosis agents. Rev Inst Med Trop
distinguishing species with polymorphic conidiogeneses or Sao Paulo. 2008;50(5):269–272.

23. Yamagishi Y, Kawasaki K, Ishizaki H. Mitochondrial 26. Zeng JS et al., Spectrum of clinically relevant Exophiala species
DNA analysis of Phialophora verrucosa. Mycoses in the United States. J Clin Microbiol. 2007; 45:3713–3720.
1997;40:329–334. 27. Desnos-Ollivier M et al., Molecular identification of black-grain
24. Abliz P et al., Identification of pathogenic dematiaceous fungi mycetoma agents. J Clin Microbiol. 2006; 44(10):3517–3523.
and related taxa based on large subunit ribosomal DNA D1/D2 28. White TJ et al., Amplification and direct sequencing of fun-
domain sequence analysis. FEMS Immunol Med Microbiol. gal ribosomal RNA genes for phylogenetics. In: Innis, M.A.,
2004;40(1):41–49. Gelfand, J.J., Sninsky, J.J., and White, J.T., Eds., 1990. PCR
25. Pounder JI, Discovering potential pathogens among fungi Protocols: A Guide to Methods and Applications, Academic
identified as nonsporulating molds. J Clin Microbiol. Press, Inc., San Diego, CA, pp. 315–322.
2007;45(2):568–571.

43 Rhinocladiella
Dongyou Liu
Contents
43.1 Introduction.......................................................................................................................................................................351
43.1.3 Diagnosis.............................................................................................................................................................. 353
43.2 Methods............................................................................................................................................................................ 354
43.2.2.1 Sequence Analysis of ITS Regions........................................................................................................ 354
43.2.2.2 Sequence Analysis of SSU rRNA Gene................................................................................................ 355
43.3 Conclusion........................................................................................................................................................................ 355
References.................................................................................................................................................................................. 355
43.1 Introduction cellaris (→ Zasmidium cellare), Rhinocladiella compac-

tum (→ Fonsecaea compacta), Rhinocladiella cristaspora
43.1.1 Classification and Morphology (→ Ardhachandra cristaspora), Rhinocladiella elatior
The genus Rhinocladiella is a dematiaceous (dark-walled) (→ Leptodontidium elatius var. elatius), Rhinocladiella ellisii
fungus belonging to the mitosporic Herpotrichiellaceae (→ Zasmidium cellare), Rhinocladiella mansonii (synonym:
group, family Herpotrichiellaceae, order Chaetothyriales, Exophiala mansonii), Rhinocladiella pedrosoi (→ Fonsecaea
class Eurotiomycetes, subphylum Pezizomycotina, phy- pedrosoi), Rhinocladiella peruviana (→ Dicyma peruviana),
lum Ascomycota, and kingdom Fungi. The mitosporic Rhinocladiella schulzeri (→ Ramichloridium schulzeri),
Herpotrichiellaceae group encompasses 13 genera: Rhinocladiella selenoides (→ Ardhachandra selenoides),
Blastacervulus, Brycekendrickomyces, Cladophialophora, and Rhinocladiella spinifera (→ Exophiala spinifera) [4].
Cladoriella, Cyphellophora, Exophiala, Heteroconium, Rhinocladiella colonies are slow growing, moist in tex-
Melanchlenus, Metulocladosporiella, Phaeococcomyces, ture, and dark olivaceous brown in color. Submerged hyphae
Rhinocladiella, Thysanorea, and Veronaea [1]. are hyaline to pale olivaceous and smooth; aerial hyphae
Currently, the genus Rhinocladiella consists of are more darkly pigmented. Conidial apparatus (consisting
eight recognized species: Rhinocladiella anceps (syn- of either tips of ascending hyphae or septate conidiophores)
onyms: Ramichloridium anceps and Veronaea parvis- is branched and olivaceous brown. Conidiogenous cells are
pora), Rhinocladiella aquaspersa (obsolete synonym: intercalary or terminal, polyblastic, cylindrical to acicular,
Ramichloridium cerophilum), Rhinocladiella atrovirens, with a sympodially proliferating and subdenticulate rachis;
Rhinocladiella basitona (synonym: Ramichloridium basito- scars are unthickened and non-pigmented to darkened refrac-
num), Rhinocladiella fasciculata (basionym: Ramichloridium tive. Conidia are solitary, hyaline to subhyaline, aseptate, thin
fasciculatum), Rhinocladiella mackenziei (obsolete synonyms: walled, smooth, and subglobose, with a slightly pigmented
Ramichloridium mackenziei, Ramichloridium obovoidea, hilum; conidial secession is schizolytic [4].
and Ramichloridium obovoiedum), Rhinocladiella pha- At the species level, Rhinocladiella aquaspersa (obsolete
eophora, and Rhinocladiella similis, in addition to 24 unas- synonyms: Acrotheca cerophila, Cladosporium cerophilum,
signed species [1–4]. Of these, Rhinocladiella aquaspersa, and Ramichloridium cerophilum) colonies grow slowly, reach-
Rhinocladiella atrovirens, and Rhinocladiella mackenziei ing a diameter of 12 mm after 14 days at 24°C on malt extract
have been shown to infect humans [5]. Rhinocladiella mack- agar (MEA). Colonies are velvety to hairy, with entire mar-
enziei is a serious human pathogen, causing cerebral pha- gin, and surface is dark olivaceous gray, with black gelatinous
eohyphomycosis while Rhinocladiella aquaspersa is often exudate droplets on oatmeal agar (OA). Submerged hyphae
associated with localized chromoblastomycosis lesions [6–8]. (1.5–3 μm wide) are pale olivaceous brown and smooth or
A number of former Rhinocladiella species have been slightly rough; aerial hyphae are olivaceous brown, smooth
transferred to other genera. These include Rhinocladiella or slightly rough, and somewhat narrower and darker than
apiculata (→ Ramichloridium apiculatum), Rhinocladiella the submerged hyphae. Conidiophores (2–3 μm × 50 μm) are
351

unbranched, arising vertically from creeping aerial hyphae, in diameter) with hardly prominent, slightly pigmented,
dark brown, thick walled, smooth or verruculose, hardly not thickened scars. Conidia (2.5–6 μm × 2–3 μm) are soli-
tapering toward the apex, with up to three additional septa. tary, smooth, thin walled, subhyaline, and ellipsoidal, with
Conidiogenous cells are integrated, terminal, proliferating truncate, slightly pigmented hilum (0.5 μm in diameter).
sympodially, rachis short and straight, with crowded, promi- Synanamorph forms on torulose hyphae originating from
nent, pigmented unthickened scars, minute, about 0.5 μm in giant cells; compact heads of densely branched hyphae form
diameter. Conidia (4–11 μm × 2–3 μm) are solitary, fusiform thin-walled, lateral, subglobose cells, on which conidiog-
to clavate, thin walled, smooth, 0–1 septate, and subhyaline, enous cells are formed; conidiogenous cells (12 × 1–1.5 μm)
with a conspicuous hilum, about 0.5 μm in diameter, slightly proliferate percurrently, giving rise to tubular annellated
raised with an inconspicuous marginal frill. Conidia some- zones with inconspicuous annellations. Conidia (2–2.5 μm in
times produce one to four short secondary conidia [4]. diameter) are smooth, thin walled, aseptate, subhyaline, and
Rhinocladiella atrovirens colonies are restricted, vel- globose [4].
vety or lanose, olivaceous, and slightly mucoid at the center; Rhinocladiella mackenziei (basionym: Ramichloridium
reverse is dark olivaceous green to blackish. Conidiophores mackenziei) colonies attain a diameter of 5 mm on MEA
are short, brown, and thick walled. Conidiogenous cells after 14 days at 24°C, with entire, smooth, sharp margin;
(9–19 μm × 1.6–2.2 μm) are cylindrical and intercalary or mycelium is densely lanose and raised in the center and oliva-
free; denticulate rachis (up to 15 μm long) shows crowded, ceous green to brown; reverse is dark olivaceous. Submerged
flat or butt-shaped, unpigmented conidial denticles. Conidia hyphae (2–3 μm wide) are subhyaline, smooth, and thin-
(3.7–5.5 μm × 1.2–1.8 μm) are hyaline, thin and smooth walled; aerial hyphae are pale brown and slightly narrower.
walled, and short cylindrical, with truncate basal scars. Conidiophores (10–25 μm × 2.5–3.5 μm) are slightly or not
Budding cells (3.0–4.3 μm × 1.7–2.5 μm) are hyaline, thin differentiated from vegetative hyphae, arising laterally
walled, and broadly ellipsoidal. Germinating cells (4.5– from aerial hyphae, with one or two additional septa, often
6.0 μm) are inflated and spherical to subspherical. An annel- reduced to a discrete or intercalary conidiogenous cell, pale-
lidic Exophiala synanamorph may be present [4]. brown. Conidiogenous cells (5–15 μm × 3–5 μm) are terminal
Rhinocladiella basitona (synonym: Ramichloridium or intercalary, variable in length, occasionally slightly wider
basitonum) colonies are smooth, compact, and slightly raised than the basal part, pale brown, rachis (0.5 μm in diameter)
at the center, flat toward margin, locally with some sub- with slightly prominent, unpigmented, non-thickened scars.
merged mycelium, and olivaceous black with black reverse. Conidia (5–12 μm × 2–5 μm) are golden-brown, thin walled,
Hyphae (2 μm wide) are thick walled, olivaceous brown, smooth, ellipsoidal to obovate, and subcylindrical, with
and septate every 15–20 μm. The conidial apparatus is pro- darkened, inconspicously thickened, protuberant or truncate
fusely branched with flexuose cells arising at acute angles, hilum (<1 μm in diameter) [4].
the lower cells often being shorter than the ultimate ones Rhinocladiella similis colonies are restricted, mostly dry
and concolourous with the hyphae. Conidiogenous cells are or initially with some black slime at the centre, velvety, and
cylindrical, with the apical part of variable length, producing olivaceous gray with olivaceous black reverse. Budding cells
numerous conidia in sympodial sequence; denticles are trun- (5 μm × 3 μm) are abundant, pale olivaceous, broadly ellip-
cate, with a slightly darkened scar without a hilum. Conidia soidal, and without capsule in India ink, often inflating and
(3.5–4.5 μm × 2.2 μm) are hyaline, smooth walled and thin developing into broadly ellipsoidal brown germinating cells
walled, and triangular with a rounded apex and with a clearly (5 μm × 4 μm) that often bear a clearly discernible truncate
discernible basal scar. Rhinocladiella basitona differs from extension, which bears a very short annellated zone. Hyphae
Rhinocladiella anceps by its basitonously branched conidio- (1.5 μm wide) are pale olivaceous to brown and regularly
phores and triangular conidia [9]. septate every 20–40 μm. Conidiogenous cells arise at acute
Rhinocladiella fasciculata (basionym: Ramichloridium angles in a profusely branched conidial apparatus, which is
fasciculatum) colonies attain a diameter of 8 mm after 14 days brown, somewhat darker than the sterile hyphae; conidioge-
at 24°C on MEA, with entire, smooth, sharp margin; myce- neous cells are cylindrical, 12–20 × 2 μm apically, with an
lium is velvety, becoming farinose in the center (due to abun- elongating sympodial part bearing conidia on small denticles
dant sporulation), olivaceous green to brown; reverse is dark mainly at the apices of the cells. Conidia (4–7 × 1.5 μm) are
olivaceous. Blackish droplets produced at the center contain subhyaline, noncatenate, cylindrical, and narrowed toward
masses of Exophiala conidia. Submerged hyphae (2–2.5 μm the base, with a small but clearly visible scar. Rhinocladiella
wide) are subhyaline, smooth, and thickwalled; aerial hyphae similis shows preponderantly sympodial conidiogenesis and
are pale brown. Conidiophores (220 μm × 2–3 μm) arise ver- possesses a profusely branched conidial apparatus of the
tically from ascending hyphae in loose fascicles and are same texture and pale-brown pigmentation as its mycelium.
unbranched or loosely branched at acute angles, cylindri- This feature is the hallmark of Rhinocladiella [9].
cal, smooth, brown, and thick walled at the base, with 0–5 The main feature to distinguish Rhinocladiella from
thin additional septa. Conidiogenous cells (30–100 μm long) Ramichloridium is the presence of exophiala-type bud-
are terminal, cylindrical, thin walled, smooth, pale brown, ding cells in species of Rhinocladiella. Although both
and fertile part as wide as the basal part, up to 2 μm wide, Rhinocladiella and Veronaea produce sympodial conidiog-
proliferating sympodially, giving rise to a rachis (<0.5 μm enous cells, Veronaea is differentiated from Rhinocladiella

Rhinocladiella 353
by its absence of exophiala-type budding cells and its pre- A fungus grew in culture and identified as R. aquaspersa on
dominantly one-septate conidia (i.e., two-celled conidia) (in the basis of the characteristic conidiation. Badali et al. [23]
comparison with one-celled conidia of Rhinocladiella) [4]. reported an R. aquaspersa-related case of chromoblastomy-
cosis in a 56-year-old male, who presented with warty nod-
ules and lymphatic distribution on the forearm, resembling
sporotrichosis. Mycological and histopathological investiga-
Rhinocladiella mackenziei (formerly Ramichloridium tion of exudates and biopsy tissue samples demonstrated a
mackenziei, Ramichloridium obovoideum). Rhinocladiella granulomatous lesion with muriform cells, the hallmark of
mackenziei to the second most common cause (next is chromoblastomycosis. The causative agent was identified
Cladophialophora bantiana) of central nervous system as Rhinocladiella aquaspersa. A second case of chromo-
(CNS) phaeohyphomycosis due to darkly pigmented fungi. blastomycosis caused by this fungus involved a 62-year-old
Rhinocladiella mackenziei infections cause high mortality Brazilian female. In this case, R. aquaspersa hyphae instead
and are diagnosed exclusively in patients from the Middle of muriform cells were observed in tissue.
East, with more than one-half of the cases occurring in Dematiaceous (melaninized) fungi responsible for cere-
patients with no known underlying immunodeficiency bral phaeohyphomycosis (e.g., Rhinocladiella mackenziei)
[10–18]. are environmental organisms that may enter human host
Podnos et al. [19] reported a case of cerebral Rhinocladiella via inhalation [24–27]. These fungi may induce an initial
mackenziei (R. obovoideum) in a 58-year-old Kuwaiti woman, subclinical pulmonary focus before causing CNS infection
with a history of chronic renal failure requiring hemodialysis. through hematogenous route [28,29]. Melanin present in
The patient presented with a 3 day history of left frontal head- their cell walls not only imparts the characteristic dark color
ache, blurry vision, dizziness, and right-sided clumsiness. A to their conidia and hyphae but also acts as virulence fac-
computed tomography-guided needle biopsy of the parieto- tor that plays an important role in the pathogenesis of infec-
occipital lesion yielded 10 mL of dark caseous fluid, which tions and possibly in their CNS localization [30]. Receptors
contained long, branching, septate hyphae. Culture of the recognizing melanin or its biochemical byproducts may
biopsy yielded Rhinocladiella mackenziei (R. obovoideum). allow these fungi to cross the blood–brain barrier and enter
Despite treatment with amphotericin B and itraconazole, the the brain parenchyma. Melanin may also confer a protec-
patient succumbed to the infection. In a separate study, Kanj tive advantage by scavenging free radicals and hypochlorite
et al. [20] described two cases of brain abscesses caused by that are produced by phagocytic cells in their oxidative burst
Rhinocladiella mackenziei (Ramichloridium mackenziei) in and that would normally kill most organisms. Additionally,
the Middle East. One patient had chronic myelomonocytic melanin may bind to hydrolytic enzymes, thereby prevent-
leukemia while the other patient was a normal host. Both ing their action on the plasma membrane. Hayakawa et al.
cases had a fatal outcome despite aggressive antifungal [31] showed that complement-mediated phagocytosis of the
therapy and surgical intervention. Alkhunaizi et al. [18] also fungus by macrophages represents an important host defense
described a Rhinocladiella mackenziei (Ramichloridium mechanism against Rhinocladiella aquaspersa.
mackenziei)-related CNS infection in a patient who under-
went living unrelated renal transplantation (LURTX) and
43.1.3 Diagnosis
who developed hemiplegia and was debilitated.
Rhinocladiella aquaspersa. Rhinocladiella aquaspersa is Because dematiaceous fungi are common soil inhabitants
occasional cause of human chromoblastomycosis, which is and are often considered as laboratory contaminants, their
characterized by the slow development of polymorphic skin isolations in culture media provide a preliminary indication
lesions (nodules, verrucas, tumores, plaques, and scar tissue). of their causal roles in phaeohyphomycosis and chromoblas-
Inside the host, infectious propagules adhere to epithelial tomycosis. Microscopic observation of moniliform hyphae or
cells and differentiate into sclerotic forms, which effectively irregularly swollen hyphae with yeast-like structures (com-
resist destruction by host effector cells and allow onset of pared to septate, acutely branching, straight-walled hyphae
chronic disease. Aspergillus species) or muriform cells in tissue biopsy is crit-
Arango et al. [21] documented an unusual case of chro- ical for confirming the involvement of Rhinocladiella mack-
moblastomycosis due to R. aquaspersa in a 60-year-old male enziei or Rhinocladiella aquaspersa in the disease process.
urban resident. The patient presented with darkly pigmented, Rhinocladiella mackenziei (classified under Eurotiomy
infiltrative, and crusty lesion in the ear, and the fungus was cetes), the causative agent for severe cerebral phaeohypho-
recovered in culture and identified as R. aquaspersa on the mycosis in humans, may be occasionally confused with
basis of the characteristic sporulation. Itraconazole therapy Pleurothecium obovoideum (classified under Sordariomycetes)
resulted in complete healing. Marques et al. [22] also reported morphologically. However, P. obovoideum clusters with sexual
an unusual case of chromoblastomycosis lesions localized in species of Carpoligna that have Pleurothecium anamorphs.
three different sites in a 52-year-old male farm worker from Because of the lengthy time and specialized skills that
Brazil. Physical examination showed extensive plaques situ- are needed for macroscopic and microscopic identification
ated on the left leg, left arm, forehead, and left side of the of dematiaceous (melanized) fungi, molecular techniques are
face. Direct examination of biopsies revealed sclerotic cells. increasingly utilized for improved speciation and detection

of these organisms [32–34]. Spatafora et al. [35] undertook Isolation Kit (Mo Bio Laboratories) from mycelium taken
a cladistic analysis of 1050 bp of the genes coding for small- from fungal colonies on MEA [37].
subunit ribosomal RNA (SSU rRNA) to clarify the phyloge-
netic relationship among species of Exophiala, Fonsecaea,
Phialophora, Ramichloridium, and Rhinocladiella. It was
noted that the conventional categories of these fungi based on 43.2.2.1 Sequence Analysis of ITS Regions
asexual states are not supported by phylogenetic analysis of Arzanlou et al. [4] utilized the universal primers ITS1 and
SSU rRNA sequences. Isolates exhibiting annellidic modes ITS4 to amplify the ITS region of the nuclear ribosomal
of blastic conidiogenesis (e.g., Exophiala spp.) are not mono- RNA operon, including the 3′ end of the 18S rRNA gene, the
phyletic and are placed as sister taxa to isolates that produce first ITS region (ITS1), the 5.8S rRNA gene, the second ITS
phialides or sympodulae. The results indicated very close region (ITS2), and the 5′ end of 28S rRNA gene. Subsequent
relationships between isolates of Wangiella dermatitidis sequencing analysis allows identification of fungal organ-
and Exophiala mansonii as well as between Rhinocladiella isms including Chaetomium species.
aquaspersa and Exophiala jeanselmei. The etiological agents
of chromoblastomycosis form a closely related group (clade), Procedure
while the agents of phaeohyphomycosis display a broader
distribution on the SSU rRNA tree. In a separate study, Zeng 1. Polymerase chain reaction (PCR) mixture (25 μL) is
and de Hoog [36] exploited the sequence diversity of the composed of 0.5 U Taq polymerase (Bioline), 1× poly-
internal transcribed spacer (ITS) region of rRNA for reliable merase chain reaction (PCR) buffer, 0.5 mM MgCl2,
identification of dematiaceous fungi. 0.2 mM of each dNTP, 5 pmol of each primer, and
approximately 10–15 ng of fungal genomic DNA.
2. Amplification is performed on a GeneAmp PCR
43.2 Methods System 9700 (Applied Biosystems) with primary
denaturation at 96°C for 5 min; 36 cycles of 96°C for
30 s, 52°C for 30 s, and 72°C for 60 s; a final exten-
Clinical specimens are examined directly under microscope sion at 72°C for 7 min.
with a fungal stain. Portions of the samples are inoculated 3. The amplicons are sequenced using BigDye
on Sabouraud glucose agar without or with antibiotics. The Terminator v. 3.1 (Applied Biosystems,) or
resulting isolates are identified on the basis of macroscopical DYEnamicET Terminator (Amersham Biosciences)
and microscopical features. For easy microscopic observation, Cycle Sequencing Kits and analyzed on an ABI
slide culture technique may be utilized. Slide cultures are set Prism 3700 (Applied Biosystems).
up in Petri dishes containing 2 mL of sterile water, into which 4. Newly generated sequences are subjected to a Blast
a U-shaped glass rod is placed, extending above the water search of the GenBank databases; sequences with
surface. A block of freshly growing fungal colony, approx. high similarity are downloaded from GenBank, and
1 cm2, is placed onto a sterile microscope slide, covered with a comparisons are made on the basis of the alignment
somewhat larger, sterile glass cover slip, and incubated in the of the obtained sequences.
moist chamber. Fungal sporulation is monitored over time, 5. Phylogenetic analysis is performed with PAUP
and when optimal, images are captured by means of a Nikon (Phylogenetic Analysis Using Parsimony) v. 4.0b10
camera system (Digital Sight DS-5M, Nikon Corporation) [4]. using the neighbor-joining algorithm with the
For fungal DNA extraction, 50 mL of Sabouraud dex- uncorrected (“p”), the Kimura 2-parameter and the
trose (SAB) broth is inoculated by needle with conidia HKY85 substitution models. Alignment gaps longer
from a 7 day culture in SAB agar and incubated for 72 h than 10 bases are coded as single events for the phy-
at 30°C. The hyphae are recovered on a 0.45 μm-pore-size logenetic analyses; the remaining gaps are treated
filter and washed with sterile saline. Aliquots of the fungal as missing data. Any ties are broken randomly when
hyphae are stored frozen at −70°C until use. Prior to lysis, encountered.
the hyphae are thawed and suspended in 400 μL of DNA 6. Phylogenetic relationships are also inferred with the
extraction buffer (1 mM EDTA pH 8.0, 1% sodium dodecyl parsimony algorithm using the heuristic search option
sulfate, 10 mM Tris–HCl pH 7.6, 100 mM NaCl, and 2% with simple taxon additions and tree bisection and
Triton X-100). Microcentrifuge tubes (1.5 mL) containing reconstruction (TBR) as the branch-swapping algo-
hyphae and buffer are sonicated in a water bath (Branson; rithm; alignment gaps are treated as a fifth character
model 2210) for 15 min, followed by heating at 100°C for state and all characters are unordered and of equal
5 min. Following lysis, DNA is purified using the QIAmp weight. Branches of zero length are collapsed and all
blood kit (Qiagen) and protocols for crude cell lysates sup- multiple, equally parsimonious trees are saved.
plied by the manufacturer. The purified DNA is stored at 4°C 7. Other measures calculated include tree length, con-
until tested. Alternatively, DNA is extracted with a FastDNA sistency index, retention index, and rescaled con-
kit (Qbiogene) from mycelium grown for 3–5 days in liquid sistency index (TL, CI, RI, and RC, respectively).
complete medium or with the UltraCleanTM Microbial DNA The robustness of the obtained trees is evaluated

Rhinocladiella 355
by 1000 bootstrap replications. Bayesian analysis is microscopic characteristics is time-consuming and techni-
performed. The best nucleotide substitution model cally demanding. The use of molecular procedures such
is determined using MrModeltest v. 2.2. MrBayes as PCR and sequencing analysis of ribosomal rRNA genes
v. 3.1.2 is used to perform phylogenetic analyses, and internal transcribed spacer regions provides a rapid and
using a general time-reversible (GTR) substitution accurate approach for the discrimination of dematiaceous
model with inverse gamma rates, dirichlet base fre- fungi including Rhinocladiella species.
quencies, and the temp value set to 0.5.
References
Note. Part of the large-subunit 28S rRNA (LSU) gene may
be also amplified with primers LR0R and LR5 followed by 1. The UniProt Consortium. Available at http://www.uniprot.
sequencing analysis. org/, accessed on August 1, 2010.
2. de Hoog GS. Rhinocladiella and allied genera. Stud Mycol.
1977;15:1–140.
43.2.2.2 Sequence Analysis of SSU rRNA Gene
3. McGinnis MR, Schell WA. The genus Fonsecaea and its
Spatafora et al. [35] utilized primers NS1 and NS4 to amplify a relationship to the genera Cladosporium, Phialophora,
1150 bp of the SSU rRNA for phylogenetic analysis of demati- Ramichloridium, and Rhinocladiella. Pan Am Health Organ
aceous fungi including Exophiala, Fonsecaea, Phialophora, Sci Publ. 1980;396:215–234.
Ramichloridium, Rhinocladiella, and Wangiella. 4. Arzanlou M et al. Phylogenetic and morphotaxonomic revi-
sion of Ramichloridium and allied genera. Stud Mycol.
Procedure 2007;58:57–93.
5. del Palacio-Hernanz A et al. Infection of the central ner-
1. Culture is grown in potato dextrose liquid media for vous system by Rhinocladiella atrovirens in a patient with
1–2 weeks, depending on growth rates. Isolates are acquired immunodeficiency syndrome. J Med Vet Mycol.
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confirmed by microscopic examination. Mycelium
6. Ajello L. Hyalohyphomycosis and phaeohyphomycosis: Two
is collected, frozen in liquid nitrogen, and stored at global disease entities of public health importance. Eur J
−70°C prior to DNA extractions. Epidemiol. 1986;2(4):243–251.
2. PCR amplification is conducted with 1 cycle of 7. Campbell CK, Al-Hedaithy SSA. Phaeohyphomycosis of the
94°C for 3 min; 40 cycles of 94°C for 1 min, 53°C brain caused by Ramichloridium mackenziei sp nov in Middle
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5 min. 8. Brandt ME, Warnock DW. Epidemiology, clinical manifesta-
tions, and therapy of infections caused by dematiaceous fungi.
3. PCR products are purified by using microcen-
J Chemother. 2003;15(Suppl 2):36–47.
trifuge ultrafiltration cartridges (UFC3 THK 9. de Hoog GS et al. Species diversity and polymorphism in
00; Millipore) and concentrated to a final vol- the Exophiala spinifera clade containing opportunistic black
ume of 25 μL. Purified products are sequenced yeast-like fungi. J Clin Microbiol. 2003;41:4767–4778.
using primers NS1, NS2, and NS4 and SR11R 10. Naim UR, Mahgoub ES, Chagla AH. Fatal brain abscesses
(5′-GGAGCCTGAGAAACGGCTAC-3′) and SR7R. caused by Ramichloridium obovoideum: Report of three
4. Sequence alignments are analyzed cladistically with cases. Acta Neurochir (Wien). 1988;93:92–95.
the software package PAUP. Informative characters 11. Sutton DA et al. US case report of cerebral phaeohyphomy-
cosis caused by Ramichloridium obovoideum (R. macken-
are analyzed by using the tree bisection–reconnection
ziei): Criteria for identification, therapy, and review of other
with random sequence addition option; 25 replica- known dematiaceous neurotropic taxa. J Clin Microbiol.
tions are performed. Support for inferred groups is 1998;36:708–715.
estimated by the bootstrapping technique. Five hun- 12. Al Abdely HM et al. SCH 56592, amphotericin B, or itraconazole
dred bootstrap replications are performed for the therapy of experimental murine cerebral phaeohyphomycosis
informative characters alone, using the tree bisection– due to Ramichloridium obovoideum (“Ramichloridium mack-
reconnection with random sequence addition option. enziei”). Antimicrob Agents Chemother. 2000;44:1159–1162.
13. Al-Abdely HM et al. Successful therapy of cerebral phaeohy-
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43.3 Conclusion triazole posaconazole. Med Mycol. 2005;43(1):91–95.
14. Kashgari TQ et al. Cerebral phaeohyphomycosis caused by
The genus Rhinocladiella currently comprises 12 distinct Ramichloridium mackenziei in the Eastern Province of Saudi
dematiaceous fungal species that are widely distributed in the Arabia. Ann Saudi Med. 2000;20:457–460.
environments, among which Rhinocladiella mackenziei is an 15. Khan ZU et al. Additional case of Ramichloridium macken-
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blastomycosis lesions in various parts of the world. Due to Infect Dis. 2002;34:467–476.
the morphological resemblances among Rhinocladiella, 17. Revankar SG, Sutton DA, Rinaldi MG. Primary central ner-
Veronaea, Ramichloridium, and melanized fungi, identifica- vous system phaeohyphomycosis: A review of 101 cases. Clin
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18. Alkhunaizi AM, Amir AA, Al-Tawfiq JA. Invasive fungal 29. Horre R, de Hoog GS. Primary cerebral infections by mela-
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44 Trichophyton
Sharon C.-A. Chen, David Ellis, Tania C. Sorrell, and Wieland Meyer
Contents
44.1 Introduction...................................................................................................................................................................... 357
44.1.1 Classification......................................................................................................................................................... 357
44.1.1.1 Morphological-Based Classification...................................................................................................... 357
44.1.1.2 Molecular-Based Taxonomy.................................................................................................................. 358
44.1.2 Ecology and Epidemiology................................................................................................................................... 358
44.1.2.1 Species Distribution............................................................................................................................... 358
44.1.2.2 Disease Transmission............................................................................................................................. 358
44.1.3 Specific Virulence Determinants......................................................................................................................... 362
44.1.4 Clinical Manifestations........................................................................................................................................ 362
44.1.4.1 Tinea Corporis and Tinea Cruris........................................................................................................... 362
44.1.4.2 Tinea Pedis............................................................................................................................................. 363
44.1.4.3 Tinea Capitis.......................................................................................................................................... 363
44.1.4.4 Tinea Unguium (Onychomycosis).......................................................................................................... 363
Invasive Forms of Disease.................................................................................................................................... 363
44.1.5 Diagnosis of Trichophyton Infection.................................................................................................................... 363
44.1.5.1 Phenotypic-Based Methods................................................................................................................... 363
44.1.5.2 Molecular-Based Diagnostic Methods................................................................................................... 367
44.2 Methods............................................................................................................................................................................ 368
44.2.1.1 Sample Collection for Phenotypic and Molecular-Based Identification................................................ 368
44.2.1.2 Media and Culture Conditions............................................................................................................... 369
44.2.1.3 DNA Extraction Protocols..................................................................................................................... 369
44.2.2.1 Species Identification Using ITS Sequence Analysis............................................................................ 370
44.2.2.2 Genotyping of Trichoohyton spp........................................................................................................... 371
44.3 Conclusion and Perspectives............................................................................................................................................. 372
References.................................................................................................................................................................................. 372
44.1 Introduction as Achorion schoenleinii and, in 1847, as Trichophyton

mentagrophytes.1,7 The early literature is confused by the
Fungi of the genus Trichophyton are the commonest agents description of many new species often based on trivial differ-
of dermatophytoses. They are especially significant in nail ences in colony morphology. The discovery of Arthroderma
infections but also invade as keratinized skin and hair caus- uncinatum, the teleomorph of Trichophyton ajelloi, in 1959
ing ringworm infection.1,2 Because of differences in clinical led to the descriptions of the sexual states of many derma-
associations and geographic distribution between species,3,4 tophytes.7 In 1986, the teleomorph states of Microsporum
accurate identification is important for disease management and Trichophyton were consolidated into a single teleo-
and for epidemiological purposes including the investigation morph genus Arthroderma (family Arthrodermataceae;
of case clusters.5,6 order Onygenales). Based on morphological characteristics,
Arthroderma was reported to contain four anamorph gen-
44.1.1 Classification era: Chrysosporium, Epidermophyton, Microsporum, and
Trichophyton.8 A masterful review of the early literature,
44.1.1.1 Morphological-Based Classification featuring many of the great names of dermatology/medical
The genus Trichophyton was first established in 1845 with mycology such as David Gruby, Raymond Sabouraud, and
the description of Trichophyton tonsurans. Other species Chester Emmons, is provided by Rippon7; 22 Trichophyton
descriptions quickly followed; in the same year, Trichophyton species were accepted representing the “state-of-the-art”
schoenleinii was described as Oidium schoenleinii and phylogeny based on morphology. However, from this time
357

on, molecular biology has played the more important role in and Trichophyton ajelloi are highly conserved and recently
delineating phylogenetic relationships (Table 44.1). diverged lineages.30 As such, species boundaries within
Trichophyton may require further evaluation using alterna-
44.1.1.2 Molecular-Based Taxonomy tive molecular approaches.
The application of molecular phylogenetic markers has
called into question recognition of the four morphological
44.1.2 Ecology and Epidemiology
anamorph genera of Arthroderma (as above). Specifically,
the phylogenetic species concept has provided data, whereby Pathogenic Trichophyton may be anthropophilic (primar-
the genus Trichophyton is split up by those of Microsporum ily affecting humans) or zoonotic (primarily infecting ani-
and Epidermophyton,9,10 with some Chysosporium spp. mals, e.g., cats, horses, cattle, sheep, and camels). Geophilic
interspersed among geophilic Trichophyton spp. The close species, for example, T. ajelloi and the recently described
genetic relationship between these anamorphic genera (base Arthroderma olidum/Trichophyton eboreum, associated with
genomic DNA composition 48.7–50.0 mol%)11 indicates that keratinous hair and feather material, rarely cause disease.37,38
species distinctions based on morphological characteristics The epidemiology of Trichophyton infections is comprehen-
may not be accurate. It is clear that many species previ- sively reviewed elsewhere1,4,39,40 but given its continuing evo-
ously considered unique on the basis of morphological dif- lution, the major epidemiological trends are presented.
ferences are not distinct species at the molecular level. For
example, the common Trichophyton rubrum and endemic 44.1.2.1 Species Distribution
African Trichophyton soudanense agents have identical Species distribution of Trichophyton varies with geography.
internal transcribed spacer (ITS) sequences within the ribo- Nonetheless, global trends have seen the progressive replace-
somal DNA (rDNA) gene region, as have Trichophyton rau- ment of zoophilic species, for example, Trichophyton verru-
bitschekii, Trichophyton fischeri, and Trichophyton kane.12–15 cosum, by anthropophilic species, in particular T. rubrum.
Conversely, taxa formerly designated as single species are now The rise of T. rubrum as the commonest cause of tinea pedis
considered as species complexes, for example, Trichophyton (foot ringworm), nail infection (onychomycosis), tinea cru-
mentagrophytes sensu Emmons comprises four distinct spe- ris (ringworm of the groin), and tinea corporis (body ring-
cies: T. mentagrophytes var. interdigitale → Trichophyton worm) is linked to urbanization, sociocultural practices, and
interdigitale (antropophilic), T. mentagrophytes var. menta- immigration. Persistence of zoophilic species is associated
grophytes → T. interdigitale (zoophilic), T. mentagrophytes with epidemics of infection in domestic or stray animals.
var. quinckeanum → T. mentagrophytes, and T. mentogor- T. rubrum is the predominant species in North and Central
phytes var. erinacei → Trichophyton erinacei.12,16 Europe, United States, and other developed countries, where
Polymorphisms within numerous gene loci have been the prevalence of tinea pedis and onychomycosis is high.1,4 In
exploited to define phylogenetic relationships among contrast, T. verrucosum and Trichophyton violaceum are the
Trichophyton spp. including the mitochondrial (mt) DNA,17,18 most frequent species in the Middle East and North Africa
small subunit (SSU),19 ITS,12,13,15,20–23 large subunit (LSU),24,25 with T. soudanense typically endemic to Central Africa.4
and intergenic spacer (IGS) regions of the rDNA gene,26 actin Species presence also varies with the site of infection.
gene,23 chitin synthase 1 (CHS1) gene,10,27 DNA topoisomer- Around 50%–90% of tinea capitis in the United Kingdom,
ase II (TOP2) gene,23,28,29 manganese superoxide dismutase United States, Brazil, and Jamaica is caused by T. tonsurans
gene,16 and, most recently, whole mt genomes (Table 44.2).30 while, in western China, T. violaceum accounts for 41% of
Analyses of these loci have resulted in ongoing taxonomic infections. T. violaceum is frequently isolated from hair/
refinements, with many morphologically based species now scalp samples of African immigrants in developed countries;
either “split” as distinct species or “synonymized” as iden- infections due to T. soudanense likewise have been reported
tical species (Table 44.1).17–36 High degrees of clonality is among migrants.39,41,42 Tinea capitis due to T. schoenleinii
found in some species, for example, species within the T. and Trichophyton concentricum is endemic to South Africa
rubrum complex, leading to the treatment of such clonal enti- and the Middle East and Southeast Asia and Polynesia,
ties as separate species.31 respectively.
Combining morphological, biological (mating, ecologi-
cal, and host-specific), and phylogenetic species concepts in 44.1.2.2 Disease Transmission
a polyphasic approach, Gräser et al.9 recommend a new tax- Tinea capitis (usually in children) is acquired from infected
onomy for anthropophilic and zoophilic Trichophyton spp. humans or their fomites.1,40 Many patients become carriers
based on four major species complexes (Table 44.1); three and intermittently shed viable organisms. Minority com-
of these four have associations with the teleomorph state munities in urban areas are particularly affected through the
Arthroderma, while the T. rubum complex is exclusively asex- sharing of combs, hats, and bedding. Travelers to endemic
ual. Notably, phylogenetic relationships between certain spe- areas may develop infection after contact with stray animals.
cies complexes, for example, those within the Arthroderma T. tonsurans infection (usually tinea corporis) is more com-
vanbreuseghemii and Arthroderma simii, vary according to mon in adults, in adolescents in close contact with children or
the genetic locus examined.21,23 In addition, studies of whole in contact with sport enthusiasts and wrestlers. Known risk
mt genomes have shown that T. mentagrophytes, T. rubrum, factors for tinea corporis, tinea cruris, and tinea pedis (mainly

Trichophyton 359
TABLE 44.1
Molecular-Based Classification of Trichophyton Species and Species Complexes of the Major Anthropophilic
and Zoophilic Species and Selected Geophilic Species and Their Corresponding Synonymized Species
Designations
GenBank Accession No. of
Species/Species Complex Corresponding Synonymized Species ITS Reference Sequence
Arthroderma vanbreuseghemii complex (supported by analysis of ITSa, LSU, and hspb intron genes and PCR fingerprinting)15,22,29,32,33
Arthroderma vanbreuseghemic Z98014
Trichophyton equinumd,e All varieties of T. equinumd,e EF067316
Trichophyton interdigitaled,f Trichophyton mentagrophytes var. geotziid,f AF168124
T. mentagrophytes var. interdigitaled,f
T. mentagrophytes var. nodulared,f
Trichophyton krajdeniid,f
Trichophyton interdigitaled,e T. mentagrophytes var. mentagrophytesd,e AY062119
Trichophtyon verrucosum var. autotrophicumd,e
Trichophyton tonsuransd,f All varieties of T. tonsuransd,f EF043270
Arthroderma simii complex (supported by ITSa, MLSTg, and RAPDh analysis)20,21,32,33
Arthroderma simiic,e Trichophyton simiid,e Z98017
T. mentagrophytesd,e T. mentagrophytes var. quinckeanumd,e Z97995
Trichophyton langeroniid,e
Trichophyton sarkisovid,e
Trichophyton schoenleiniid,f Z98011
Arthroderma benhamiae complex (supported ITSa, IGSi, and mtj DNA analysis)15,20,21
Arthroderma benhamiaec Z98015
Unnamed species (Trichophyton anamorph of A. benhamiaec) T. mentagrophytes var. granulosumd,e Z98016
Trichophyton concentricumd,f Z98012
Trichophyton erinaceid,e T. mentagrophytes var. erinaceid,e Z97997
T. verrucosumd,e All varieties of T. verrucosumd,e except Z98003
T. verrucosum var. autotrophicumd,e
Trichophyton rubrum complex (supported by multiple gene and microsatellite analysis)14,25–28,33–36
Trichophyton rubrumd,f All varieties of T. rubrumd,f Z97993
(Population 1: worldwide) Trichophyton fischerid,f
Trichophyton kaned,f
Trichophyton raubitschenkiid,f
T. rubrumd,f Trichophyton soudanensed,f AF170473
(Population 2: African) Trichophyton gourvilliid,f
Trichophyton megniniid,f
Trichophyton violaceumd,f All varieties of T. violaceumd,f AJ270811
T. yaoundeid,f
Selected geophilic Trichophyton species (soil keratinophilic fungi [supported by ITSa]9)
Arthroderma flavescensc Trichophyton flavescensd AJ877219
Arthroderma gertleric Trichophyton vanbreuseghemiid AJ877210
Arthroderma gloriaec Trichophyton gloriaed AJ877209
Arthroderma insingularec Trichophyton terrestred AJ000606
Arthroderma lenticularec T. terrestred AJ877211
Arthroderma quadrifidumc T. terrestred AJ877214
Arthroderma uncinatumc Trichophyton ajelloid AJ877212
Trichophyton phaseoliformed Epidermophyton stockdaleaed AJ970152
a ITS, internal transcribed spacer.

b hsp, heat-shock protein.
c Teleomorph (sexual stage) species.
d Anamorph (asexual stage) species.
e Commonly isolated from animal infection (zoophilic).
f Commonly isolated from human infection (anthropophilic).
g MLST, multi-locus sequence typing.
h RAPD, random amplification of polymorphic DNA.
i IGS, intergenic spacer region.
j mt, mitochondrial gene.

360
TABLE 44.2
Characteristics of Dermatophyte-Specific PCR-Based Assays to Detect and Identify Trichophyton Species
Cultures/Specimens
Reference (No. Tested) Gene Target Primers Primers and Primer Sequence (5′–3′) Trichophyton Species or Genera Identified
PCR–RFLP
[26] Cultures (53) IGS(NTS) rDNA NA NA Trichophyton rubrum
ITS1/2 rDNA ITS1 TCCGTAGGTGAACCTGCGG Trichophyton terreste
ITS4 TCCTCCGCTTATTGATATGC Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton
violaceum,
Trichophyton soudanense/T. rubrum, Trichophyton tonsurans/
Trichophyton equinum, Trichophyton quickeanum/Trichophyton
schoenlenii
[76] Cultures (26) ITS1/2 rDNA ITS1 TCCGTAGGTGAACCTGCGG T. tonsurans
[67] Cultures TOP2 dPsD1 See Kanbe et al.67 T. rubrum, T. violaceum, T. erinacei,
(review) PsT T. tonsurans, T. mentagpphytes var. itnerdigitale, T. mentagrophytes var.
PsME mentagrophytes, Arthroderma spp.
sets
[80] Nails (NA) 18S rDNA TR1 GTTTCTAGGACCGCCGTA Trichophyton spp., T. rubrum,
TR2 CTCAAACTTCCATCGACTTG Candida spp., Aspergillus spp.
NS5 AACTTAAAGGAATTGACGGAAG
NS6 GCATCACAGACCTGTTATTGCCTC
[73] Nails (879) 28S rDNA LSU1 GATAGCGMACAAGTAGAGTG T. rubrum, T. mentagrophytesa
LSU2 GTCCGTGTTTTCAAGACGGG

[77] Skin scales/swabs 18S rDNA Multiple primer sets used77 Trichophyton spp.
(38) Real-time PCR
Single- or multiplex PCR

[82] Cultures (5) ITS1 rDNA tonsF1 CGGAGGCCGGCCCCCAG T. tonsurans
tonsR1 GGTCCAGCGTTGAGCCGCTA
[74] Nails (118) ITS2 rDNA panDerm1 GAAGAAGATTGTCGTTTGCATCGTCTC T. rubrum
CHS1 panDerm2 CTCGAGGTCAAAAGCACGCCAGAG Pan-dermatophyte
(multiplex) Univ 1 TCTTTGAACGCACATTGCGCC
Truburm rev CGGTCCTGAGGGCCCTGAA
[70] Nails (62) Actin F-actin CGAACCGTG AGAAGATGACC T. rubrum (cross-reaction with T. violaceum)
ITS1 rDNA R-actin GAACCACCGATCCAGACGGAGTA
(double round) ITS1 TCCGTAGGTGAACCTCCGG
[65] Nails (62/75) CSH1 CHS11S CATCGAGTACATGTGCTCGC Trichophyton spp.
(nested PCR) CHS11R CTCGAGGTCAAAAGCACGCC
JF2 GCAAAGAAGCCTGGAAGAAG
JR2 GGAGACCATCTGTGAGAGTTG
Trichophyton
DNA sequencing
[87] Cultures (NA) ITS/2 rDNA SR6R AAGTATAAGTCGTAACAAGG T. rubrum, T. tonsurans, T. violaceum, T. mentagrophytes, T. soudanense
LR1 GGTTGGTTTCTTTCCT
[21] Cultures (23) ITS1 rDNA 18SF1 AGG TTT CCG TAG GTG AACCT T. rubrum, T. mentagrophytes,
58SR1 TTC GCT GCG TTC TTC ATCGA T. violaceum, T tonsurans,
T. schoenlenii, T. terreste,
T. verrucosum
[15] Cultures (47) ITS1/2 rDNA SR6R AAGTATAAGTCGTAACAAGG Multiple species and variation within species15
LR1 GGTTGGTTTCTTTCCT
[25] Cultures 18S rDNA As per kit MicroSeq D2 LSU Fungal Sequencing Kit (Applied T. rubrum. T. mentagrophytes,
ITS/2 rDNA SR6R Biosystems, Foster City, CA) T. interdigitale, T. violaceum,
LR1 AAGTATAAGTCGTAACAAGG T. tonsurans, T. soudanense
GGTTGGTTTCTTTCCT
[72] Nails (195) (GT) repeat T1for TGGTCTGCCCTTGACTGACC T. rubrum
microsatellite T1rev GTAAGGATGGCTAGTTAGGGGG Trichophyton spp.
ITS rDNA LSU266 GCATTCCCAAACAACTCGACTC T. ruburm, T. interdigitale
V9D TTACGTCCCTGCCCTTTGTA
[73] Nail (879) 28S rDNA LSU1 GATAGCGMACAAGTAGAGTG T. rubrum. T. mentagrophytesa
LSU2 GTCCGTGTTTTCAAGACGGG
[81] Skin, hair (7) CHS1 CHS11S CATCGAGTACATGTGCTCGC T. mentagrophytes
CHS11R CTCGAGGTCAAAAGCACGCC
Probe-based methods
[79] Cultures ITS1/2 rDNA ITS1 TCCGTAGGTGAACCTCCGG T. rubrum, T. schoenleinii,
microarray ITS2 GCTGCGTTCTTCATCGATGC T. violaceum, T. mentagrophytes,
ITS3 GCATCGATGAAGAACGCAGC T. soudanense, T. verrucosum,
ITS4 TCCTCCGCTTATTGATATGC T. terreste, T. tonsurans
[78] Cultures (36) ITS1/2 rDNA SR6RL AAGTATAAGTCGTAACAAGG T. rubrum, T. tonsurans,
padlock probes LR1 GGTTGGTTTCTTTCCT T. violaceum, T. mentagrophytes, T. soudanense
[61] Hair (32) IGS Forward GTTTTCTAGAAATGGACCCTCTGG T. tonsurans
real-time PCR Reverse GCGATTTGG ACC ACT T
[84] Skin (71) ITS1/2 rDNA Multiple primers used84 T. rubrum, T. mentagrophytes complex/T.
Hair, nails (21) real-time PCR verrucosum, T. schoenlenii, T. violaceum,
T. tonsurans, T. equinum, T. soudanense
[71] Nails (549) 18S, 5.8S rDNA DERMF1 GGA AAC GAC CGC CCA GG T. rubrum, T. mentagrophytes,
Skin (230) PCR–RLB, BDERMP-2 CGG AAT TCT GCA ATT CAC ATT ACT T. interdigitale, T. verrucosum,
Hair (20) sequencing T. violaceum, T. tonsurans,
T. erinacei (by sequencing only)
Abbreviations: CHS1, chitin synthetase 1; hsp, heat-shock protein; IGS, intergenic spacer region; ITS, internal transcribed spacer; mt, mitochondrial gene; MLST, multi-locus sequence typing; NST,
non-transcribed spacer; RAPD, random amplification of polymorphic DNA; RFLP, restriction fragment length polymorphism; RLB, reverse line blot; TOP2, DNA topoisomerase II.
a T. mentagrophytes types I and II.
361
T. rubrum and T. interdigitale) include foot dampness and the PACC gene hinders both protease secretion and fungal
abrasion combined with exposure to high fungal inocula in growth. Delayed and immediate hypersensitivity immune
communal baths and aquatic facilities, exchange of linen via responses as well as the production of Th2 cytokines are
communal laundering, exposure to “house dust,” for exam- also important in pathogenicity. Trichophyton spp. coun-
ple, floor carpets, and wearing of tight clothing. Immigration ter the host immune response in several ways: lymphocyte
has led to the introduction of endemic anthropophilic spe- inhibition by cell wall mannans (T. mentagrophytes and T.
cies, for example T. soudanense to non-endemic regions. rubrum), alteration of macrophage function and the differen-
Nosocomial transmission has occurred sporadically in adult tial activation of keratinocytes, and secretion of proteases. T.
and pediatric institutions usually involving T. tonsurans or T. rubrum proteases exhibit their effect either by their surface
violaceum.5,6,33–46 Table 44.3 presents the major case clusters. antigen properties or directly on immune mediators. Host–
Both acute and long-term care facilities have been implicated fungus interactions are detailed elsewhere.47
where staff may transmit the fungus among patients. Rapid
identification of the pathogen as well as strain typing is criti-
44.1.4 Clinical Manifestations
cal to inform infection control practices.
Descriptions of the clinical manifestations of Trichophyton
infections are beyond the scope of this chapter but can be
44.1.3 Specific Virulence Determinants
found in a number of expert texts.1,2,52 Clinical forms of dis-
Infection results from destruction of keratin in conjunction ease vary with the causative pathogen (zoophilic species
with the host inflammatory response. Whereas infection may cause a more intense inflammatory reaction than anthro-
be eliminated though the host immune response, some species, pophilic species), the body site affected, and host immune
for example, T. rubrum, reach a high degree of host adapta- status. Infections restricted to epidermis/skin structures are
tion and result in chronic infection with few symptoms. The typical but deep-seated infections may occur. Infections are
almost exclusive localization of Trichophyton spp. in keratin- classified according to body site affected and degree of ker-
ized tissues and finding of in vitro keratinolytic activity have tinization at that site.
focused research on fungal secreted proteases. Relatively few
data are described for other putative virulence factors.47 44.1.4.1 Tinea Corporis and Tinea Cruris
Time-dependent increase (maximal ≈ 12 h) in the number All Trichophyton spp. cause infection of the glabrous areas of
of adhering spores followed by germination and invasion the body, groin, and face (tinea faciei) but the prevailing fungi
of the stratum corneum (3 days) has been shown using in of a given region are most likely to be recovered. Overall,
vitro models. Candidate mediators include carbohydrate- T. rubrum is the commonest species. Other common patho-
specific adhesions on microconidial surfaces (T. rubrum) gens are the T. mentagrophytes complex and T. tonsurans.
and secreted proteases (e.g., the dipeptidyl-peptidase IV of T. concentricum causes the tinea imbricate variant of tinea
T. rubrum.48,49 Keratinolytic protease activity, in conjunction corporis (as above).53 Tinea corporis may affect the head,
with reduction of disulfide bridges in skin protein to render neck, and arms, while the source of tinea faciei is usually
it accessible to proteases, is important for pathogenicity. a zoophilic reservoir or extension from infection elsewhere.
Trichophyton secretes multiple serine endoproteases (sub- Characteristic lesions are circular, sharply marginated, and
tilins) and metalloendoproteases. Protease activity may be erythematous with raised edges. Central clearing occurs with
induced by restricted nutrient supply and regulated by zinc- scaling of the advancing border. Crusting, vesicles, and pus-
finger transcription factors.50 In T. rubrum, endoprotease tules may develop. In chronic T. rubrum infection, central
expression is upregulated by the transcription factor PACC, clearing is absent with little redness/scaling.52 Lesions in
which in turn is activated by elevated pH.51 Disruption of acute tinea cruris are exudative and resemble eczema.1
TABLE 44.3
Major Reported Nosocomial Dermatophyte Outbreaks
Reference Setting Age (Years) No. Infected Location Organism
[5] Nursing home 26–95 7 Ottawa, Canada Trichophyton tonsurans
[44] Pediatric surgical unit 23–50 10 Illinois, USA T. tonsurans
[43] Pediatric rehabilitation unit NAa 4 Illinois, USA T. tonsurans
[45] Psychiatric ward NA 13 New Zealand T. tonsurans
[46] Neonatal ICUb Neonates born at 24–28 5 New York, USA Microsporum canis
weeks gestation
[6] Pediatric medical unit 16–48 21 Missouri, USA T. tonsurans
a NA, not available.

b ICU, intensive care unit.

Trichophyton 363
44.1.4.2 Tinea Pedis

Three types of infection occur in the horny layer of the skin
of the soles (and palms): interdigital (athelete’s foot; the com-
monest form affecting the web spaces), vesicular (dyshi-
drotic), and chronic noninflammatory (moccasin). T. rubrum
typically causes chronic dry erythematous reactions and T.
interdigitale inflamed bullous lesions. Combined tinea pedis
and hand infection (tinea manum) infrequently occurs; this is
usually due to T. rubrum.
44.1.4.3 Tinea Capitis

Tinea capitis affects the scalp, eyebrows, or eyelashes.
Infection ranges from mild patchy scales to highly inflam-
matory reactions with folliculitis, kerion (pus) formation,
extensive scarring, and alopecia. Hair infection may be FIGURE 44.1 Disseminated Trichophyton infection with wide-
ectothrix (arthroconidia on outside of hair shaft) or endothrix spread skin lesions in an immunocompromised patient. (Matthew O’
Sullivan and David Looke, Personal communication with permission.)
(arthroconidia within the shaft). The endothrix-growing T.
tonsurans is the predominant pathogen in the United States
with T. violaceum and T. soudanense restricted to other 44.1.5 Diagnosis of Trichophyton Infection
regions (see above). Dry inflammatory patches are typical. Laboratory diagnosis has traditionally required the associa-
Black dots are seen as hair break off at the opening of the tion of characteristic hyphae visualized in clinical specimens
follicle. Kerions often form in ectothrix infection caused with culture isolation of the organism.58–62 Organisms in
by T. verrucosum and T. mentagraphytes. A unique form of histopathological sections stain with periodic acid-Schiff or
the disease due to T. schoenleinii manifests as yellow cup- Grocott methanamine silver (not further discussed). Standard
shaped crusts or scutulae around the hair shaft with scarring culture-based identification methods are slow (2–4 weeks),
alopecia; crusts enlarge to form a crusting mass or favus.1 often imprecise due to fungal phenotypic pleomorphism and
require considerable expertise and the maintenance of refer-
44.1.4.4 Tinea Unguium (Onychomycosis) ence “tester” strains.63,64 Nonetheless, such methods remain
Nail plate invasion is referred as tinea unguium, although widely used in the diagnosis although molecular methods are
the general term “onychomycosis” is often used. Toenails also increasingly used, both to provide rapid species identi-
are more often affected than fingernails. T. rubrum accounts fication of cultures and to directly detect Trichophyton DNA
for 70% of cases and T. interdigitale for 20%. There are five in clinical specimens. Proper sample collection is essential to
types of infection: invasive subungual (proximal and dis- optimize diagnostic yield.
tal subtypes), endonyx (diffuse milky-white discoloration
caused by T. soudanense), superficial white onychomycosis 44.1.5.1 Phenotypic-Based Methods
usually confined to toenails and due to T. interdigitale, and Phenotypic-based identification of dermatophytes is based on
total dystrophic disease.1 microscopic morphology and culture characteristics of the
anamorphic state of the fungus (Table 44.4). However, the
Invasive Forms of Disease site and appearance of lesion(s), geographic location, history
Rarely Trichophyton spp. cause an aggressive, invasive of travel, and animal contact and ethnicity of patient must
infection, usually in patients with chronic tinea. T. rubrum be taken into consideration, especially in identifying rare or
is the usual pathogen but disease due to the T. mentagro- non-sporulating species.
phytes complex, T. violaceum, and T. verrucosum has also Trichophyton spp. have smooth-walled macro- and
been reported.54,55 There are three patterns of infection: microconidia. Macroconidia are mostly borne laterally
(i) majocchi’s granuloma (nodular granulomatous perifol- directly on hyphae or on short pedicles, and are thin or thick
liculits) characterized by localized dermal/subcutaneous walled, clavate to fusiform, and range from 4–8 × 8–50 mm
infection due to disruption of hair follicles and spillage of in size; however, they may be few or absent. Microconidia
fungi into the dermis with resultant granulomatous inflam- (2–3 × 2–4 mm) are spherical, pyriform to clavate but may
mation; in immunocompetent patients, infection is often be irregular in shape. The presence of microconidia distin-
restricted to the extremities following trauma but immune guishes this genus from Epidermophyton and the smooth-
compromised patients may develop more generalized skin walled, mostly sessile macroconidia separate it from
lesions; (ii) locally invasive infection with subcutaneous Microsporum. Differentiation of the major pathogenic spe-
but without hair follicle involvement; patients are usually cies based on phenotypic criteria are summarized in Table
immunocompromised 56,57; and (iii) very rarely, dissemina- 44.4 and illustrated by Figures 44.2 through 44.7. Good qual-
tion to bone, lymph nodes, liver, and the nervous system ity slide preparations and procedures to ensure stimulation
occurs (Figure 44.1).55 of sporulation are required. Surface texture, topography, and

364
TABLE 44.4
Phenotypic-Based Identification of the Major Pathogenic Trichophyton Species (See Figures 44.2 through 44.7)
Species Microscopy Culture Comments
Trichophyton. Macroconidia may be less distinct or absent. Microconidia are typically present. Note their shape, size, and arrangement.
Trichophyton rubrum Most cultures show scanty to moderate numbers of Colonies are flat to slightly raised, white to cream, On primary isolation, some cultures may lack
(Population 1: worldwide) slender clavate to pyriform microconidia. Macroconidia suede-like to downy, with a yellow-brown to wine-red reverse pigmentation and fail to produce
Anthropophilic when present are smooth, thin-walled, and cylindrical. reverse. microconidia. Key features are culture and
microscopic characteristics and failure to
perforate human hair in vitro.
T. rubrum Most cultures do not produce conidia; the hyphae may Colonies are often slow-growing with a flat to folded, Key features are clinical history, culture
(Population 2: African) show reflexive or right-angle branching. Some strains suede-like surface. Surface mycelium and reverse characteristics, and microscopic morphology
Anthropophilic. produce pyriform microconidia; chlamydoconidia may pigment range from white to pink, deep apricot-orange and endothrix invasion of human hair.
be found in older cultures. to deep red.
Trichophyton interdigitale Numerous subspherical to pyriform microconidia, Colonies are usually flat, white to cream in color with a Key features include culture characteristics,
Anthropophilic form occasional spiral hyphae, and spherical chlamydoconidia powdery to suede-like surface and yellowish and microscopic morphology, and in vitro
are present (the latter are more abundant in older pinkish-brown reverse pigment, often becoming a darker perforation of human hair.
cultures). Occasional slender-clavate, smooth-walled, red-brown with age.
multi-septate macroconidia are seen in some cultures.
T. interdigitale Numerous single-celled, spherical to subspherical Colonies are generally flat, white to cream in color, with a Key features include culture characteristics,
Zoophilic form microconidia are formed, often in dense clusters. powdery to granular surface. Some cultures show central microscopic morphology, and clinical disease
(mice, guinea-pigs, kangaroos, cats, Varying numbers of spherical chlamydoconidia, spiral folding or develop raised central tufts or pleomorphic with known animal contacts.
horses, sheep, and rabbits) hyphae, and smooth, thin-walled, clavate shaped, suede-like to downy areas. Reverse pigmentation is

multi-celled macroconidia may also be present. usually a yellow-brown to reddish-brown color.
Trichophyton tonsurans Hyphae are broad, irregular, branched with numerous Colonies show considerable variation in texture and color. Key features include microscopic morphology,
Anthropophilic septa. Numerous microconidia varying in size and shape They may be suede-like to powdery, flat with a raised culture characteristics, endothrix invasion of
(long clavate to broad pyriform) are borne at right angles center or folded, often with radial grooves. Color may hair, and partial thiamine requirement.
to the hyphae that often are unstained by lactophenol vary from pale buff to yellow (the sulfureum form which
cotton blue. Very occasional smooth, thin-walled, resembles Epidermophyton floccosum), to dark brown.
irregular, clavate macroconidia may be present. Swollen The reverse color varies from yellow-brown to
giant forms of microconidia and chlamydoconidia are red-brown to deep mahogany.
produced in older cultures.
Trichophyton. equinum Abundant microconidia that are clavate to pyriform and Colonies are usually flat but may develop gentle folds or Key features include microscopic morphology,
Zoophilic sessile or spherical, and are stalked/formed laterally radial grooves, white to buff in color, suede-like to culture characteristics, and clinical lesions in
(horses) along the hyphae. Macroconidia are rarely produced but downy in texture. Cultures usually have a deep-yellow horses. Most strains require nicotinic acid for
when present are clavate, smooth, thin walled, and of submerged fringe and reverse, which later becomes dark growth except those from Australia and New
variable size. Occasional nodular organs may be present red in the center. Zealand, which are autotrophic. Invaded hair
and the microconidia often undergo a transformation to show an ectothrix infection but do not
produce abundant chlamydoconidia in old cultures. fluoresce under Wood’s ultra-violet light.
Trichophyton
Trichophyton mentagrophytes Numerous microconidia, predominantly slender clavate Colonies are flat or slightly raised and folded, white to Key features include microscopic morphology,
Zoophilic when young, are borne laterally along the sides of cream, suede-like in texture with a pale yellow-brown to culture characteristics, and a rapid urease test
(mice) hyphae. With age, the microconidia become broader and pinkish-brown reverse. A characteristic pungent (within 2–3 days).
pyriform with some subspherical forms. Occasional to “mousy” odor may be present.
moderate numbers of smooth-walled, multi-septate,
clavate macroconidia may be present.
Trichophyton erinacei Numerous subspherical to pyriform microconidia, Colonies are usually flat, white to cream in color with a Key features include microscopic morphology
Zoophilic (hedgehogs) occasional spiral hyphae, and spherical chlamydospores powdery to suede-like surface and yellowish and and culture characteristics; slender clavate
are present, the latter more abundant in older cultures. pinkish-brown reverse pigment, often becoming a darker microconidia and brilliant lemon yellow
Occasional slender, clavate, smooth-walled, multi- red-brown with age. reverse pigment on Sabouraud’s agar; and
septate macroconidia may be present. negative hydrolysis of urea.
Nonsporulating Trichophyton species. No conidia are present and colonies are sterile. Chlamydoconidia or other hyphal structures may be present but are non-diagnostic. Common species include
T. verrucosum and T. violaceum. Less common species include T. concentricum and T. schoenleinii.
Trichophyton schoenleinii No macroconidia and microconidia are seen in routine Colonies are slow growing, waxy, or suede-like with a Key features include clinical history, culture
Anthropophilic cultures but numerous chlamydoconidia may be present deeply folded honey-comb-like thallus. The thallus is characteristics, and microscopic morphology
in older cultures. Characteristic antler “nail head” cream colored to yellow to orange-brown. Cultures are showing favic chandeliers. Invaded hairs
hyphae or “favic chandeliers” may be observed. Sparse difficult to maintain in their typical convoluted form and remain intact and fluoresce pale greenish-
distorted clavate microconidia may be formed when rapidly become flat and downy. No reverse pigmentation yellow under Wood’s ultra-violet light.
grown on polished rice grains. is present.
Trichophyton violaceum Hyphae are broad, tortuous, and branched. No conidia are Colonies are very slow growing, glabrous or waxy, T. violaceum has a partial nutrient requirement
Anthropophilic usually seen but occasional pyriform microconidia may heaped and folded, and deep violet in color. Cultures for thiamine that separates this organism from
be present on enriched media. Numerous often become pleomorphic, forming white sectors. T. gourvillii, T. rubrum, and other species that
chlamydoconidia are present in older cultures. Occasional non-pigmented strains may occur. may produce purple pigmented colonies.
Trichophyton concentricum Cultures consist of broad, much-branched, irregular, often Colonies are slow growing, raised, and folded, glabrous Key features include association with chronic
Anthropophilic segmented, hyphae, which may have “antler” tips becoming suede-like, mostly white to cream colored, but widespread noninflammatory tinea imbricata
resembling T. schoenleinii. Chlamydospores are present sometimes orange-brown colored, often deeply folded with concentric rings of scaling. The species
in older cultures. Microconidia and macroconidia are into the agar that may produce splitting of the medium is not known to invade human hair.
not usually produced but some isolates will produce in some cultures. Reverse is buff to yellow-brown to NB: Hyphal segments may artificially resemble
occasional clavate to pyriform microconidia. brown in color. macroconidia.
Trichophyton verrucosum All strains produce chains of chlamydoconidia referred to Colonies are slow growing, small, button, or disk-shaped, Key features include culture characteristics and
Zoophilic as “chains of pearls” when grown in brain heart infusion white-cream colored, with a suede to velvety surface, requirements for thiamine and inositol, large
(cattle) broth containing para-aminobenzoic acid and agar raised center and flat periphery with submerged ectothrix invasion of hair.
at 37°C. growth. The reverse color varies from non-pigmented to
yellow.
Sources: Ellis, D. et al., Descriptions of Medical Fungi, 2nd edn., Mycology Unit, Women’s and Children’s Hospital, North Adelaide, Australia, 2007; Suhonen, R.E. et al., Fungal Infections of the Skin, Hair and
Nails, Martin Dunitz, London, U.K., 1999, p.132.
365
FIGURE 44.2 Culture characteristics of T. rubrum (population 1: worldwide form).
FIGURE 44.3 Culture characteristics of T. rubrum (population 2: African).
FIGURE 44.4 Culture characteristics of T. interdigitale anthropophlic (left and middle) and zoophilic (right) forms.
FIGURE 44.5 Culture characteristics of T. mentagrophytes (left and middle) and T. erinacei (right).
pigmentation characteristics are variable and are less reliable not usually produce conidia; chlamydoconidia or other
criteria for identification.63 hyphal structures may be present but microscopy is often
In practice, two groups of Trichophyton may be recog- non-diagnostic, that is, T. verrucosum, T. violaceum, T.
nized on microscopy (Table 44.4): (i) species that usually concentricum, T. schoenleinii, and T. soudanense. These
produce microconidia but which macroconidia may/may not non-sporulating species are identified by a combination of
be present, that is, T. rubrum, T. interdigitale, T. mentag- growth and colony morphology features on various media.
rophytes, Trichophyton equinum, T. erinacei, T. tonsurans, Many Trichophyton have highly variable or overlapping mor-
Trichophyton terrestre, and sometimes, T. verrucosum phological characteristics and the final identification is often
(produces conidia on some media) and (ii) species that do based on the expert opinion of laboratory staff.

Trichophyton 367
FIGURE 44.6 Culture characteristics of T. schoenleinii, T. violaceum, and T. concentricum.
FIGURE 44.7 Culture characteristics of T. tonsurans, T. equinum, and T. verrucosum.
44.1.5.2 Molecular-Based Diagnostic Methods many PCR assays are only moderately sensitive74,80 or are
Molecular techniques have been developed to accurately unable to provide identification to species level.65,75,77,81 Since
identify culture isolates, directly detect Trichophyton DNA skin, hair, and nails are not sterile sites, results of PCR tests
in clinical specimens, and study strain variation for epide- should be interpreted in the clinical setting. The various
miological purposes.64–75 Factors influencing the detection methodology formats, gene targets, primers used, and target
of Trichophyton DNA in clinical samples include correct species of the major PCR assays are summarized in Table
specimen processing, the DNA extraction protocol, and PCR 44.2. Comparison of assay performance is problematic since
design (target selection and amplicon specification method). the “gold standard” culture method is insensitive.
44.1.5.2.1 Molecular Targets 44.1.5.2.2.1 PCR–RFLP PCR–RFLP analyses have been

Target selection is critical for assay sensitivity and for the used to detect and/or identify Trichophyton spp. (Table
level of identification (to genus or species). Assays that tar- 44.2). Analysis of the ITS1/2 region using the restriction
get multi-copy, rather than single-copy, genes are generally endonucleases MvaI and HinfI has been able to differentiate
more sensitive. The most widely used target is the rDNA between the main Trichophyton species.26,76 Other restriction
gene complex. Although the variable regions of the SSU enzymes useful in species identification include RsaI and
rDNA locus enables phylogenetic delineation, it is the highly Sau3A.67,73 PCR–RFLP analysis of the TOP2 gene has also
variable ITS1/2 region and its intermediary 5.8S rDNA been employed to identify a number of Trichophyton as well
locus that are especially useful for species identification of as Arthroderma spp. using HinfI and HincII (as summarized
Trichophyton12,13,15,20,21,31; the multi-copy mt gene has also in Kanbe, 2008).67 Baek et al. used a HaeIII enzyme-based
been used.17,18,22 Where single-copy (e.g., CHSI, TOP2, and RFLP assay with good sensitivity and specificity to differen-
alkaline protease 1 [ALP1]) genes have been targeted, PCR tiate Trichophyton spp. including T. rubrum from Candida
has often been combined with methods such as restriction or Aspergillus spp. in onychomycotic nails.80 RFLP analysis
fragment length polymorphism (RFLP) analysis (Table 44.2) has also been combined with real-time PCR to detect der-
for sufficient sensitivity and specificity.27,65–67 matophytes in skin scales and swabs (Table 44.2).77 Its more
widespread use, however, is limited by the requirement for
44.1.5.2.2 Overview of Methods for the Detection standard patterns for each species. Only a small number of
and/or Identification of Trichophyton spp. species may be identified and certain species may not be able
Techniques developed for the identification of Trichophyton to be resolved (Table 44.2).
cultures include PCR–RFLP analysis,26,67,76 PCR ampli-
fication of specific genetic loci followed by sequence 44.1.5.2.2.2 Single-Step and Multiplex PCR-Based
analysis,9,12,15,26 real-time PCR,77 and other probe-based Methods A number of single- and multiplex PCR meth-
methods.71,78,79 Similar techniques have been employed to ods, targeting the ITS1/2 region of the rDNA gene cluster
detect Trichophyton DNA in clinical specimens, although as well as the single-copy actin and CHS1 genes, have been

tested for their ability to identify, and to directly detect the diagnosis of onychomycosis with a sensitivity of 83.6%
Trichophyton spp. in clinical specimens (Table 44.2). Assays (range 75%–100%), specificity of 100% (when compared
targeting single-copy genes may be nested.65,70 Due to the against nails from healthy controls), and a turn-around-
large number of potential pathogens, multiple primers for time of 48 h.75 This system does not provide species
each species are necessary and assays are limited in the identification.
number of species they are able to detect and identify (Table
44.2). Yoshida et al. evaluated an ITS1-targeted PCR assay 44.1.5.2.2.4 Sequence-Based Identification Meth
that detected and discriminated T. tousurans from other der- ods Sequence-based PCR assays have remained the back-
matophytes in a single PCR amplification; however, other bone of molecular identification methods. Most published
Trichophyton spp. were not able to be identified.82 Others assays have targeted the 18S, ITS including the 5.8S, 28S
have reported a rapid technique whereby DNA in diseased rDNA regions, or the CHS1 gene for the detection and iden-
nails was amplified using a combination of two primer sets— tification of Trichophyton species.9,12,27,72,73,85 ITS sequencing
a pan-dermatophyte set specific to CHS1 and a T. rubrum- is the current recommended gold standard method for the
specific set directed at the ITS2 locus. Dermatophtyes were identification of Trichophyton cultures, especially for atypi-
identified to genus, and T. rubrum to species, level within 5 h cal isolates or uncommon species. This gene locus is the only
(Table 44.2).74 Broad range primers offer an alternative but molecular marker for which a relatively complete dataset of
require subsequent DNA sequencing for species identifica- sequences are available. Reviews of sequence-based identifi-
tion (44.1.5.2.2.4). cation methods that have been used for the identification of
Trichphyton spp. are recently published.67,86
44.1.5.2.2.3 Probe-Based Identification Methods Probe- Monod et al. amplified template DNA of the LSU (28S)
based approaches have likewise been developed to assist with rRNA gene in nails using a universal primer set, followed by
species identification. Early studies demonstrated the potential DNA sequencing to detect T. rubrum and T. mentagrophytes
of SSU rDNA-directed species-specific probes to differentiate species complex.73.Others have detected T. rubrum and T.
dermatophytes from Candida in nail and skin specimens. In interdigitale DNA in infected nails by an ITS-targeted PCR
one study, the sensitivity of such an approach was 92% (cf. assay as well as by using the microsatellite marker T1 spe-
73% sensitivity for culture).83 Sugita et al. reported a real-time cific to the T. ruburm/T. violaceum clade (Table 44.2). There
TaqMan assay targeting the IGS rDNA region for detecting are anecdotal reports of ITS-targeted PCR assays identifying
T. tonsurans in hair (Table 44.2).61 Another real-time assay Trichophyton spp. in tissue specimens.55 Sequencing of the
was tested for its ability to detect dermatophytes in 92 clini- CHS1 gene has been successfully employed to detect T. men-
cal samples. Several species could not be identified, T. ver- tagrophytes in the skin and hair.81
rucosum was not differentiated from the T. mentagrophytes
complex and members of this complex were not distinguished
44.2 Methods
from each other.84 The assay (detection limit 2–20 cells) was
limited by high costs and the need for three separate real-time 44.2.1 Sample Preparation
reactions (two to detect all relevant species and one for the
detection of an internal control). 44.2.1.1 Sample Collection for Phenotypic and
Recently, an oligonucleotide array targeted at ITS1/2 Molecular-Based Identification
rDNA polymorphisms was developed to detect dermato- Specimens should be harvested by experienced staff in suf-
phytes including Trichophyton species (sensitivity 99.5% and ficient amounts. As a result of electrostatic attraction, plastic
specificity 97.8%; Table 44.2).79 In another study, circular- containers such as Petri dishes are unsuitable. Instead, sterile
ized ITS-targeted padlock probes were employed in a highly glass containers should be used to collect specimens.58
specific rolling circle amplification-based assay to identify Skin: Skin lesions should be scraped from the edge or in
five major Trichophyton species, including the closely related entirety using a dermal curette but scalpel blades and glass
T. rubrum and T. soudanense.78 A sensitive (detection limit slides may also be used. Swabs should be taken from highly
2–20 cells) PCR/reverse line blot (RLB) assay has also been inflammatory lesions. Additionally, the use of vinyl tape skin
reported to detect Trichophyton spp. in skin, hair, and nails stripping improves patient compliance during the procedure
using ITS-based probes.71 Six species were reliably identified as well as the diagnostic yield, especially in young children
in 93.6% of culture- and microscopy-positive samples (Table or in sensitive areas.59
44.2). The assay could not differentiate between T. souda- Nails: In distal subungual infection, nails should be
nense and T. rubrum, and a group-specific T. mentagrophytes clipped and then scraped with a small curette or scalpel blade
probe detected both T. mentagrophytes and T. schoenleinii. from the nail bed and the lower nail table at the edge of the
The RLB assay is simple, rapid (48 h), two to three times less lesion. In proximal disease, it is essential to pare the healthy
expensive than DNA sequencing and suited for batch testing. upper table prior to collecting material from the infected
Within each RLB run, up to 45 samples (45 probes) can be lower table. Nail plate subungual microdrilling consistently
analyzed.71 provides better yield of fungal DNA than conventional nail
Finally, an ELISA-based approach (Onychodiag; Bio clipping/subungual scraping.60 It is essential that nails are cut
Advance, Bussy-Saint-Martin, France) was evaluated for into fine small pieces or mechanically ground.

Trichophyton 369
Hair: Hair roots/crusts should be plucked from the infected may be found in some commercial enzymes (e.g., lyticase),
area or its edge, and hair brushed from patients using a bris- which employ yeast vector systems to release fungal DNA.68
tle hairbrush and impressed on culture medium.61 Animal or Automated systems (e.g., the MagNAPure LC instrument,
human carriers can be detected by rubbing the whole scalp Roche) potentially enable high throughput extraction.69
or hair with a sterile piece of carpet, a swab humidified with Skin, hair, and nail specimens: Combined mechanical
distilled water, or a toothbrush.55 The brush/carpet square is and chemical disruption (usually overnight incubation) with/
combed through the hair and as above, impressed on culture without commercial DNA extraction kits have also been used
medium. to extract Trichophyton DNA.65,70–72 Some experts recom-
Skin biopsy and other tissues: For histological exami- mend an additional overnight incubation of nail pieces in a
nation, skin and tissue samples may be placed in forma- dissolvent such as sodium sulfide prior to DNA extraction.73
lin. However, specimens intended for diagnostic PCR tests Of note, a rapid extraction method from nails simply com-
should be collected into formalin-free containers as formalin prising a 10 min incubation at 95°C in “extraction” buffer fol-
is highly inhibitory to the action of Taq DNA polymerase. lowed by the addition of 2% bovine serum albumin (BSA) to
Specimens for reference facilities may be stored at −20°C or counter inhibitors of PCR has also been successfully used.74
−70°C before PCR. For paraffin-embedded (PE) samples, One other study used only the high pure PCR template prepa-
PCR may be performed although with reduced sensitivity; ration kit (Roche) to extract DNA.75
several 10 μm sections should be submitted for analysis.62 We have selected for more detailed description three
DNA extraction protocols on the basis of their practicality,
44.2.1.2 Media and Culture Conditions performance, and potential to be incorporated into a clinical
For primary isolation, specimens are usually inoculated onto laboratory. Although commercial DNA extraction kits have
Sabouraud’s dextrose agar (SDA) containing cycloheximide been successfully used, we have not included protocols that
(actidione), chloramphenicol, gentamicin, and yeast extract. employ these, since comparison of kit performances has not
Other suitable media include dermasel agar, mycobiotic agar, been performed and it is not possible to recommend one kit
and dermatophyte test medium. It is useful to cut skin scales over another.
or nail pieces into the agar.63 Cultures should be incubated at
26°C–30°C for 4 weeks and examined regularly for fungal 44.2.1.3.1 DNA Extraction from Trichophyton Cultures
growth. Details of media conditions and test procedures can Several DNA extraction methods have been applied to
be found in expert texts.63,64 extract genomic DNA from Trichophyton cultures.12,15,74,79
When identifying individual isolates, most laborato- Two methods are presented.
ries use additional media and employ confirmatory tests
to (1) enhance sporulation and pigmentation, for example, 44.2.1.3.1.1 Protocol of Brillowska-Dabrowska
Lactritmel and potato dextrose agar; (2) assess growth and et al.74 Materials: 7- to 10-day old Trichphyton cultures;
colony morphology, for example, Sabouraud’s salt agar, SDA plates containing chloramphenicol (0.05%) and cyclo-
Littman oxgal agar, Bromcresol purple milk solids glucose heximide (0.5%); lysis buffers, and other solutions and sol-
agar, 1% peptone agar; (3) assess hair perforation; and (4) vents as specified in the procedure given below.
assess physiological characteristics (e.g., the urea hydroly- Procedure
sis test) and nutritional growth requirements (e.g., using the
Trichophyton Nos. 1–7 agars).7,63,64 1. Grow cultures on SDA containing chloramphenicol
and cycloheximide (or in 2 mL of SD broth) for 7–10
44.2.1.3 DNA Extraction Protocols days at 22°C–27°C in air.
Efficient fungal cell wall lysis and DNA purification is essen- 2. Harvest cells by centrifugation and suspend the
tial for PCR sensitivity and reproducibility. There are no cell pellet in 500 μL lysis buffer (400 mM Tris
standardized extraction protocols for isolating Trichophyton hydrochloride [HCl] pH 8.0, 60 mM EDTA pH 8.0,
DNA, either from Trichophyton cultures or directly from 150 mM sodium chloride, 1% SDS) to incubate at
skin, hair, and nail samples. room temperature for 10 min.
Cultures: Treatment with glass or zirconium silicon beads, 3. Add a small volume (150 μL) of potassium acetate
mechanical grinding, or repeated freeze-thawing using liq- (pH 4.8), vortex tubes, and then centrifuge for 1 min
uid nitrogen are still often used followed by chemical lysis at 12,000 × g.
(usually with one or more of sodium dodecyl sulfate (SDS), 4. Transfer supernatant to a clean tube and add an
dithiothreitol (DTT) and proteinase K solutions). Commercial equal volume of isopropyl alcohol.
DNA extraction kits increase DNA isolation efficiency but 5. Wash the DNA in 70% alcohol. Resuspend the DNA
still require fungal cell wall lysis. Various kits including pellet in 50 μL of TE (10 mM Tris, 1 mM EDTA
the QIAmp DNA Mini kit (Qiagen), GenElute mammalian buffer) and store at −20°C.
genomic DNA kit (Sigma-Aldrich), and High pure PCR tem-
plate preparation kit (Roche, Mannheim, Germany) have been 44.2.1.3.1.2 Cetyltrimethylammonium Bromide (CTAB)
used with good efficiency, although comparison between Protocol of Gräser et al.12,34 Materials: 7- to 10-day-
kits has not been performed. Contaminating fungal DNA old Trichophyton cultures; SDA agar plates containing

chloramphenicol (0.05%) and cycloheximide (0.5%); Mortar 44.2.2 Detection Procedures

and liquid nitrogen supply; 2% CTAB, extraction buffer, and
other solutions and solvents as described below. 44.2.2.1 Species Identification Using
ITS Sequence Analysis
Procedure DNA sequencing is currently the most accurate method for
the definitive identification of Trichophyton spp. A suggested
1. Grow cultures on SDA containing chlorampheni- protocol for ITS sequence analysis is described below.
col and cycloheximide for 7–10 days at 22°C–27°C Materials: Genomic DNA (10 ng/μL); 10× PCR buf-
in air. fer (100 mM Tris-hydrochloride, pH 8.3, 500 mM potas-
2. Cut one to two fungal colonies from the agar plate, sium chloride, 15 mM magnesium chloride, 0.01% [w/v]
transfer to a mortar, and grind in liquid nitrogen. gelatine); 10× dNTPs (dinucleotide triphosphates) (2 mM
Suspend mycelial powder in 1 mL extraction buffer dATP, dCTP, dGTP, dTTP); forward primer SR6R: 5′
(100 mM Tris-HCl [pH 8.0], 1.4 M sodium chloride, AAGTATAAGTCGTAACAAGG 3′ (10 ng/μL)87; reverse
30 mM EDTA, 2% CTAB, 2% β-mercaptoethanol). primer LR1: 5′ GGTTGGTTTCTTTCCT 3′ (10 ng/μL)87;
Subject cells to three rounds of freezing and sterile distilled water; DNA Taq polymerase (1 U/μL).
thawing.
3. To the cells, add a small volume (e.g., 50–100 μL)
of proteinase K (final concentration 50 μg/mL) and
Procedure
incubate the mixture for 1.5 h at 60°C.
4. Centrifuge for 5 min at 13000 × g and add one
1. Prepare master mix:
volume of phenol: chloroform: isoamyl alcohol
(25:24:1) to the supernatant, and then vortex and For one reaction 10 μL 10× PCR buffer
centrifuge again for 15 min. Remove the upper 10 μL 10× dNTPs
phase. Repeat the procedure four times; for the last 5 μL forward primer SR6R
treatment, add only one volume of chloroform 5 μL reverse primer LR1
5. Precipitate the DNA with 1/10 volume of sodium 0.5 μL DNA Taq polymersase
acetate (4.2 M, pH 5.2) and 1 mL of cold isopropyl
alcohol a. To prepare 100 μL reaction/PCR tube:
6. Incubate for 16 h at −20°C, centrifuge the DNA i Pipette 10μL of 10 ng/μL genomic DNA
solution for 10 min and wash the DNA in 70% alco- into a PCR tube
hol. Air dry ii Add 53.5 μL of sterile water
7. Resuspend the DNA pellet in 50 μL of 1× TE buffer iii Add 36.5 μL of the master mix (see above)
(10 mM Tris, 1 mM EDTA pH 8.0) and then treat iv Vortex and place the PCR tubes in the ther-
with RNase (final concentration 100 μg/mL) for mal cycler
15 min at 37°C. Store DNA at −20°C. v Start the reaction
2. Conduct PCR amplification with the following
44.2.1.3.2 DNA Extraction from Nails, conditions
Skin, and Hair Samples First step (initial denaturation): 1 cycle of
The method presented is adapted from Brillowska-Dabrowska
97°C 3 min
et al.74
Linked to second step
Materials: 7- to 10-day old Trichphyton cultures; SDA Second step: 20 cycles of
plates containing chloramphenicol (0.05%) and cyclohexi- Denaturation (1) 97°C 35 s
mide (0.5%); lysis buffers and other solutions and solvents as Annealing (2) 50°C 55 s
described below. Extension (3) 72°C 45 s
Auto-extension of segment (3)
Procedure Extension per cycle: 4 s
Linked to third step
1. Cut nails with scalpel or grind and cut hair or skin Third step: 10 cycles of:
pieces into 3–5 mm fragments and place in 1.5 mL Denaturation (1) 97°C 45 s
Eppendorf tubes. Annealing (2) 50°C 55 s
2. Add 100 μL extraction buffer (60 mM sodium bicar- Extension (3) 72°C 2 min
bonate, 250 mM potassium chloride, and 50 mM Auto-extension of segment (3)
Tris hydrochloride pH 9.5) and incubate at 95°C for Extension per cycle: 4 s
10 min. Linked to fourth step
3. Add 100 μL 2% BSA. Vortex gently. Fourth step (final extension): 1 cycle of:
4. The DNA-containing solution may be used for PCR. 72°C 6 min

Trichophyton 371
3. Check the obtained PCR products on a 1.4% aga- assigned strains into two major clusters, clusters A and B:
rose gel. 30% and 70% of Japanese isolates belonged to clusters A and
4. Sequence the ITS1/2 rDNA region: (i) clean up the B, respectively, while 91% of Chinese isolates belonged to
obtained PCR products using a commercial puri- cluster A.99 RAPD analysis is also reported to demonstrate
fication kit and prepare the sequencing template strain variation within T. verrucosum, including distinguish-
according to the instructions of the commercial ing wild type from vaccine strains,90 and T. interdigitale (23
sequencing service being used; (ii) sequence the profiles among 33 isolates).103 Prior knowledge of gene poly-
PCR product in both directions using the forward morphisms is required prior to analysis. RAPD and AP-PCR
(SR6R) or reverse primer (LR1); edit the sequences methods require careful standardization and may suffer from
manually; (iii) conduct a BLASTn search against low inter-laboratory reproducibility due to the use of differ-
one of the two quality-controlled curated ITS data- ent assay conditions.
bases, either at the CBS Fungal Biodiversity Center, The high degree of clonality within T. rubrum is fur-
Utrecht, the Netherlands (http://www.cbs.knaw. ther demonstrated by PCR fingerprinting using the minis-
nl) or at the Sydney Medical School—Western of atellite primers (AC)10 and (5′GTATTGCCCT3′).12,94 The
the University of Sydney, Sydney, Australia (http:// primer (GACA)4 was used to genotype T. mentagrophytes, T.
www.mycologylab.org); or use the sequencing data- rubrum, and T. violaceum; few strain differences were found
base at GenBank/European Molecular Biology but different fingerprint profiles distinguished between spe-
Laboratory Nucleotide Sequence Database/DNA cies as well as between the varieties of T. mentagrophytes.95
Data Bank of Japan (http://www.ncbi.nlm.nih. PCR fingerprinting has the same limitations as RAPD and
gov, http://www.ebi.ac.uk/embl/, http://www.ddbj. AP-PCR methods even though primers are directed toward
nig.ac.jp/). Note that GenBank, although the most specific fungal DNA repeats. Microsatellite polymorphisms,
widely used gene repository, contains unedited and for example, those within the marker T1 (GT)8–10, can also
nonvalidated sequences with errors of up to 20%,88 be used to study intraspecies genetic variation. Within T.
which may lead to misidentifications. rubrum species complex, four polymorphic alleles were
identified among 130 strains in one study. There was an asso-
ciation between genetic patterns and geographic region, sug-
44.2.2.2 Genotyping of Trichoohyton spp. gesting an African origin for the complex but with emergence
Determination of intraspecies variation is essential to answer of a new genotype in Asia which spread to Europe and North
questions in epidemiology and population genetics and in America.3 These results have been confirmed by analysis of
strain tracking. Strategies have targeted (1) undefined genetic 200 global T. rubrum strains using multilocus multisatellite
markers, as in random amplified polymorphic DNA analysis typing (MLMT).34 Limitations of employing microsatellite
(RAPD) analysis,89–91 arbitrarily primed PCR (AP-PCR),92,93 polymorphisms include hypervariability of genetic patterns.
PCR fingerprinting,94,95 amplified fragment length poly- In addition, stepwise gene mutations may create alleles that
morphism (AFLP) analysis,12 combined random amplified manifest at identical positions on gel electrophoresis but
monomorphic DNA (RAMD)/single-strand conformation which are homoplaseous (i.e., alleles observed are the same
polymorphism (SSCP) analysis,12 and microsatellite typas a result of parallel or convergent evolution (e.g., mutation)
ing3,34 or (2) known genetic markers as in RFLP analysis of rather than a common ancestry). This can be minimized by
the mt gene and IGS rDNA region26,35,96–100 and single-nucle- examining such multiple loci.
otide polymorphism (SNP) analysis of specific gene loci.6,101
44.2.2.2.2 Strategies Targeting Defined Gene Regions
44.2.2.2.1 Strategies Targeting Undefined Specific genetic loci are increasingly targeted to study
Genomic Regions intraspecies genetic variation. The ITS rDNA loci are too
Early reports indicated that AP-PCR and RAPD methods were conserved for this purpose but other loci have been useful.
not sufficiently sensitive to discriminate within Trichophyton Nontranscribed spacer: The IGS or non-transcribed
spp., particularly species with a high degree of clonality such spacer (NTS) rDNA region contains SNPs and large repeti-
as T. rubrum.89,92 In one study, 22 of 30 T. rubrum isolates tive regions that differ between Trichophyton strains. In a
were identical by RAPD analysis and the remainder exhib- combined RFLP/NTS-directed probe-based approach, 14
ited minor variation89; similarly, AP-PCR analysis of eight hybridization patterns among 50 T. rubrum isolates were
strains revealed little variation.92 A combined PCR finger- found,26 while analysis based on two tandem repeats in the
printing (using the simple repeat primer (AC)10), AFLP, and NTS region (TRS-1 and TRS-2) has identified 23 genotypes
RAMD/SSCP analysis approach also revealed few poly- among 101 strains.35 IGS-based genetic types may vary with
morphisms.12 However, recent analyses using two random geography. Twenty IGS genotypes were identified among
primers identified five distinct RAPD patterns among 10 49 T. rubrum isolates from three regions in China with spe-
Brazilian T. rubrum isolates,91 and subsequently, 12 patterns cific hybridization patterns obtained for strains from two
among a larger number (n = 67) of strains.102Analysis of 150 regions.99,104 Correlation between genotype and increased
Japanese and Chinese isolates using the same two primers minimal inhibitory concentration (MICs) to antifungal drugs

has also been observed.100 NTS-based RFLP analysis using References

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45 Veronaea
Dongyou Liu
Contents
45.1 Introduction...................................................................................................................................................................... 377
45.1.3 Diagnosis.............................................................................................................................................................. 379
45.2 Methods............................................................................................................................................................................ 379
45.3 Conclusion........................................................................................................................................................................ 380
References.................................................................................................................................................................................. 380
45.1 Introduction are erect, straight or flexuose, unbranched or occasion-

ally loosely branched, geniculate, and smooth walled, with
45.1.1 Classification and Morphology pale to medium or olivaceous brown color. Conidiogenous
The genus Veronaea is a dematiaceous mold belonging to cells are terminally integrated, polyblastic, occasionally
the mitosporic Herpotrichiellaceae group, family Herpotri intercalary, cylindrical, later septate, and pale brown, with
chiellaceae, order Chaetothyriales, class Eurotiomycetes, subhyaline fertile part and faintly pigmented, unthickened
subphylum Pezizomycotina, phylum Ascomycota, king- scars. Conidia are solitary, smooth, cylindrical to pyriform,
dom Fungi. The mitosporic Herpotrichiellaceae group con- rounded at the apex and truncate at the base, pale brown, and
sists of 13 genera: Blastacervulus, Brycekendrickomyces, 1–2 septate; conidial secession is schizolytic [4].
Cladophialophora, Cladoriella, Cyphellophora, Exophiala, At the species level, Veronaea botryosa colonies attain a
Heteroconium, Melanchlenus, Metulocladosporiella, Phaeo diameter of 30 mm on malt extract agar (MEA) after 14 days
coccomyces, Rhinocladiella, Thysanorea, and Veronaea. In at 24°C, with entire, sharp margin; surface is velvety and
turn, the genus Veronaea is separated into three species: slightly raised in the center, with grayish-brown to blackish-
Veronaea botryosa (obsolete synonyms: Veronaea cop- brown color; and reverse is greenish black. Submerged hyphae
rophila and Sympodina coprophila), Veronaea compacta, are hyaline to pale olivaceous and smooth while aerial hyphae
and Veronaea japonica [1–3]. A former Veronaea species, are more darkly pigmented. Conidiophores (200 μm × 2–3 μm)
that is, Veronaea musae (obsolete synonym: Ramichloridium are erect, straight or flexuose, occasionally branched, usu-
musae), is now known as Periconiella musae. The type spe- ally geniculate (due to the sympodial development of the
cies of the genus is Veronaea botryosa, which was first iden- conidia), and smooth walled, with a pale brown to olivaceous-
tified in 1957 from olive slag in Italy and which is the only brown color. Conidiogenous cells (10–100 μm long) are ter-
Veronaea species implicated in human infection. minal, occasionally intercalary, cylindrical, pale brown,
Classification and identification of Veronaea spp. are later often becoming septate, fertile part subhyaline, rachis
largely based upon their conidial size, septations, shape, with crowded, flat to slightly prominent, faintly pigmented,
roughness, and conidiophore features. Veronaea resem- unthickened scars. Conidia (3–12 μm × 1.5–3 μm) are solitary,
bles Rhinocladiella as both produce sympodial conidiog- smooth walled or slightly verrucose, cylindrical to pyriform,
enous cells. However, Veronaea is distinguishable from rounded at the apex and truncate at the base, pale brown, and
Rhinocladiella by its absence of exophiala-type budding 1–2 septate (mostly one septate or two celled), with a faintly
cells and its predominantly one-septate conidia (i.e., two- darkened, unthickened hilum (0.5 μm in diameter) [4].
celled conidia) (in comparison with one-celled conidia of Veronaea compacta colonies are slow growing, attaining
Rhinocladiella) and by its production of flat, barely promi- a diameter of 15 mm on MEA after 14 days at 24°C; surface is
nent conidiogenous loci. velvety to lanose, slightly raised in the center, and pale gray to
Veronaea colonies are moderately fast growing, velvety pale brownish gray in color; reverse is dark gray. Submerged
in texture, and pale olivaceous brown in color. Submerged hyphae (1.5–3 μm wide) are subhyaline, smooth, and thin
hyphae are hyaline to pale olivaceous and smooth while walled while aerial hyphae are thick walled and pale brown.
aerial hyphae are more darkly pigmented. Conidiophores Conidiophores (60 μm × 4 μm) are slightly differentiated
377

from vegetative hyphae, lateral or occasionally terminal, and China, India, Australia, New Zealand, Italy, Poland, Egypt,
unbranched or branched at acute angles, with 1–3 additional New Guinea, and Brazil. Human subcutaneous mycoses due
septa. Conidiogenous cells (up to 10 μm in length) are termi- to V. botryosa have been reported in China, the Philippines,
nal, occasionally intercalary, pale brown, and cylindrical to Libya, France, and the United States [8–12].
doliiform or flask shaped, with hardly prominent denticles; Matsushita et al. [13] documented a case of V. botryosa
scars (0.5 μm in diameter) are flat, slightly pigmented, and skin infection in a 12-year-old boy from China, who received
not thickened. Conidia (4–9 μm × 2–3 μm) are solitary, pale a scratch on his right elbow 6 years earlier. Later, pap-
brown, smooth, thin walled, ellipsoidal to ovoid, and 0–1 ules (10–12 mm × 6–8 mm) with exudates appeared on his
septate, often constricted at the septa, with a round apex extremities and other parts of the body (i.e., face, hands, legs,
and truncate base; hilum (0.5 μm in diameter) is prominent, scrotum, and buttocks). The lesions became nodular, form-
slightly darkened, and unthickened [4]. ing thick, crusted areas, with adjacent lesions coalescing to
Veronaea japonica colonies are slow growing, attaining a form plaques. Hematoxylin- and eosin-stained biopsy tissue
diameter of 7.5 mm on MEA after 14 days at 24°C; surface and KOH-treated lesion scraping revealed septate, branched,
is velvety to lanose, slightly raised in the center, and oliva- yellowish-brown-pigmented hyphae (2.5–4.0 μm wide).
ceous brown, with entire margin; reverse is dark olivaceous. Culture of scrapings from the crusty lesions and biopsy tis-
Submerged hyphae (1.5–3 μm wide) are subhyaline, smooth, sue on Sabouraud dextrose agar containing chloramphenicol
and thin walled, while aerial hyphae are slightly narrower (Sab+C) and Sab+C containing cycloheximide yielded vel-
and pale brown; hyphal cells later become swollen, thick vety, olivaceous gray colonies after 7–10 days of incubation
walled, dark brown, and often aggregated. Conidiophores at 25°C and 37°C in the dark. Colonies on Sab+C and potato
(65 μm × 2–3 μm) are slightly differentiated from aerial veg- dextrose agar were velvety to lanate and grayish brown to
etative hyphae, lateral, or terminal, often wider than the sup- black, reaching diameters of 22–25 mm in 10 days at 25°C.
porting hypha, unbranched or occasionally branched, pale Growth at 37°C was slow, reaching diameters of 9–11 mm
brown, thin walled, and smooth, with 1–3 additional septa. after 2 weeks. No growth was evident at 40°C. Slide cultures
Conidiogenous cells (up to 15 μm long) are terminal, occasion- on potato dextrose agar at 25°C for 10 days showed septate,
ally intercalary, variable in length, pale brown, and cylindri- branched, melanized hyphae (2.5–4.5 μm wide), bearing
cal to clavate, with hardly prominent denticles; scars (0.5 μm lateral and terminal conidiophores, as stained by lactophe-
diameter) are flat, slightly pigmented, and not thickened. nol cotton blue. Conidiophores (200–250 μm × 2.5–4.0 μm)
Conidia (6–10 μm × 2–4 μm) are solitary, pale brown, smooth, were erect, straight, or flexous, unbranched or occasion-
thin walled, ellipsoidal to ovoid, and one septate, often con- ally branched, rarely geniculate, smooth walled, septate,
stricted at the septum, with a round apex and truncate base; and pale brown. Conidiogenous cells were terminal or lat-
hilum (1 μm in diameter) is unthickened but slightly darkened. eral and cylindrical in the apical area, bearing numerous
Morphologically, V. japonica is similar to V. compacta; but it scars. Sympodially produced conidia (4.5–10 μm × 2–4 μm)
can be distinguished by the presence of dark brown, swollen were hyaline to pale brown, 0–3 septate (mostly one sep-
hyphal cells in culture, which are absent in V. compacta [4]. tate), smooth walled, and cylindrical, with rounded apices
and truncate at the bases. The isolate was thus identified as
Veronaea botryosa.
Sutton et al. [14] reported a Veronaea botryose-related
Veronaea is one of the 70 dark-walled (brown-pigmented, case involving a 62-year-old orthotopic cardiac transplant
dematiaceous, phaeoid, or melanized) fungal genera (includ- recipient, who presented with chronic induration, mild ery-
ing 130 species) that occur as soil saprophytes, plant patho- thema, and intense tenderness over the dorsum of the right
gens, and environmental contaminants and that are responsible hand after an attempted intravenous line insertion several
for cutaneous, subcutaneous, corneal, and systemic diseases months earlier. A 2 × 1.1 cm area of skin was excised along
(often referred to as phaeohyphomycosis or chromoblastomy- with a separate 4 × 2 × 1 cm pink–gray cyst. Histopathology
cosis) in humans (especially immunocompromised individu- of the excised lesion showed acute inflammation, microab-
als) and animals [5]. Among the more prominent dematiaceous scess formation, and brown moniliform hyphal elements
human pathogens are members of Alternaria, Bipolaris, with hematoxylin and eosin stain. Special fungal stains, the
Cladophialophora, Curvularia, Exophiala (Wangiella), Gomori methenamine silver stain, and the Fontana-Masson
Fonsecaea, Madurella, Phialophora, Scedosporium, and stain for melanized hyphae were also positive. Culture of
Scytalidium genera [6]. While some thermophilic dematia- the excised tissue on Sabouraud dextrose agar plate, Sabhi
ceous fungi (with growth at 40°C or higher) are neurotropic agar slant, and Sabhi agar slant with chloramphenicol and
and have a predilection for central nervous system (CNS) gentamicin yielded dematiaceous colonies after 3 days of
tissue causing brain lesions and/or abscesses, other less ther- incubation. A potato dextrose agar slide culture revealed a
motolerant fungal species (e.g., Ramichloridium schulzeri, “Rhinocladiella-like” type of conidiation. Colonies were
Rhinocladiella aquaspersa, and Veronaea botryosa) are non- velvety to woolly and grayish or blackish brown centrally,
neurotropic and often induce mainly skin infections [7]. with a dark green periphery. The reverse was olivaceous
Veronaea botryosa has been isolated from soil, plant, black. At 25°C on a 60 mm-diameter potato flake agar plate,
water, and air samples in many parts of the world including the growth rate was moderately rapid, reaching 30 mm in 2

Veronaea 379
weeks (2.1 mm/day); slower growth occurred at 35°C (sug- 1 year and pain of 6 month duration following minor trauma.
gesting its potential for invasive disease), and no growth Fine-needle aspiration (FNA) was performed and the smears
was noted at 40°C. Conidiophores (300 μm × 2.5–4 μm) revealed multinucleated giant cells, chronic inflammatory
were brown, septate, smooth, straight or flexuous, and cells, and branching, septate hyphae. PAS stain showed
branched or unbranched, with the apical area being some- bright magenta-colored hyphae with branching septae.
what darker. Smooth-walled, hyaline to pale brown, mostly Masson-Fontana stain demonstrated brownish black branch-
two-celled conidia (2–4 μm × 5–12 μm, but predominately ing and septate hyphae. The occupational, clinical, and
3.5 μm × 8 μm) with rounded apices and truncate bases were histomorphological features suggested Veronaea botryosa
borne from the geniculate apical conidia-bearing portion of phaeohyphomycosis.
the conidiogenous cell. The organism was clearly Veronaea
botryosa. Antifungal therapy with itraconazole and then
voriconazole resolved the lesions after 10 months. 45.1.3 Diagnosis
Da Cunha Filho et al. [15] described two cases of pha- Conventional laboratory identification of Veronaea botryosa
eohyphomycosis caused by Veronaea bothryosa. The first is dependent on observation of the characteristic microscopic
case involved a 44-year-old kidney transplant recipient, who and macroscopic features of the fungus. Scrapings from skin
developed a lesion on the dorsal aspect of the left foot after lesions and biopsy are first examined under microscope with
wooden splinter trauma several years earlier. The lesion was various stains (e.g., KOH, lactophenol cotton blue, Gomori
painful to touch, with an erythematous-violet color. Grocott– methenamine silver stain, PAS stain, Fontana-Masson stain,
Gomori methenamine staining and direct KOH examina- and hematoxylin and eosin stain) for hyphal elements and
tion of exudation demonstrated dematiaceous hyphae. On septate hyphae. Fontana-Masson stain is particularly useful
Sabouraud dextrose agar, colonies were raised, velvety, for detection of melanized hyphae. Portions of samples are
initially gray, and later black. These features were typical cultured on mycological media (e.g., Sabouraud dextrose agar
of Veronaea bothryosa. Following therapy of itraconazole containing chloramphenicol [Sab+C] and Sab+C containing
200 mg daily for 10 months along with tapering dose of cycloheximide, and potato dextrose agar) at 25°C and 37°C in
immunosuppressor drugs, there was reduction of pain. The the dark for isolation of colonies with distinct morphology and
patient died due to complications arising from an accidental structures. Slide cultures of Veronaea isolate on potato dextrose
cerebral vascular hemorrhage. The second case concerned agar may be prepared and incubated at 25°C for identification
a 64-year-old patient, who presented with an asymptomatic of characteristic hyphae, conidiophores, conidiogenous cells,
lesion with a small erythematous-violet papule on the dorsal and conidia [13,14]. To speed up the diagnosis, molecular tech-
aspect of the right foot, resulting from a trauma from a corn niques such as polymerase chain reaction (PCR) and sequenc-
husk. Direct KOH examination of the lesion biopsy showed ing analysis of small and large subunit rRNA genes as well as
dematiaceous hyphae. Culture of the lesion sample yielded internal transcribed spacer (ITS) regions may be employed.
dark-gray colonies, with a velvety surface in the middle.
These characteristics were indicative of Veronaea bothryosa.
Chen et al. [12] reported a Veronaea botryose-related 45.2 Methods
case in a 76-year-old male with a history of diabetes mel-
litus, coronary artery disease, and Cushing’s syndrome (due
to his long-term use of oral corticosteroids). The patient pre- Clinical specimens are examined directly under microscope
sented with recurrent multifocal, crusted, brownish-red nod- with a fungal stain. Portions of the samples are inoculated
uloplaques on the right forearm, left upper limb, and right on Sabouraud glucose agar without or with antibiotics. The
knee. Grocotle’s methenamine silver nitrate (GMS) staining resulting isolates are identified on the basis of macroscopical
of skin biopsy showed brownish hyphae and yeast-like cells and microscopical features. For easy microscopic observation,
scattered in granulomatous infiltrates. The hyphae and yeast- slide culture technique may be utilized. Slide cultures are set
like cells stained black and pink with GMS and periodic acid up in Petri dishes containing 2 mL of sterile water, into which
Schiff (PAS) stains, respectively. They also stained black a U-shaped glass rod is placed, extending above the water
with Masson-Fontana stain, indicating the presence of mela- surface. A block of freshly growing fungal colony, approx.
nin. Culture of the specimen on Sabouraud dextrose agar and 1 cm2, is placed onto a sterile microscope slide, covered with a
potato media at room temperature yielded grayish-brown, somewhat larger, sterile glass cover slip, and incubated in the
velvety colonies after 2 weeks. Slide cultures from the colo- moist chamber. Fungal sporulation is monitored over time,
nies revealed erect and straight conidiophores with conidia and when optimal, images are captured by means of a Nikon
borne terminally and laterally on the apices. The conidia are camera system (Digital Sight DS-5M, Nikon Corporation) [4].
pale brown, smooth walled, two celled, and cylindrical with For fungal DNA extraction, 50 mL of Sabouraud dextrose
round tops and truncated bases. The fungus was identified as (SAB) broth is inoculated by needle with conidia from a
Veronaea botryosa. 7 day culture in SAB agar and incubated for 72 h at 30°C.
Kumar and Hallikeri [16] presented a case of phaeohy- The hyphae are recovered on a 0.45 μm-pore-size filter and
phomycosis in a 29-year-old male agriculturist, who com- washed with sterile saline. Aliquots of the fungal hyphae are
plained of painful swelling of the right supraorbital region of stored frozen at −70°C until use. Prior to lysis, the hyphae

are thawed and suspended in 400 μL of DNA extraction buf- algorithm; alignment gaps are treated as a fifth
fer (1 mM EDTA pH 8.0, 1% sodium dodecyl sulfate, 10 mM character state and all characters are unordered and
Tris–HCl pH 7.6, 100 mM NaCl, and 2% Triton X-100). of equal weight. Branches of zero length are col-
Microcentrifuge tubes (1.5 mL) containing hyphae and buf- lapsed and all multiple, equally parsimonious trees
fer are sonicated in a water bath (Branson; model 2210) for are saved.
15 min, followed by heating at 100°C for 5 min. Following 7. Other measures calculated include tree length, con-
lysis, DNA is purified using the QIAmp blood kit (Qiagen) and sistency index, retention index, and rescaled con-
protocols for crude cell lysates supplied by the manufacturer. sistency index (TL, CI, RI, and RC, respectively).
The purified DNA is stored at 4°C until tested. Alternatively, The robustness of the obtained trees is evaluated
DNA is extracted with a FastDNA kit (Qbiogene) from myce- by 1000 bootstrap replications. Bayesian analysis is
lium grown for 3–5 days in liquid complete medium. performed. The best nucleotide substitution model
is determined using MrModeltest v. 2.2. MrBayes v.
45.2.2 Detection Procedures 3.1.2 is used to perform phylogenetic analyses, using
a general time-reversible (GTR) substitution model
Arzanlou et al. [4] utilized the universal primers ITS1 and with inverse gamma rates, dirichlet base frequen-
ITS4 to amplify the internal transcribed spacer region (ITS) cies, and the temp value set to 0.5.
of the nuclear ribosomal RNA operon, including the 3′ end
of the 18S rRNA gene, the first internal transcribed spacer
Note. Part of the large subunit 28S rRNA (LSU) gene may
region (ITS1), the 5.8S rRNA gene, the second internal tran-
be also amplified with primers LR0R and LR5 followed by
scribed spacer region (ITS2), and the 5′ end of 28S rRNA
sequencing analysis.
gene. Subsequent sequencing analysis allows identification of
fungal organisms including Chaetomium species.
Procedure 45.3 Conclusion
Veronaea is one of the 70 dematiaceous fungal genera that
1. Polymerase chain Reaction (PCR) mixture (25 μL) can take advantage of temporary weakness in the host
is composed of 0.5 U Taq polymerase (Bioline), immune system and traumatic injury, causing a variety of
1 × PCR buffer, 0.5 mM MgCl2, 0.2 mM of each clinical diseases collectively referred to as phaeohyphomyco-
dNTP, 5 pmol of each primer, and approximately sis, chromoblastomycosis, or eumycetoma. Of the eight spe-
10–15 ng of fungal genomic DNA. cies within the genus Veronaea, only Veronaea botryosa has
2. Amplification is performed on a GeneAmp PCR been associated with human skin infections. Conventional
System 9700 (Applied Biosystems) with primary laboratory identification of V. botryosa is based on detec-
denaturation at 96°C for 5 min; 36 cycles of 96°C for tion of its characteristic phaeoid septate hyphae (3–4.5 μm
30 s, 52°C for 30 s, and 72°C for 60 s; a final exten- wide), geniculate, unbranched, pale-brown conidiophores,
sion at 72°C for 7 min. scar-bearing conidiogenous cells, and sympodially pro-
3. The amplicons are sequenced using BigDye duced, bicellular conidia with round tops and truncated bases
Terminator v. 3.1 (Applied Biosystems) or through direct mycologic examination and culture micros-
DYEnamicET Terminator (Amersham Biosciences) copy. However, this diagnostic scheme is notably lengthy
Cycle Sequencing Kits and analyzed on an ABI and requires considerable technical expertise. With the
Prism 3700 (Applied Biosystems). application of PCR and sequencing procedures, the identity
4. Newly generated sequences are subjected to a Blast of V. botryosa and other fungi can be rapidly and precisely
search of the GenBank databases, sequences with determined, contributing to the more effective treatment and
high similarity are downloaded from GenBank, and c ontrol of human phaeohyphomycosis.
comparisons are made on the basis of the alignment
of the obtained sequences.
5. Phylogenetic analysis is performed with PAUP References
(Phylogenetic Analysis Using Parsimony) v. 4.0b10 1. The UniProt Consortium. Available at http://www.uniprot.
using the neighbor-joining algorithm with the uncor- org/, accessed on August 1, 2010.
rected (“p”), the Kimura 2-parameter and the HKY85 2. de Hoog GS, Rahman MA, Boekhout T. Ramichloridium,
substitution models. Alignment gaps longer than 10 Veronaea, and Stenella: Generic delimitation, and new
bases are coded as single events for the phylogenetic combinations and two new species. Trans Br Mycol Soc.
analyses; the remaining gaps are treated as missing 1983;81:485–490.
data. Any ties are broken randomly when encountered. 3. Kharwar RN, Singh RK. Additions to the hyphomycete
genus Veronaea as phytoparasitic species. Microbiol Res.
6. Phylogenetic relationships are also inferred with
2004;159(2):103–111.
the parsimony algorithm using the heuristic search 4. Arzanlou M et al. Phylogenetic and morphotaxonomic revi-
option with simple taxon additions and tree bisection sion of Ramichloridium and allied genera. Stud Mycol.
and reconstruction (TBR) as the branch-swapping 2007;58:57–93.

Veronaea 381
5. Chakrabarti A. Epidemiology of central nervous system 12. Chen YT et al. Cutaneous phaeohyphomycosis caused by an
mycoses. Neurol India 2007;55:191–197. Itraconazole and Amphoterecin B resistant strain of Veronaeae
6. Brandt ME, Warnock DW. Epidemiology, clinical manifesta- botryosa. Int J Dermatol. 2006;45(4):429–432.
tions, and therapy of infections caused by dematiaceous fungi. 13. Matsushita A et al. Subcutaneous phaeohyphomycosis caused
J Chemother. 2003;15(Suppl. 2):36–47. by Veronaea botryosa in the People’s Republic of China.
7. Kondo Y et al. Cutaneous phaeohyphomycosis caused by J Clin Microbiol. 2003;41:2219–2222. Erratum in: J Clin
Veronaea botryosa observed as sclerotic cells in tissue. Int J Microbiol. 2003;41(7):3462.
Dermatol. 2007;46(6):625–627. 14. Sutton DA, Rinaldi MG, Kielhofner M. First U.S. report of
8. Zhang HE, Wang DL, Li RY. Report of the case of phaeohy- subcutaneous phaeohyphomycosis caused by Veronaea bot-
phomycosis caused by Veronaea botryosa. Chin J Dermatol. ryosa in a heart transplant recipient and review of the litera-
1990;23:96–98. ture. J Clin Microbiol. 2004;42(6):2843–2846.
9. Wang DL et al. Studies on Veronaea botryosa agent of the first 15. Da Cunha Filho, RR et al. Subcutaneous phaeohyphomycosis
human case. Acta Mycol Sin. 1991;10:159–165. caused by Veronaea bothryosa: Report of 2 cases. An Bras
10. Ayadi A, Huerre MR, de Bievre C. Phaeohyphomycosis Dermatol. 2005;80:1.
caused by Veronaea botryosa. Lancet 1995;346:1703. 16. Kumar KK, Hallikeri K. Phaeohyphomycosis. Indian J Pathol
11. Foulet F et al. Cutaneous phaeohphomycosis caused by Microbiol. 2008;51:556–558.
Veronaea bothryosa in a liver transplant recipient successfully
treated with Itraconazole. Clin Infect Dis. 1999;29:689–690.



46 Acremonium
Dongyou Liu, Xianghong Du, and Song Weining
Contents
46.1 Introduction...................................................................................................................................................................... 385
46.2 Methods............................................................................................................................................................................ 387
46.2.2.1 Sequencing Analysis of ITS and Partial 28S rRNA Gene.................................................................... 388
46.2.2.2 PCR-RFLP of ITS2 Region................................................................................................................... 388
46.3 Conclusion........................................................................................................................................................................ 388
References.................................................................................................................................................................................. 388
46.1 Introduction Haematonectria, and Neocosmospora), and the new combi-

nation Fusarium falciforme was proposed for Acremonium
46.1.1 Classification and Morphology falciforme [4].
The genus Acremonium is classified within the mitosporic Teleomorphs of Acremonium are found in several gen-
Hypocreales group, order Hypocreales, class Sordariomycetes, era of ascomycetes such as Emericellopsis, Hapsidospora,
subphylum Pezizomycotina, phylum Ascomycota, and king- Nectria, Nectriella, Neocosmospora, Pronectria, and
dom Fungi. The mitosporic Hypocreales group encompasses Thielavia (Sordariales), reflecting the polyphylic nature of
24 genera: Acremonium, Acrostalagmus, Cephalosporium, the Acremonium genus. For example, Acremonium alabam-
Chaetopsina, Cylindrocladiella, Escovopsis, Fusarium, ense is the anamorph of Thielavia terrestris, a member of
Gliocladiopsis, Gliocladium, Hobsonia, Illosporium, Sordariales. It has been proposed that the name Acremonium
Myrothecium, Parasarcopodium, Polycephalomyces, should be restricted to only the anamorphs of the family
Rotiferophthora, Sesquicillium, Solheimia, Stachybotrys, Hypocreaceae, and a new genus should be established to
Stilbella, Trichothecium, Tubercularia, Ustilaginoidea, accommodate other species of Acremonium, such as grass
Verticillium, and Xenocylindrocladium [1]. endophytes and others related to the Clavicipitaceae.
In turn, the genus Acremonium consists of 33 recog- Acremonium spp. are filamentous fungi that are com-
nized species: Acremonium alabamense, A. atrogriseum, monly isolated from soil, plant debris, rotting mushrooms,
A. alcalophilum, A. alternatum, A. antarcticum, A. blochii, etc. in Europe, Asia, Egypt, and North and Central America.
A. breve, A. brunnescens, A. butyri, A. collariferum, A. cro- Several Acremonium species have been implicated in human
tocinigenum, Acremonium cucurbitacearum, Acremonium infections, including Acremonium falciforme, Acremonium
curvulum, Acremonium exuviarum, A. fuci, A. falciforme kiliense, Acremonium strictum, and Acremonium recifei.
(obsolete synonym: Cephalosporium falciforme), A. hya- Acremonium grows moderately. After 7 day incubation
linulum, A. kiliense (obsolete synonyms: Cephalosporium at 25°C on potato glucose agar, colonies measure 1–3 cm in
granulomatis, C. infestans, and C. madurae), A. murorum, diameter and are compact, flat or folded, and slightly raised
A. nepalense, A. obclavatum, A. potronii, A. recifei (obso- in the center. Colonies are white to pale gray, velvety, pow-
lete synonym: Cephalosporium recifei), A. restrictum, dery, and membrane-like initially and become cottony or fas-
A. roseogriseum, A. rutilum, A. sclerotigenum, A. spinosum, ciculate (showing spiky aggregates of hyphae) with age. On
A. strictum, A. stromaticum, A. thermophilum, A. zeae, and the front, colonies are white, pale gray, cottony, or pale pink
Cephalosporium acremonium, in addition to over 30 unas- in color and, on the reverse, they are either uncolored or a
signed species [1–3]. pink-to-rose-colored pigment production.
A recent study showed that Acremonium falciforme Acremonium spp. possess hyaline, narrow, and sep-
shares considerable sequencing similarity in the ribosomal tate hyphae, with vegetative hyphae forming hyphal ropes.
large subunit (LSU) with members of the Fusarium solani Unbranched, solitary, awl-shaped erect phialides are located
species complex (i.e., the F. solani clade containing F. solani, on the hyphal tips, the hyphal ropes, or both. The phialides
385

are separated from hyphae by a septum and taper toward immunosuppressive therapy, and anatomic disorders. The fact
their apices. Conidia (1–3 × 3–8 μm) are usually one celled that A. kiliense infection often presents as a cutaneous papu-
(ameroconidia), hyaline or pigmented, globose to cylindrical, lar eruption without other clinical signs in immunocompetent
and mostly aggregated in slimy heads at the apex of each patients suggests its relatively low pathogenicity.
phialide (which may or may not possess collarettes). Bound Acremonium strictum is another species commonly
by a gelatinous material, conidia may be single or multicel- identified in human diseases [13,15,20–26]. For example, a
lular, fusiform with a slight curve (crescent-like). 59-year-old male who had undergone nonmyeloablative con-
A. falciforme often produces crescentic, nonseptate ditioning therapy and bone marrow transplantation after an
conidia, sometimes being two or three celled; A. kiliense initial diagnosis of acute myelogenous leukemia presented
forms solitary hyphae, chlamydospores (especially in oatmeal gastrointestinal graft-versus-host disease (GVHD) with
agar), and short straight cylindrical conidia; A. recifei usu- severe gastrointestinal bleeding. A fungus was isolated in
ally shows crescentic and nonseptate conidia; and A. strictum blood cultures and also a fungal organism was identified in
colonies may appear moist, pink, or salmon color, resembling multiple organs by histopathology at autopsy. Gram stains of
those of Lecythophora hoffmannii. positive blood culture bottles showed both yeast-like forms
with hyphal elements and fully formed hyphal masses sug-
gestive of a sporulating mold. On Sabouraud agar subculture
at 30°C, colonies measured 2.2 cm in 7 days. Young colo-
Being mainly environmental saprophytes, Acremonium nies were smooth, moist, and pink, with a colorless reverse;
species often gain entry to human host following penetrat- mature colonies were velvety and raised in the center.
ing injuries or surgery procedures [5,6]. Besides being one Lactophenol aniline blue preparations revealed conidia and
of the causative agents of eumycotic white grain mycetoma septate hyphae. The conidia (of 3–4 μm × 1–1.5 μm in size)
(principally by A. falciforme, A. kiliense, and A. recifei), were one celled, cylindrical, smooth, hyaline to slightly pink,
Acremonium spp. are also responsible for rare cases of ony- and grouped in slimy heads. The conidiophores were simple,
chomycosis, keratitis, endophthalmitis, endocarditis, gastritis, slender, and erect phialides with basal septa arising from
fungemia, meningitis, diffuse cerebritis, peritonitis, invasive the vegetative hyphae. Based upon micro- and macroscopic
pulmonary disease, and osteomyelitis [7–13]. These infections characteristics, the fungus was identified as A. strictum [27].
are often diagnosed in patients with predisposing conditions The next common Acremonium species associated with
(e.g., Addison’s disease, neutropenia, immune suppression, human infections is A. falciforme [7,9,10,28]. In addition, a
burns, organ transplantation, artificial implants, intravenous number of clinical cases due to Acremonium spp. have also
drug abuse, splenectomy, bone marrow transplantation, and been reported [29–31]. A patient with leukemia presented
diabetes mellitus) and preterm infants [14,15]. Neonates who painful cutaneous nodules and severe myalgia indicative of
receive mechanical ventilation, umbilical vein catheterization, pyomyositis. Culture of an aspiration grew an Acremonium
previous treatment with antibacterial agents, and prior use of species organism. After surgical drainage and treatment with
parenteral nutrition such as intravenous lipid show increased amphotericin B and granulocyte colony-stimulating factor
susceptibility to Acremonium infections. Acremonium endo- (G-CSF), the patient recovered fully [32]. In another case, a
phthalmitis may develop after penetrating keratoplasty and 64-year-old woman developed chronic uveitis in her left eye, 2
retinal detachment surgery, with intraocular inoculations weeks after an uncomplicated cataract extraction. Potassium
from irrigation solutions, and display clinical symptoms of hydroxide (KOH)–Calcofluor staining and Gram stain-
mild pain, redness, floaters, and slightly decreased visual acu- ing of the vitreous fluid uncovered septate fungal hyphae of
ity. Invasive Acremonium infection is difficult to treat and the Acremonium species. Subsequent culture of the vitreous fluid
outcome is generally poor. Acremonium species was reported grew an Acremonium organism. The patient was given intra-
to cause invasive allergic fungal sinusitis (AFS) or eosino- venous ampotericin B daily for 5 days and oral voriconazole
philic fungal rhinosinusitis (EFRS) in an immunocompetent medication for 4 weeks. During the postoperative 18 month
patient, leading to unilateral visual loss [16]. follow-up, she was stable without significant relapse of uveitis,
Among the Acremonium species of clinical interest, and her visual acuity improved from 20/40 to 20/20 [33].
Acremonium kiliense is most important and commonly Acremonium species are generally sensitive to ampho-
described [17–19]. Examined by light microscopy, A. kiliense tericin B and ketoconazole. Amphotericin B therapy in
is stained Gram-positive and is characterized by the appear- combination with ketoconazole or another new azole or
ance of septate hyphae and chlamydospores. The fungus grows allylamine may be used for treatment [34]. Patients experi-
slowly in specific culture (e.g., Sabouraud agar, glucose, and encing failure of amphotericin B treatment may be treated
malt extract). A range of clinical diseases has been attributed with a triazole derivative voriconazole. Voriconazole
to A. kiliense, including dermatophytoses, kerion, onycomyco- achieves a sufficient therapeutic level in aqueous and vit-
ses, keratitis, and mycetomas in immunocompetent individuals reous liquids by oral administration for eradication of
and pneumonia, arthritis, osteomyelitis, endocarditis, perito- Acremonium osteomyelitis [35].
nitis, meningitis, and sepsis in immunocompromised patients. Acremonium chrysogenum is the production host
Risk factors for A. kiliense infection are prosthesis, catheters, employed in the fermentation process of manufacturing

Acremonium 387
cephalosporins with broad activity against Gram-positive 46.2 Methods

and Gram-negative bacteria, while application of cephalo-
sporins is preferred in a wide range of cases due to microbial 46.2.1 Sample Preparation
resistance toward penicillins [36,37]. Culture of mycetoma lesion is performed by the collection of
the abscess or the fistula secretion or by tissue biopsy. The
46.1.3 Laboratory Diagnosis samples are cultured in media such as Sabouraud agar or
mycobiotic agar to isolate fungi and/or blood agar to isolate
Acremonium is a ubiquitous, saprophytic fungus that is char- bacteria. The etiologic agents are identified according to their
acterized by the formation of narrow hyphae with solitary, macroscopic and microscopic features.
slender (2 μm), unbranched, awl (needle)-shaped phialides Blood culturing is performed using the BACTEC 9240
(or weakly branched conidiophores) arising from vegetative automated blood-culturing system (Becton Dickinson), with
hyphae and producing clusters (slimy messes) or chains of each culture consisting of one each Plus Aerobic/F, Lytic/10
small, one-celled conidia mostly aggregated at the apex of Anaerobic/F, and Myco/F Lytic bottles. Aerobic and anaer-
each phialide. In tissue sections, Acremonium often shows obic media are held in the BACTEC cabinet for 5 days;
hyaline, septate hyphae and characteristic reproductive Myco/F bottles are held for 28 days. Aerobic and anaerobic
structures known as phialides and phialoconidia. The use of bottles positive for yeast-like fungi are subcultured to choco-
specific fungal stains such as Grocott methenamine-silver late, bromcresol green, and inhibitory mold agar plates and
and Giemsa stains aids the identification of Acremonium, incubated at 35°C supplemented with CO2 to 5%. Other fun-
although 10% KOH and Gram stain offer alternative detec- gal cultures are performed using Sabouraud dextrose agar
tion methods. (SAB; Emmon’s modification); brain heart infusion agar
Acremonium shows a high degree of morphologi- with blood, chloramphenicol, cycloheximide, and gentami-
cal similarity to Fusarium, Verticillium Lecythophora, cin; and inhibitory mold agar incubated at 30°C. Subcultures
Phialemonium, Gliomastix, and Cylindrocarpon as well as for morphological studies are made on potato dextrose agar
Candida. Strains of Fusarium, which do not produce mac- and incubated at 30°C [27].
roconidia, is differentiated from those of Acremonium by For DNA extraction from fungal strains, a loopful from
their faster growth and production of deeply woolly colo- individual colonies of each fungus culture is homogenized
nies. In comparison with Acremonium, Lecythophora and in 500 μL of lyticase lysis buffer (LLB; 50 mM Tris pH
Phialemonium phialides are not separated from hyphae by a 7.6, 1 mM EDTA pH 8.0, 0.2% β-mercaptoethanol, and
septum; while Gliomastix generates olive-green to greenish- 1 U/100 μL recombinant lyticase [Sigma]) and incubated
black colonies and chains or balls of dark conidia. at 37°C for 1 h. After incubation, acid-washed glass beads
Acremonium is differentiated from hyaline isolates of 710–1180 μm in diameter (Sigma) are added and the solution
Phialophora by the absence or very limited development of is vortexed thoroughly for 2 min. Amounts of 400 μL of the
a collarette on the phialide and the predominant formation supernatant are used for DNA extraction on a MagNA Pure
of awl-shaped phialides with a basal septum. Compared to compact instrument using a MagNA Pure compact nucleic
Acremonium, microconidial Fusarium isolates usually grow acid isolation kit I (Roche Diagnostics). DNA concentrations
faster and have colonies with a characteristic fluffy appear- are determined by using a PicoGreen double-stranded DNA
ance. Potato dextrose agar and cornmeal agar are the most quantification kit (Molecular Probes) on an F-2500 fluores-
suitable media for their identification, and exposure to day- cence spectrophotometer (Hitachi) [41].
light maximizes their culture color characteristics. For peripheral blood specimens, after hypotonic lysis of
Given the length of time for fungal culture and special- the erythrocytes using red blood cell lysis buffer (10 mM Tris
ist skills for macroscopic and microscopic identification pH 7.6, 5 mM MgCl2, 10 mM NaCl), the leukocytes are pel-
of Acremonium from other morphological similar fungal leted and resuspended in 470 μL LLB. The subsequent steps
organisms, nucleic acid amplification and sequence analy- were identical to the extraction protocol described above. For
sis have been increasingly applied for their characteriza- blood culture specimens, 200 μL aliquots derived from blood
tion and speciation. In particular, small subunit (SSU) 18S cultures previously shown to be fungus positive are trans-
and LSU 28S ribosomal RNA (rRNA) genes as well as their ferred to Falcon tubes and red blood cell lysis buffer is added.
internal transcribed spacer (ITS) regions provide valuable The subsequent procedure is as described above. For plasma
target for molecular detection of fungal organisms includ- containing white blood cells, peripheral blood specimens
ing Acremonium spp. [38,39]. For example, PCR amplifica- anticoagulated with EDTA are kept at 4°C for at least 4 h to
tion of partial 5S rRNA gene and internal transcribed spacer sediment the red blood cells. The entire supernatant, that is,
region 2 (ITS2) followed by digestion of the amplicon with plasma containing white blood cells, is used for DNA extrac-
restriction enzymes Hinfl and Sau3AI divides Acremonium tion. The samples are centrifuged at 15,000 × g for 10 min.
spp. into distinct restriction fragment length polymorphism Most of the plasma is removed, leaving a residual volume of
(RFLP) groups, with A. kiliense and A. strictum forming 100 and 430 μL of LLB is added. The DNA extraction is per-
similar subgroups on the basis of morphological differences formed as described above. For specimens from respiratory
and distinct RFLP patterns [40]. tract and lung biopsies, solid material is cut into small pieces

and homogenized in 430 μL of LLB. The ensuing steps are followed by the initial activation of DNA polymerase at 96°C
as described above [41]. for 12 min; (ii) 40 cycles consisting of a denaturation step
Alternatively, fungal DNA can be extracted from mature for 10 s at 96°C, an annealing step for 10 s at 58°C, and an
colonies, grown on SAB agar with chloramphenicol and gen- extension step for 30 s at 72°C; and (iii) the final extension at
tamicin (Remel) at 30°C, using the QIAmp Mini Kit (Qiagen) 72°C for 4 min.
following the manufacturer’s tissue extraction protocol [27]. Restriction analysis of amplified PCR products is performed
by HaeIII, endonuclease digestion (New England BioLabs).
46.2.2 Detection Procedures The restriction fragments are separated on a 3% (wt/vol) aga-
rose gel, stained with ethidium bromide, visualized by UV
46.2.2.1 Sequencing Analysis of ITS and transillumination (312 nm), and analyzed by ULTRA LUM
Partial 28S rRNA Gene (Ultra-Lum, Inc.) gel detection and analysis system.
Novicki et al. [27] employed the ITS1 (5′-TCC
GTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTT
ATTGATATGC-3′) primers to amplify the intergenic tran- 46.3 Conclusion
scribed spacer 1 (ITS1) and ITS2 regions of the rRNA operon, The anamorphic genus Acremonium consists of a large num-
flanking the 5.8S rRNA gene [42]. In addition, the D1–D2 vari- ber of saprophytic filamentous fungal species that have hya-
able domain of the 28S rRNA gene is amplified with the NL-1 line, septate hyphae and reproduce by phialidic conidiation.
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4 Several Acremonium spp. are involved in eumycotic myce-
(5′-GGTCCGTGTTTCAAGACGG-3′) primers. toma and other focal infections in otherwise healthy indi-
PCR mixture (25 μL) is made up of GeneAmp 1× PCR viduals. In addition, they have been increasingly implicated
buffer II (Applied Biosystems), 2.5 mM MgCl2, 0.2 mM in invasive systemic mycotic diseases in immunosuppressed
deoxynucleotide triphosphate, 400 nM each of primers ITS1 individuals with underlying conditions such as malignancy
and ITS4, or primers NL-1 and NL-4, 2.5 U AmpliTaq DNA and other illnesses. Given their similar morphological fea-
polymerase (Applied Biosystems), and 5 μL of template tures to a number of fungal organisms that may display var-
DNA. ied sensitivity to antifungal agents, it is important to identify
PCR amplification is performed with an initial 95°C for Acremonium to species level. Unfortunately, identification of
10 min (polymerase activation); 35 cycles of 95°C for 30 s, Acremonium spp. by macroscopic and microscopic examina-
55°C for 30 s, and 72°C for 1 min; and a final extension at tion is not only time-consuming but also challenging. This
72°C for 10 min. is especially so with some Acremonium species that do not
The resulting amplicons are sequenced and compared with develop a teleomorphic stage in vitro. Therefore, applica-
those at the National Center for Biotechnology Information tion of molecular detection procedures such as PCR and
GenBank nucleotide database using the nucleotide–nucleotide sequencing analysis is critical for accurate determination of
BLAST program for homologous sequences. The sequences Acremonium species identity and assists effective treatment
are then aligned and phylogenetic trees are drawn with and control of Acremonium infections.
Clustal X using the neighbor-joining method. The aligned
sequences are edited with Jalview version 1.3b. Phylogenetic
trees are displayed using Treeview version 1.6.6. This allows References
precise identification of fungal organisms of interest includ- 1. The UniProt Consortium. Available at http://www.uniprot.
ing Acremonium spp. org/, accessed on August 1, 2010.
2. Glenn AE et al. Molecular phylogeny of Acremonium and its
46.2.2.2 PCR-RFLP of ITS2 Region taxonomic implications. Mycologia 1996;88:369–383.
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47 Beauveria
Dongyou Liu
Contents
47.1 Introduction...................................................................................................................................................................... 391
47.1.2.1 Beauveria bassiana............................................................................................................................... 392
47.1.2.2 Beauveria brongniartii.......................................................................................................................... 392
47.1.2.3 Beauveria alba....................................................................................................................................... 393
47.1.3 Diagnosis.............................................................................................................................................................. 393
47.2 Methods............................................................................................................................................................................ 393
47.2.2.1 Standard PCR Detection........................................................................................................................ 393
47.2.2.2 Real-Time PCR Detection..................................................................................................................... 394
47.2.2.3 Analysis of ITS Sequence...................................................................................................................... 394
47.3 Conclusion........................................................................................................................................................................ 394
References.................................................................................................................................................................................. 395
47.1 Introduction bark beetles) [5]. Beauveria spp. are occasionally isolated
in the clinical laboratory as saprophytic contaminants; and
47.1.1 Classification and Morphology B. bassiana and possibly B. brongniartii have been impli-
The genus Beauveria is a filamentous fungus belonging to the cated in human hyalohyphomycosis.
mitosporic Cordycipitaceae group, family Cordycipitaceae, Beauveria spp. grow moderately rapidly, producing colo-
Order Hypocreales, class Sordariomycetes, subphylum nies of 1–3 cm in diameter on potato glucose agar after incu-
Pezizomycotina, phylum Ascomycota, and kingdom Fungi. bation at 25°C for 7 days. Colonies are cottony to powdery
The genus currently comprises 11 recognized species: or mealy in texture, initially white and later becoming yel-
Beauveria alba, Beauveria amorpha, Beauveria bassiana, lowish white or pale pinkish in color. The reverse is white
Beauveria brongniartii, Beauveria caledonica, Beauveria or pale. Hyphae (2–8 μm in width) are hyaline, septate, and
felina, Beauveria malawiensis, Beauveria tenella, Beauveria narrow, with random branches. Conidiogenous cells on the
velata, Beauveria vermiconia, and Beauveria virella, in hyphae are flask shaped with an inflation at the base and
addition to an unassigned species: Beauveria cf. caledonica narrow zigzagging filaments at the apex. Laterally from
ARSEF 2251. The teleomorphs of the genus Beauveria are the filament, conidia are produced from each bending point
found in Cordyceps (e.g., Cordyceps bassiana and Cordyceps (which is termed sympodial geniculate growth). Conidia
scarabaeicola) [1,2]. (2–4 μm in diameter) are hyaline, one celled, and globose to
Beauveria spp. are entomogenous fungi that infect insects ovoid in shape. As conidiogenous cells tend to form dense
and other arthropods. They have the ability to survive sap- clusters, which appear as small powdery balls in the aerial
rotrophically in the soil environment for extended time peri- hyphae under microscope, and make it difficult to visualize
ods and to form an endophytic association with plants (e.g., the arrangement and structure of conidia, young cultures are
corn) [3,4]. These organisms can be dispersed by sporulating preferred for optimal microscopic examination [6].
cadavers as infections in migrating insect hosts or by infec-
tious conidia on wind currents. 47.1.2 Clinical Features
Beauveria bassiana was first identified in the early nine- Beauveria spp. are insect pathogens with ubiquitous distri-
teenth century by the Italian scientist Agostino Bassi as the bution. B. bassiana, B. brongniartii, and B. alba have been
causative agent for white muscardine disease in silkworms shown to cause keratitis, deep tissue infection, disseminated
(Bombyx mori). Subsequently, B. bassiana and B. brongniar- infection, and other diseases in humans, especially those
tii as well as Metarhizium anisopliae have been exploited as with suppressed immune system and/or underlying condi-
mycopesticides for biological control of insect pests (e.g., tions (e.g., steroid treatment, prolonged granulocytopenia
391

following chemotherapy, and hematological malignancies) as along with a slight infiltration and edema. Direct micro-
well as after accidental injury [7–11]. scopic examination of corneal scrapings revealed Gram-
positive septate fungal hyphae with budding. Slide culture
47.1.2.1 Beauveria bassiana of the corneal scraping grew a whitish yellow colony, with
Tucker et al. [12] documented a case of disseminated Beauveria the fungus exhibiting zigzag rachis and oval conidia, char-
bassiana infection in a 44-year-old woman with acute lym- acteristics of B. bassiana. Topical voriconazole with micon-
phoblastic leukemia. After undertaking prophylactic treatment azole, pimaricin, and oral itraconazole were effective, and the
with ciprofloxacin and fluconazole, the patient developed skin lesion disappeared leaving only a mild scar after 2 months.
lesions, sinusitis, headache, and facial pain. Histopathological It is noteworthy that the keratomycosis caused by B. bassi-
examination of the skin biopsy specimen revealed areas of ana is restricted to the superficial cornea, as the pathogenesis
necrosis heavily permeated by fungal hyphae. Cultures of the for B. bassiana is relatively weak and does not cause severe
tissue biopsy sample on blood agar and Sabouraud glucose stromal infiltration or anterior chamber inflammation. Since
agar (SGA) plates at 30°C yielded a pure growth of a white the lesion caused by B. bassiana extends horizontally and
mold. On potato dextrose agar (PDA) at 30°C, colonies reached not vertically within the stroma, endothelial plaque and ante-
diameters of 4.5 cm in 13 days and appeared densely cot- rior inflammation are not evoked. On the other hand, other
tony to flocculent, yellowish white, raised, and dome shaped. filamentous fungi of keratomycosis, such as Aspergillus and
Conidiogenous cells in sporodochial clusters were slightly Fusarium, invade deeply into the corneal stroma and induce
swollen at the base and narrowed at the tip to form a zigzag endothelial plaque and hypopyon. In another study, Pariseau
rachis. Conidia (2–3 μm × 1.5–2 μm) were oval to subglobose et al. [15] described two contact lens-associated Beauveria
and apiculate. However, stab inoculations of the patient isolate keratitis cases involving a 55-year-old diabetic woman and a
and a control B. bassiana isolate onto PDA failed to grow at 31-year-old healthy woman in United States. Cultures of cor-
35°C. Sequence of the nuclear ribosomal internal transcribed neal scrapings from these patients grew B. bassiana. These
spacer (ITS) region from the case isolate demonstrated the isolates appeared to be unrelated to each other and also dis-
highest homology (98% identity) to the GenBank sequences tinct from that used in Beauveria-based biopesticides sold in
of B. bassiana and its teleomorph Cordyceps bassiana. After the United States as analyzed using morphological features,
treatment with liposomal amphotericin (AmBisome) in com- DNA sequencing, and random amplified polymorphic DNA
bination with itraconazole, her skin lesions healed with some (RAPD). The patients responded to natamycin, ketoconazole,
scarring but no recurrence of fungal infection. amphotericin, and voriconazole treatments.
Kisla et al. [13] described a case of Beauveria bassiana
keratitis in an 82-year-old woman, who underwent surgical 47.1.2.2 Beauveria brongniartii
repair of a graft wound dehiscence and anterior vitrectomy Henke et al. [16] documented a case of human deep tissue
on her right eye following blunt trauma. Seven months later, infection due to an entomopathogenic Beauveria species in
the patient complained of decreased vision, a foreign body a 38-year-old woman receiving immunosuppressive therapy.
sensation, and mild aching in her right eye. The corneal graft The patient presented with pain in the right upper abdomen,
was diffusely edematous; and a stromal infiltrate was noted in severe dyspnea, dry cough, and a fever of 38.5°C. Computed
the area of the epithelial defect. Smears stained with Giemsa tomography (CT) scan of the thorax revealed a discrete
revealed the presence of septate hyphae. Samples were inocu- interstitial infiltrate. Allergic alveolitis was histologically
lated onto blood, chocolate, brain–heart infusion agars, and confirmed by transbronchial lung biopsy. An ultrasonogra-
eugonic broth for anaerobes. While bacterial cultures yielded phy of the abdomen revealed multiple lesions in the liver and
no growth, fungal cultures grew 14 colonies of a white mold. spleen, suggesting systemic fungal infection. CT scans of the
Colonies on PDA were yellowish white, moderately fast grow- abdomen confirmed several focal hypodense lesions with a
ing, dense, and from powdery to woolly in texture. Conidia maximum diameter of 1.8 cm in the liver and in the spleen.
(asexual spores) were produced from small pegs formed in Hematoxylin-eosin-stained sections of the hypodense liver
a zigzag arrangement at the tips of the conidiogenous cells specimen obtained by the CT-aided liver biopsy showed that
(spore-bearing cells) that are swollen at the base. These mac- the liver tissue was damaged in the center of the lesion with
roscopic and microscopic features confirmed the fungus as extensive focal necrosis. Grocott Gomori methenamine sil-
Beauveria bassiana. Following treatment with natamycin 5% ver stain and calcofluor white stain from a specimen obtained
suspension and fluconazole for 3 weeks, the infiltrate progres- from the center of the focus revealed infiltrating hyphae.
sively improved, the epithelial defect became smaller, and the The specimen was homogenized and cultured on SGA,
patient recovered without recurring infection. malt extract agar (MEA), and Sabouraud glucose broth (10 g of
Sonoyama et al. [14] reported a superficial keratomycosis peptone/L and 40 g of glucose/L, pH 5.6) at 37°C and 26°C. A
due to Beauveria bassiana in an 80-year-old woman, who hyphomycete was obtained on all SGA and MEA plates incu-
presented with ocular pain and hyperemia 9 days after an bated at 26°C, but not at 37°C. Subcultures of the Sabouraud
ocular injury by the frame of her glasses. The patient had glucose broth were performed on SGA and MEA at 37°C and
recurrent diabetic iritis and continuously used topical antibi- 26°C. The isolate grew moderately rapidly at 26°C, form-
otics and corticosteroids. A slit-lamp examination indicated ing a procumbent to flocculent mycelium with a powdery
a corneal ulcer confined within the superficial stromal layer, surface, which was white at first and was later pale yellow.

Beauveria 393
Conidiogenous cells of the clinical isolate were aggregated in with 0.1% Tween 80 only. Larvae are observed for 10 days. In
moderately dense clusters alongside hyphae (1.5–3.0 μm wide) a control assay, the highly insect-virulent culture collection
and were subspherical to ampulliform with peg-like, narrow, strain B. bassiana ARSEF No. 252 is used as Ref. [18].
denticulate rachides that elongated in a zigzag manner. Conidia The susceptibility of Beauveria strains to itraconazole and
(2–3 μm in size) were smooth walled, single, and spherical to other antifungal drugs may be determined in a microdilu-
subspherical. The clinical isolate grew as an extremely floc- tion assay. A serial twofold dilution of itraconazole in RPMI
cose and velvety colony producing a low number of conidia, broth supplemented with 2% glucose is inoculated with 104
which is atypical for B. bassiana. On the basis of the snow- conidia per milliliter. After 72 h, the minimum inhibitory
ball-like appearance of conidiogenous cells and conidia, the concentration (MIC) is determined spectrophotometrically.
strain was considered as Beauveria, close to the generic type Molecular techniques are increasingly utilized for detec-
species, B. bassiana, and to its relative, Beauveria brongniar- tion, identification, and phylogenetic analysis of Beauveria
tii. Analysis of the nucleotide sequences of the ITS regions strains [19–27]. Because the nucleotide sequences of the ITS
(ITS1 and ITS2) of the nuclear ribosomal DNA showed that regions (ITS1 and ITS2) of the nuclear ribosomal RNA evolve
the patient isolate was identical, except for a single mutation in relatively quickly, they provide attractive chronometers for
the ITS2 sequence, to three strains deposited in GenBank as determining the relationship of genotypically closely related
Beauveria tenella (B. tenella is a synonym of B. brongniartii). species. The large- and small-subunit rRNA genes and the
Following microbiological and pathological findings, elongation factor 1α gene offer useful targets for Beauveria
antifungal therapy was initiated with 200 mg of itraconazole identification [28–30].
orally twice daily 1 week after admission. Three weeks after
the start of therapy, the patient recovered, and the antifungal
47.2 Methods
therapy was stopped after 3 months.
47.1.2.3 Beauveria alba
McDonnell et al. [17] reported a case of mycotic kerati- Clinical specimens are examined under microscope with a
tis caused by Beauveria alba in a 70-year-old white man fungal stain. Portions of the samples are inoculated on SGA
after phacoemulsification and intraocular lens implanta- without or with antibiotics. The resulting isolates are identi-
tion. A saprophytic fungus, Beauveria alba (also known fied on the basis of macroscopical and microscopical features.
as Engyodontium album), was demonstrated in the cor- For easy microscopic observation, slide culture technique
neal button by histopathologic examination and isolated. may be utilized. Slide cultures are set up in Petri dishes con-
Microscopically, conidiogenous cells formed in whorls or taining 2 mL of sterile water, into which a U-shaped glass
in aggregations are basally swollen in B. bassiana and are rod is placed, extending above the water surface. A block of
solitary and non-swollen in B. alba. Extensive therapeutic freshly growing fungal colony, approx. 1 cm2, is placed onto
surgery was necessary to control the infection. a sterile microscope slide, covered with a somewhat larger,
sterile glass cover slip, and incubated in the moist chamber.
Fungal sporulation is monitored over time, and when opti-
47.1.3 Diagnosis mal, images are captured by means of a Nikon camera sys-
Conventional techniques for fungal identification rely on tem (Digital Sight DS-5M, Nikon Corporation).
the detection of macroscopic and microscopic features. For DNA extraction, a small amount of fungal pellet is sus-
Beauveria spp. are characterized by their sympodial develop- pended in 600 μL extraction buffer (200 mM Tris–HCl, pH
ment of single-celled conidia (ameroconidia) on a geniculate 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate [SDS],
or zigzag rachis; their formation of flask-shaped, rachiform and 250 mM NaCl) and vortexed for 15 s. The mixture is
conidiogenous cells that proliferate sympodially and aggre- then incubated at 100°C for 15 min, chilled on ice for 60 min,
gate into sporodochia or synnemata; their production of and centrifuged at 13,000 rpm for 15 min. The supernatant is
hyaline and globose or ovoid conidia. These morphological transferred to a new tube, and the solution is extracted with
characteristics are useful for discrimination of Beauveria, phenol–chloroform–isoamyl alcohol (25:24:1 v/v). DNA is
Aspergillus, Fusarium, and other fungi, ensuring successful precipitated with cold isopropanol (−20°C), air dried, and
treatment with antimycotic agents. resuspended in 100 μL distilled water. Alternatively, genomic
The virulence of Beauveria strains may be assessed DNA is extracted from colonies grown on 2% MEA, Difco,
in bioassays using larvae of the Colorado potato beetle using the FastDNA kit (BIO101).
(Leptinotarsa decemlineata) and conducted on potato leaf.
Briefly, the surface of the leaf disks (diameter, 8 mm), laying 47.2.2 Detection Procedures
on water agar, is treated with 10 μL of 106 conidia mL−1 in
a first experiment and 5 × 106 conidia mL−1 in 0.1% Tween 47.2.2.1 Standard PCR Detection
80 in a second experiment. Instar larvae of L. decemlineata Hegedus and Khachatourians [22] reported the use of
are put on the treated leaves for 2 days at 25°C (16 h light primers P1 (5′- AAGCTTCGACATGGTCTG-3′) and P3
and 8 h dark each day) and then are transferred to untreated (5′-GGAGGTGGTGAGGTTCTGTT-3′) for specific poly-
potato leaves. Control larvae are fed with leaf disks treated merase chain reaction (PCR) detection of Beauveria bassiana.

Procedure analysis for fungal detection using pan-fungal primers ITS1

1. PCR mixture (50 μL) consists of 20 mM Tris–HCl reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM each of the fungi is verified by subsequent sequencing analysis.
of dNTP, 0.75 μM of each primer, 100 ng of DNA,
and 1.25 U Taq DNA polymerase (Gibco BRL). Procedure
2. Amplification is performed with initial denaturation
at 94°C for 3 min; 40 cycles of 94°C for 30 s, 55°C 1. PCR mixture is composed of 1× Lightcycler
for 30 s, and 72°C for 45 s; and a final 10 min exten- FastStart DNA Master Hybridization Probes mix-
sion at 72°C. ture (Roche Applied Science) containing deoxy-
3. PCR products are separated on 1.0% (wt/vol) nucleoside triphosphates, FastStart Taq DNA
agarose-TBE (0.09 M Tris, 0.09 M boric acid, and polymerase, and 1 mM MgCl2 (additional MgCl2 is
0.02 M EDTA) gels with ethidium bromide stain added to a final concentration of 4.6 mM), 0.4 μM
and visualized under UV light. each of ITS1 forward and ITS4 reverse primers,
1× SYBR green (Molecular Probes), and 3 μL tem-
Note. A specific product of 524 bp product is expected from plate DNA.
B. bassiana. 2. Thermal cycling parameters include 95°C for
47.2.2.2 Real-Time PCR Detection and 76°C for 30 s; and a final extension at 72°C for
Castrillo et al. [30] designed primers and probe target- 2 min.
ing a 445 bp sequence characterized amplified region 3. The quality of the amplicon is determined using the
(SCAR) fragment for real-time PCR detection of derivative of the melt analysis curve (55°C–99°C,
Beauveria bassiana. The primers GHTqF1 (5′-TTTTCAT 45 s hold at 55°C, and 5 s/°C) using the RotorGene
CGAAAGGTTGTTTCTCG-3′) and GHTqR1 (5′-CTGT 3000 (Corbett Robotics, Inc).
GCTGGGTACTGACGTG-3′) amplify a 96 bp region of the 4. The amplified product is purified for bidirectional
SCAR fragment; and the probe GHTqP1 (5′-TTCCGTTCCG sequencing using ExoSAP-IT (USB Corp). Five
CTCCGCCAAAAGCC-3′) is labeled at the 5′ end with fluo- microliters of Big Dye Terminator Ready Reaction
rescent FAM reporter dye and at the 3′ end with tetramethyl- Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
6-carboxyrhodamine (dye) (TAMRA) quencher dye. each primer (0.8 pmol/μL) and 3 μL of purified PCR
Procedure thermal cycler (ABI), using 25 cycles of 96°C for
1. PCR mixture (25 μL) is composed of 1× iQ Supermix
(BioRad), 0.5 μM of each primer, 0.125 μM of
probe, 0.8 μg/μL (final concentration) of bovine
serum albumin (BSA) (Ambion), and 2 μL of each
of the appropriately diluted sample DNA. (BSA
50 cm capillary array.
helps reduce PCR inhibition from co-extracted con-
taminants from environmental samples, especially
humic acids from soil.)
2. Each assay includes a standard curve (five or six
series 10-fold dilutions from 40 ng to 4 or 0.4 pg
of control B. bassiana GHA DNA) for determin-
ing starting DNA concentration of unknowns and a
non-template control.
Note. If real time PCR instrument is unavailable, standard
3. Thermal cycling conditions are as follows: initial
denaturation at 95°C for 10 min; 40 cycles of dena-
the resulting amplicon is sequenced with the same primers.
turation at 95°C for 15 s and single-step annealing
Sequence-based identifications are defined by percent iden-
and extension at 60°C for 1 min.
tity: species, ≥99%; genus, 93%–99%; and inconclusive,
≤93%.
Note. This assay may be adapted for standard PCR detection
with the following cycling parameters: initial denaturation
at 94°C for 4 min and 30 cycles of 94°C for 1 min, 55°C for
1 min, and 72°C for 1 min.
47.3 Conclusion
Beauveria spp. are entomopathogenic fungi that are com-
47.2.2.3 Analysis of ITS Sequence monly present in the environments and are utilized as biolog-
Pounder et al. [31] described a real-time PCR with SYBR ical control agents for insect pests. Several Beauveria species
green DNA-binding dye and amplicon-melting temperature (e.g., B. bassiana, B. brongniartii, and B. alba) have been

Beauveria 395
shown to cause hyalohyphomycosis in humans, with a variety 16. Henke, M.O. et al. 2002. Human deep tissue infection with
of clinical symptoms (e.g., keratitis, deep tissue infection, and an entomopathogenic Beauveria species. J. Clin. Microbiol.,
disseminated infection). The distinct morphological features 40:2698–2702.
17. McDonnell, P.J. et al. 1984–1985. Mycotic keratitis due to
of the Beauveria genus include (i) the sympodial develop-
Beauveria alba. Cornea, 3(3):213–216.
ment of single-celled conidia (ameroconidia) on a geniculate 18. Safavi, S.A. et al. 2007. Effect of nutrition on growth and vir-
or zigzag rachis; (ii) the formation of flask-shaped, rachiform ulence of the entomopathogenic fungus Beauveria bassiana.
conidiogenous cells that proliferate sympodially and aggre- FEMS Microbiol. Lett., 270:116–123.
gate into sporodochia or synnemata; and (iii) the presence 19. Kosir, J.M. and J.M. MacPherson, and G.G. Khachatourians.
of hyaline and globose or ovoid conidia. Although in vitro 1991. Genomic analysis of virulent and less virulent strain
culture and microscopic examination detecting these charac- of the entomopathogenic fungus Beauveria bassiana using
teristics are useful for differentiation of Beauveria spp. from RFLP. Can. J. Microbiol., 37:534–541.
20. Hegedus, D.D. and G.G. Khachatourians. 1993a. Construction
other fungi, the whole process can be lengthy and techni- of cloned DNA probes for the specific detection of the ento-
cally demanding. The use of PCR and sequencing analysis mopathogenic fungus Beauveria bassiana in grasshoppers.
provides a much faster and more accurate alternative to the J. Invertebr. Pathol., 62:233–240.
identification of Beauveria spp. 21. Hegedus, D.D. and G.G. Khachatourians. 1993b. Identification
of molecular variants in mitochondrial DNAs of mem-
bers of the genera Beauveria, Verticillium, Paecilomyces,
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1. The UniProt Consortium. Available at http://www.uniprot. 59:4283–4288.
org/, accessed on August 1, 2010. 22. Hegedus, D.D. and G.G. Khachatourians. 1996. Detection
2. Li, Z. et al. 2001. Discovery and demonstration of the teleo- of the entomopathogenic fungus Beauveria bassiana within
morph of Beauveria bassiana (Bals.) Vuill., an important infected migratory grasshoppers (Melanoplus sanguinipes)
entomogenous fungus. Chin. Sc. Bull., 46:751–753. using polymerase chain reaction and DNA probe. J. Invertebr.
3. Wagner, B.L. and L.C. Lewis. 2000. Colonization of corn, Zea Pathol., 67:21–27.
mays, by the entomopathogenic fungus Beauveria bassiana. 23. Shih, H.L. et al. 1995. Complete nucleotide sequence of
Appl. Environ. Microbiol., 66:3468–3473. Beauveria bassiana 5.8s rRNA coding gene and flanking
4. Shimazu, M., H. Sato, and N. Maehara. 2002. Density of the internal transcribed spacers. DNA Seq., 5:381–383.
entomopathogenic fungus, Beauveria bassiana Vuillemin 24. De Muro, M.A., S. Mehta, and D. Moore. 2003. The use of
(Deuteromycotina: Hyphomycetes) in forest air and soil. Appl. amplified fragment length polymorphism for molecular anal-
Entomol. Zool., 37:19–26. ysis of Beauveria bassiana isolates from Kenya and other
5. Strasser, H., T.M. Butt, and A. Vey. 2000. Are there any risks countries and their correlation with host and geographic ori-
in using entomopathogenic fungi for pest control, with par- gin. FEMS Microbiol. Lett. 229:249–257.
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Tolypocladium and Beauveria species? Biocontrol Sci. relatedness and population structure of Beauveria bassiana.
Technol., 10:717–735. Environ. Microbiol., 5:908–915.
6. De Hoog, G.S. et al. 2000. Atlas of Clinical Fungi. 26. Wang, C. et al. 2004. Molecular monitoring and evalu-
Centraalbureau voor Schimmelcultures, Utrecht, the ation of the application of the insect-pathogenic fungus
Netherlands. Beauveria bassiana in southeast China. J. Appl. Microbiol.,
7. Sachs, S.W., J. Baum, and C. Mies. 1985. Beauvaria bassiana 96:861–870.
keratitis. Br. J. Ophthalmol., 69:548–550. 27. Rehner, S.A. and Buckley, E. 2005. A Beauveria phylogeny
8. Low, C.D., P.R. Badenoch, and D.J. Coster. 1997. Beauveria inferred from nuclear ITS and EF1-α sequences: Evidence for
bassiana keratitis cured by deep lamellar dissection. Cornea, cryptic diversification and links to Cordyceps teleomorphs.
16:698–699. Mycologia, 97:84–98.
9. Gürcan, S. et al. 2006. First case report of empyema caused by 28. Neuvéglise, C., Y. Brygoo, and G. Riba. 1997. 28s rDNA
Beauveria bassiana. Mycoses, 49(3):246–248. group-I introns: A powerful tool for identifying strains of
10. Tu, E.Y. and A.J. Park. 2007. Recalcitrant Beauveria bassiana Beauveria brongniartii. Mol. Ecol., 6:373–381.
keratitis: Confocal microscopy findings and treatment with 29. Castrillo, L.A., J.D. Vandenberg, and S.P. Wraight. 2003.
posaconazole (Noxafil). Cornea, 26(8):1008–1010. Strain-specific detection of introduced Beauveria bassiana in
11. Oh, J.Y. et al. 2009. A case of necrotizing sclerokeratitis agricultural fields by use of sequence-characterized amplified
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J. Ophthalmol., 53(5):551–553. 30. Castrillo, L.A., M.H. Griggs and J.D. Vandenberg. 2008.
12. Tucker, D.L. et al. 2004. Disseminated Beauveria bassiana Quantitative detection of Beauveria bassiana GHA
infection in a patient with acute lymphoblastic leukemia. (Ascomycota: Hypocreales), a potential microbial control
J. Clin. Microbiol., 42(11):5412–5414. agent of the emerald ash borer, by use of real-time PCR. Biol.
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bassiana keratitis. Cornea, 19:405–406. 31. Pounder, J.I. et al. 2007. Discovering potential pathogens
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antimycotic agents. Clin. Ophthalmol., 2(3):675–678.
15. Pariseau, B. et al. 2010. Beauveria keratitis and biopesticides:
Case histories and a random amplification of polymorphic
DNA comparison. Cornea, 29(2):152–158.

48 Chaetomium
Contents
48.1 Introduction...................................................................................................................................................................... 397
48.1.3 Diagnosis.............................................................................................................................................................. 398
48.2 Methods............................................................................................................................................................................ 398
48.3 Conclusions....................................................................................................................................................................... 399
References.................................................................................................................................................................................. 399
48.1 Introduction ostioles (small-rounded openings) and contain asci and

ascospores inside. Asci are clavate to cylindrical in shape
48.1.1 Classification and Morphology and rapidly dissolve to release their ascospores (4 to 8 in
The genus Chaetomium consists of dematiaceous (dark-walled) number). Ascospores are one celled, olive brown, and lemon
fungi classified in the family Chaetomiaceae, order Sordariales, shaped [2].
class Sordariomycetes, subphylum Pezizomycotina, phylum Features fundamental to the identification of Chaetomium
Ascomycota, and kingdom Fungi. Of the 35 recognized and species include the presence of hairs or setae covering the
69 unassigned species within the genus, the following are most ascomata, the size and shape of ascospores, and the presence
common: Chaetomium atrobrunneum (obsolete synonym: and position of germ pores. Among the similar Chaetomium
C. fusisporale and C. rectopilium), Chaetomium funicola, species (i.e., C. gangligerum, C. raii, C. jodhpurense, and
Chaetomium globosum (obsolete synonyms: C. caprophilum C. fusisporum), C. gangligerum produces broader asco-
and C. cinnamomeum), Chaetomium perlucidum, Chaetomium spores (12–15 μm × 7.5–9.5 μm) and usually forms chla-
piluliferum (a teleomorph of Botryotrichum piluliferum), and mydospores; C. raii has smaller ascospores (10–13 μm ×
Chaetomium strumarium (obsolete synonyms: Achaetomium 5.5–7.5 μm), and C. jodhpurense and C. fusisporum have
cristalliferum, Achaetomium strumarium, C. spinulosum, and larger ascospores (14–19 μm × 6–8 μm and 14–17 × 7–8 μm,
C. sulphureum) [1,2]. respectively).
Chaetomium species are saprobic ascomycetes with C. strumarium is distinguished by its formation of
worldwide distribution and are commonly found in dung, ascocarps covered with pale, thin-walled, flexuous hairs, a
straw, paper, bird feathers, seeds, plant debris, soil, and air. feature contributing to its original placement in the genus
Some Chaetomium spp. are thermophilic and neurotropic in Achaetomium. Presence of pinkish exudate droplets and/or
nature and are among the fungi causing phaeohyphomyco- crystals associated with hyphae or ascocarps, good growth
sis in humans, with clinical presentations ranging from brain at 42°C, and production of small conidia further separate
abscess, peritonitis, cutaneous lesions, onychomycosis, to C. strumarium from other species [3].
fatal deep mycoses. Furthermore, C. perlucidum produces globose (spherical)
Chaetomium species grow rapidly at between 25°C and to subglobose or ovoid ascomata (108–220 μm × 90–200 μm),
35°C, with some species responsible for invasive human with undulate hairs and a wide ostiole (30–50 μm in diame-
disease growing well at 35°C–37°C, and others showing a ter) and eight-spored asci (20–40 μm × 7–18 μm). The asco-
predilection for the central nervous system growing at up spores (12.0–15.0 μm × 6–7.5 μm) are elliptical and olive
to 42°C–45°C. Chaetomium colonies are cottony and white brown and contain a subapical germ pore. These features
initially and become gray to olive with age. The colony help differentiate C. perlucidum from other invasive species
reverse is tan to red or brown to black. Hyphae are septate. of Chaetomium [4].
Perithecia are large, dark brown to black, fragile, and glo- C. globosum is distinguished by its lack of growth at
bose to flask shaped and have filamentous, hair-like, brown elevated temperatures (>35°C) and its generation of lemon-
to black appendages (setae) on their surface. Perithecia have shaped ascospores.
397

48.1.2 Clinical Features species. However, this is contradicted by reports of melanin

from Aspergillus (e.g., Aspergillus fumigatus) [17], and so the
Despite being saprophytic ascomycetes with only occasional information obtained may have limited value in differentiat-
involvement in human disease processes, Chaetomium spe- ing the causal fungi.
cies are capable of inducing a broad spectrum of mycoses In vitro culture techniques offer a slow but valuable way to
including onychomycosis, sinusitis, empyema, pneumonia, isolate and propagate Chaetomium organisms for subsequent
and fatal disseminated cerebral disease, especially in immu- macroscopic and microscopic characterization. Inoculation
nocompromised patients and intravenous drug users [5–9]. of sterilized plant material with ascospore suspension may
Chaetomium atrobrunneum is a notably invasive, neuro- enhance induction of mature perithecia, leading to the pro-
tropic species, and its ability to grow at elevated temperatures duction of well-developed ascomata on the surface of the
may contribute to its neurotropism [10–13]. Thomas et al. [9] substrate.
described a case of fatal brain abscess due to C. atrobrun- Molecular methods such as (i) PCR and (ii) sequence anal-
neum in a bone marrow transplant patient. The rapid pro- ysis of rRNA and internal transcribed spacer (ITS) regions
gression of cerebral infection indicates that the brain tissue provide an approach for the rapid and accurate identification
provides a favorable environment for growth and prolifera- of Chaetomium species from other fungi.
tion of the fungus.
Chaetomium strumarium is another invasive, neurotropic
species. Abbott et al. [3] reported three C. strumarium- 48.2 Methods
related cases of fatal cerebral mycosis in males with prior
histories of intravenous drug use from the United States and 48.2.1 Sample Preparation
Australia. C. strumarium was detected by histopathology Clinical specimens are examined by microscopy with a mel-
and isolated from the brain tissue. anin-specific stain (i.e., the Masson-Fontana stain). However,
Chaetomium perlucidum was recently confirmed as a as mentioned above, a melanin stain may not be particularly
neurotropic species. Barron et al. [4] documented the first useful. Portions of the samples are inoculated on Sabouraud
two cases of invasive human mycoses caused by this phaeoid glucose agar with or without antibiotics. The resulting iso-
ascomycete. The first case concerned a 45-year-old female lates are identified on the basis of macroscopical and micro-
patient with acute myelogenous leukemia, who had an unre- scopical features.
lated, 4/5 HLA-matched umbilical cord blood transplant. For DNA extraction, a small amount of fungal pellet is
The patient became disoriented and febrile, and computed suspended in 600 μL extraction buffer (200 mM Tris–HCl,
tomography of the chest revealed a 3 × 2 cm mass in the right pH 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate
lower lobe. After suffering a massive right-sided intrapa- [SDS], and 250 mM NaCl) and vortexed for 15 s. The mix-
renchymal hemorrhage, the patient died. Autopsy revealed ture is then incubated at 100°C for 15 min, chilled on ice
disseminated invasive fungal infection in the lungs, brain, for 60 min, and centrifuged at 13,000 rpm for 15 min. The
and myocardium; and cultures from the surgically obtained supernatant is transferred to a new tube, and the solution is
lung tissue yielded C. perlucidum. The second case involved extracted with phenol–chloroform–isoamyl alcohol (25:24:1
a 78-year-old female with a history of asthma and chronic v/v). DNA is precipitated with cold isopropanol (−20°C), air
bronchiectasis. The patient underwent a lobectomy due to dried, and resuspended in 100 μL distilled water.
worsening symptoms, and cultures from the lung tissue grew
C. perlucidum. The patient showed no further manifestations
of disease after the lobectomy. 48.2.2 Detection Procedures
Chaetomium globosum is an occasional agent of onycho-
mycosis [14,15]. In addition, Teixeira et al. [16] reported that Arzanlou et al. [18] utilized the universal primers ITS1 and
C. globosum was responsible for a systemic infection with ITS4 to amplify the ITS region of the nuclear ribosomal
enlargement of the axillary and cervical lymph nodes in a RNA operon, including the 3′ end of the 18S rRNA gene, the
chronic myeloid leukemia patient, who underwent an allo- first ITS region (ITS1), the 5.8S rRNA gene, the second ITS
geneic sibling-matched bone marrow transplant. Culture of region (ITS2), and the 5′ end of 28S rRNA gene. Subsequent
the aspirates from both lymph nodes resulted in the growth sequencing allows identification of the Chaetomium species.
of C. globosum. The infection was successfully treated with Procedure
amphotericin B.
1. PCR mixture (25 μL) is composed of 0.5 U Taq
polymerase (Bioline), 1× PCR buffer, 0.5 mM
48.1.3 Diagnosis
MgCl2, 0.2 mM of each dNTP, 5 pmol of each
Considerable similarities exist between mycosis caused by primer, and approximately 10–15 ng of fungal
Chaetomium and Aspergillus from radiographical and his- genomic DNA.
topathological standpoints. A melanin-specific stain (i.e., the 2. Amplification is performed on a GeneAmp PCR
Masson-Fontana stain) is helpful reputedly for distinguishing System 9700 (Applied Biosystems) with primary
the melanin-containing Chaetomium from most Aspergillus denaturation at 96°C for 5 min; 36 cycles of 96°C for

Chaetomium 399
30 s, 52°C for 30 s, and 72°C for 60 s; a final exten- Chaetomium spp. require lengthy incubation and demand
sion at 72°C for 7 min. specialized skill, molecular procedures have been increas-
3. The amplicons are sequenced using BigDye ingly applied for culture-independent detection and differen-
Terminator v. 3.1 (Applied Biosystems,) or tiation of Chaetomium spp. The use of melanin stains alone
DYEnamicET Terminator (Amersham Biosciences) may be inconclusive.
Cycle Sequencing Kits and analyzed on an ABI
Prism 3700 (Applied Biosystems).
References
4. Newly generated sequences are subjected to a Blast
search of the NCBI databases, sequences with high 1. The UniProt Consortium. Available at http://www.uniprot.
similarity are downloaded from GenBank, and org/, accessed on August 1, 2010.
comparisons are made on the basis of the alignment 2. von Arx, J. A., J. Guarro, and M. J. Figueras. 1986. The
ascomycete genus Chaetomium. Beih. Nova Hedwigia
of the obtained sequences. 84:1–162.
5. Phylogenetic analysis is performed with PAUP 3. Abbott, S. P. et al. 1995. Fatal cerebral mycoses caused by
(Phylogenetic Analysis Using Parsimony) v. 4.0b10 the ascomycete Chaetomium strumarium. J. Clin. Microbiol.
using the neighbor-joining algorithm with the 33:2692–2698.
uncorrected (“p”), the Kimura 2-parameter, and the 4. Barron, M. A. et al. 2003. Invasive mycotic infections caused
HKY85 substitution models. Alignment gaps longer by Chaetomium perlucidum, a new agent of cerebral phaeohy-
than 10 bases are coded as single events for the phy- phomycosis. J. Clin. Microbiol. 41(11):5302–5307.
5. Hoppin, E. C., E. L. McCoy, and M. G. Rinaldi. 1983.
logenetic analyses; the remaining gaps are treated
Opportunistic mycotic infection caused by Chaetomium in a
as missing data. Any ties are broken randomly when patient with acute leukemia. Cancer 52:555–556.
encountered. 6. Anandi, V. et al. 1989. Cerebral phaeohyphomycosis caused
6. Phylogenetic relationships are also inferred with by Chaetomium globosum in a renal transplant recipient.
the parsimony algorithm using the heuristic search J. Clin. Microbiol. 27:2226–2229.
option with simple taxon additions and tree bisec- 7. Yeghen, T. et al. 1996. Chaetomium pneumonia in a patient
tion and reconstruction as the branch-swapping with acute myeloid leukaemia. J. Clin. Pathol. 49:184–186.
algorithm; alignment gaps are treated as a fifth 8. Aru, A., L. Munk-Nielsen, and B. H. Federspiel. 1997. The
soil fungus Chaetomium in the human paranasal sinuses. Eur.
character state and all characters are unordered and Arch. Otorhinolaryngol. 254:350–352.
of equal weight. Branches of zero length are col- 9. Thomas, C. et al. 1999. Fatal Chaetomium cerebritis in a bone
lapsed and all multiple, equally parsimonious trees marrow transplant patient. Hum. Pathol. 30:874–879.
are saved. 10. Guarro, J., L. Soler, and M. G. Rinaldi. 1995. Pathogenicity
7. Other measures calculated include tree length, con- and antifungal susceptibility of Chaetomium species. Eur. J.
sistency index, retention index, and rescaled consis- Clin. Microbiol. Infect. Dis. 14:613–618.
tency index. The robustness of the obtained trees is 11. Segal, R. and S. Kusne. 1999. Cerebral fungal infections
in the immunocompromised host: A literature review and
evaluated by 1000 bootstrap replications. Bayesian
a new pathogen-Chaetomium atrobrunneum: Case report.
analysis is performed. The best nucleotide substitu- Neurosurgery, 45:200.
tion model is determined using MrModeltest v. 2.2. 12. Guppy, K. H. et al. 1998. Cerebral fungal infections in
MrBayes v. 3.1.2 is used to perform phylogenetic the immunocompromised host: A literature review and a
analyses, using a general time-reversible substitu- new pathogen—Chaetomium atrobrunneum: Case report.
tion model with inverse gamma rates, dirichlet base Neurosurgery 43:1463–1469.
frequencies, and the temp value set to 0.5. 13. Rock, J. P. 1998. Cerebral fungal infections in the immu-
nocompromised host: A literature review and a new patho-
gen—Chaetomium atrobrunneum: Case report—Comment.
Note. Part of the large-subunit 28S rRNA (LSU) gene may Neurosurgery 43:1469.
be also amplified with primers LR0R and LR5 followed by 14. Stiller, M. J. et al. 1992. Onychomycosis of the toenails
sequencing analysis. caused by Chaetomium globosum. J. Am. Acad. Dermatol.
26:775–776.
15. Lesire, V. et al. 1999. Possible role of Chaetomium globosum
48.3 Conclusions in infection after autologous bone marrow transplantation.
The genus Chaetomium consists of a large group of dema- Intensive Care Med. 25:124–125.
tiaceous (dark-walled) fungi that are present in a variety of 16. Teixeira, A. B. et al. 2003. Phaeohyphomycosis caused by
Chaetomium globosum in an allogeneic bone marrow trans-
environments, including dung, straw, paper, bird feathers,
plant recipient. Mycopathologia 156(4):309–312.
seeds, plant debris, soil, and air. Several Chaetomium spp. 17. Youngchim, S. et al. 2004. Production of melanin by
are opportunistic human pathogens, causing phaeohypho- Aspergillus fumigatus. J. Med. Microbiol. 53:175–181.
mycosis, with clinical manifestations of brain abscess, peri- 18. Arzanlou, M. et al. 2007. Phylogenetic and morphotaxonomic
tonitis, cutaneous lesions, onychomycosis, and fatal deep revision of Ramichloridium and allied genera. Stud Mycol.
mycoses. Since conventional methods for identification of 58:57–93.

49 Colletotrichum
M.R. Shivaprakash, Abhishek Baghela, and Arunaloke Chakrabarti
Contents
49.1 Introduction...................................................................................................................................................................... 401
49.1.1.1 Classification.......................................................................................................................................... 401
49.1.1.2 Morphology............................................................................................................................................ 402
49.1.1.3 Epidemiology......................................................................................................................................... 402
49.1.3 Diagnosis.............................................................................................................................................................. 406
49.2 Methods............................................................................................................................................................................ 407
49.2.2.1 PCR-ITS Region.................................................................................................................................... 407
49.2.2.2 Sequencing of rRNA-ITS Region.......................................................................................................... 407
References.................................................................................................................................................................................. 408
49.1 Introduction as ascomycetous sporulation and described under the Genus

Glomerella. However, with the exception of C. falcatum,
Colletotrichum species are well-known plant pathogens teleomorph stage is not reported from the field.4
in tropical and subtropical countries. They are known to Taxonomy and nomenclature of Colletotrichum is confus-
cause important plant disease, anthracnose, in a wide range ing, even to scientists working in this field.5 Initially the spe-
of plants including cereals, legumes, vegetables, peren- cies under the Genus Colletotrichum were identified based
nial crops, and tree fruits. They have tremendous economic on the host specificity, location, and the morphology of the
impact.1 Recently, Colletotrichum species have also been conidia. However, there was duplication in the names, as the
recognized as emerging human pathogens causing oph- same species were referred to by different names. Few spe-
thalmic lesion. Occasionally, they may cause subcutaneous cies with curved spores were classified under Vermicularia.
infections in humans. Von Arx, with his seminal works on morphological char-
acters, could reduce the number of Colletotrichum species
49.1.1 Classification, Morphology, from several hundred to 11 species. A vast majority of those
and Epidemiology species was reduced as synonymy to C. gloeosporioides.
Subsequently, based on morphological characters, species
49.1.1.1 Classification under the Genus Colletotrichum was classified into 22 species
In the first description, the genus was known as Vermicularia by Sutton (1980),6 40 by Sutton (1992),7 and 60 by Dictionary
(described by Tode in 1790). The name Colletotrichum of the Fungi.8 Recently, due to the difficulty to differenti-
was introduced by Corda in 1831.2 Traditionally, these ate the Colletotrichum species based on the morphological
fungi are included in the Form–class Coelomycete, order characters and host range (in plants), phylogenetic analysis
Melanoconiales, and are characterized by the formation on the basis of nucleic acid sequences is successfully used.
of mitosporic, asexual fruiting bodies known as acervular The need for the polyphasic approach for the identification of
conidiomata (a cup-shaped fruiting body covered with the the species under this genus has been highlighted by Cai et
sterile pigmented and unbranched hyphae called setae). The al.9 Hyde et al. provided an overview of 66 commonly known
conidiomata bearing the conidia is better seen while the fun- Colletotrichum species along with 19 doubtfully classified
gus parasites the plant tissue bearing the conidia within the species.5 In spite of many known plant pathogenic species
structures.3 The sexual state of the fungi has been identified of Colletotrichum, human infections are reported due to few
401

species only and those include C. dematium,10–15 C. coccodes sizes are described by the researchers. Colonies of putative
(reported as C. atramentarium),16,17 C. gloeosporioides,12,18,19 C. dematium strains are also variable7 (Table 49.1). According
C. graminicola,20 C. crassipes,21 C. capsici22 (currently to Sutton,6 the conidia of C. dematium are strongly curved
named as C. truncatum), C. truncatum,23 and an unknown and less than 3 μm wide and this description helps to dis-
species.24 tinguish them from C. capsici.23 By multi-locus phylogenies
and morphology, Colletotrichum capsici is synonymized
49.1.1.2 Morphology with C. truncatum and C. Curvatum.23 However, there is no
Traditionally, the identification of Colletotrichum relies on consensus among researchers working in this field on this
the morphological and cultural characteristics. The important synonymy proposed by Damm et al.5 Sutton6,7 proposed that
morphological character that helps in speciation includes size C. crassipes is similar to C. gloeosporioides, though the
and shape of condiomata, conidia, conidiophores, appresso- conidia of C. crassipes is wider and longer, and the fungus
ria, and setae on culture or on natural substrate (Figures 49.1 has lobed appressoria. The identification of C. graminicola,
and 49.2). However, the most common human pathogenic a grass-associated fungus and isolated from a case of human
species, Colletotrichum dematium, is difficult to recognize keratitis,20 is not clear due to frequent inaccurate naming for
on the basis of morphological characters, as different conidia this fungus.4
Colletotrichum species are basically plant pathogens.
Few species like C. dematium,10–15 C. coccodes (reported
as C. atramentarium),16,17 C. gloeosporioides,12,18,19
C. graminicola,20 C. crassipes,21 C. capsici22 (currently
named as C. truncatum), and C. truncatum23 are reported to
cause human infection. Among those species, Colletotrichum
capsici (C. truncatum) is known to cause anthracnose on
chili pepper, cotton, tomatoes, and a wide range of legume
(a) (b) species.27 It has also been associated with symptoms of leaf
tip die-back, foliar blight, leaf spot, leaf lesions, and boll
rot of various plants.27 Colletotrichum crassipes was origi-
nally described from fruit of Vitis vinifera (grape) from
Conegliano, Italy.5 Colletotrichum truncatum has a wide
host range and causes anthracnose diseases of many legumi-
nous and solanaceous plants.7,23,27 Colletotrichum dematium
is claimed to cause several important plant diseases, such as
(c) (d) (e) leaf blight of Japanese radish seedlings and mulberry and
cowpea anthracnose. Colletotrichum gloeosporioides is pos-
Figure 49.1 Colletotrichum truncatum (C. capsici): (a)–(b): sibly the most common and widely distributed plant patho-
Colony morphology on PDA. (c)–(d): Conidia. (e): Conidiogenous gen of the world and has been associated with at least 470
cells. Scale bars: (a)–(b) = 9 mm; (c) = 10 μm; (d)–(e) = 5 μm. different host genera. C. gloeosporioides is prevalent in the
(Photographs provided kindly by Shenoy, B.D. et al., Fungal tropics though the fungus has also been isolated from a wide
Divers., 27, 197, 2007.) range of temperate and subtropical habitats.5–7,28 C. gramini-
cola emerged as an important pathogen of corn crops during
the 1970s and 1980s. However, with the development of the
resistant cultivars the impact due to this fungus has reduced
drastically. C. graminicola is the first Colletotrichum spe-
cies to have its entire genome sequenced.5 C. coccodes is
known to cause potato black dot and anthracnose of tomato,
chili, and mint.2
The burden of human infection due to Colletotrichum
species is clearly not known due to lack of awareness of
this agent in causing disease and availability of few reports.
However, three series of mycotic keratitis reported by
Fernandez et al.,12 Kaliamurthy et al.,29 and Marangon et
al.30 had shown that Colletotrichum was the causative agent
in 2.8% (10 of 360), 1.9% (7 of 378), and 3.0% (14 of 467)
of cases, respectively. Reported human infection due to
Figure 49.2 Colletotrichum dematium. Conidiomata with Colletotrichum species is summarized in Table 49.2. Most
abundant setae and falcate conidia. patients with Colletotrichum keratitis are rural agricultural

Colletotrichum 403
Table 49.1
Characteristics Features of Colletotrichum Species Implicated in Human Infection
Species Conidial Dimensions
(Teleomorph) Colony Morphology Conidial Morphology (in μm) References
C. dematium Extremely variable, initially white to Abundant setae and black conical sclerotia; Conidia 18–26 × 2.0–3.0 [6,25]
(unknown) pale mouse gray mycelium, later conidia hyaline smooth walled, aseptate Appressoria 8.0–11.5 ×
mouse gray, felt-like, reverse black. falcate, fusiform, and gradually tapered at 6.5–8.0
each end; appressoria medium brown,
clavate to circular, margin entire and
slightly irregularly lobed aseptate, rarely
septate (Figure 49.2).
C. gloeosporioides Extremely variable, effuse, gray to Conidia born of elongated phialides in Conidia 8–11 × 3.0–4.5 [6,25,26]
(Glomerella black, with pinkish patches; reverse acervular conidiomata; conidia cylindrical, Appressoria 4.5–10 ×
cingulata) dark brown with vinaceous stains. obtuse end, and slightly tapered; 4–7.5
appressoria circular with slightly irregular,
pale to medium brown.
C. coccodes Dark brown, with black sclerotia Sclerotia setose, spherical; conidia straight Conidia 16–22 × 3–4 [6,25]
(unknown) occasionally sparse white aerial fusiform attenuated at the ends; appressoria Appressoria 11.0–16.5 ×
mycelium; conidial mass honey clavate brown. 6.0–9.5
colored; reverse dark brown.
C. graminicola Colony effuse gray to brown, with Stromata formed from dendroid brown Conidia 23–29 × 3.5–5 [6,25]
(Glomerella salmon patches of conidial slime; hyphae; setae abundant; conidia fusiform to Appressoria 17–20 ×
graminicola) reverse vinaceous to purple. falcate; appressoria brown and irregular. 12–14
C. truncatum (C. Colonies on oatmeal agar flat with entire Hyphae hyaline, septate; Conidiomata Conidia 20–23.5 × 3.5–4 [6,23,25,27]
capsici)a margin, no aerial mycelium, surface acervular; Setae hyaline to pale brown, Appressorium
(Glomerella buff, covered with olivaceous-gray to smooth to verruculose, 80–150 μm long, 6.5–13 × 5.5–7.5
truncate) iron-gray acervuli, and reverse buff to two- to five-septate, tapering toward the tip;
pale olivaceous-gray (Figure 49.1). Conidia hyaline, smooth-walled to
verruculose, aseptate, long central part of
conidia usually slightly curved with parallel
walls, and ending abruptly at the round and
truncate base, while tapering toward the
acute and more strongly curved apex;
Appressoria light to medium brown, entire
edge to lobed, outline roundish to
ellipsoidal (Figure 49.1).
C. crassipes On potato carrot agar, cottony, Setae acicular, septate, brown to dark brown, Conidia 11–18 × 6.5–8 [21]
(unknown) olivaceous brown black reverse. thick walled to dark brown; Conidia were Appressorium 18 × 10
slimy hyaline, mostly cylindrical;
Appressorium-irregular, darkbrown with
crenate or deeply lobed walls.
a C. capsici is now considered as C. truncatum.
workers. Although precedent history of injury to the eye is tissues. The predominant infection caused by this agent
obvious in the majority of cases, occasional minor unnoticed is keratitis. The major risk factor that predisposes to the
trauma may be the possible reason in a few cases. Colletotrichum keratitis is corneal trauma. Other reported
risk factors include use of corticosteroids or antiviral drugs
on eye, or the presence of diabetes mellitus. The infection
49.1.2 Pathogenesis and Clinical Features
due to this fungus has also been reported in post-cataract
Like other Coelomycetes, Colletotrichum are not ubiqui- surgery.19,20 Colletotrichum causing endophthalmitis has
tous airborne organisms, and there is no report of acquisi- been reported after subconjunctival injection of triamcino-
tion through respiratory tract. Most infections reported are lone acetonide for treatment of anterior scerelitis.31 The
due to traumatic implantation of the fungus from plant/ next group of infections caused by Colletotrichum species
wood material or the soil or acquisition through abrasions, is subcutaneous infections in the form of phaeohyphomyco-
wounds, lacerations, or other trauma. This process helps the sis among immunosuppressed patients.21,32,33 Midha et al.24
entry of the fungus in the eye and cutaneous or subcutaneous have reported a lethal case of systemic fungal infection in a

Table 49.2
List of Reported Human Infections due to Colletotrichum
S. References Species Age/
No. (Nos) Identification Sex Risk Factors Clinical Presentation
Subcutaneous infections
1 O’Quinn et al.33
Case 1 C. gloeosporioides 34/M Acute lymphocytic leukemia on A tender erythamatous nodule measuring
chemotherapy and relapse. Injury 1.2 × 1.2 cm with minimal superficial scaling
(fallen on cactus). on right forearm.
Case 2 C. coccodes 47/M Stage III non-hodgkins lymphoma of 8 Enlarged, tender, and erythematous nodule on
years duration. Undergone left arm measuring 1 × 1.5 cm with a central
autologous peripheral stem cell pustule and superficial crusting.
transplantation.
Case 3 C. coccodes 58/M Stage I non-hodgkins lymphoma, A tender erythematous papule measuring
allogenic bone marrow transplantation. 0.5 × 0.5 cm with a central pustule.
2 Guarro et al., C. gloeosporioides 56/M Diabetes mellitus. Injury by rotten Erythematous, violaceous, and tuberose
199832 wood. nodular lesions on his left forearm and elbow
measuring 1 × 3 cm in diameter.
3 Castro et al.21 C. crassipes 34/M Renal transplant recipient. Nodular and purulent cysts with thick walls on
the right leg.
Eye infections
1 Fernandez et al.12
Case 1 C. gloeosporioides 68/M IDDM NA
Case 2 Colletotrichum spp. 34/M IDDM, Trauma
Case 3 Colletotrichum spp. 78/F Corneal erosion
Case 4 C. gloeosporioides 74/M IDDM
Case 5 Colletotrichum spp. 69/M Trauma by tree branch, topical steroids
Case 6 C. dematium 40/M Sand in eye
Case 7 Colletotrichum spp. 35/M Trauma by dust and prednisolone use
Case 8 C. dematium 28/M Injury by plant liquid
Case 9 C. dematium 28/M Injury by plant liquid
Case 10 C. gloeosporioides 78/M Facial injury and exposure keratopathy
2 Kaliamurthy et al.
Case 1 C. dematium 47/M Topical chloramphenicol ointment Corneal ulcer irregular edges, stromal
infiltration, flare and cells in anterior chamber.
Case 2 C. dematium 19/M Injury by stick Paracentral ulcer with dendritic pattern, stromal
infiltrate.
Case 3 C. dematium 30/F Fall of insect, removed by finger Central corneal ulcer with stromal infiltrate.
Case 4 C. dematium 41/M Acyclovir ointment Paracentral corneal ulcer, serrated
marginstromal infiltration, and hypopyon.
Case 5 C. dematium 50/F NA Central corneal ulcer, flare.
Case 6 Colletotrichum spp. 80/F NA Central ulcer, hypopyon, and stromal
infiltratation.
Case 7 Not done 70/M Mud particles in eye Central ulcer, hypopyon, and stromal
infiltratation.
3 Yamamoto et al.19 C. gloeosporioides 82/M Myelodysplastic syndrome, cataract NA
surgery
4 Ritterband et al.20 C. graminicola 24/M Cataract surgery NA
5 Upadhyay et al.22 C. capsici NA NA NA
6 Matsuzaki et al.50
C. gloeosporioides NA Injury, topical steroid NA
C. gloeosporioides NA Injury by leaf of orange tree NA
7 Liao et al.14 C. dematium Injury NA
8 Shukla et al.18 G. lomerella cingulata Injury NA
9 Liesegang and C. coccodes
Forster17

Colletotrichum 405

List of Reported Human Infections due to Colletotrichum
S. References Species Age/
No. (Nos) Identification Sex Risk Factors Clinical Presentation
1 Joseph et al.51 C. dematium 25/F Trauma with stones Central ulcer, corneal oedema, and stromal
infiltratation.
2 Mendiratta et al.15 C. dematium 27/M Injury with soybean branch Paracentral ulcer irregular margin and leathery
slough at base and stromal infilatrate.
3 Giaconi et al.13 C. dematium 59/M Injury after work at grinding wheel, NA
topical antiviral and steroids
4 Mitani et al.52 C. gloeosporioides 80/F Traumatic injury while working in field Corneal ulcer with grayish stromal infiltrate,
indistinct margins, and Hypopyon.
5 Singh et al.31 C. dematium 38/M Acute nodular scerelitis, Fungal endophthalmitis.
subconjunctival injection of
triamcinolone acetonide
neutropenic patient possibly caused by an unidentified spe- lesion. The nodule may be solitary or multiple satellite nod-
cies of Colletotrichum. ules around primary lesion.
Colletotrichum keratitis may present with the symptoms In addition to one possible case of systemic infection
similar to other fungal keratitis like ocular pain, redness, reported by Midha et al.,24 one out of three cases of pha-
decreased vision, photophobia, and discharge. Corneal ulcer eohyphomycosis reported by O’Quinn et al.33 died due to
caused by this agent may be central or paracentral with fulminating infection in the lungs suggesting possible dis-
irregular or serrated edges with varying amounts of stro- semination due to C. coccodes.
mal infiltration and edema. Hypopyon may also be present. In general, most cases of keratitis caused by Colletotrichum
Sometimes the ulcer on presentation may exhibit dendritic species appear to respond well to the topical natamycin alone
pattern resembling viral keratitis. Like other melanized or in combination with amphotericin B. Different combina-
fungi, Colletotrichum keratitis progresses slowly and less tions of drugs like topical natamycin and oral itraconazole,
aggressively, in contrast to Aspergillus or Fusarium kerati- intravitreal amphotericin B and fluconazole, and amphotericin
tis. Macroscopic brown pigmentation of the corneal infiltrate B and topical miconazole were used successfully for the man-
may also be noticed when keratitis is caused by this mela- agement of keratitis.12,15,18–20,29,33 Kaliamurthy et al. reported
nized fungus.34 successful resolution of corneal lesion in five cases using topi-
Disease caused by Collectotrichum belongs to the group of cal natamycin and ciprofloxacin for a mean duration of 47 ± 14
infections called “phaeohyphomycosis,” the name proposed days.29 Topical antifungal alone may not be sufficient for the
by Ajello et al.35 in 1974, as the fungus produces melanin. resolution of the lesion and may require surgical procedure
A case of subcutaneous infection due to C. gloeosporioides like therapeutic keratoplasty (TPK).29 Treatment data with the
has been reported as hyalohyphomycosis by Guarro et al.,32 use of newer triazoles are not available but one case of kera-
as the hyphae were hyaline on direct microscopic examina- titis caused by C dematium was treated with topical voricon-
tion of the tissue. In such a situation, Fontana-Masson stain azole (1%). The lesion did not respond to the therapy though
may correctly identify the presence or absence of melanin. the strain was susceptible by in vitro antifungal susceptibility
The subcutaneous infection is due to the traumatic implanta- testing (1 μg/mL).30 Subcutaneous phaeohyphomycosis due to
tion of the fungus, though the history of the trauma may not C. gloeosporioides and C. coccodes was successfully treated
be elicited in all patients, especially in prolonged course of with the combination of amphotericin B and itraconazole.33
the progression of the disease. The presence of the fungus in Reports of antifungal susceptibility testing are few.
the wood and soil and frequent occurrence of lesions on the Guarro et al. tested 16 isolates of Colletotrichum (C. gloeo-
exposed part of the body supports traumatic acquisition of sporioides—7, C. coccodes—5, C. dematium—4) against
infection. The subcutaneous infection due to Colletotrichum azoles (fluconazole, itraconazole, ketoconazole, and micon-
spp. is usually seen in the warm climates and particu- azole), amphotericin B, and flucytosine and showed low
larly in immunocompromised individuals.36 Subcutaneous minimum inhibitory concentration (MIC) for all antifungal
Colletotrichum infections may present as painless or tender agents tested except flucytosine, though high MICs were
nodule on the extremities with or without obvious history expected due to the extensive use of sublethal concentration
of trauma at the site of origin. The size of the nodule may of azoles in the agricultural practice.32 Experimental demon-
vary. The overlying skin may be normal without any signs stration indicated possible additive effect while combinations
of inflammation or may be accompanied with erythematous of azoles and caspofungin were used.37

49.1.3 Diagnosis some Colletotrichum spp. may be misidentified as Fusarium

due to the close resemblance of the falcate conidia in both
49.1.3.1 Conventional Techniques these fungi. However, the conidia of Colletotrichum species
49.1.3.1.1 Keratitis are nonseptate. Further, the presence of appressoria and in
The diagnosis of keratitis due to Colletotrichum species the later stage acervulus with setae may help in identification.
includes clinical history, clinical examination, and accu-
rate identification of causative agent. Isolation of the caus- 49.1.3.2 Molecular Techniques
ative agent is important for authoritative identification and Due to the inadequacies and plasticity of morphological
appropriate antifungal therapy. The corneal scrapings should characters, nucleic acid sequence analysis has been regarded
preferably be collected by the ophthalmologists and directly as more reliable method for the identification and classifica-
inoculated at the bedside or transported to the laboratory tion of Colletotrichum species.4,7,23,26,28 Even the application
between two sterile glass slides (one for microscopy and of the molecular techniques directly on the clinical samples
the other for culture). Swabs or samples collected by labo- may help in rapid diagnosis.
ratory loop are not good specimens and should be avoided.
The direct examination of potassium hydroxide wet mount 49.1.3.2.1 Molecular Diagnosis
of the corneal scrapings helps in early diagnosis. The use The utility of polymerase chain reaction (PCR) has been
of calcofluor white stain and examination under fluorescent established for the detection of many difficult organisms
microscope would increase the sensitivity and rapidity in like Mycobacteria, Microsporidia, and Acanthamoeba
detection of fungal elements. Isolation of the fungus can be causing eye infections. But the same for the diagnosis of
attempted by inoculating the corneal scrapings on the blood Colletotrichum is not well established. Kumar and Shukla
agar/Sabourauds dextrose agar by using “C” streak. Only developed a PCR targeting internal transcribed spacer (ITS)
colonies appearing on the “C” streak should be considered regions and single-stranded conformation polymorphism of
as corneal pathogen. rRNA genes for rapid diagnosis of mycotic keratitis.38 One
of the three fungi identified by this method is Colletotrichum
49.1.3.1.2 Subcutaneous Phaeohyphomycosis state of Glomerella cingulata. In another study by Vengayil
Excised tissue or biopsy or the aspirated fluid from the lesion et al., PCR was proved not only as an effective rapid method,
are the ideal samples for the laboratory confirmation of the but also as a sensitive method for the diagnosis of fungal
infection. Direct microscopic examination of the potassium keratitis compared to KOH wet mount and Gram’s smear.39
hydroxide (KOH) or calcofluor wet mount, histopathology, However, the sample collection and DNA extraction proce-
and isolation of the fungus should be attempted from the dure may interfere with the PCR test sensitivity. To overcome
sample. The presence of pigmented or dark-colored hyphae this problem, Menassa et al. recently developed a DNA-
in the KOH wet mounts or on histopathology slides helps in stabilizing Whatman (FTA) filter paper method for specimen
the diagnosis of phaeohyphomycosis cases. However, occa- collection. It can be used as a single-step, non-nested PCR for
sionally pigment may not be noticed. Fontana-Masson stain fungal keratitis without the need of DNA extraction.40 Thus
may confirm the diagnosis in such situations. This stain spe- PCR is a promising tool for rapid diagnosis of fungal keratitis
cifically stains the melanin present in the fungal cell wall and caused by Colletotrichum spp.
appears black to brown. Various other special fungal stains
like periodic acid schiff stain (PAS) and Gomori’s methana- 49.1.3.2.2 Molecular Identification
mine silver stain (GMS) may be performed on histopathology Due to certain limitations of the morphological identifica-
sections. Materials from suspected cases should be cultured tion of Colletotrichum species, molecular techniques seem
to isolate the fungus. The fungus grows within 4–6 days. to be an important tool for species determination. The
rRNA-ITS sequencing holds the potential to identify most
49.1.3.1.3 Identification of Fungus of the clinically important species of Colletotrichum. Cano
Identification of Colletotrichum to the species level may be et al. demonstrated the utility of ITS and D1–D2 region of
important for epidemiological considerations. Appearances rRNA gene to identify the most relevant clinical species of
of conidia, appressoria, and acervulus on the routine media Colletotrichum causing human infections.41 Though the ITS
may take longer time and this may lead to difficulty in mor- region is widely sequenced region, there are some concerns to
phological identification. Cultures are usually plated onto use ITS sequence data for species identification. Crouch and
potato dextrose agar (PDA) and incubated at 25°C. Five 4 mm Beirn reported a high error rate (86%) while comparing ITS
plugs may be cut from the actively sporulating areas near sequence data within the C. graminicola species complex.4
the growing edge of a 5-day-old culture using a sterile cork Entry of sequence data under an incorrect specific name in
borer. Each plug may be placed on PDA plates and grown public domain has created more confusion. According to one
by alternating 12 h under ultraviolet light or 12 h in dark at analysis of 343 ITS sequences named C. gloeosporioides
25°C to induce characteristic morphological characters.6 (accessed on September 6, 2009) more than 86% had con-
Identification of medically important Colletotrichum species siderable evolutionary divergence from the type species of
is usually performed on the basis of morphological features C. gloeosporioides, and most likely the strains are other
as shown in Table 49.1. It is noteworthy to mention here that than Colletotrichum species.28 However, as the spectrum

Colletotrichum 407
of species of Colletotrichum causing human infection is is recovered by filtration and washed with sterile normal
expanding, it might not be possible to identify all the clini- saline. About 0.2–0.3 g of the mycelial mat is grinded in the
cally relevant species of Colletotrichum just by rRNA-ITS presence of liquid nitrogen, and the resultant powder is trans-
sequence. Thus multigene phylogenetics is employed to sys- ferred to a 1.5 mL microcentrifuge tube containing 600 μL
tematically characterize Colletotrichum species.4,23,26,42–44 of lysis buffer (100 mM Tris–HCl pH8.0, 50 mM EDTA,
Prihastuti et al. used six genes, the nuclear rDNA ITS region, 3% sodium dodecycl sulphate (SDS)). After vortexing the
partial Actin (ACT), β-tubulin (TUB2), Calmodulin (CAL), tube briefly proteinase K is added to a final concentration
Glutamine synthetase (GS), and Glyceraldehyde 3-phos- of 20 μg/mL. The tube is incubated at 56°C for 1 h. Finally,
phate dehydrogenase (GPDH) to study a few closely related DNA can be extracted using phenol:chloroform extraction
Colletotrichum species (C. gloeosporioides sensu lato) and procedure. The DNA precipitation is done with equal volume
established that species relationships could well be resolved of isopropanol in the presence of 3M sodium acetate. The
by the same procedure.26 Multigene phylogenetics is an accu- pellet is washed with 70% alcohol and dissolved in 100 μL
rate and reliable method for the diagnosis of Colletotrichum of TE (10 mM Tris–HCl pH 7.5, 1 mM EDTA), and stored at
species, but it is neither very efficient nor economical. It is −20°C for further use. Alternatively the commercial DNA
currently impractical to apply multiple gene phylogenetics to isolation kit may be used for the extraction of the DNA from
identify each clinical isolate. It is paramount that sequence the culture.
data be generated from type species and used in species com-
parisons and phylogenetic analysis. However, the ITS region
is still useful in some cases for reconstruction of interspe- 49.2.2 Detection Procedures
cific relationships, although it is not ideal for inferring intra-
49.2.2.1 PCR-ITS Region
specific relationships. Currently, ITS is the only gene region
that is available from all the ex-type or ex-epitype cultures of The rRNA gene-ITS can be amplified by PCR in the pres-
Colletotrichum species. ence of 2 mM MgCl2, 200 μM of dNTP, 0.25 μM of primer
(ITS1—GCATATCAATAAGCGGAGGAAAAG and
49.1.3.2.3 Epitypification in Colletotrichum ITS4—GGTCCGTGTTTCAAGACGG), 0.25 U of Taq
According to article 9.7 of the International Code of polymerase, and 5–10 ng of fungal genomic DNA in a total
Botanical Nomenclature (Vienna Code) volume of 10 μL. The amplification reaction is performed in
a thermalcycler. The PCR cycling conditions consist of an
An epitype is a specimen or illustration selected to serve as initial denaturation step for 5 min at 94°C, followed by 35
an interpretative type when the holotype, lectotype, or previ- cycles of denaturation at 94°C for 1 min, annealing at 55°C
ously designated neotype, or all original material associated
for 30 s and 72°C for 1 min, and a final extension step at 72°C
with a validly published name, is demonstrably ambiguous
and cannot be critically identified for purposes of the precise for 5 min.
application of the name of a taxon.45–47 When an epitype is
designated, the holotype, lectotype, or neotype that the epi- 49.2.2.2 Sequencing of rRNA-ITS Region
type supports must be explicitly cited. To extract the gene fragment, agarose gel can be excised with
It has become relatively common to epitypify fungi.27,42,46,48 a clean, sharp scalpel. To the weighed gel slice, three times
One of the major reasons to epitypify is that the type mate- the volume of buffer, Qiagen (QG) is added and incubated
rial is lost or is in poor condition. Even if the type material at 50°C for 10 min or until the gel slice is dissolved com-
is in relatively good condition, epitypification is needed for pletely. To accelerate dissolution, the gel is vortexed every
gene research. Another reason is that mycologists may have 2–3 min during incubation. One gel volume of isopropanol
seen good type material and their understanding of a taxon/ is added to the sample and mixed. The Q/A quick spin col-
genus/family may be based on literature or drawings of the umn (Qiagen, QIA quick) is placed in 2 mL collection tube.
type or representative (possibly misidentified) collections. To bind DNA, the sample is applied to the Q/A quick column
The individual understanding may therefore be questionable. and centrifuged for 1 min. The flow-through is discarded and
So epitypification can solve many taxonomic problems and the Q/A quick column is placed back into the same tube. To
stabilize the understanding of species, genera, families, or wash 0.75 mL of buffer, Qiagen (PE) is added to Q/A quick
orders in general and particularly in Colletotrichum. column and centrifuged for 1 min. The flow-through is dis-
carded. The column is centrifuged in a 2 mL collection tube
for 1 min at 13,000 rpm. To elute DNA, 30–50 μL of elution
49.2 Methods buffer (10 mM Tris–HCl pH 8.5) is added to the center of the
Q/A quick membrane and centrifuged for 1 min. The elute is
used as the purified gene product. Sequencing reactions can
Whole cell DNA from the mycelia can be extracted fol- be performed in any sequencing platform, but are more com-
lowing a slightly modified protocol of small-scale fun- monly performed with Big Dye Terminator Cycle Sequencing
gal DNA extraction method by Lee and Taylor.49 Briefly, Kit, Version 3.1/1.1 (Applied Biosystems). The sequencing
Colletotrichum species is allowed to grow on PDA at 37°C reactions may be analyzed on ABI Genetic Analyzer (Applied
on a rotary shaker at 120 rpm for 3–5 days. The mycelial mat Biosystems) or other software depending upon the sequencing

kit used. The identity of the isolate can be found by compar- 12. Fernandez, V. et al., Colletotrichum keratitis. Am. J.
ing the sequence with the deposited sequence in the public Ophthalmol., 134, 435, 2002.
databases like GenBank. 13. Giaconi, J.A. et al., Voriconazole and fungal keratitis: A report
of two treatment failures. J. Ocul. Pharmacol. Ther., 22, 437,
2006.
49.3 C
onclusion and Future 14. Liao, W.Q., Shao, J.Z., and Li, S. Q., Colletotrichum dema-
tium caused keratitis. Chinese Med. J., 96, 391, 1983.
Perspectives 15. Mendiratta, D.K. et al., Keratitis due to Colletotrichum
Colletotrichum species, basically the plant pathogen, is dematium—A case report. Indian J. Med. Microbiol., 23, 56,
recently emerging as an opportunistic agent causing eye infec- 2005.
16. Rosa, R.H. Jr., Miller, D., and Alfonso, E.C., The changing
tions and subcutaneous infections in humans. Though very
spectrum of fungal keratitis in south Florida. Ophthalmology,
few human cases are reported in the literature, exact number 101, 1005, 1994.
may be much higher due to lack of awareness and difficulty 17. Liesegang, T.J. and Forster, R.K., Spectrum of microbial kera-
in identification of these agents in the routine clinical mycol- titis in South Florida. Am. J. Ophthalmol., 90, 38, 1980.
ogy laboratory. Among many species of Colletotrichum, 18. Shukla, P.K. et al., Clinical and experimental keratitis caused
C. dematium, C. coccodes, C. gloeosporioides, C. gramini- by the Colletotrichum state of Glomerella cingulata and
cola, C. crassipes, and C. truncatum are known to cause Acrophialophora fusispora. Sabouraudia, 21, 137, 1983.
human infections. As this fungus takes longer time to pro- 19. Yamamoto, N., Matsumoto, T., and Ishibashi, Y., Fungal kera-
titis caused by Colletotrichum gloeosporioides. Cornea, 20,
duce conidia, appressoria, and acervulus on the routine cul- 902, 2001.
ture media, morphological identification becomes difficult or 20. Ritterband, D.C., Shah, M., and Seedor, J.A., Colletotrichum
delayed. Molecular techniques, especially the sequencing of graminicola: A new corneal pathogen. Cornea, 16, 362, 1997.
ITS and D1–D2 region of rDNA, may help in rapid identifi- 21. Castro, L.G. et al., Phaeohyphomycotic cyst caused by
cation of the fungi. However, many sequences deposited in Colletotrichum crassipes. J. Clin. Microbiol., 39, 2321, 2001.
GenBank are wrongly labeled due to multiple names of the 22. Upadhyay, M.P. et al., Epidemiologic characteristics, predis-
same species or misidentification of the fungus on the basis posing factors, and etiologic diagnosis of corneal ulceration in
Nepal. Am. J. Ophthalmol., 111, 92–99, 1991.
of morphological characters. So epitypification of all the
23. Damm, U. et al., Colletotrichum species with curved conidia
Colletotrichum species may resolve many taxonomic prob- from herbaceous hosts. Fungal Divers., 39, 45, 2009.
lems and may help in accurate identification. 24. Midha, N.K., Mirzanejad, Y., and Soni, M., Colletotrichum
Sp.: Plant or human pathogen? Antimicrob. Infect. Dis.
Newsl., 15, 26, 1996.
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50 Cylindrocarpon
Dongyou Liu
Contents
50.1 Introduction.......................................................................................................................................................................411
50.1.2.1 Cylindrocarpon lichenicola (Fusarium lichenicola)..............................................................................412
50.1.2.2 Cylindrocarpon destructans...................................................................................................................412
50.1.2.3 Cylindrocarpon cyanescens (Phialophora cyanescens)........................................................................412
50.1.3 Diagnosis...............................................................................................................................................................413
50.2 Methods.............................................................................................................................................................................413
50.2.2.1 Sequencing Analysis of ITS Regions......................................................................................................414
50.2.2.2 Sequencing Analysis of rRNA LSU.......................................................................................................414
50.3 Conclusion.........................................................................................................................................................................414
References...................................................................................................................................................................................415
50.1 Introduction and conidiophores are long and simple or poorly branched.
Subulate (slender and tapering to a point) conidiogenous cells
50.1.1 Classification and Morphology (phialides) (of 38–50 μm × 3–5 μm) sometimes have a distinct
The genus Cylindrocarpon (obsolete synonyms: Allantospora, collarette at the apices. Macroconidia (19.6–32 μm × 5 μm)
Coleomyces, Fusidium, and Moeszia) belongs to the family are borne singly and in clusters at the apices of phialides, pre-
Nectriaceae, order Hypocreales, class Sordariomycetes, sub- dominately three septate but occasionally up to five septate,
phylum Pezizomycotina, phylum Ascomycota, and kingdom hyaline, straight or curved, cylindrical to fusiform, rounded
Fungi. Depending upon the presence or absence of chla- at apex, and distinctly truncate at the base with offset basal
mydoconidia and microconidia, the 35 species in the genus pedicels. Septations appear as rings around the macroco-
Cylindrocarpon are arranged into four groups [1]. nidia. Microconidia may be absent or one celled if present.
Cylindrocarpon spp. inhabit a wide range of woody Chlamydospores (chlamydoconidia) (of 8–15 μm in diame-
and herbaceous plants and are commonly present in soil ter) may form in old cultures, occurring singly, in chains or in
[2]. Several Cylindrocarpon spp. also infect humans and clumps, intercalary, or terminal, and appear distinct hyaline
animals, resulting in keratitis, mycetoma, athlete’s foot, to pale brown, smooth to spinulose, globose cells within the
peritonitis, and disseminated infection in human patients multicellular macroconidia. C. lichenicola grows poorly at
[3]. The human-infecting species include Cylindrocarpon 35°C and shows no growth at 42°C [7].
cyanescens (obsolete synonym: Phialophora cyanescens), Cylindrocarpon lichenicola (Fusarium lichenicola) pro-
Cylindrocarpon destructans, Cylindrocarpon lichenicola duces relatively long and narrow, several-celled, cylindri-
(synonym: Fusarium lichenicola; obsolete synonym: C. cal conidia with typical rounded apices, which deviate only
tonkinense), and Cylindrocarpon vaginae [4–6]. slightly from the macroconidia of other Cylindrocarpon spe-
Morphologically, all Cylindrocarpon species produce cies with straight conidia by having a conspicuously protuber-
slimy macroconidia in basipetal succession (the youngest ant, symmetrical, truncate base. The type species of the genus
conidium at the base), which do not adhere in chains. At Cylindrocarpon, Cylindrocarpon cylindroides (teleomorph
the species level, Cylindrocarpon lichenicola (synonym: Neonectria neomacrospora) is phylogenetically distinct from
Fusarium lichenicola) colonies grow rapidly on potato flakes C. lichenicola (F. lichenicola) and other members of the genus
agar (PFA), reaching a diameter of 35 mm in 6 days at 25°C. Fusarium. The medically important and plant-pathogenic
Colonies are velvety to floccose, initially white, then yel- Cylindrocarpon destructans (teleomorph Neonectria radici-
low, and finally pale brown at maturity. The reverse ranges cola) also clusters phylogenetically in a group of organisms
from buff at the periphery to a darker brown centrally, with well segregated from the F. solani and Gibberella clades [6].
a brown-diffusing pigment. Hyphae are septate and hyaline, Cylindrocarpon (Phialophora) cyanescens is characterized
411

by its phialidic conidia, chlamydospores in aggregations, and molds rarely produce conidia in tissues and only in lesions
intense diffusing pigment [8]. exposed to ambient air, the presence of the fungus on the
Cylindrocarpon species resemble Fusarium species cutaneous surface and therefore exposure to ambient air
morphologically and taxonomically, with both sharing in this case may have allowed the Cylindrocarpon organ-
teleomorphs in the genus Nectria; and Cylindrocarpon isms to produce conidial structures. Multiple positive fun-
differs from Fusarium by lacking an asymmetrical foot gal cultures from the biopsied tissue permitted subsequent
cell on the macroconidia [9]. Through sequencing analy- microscopic and macroscopic identification of the fungus
sis of the ribosomal large subunit (LSU), it was shown as C. lichenicola. The infection resolved following marrow
recently that despite their deviating conidial morphologies, regeneration, aggressive debridement of the affected tissue,
Cylindrocarpon lichenicola and Acremonium falciforme and treatment with amphotericin B.
constitute members of the Fusarium solani species complex Kaliamurthy et al. [17] described a fungal keratitis due
(i.e., the F. solani clade containing F. solani, Haematonectria, to Cylindrocarpon (Fusarium) lichenicola in a farmer,
and Neocosmospora) [10]. Thus, the original name Fusarium who presented with pain, redness, and irritation of the left
lichenicola is reestablished for Cylindrocarpon lichenicola, eye following ocular injury caused by hay 5 days earlier.
and the new combination Fusarium falciforme is proposed Slit-lamp examination of the affected eye showed a cor-
for Acremonium falciforme [6]. neal ulcer (6 × 5 mm) with irregular margins, raised, with
While F. lichenicola, F. falciforme, and members of the necrotic slough and infiltration. The lens was cataractous
F. solani clade all produce long, slender, cylindrical conidio- and visual acuity was restricted to “hand movements” only.
phores and integrated, terminal phialides, clearly reflecting Scrapings from the base and edges of the corneal ulcer con-
their biological unity, other Fusarium groups tend to form tained septate fungal hyphae as examined by microscopy
shorter, discrete monophialides (phialides with only a single after staining with lactophenol cotton blue and Gram stain.
fertile opening) that are subulate (awl or candle shaped, i.e., Histopathological examination of the infected corneal button
rigid looking, tapered, and narrow at the apex) or inflated, removed at therapeutic penetrating keratoplasty (TPK) also
or form polyphialides (with each phialide containing several showed septate fungal hyphae. Scrapings and corneal but-
fertile openings). In addition, F. lichenicola, F. falciforme, ton are inoculated onto Sabouraud glucose-neopeptone agar,
and F. solani have two- to several-celled conidia that are and sheep blood agar grew cottony, white-red fungal colo-
significantly blunter at the apex and proportionately broader nies within 48 h, with brown pigmentation on reverse. The
than the macroconidia of fusaria in the Gibberella clade. fungus was identified as Cylindrocarpon lichenicola since
However, F. solani conidia demonstrate a classical fusarial it displayed branched, septate, hyaline hyphae; phialides on
macroconidial form (curved phragmoconidia with somewhat simple or sparsely branched conidiophores; cylindrical to
pointed apices and basal foot cells), whereas F. lichenicola fusiform, smooth-walled macroconidia, each with three to
conidia do not; F. falciforme conidia may vary from strain six septa, a blunt rounded apex, and distinctly truncate base;
to strain. Furthermore, despite showing varied colony color- and smooth-walled chlamydoconidia on short branches.
ations, F. lichenicola, F. falciforme, and F. solani colonies
show similar chestnut red-brown to purplish reverse pig- 50.1.2.2 Cylindrocarpon destructans
ments. The violet-purple reverse color in F. falciforme over- Cylindrocarpon destructans is one of the fungal species
laps with that in some F. solani isolates [6]. (e.g., Acremonium [Fusarium] falciforme, Acremonium kil-
iense, Acremonium recifei, Cylindrocarpon destructans,
50.1.2 Clinical Features Fusarium moniliforme, Fusarium solani, Neotestudina
rosatii, Polycytella hominis, and Pseudallescheria boydii)
50.1.2.1 C ylindrocarpon lichenicola that are known to cause white grain eumycetoma. Being
(Fusarium lichenicola) a slowly progressing infection, eumycetoma develops in
Resembling Fusarium solani, a common agent of keratomy- individuals usually after traumatic implantation of fungus-
coses and disseminated disease in neutropenic and/or immu- contaminated plant material. Zoutman and Sigler [18] docu-
nocompromised hosts, C. lichenicola (F. lichenicola) has mented a Cylindrocarpon destructans-related eumycetoma
been shown to cause keratitis, localized cutaneous invasive in a 39-year-old male, originally from Antigua, West Indies.
disease, disseminated infection, and peritonitis in humans The patient presented with a 12-year history of swelling of
[3,11–16]. Patients from areas with warm climates may the left foot with pus and grains expressed from a draining
develop a distinctive fusarial intertrigo caused by Fusarium sinus tract, from which Cylindrocarpon destructans was iso-
solani, Cylindrocarpon lichenicola (Fusarium lichenicola), lated in pure culture. The fungus was identified by its micro-
or Fusarium oxysporum [6]. conidial morphology, the presence of chlamydospores, and
Iwen et al. [7] reported a case of trauma-related invasive an intense brown diffusible pigment.
Cylindrocarpon lichenicola infection localized to the right
hand in a 53-year-old male patient with acute myelogenous 50.1.2.3 C
ylindrocarpon cyanescens
leukemia (AML). Histological examination of skin biopsy (Phialophora cyanescens)
showed septate, branching hyphae along with globular struc- Cylindrocarpon (Phialophora) cyanescens has also
tures (8–11 μm in diameter) resembling conidia. As invasive been shown to cause white grain eumycetoma [5,18].

Cylindrocarpon 413
Cylindrocarpon cyanescens produces cylindrical phialides absent in C. lichenicola), and its macroconidia-forming
with nonflaring collarettes. Hemashettar et al. [19] reported a conidiophores are somewhat shorter. On the other hand, the
case of white grain eumycetoma on the foot of a 57-year-old macroconidia-forming conidiophores in C. lichenicola are
Indian male, who sustained a trauma by a thorn 10–12 years typically elongate. The conidiophores in A. falciforme may
earlier and developed a pustule at the site after thorn removal. be similar in shape to the conidiophores of other members
The pustule discharged serosanguineous fluid and eventually in the F. solani clade but are relatively strongly septate [6].
healed. Then, the patient was admitted to the hospital with Cylindrocarpon (Fusarium) lichenicola is further differ-
slight swelling of the left foot, pain, and draining sinuses. entiated macroscopically from F. solani by forming macro-
Histologic examination of a biopsy tissue specimen revealed conidia, which are predominately straight rather than curved,
oval, lobular, white granules (0.5–1.0 mm in diameter) com- by having apical cells that are rounded rather than tapering,
posed of hyaline, septate hyphae and thick-walled chlamydo- by having basal cells with truncate and offset rather than
spores (up to 15 μm in diameter). Culture of granules from a attenuated pedicels (foot cells), by lacking microconidia, by
draining sinus on Sabouraud dextrose agar (SDA) with chlor- having pigmented chlamydoconidia, and by having a brown
amphenicol (SDA+C) at room temperature (25°C–30°C) after color rather than cream color on reverse of SDA medium [6].
12 days produced compact, very-slow-growing, and poorly Granules produced by Cylindrocarpon species cannot be
sporulating colonies with a strong reddish brown pigment distinguished from each other, nor from those of Acremonium
that diffused into the medium. Colonies on Sabouraud dex- spp. and Fusarium verticillioides (F. moniliforme) without
trose agar were raised in the center, downy, and white at first, cultural or immunofluorescence studies. However, granules
becoming grayish buff after 2 weeks, measuring 6–7.5 mm in of Cylindrocarpon species may be differentiated from those
diameter at 25°C and 3.5–4.5 mm in diameter at 37°C. The of P. boydii by having less prominent, fewer, and smaller
isolate failed to grow at 40°C. Microscopic examination of chlamydospores.
the slide culture preparations on potato dextrose agar after 3 Molecular methods have offered an alternative means to
weeks of incubation at 25°C showed sterile, septate, hyaline identify fungal organisms including Cylindrocarpon species.
hyphae (2.5–5.0 μm wide), producing chlamydospores but Amplification and sequencing analysis of the internal tran-
devoid of conidia. Conidiation occurred sparsely and best scribed spacer (ITS) ITS1-5.8S-ITS2 region allows differen-
on Takashio and oatmeal salts agars after prolonged incu- tiation of Cylindrocarpon species from other fungi [21–26].
bation (3–14 weeks at room temperature) and consisted of
zero- to one-septate, cylindrical, sometimes slightly curved
conidia produced from unbranched septate phialides with- 50.2 Methods
out collarettes. Single-celled conidia measured 9–14 μm
long and 2–2.5 μm wide, and two-celled conidia measured
17–25 μm long × 2–3 μm wide. Chlamydospores were mostly Clinical samples are cultured on Sabouraud dextrose agar or
solitary, intercalary, or terminal and were subglobose and other mycological media. Portions of samples are examined
smooth to slightly roughened. The fungus was identified as under microscope for mycotic elements using fungal stains.
a Cylindrocarpon sp. on the basis of the development of rare Colonies are grown on synthetic nutrient agar, oatmeal agar,
cylindrical conidia borne from solitary phialides lacking col- and malt extract agar for 7–10 days at room temperature
larettes, in addition to chlamydospores formed singly or in (22°C). To induce conidiation, isolate is subcultured onto
short chains. It appeared highly similar to C. cyanescens in PFA plates and PFA slide cultures, both incubated at 25°C.
its strong production of reddish brown diffusible pigment and The microscopic morphology is examined from colonies on
restricted growth. While C. cyanescens did not invade bone, PFA plates and slide culture mounts after 6 days of incuba-
C. destructans caused lytic bone destruction of the tarsals tion. Temperature studies are performed on PFA slants to
and the base of the third metatarsal. evaluate the growth rate at 25°C, 35°C, and 42°C [7].
For fungal DNA extraction, 50 mL of Sabouraud dextrose
(SAB) broth is inoculated by needle with conidia from a
50.1.3 Diagnosis
7 day culture in SAB agar and incubated for 72 h at 30°C.
Like their Fusarium relatives, Cylindrocarpon species are The hyphae are recovered on a 0.45 μm-pore-size filter and
anamorphs of ascomycetes belonging to the Hypocreaceae washed with sterile saline. Aliquots of the fungal hyphae are
(teleomorph Nectria) and are cosmopolitan soil- and plant- stored frozen at −70°C until use. Prior to lysis, the hyphae
associated fungi. Rapid and accurate determination of are thawed and suspended in 400 μL of DNA extraction buf-
Cylindrocarpon species is vital for implementing appropri- fer (1 mM EDTA pH 8.0, 1% sodium dodecyl sulfate, 10 mM
ate treatment and control strategies for these organisms [20]. Tris–HCl pH 7.6, 100 mM NaCl, and 2% Triton X-100).
Both Cylindrocarpon (Fusarium) lichenicola and mem- Microcentrifuge tubes (1.5 mL) containing hyphae and buf-
bers of the F. solani clade produce elongate, filiform, often fer are sonicated in a water bath (Branson; model 2210) for
several-celled conidiophores incorporating terminal mono- 15 min, followed by heating at 100°C for 5 min. Following
phialides (up to 40 μm in length) with a relatively broad, lysis, DNA is purified using the QIAmp blood kit (Qiagen)
blunt apex. The elongate conidiophores in F. solani isolates and protocols for crude cell lysates supplied by the manufac-
are mainly associated with microconidia (which are often turer. The purified DNA is stored at 4°C until tested [6].

Alternatively, DNA is extracted with a FastDNA kit or partial ITS sequences available in GenBank.
(Qbiogene) from mycelium grown for 3–5 days in liquid Comparison of sequences from referenced iso-
complete medium. lates, clinical isolates, and GenBank sequences is
performed using a nongapped, advanced BLAST
search. The similarities of the sequences are deter-
50.2.2 Detection Procedures mined with the expectation frequency minimized to
50.2.2.1 Sequencing Analysis of ITS Regions 0.0001 [28].
Iwen et al. [7] described the use of primers ITS1 (5′-TCC 50.2.2.2 Sequencing Analysis of rRNA LSU
GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC
Summerbell and Schroers [6] employed primers V9G and
GCT TAT TGA TAT G-3′) from the conserved regions of the
LR5 to amplify the LSU region (encompassing domain D1
18S (ITS1) and the 28S (ITS4) ribosomal RNA (rRNA) genes,
and most of domain D2, nucleotides 116–615) of rRNA
respectively, to amplify the intervening 5.8S rRNA gene,
followed by sequencing for identification and phylogenetic
ITS1 region, and ITS2 noncoding regions [27]. Subsequent
analysis of Cylindrocarpon and Fusarium species.
sequencing analysis of the resulting amplicon enables identi-
The PCR cycling program consists of an initial denatur-
fication of Cylindrocarpon species.
ation at 94°C for 1 min; 35 cycles of 94°C for 35 s, 55°C for
Procedure 50 s, and 72°C for 3 min; and a final elongation at 72°C for
6 min. (final elongation). The PCR products are purified with
1. PCR mixture (50 μL) is made up of 5 μL sam- a GFX purification kit (Amersham Pharmacia Biotech) and
ple DNA, 20 mM Tris–HCl pH 8.4, and 50 mM visualized on an electrophoresis gel after ethidium bromide
KCl; 0.1 mM (each) dNTP, 1.5 mM MgCl2, and staining. The rRNA is sequenced with a BigDye terminator
0.3 μM (each) primer; and 1.5 U of Platinum Taq cycle sequencing kit (Applied Biosystems) and analyzed on
high-fidelity DNA polymerase (Gibco BRL, Life an ABI Prism 3700 instrument (Applied Biosystems). The
Technologies). primers used in the sequence reaction are ITS1 and ITS4,
2. Amplification is conducted in a Stratagene NL1 and NL4, and LR5.
Robocycler model 96 thermocycler with initial Sequence chromatographs are assembled and edited with
denaturation at 95°C for 4.5 min; 40 cycles of 95°C SeqmanII software (DNAStar) and aligned with sequences
for 30 s, 50°C for 30 s, and 72°C for 1 min; and a downloaded from GenBank. The alignment is initially per-
final extension at 72°C for 3 min. formed with the ClustalX program (version 1.8; ftp://ftp-
3. Amplicons are separated by agarose gel electropho- igbmc.u-strasbg.fr/pub/ClustalX) and adjusted manually with
resis, purified, and ligated into the PCR 2.1 plas- the Megalign program (DNAStar). The phylogenetic analysis
mid vector (Invitrogen). Competent INVαF′ One is performed with a part of the LSU rRNA available for all
Shot cells are transformed using standard protocols. accessions. A parsimony analysis is performed with the soft-
Colonies are isolated and purified with a Qiagen ware package PAUP (version 4.0b8). Gaps are coded as miss-
miniprep spin kit. An aliquot of purified plasmid is ing data; characters are defined as unordered and equally
digested with EcoRI endonuclease (New England weighted; characters not informative to the parsimony analy-
Biolabs) and screened by agarose gel electrophoresis are excluded; heuristic searches of parsimonious trees are
sis for a 300 bp doublet, indicating the presence of performed for all sequences with random sequence addition
the EcoRI cleavage site GAATTC within the 5.8S and 1000 replicates, using the starting trees from the step-
sequence. wise addition, tree bisection–reconnection as the swapping
4. Selected plasmids are analyzed by automated dye algorithm, and all optimal trees for the next swapping round;
termination sequencing procedure on a Perkin- and branch robustness is tested by the use of 100 replications
Elmer/ABI model 373 DNA sequencer. For the of such searches based on bootstrapped data sets with ran-
sequencing of cloned fragments, both strands of the dom sequence addition and 10 replicates per search.
plasmid containing the fungal insert are sequenced
with universal M13 forward and reverse sequencing
50.3 Conclusion
primers. For direct sequencing of noncloned ampli-
cons, PCR products are directly sequenced using Cylindrocarpon spp. are common plant pathogens that are
the ITS1 and ITS4 PCR primers. widely distributed in the environment. Some Cylindrocarpon
5. The resultant nucleotide sequences are aligned with spp. may infect humans and animals occasionally, result-
the MacVector sequence analysis software, version ing in keratitis, mycetoma, athlete’s foot, peritonitis, and
6.5 (Oxford Molecular Group, Inc., Campbell, CA), disseminated infection. Among the human-infecting
and the Clustal W alignment algorithm. Intraspecies Cylindrocarpon species, Cylindrocarpon (Fusarium)
sequence similarity and variation are determined by lichenicola is the only one known to induce invasive dis-
the MacVector software and are visually confirmed eases besides localized infections, whereas Cylindrocarpon
using pairwise nucleotide alignments. Sequences cyanescens (obsolete synonym: Phialophora cyanescens)
from referenced isolates are aligned to complete and Cylindrocarpon destructans are mainly responsible

Cylindrocarpon 415
for white grain eumycetoma. As conventional laboratory 13. Sharma R et al. Peritonitis in continuous ambulatory peri-
identification of Cylindrocarpon species is largely based on toneal dialysis due to Cylindrocarpon lichenicola infection.
macroscopic and microscopic characteristics of these organ- Nephrol Dial Transplant. 1998;13:2662–2664.
14. Mangiaterra M et al. Keratomycosis caused by Cylindrocarpon
isms, it is not only time-consuming but also technically chal-
lichenicola. Med Mycol. 2001;39:143–145.
lenging. Application of molecular techniques such as PCR 15. Rodríguez-Villalobos H et al. Disseminated infection due
and sequencing analysis of rRNA genes and ITS regions to Cylindrocarpon (Fusarium) lichenicola in a neutro-
offers a rapid, precise, and sensitive tool for Cylindrocarpon penic patient with acute leukaemia: Report of a case and
determination. review of the literature. Eur J Clin Microbiol Infect Dis.
2003;22(1):62–65.
16. Chazan B et al. Mycetoma of the foot caused by Cylindro
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lings of Phellodendron amurense in Japan. Mycologia 1855–1859.
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3. Booth C, Clayton CY, Usherwood M. Cylindrocarpon species Cylindrocarpon sp. in India. J Clin Microbiol. 2000;38(11):
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1997;34:65–67. J Clin Microbiol. 2000;38:1510–1515.

51 Fusarium
Palanisamy Manikandan, László Galgóczy, Kanesan Panneer Selvam,
Coimbatore Subramanian Shobana, Sándor Kocsubé, Csaba Vágvölgyi,
Venkatapathy Narendran, and László Kredics
Contents
51.1 Introduction.......................................................................................................................................................................417
51.1.1 Classification..........................................................................................................................................................417
51.1.2 Epidemiology.........................................................................................................................................................418
51.1.4 Diagnosis.............................................................................................................................................................. 424
51.2 Methods............................................................................................................................................................................ 425
51.2.2.1 Morphological Identification of Fusarium Species............................................................................... 425
51.2.2.2 Molecular Identification of Fusarium Species...................................................................................... 425
References.................................................................................................................................................................................. 428
51.1 Introduction In humans, Fusarium species cause four patterns of inva-

sive infections predominantly in immunocompromised
Fusarium species are widely distributed filamentous fungi patients2,12–14: refractory fever of unknown origin, sinopulmo-
that commonly occur in soil, water environments, and plants. nary infection, disseminated infection, and a variety of focal
The genus includes both plant pathogenic and soil sapro- single-organ infections15 such as ocular infections, usually
phytic representatives.1 During the past decades, Fusarium keratitis or endophthalmitis.16–18 The mortality rate is greater
species have emerged as increasingly important causal agents than 70% in systemic infections.9 In addition, an array of sec-
for opportunistic infections in human population.2 ondary metabolites is produced by Fusarium spp., which are
associated with cancer and other growth defects in humans
and animals. Interestingly, some of these secondary metabo-
lites are used commercially either directly or as the starting
Fusarium, first described by Link in 1809,3 is a genus clas- material for chemical synthesis of plant and animal growth
sified under the order Hypocreales, class hyphomycetes promoters in both first-world and second-world settings,
(Ascomycetes). Formerly included in the Deuteromycetes, and the mycotoxins produced by some of these fungi were
the genus contains about 100 recognized species,4 many of reported to be used as biological weapons.19–23 Few species
which can induce diseases in several agriculturally impor- such as F. pallidoroseum can be directly used as a mycoher-
tant crops and cause diseases in humans and domestic ani- bicide against water hyacinth.24
mals.5–7 Fusarium spp. are ubiquitous filamentous fungi in The Fusarium classification system is developed mainly
all major geographic regions of the world and are routinely based on the morphology of the conidia produced by the
isolated from environmental sources such as soil, plant roots, representatives of the genus. Macroscopic and microscopic
plant debris, and water systems.2,8 Most of the species of features, such as color of the colony; length and shape of the
the genus have both an anamorphic (asexual) and teleomor- macroconidia; the number, shape, and arrangement of micro-
phic (sexual) life cycle and exist in soils as saprophytes or conidia; and the presence or absence of chlamydospores
“chlamydospores.”4 are the key features for the differentiation of Fusarium spp.,
As a plant pathogen, Fusarium has been reported as the and the main taxonomic systems have organized sections
causal agent of major devastating diseases in most economi- from the species sharing common morphological charac-
cally important plants, e.g., banana,9,10 wheat, and barley.11 teristics (Figures 51.1 through 51.3). However, speciation
417

may be difficult due to the variability between isolates and

because the features that are required are not always well
developed. This would best describe why although more than
100 species have been recognized so far based on the various
identification concepts, only 70 species are well described.25
The morphologically similar Fusarium species are
grouped together as species complexes (SCs). For instance,
the Fusarium solani species complex (FSSC) includes mor-
phologically identical isolates. Further genotypic character-
ization of isolates from this complex is laborious and usually
not routinely performed in clinical laboratories. Members of
this SC are usually reported in the literature as Fusarium
solani.26 During the past few decades, there are controversies
amongst researchers that the taxonomic system based on the
Figure 51.1 Colony morphology of Fusarium (white to violet sections sharing common morphological characteristics is of
color) from patients with Fusarium keratitis on PDA. poor reliability compared to advanced molecular investiga-
tion methods such as random amplified polymorphic DNA
(RAPD), amplified fragment length polymorphism (AFLP),
restriction fragment length polymorphism (RFLP), and DNA
sequencing.9 For instance, O’Donnell et al. reported that the
Gibberella fujikuroi species complex (GFSC) revealed 45
species using molecular methods for several gene targets, of
which 23 were never reported before.27 From the clinical point
of view, identification at the species level is important for epi-
demiological studies and may also be very important because
some new antifungal agents exhibit variable activities against
Fusarium isolates belonging to different species.28,29
Together, in spite of the key work done by Wollenweber
and Reinking30 for developing a main taxonomic system, and
the contributions during the last few decades by a significant
number of scientists in Fusarium taxonomy, the systematic
classification of Fusarium species has still a number of open
question marks that need to be solved.9
Figure 51.2 Differential interference contrast microphoto- 51.1.2 Epidemiology

graph showing chlamydospores produced by Fusarium solani
(magnification 40×). A variety of human and animal infections due to Fusarium
spp. are widely reported from all over the world as the genus
does not require any strict environmental conditions for sur-
vival. For instance, Fusarium can be recovered from diverse
sources including soil, decomposing organic matter, plant
roots, woody plants, trees, agricultural, and non-agricultural
substrates and are found in various geographic regions with
hot, temperate, or cool climates. Fusarium infections are
being increasingly recognized as life-threatening mycotic
infections worldwide.31–33 The first documented case of dis-
seminated invasive Fusarium infection was reported in
1973 in a child with acute leukaemia.34 Infections caused by
Fusarium species in different organs are collectively known
as fusariosis. The epidemiological distribution of fusariosis
is controversial,35 as 85% of the cases until the year 2000
were reported from Western and Mediterranean countries
such as United States, France, Italy, and Brazil,35 and a large
number of institutions in the world have only occasionally
or have never documented invasive Fusarium infections.35
Figure 51.3 Gram smear showing macroconidia from corneal The distribution of these cases only among such geographi-
scraping of patients with Fusarium keratitis. cal areas cannot yet be ruled out. Among the well-described

Fusarium 419
70 Fusarium spp., the taxa implicated in almost half (50%) In the retrospective studies available, the proportion of
of the disease cases include F. solani, followed by F. oxys- farmers and agricultural workers among the affected patients
porum (14%), F. verticillioides (11%), F. moniliforme (10%), was relatively high (16%–86%). Male patients were gener-
and F. proliferatum (5%). Further common human pathogens ally more frequent than females: male:female ratios were
are F. dimerum, F. chlamydosporum, F. nygamai, F. napi- between 1.4:1 and 3.5:1, with the exception of a study from
forme, F. semitectum, and F. equiseti.35 Nepal, where both sexes were equally affected.56 The average
It is likely that Fusarium spp. colonize patients prior to age of the patients in the studies ranged from 35.8 to 59 years.
hospital admission and the subsequent immunosuppres- Among the factors predisposing for keratomycosis, cor-
sion and neutropenia could then result in a variety of infec- neal trauma is considered the most common, with an inci-
tions.31,36 Although skin, blood, lung, and sinus infections are dence between 31.6% and 89.9%, apart from a study from
most common, other body organ systems can also be affected. Pennsylvania, in which only 8.3% of the patients reported
Fusarium species have been documented as etiological agents a recent trauma.78 The reported injuries were caused by dif-
in localized tissue infections, including keratitis, endophthal- ferent traumatizing agents including plant material (corn
mitis, septic arthritis, cystitis, peritonitis, brain abscesses, and stalks, grass, ground nuts, hay, kernel, onions, paddy, palm
breast abscess.37–39 F. solani breast abscess was reported from leaf, sugar cane, thorn, tree branch, and vegetable matter),
India in an uncontrolled diabetic patient who was a paddy animal matter (cat scratch, cow dung, cow’s tail, hair, hen
grower.38 Disseminated fusariosis with heavy involvement of peck, and insects), chemical gas, dust, fingernails, electric
liver and lungs was reported in a healthy farmer.40 Although welding light, glass, heat injury, metal objects, mud, soil,
infrequent, post-transplant invasive fusariosis is reported in stones, or physical violence. In Tanzania, positive correlation
Asian countries like India.41 In systemic fusariosis, Fusarium was found with HIV carriage: 81.2% of patients with fungal
solani is the predominantly isolated taxon, followed by keratitis were HIV positive.14 Further predisposing factors
F. moniliforme, F. oxysporum, and F. proliferatum.31 include preexisting ocular diseases (e.g., atopic conjunctivi-
Fusarium species are isolated and reported in large num- tis, chronic dacryocystitis, dry eyes, lagophthalmos, recurrent
bers from eye infections in developing countries such as corneal erosion, corneal scaring, or ulcer), the use of topical
India.42 Based on the reported case numbers, keratomycosis is corticosteroids, previous eye surgery, systemic diseases (e.g.,
the most frequent human infection caused by Fusarium. Most diabetes mellitus, leprosy, or rheumatoid arthritis), as well as
of the studies about fungal keratitis report the occurrence of contact lens wear. During the past decade, the epidemiology
Fusarium at the genus level only; however, identification of the of contact lens solution-related Fusarium keratitis has been
Fusarium isolates at the species level would be of great impor- studied in detail.
tance as it is gaining consequences on clinical outcome.43 Risk factors associated with microbial keratitis in con-
The literature provides a large amount of data about the epi- tact lens wearers include continuous overnight wear, lower
demiology of Fusarium keratitis. The incidence and epidemi- socioeconomic class, smoking, hypoxia, poor lens hygiene
ological pattern of Fusarium spp. among culture-proven cases practice, blepharitis, diabetes mellitus, epithelial trauma,
of keratomycosis is different from country to country (Tables and steroid use, specifically in daily wear lenses.110 Contact
51.1 and 51.2).2,5,14,44–107 According to a series of retrospective lens wearers have an elevated risk for fungal keratitis, but
studies, Fusarium spp. can be the predominant causal agents the incidence of Fusarium keratitis is quite rare among this
of keratomycosis5,14,45,46,53–55,58–62,65–67,70,73,76,79,80,82,86,87,91,92,97,100 group of patients. There has been no report until the begin-
besides Aspergillus spp.,47,51,56,71,83,84,88,94–96,98,99,102–104,106 ning of 2006 on the association of multipurpose contact lens
Candida spp.,44,68,78,83 and Acremonium spp.74 Tropical and solutions with fungal keratitis. Before June 2005, the num-
subtropical countries are the most affected, indicating that ber of cases with contact lens-related fungal keratitis was
climate plays an important role in determining the predomi- about three per year in Hong Kong. In late August 2005, the
nance of certain species in fungal keratitis. Certain regions Centre for Health Protection (CHP), Department of Health
of different continents, e.g., southern Florida, Ghana, and of Hong Kong, initiated an investigation because of the sud-
southern India, have similar climates that are favoring the den increase in the number of cases of contact lens-related
predominance of Fusarium spp.55,79,86,100 The incidence of microbial keratitis. The initial investigation suggested that
Fusarium spp. in keratomycosis may also vary with cli- many fungal keratitis cases were caused by Fusarium spp.
matic conditions within a single country, e.g., between dif- Great majority of the patients were disposable contact lens
ferent parts of China,59,60,62,63 Ghana,55 and India (Table users and had reported of using a commercial multipur-
51.2).55,86,87,88,94,98–100,103,104 Keratitis caused by Fusarium is less pose contact lens solution, namely Bausch & Lomb ReNu.
frequent in regions with temperate climates like European Up until May 31, 2006, a total of 33 cases of contact lens-
countries: only four cases have been reported from Paris, related Fusarium keratitis were reported to the CHP. Sixty-
France, in 8 years,68 and only a single case has been diag- four percent (21/33) were female, and the age range of all
nosed in Hungary.108 The most frequent taxon of Fusarium cases was 16–51 years (mean 28 years). In a retrospective
reported from corneal infections is the FSSC.14,58,63,64,79,86,87,109 unmatched case–control study, Ma et al.111 focused on the
Fusarium keratitis may occur as a mixed infection risk factors playing a role in the development of Fusarium
with bacteria, mainly Streptococcus and Staphylococcus keratitis among disposable soft contact lens users. They suc-
spp.73,74,86,91,109 or herpes simplex virus.109 cessfully interviewed 32 patients, and they choose 24 from

TABLE 51.1
Incidence of Fusarium spp. among Culture-Proven Cases of Fungal Keratitis (Based on
Literature Search)
Number of Number of
Country Studied Period of Study Fungal Ulcers Fusarium Isolated Reference
Australia July 1996–May 2004 35 5 (14.3%) [44]
Australia 1998–2008 16 8 (50.0%) [45]
Australia October 1999–September 2004 13 7 (53.8%) [46]
Bangladesh 11 months 51 10 (19.6%) [47]
Bangladesh 1987 7 1 (14.0%) [48]
Bangladesh 1991 107 NA (28%) [49]
Bangladesh Unknown 63 22 (34.9%) [50]
Brazil 1975–2003 265 137 (58.8%) [51]
Brazil 1983–1997 49 12 (32.0%) [52]
Brazil January 1994–December 1999 20 12 (60.0%) [53]
Brazil 2000–2004 66 44 (66.7%) [54]
Ghana, Accra (south) June 1999–May 2001 43 27 (63.0%) [55]
Nepal 1985–1987 68 8 (11.8%) [56]
Nepal August 1998–July 2001 145 45 (22.0%) [57]
Thailand (central) January 1988–December 2000 35 12 (34.3%) [5]
Nigeria 1974–1977 42 14 (33.3%) [58]
Tanzania October 1994–October 1995 32 24 (75.0%) [14]
China, Beijing (north) January1995–October 2000 498 321 (64.5%) [59]
China, Zhengzhou (central) January 1975–June 1997 615 NA (65.0%) [60]
China, Zhengzhou (central) January 2000–March 2009 1458 076 (73.8%) [61]
China, Quingdao (north) January 1996–December 1999 97 63 (64.9%) [62]
China, North January 1999–December 2004 596 437 (73.3%) [63]
China, North January 2001–December 2006 549 426 (77.6%) [64]
China 1989–2000 775 455 (58.7%) [65]
China January 2001–December 2004 681 394 (57.9%) [66]
China, Zhejiang September 2002–July 2004 61 33 (54.1%) [67]
France, Paris January 1993–January 2001 19 4 (21.1%) [68]
Iran May 2004–March 2005 7 1 (14.3%) [69]
Iran 1998–1999 29 10 (34.5%) [70]
Iran 1982–2001 27 2 (7.4%) [71]
Malaysia January 2004–April 2005 4 2 (50.0%) [72]
Paraguay April 1988–April 1989 26 11 (42.3%) [73]
Paraguay 1988–2001 136 41 (15.0%) [74]
Paraguay (children) 1988–2002 35 5 (14.3%) [75]
Singapore January 1991–December 1995 29 15 (52.0%) [76]
Thailand January 2001–December 2004 49 13 (26.5%) [77]
Pennsylvania, USA January 1991–March 1999 24 6 (25.0%) [78]
South Florida, USA January 1982–January 1992 127 79 (62.2%) [79]
South Florida, USA January 1969–December 1977 133 82 (61.6%) [80]
Florida, USA January 2004–December 2005 122 66 (54.1%) [81]
Florida, USA January 1999–June 2006 59 24 (40.7%) [82]
Minneapolis, USA January 1971–January 1981 19 3 (15.7%) [83]
Massachusetts, USA January 2004–November 2007 46 19 (41.3%) [84]
them based on their case criteria. The mean age was 30.3% hygiene practice was also characteristic among the patients.
and 29.2% (7/24) were male. Twenty-three of 24 used Bausch In late February 2006, the Bausch & Lomb Company vol-
& Lomb contact lens solution and 21 could specify it as ReNu untarily stopped sales of ReNu solution from the markets in
MoistureLoc. Twenty-one of the 24 stored their solutions in Hong Kong. In Singapore, Wong et al.76 conducted a retro-
the bathroom or kitchen. Based on these data it can be stated spective case series study on fungal keratitis in the period
that ReNu MoistureLoc was strongly connected to the cases 1991–1995 at the Singapore National Eye Centre. They
of Fusarium keratitis in Hong Kong. However, poor lens identified 29 cases of fungal keratitis. Among these cases,

Fusarium 421
TABLE 51.2
Incidence of Fusarium spp. in India among Culture-Proven Cases of Fungal
Keratitis (Based on Literature Search)
Number of Number of
State Studied Period of Study Fungal Ulcers Fusarium Isolated Reference
Tamilnadu
Coimbatore (children) February 1997–January 2004 37 17 (16.8%) [85]
Madurai (south) January 1994–March 1994 155 73 (47.1%) [86]
Tiruchirapalli (south) June 1999–May 2001 353 141 (39.9%) [55]
Tiruchirapalli (south) July 1985–November 1985 40 19 (47.5%) [87]
Madras (south) 1980–1982 68 8 (11.8%) [88]
Vellore NA 7 3 (42.8%) [89]
Chidambaram July 2002–June 2005 230 74 (32.0%) [90]
Tirunelveli September 1999–March 2001 554 254 (45.8%) [91]
Tirunelveli September 1999–August 2002 1100 471 (42.8%) [92]
Tirunelveli September 1999–August 2002 1138 511 (41.6%) [93]
Rest of India
New Delhi (north) January 1999–December 2001 215 23 (10.7%) [94]
New Delhi January 2000–December 2004 77 6 (7.8%) [95]
New Delhi January 1999–June 2001 191 24 (12.5%) [96]
New Delhi NA 31 10 (32.3%) [97]
Chandigarh (north) 6 years 61 10 (16.4%) [98]
Chandigarh (north) January 1999–December 2003 34 8 (23.5%) [99]
Hyderabad (south) January 1991–December 2000 1360 506 (37.2%) [100]
Hyderabad January 1991–December 1996 557 210 (37.6%) [101]
Hyderabad February 1991–June 1995 21 3 (4.1%) [102]
Patna (east) 2 years 76 6 (7.8%) [103]
Mumbai (west) 1988–1996 387 33 (8.5%) [104]
Goa February 1993–January 1994 16 2 (12.5%) [105]
Kolkata January 2001–December 2003 623 132 (21.2%) [106]
Aurangabad NA 12 4 (33.3%) [107]
15 were due to Fusarium species (Table 51.1). Twelve fur- without the use of goggles. A case study—based on 61 cases
ther cases were observed in this institute from 2001 to 2004 and 367 controls—carried out by Saw et al.112 showed that
(unpublished data). Since March 1, 2005, there was a sig- the risk of Fusarium keratitis was much higher in cases com-
nificant increase of Fusarium-related eye infections among pared with control group for ReNu with MoistureLoc than
contact lens users. By May 2006, there were 66 reported for ReNu MultiPlus. Soft monthly disposable contact lenses
cases nationwide, and 12 of these cases occurred between also increased the risk of Fusarium keratitis. They revealed
March and May, 2006. In a case study, Khor et al.17 inter- that the use of contact lenses past the replacement date
viewed these patients. The patients were equally distributed increased the risk of Fusarium keratitis. Other factors such
by sex and the mean age was 27.1 years. Except from one, the as washing of hands before replacing the lens, leaving the cap
patients wore disposable contact lenses. A total of 62 patients of the solution bottle or the lens case open, or outdoor activ-
reported using multipurpose solutions from the same brand ity were not significantly associated with Fusarium keratitis.
(ReNu, Bausch & Lomb, Rochester, NY). Forty-two patients On February 17, a news release warned contact lens wear-
(63.6%) reported using ReNu with MoistureLoc, 6 (9.1%) ers about the increasing incidence of fungal keratitis. On that
reported using ReNu MultiPlus, and 11 (16.7%) reported same day, Bausch & Lomb voluntarily stopped all sales of
using an unspecified ReNu multipurpose cleaning solution. ReNu products in Singapore. As a result of actions taken by
Based on the interview with the patients, several risk factors the Ministry of Health and Bausch & Lomb, the number of
have been revealed. Isolates derived from these cases showed cases appeared to be decreasing after March.
100% identity with Fusarium solani CBS490.63 based on the The Fusarium isolates responsible for the cases of con-
28S rRNA gene sequence. The main risk factors were the tact lens-related microbial keratitis17,112,113 showed distinct
extended use of contact lenses after the planned replacement genotypes. This observation suggested that it is unlikely
date (43.9%), the overnight use of daily wear contact lenses that common or clonal strains were the cause of the infec-
(19.7%), and swimming with contact lenses (30.3%) with or tions. The analysis of the F. solani and F. oxysporum isolates

showed that these strains are capable of forming biofilms on involving the legs.15 Outbreaks of nosocomial fusariosis have
soft contact lenses under permissive conditions, but not in also been reported. Fusarium spp., in hospital water distribu-
the presence of, or after recommended 4 h treatment with, tion system, may result in disseminated fusariosis in immu-
MoistureLoc solution. These studies also concluded that nosuppressed patients.130
there are no significant differences between the effectiveness The possible association of the fusariosis of hospitalized
of a freshly opened or an aged MoistureLoc solution if the patients with the colonization of a hospital water system by
conditions of usage are adequate. Fusarium was reported by Anaissie et al.131 The authors
Investigation carried out by both the U.S. Food and Drug applied molecular typing methods, including RAPD, RFLP,
Administration (FDA) and Bausch & Lomb found no point and interrepeat (IR) PCR, and demonstrated that two patients
source of contamination in the factory. No contamination were infected by F. solani strains with genotypes identical
was found in any bottles, and all the tested products were to that of certain environmental isolates, while the isolates
found stable and effective. However, an analysis by Bausch deriving from six patients matched the isolate of another
& Lomb showed that the solution can induce a breach in patient. In a similar study, there was no identity of RAPD
the corneal epithelium that can lead the entry of Fusarium patterns between clinical isolates and strains isolated from
into the cornea. Bausch & Lomb concluded that ReNu with water samples from the hospital environment, suggesting that
MoistureLoc’s formulation could create biofilms that shield the most likely source of fusariosis was the external environ-
the fungus from the sterilizing agent.114,115 In the review of ment, rather than the hospital water system.132 In the study
Epstein,116 the five principal elements of the Fusarium out- by Anaissie et al.,131 the two clinical isolates matching envi-
break were specified as the decrease of the antimicrobial ronmental strains were collected several months before the
effect due to biocide uptake by soft contact lenses, selection environmental isolates; therefore it cannot be excluded that
of Fusarium due to persistence, the supported growth of the patients may have contaminated the water systems.132
microbes by MoistureLoc biofilm-forming capabilities and Fusarium spp. may also exist in the soil of potted plants in
the nutritive properties of cellulose, chemical trauma (cor- hospitals. These plants constitute a hazardous mycotic reser-
neal staining) upon lens insertion due to biocide release, and voir for nosocomial fusariosis.133
the blocking of host inflammatory response by lens. In 2006, The reported major risk factors for invasive fusariosis
the FDA initiated an inspection against Bausch & Lomb’s include acute leukemia, bone marrow transplantation, immu-
manufacturing site in Greenville, South Carolina. They nosuppressed state, particularly neutropenia, and the use of
found that the company failed to regulate the storage and corticosteroids.134,135 The modes of entry are the respiratory
transport temperatures in and beyond the plant. Bullock et tract, the skin, and injuries of the corneal epithelium in the
al.117 demonstrated the in vitro loss of antimicrobial activity case of mycotic keratitis. Trauma remains the major predis-
of ReNu MoistureLoc against clinical isolates of Fusarium posing factor for the development of cutaneous and corneal
when the product was exposed for a prolonged time (4 week) infections caused by Fusarium in immunocompetent hosts.37
to a temperature of 60°C. With this study, they simulated the Although many studies reported that skin (vascular catheters,
possible conditions to which some of the manufacturer’s bot- periungual regions, or burns) is the main portal of entry in
tles may have been exposed during storage and transport, or hematologic cancer with neutropenic patients, other portals
even, perhaps, after purchase. They also concluded that ReNu of entries such as inhalation into the lungs or upper airways
MoistureLoc was effective against Fusarium isolates after a have also been documented.136 Intake of contaminated grain
10 min boiling. This suggests that the disinfectant alexidine foods involves gastrointestinal route of infection.137 In par-
inactivation is both time and temperature dependent. ticular, fusariosis in patients with cancer is predominantly a
Fusarium was also reported to be the cause of mycotic community-acquired infection, usually transmitted via the
otitis of the external ear in Gabon, Central Africa,118 and airborne route from the outdoor environment.131
was also found to germinate in the middle ear of agricul- Fusarium spp. are the only opportunistic molds that can be
tural workers.119 Four of five cases of Fusarium osteomyeli- easily recovered from the blood stream.128 Histopathologically,
tis were reported in healthy individuals following surgery or Fusarium infection may mimic any other mycoses exhibiting
trauma.120 Skin lesions are present in 80% of the cases of dis- moniliaceous, septate branching, or non-branching hyphae.
seminated infection and they may be primary or metastatic. Because of these morphological similarities, identification
They are important for early diagnosis because of accessi- of the fungus obtained from cultures is required to establish
bility for biopsy and culture. Typical skin lesions are pain- fusarial aetiology.138
ful, erythematous, subcutaneous nodules and plaques, which Seasonal variation has also been observed in fusari-
later undergo central necrosis.121 osis. Among patients living in rural areas, most infections
Fusarium species can cause localized infections of the occurred between June and September. Fifty percent of
nails and skin.122 Fusarium can also cause eumycetoma123 the eye infections and pulmonary infections peaked during
and infection in burn wounds, colonization in burn wounds,124 August15,31 and 62% of the fusariosis occurred during sum-
granulomas, ulcers, and panniculitis.2 Fusarium oxyspo- mer months, June through August.139 Fusariosis occurs more
rum mainly causes infection in fingernail and toenail125–128; commonly in males in all age group ranging from 2 to 78.31
however, F. solani is more frequently isolated from toenails Fusarium can also be isolated from the conjunctival sac and
only.35,128,129 More than 50% of the fusarial infections is from pharynx as a normal flora.140

Fusarium 423
51.1.3 Clinical Features and Pathogenesis 30.159 Skin lesions are important potential sources of diag-
nostic tissue in some patients with fusariosis.37
The clinical features and manifestations of severe invasive When four different taxa of Fusarium were tested in
fusariosis in immunosuppressed patients mimic those seen murine model, F. solani was found to be the most viru-
in patients with aspergillosis and are always nonspecific.141 lent species.39,160 The production of secondary metabolites,
This can lead to inappropriate treatment regimen by the cli- mycotoxins are mainly associated with the pathogenesis.2
nicians.142 Also, nosocomial fusariosis has a longer latency Common mycotoxins produced by Fusarium spp. include
period than community-acquired infection131; therefore the moniliformin, zearalenone, trichothecenes (deoxynivalenol,
progression of disease may be unnoticed. However, the most nivalenol, and diacetoxyscirpenol), fumonisins, T-2 toxin,
common findings at the initial presentation are persistent and fusaric acid.161 Among these, trichothecenes, zearele-
fever, sinusitis, or skin lesions with a black necrotic center.31 nones, and fumonisins are the most notorious.162 Ingestion
The neutropenic patients, especially those with acute leu- of grains contaminated with these toxins may give rise to
kemia, allogeneic bone marrow transplant recipients, as well allergic symptoms or be carcinogenic in long-term consump-
as the patients with extensive burns or with chronic renal fail- tion. They are known to cause myelosuppression through
ure15 are at increased risk of disseminated fusariosis during toxin production.126 Furthermore, exposure to fumonisin may
hospitalization.122,143–146 In particular, patients with hemato- lead to human birth defects.163 The best example is alimen-
logical malignancies account for approximately 90% of the tary toxic aleukia, which has been associated with the inges-
reported cases of disseminated fusariosis.147 Infection typi- tion of overwintered cereal grains colonized by the toxigenic
cally occurs during a prolonged period of neutropenia, last- F. sporotrichioides and F. poae.164 The toxin produced by
ing up to 65 days.31,128,144,148 In neutropenic cancer patients, these organisms, called T-2 toxin, is considered as the princi-
disseminated infections by Fusarium spp. usually present pal component responsible for the acquisition of alimentary
as persistent fever, fungemia, myalgia, and unresponsive- toxic aleukia. The first recognized trichothecene mycotoxi-
ness to a wide spectrum of antibiotics. In immunocompro- cosis was alimentary toxic aleukia in the USSR in 1932 and
mised patients, Fusarium spp. may also cause eumycetoma, the mortality rate was 60%.165 Common manifestations of
cause onychomycosis, or harmlessly colonize the ulcers.39,138 trichothecene toxicity are depression of immune responses
Upon recovery from myleosuppression, the infection may and nausea. In vitro, they impair cellular immunity and
either resolve completely or become chronic and localized to decrease the humoral response to T-dependent antigens.166
sinuses, lungs, eye, brain, joint, or muscle149 with the potential Experimental injection of T-2 toxin resulted in cardiomyopa-
for relapse and dissemination upon reinstitution of cytotoxic thy.167 Zearalenone is mainly produced by F. graminearum,
chemotherapy.128 Other presentations of invasive fusariosis and it produces estrogenic effects such as infertility, vul-
in compromised hosts include osteomyelitis, septic arthritis, val edema, vaginal prolapse, and mammary hypertrophy
myositis, foot abscesses, endocarditis, myocarditis, external in females and feminization of males—atrophy of testes
otitis, peritonitis, brain abscesses, cystitis, meningoencepha- and enlargement of mammary glands. F. moniliforme and
litis, and chronic hepatic infection.2,32,134,120,150–156 F. proliferatum produce fumonisin toxin in maize which
As mentioned previously, infections caused by this fun- may cause esophageal cancer.168 In India, a single outbreak of
gus can mimic aspergillosis.122 However, some distinct acute foodborne disease possibly caused by fumonisin B1 has
differences can be seen, such as an increased incidence of been reported. The main features of the disease were tran-
skin and subcutaneous lesions and a positive blood culture, sient abdominal pain, borborygmus, and diarrhea.169 Studies
mainly in the first days of fever, for patients with dissemi- on reduction or elimination of Fusarium spp. and mycotoxins
nated fusariosis.136,143 In contrast to invasive aspergillosis, from contaminated agricultural and food commodities are in
the blood cultures are positive in disseminated fusariosis in progress.170–172 As a saprophytic nature of the genus, it can
up to 50%–70% of the cases.32,145,157 Skin lesions, the hall- easily invade in the neutropenic patient and develop invasive
marks of disseminated fusariosis, occur in 60%–90% of the fusariosis. In addition, prolonged usage of corticosteroids
cases, compared with a rare occurrence (10%) of such lesions and antibiotics in patients with organ transplantation, silastic
in disseminated aspergillosis.2,158,159 Patients with neutro- catheters, and infections of central venous catheters (CVCs),
penia have a higher rate of disseminated skin lesions com- continuous ambulatory peritoneal dialysis (CAPD) catheters,
pared with nonneutropenic immunocompromised patients.37 and contact lenses have been reported.2
Subcutaneous nodules, palpable and nonpalpable purpura, Keratomycosis, one of the most frequent human infec-
red or gray macules, red or gray papules, macules or papules tions caused by Fusarium, is a suppurative, usually ulcerative
with progressive central necrosis with central, flaccid pus- corneal disease, most frequently occurring as a localized
tules, vesicles, and hemorrhagic bullae are types of lesions infection.173 Infection is exogenous in most of the cases, the
seen in patients with disseminated fusariosis.2,148,159 These pathogen is entering through the epithelium of the cornea.
skin lesions can involve any site, with predominance on the Patients usually present with photophobia and discharge
extremities.37,159 The lesions, especially the subcutaneous from the eyes. A persistent infiltrate is often present at the
nodules, are often tender159 and most patients have lesions at site of superficial injury, which gradually increases in size
various stages of evolution.31,159 The number of disseminated and density. The cornea becomes slightly thickened, and
skin lesions is variable and ranges between 4 and more than satellite lesions may develop peripheral to the focal area of

infiltrations. The signs of inflammation are minimal in com- and squat to highly curved and elongate with needle-like
parison with bacterial keratitis. spores.189 Further key macroconidial characters are the num-
Fusarium species are often resistant to most of the antifun- ber of septa, and the shape of the apical and basal cells: the
gal agents, and F. solani is the most resistant species within apical cell of the macroconidium may be rounded, needle-
the genus.29,174,175 In vitro resistance has been documented like, or whip-like and the basal cell may be barely notched
against the antifungal agents miconazole, ketoconazole, or resemble an upside-down foot.189 The presence or absence
5-fluorocytosine, fluconazole, itraconazole, and nikkomycin of microconidia with various shapes and sizes (which are
Z.28,122,128,176–181 However, amphotericin B (AMB) has been formed in aerial mycelium) is a primary distinguishing char-
shown to be the mainstay in the treatment of fusariosis.182 acter in Fusarium taxonomy. They may be produced singly,
In view of the inherent resistance of Fusarium spp. to most in false heads only, or in false heads and chains.2,189 Their
antifungal agents, AMB-based combination regimens have shapes may be napiform, oval, pyriform, clavate, fusiform,
also been suggested or used for fusariosis.128 Natamycin or globose.2 The microconidiophores can be monophialidic
is also active against Fusarium spp. both in vitro and in only or both mono- and polyphialidic.2,189 Monophialides
vivo,25,26,123,124 and in combination with AMB it has been the have a single opening in the conidiogenous cell, while
mainstay of treatment for Fusarium keratitis.123 However, its polyphialides have two or more.189 Chlamydospores are more
toxicity precludes its systemic use in clinical practice although common in the older cultures, their presence or absence is
topical application in powder form could halt the progres- a distinguishing character. These thick-walled, verrucose,
sion of fusariosis in pediatric patients with severe burns.125 light-colored spores filled with lipid-like material can be
Ketoconazole, miconazole, fluconazole, flucytosine, and itra- formed singly, in pairs, in clumps, or in chains in the hyphae
conazole have no in vitro activity against Fusarium spp.29,174 above or below the agar surface.2,189 Their outer wall can be
However, the newer broad-spectrum triazoles, voriconazole smooth or rough.2 Pseudochlamydospores were described by
(VRC), posaconazole, and ravuconazole have variable Marasas et al.164 in the case of F. andiyazi. They are thin and
in vitro activities against Fusarium spp. and show prom- smooth walled and can be found singly or in short chains in
ise for the management of fusariosis.29,126–129,174,175,178,183–187 the hyphae.164
Posaconazole, a broad-spectrum triazole, is active against A lot of secondary characters can be used in Fusarium
Fusarium species both in vitro and in animal models.29,174 identification, e.g., the presence or absence of sporodochia,
Overall, due to the inherent resistance of Fusarium spp. sclerotia, and stroma2; toxins and other metabolites192; or
against most of the current group of antifungal agents and the odor.189 Their usage is limited, as these data are not com-
profound solid state of immunosuppression in patients who monly available for routine diagnoses.189
typically develop fusariosis, the currently available therapeu- Beyond morphology, the studies of sexual cross-fertility
tic strategies for invasive fusariosis particularly in heavily can be used for the identification of Fusarium species.189,193
immunocompromised patients have less solutions.2,5–9,25 Due to the disadvantages of this technique (that it is slow and
needs adequate incubator space), it is not recommended for
51.1.4 Diagnosis the purposes of clinical diagnosis.
51.1.4.1 Conventional Techniques 51.1.4.2 Molecular Techniques

Identification of Fusarium species is traditionally carried out Molecular techniques may help us to avoid the misidentifi-
by the investigation of macro- and micromorphology. The cation of Fusarium based on their macro- and microscopic
most commonly used macromorphological characters are observation. The main advantage of the identification by
the colony morphology on the upper and lower surface of molecular techniques is that they do not require viable organ-
the medium, the linear or radial growth rates, and the pig- ism or sporulation; therefore, they enable a rapid diagnosis.194
mentation.2,188,189 These characters are useful for describing a Several methods are available for molecular identifica-
species under standard environmental conditions (e.g., light, tion of Fusarium species, and they have been continuously
temperature, and substrates)2,190; however, they should be improving by new methods. Fingerprint techniques, like
applied with caution as they may vary between the isolates RFLP, AFLP, etc., are useful to identify an unknown strain,
of a certain species, especially among clinically important if a set of reference strains are available.25,189 Their advan-
ones.2 tages are that DNA sequencing is not required and these
The micromorphological characters of the three types of methods are also appropriate to reveal the evolutionary dis-
spores (macroconidia, microconidia, and chlamydospores) tance between the investigated isolates.25,189 DNA sequences
are more suitable criteria for the differentiation of Fusarium of the internal transcribed spacer (ITS) regions of the ribo-
species.2,189 The absence or presence of one type of them is somal RNA gene cluster are not very useful in distinguish-
a key characteristic. The shape of macroconidia (which are ing Fusarium species.195 The sequencing of partial fragments
formed in sporodochia) is the main distinguishing character, from other amplified genes and their comparison with simi-
as it is relatively consistent and stable on natural substrates lar sequences available in the GenBank database is a com-
and under standard environmental conditions, in contrast to monly followed strategy for Fusarium identification.25,189,196
the size of macroconidia, which is varying within an indi- The most frequently used targets are the β-tubulin gene, the
vidual species.2,189,191 Macroconidia vary in shape from short translation elongation factor 1α gene (tef1), and the histone

Fusarium 425
gene.25,189,196 For rapid group- or species-level identification, 51.2.2 Detection Procedures

specific primer pairs for PCR are described in the litera-
ture.197,198 Real-time PCR detection protocols were evaluated 51.2.2.1 Morphological Identification
for fast detection and identification based on the differences of Fusarium Species
between the tef-encoding genes199 and the genes involved in The steps of identification based on the macro- and micro-
mycotoxin biosynthesis; however, this approach is not yet morphological characters were described by Summerell
available for all species.200–204 A DNA microarray was also et al.189 and Leslie and Summerell.25
developed for the easy and fast detection and identification Procedure
of the fungal genus Fusarium based on the recent phyloge-
netic analyses of tef1.205 Investigation of the intergenic spacer 1. Plate the strain onto PDA, CLA, and SNA medium
(IGS) regions with molecular methods is useful to resolve the and incubate it at 20°C–25°C for 7–10 days in the
identified species into various subgroups.206 presence of light.
2. Observe the colony morphology, the growth rates,
the presence of sporodochia, and the color of the
51.2 Methods
culture and of the produced pigments in PDA.
51.2.1 Sample Preparation 3. Examine under light microscope the shape and size
of macroconidia and chlamydospore production on
In the case of filamentous fungi, conventional DNA extraction CLA.
methods are well suited for the preparation of total genomic 4. Investigate under light microscope the shape, size,
DNA.207 Disadvantages of these techniques are that they are and formation of microconidia, their conidiogenous
slow, and relatively high amount of sample is needed. In the cells, and chlamydospore production on SNA.
studies reviewed in the previous sections, commercial DNA 5. Examine the growth rates based on linear growth in
purification kits were used for rapid and safe sample preparation. a race tube as described by Ryan et al.,211 and/or in a
Media and culture conditions: The following three media Petri-dish as described by Burgess et al.212
are preferred for morphological identification of Fusarium 6. Organize the data with the use of a recording
species: potato dextrose agar medium (PDA, 50% potato fil- sheet.25,212
trate obtained by boiling 300 g diced potato in 500 mL water 7. Make the identification using a manual. The four
and filtering, 20 g sucrose) for the investigation of colony mor- most commonly used manuals for morphology iden-
phology and pigmentation; and for measuring of the growth tification were written by Gerlach and Nirenberg,213
rates189; carnation leaf-piece agar medium (CLA; sterile Nelson et al.,191 Burgess et al.,212 and Leslie and
3–5 mm carnation leaf pieces in agar) for the investigation of Summerell.25
macroconidia208; and “Spezieller Nährstoffarmer agar” (SNA,
1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4 × 7 H2O, 0.5 g KCl, 0.2 g The main macromorphological characteristic features
glucose, 0.2 g sucrose) for the investigation of microconidia are25,191,212,213
and chlamydospores209,210 (all media are given for 1 L and con- a. Abundance and color of aerial mycelium
tain 20 g agar). After incubation at 20°C–25°C for 7–10 days b. Pigmentation
in the presence of light, particularly some exposure to UV c. Growth rate
(black) light (λ = 310–360 nm), Fusarium isolates grow well The main micromorphological characteristic features
and form conidia.189 Artificial daylight (12 h light/12 h dark) are25,191,212,213
is often used, although total darkness or continuous light is d. Macroconidia: size, shape, number of septa, shape
necessary for the evaluation of critical diagnostic characters of apical and basal cell
of some species.209,210 In the case of PDA cultures, the pres- e. Microconidia: size, shape, number of cells, for-
ence of light is not essential, but it is increasing the produc- mation, nature of conidiogenous cells, and
tion and total amount of pigment. On the other hand, light is conidiophores
important for CLA cultures as it increases the production of f. Chlamydospores: presence or absence, formation
sporodochia.189 Genomic DNA can be extracted from mycelia
grown either on solid or in liquid malt extract medium after
incubation at 20°C–25°C for 7 days. 51.2.2.2 Molecular Identification of Fusarium Species
Microscopy sample preparation: Microscopy samples can 51.2.2.2.1 Genus-Specific Identification
be made from colonies grown on PDA, CLA, and SNA after 51.2.2.2.1.1 PCR Protocol of Hue et al.197 PCR meth-
incubation at 20°C–25°C for 7–10 days in the presence of light. ods for rapid and reliable identification of Fusarium spe-
Genomic DNA preparation: Genomic DNA can be cies isolated from clinical samples were evaluated by Hue et
extracted from mycelia grown either on solid or in liquid al.197 and Hennequin et al.214 Hue et al.197 described a method
PDA medium after incubation at 20°C–25°C for 5–7 days. appropriate for the preparation of PCR templates from
Beyond conventional DNA preparation methods,25,194 com- Fusarium-containing blood and performed PCR with prim-
mercial DNA purification kits can be used for rapid sample ers designed for the ribosomal RNA genes (rDNAs) based on
preparation. a large number of isolates belonging to the genus Fusarium.

Primers P28SL (5′-ACA AAT TAC AAC TCG GGC CCG cycles of 94°C for 30 s, 64°C for 90 s, and 72°C for
AGA-3′) and P58SL (5′-AGT ATT CTG GCG GGC ATG 90 s; and a final 72°C for 10 min.
CCT GT-3′) amplified a fragment of 329 bp containing ITS2 3. Visualize the PCR products after electrophoresis on
and a portion of 5.8S and 28S rDNA. To avoid false-negative 1% agarose gels stained with ethidium bromide and
results, a positive internal control was used from a part of λ run in 1 × TAE buffer.
phage DNA. It was amplified by two primers (C1, 5′-ACA
AAT TAC AAC TCG GGC CCG AGA CCA CAG CGC- 51.2.2.2.2 Species-Specific Identification
3′ and C2, 5′-AGT ATT CTG GCG GGC ATG CCT GTG Fusarium species can be identified at the species level by
TAC AAC TGG-3′), whose 3′ ends correspond to the λ DNA sequencing of amplified gene fragments and comparing the
while the 5′ ends correspond to the primers used in the PCR sequences with similar ones available in sequence databases
amplification. The PCR reaction resulted in a 517 bp frag- (FUSARIUM-ID, GenBank, EMBL, and DDBJ) using a
ment which contains the Fusarium sequence at the ends, then Basic Local Alignment Search Tool (BLAST) search.
it was amplified with the Fusarium primers.
51.2.2.2.2.1 β-Tubulin Gene PCR Protocol 1206 Yli-
Procedure197:
Mattila et al.206 used a universal forward primer (T1: 5′-ATG
CGT GAG ATT GTA AGT-3′)215 and a specific reverse
1. Lyze the cells with a commercial genomic blood
primer (tub-conrev T22: 5′-TGA CCG AAA ACG AAG TTG
DNA purification kit. Mix it with 200 μL of
TC-3′) for amplifying a fragment with the first two introns of
enzyme buffer (0.9 M sorbitol, 0.1 M Tris, 0.1 M
the β-tubulin-encoding gene.206
EDTA) and 20 μL of lyticase and incubate the
samples at 37°C for 90 min. Treat the sample with
1. Prepare the reaction mixture containing 5–50 ng
proteinase K (10 μL, 20 mg/mL) and incubate it at
genomic DNA, 2.5 mM MgCl2, 10 mM Tris–HCl
55°C for 30 min. Add 5 μL of RNase and incubate
(pH 8.8), 50 mM KCl, 0.1% (w/v) Triton X-100,
the sample at 37°C for 30 min. Extract the DNA
0.4 μM of each primer, 0.12 mM of each dNTP, 3 U
with a commercial genomic blood DNA purifica-
of Taq polymerase.
tion kit.
2. Perform the PCR in a thermocycler using the fol-
2. Prepare the reaction mixture according to the man-
lowing reaction conditions: 1 min at 94°C; 30 cycles
ufacturer’s instructions. Use 1 ng of template DNA
of 94°C, 51°C, and 74°C for 1 min each; and a final
per reaction mixture.
74°C for 7 min.
3. Perform the first PCR in a thermocycler using the
3. Visualize the PCR products after electrophoresis on
following reaction conditions: 94°C for 5 min; 40
1% agarose gels stained with ethidium bromide and
cycles of 94°C, 68°C, and 72°C for 1 min each; and
run in 1 × TAE buffer.
a final 72°C for 10 min. Include the positive internal
4. Purify the PCR products with a commercial PCR
control.
DNA purification kit.
5. After the sequencing of the PCR product, compare
2% agarose gel stained with ethidium bromide and
it with the β-tubulin sequences available in the
run in 1 × TAE (Tris-acetate-EDTA) buffer.
GenBank database using a BLAST search.
51.2.2.2.1.2 PCR Protocol of Hennequin et
Amplification of a partial β-tubulin gene fragment was
al.214 Hennequin et al.214 designed a primer pair based on
carried out by Chung et al.216 using a specific primer pair,
the 28S rDNA sequences of Fusarium species associated
FU-tubulin3 (5′-CGA GCC CGG TAC CAT GGA CG-3′) and
with human infections. In contrast to the DNA from unre-
FU-tubulin2 (5′-GGT CGC CGT AAG AGG GGT TGG-3′).
lated genera, the primers Fus1 (5′-TGA AAT CTG GCT
CTC GGG-3′) and Fus2 (5′-CAT GCG CGA ACC TCA 51.2.2.2.2.2 β-Tubulin Gene PCR Protocol 2216
GTC-3′) amplified a 480 bp fragment from the DNA extracts
of Fusarium strains and of the members of related genera 1. Prepare the reaction mixture in a final volume of
Acremonium and Cylindrocarpon. 20 μL containing 5–50 ng genomic DNA, 0.2 μM of
Procedure214: each primer, 0.25 mM of each dNTP, 1 U of Ex Taq
polymerase and Ex Taq reaction buffer (containing
1. Prepare the reaction mixture in a final volume of 2 mM MgCl2).
50 μL containing 5 μL genomic DNA template, 2. Perform the PCR in a thermocycler using the fol-
10 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2, lowing reaction conditions: 94°C for 3 min; 35
0.01% (w/v) gelatin, 5 μL of DNA, 0.25 μM of each cycles of 94°C for 1 min, 60°C for 30 s, and 72°C for
primers, 0.2 mM of each deoxynucleoside triphos- 2 min; and a final 72°C for 5 min.
phates (dNTPs), and 0.3 U of Taq polymerase. 3. Visualize the PCR products after electrophoresis on
2. Perform the PCR in a thermocycler using the fol- 1% agarose gels stained with ethidium bromide and
lowing reaction conditions: 94°C for 10 min; 35 run in 1 × TAE buffer.

Fusarium 427
4. Purify the PCR products with a commercial PCR 51.2.2.2.2.5 ITS Sequence PCR Oechsler et al.220
DNA purification kit. designed primers for the end of the 18S ribosomal DNA
5. After the sequencing of the PCR product, compare (F18A: 5′-GCG GAG GGA TCA TTA CCG AGT T-3′) and
it with the β-tubulin sequences available in the the beginning of the 28S rRNA (F28S: 5′-CAG CGG GTA
GenBank database using a BLAST search. TTC CTA CCT GATC-3′) of the target Fusarium species,
and amplified with them the ITS region comprising ITS1,
51.2.2.2.2.3 Translation Elongation Factor 1α Gene (tef1) 5.8S rRNA, and ITS2. Although previous ITS analyses were
PCR The tef1 gene has high phylogenetic utility at the spe- not very useful for distinguishing Fusarium species, the
cies level in Fusarium.196 Several universal primers have sequence data from this study correlated well with the mor-
been designed that work across the phylogenetic breadth of phologic classification. The authors suggested the feasibility
the genus.196,217 Geiser et al.196 created the first generation of of Fusarium detection and identification at the species level
a database (FUSARIUM-ID, http://isolate.fusariumdb.org/ from ocular sources using the sequence of the ITS region.
intro.php), which contains more than 400 sequences repre-
Procedure220
senting a phylogenetically diverse selection of tef1 sequences
from the Fusarium genus.217
1. Prepare the reaction mixture for a standard PCR
Procedure196,217 protocol in the final volume of 25 μL containing
200 ng DNA template.
1. Perform a standard PCR protocol with an anneal- 2. Perform the PCR in a thermocycler using the follow-
ing temperature of 53°C to amplify the tef1 gene ing reaction conditions: 95°C for 3 min; 45 cycles of
region using forward primer ef1 (5′-ATG GGT 95°C for 30 s, 55°C for 30 s, and 68°C for 2 min.
AAG GA(A/G) GAC AAG AC-3′) and reverse 3. Visualize the PCR products after electrophoresis in
primer ef2 (5′-GGA (G/A)GT ACC AGT (G/C)AT 1% agarose gels stained with ethidium bromide and
CAT GTT-3′). run in 1 × TAE buffer.
2. Visualize the ∼700 bp PCR products after electro- 4. Purify the PCR products with a commercial PCR
phoresis on 1% agarose gel stained with ethidium DNA purification kit.
bromide and run in 1 × TAE buffer. 5. After the sequencing of the PCR product, compare
3. Purify the PCR products with a commercial PCR it with the ITS sequences available in the GenBank
DNA purification kit. database using a BLAST search.
it with the tef1 sequences available in the Fusarium 51.2.2.2.2.6 F. oxysporum- and F. solani-Specific
database (http://isolate.fusariumdb.org/) using a PCR There are a lot of species-specific primer pairs in the
BLAST search. literature for rapid and reliable identification of Fusarium
species.198,214,217,221 Ghignone and Migheli198 collected the
51.2.2.2.2.4 Histone-Encoding Gene PCR Steenkamp species-specific primers for the detection of phytopathogenic
et al.218 performed PCR for amplification of a histone H3 fungi and created a continuously improving database from
gene fragment ranging from 519 to 527 bp with the primers them (http://www.sppadbase.com/). This database contains
H3-1a (5′-ACT AAG CAG ACC GCC CGC AGG-3′) and primers for identification of some human pathogenic mem-
H3-1b (5′-GCG GGC GAG CTG GAT GTC CTT-3′).219 bers of the Fusarium genus and references where the proto-
cols are described.
Procedure218 Hue et al.197 designed primer pairs for specific detection
of five clinically important Fusarium species derived from
1. Prepare the reaction mixture containing 0.25 ng
human sources. Only the OX 31 (5′-TGA CTT GGA TGA
genomic DNA/0.05 U Taq polymerase, 2.5 mM
GAC CTT GGC G-3′) and OX 32 (5′-CAG GAT TTA CCG
MgCl2, 1 × reaction buffer, 0.2 μM of each primer,
ACA CAG CTT TTG-3′) primer pair was specific for the inves-
0.25 mM of each dNTPs.
tigated F. oxysporum strains (annealing temperature = 66°C),
while the SOL 31 (5′-GCT ACC GAG GCC ATC AAT TCA
lowing reaction conditions: 92°C for 1 min; 30
TG-3′) and SOL 32 (5′-TGA TGT TGT ACT TCT CCT TGC
cycles of 92°C, 68°C, and 72°C for 1 min each; and
CC-3′) primer pair was specific for four of the five tested
a final 72°C for 5 min.
F. solani strains (annealing temperature = 66°C). The proce-
dure and methodology are described in Section 51.2.2.2.1.
1% agarose gel stained with ethidium bromide and
run in 1 × TAE buffer.
4. Purify the PCR products with a commercial PCR 51.3 C
Perspectives
it with the histone H3 sequences available in the As human and animal diseases due to Fusarium spp. have
GenBank database using a BLAST search. become increasingly common, there is an urgent need to

develop rapid, sensitive, and specific diagnostic methods. 11. Windels, C.E., Economic and social impacts of Fusarium
Correct identification of Fusarium to species/clonal level head blight: Changing farms and rural communities in the
is essential for the control of human and plant fusarial dis- Northern Great Plains, 91st Annual Meeting of The American
Phytopathological Society August 8, 1999, Montreal, Quebec,
eases. Considering the fact that the morphological features
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1997.

52 Microascus, Including Scopulariopsis
Jouni Issakainen and Dongyou Liu
Contents
52.1 Introduction...................................................................................................................................................................... 435
52.1.2.1 Nails....................................................................................................................................................... 436
52.1.2.2 Skin........................................................................................................................................................ 437
52.1.2.3 Ears........................................................................................................................................................ 437
52.1.2.4 Respiratory Tract................................................................................................................................... 437
52.1.2.5 Central Nervous System........................................................................................................................ 437
52.1.2.6 Heart and Other Organs......................................................................................................................... 437
52.1.3 Pathogenesis.......................................................................................................................................................... 438
52.1.4 Diagnosis.............................................................................................................................................................. 438
52.2 Methods............................................................................................................................................................................ 439
52.2.1.1 Sample Collection, Transportation, and Handling................................................................................ 439
52.2.1.2 DNA Extraction..................................................................................................................................... 440
52.2.2.1 Specific Detection of the Family Microascaceae.................................................................................. 440
52.2.2.2 Microascus-Specific Detection.............................................................................................................. 440
References.................................................................................................................................................................................. 442
52.1 Introduction incorporates here practically all the few molecular clades of
fungi that are known to have sexual fruiting bodies described
52.1.1 Classification, Morphology, and Biology as Microascus. No position is taken here whether this clade
The meiosporic (i.e., sexually sporulating) genus Microascus should be split into smaller genera, or whether also other, less
includes some less common but regular nail pathogens. In studied meiosporic genera will be shown to be located inside
rare occasions, several species have caused lethal infections the same phylogenetic clade. The definition also means that
in immunocompromised patients. all known members of Pseudallescheria and Scedosporium
The genus belongs in the family Microascaceae. The same remain outside Microascus.
family also covers some more important human pathogenic More importantly, this delimitation deals the various mito-
genera, namely, the meiosporic genus Pseudallescheria sporic stages of the same molecular clade simply as members
and the mitosporic (i.e., asexually sporulating) genus of the same large meiosporic genus Microascus, regardless
Scedosporium. These are dealt with in other chapters of of whether the entire life cycle between the meiosporic and
the present book. The whole family is a part of the subclass mitosporic stages has been experimentally solved, or whether
Hypocreomycetidae [1]. Due to the small number of DNA- the name of the mitosporic stage (anamorph name) has been
based taxonomic studies, however, the taxonomy of these validly combined to any name of a meiosporic stage (teleo-
clades of fungi is far from being settled. Literature concern- morph name), in terms of current taxonomic rules.
ing the whole family as a medical mycological problem has The clinically most relevant consequence of the above
been recently reviewed [2]. delimitation is that the mitosporic genus Scopulariopsis
For simplicity, the name Microascus is used in the pres- s.l. is handled here simply as a part of the meiosporic genus
ent chapter in a wide sense (sensu lato, s.l.), referring to Microascus s.l. As the phylogenetic work of Issakainen et al.
the whole provisional “Microascoid clade” of Issakainen [3] shows, the morphology-based meiosporic genus and the
et al. [3]. Using this technical delimitation, the genus morphology-based mitosporic genus broadly correspond with
435

each other in molecular terms. In addition to Scopulariopsis Microascus longirostris is the taxonomical type species
anaomorphs however, some members of Microascus s.l. are of the genus, described by Zukal in 1885 [7]. Thus, if some
known to produce other types of asexual sporulation. Some clades of the genus will be split into new genera, the genus
of these mitosporic forms are so distinctive that they have with M. longirostris will retain the name Microascus.
been given separate generic names such as Doratomyces and The difficult task of taxonomically connecting each
Wardomyces [3]. meiosporic species with the mitosporic stages has contin-
The sexual fruiting bodies of Microascus are dark and ued for more than a century and will do so in the future.
small, measuring 0.05–0.3 mm in diameter [4]. They are For some species, the connection has been observed a long
usually bottle-like having an opening through the neck part time ago since they produce large amounts of both sporulat-
(then called perithecia), but more or less spherical and closed ing stages in the same colony. This applies to, for example,
(cleistothecia) in some species, especially in those formerly Microascus longirostris, M. cinereus, M. desmosporus, and
classified as Pithoascus. The wall of the fruiting body con- M. trigonosporus [7].
sists of angular cells (textura angularis). For a few species, the connection has been found only
As the name implies, species of Microascus have small recently. This is true especially for the clinically most
asci (singular: ascus, the ascospore-bearing cells, which are important species Scopulariopsis brevicaulis. In spite of
formed within the perithecium). The eight ascospores within the common cultivation of this fungus all around the world,
each ascus are smooth, one-celled, and more or less curved. the diligent study of Abbott et al. [8] revealed the sexual
As compared with Pseudallescheria, the ascospores are paler stage as late as in the 1990s. The species was thus renamed
brown and the single germ pore of the spore is less prominent as Microascus brevicaulis, indicating its whole life cycle.
or missing [4]. Similar studies have been performed in some other species
Most mitosporic forms of Microascus sporulate by means as well [9,10].
of annelloconidia. This type of conidiation is characterized Despite the apparent overlap between Scopulariopsis
by consecutive ring-like scars near the tip of each conidiog- and Microascus, these two genera cannot be treated simply
enous hypha. Each scar is a mark left by a detached, broad- as different stages of the same taxon. Scopulariopsis ana-
based conidium. Of the anamorph genera of Microascus, morphs and related synnematous forms occur in Kernia as
annelloconidia are especially typical to Scopulariopsis and well. More rarely, Scopulariopsis or very similar anamorphs
to its synnematal counterparts, Doratomyces and Trichurus. have also been reported in other genera such as Petriella and
Annelloconidia of Microascus are formed in dry chains Pithoascus [4,11]. Some extant Microascus species may be
which eventually break and let the spores be dispersed by completely unable to form a Scopulariopsis stage and vice
wind. This contrasts with the insect-dispersion of the slimy versa.
Scedosporium conidia. As compared with the oval and Microascus species can be found on a wide variety of
smooth Scedosporium spores, the conidia of Microascus organic substrates such as soil, rotten plants or paper. They
species have a broader base and are more dome-shaped or can be termed as saprotrophs. Many species prefer more pro-
bullet-shaped, sometimes conspicuously warted. Only in teinous or otherwise nutrient-rich substrates such as animal
some clinically rare anamorphs of Microascus, notably the remains or dung, and the fruiting bodies are often formed on
mitosporic genus Wardomyces, each conidium detaches by these. The human body, often in various debilitated condi-
breaking and has a longitudinal germ slit [2,5]. tions, occasionally serves as a substrate for the genus, leading
Planning of molecular diagnostic tools for Microascus to various types of infection [2,7,12–15].
is currently hampered by the fact that there are many more
poorly known genera and species that may prove to be parts of
the natural Microascus. At least, such genera may be so close
relatives of Microascus that their molecular characters must Regarding their clinical significance, Microascus species
be considered in, for example, designing of primers [6]. Such can be broadly characterized by their two main roles: as less
potentially related genera include the meiosporic Lophotrichus, common but regular pathogens of the keratinous tissues and
Enterocarpus, and Canariomyces, and the mitosporic form as very rare but serious opportunistic agents in deep tissues
genera Wardomycopsis, Humicola, and Mammaria [4]. of the body. A short overview of the infections and of most
Because of the mentioned taxonomical problems and of relevant Microascus species in each body site follows.
limited strain collecting, the number of extant Microascus
species is not known. The number of currently described 52.1.2.1 Nails
species is most likely an underestimation but can be The most common clinical phenomenon associated with
used as a starting point: more than 15 Microascus species, Microascus is a chronic nail infection. Patients are typically
20 Scopulariopsis species, 5 Wardomyces species, and mid-aged to older people whose big toe nail, seldom other
3 Doratomyces species are known. No position is taken, here, nails, shows various symptoms of tissue damage including
to the delimitation of single species. Inferring from the much fragility, thickening, and discoloration of nails, nail deforma-
more easily detected macrofungi, it is reasonable to assume tions, problems in walking with shoes on, or nail loss [16].
that the real species number of this clade is in the magnitude The mechanisms of this benign but often disturbing infection
of hundreds, at least. are still poorly known.

Microascus, Including Scopulariopsis 437
M. brevicaulis is by far the most common Microascus infection [29]. Microascus was found to be a stronger genus
species to be encountered in nails. In a Finnish material, than Pseudallescheria in infecting the external ear channel.
80% of the cases suggestive of Microascus nail infection Its ecological range as an ear pathogen extended to post-
concerned this species, followed by Scopulariopsis candida operational cavities of the middle ear. In these deeper parts,
and S. fusca [16]. Scopulariopsis species have been shown however, P. boydii was equally common [6].
to grow in symptomatic nails more often than other environ-
mental fungi [16–20]. 52.1.2.4 Respiratory Tract
Genuine vegetative growth of Microascus in the nail (in In other than keratinous tissues, Microascus species are
contrast to external contamination) is often shown by typical primarily seen in patients whose immune system has been
hyphae and spores in direct nail microscopy, and by repeat- suppressed due to, for example, ongoing cytostatic therapy,
edly positive culture. In a minority of the cases, common who have other underlying diseases or whose body has been
dermatophytosis (causative agents: Trichophyton stages of physically injured. When these preconditions are fulfilled to
Arthroderma) obviously predisposes the nail for Microascus. such an extent that Microascus invades deeper tissues, the
Absolute exclusion of earlier dermatohyphytosis or forgotten infection may turn very difficult to treat and run a lethal
traumas is often difficult to achieve but a great amount of course.
indirect evidence suggests that Microascus often acts as the Microascus species are known as rare causes of fungal
main infectious agent and at least participates in causing the sinusitis. In immunosuppressed individuals, sinusitis due to
symptoms [18,20,21]. Scopulariopsis spp. may become invasive. The pale-spored
M. brevicaulis is estimated to cause about 1.6% of all fun- species Scopulariopsis candida and S. acremonium have
gal nail infections in a Canadian survey [18]; Scopulariopsis been involved [30]. The Scopulariopsis candida anamorph
spp. have been observed in 6.4% of onychomycosis cases in of Microascus manginii was reported as an etiologic agent
Italy [19], in 5.7% in Denmark [22], in 5.6% in Spain [23], in in invasive sinonasal disease in a 12-year-old girl being
1.9% in Colombia [24], and in 1.5% in Estonia [25], and <1% treated for non-Hodgkin’s lymphoma. Some nasal cases have
in Belgium [26]. become invasive in susceptible hosts [30,31].
The variety of symptoms assigned to Microascus may Microascus infections of the lungs are rare. As a local-
indicate that the cases do not form a homogenous entity [16]. ized form of infection, a fungus ball in the lungs due to
López-Jodra and Torres-Rodríguez [21] listed Scopulariopsis Scopulariopsis spp. was described by Endo et al. [32]. In
spp. as typically responsible for white superficial ony- immunocompromised patients, Scopulariopsis lung infec-
chomycosis. Gupta and Gregurek-Novak [27] referred to tions are serious and have a poor prognosis. Severe pneu-
Scopulariopsis onychomycosis as reminiscent of distal and monia due to Microascus infections has been observed a
lateral subungual onychomycosis. According to Tosti et al. few times [33–35]. The species spectrum of Microascus in
[18], S. brevicaulis can cause both distal and proximal sub- the lungs may differ from that of superficial infections. The
ungual onychomycosis. On the other hand, a similar range of ubiquitous S. brevicaulis has been positively recorded in
symptoms is also seen in dermatophytosis, suggesting varia- only one case (pneumonia of a drug abuser). In three other
tion between the human hosts. cases, the species remained unidentified [32,33]. In one fatal
pneumonia case [34] the causative agent was a rare species,
52.1.2.2 Skin Microascus trigonosporus.
In addition to nails, Microascus species have for long been
known to colonize and, potentially, infect the skin. Here, it 52.1.2.5 Central Nervous System
seems that the keratinous tissue needs to be previously debili- At least five cases of severe CNS infections are known to
tated by factors such as long-term wet conditions in the boots of be caused by Microascus species. Most of the infections
soldiers [7] or deep immunosuppression. Potential predispos- have been in immunocompromised individuals [35]. In three
ing factors in less extreme living conditions include primary patients, the infection presented as an abscess of the CNS.
dermatophyte infection, sweating skin folds with candidosis, The fourth case was meningitis of an AIDS patient [36]. A
and allergic lesions [16]. M. brevicaulis is the main agent. brain infection has also been observed in an immunocompe-
tent person [37]. In this case, a large lesion of the white mat-
52.1.2.3 Ears ter was removed surgically. Histopathological observations
Microascus species have repeatedly infected the external ear were reminiscent of acute hemorrhagic leucoencephalitis.
channel. Considering the thin keratinous epidermis and moist Fungal growth proceeded around the operation site. As in
conditions in the ear, these infections may be comparable to lungs, the rare CNS infections may have a different overall
those in the wetted skin. Details, however, are not known. species spectrum than the keratinous tissues: M. brevicaulis
One of the earliest records of Microascus in the ears does not have a dominant role and at least M. cinereus and
appears to be a case with Microascus desmosporus [7]. S. brumptii have been seen as causative agents.
Microascus (Scopulariopsis) brevicaulis was documented
to cause external otitis in a patient with cholestematous 52.1.2.6 Heart and Other Organs
otitis media [28]. In an immunocompromised patient, a Serious endocarditis due to M. brevicaulis has occurred in a
Scopulariopsis ear infection led to mastoiditis and fatal few heart surgery patients, mostly prosthetic valve recipients

[33,38]. Other types of infection include peritonitis, eye infec- brighteners (e.g., Calcofluor White or Blankophor) or older
tions, and various soft tissue infections [6,33]. These have chemical stains, according to facilities and the experience of
occurred in immunocompetent persons and been generally the worker.
curable with surgery and/or antifungal treatments. Isolation Some details apply specifically to Microascus. The
of Microascus from blood is a very rare event [39]. genus includes species with brown cell walls (dematiaceous
hyphae) and species with clear, transparent walls (hyaline
52.1.3 Pathogenesis hyphae), and intermediates in between. This may mislead
a less trained microscopist since in some literature sources,
Nail infection due to Microascus may not be actually oppor- these color categories are treated as separate entities (pha-
tunistic. However, several poorly understood factors of the eohyphomycosis and hyalohyphomycosis, respectively). The
patient’s genotype, of his or her age or clinical history and most common pathogenic Microascus species have hyaline
of the keratinolytic enzyme pattern of the fungal strain may hypha but also some darkly pigmented species have been
contribute to the initiation or progress of this disease [40]. associated with human disease [44]. In nails, Microascus
Torres-Rodríguez and López-Jodra [21] and Summerbell species may form round spores and hyphae with characteris-
et al. [20] showed that genuine Scopulariopsis growth often tic morphology [20].
occurs in the nails without the help of dermatophytes. In Histopathological staining shows the position of a fun-
addition to the cutaneous barrier breaks (bandage in a gal infection in tissues and, in trained hands, gives a wealth
catheter site) [35], the nail pathogenicity has also been of other information of the whole pathological process.
approached from the point of view of keratinolytic enzymes Accordingly, it is one of the central components of the diag-
produced [41]. nosis of deep, opportunistic mycoses [30]. It is also useful
In some experimental models, some strains of Microascus in diagnosing and confirming superficial fungal infections
were shown to be very weak utilizers of nail tissue. However, including those of nails [45]. The periodic acid-Schiff stain
another study revealed that individual strains of a given spe- (PAS) used routinely in pathological investigations also sticks
cies have significant differences in this respect. Filipello to most fungal cells and stains them red. Gomori’s methena-
Marchisio et al. [41] showed that some strains of M. brevicaulis mine-silver stain renders fungal cell walls black, against light
are genuinely keratinolytic, not merely keratinophilic. In green background. The latter dye gives better resolution in
other words, these fungi do enzymatically invade healthy morphological details and should be used to confirm the PAS
keratinous tissues, not merely utilize secondary breakdown result especially in serious infections.
products of keratin. The observed enzymatic activity of
Microascus was slower than that of dermatophytes but suf- 52.1.4.1.2 Fungal Cultures
ficient for giving some strains the status of pathogens [41]. A central method for diagnosing most fungal infections is to
Infections due to Microascus in non-onychomycosis cases recover the living fungal strain from the infected tissue. This
are usually associated with some debilitating factors. These applies to Microascus, as well. An example of possible cul-
include immunosuppressive therapies due to transplantation, turing procedures and growth media is illustrated as a flow
local barrier breaks such as catheter sites or moist bandages. chart by de Hoog et al. [13,14]. Other variations appear in
Details of specific pathogenesis of Microascus in deep infec- numerous handbooks such as Larone [46].
tions are not known. A few cases have been associated with In general, a clinical specimen is cultured simultane-
just local factors, or with no observed factors at all [35,42,43]. ously on several (2–5) general-purpose culture media (e.g.,
Sabouraud Dextrose Agar, Malt Extract Agar, and/or Brain
52.1.4 Diagnosis Heart Infusion Agar) supplemented with antibacterial agents
such as streptomycin and penicillin, and incubated at tem-
52.1.4.1 Conventional Techniques peratures between 24°C and 30°C. This allows the growth
Infections due to Microascus are mostly diagnosed by of a wide variety of pathogenic fungi, including Microascus
microscopy of human tissues and by isolating the causative [13,14].
agents on culture media. Identification of the isolated strains Fungal cultures are usually incubated for several weeks
by conventional techniques is mainly based on morphologi- (up to about 1 month) and screened several times during the
cal analysis of the sporulating structures [13,14]. incubation. For most strains of pathogenic Microascus, the
routineous culture conditions pose no problem, since they
52.1.4.1.1 Microscopy commonly form visible colonies usually in a few days or,
Direct microscopy of Microascus in human tissue does not at latest, in about 1 week. Some tissue-adapted pathogenic
differ from that of other opportunistic fungi. In general, a strains, may grow slower, however, or fail to grow at all. This
portion of the sample is mounted on a glass slide, covered phenomenon is known, for example, in Pseudallescheria but
with a coverslip in a suitable mounting medium such as lacto- may apply to Microascus as well. The related but apatho-
phenol solution, and then screened under a transmitted light genic genus Kernia is also known as difficult to culture and
microscope with a total magnification of about 200–400 preceding treatments or host defense may affect the viability
times. The image may be enhanced by various methods such of any strain. Therefore, the recovery of a given pathogenic
as phase contrast, Nomarski interference contrast or optical strain is never self-evident.

Regarding culture methods, there are some points which optimal growth occurs with 3%–6% NaCl in the medium.
specially affect Microascus. A medium supplement, cyclo- In addition to cellulose and keratin, it can typically utilize
heximide (also called actidione) is added in some culture arsenic compounds. Some Microascus species compete with
media to suppress contaminating molds and yeasts to make dermatophytes by producing antifungal agents [50].
the medium selective for dermatophytes. This seemingly Serological tests have the capacity to identify some mem-
attractive medium for fungal cultures of the skin gives bers of the Microascaceae directly from a clinical specimen
unpredictable and directly misleading results when infec- [51,52]. However, considering that immunological tests are
tions due to Microascus and related genera are concerned. usually aimed at only a limited number of pathogenic spe-
For instance, the concentration of 0.5 g/L of cycloheximide cies, and that there exist a large number of opportunistic fun-
allows some members of the family to grow but suppresses gal species [13,14], screening of all potential patients with
others [13,47]. At least M. brevicaulis is often found growing immunological tests is currently not cost-effective.
in cycloheximide-containing media but it is not known how
consistently all species and strains of Microascus react to 52.1.4.2 Molecular Techniques
this substance. Another chemical with possible selective use As in other groups of fungi, molecular techniques are use-
in Microascus is benomyl. Members of the Microascaceae ful for identification and differentiation of Microascaceae
are usually resistant to this substance [47,48]. as well. The conserved rRNA coding genes and the more
For colony characters and microscopic morphology of variable internally transcribed spacer (ITS) regions between
Microascus, special literature should be consulted [4,7,13]. In them are most frequently targeted for phylogenetic studies
general, the colony colors of the genus vary between black, [13,53]. Naturally, sequences of several protein-encoding
gray, whitish, and buff tones (the latter especially for M. brevi- genes also have the potential to be used with Microascus but
caulis). Green and pink colors, common in some other gen- have not been applied to this genus, as yet. Such study targets
era, are not encountered. The colony texture is mold-like (as include β-tubulin, actin, chitin synthase, acetyl coenzyme A
opposite to yeast) but may show various types of moderately synthase, glyceraldehyde-3-phosphate dehydrogenase, and
fluffy mycelium or powdery granules or masses of conidia. lignin peroxidase or orotidine-5′-monophosphate decarbox-
In some strains, perithecia are clearly visible as small black ylase genes [1].
dots against the mycelium. In clinical laboratories, identifica- Molecular studies on this family are mainly concentrated
tion of Microascus is usually based on the mitosporic stage on Pseudallescheria [54–56]. Molecular relationships within
since the meiosporic stage may appear slowly, only after mat- the genus Microascus have thus far been explored by only
ing with a compatible strain, or may be totally missing [8,9]. one study limited to a 350 bp gene region of the large subunit
Microscopically, the key characters of the most common ribosomal RNA gene (LSU rDNA) [3]. Based on the analy-
Scopulariopsis stages include slightly to clearly branched ses, the family was provisionally divided into 12 molecular
to synnematal conidiophores with often sharp branching lineages, seven of which were within Microascus. Several
angles reminiscent of the common but non-related genus opportunistic human pathogenic species, including M. brevi-
Penicillium. Using high magnifications, the densely packed caulis, were placed in one clade, together with M. manginii.
annellides can be seen near the tips of the conidiogenous Some deviating sporulation forms such as the synnematous
hyphae. The conidia are one-celled and vary according to Doratomyces and the germ-slitted Wardomyces were concen-
species but are generally dome-shaped to broadly bullet- trated in another clade, together with M. albonigrenscens. An
shaped. Their colors vary from hyaline to dark-walled, via extensive molecular taxonomic revision of the genus is sorely
brownish or gray tones. Their surface ornamentation varies needed.
from smooth to coarsely warty. Ornamentation varies gradu-
ally even in a single chain, depending on the age and position
52.2 Methods
of the conidium. In some species and strains, gradual transi-
tions occur from the normal scarcely branched Microascus 52.2.1 Sample Preparation
conidiophores to large synnemata assigned to Doratomyces.
In clinical samples, however, the latter are rarely seen [13,14]. 52.2.1.1 Sample Collection, Transportation,
and Handling
52.1.4.1.3 Biochemical Methods Core criteria for diagnostic medical mycology include that
No biochemical culture methods are routineously used for the sample is representative for the infection and contains
the diagnosis of Microascus. Subcultures of the strains on (preferably living) pieces of the causative fungus. Dying of
different growth media, or at different temperatures may be the fungus during the transportation is usually not the prob-
used according to need. Fungal growth may be character- lem but the increase of contaminants may be.
istically inhibited by the selected conditions, or sporulation Tissue types and consistence of good samples vary
may be accelerated. Members of the family show variable widely, depending on the infected body site, for instance.
resistance against cycloheximide [49]. M. brevicaulis grows Typical samples for diagnostic mycology include scrapings
optimally in temperatures from 24°C to 30°C, but tolerates of (for culture, properly disinfected) skin, pieces taken from
the human body temperature of 37°C. M. brevicaulis prefers deep layers of nails, surgically obtained biopsies from deep
somewhat saline or otherwise osmotically dry environments: tissues, as well as body liquids or excretions [57]. Also the

transport system varies widely, ranging from sterile test 52.2.2 Detection Procedures
tubes (for moist samples) to empty, sealed Petri dishes or
even small paper envelopes (for dry skin and nail samples). 52.2.2.1 Specific Detection of the
When a specimen arrives in the medical mycology labo- Family Microascaceae
ratory, it is traditionally divided into at least two sections, Principle: DNA bases or primers reliably specific to the
one for direct microscopy and one for culturing. However, whole family Microascaceae have not been designed. This
histopathological samples are often taken and submitted is partly because the family itself is deficiently studied and,
to the relevant histological laboratory separately. Separate secondly, because the phylogenetic clade of Microascaceae
planning can also be recommended for obtaining and han- is surrounded by an ocean of other, much less known taxa.
dling of possible molecular samples. Procedures which Even from the clinical point of view, there is a clear need to
prepare the specimen for microscopy, for instance, may chart the whole surrounding of the family, not to speak of the
destroy the DNA. Very little work has been done spe- general needs of the very poorly funded basic research on
cifically on Microascus from the point of view of sample fungal diversity.
handling. Due to the current lack of information, one can identify a
given strain in the natural Microascaceae only by its relative
52.2.1.2 DNA Extraction molecular phylogenetical resemblance to some known spe-
A number of procedures have been developed and applied cies, not based on any defined molecular borders or current
for isolation of fungal DNA [58–65]. In general, chemical definitions of taxa.
lysis and/or mechanical breaking (e.g., mortar, agitated glass Procedure: To identify a given strain as a member of the
beads, sonication and/or rapid deep-freezing using liquid family Microascaceae, one should sequence sufficient lengths
nitrogen) has been utilized to disrupt the rigid cell walls of of its genome, for example, parts of LSU, ITS, and comple-
fungi. Proteinaceous cell components are eliminated enzy- menting protein-coding genes. The sequences should be
matically (using proteinase K). Suitable buffers such as analyzed with proper phylogenetic methods, including par-
Tris-EDTA (Tris-hydroxymethylaminomethane and ethyl- simony analysis, with all data existing in public data banks.
enediamine tetraacetic acid) are used to stabilize the condi- Note: Naturally, all data available by traditional meth-
tions. In addition, several commercial kits are available for ods such as conidiogenesis must be evaluated together with
DNA extraction [60,62,65]. the molecular tests. With normally sporulating strains, the
In DNA extraction, one must naturally consider whether typical conidia may reveal the broad affinities in a couple of
the sample is taken directly from the infected human tissue days, and in some cases may be already present in human
which contains large amounts of human DNA and perhaps tissue a assessed by direct microscopy. Therefore, prelimi-
other microbes or, supposedly, a pure culture. Even in the lat- nary screening by microscopy provides a cost-effective,
ter case, however, pieces of human tissue debris and contami- genus-level identification that can be subsequently verified
nating microbes may hide on agar under the fungal mycelium by molecular procedures such as DNA sequencing.
and be inadvertently taken in the sample. The toughness and
other physical characters of the sample may also contribute 52.2.2.2 Microascus-Specific Detection
to the outcome. Principle: LSU bases preliminarily characteristic to the main
Typically, pure fungal cultures (either agar- or broth- subclades of Microascaceae have been proposed by Issakainen
based) are minced at room temperature (or in liquid nitrogen et al. [3]. These, when continuously complemented with
if RNA is to be isolated). Fungal DNA is purified by pro- recent and future sequencing results, can be used for specific
teinase K-digestion in the presence of sodium dodecyl sulfate detection in the approximate levels of genus and subgenus.
(SDS) followed by phenol-chloroform extraction together Due to lack of basic molecular mapping and delimitation,
with 0.5 M KCl. The extract is then treated with RNase the molecular detection of individual Microascus species is
(Sigma) for 30 min at 37°C, extracted again with phenol- still very unreliable. This situation will certainly improve as
chloroform, precipitated by isopropanol and resuspended in soon as studies based on ITS regions and on complement-
a final concentration of 10 μg/mL. ing protein genes will cover some hundreds of strains from
To isolate DNA from nails, commercial kit based on glass Microascus and from all the closest genera.
beads can be utilized. Namely, 0.5–3 mg of nail material in Kardjeva et al. [66] described a PCR assay for S. brevi-
300 μL cell lysis solution is added with 5–10 mg glass beads caulis targeting the large subunit of the rRNA gene with
(0.5 mm in diameter; BioSpec Products Inc.). The mixture is the SCOP primer pair (5′-GAA TGC TGC TCA AAA TGG
vortexed for 1 min. After incubation for 1 h in a 65°C, 3 μL GAG-3′ and 5′-CAT TAC GCC AGC ATC CTTG C-3′),
proteinase K solution (20 mg/mL) is added. The tube is incu- which results in the amplification of a specific 336 bp frag-
bated overnight at 55°C and vortexed again. Then, 100 μL ment. Given that M. brevicaulis, Microascus manginii
of protein precipitation solution is added. The tube is mixed (anamorph: Scopulariopsis candida), and S. koningii share
a few times by inversion and centrifuged for 5 min. After sequence homology in the region concerned, a positive result
incubation at −20°C overnight, the tube is centrifuged for with the SCOP primers could indicate any of these three spe-
10 min. The DNA pellet is dissolved in 10–30 μL hydration cies. On the other hand, S. brevicaulis is more common in
solution [66]. association with nails than either of the other two species

Procedure primer design and interpretation of results can be made only

after such data is at hand.
1. The PCR mixture (25 μL) is comprised of 2.5 U Taq Development of routineous, affordable molecular tests
DNA polymerase and 1× PCR buffer (containing even for Microascus species and their pathogenic strains
10 mM Tris–HCl pH 9.0, 50 mM KCl, and 1.5 mM would help early confirmation of the infecting fungal spe-
MgCl2), 200 μM of each deoxynucleoside triphos- cies in clinical samples [71]. This would facilitate prompt
phate, 15 ng of template DNA, 20 pmol each of the implementation of appropriate treatment regimens that would
SCOP primers and distilled water (to 25 μL). reduce suffering not only in chronic nail infections but also
2. The PCR amplification is conducted in a thermocycler in the rare, severe infections of the immunocompromised
with the following program: 1 cycle of 94°C for 3 min; patients [33,35,73–82]. Clinically, the whole family shares the
35 cycles of 94°C for 45 sec, 69°C for 1 min, and 72°C feature of being resistant to many common drugs, especially
for 1 min; and a final cycle of 72°C for 10 min. in the serious infections of immunocompromised patient.
3. Five microliters of each reaction is loaded on 1.2% In these, even Microascus species are lethal. A family-level
agarose gels and electrophoresed for 1.5 h at 100 V diagnostic primer of Microascaceae incorporated in multi-
in 0.5× TBE buffer (89 mM Tris-borate, 2.5 mM microbial screening chips might thus be of clinical interest.
EDTA pH 8.3). The gels are stained with ethidium Assuming that other indicators of a deep fungal infection are
bromide and photographed. present, a positive result on this test would suggest, for exam-
ple, that Amphotericin B is not the drug of choice.
Note: The SCOP primers are able to detect as little as Regarding a family-level test, Microascaceae seems to be
150 pg of template DNA. Given the fact that 10 pg of target a natural molecular taxonomic entity which has a rather long
DNA generally corresponds to circa 25 fungal cells or colony evolutionary distance from other groups of pathogenic fungi
forming units per clinical specimens, the detection limit of [1,83,84]. However, the evolutionary distance only applies to
the SCOP assay should correspond to 375 or more cells per the pathogenic species. In the phylogenetic trees, each patho-
clinical specimen. Sequences derived from the 336 bp PCR genic species is closely surrounded by an unknown number of
products shows a 100% identity with the three species that environmental species which have not appeared as pathogens,
are identical in this region. at least not yet. The same is true even within a single species,
considering the varying pathogenities of separate Microascus
52.3 C
onclusion and Future strains [41]. In any event, molecular tests offer very effective
new tools, as compared with the conventional methods.
Perspectives
Within Microascus, more basic research is definitely
Microascus, together with some less known saprophytic gen- needed. Use of shotgun molecular methods and metage-
era, constitutes more than a half of the family Microascaceae nomic approaches will probably dig out a big number of new
as currently outlined [2,4,7,11,14,54,67,69]. A smaller part of related fungi from soils. Therefore, it would make sense to
the family consists of a separate clade which contains other design an initial molecular test above the approximate rank
human pathogenic genera, especially Pseudallescheria (see of the family and then, stepwise or by nested tests, refine the
Chapter 58 in this book) [68]. study to species.
Regarding the clinical importance and molecular detec- Regarding the development of test procedures, it is rel-
tion of Microascus, M. brevicaulis stands out as a regular evant to find out whether a given test can be performed of
nail pathogen [20]. Kardjeva et al. [66] has designed a LSU a pathologists’ fixed microscopy slide, of a separate fresh
rDNA-based molecular test, which seems to be specific for tissue specimen, or of a pure fungal culture on a plate—or
this clade but not for species. Tests based on ITS or other several of these. Molecular test applicable to histological
variable areas can be designed in near future for species level. stainings would elucidate the location and pathogenic role of
However, the work of Filipello Marchisio et al. [41] point out Microascus in nails, for instance.
the need for molecular or other tests which are targeted to the Speculating on a DNA-based clinical diagnosis of
enzyme armamentarium of a given Microascus strain, since Microascus-related fungi within 10 years, for instance, the
this could directly differentiate between nail pathogenic and specimens could perhaps be divided into (a) nails and (b) all
contaminating strains. other types of specimens including upper respiratory cavities,
With the advent of nucleic acid amplification technology eyes, ears, wounds, catheter sites, and skin abscesses. Due
along with vast improvement in DNA sequencing proce- to the repeated occurrence of M. brevicaulis in the skin and
dures, it is possible to have the identity of a fungus verified in ears, the classification of the latter specimens may be debated.
hours, instead of days or weeks. Species of Microascus were Regarding (a) Microascus infections of nails, the benign
successfully included in such tests of Kardjeva et al. [66] and course and contaminated nature of such infections do actu-
Vollmer et al. [53]. In coming years, the most limiting factor ally mask a very complex question. Based on current knowl-
will not be the technology but the effort put in basic mapping edge, an ideal molecular test would simultaneously combine
of the thousands of environmental fungal strains which may (i) in situ histological microscopy to confirm the nail tissue
seem to have no importance but which form the phylogenetic invasion; (ii) in situ identification of M. brevicaulis on species
framework out of which the pathogens stand out. Reliable level; (iii) identification of other Microascaceae (including

Pseudallescheria and Petriella) at least on family level; 8. Abbott, S.P., Sigler, L., and Currah, R.S., Microascus brevi-
(iv) presence of keratinolytic enzymes to recognize patho- caulis sp. nov., the teleomorph of Scopulariopsis brevicaulis,
genic strains; (v) simultaneous presence of dermatophytes, supports placement of Scopulariopsis with the Microascaceae.
Mycologia, 90, 297, 1998.
perhaps also of selected bacteria; (vi) perhaps unknown host
9. Abbott, S.P. and Sigler, L., Heterothallism in the Microascaceae
immunity factors; and (vii) perhaps molecular markers of demonstrated by three species in the Scopulariopsis brevicau-
drug resistance. All this should be based on conventional lab- lis series. Mycologia, 93, 1211, 2001.
oratory procedures to ensure that test results are not based on 10. Abbott, S.P., Lumley, T.C., and Sigler, L., Use of holomorph
gross contamination or other elementary pitfalls [13,14,46,85]. characters to delimit Microascus nidicola and M. soppii sp. nov.,
Regarding (b) other types of infections due to Microascus, with notes on the genus Pithoascus. Mycologia, 94, 362, 2002.
especially in immunosuppressed patients: A good minimal- 11. Malloch, D. and Cain, R.F., The genus Kernia. Can. J. Bot.,
istic molecular test for near future would be a rapid combined 49, 855, 1971.
12. Malloch, D. and Hubart, J.-M., An undescribed species of
test which extracts the following information directly from Microascus from the Cave of Ramioul. Can. J. Bot., 65, 2384,
the fresh patient tissue specimen of human tissue: (i) Family 1986.
Microascaceae (including Microascus) positive or negative; 13. de Hoog, G.S. et al., Atlas of Clinical Fungi, 2nd edn., vol.
(ii) Pseudallescheria boydii—Scedosporium apiospermum 1. Centraalbureau voor Schimmelcultures, Utrecht, the
species complex (including the new species) positive or nega- Netherlands, 2000.
tive; (iii) Scedosporium prolificans positive or negative; and 14. de Hoog, G.S. et al., Atlas of Clinical Fungi Cd-Rom
perhaps (iv) Microascus manginii clade positive or negative. (CD-ROM), 2nd edn., ASM Press, Washington, DC, 2007.
15. Mycology Online at http://www.mycology.adelaide.edu.au/,
Assuming good selectivity and sensitivity by the first three
accessed on August 1, 2010.
tests, already, one could in principle infer the risk of a seri- 16. Issakainen, J. et al., Occurrence of Scopulariopsis and
ous Microascus infection from the result that the family is Scedosporium in nails and keratinous skin. A 5-year retro-
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19. Romano, C., Gianni, C., and Difonzo, E.M., Retrospective
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agents have been either unidentified or other species than cance of nondermatophytes. Med. Mycol., 43, 39, 2005.
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22. Svejgaard, E.L. and Nilsson, J., Onychomycosis in Denmark:
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53 Myceliophthora and Thielavia
Dongyou Liu
Contents
53.1 Introduction...................................................................................................................................................................... 445
53.1.3 Diagnosis.............................................................................................................................................................. 446
53.2 Methods............................................................................................................................................................................ 447
53.3 Conclusion........................................................................................................................................................................ 448
References.................................................................................................................................................................................. 448
53.1 Introduction with more luxuriant colonies growing at the elevated tem-
peratures. Colonies are initially white and cottony and later
53.1.1 Classification and Morphology become pale brown and powdery, without a well-defined
The genus Myceliophthora is the only member of the margin. Submerged hyphae (up to 6.0 μm in width) are wider
mitosporic Chaetomiaceae group, family Chaetomiaceae, than aerial hyphae (0.8–3.0 μm). Blastoconidia are borne ter-
order Sordariales, class Sordariomycetes, subphylum minally or laterally on the hyphae, sometimes with short or
Pezizomycotina, phylum Ascomycota, and kingdom Fungi. long pedicels and occasionally with a secondary blastoco-
The family Chaetomiaceae covers the genera of Achaetomium, nidium being produced from the distal end of the first. One
Aporothielavia, Chaetomidium, Chaetomium, Corylomyces, to four blastoconidia are borne on one hyphal cell or ampul-
Corynascus, Farrowia, Thielavia, and Zopfiella as well as liform swelling. Conidia (4.5–11.0 × 3.0–4.5 μm) are obovoid
mitosporic Chaetomiaceae and unclassified Chaetomiaceae. to pyriform, hyaline, smooth, and thick walled and become
Currently, the genus Myceliophthora consists of eight spe- rough walled at maturity [3].
cies: Myceliophthora fergusii, Myceliophthora fusca, Because its hyphae lack clamp connections (which are
Myceliophthora hinnulea, Myceliophthora indica, common in fungi with basiomycetous affinities), M. ther-
Myceliophthora lutea, Myceliophthora sulphurea, mophila was transferred from the genus Sporotrichum to
Myceliophthora thermophila, and Myceliophthora velleream, Chrysosporium. However, given that Chrysosponum spp.
of which Myceliophthora thermophila (obsolete synonyms: show a maximum growth temperature of 45°C rather than
Chrysosporium thermophilum and Sporotrichum thermophi- 48°C and produce arthroconidia, the fungus was further
lum) is an anamorph of Thielavia heterothallica [1]. transferred to the genus Myceliophthora.
The genus Thielavia in the family Chaetomiaceae con- Thielavia is a teleomorph of Myceiophthora, with an opti-
sists of 20 recognized species: Thielavia appendiculata, mal growth temperature of 37°C. On potato dextrose agar
Thielavia arenaria, Thielavia australiensis, Thielavia (PDA) and Sabouroud dextrose agar (SDA), Thielavia forms
basicola, Thielavia cephalothecoides, Thielavia coacti- floccose, dark gray, broadly-spreading colonies, with a dis-
lis, Thielavia fragilis, Thielavia gigaspora, Thielavia het- tinct felt-like aerial mycelium on front and a gray to black
erothallica, Thielavia hyalocarpa, Thielavia hyrcaniae, background on reverse. Its mycelium consists of hyaline and
Thielavia intermedia, Thielavia microspora, Thielavia dark olivaceous, branched, and septated hyphae, whose walls
minuta, Thielavia ovispora, Thielavia rapa-nuensis, are smooth and measure 1.0–3.0 µm. Aleuriospores (conidia)
Thielavia subthermophila, Thielavia terrestris, Thielavia appear laterally and terminally on hyphae. Being single-
terricola, and Thielavia tortuosa as well as three unas- celled, hyaline, or light brown in color, spores are broadly
signed species (Thielavia sp. 1863, Thielavia sp. B27, and clavate with a truncate base, and measure 5 × 3 µm in diam-
Thielavia sp. MUCL 40242) [1,2]. Of these, Thielavia het- eter. In mature cultures, ascomata (cleistothecia) develop
erothallica (the ascomycetous teleomorph or perfect state within a mycelial mat measuring up to 180 µm. The wall
of Myceliophthora thermophila) and Thielavia terrestris are of ascomata consists of tectura epidermoidea coated with
relevant to human infections. dark hyphae. The broad, fusiform ascospores (16 × 9 µm) are
Myceliophthora thermophila colonies reach 40 mm in single-celled, with a chacteristic superficial germ pore (mea-
diameter after 2 weeks (25°C) on potato flake agar (PFA), suring 1.0–1.5 µm) [7].
445

53.1.2 Clinical Features and Pathogenesis the aortic outflow graft from the LVAD, and transesophageal
echocardiography (TEE) imaging allowed visualization of
Myceliophthora thermophila is a thermophilic, saprobic fun- a large mass or vegetation located at the aortic insertion of
gus that is commonly present in silage, fresh alfalfa grass for- the LVAD outflow conduit along with a severely dilated and
age, hay, wheat straw compost, wood pulp, birch chips, dry hypokinetic LV. The patient was taken to the operating room
pasture soil, and animal excreta. As a disease-causing agent for exploration and replacement of the existing LVAD for
of cultivated mushrooms, the organism occasionally infects presumed LVAD endocarditis. Histologic examination of the
humans after traumatic injuries in agricultural settings or outflow graft showed extensive fungal overgrowth. Cultures
cardiac surgery. Individuals with severe immune system of the mass yielded a rare environmental thermophilic fun-
abrogation are also vulnerable to M. thermophila infection gus, Myceliophthora thermophila. Blood cultures also dem-
primarily through environmental contamination. Clinical onstrated fungal growth after 7 days of incubation. The
manifestations range from single subcutaneous nodules, patient undertook aggressive antifungal therapy with both IV
keratitis, vegetation growth in the great vessels and heart to liposomal amphotericin B 10 mg/kg daily for 6 weeks and
disseminated disease and death [5,6]. voriconazole 400 mg twice daily orally. After explanation
Destino et al. [7] described a case of Myceliophthora of the infected LVAD, the patient’s blood cultures cleared.
thermophila knee and distal femur infection in a 4-year-old The patient was discharged 27 days after presentation and
boy following a barnyard pitchfork injury. The patient devel- remained on oral voriconazole for chronic suppression until
oped osteomyelitis of the distal femur, with severe destruc- the time of his heart transplant 2 years later.
tive osseous and cartilaginous infection. Culture of debrided Theoulakis et al. [4] reported the first case of fungal kera-
bone or cartilage yielded M. thermophila, although fungal titis due to Thielavia sp. involving a 10-year-old girl. Two
elements could be seen histologically in other surgical sam- weeks after suffering from ocular plant injury, the patient
ples. Clinical improvement required the prolonged use of presented with pain and corneal stromal infiltration with cen-
multiple antifungal agents. tral ulceration and ill-defined margins. Cultures of corneal
In a separate study, Tekkok et al. [8] documented a case scrapings and biopsy yielded a fungus that was confirmed as
of combined Clostridium perfringens and Myceliophthora Thielavia subthermophila by sequence analysis of the ribo-
thermophila infections in a 21-month-old male patient, somal internal transcribed spacer (ITS) region. The organism
who sustained a penetrating head injury from a rusty nail responded to treatment of topical amphotericin B and oral
in a barnyard. The patient developed a gas-containing left voriconazole.
parietal brain abscess and presented with high fever, galeal
swelling, and seizure. Initial cultures of wound aspira-
tions grew Clostridium perfringens. As the wound failed 53.1.3 Diagnosis
to respond to antibiotic treatment, an excised specimen was Myceliophthora thermophila is a heterothallic, thermophilic
obtained 6 weeks after the injury, which yielded a sapro- fungus with spherical, black, nonostiolate cleistothecia con-
phytic and opportunistic fungus, Myceliophthora thermoph- taining ellipsoidal, evanescent asci. Asci contain eight one-
ila. The patient recovered with en bloc resection of the lesion, celled ellipsoidal ascospores and are deep brown to black,
6 weeks of amphotericin B, and 4 months of itraconazole. with one germ pore. As a pigmented filamentous hyphomy-
Farina et al. [9] described the first isolation of a human cete and an agent of phaeohyphomycosis, melanin in its cell
pathogenic strain of M. thermophila, causing fatal vas- wall provides a distinguishing feature. However, cell walls
culitis in a 22-year-old Italian woman affected by cystic of phaeoid molds may appear hyaline or clear upon routine
medial necrosis. The patient developed fungal aortitis after microscopy despite the presence of melanin. Staining of
cardiac surgery for aortic insufficiency. After experiencing hyphal elements with Masson-Fontana melanin stain usu-
two episodes of septic embolization, the patient underwent ally demonstrates pigment, permitting identification of a
replacement of the aortic root and initial ascending aorta dark fungus. M. thermophila grows on a variety of media
by a homograft. The lumina of the ascending aorta, aortic recommended for mold identification (e.g., potato dextrose
arch, and the origin of the innominate artery were completely or 2% malt agar) with optimal temperatures at 35°C–42°C,
filled with vegetation, from which the phaeoid thermophilic although it also grows well near 50°C, reflecting its epithet
hyphomycete Myceliophthora thermophila was isolated in “thermophila.” Considering that most clinical specimens are
pure culture. The patient succumbed to the infection and its incubated at 30°C, the less than optimal growth temperature
complications. for M. thermophila may be a contributing factor for the rela-
Weitzel et al. [10] reported a case of Myceliophthora ther- tive infrequent isolation of this thermophilic fungus, which
mophila infection in a 40-year-old man after an left ventricle requires elevated temperatures for enhanced recovery.
assist device (LVAD) placement for dilated cardiomyopathy Macroscopic and microscopic identification of
and inotrope-dependent New York Heart Association class Myceliophthora thermophila can be achieved upon observation
IV heart failure as a bridge to transplantation. Two months of (i) tan to brown powdery colonies with ill-defined margins
later, the patient presented to the hospital for evaluation of when grown on potato flakes agar at 42°C; (ii) more luxuri-
fever, chills, and altered mental status. Computed tomogra- ant growth at elevated temperatures of 35°C and 42°C than at
phy imaging of his chest revealed an abnormality located at 26°C; (iii) septate vegetative hyphae with conidial production

Myceliophthora and Thielavia 447
from ampulliform swellings; and (iv) obovoid (inverted egg a 10 mg/mL concentration of proteinase K to the tube, the
shaped) or pyriform (pear shaped) conidia measuring 4.5– mixture is incubated at 65°C for 10 min. With the addition of
11.0 × 3.0–4.5 μm that are hyaline and smooth when immature, 140 μL of 5 M NaCl solution, the mixture is combined with
becoming darker and roughened at maturity [7]. 1/10 volume (∼65 μL) of CTAB (cetyltrimethylammonium
Since conventional laboratory diagnosis of fungal infec- bromide) buffer 10%, followed by incubation for another
tions is dependent on recovery of fungi from culture of clini- 30 min at 65°C. One volume (∼700 μL) of chloroform–
cal specimens for subsequent identification of the presence isoamyl alcohol (vol/vol = 24/L) is added and mixed by
of reproductive structures, the process can be lengthy (up to inversion. After incubation for 30 min at 0°C (on ice water)
21 days and inconclusive [some molds fail to sporulate]). The and centrifugation at 14,000 rpm at 4°C for 10 min, the top
development and application of rapid, accurate diagnostic layer is transferred to a clean Eppendorf tube. The sample is
techniques are essential for the initiation of targeted anti- added with 225 μL of 5 M NH4-acetate and incubated for at
fungal therapy for a widening spectrum of emerging fungal least 30 min (on ice water) and centrifuged. The supernatant
pathogens, especially in immunocompromised individuals. is transferred to a clean sterile Eppendorf tube and mixed
Independent of mold sporulation, PCR amplification and with a 0.55 volume (∼510 μL) of ice-cold isopropanol. After
sequencing of target regions within the ribosomal RNA gene centrifugation for 7 min at 14,000 rpm and 4°C (or room tem-
complex (e.g., internal transcribed spacer (ITS) regions 1 and perature), the supernatant is decanted. The pellet is washed
2 located between the highly conserved small (18S) and large with ice-cold ethanol 70% two times and dried by using a
(28S) ribosomal subunit genes in the rRNA operon) allow vacuum dryer. The powder is resuspended in 48.5 μL of Tris-
identification to the species level for many fungi includ- EDTA buffer with 1.5 μL of 10 mg of RNase/mL, incubated
ing Myceliophthora thermophila and serve as a valuable at 37°C for 15–30 min, and stored at −20°C until use [13].
approach to detect clinically relevant pathogens [11].

53.2 Methods
53.2.1 Sample Preparation DNA-binding dye and amplicon-melting temperature anal-
ysis for fungal detection using pan-fungal primers ITS1
Clinical specimens are examined with a direct calcofluor forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
white stain for septate hyphal elements. Portions of the reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
samples are inoculated on inhibitory mold agar (containing of the fungi is verified by subsequent sequencing analysis.
cyclohexamide), modified Sabouraud agars, or potato dex-
trose agar. Microscopic structures are observed on tease or Procedure
tape preparations and slide cultures for up to 21 days [12].
After the growth for 1–7 days on potato dextrose agar 1. PCR mixture is composed of 1× Lightcycler
slants, lysates are prepared from approximately 1 cm2 of FastStart DNA Master Hybridization Probes mixture
mycelia with IDI lysis kits (GeneOhm Sciences, San Diego, (Roche Applied Science) containing deoxynucleo-
CA). Briefly, in a biological safety cabinet, mycelia are col- side triphosphates, FastStart Taq DNA polymerase,
lected by scraping the slant with a sterile stick in 1 mL of and 1 mM MgCl2 (additional MgCl2 is added to a
sterile, molecular-grade H2O. The material is transferred to a final concentration of 4.6 mM), 0.4 μM each of ITS1
2 mL screw-cap tube. The tubes are centrifuged for 1 min at forward and ITS4 reverse primers, 1× SYBR green
6000 × g. If the mycelia do not pellet, the material is contained (Molecular Probes), and 3 μL template DNA.
with a pediatric blood serum filter (Porex Corp., Fairburn, 2. Thermal cycling parameters include 95°C for
GA). Supernatant is removed. The material is resuspended 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s,
in 200 μL of IDI sample buffer and transferred to the lysis and 76°C for 30 s; and a final extension at 72°C for
tube, which contained glass beads. Lysis tubes are vortexed 2 min.
on the highest setting for 5 min. The tubes are placed in a 3. The quality of the amplicon is determined using the
boiling water bath for 15 min. Tubes are centrifuged for 5 min derivative of the melt analysis curve (55°C–99°C,
at 16,000 × g. The supernatant is stored at −20°C until ampli- 45 s hold at 55°C, and 5 s/°C) using the RotorGene
fication [12]. 3000 (Corbett Robotics, Inc.).
Alternatively, about 1 cm2 of fungal material is trans- 4. The amplified product is purified for bidirectional
ferred to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) sequencing using ExoSAP-IT (USB Corp.). Five
mixture of silica gel and Celite (silica gel H, Merck 7736/ μL of Big Dye Terminator Ready Reaction Mix v.
Kieselguhr Celite 545; Machery) and 300 μL of TES buffer 1.1 (Applied Biosystems) is added to 4 μL of each
(2 g Tris (hydroxymethyl)-aminomethane, 0.38 g Na-EDTA, primer (0.8 pmol/μL) and 3 μL of purified PCR
and 2 g sodium dodecyl sulfate in 80 mL of ultrapure water product. Cycle sequencing is performed with a 9700
(pH 8)). The fungal material is ground with a micropestle for thermal cycler (ABI), using 25 cycles of 96°C for
1–2 min. The volume is adjusted by adding 200 μL of TES 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
buffer. After vigorous shaking and the addition of 10 μL of reaction products are passed through a Sephadex

G-50 fine column to remove unincorporated dye ter- References

1. The UniProt Consortium. Available at http://www.uniprot.org/,
run on an ABI Prism 3100 Genetic Analyzer with a accessed on August 1, 2010.
50 cm capillary array. 2. Moustafa AW, Abdel-Azeem AM. Thielavia gigaspora, a
5. Sequences are analyzed with the SmartGene new thermotolerant ascomycete from Egypt. Microbiol. Res.
Integrated Database Network software version 2008;163(4):441–444.
3.2.3 vr. SmartGene is a web-based software and 3. Bourbeau P et al. Fatal disseminated infection caused by
database system with reference sequences derived Myceliophthora thermophila, a new agent of mycosis: Case
from the National Center for Biological Information history and laboratory characteristics. J. Clin. Microbiol.
1992;30(11):3019–3023.
(NCBI) GenBank repository. 4. Theoulakis P et al. Keratitis resulting from Thielavia subther-
mophila Mouchacca. Cornea 2009;28:1067–1069.
Note. If preferred or real time PCR instrument is not avail- 5. Abdel-Hafez AI, el-Sharouny HM. Keratinophilic and sapro-
able, standard PCR may be performed with primers ITS1 phytic fungi isolated from students’ nails in Egypt. J. Basic
and ITS4, and the resulting amplicon is sequenced with the Microbiol. 1990;30(1):3–11.
same primers. Sequence-based identifications are defined 6. Revankar SG et al. Disseminated phaeohyphomyco-
by percent identity: species, ≥99%; genus, 93%–99%; and sis: Review of an emerging mycosis. Clin. Infect. Dis.
2002;34:467–476.
inconclusive, ≤93%. For strains producing discrepant iden-
7. Destino L et al. Severe osteomyelitis caused by Myceliophthora
tification between the methods based on phenotypic char- thermophila after a pitchfork injury. Ann. Clin. Microbiol.
acteristics and ITS sequence analysis, the D1–D2 region of Antimicrob. 2006;5:21.
the large-subunit RNA gene is amplified with primers NL1 8. Tekkok IH, Higgins MJ, Ventureyra EC. Posttraumatic
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 gas-containing brain abscess caused by Clostridium per-
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for fringens with unique simultaneous fungal suppuration by
species clarification [14]. Myceliophthora thermophila: Case report. Neurosurgery
1996;39:1247–1251.
9. Farina C et al. Fatal aortic Myceliophthora thermophila infec-
53.3 Conclusion tion in a patient affected by cystic medial necrosis. Med.
Mycol. 1998;36:113–118.
Myceliophthora thermophila is a saprobic, thermophilic pha- 10. Weitzel N et al. Left ventricular assist device outflow cannula
eoid mold that commonly occurs in pasture soil, wood chips, obstruction by the rare environmental fungus Myceliophthora
straw, moldy hay, compost piles, and other environmental set- thermophila. Anesth. Analg. 2009;108:73–75.
tings where heat is generated. It is also present in the excreta 11. Bagyalakshmi R et al. Newer emerging pathogens of ocular
and rumen of cattle. As a pathogen of cultivated mushrooms, non-sporulating molds (NSM) identified by polymerase chain
reaction (PCR)-based DNA sequencing technique target-
M. thermophila may cause localized infections in immu-
ing internal transcribed spacer (ITS) region. Curr. Eye Res.
nocompetent individuals through traumatic injury and dis- 2008;33:139–147.
seminated infections in immunocompromised patients, with 12. Pounder JI. Discovering potential pathogens among fungi
a propensity for the vascular system. Due possibly to the identified as nonsporulating molds. J. Clin. Microbiol. 2007;
fact that clinical specimens are routinely cultured at 30°C 45(2):568–571.
and that many saprobic fungi are discarded as contaminants 13. Zeng JS et al. Spectrum of clinically relevant Exophiala
without further analysis, thermophilic M. thermophila is species in the United States. J. Clin. Microbiol.
2007;45:3713–3720.
infrequently reported. With the help of molecular techniques
14. Leaw SN et al. Identification of medically important yeast
such as PCR amplification and sequencing of rRNA genes species by sequence analysis of the internal transcribed spacer
and internal transcribed spacer (ITS) regions, the true extent regions. J. Clin. Microbiol. 2006;44:693–699.
of opportunistic M. thermophila infections in humans will
become clearer in future.

54 Neocosmosporas
Palanisamy Manikandan, Csaba Vágvölgyi, Venkatapathy
Narendran, Kanesan Panneer Selvam, and László Kredics
Contents
54.1 Introduction...................................................................................................................................................................... 449
54.1.1 Classification......................................................................................................................................................... 449
54.1.2 Epidemiology and Pathogenesis........................................................................................................................... 451
54.1.4 Diagnosis.............................................................................................................................................................. 453
54.2 Methods............................................................................................................................................................................ 455
References.................................................................................................................................................................................. 456
54.1 Introduction In 1984, Cannon and Hawksworth15 performed a revision

of the genus on the basis of scanning electron microscopi-
54.1.1 Classification cal investigation of ascospore ornamentation. N. vasinfecta
Neocosmospora is a teleomorph, homothallic, filamentous and N. africana were reduced to the level of varieties of
fungal genus belonging to the Fusarium solani species com- N. vasinfecta as var. vasinfecta and var. africana, and the exis-
plex also known as Fusarium section Martiella (Ascomycota, tence of diploid and tetraploid races corresponding to small
Pezizomycotina, Sordariomycetes, Hypocreomycetidae, and large ascospore morphs was postulated. Further, all pre-
Hypocreales, Nectriaceae). The genus was introduced viously described N. vasinfecta varieties (var. tracheiphila,
in 1899 by Smith.1 He described the type species of the var. nivea, var. sesame, var. minor, and var. conidiifera) were
genus as N. vasinfecta, and differentiated N. vasinfecta rejected and N. ornamentata was reduced to synonimity with
var. vasinfecta, var. tracheiphila, and var. nivea that were N. vasinfecta. The previously described species N. tenuicris-
isolated from diseased cotton (Gossypium sp.), cowpea tata, N. parva, N. striata, as well as N. indica Wadhwani,16
(Vigna unguiculata), and watermelon (Citrullus lanatus), were accepted and included in the revised taxonomy of the
respectively. Cross-inoculation experiments revealed that genus.15 Table 54.1 shows morphological characters of these
none of these races could infect the host plants of the oth- five species.
ers; however, no other solid criteria for the separation of the In 1989 Udagawa et al.17 described further two species of
three races could be defined.1 Smith erroneously regarded the genus, N. arxii and N. boninensis. N. endophytica with a
Fusarium vasinfectum (later redefined as F. oxysporum var. Penicillifera anamorph,18 N. diparietispora,19 and N. spinu-
vasinfectum) as an anamorph of N. vasinfecta. In 1903, von losa were also introduced.20
Jaczewski2 introduced a new variety, N. vasinfecta var. ses- The introduction of molecular tools for studying the tax-
ame, from Sesamum orientale. A new taxon, N. africana, onomic relationships of fungi revealed further information
was described from Africa in 1955,3 with cerebriform rather about the phylogeny of Neocosmospora. Using sequences
than rugose ascospores. This was followed by morphologi- of 18S and 28S ribosomal DNA it was demonstrated that
cal and developmental investigations of the two taxa.4–7 Both N. vasinfecta is closely related to Nectria haematococca.21,22
species were found later in Japan.8 N. ornamentata differ- O’Donnell23 suggested that the Acremonium-like anamorph
ing in the shape and degree of rugoseness of the spores was of N. vasinfecta is comparable to the microconidial form
described by Barbosa,9 while new varieties under the names of fusaria. During the revision of Hypocreaceae in 1999,
of N. vasinfecta var. minor10 and var. conidiifera11 were also Rossman et al.24 excluded the species having green asco-
introduced. Later, Udagawa and Horie,12 Mahoney,13 and spores and Penicillifer anamorphs from Neocosmospora
Ueda and Udagawa14 described N. striata, N. parva, and and replaced them to the genus Viridispora. This revision
N. tenuicristata, respectively. accepted the species N. vasinfecta, N. boninensis, N. indica,
449

TABLE 54.1
Key Morphological Characters of Five Species from the Genus Neocosmospora
Character N. vasinfecta N. tenuicristata N. parva N. indica N. striata
Mycelium White to pale buff, White to buff Inconspicuous, hyaline Very pale buff, Inconspicuous, hyaline
floccose somewhat darker
toward the edges,
floccose
Hyphae Septate, tending to Tending to aggregate in Septate, tending to Septate, tending to Septate
aggregate in strands, strands, there aggregate in strands aggregate in rope-like
there anastomosing anastomosing strands, there
anastomosing
Ascomata Orange-brown to Orange-red to reddish Orange-brown, very Formed freely at edge of Dark brown, pyriform,
red, ± globose, glabrous brown, ± globose, fragile, ovoid to pyriform, mycelium, orange to glabrous but for a
but for a number of ± glabrous or covered glabrous but for a number red, globose, glabrous number of rhizoidal
rhizoidal hyphae, with an inconspicuous of rhizoidal hyphae, but for a number of hyphae, ostiolate,
ostiolate, neck lined web of hyaline hyphae, ostiolate, neck lined with rhizoidal hyphae, periphyses not observed
with periphyses ostiolate, neck lined periphyses ostiolate with a short
with periphyses neck lined with
periphyses
Ascomatal Several-layered, outer Several-layered, outer Several-layered, with a Several-layered, with a Pigmented outer layer,
wall pigmented wall pigmented wall of ± pigmented outer wall of pigmented outer layer almost hyaline inner
of ± thick-walled textura thick-walled textura irregular textura intricata, of textura intricata, layer, both of textura
angularis, sometimes angularis, inner layers inner layer almost and thin hyaline inner epidermoidea
with an inconspicuous hyaline, also of textura hyaline, consisting of an layer(s) of textura
covering hyphal web, angularis ill-defined intermediate angularis
inner layers ± hyaline, between textura intricata
also of textura and textura angularis
angularis
Periphysoids Present but evanescent at Present at maturity, Not observed Evanescent at an early Not observed
an early stage, composed of very stage
consisting of vertically thin-walled hyaline
oriented rows of hyaline cells
thin-walled cells
Asci Cylindrical, thin-walled, Cylindrical, thin-walled, Cylindrical to cylindric- Clavate, 38–49 × 60–85 × 6–10 μm;
stalk 8–15 μm long, stalk 5–12 (–20) μm clavate, thin-walled, 15.5–22 μm, the longer cylindrical to cylindric-
body without long, body without short-stalked, without asci tending to be clavate, thin-walled, with
discernable apical discernable apical discernible apical thinner, very a small inconspicuous
structures, not structures, not structures, not evanescent, thin-walled, without apical ring which does
evanescent, eight- evanescent, eight-spored discernible apical not stain blue in iodine,
(rarely six to seven) eight-spored structures, evanescent, eight-spored
spored eight-spored
Ascospores Uniseriately arranged, Uniseriately arranged, Obliquely uniseriately or Biseriately arranged, Obliquely uniseriately or
buff to salmon pink in yellowish-brown, partially biseriately orange brown en partially biseriately
mass, pale yellow globose-ellipsoidal to arranged, orange-yellow masse, yellow-brown arranged, very pale
individually, globose to ellipsoidal to orange-buff en masse, individually, irregularly yellow to hyaline,
ellipsoidal golden yellow ellipsoidal ellipsoidal
individually, ellipsoidal
Ascospore Germ pores absent, Without germ-pores, Without germ-pores, with Without germ-pores, Without germ-pores, some
wall apical spore commonly often somewhat a reticulate ornamentation inner edge of wall spores with a median
has an irregularly irregular in thickness, mostly obscured by a elliptical in section, transverse straight or
thickened wall with an ornamentation weakly verrucose wall very irregular in curved septum, then very
of transversely epispore layer thickness, reticulate, slightly constricted at the
arranged ridges, apical the reticulations septum with transverse
spore commonly has an obscured in irregular hyaline flanges
irregularly thickened patches by a weakly
wall verrucose epispore
layer
Source: Based on Cannon, P.F. and Hawksworth, D.L., Trans. Br. Mycol. Soc., 82, 673, 1984.

Neocosmosporas 451
N. parva, N. spinulosa, N. tenuicristata, and N. vasinfecta N. vasinfecta was considered a possible myconematicide.
var. africana. Initial studies reported that this fungus did not parazitize
In a subsequent study, O’Donnell25 used sequence data eggs of the soybean cyst nematode Heterodera glycines35
from the nuclear large subunit 28S rDNA, the nuclear ribo- and also did not measurably affect egg viability.33 In con-
somal internal transcribed spacer (ITS) region, and from trast, Chen et al.36,37 found that N. vasinfecta is capable of
introns and exons of the translation elongation factor 1α penetrating the cyst wall of H. glycines and is moderately
(tef1) gene for studying the molecular phylogeny of the pathogenic to eggs with a hatch reduction ability between
F. solani species complex. The results clearly demonstrated 21% and 56%. In a subsequent study, filtrates from N. vasin-
that Neocosmospora is deeply nested within this complex fecta and the closely related F. solani grown in malt extract
with Fusarium sp. anamorphs. This molecular phylogenetic broth were toxic to second-stage juveniles of H. glycines and
study divided the F. solani species complex into three clades, also reduced egg viability.38 The substances in the culture
with Neocosmospora belonging to clade 3. O’Donnell25 for- filtrates of the fungus may play a role themselves in coloniza-
mally suggested synonimity of the genus Haematonectria tion of eggs, or may have anti-microbial activities that enable
Samuels and Nirenberg—a taxon proposed by Rossman competition with other fungi in soil and cysts.
et al.24 to include Nectria haematococca and the closely The type species of the genus, N. vasinfecta is considered
related species N. illudens, N. ipomoeae, and N. monilifera— a weak pathogen of roots causing little crop loss, though
with Neocosmospora and noted that inclusion of morpho- it may have a significant effect in conjunction with other
logically similar but phylogenetically unrelated species like pathogens.15 N. vasinfecta is known to cause root- and fruit-
N. parva within Neocosmospora results in polyphyly of the rot and seedling damping off in Malvaceae, Leguminosae,
genus. More recent molecular phylogenetic studies examined Piperaceae, Cucurbitaceae, etc. including pepper, peanuts,
the F. solani species complex with special emphasis on its beans, pigeon pea, coconuts, Albizziac, and others.39–42
members capable of causing infections in humans.26,27 Zhang The fungus is a presumed pathogen of the medicinal plant
et al.26 first reported that all known species from clinical Astragalus membranaceus,43 while certain strains are known
and veterinary sources—including N. vasinfecta—belong to be able to infect soybean (Glycine max).8,44 Nematodes can
to clade 3 of the species complex. This was confirmed in a be suspected as possible vectors of Neocosmospora species,
study of O’Donnell et al.27 where the ITS, 28S rDNA and tef1 and may also facilitate the entering of the fungus into the
sequence data were supplemented with sequence data from host plant through nematode lesions, as suggested by Gray
the second largest subunit of the RNA polymerase II (rpb2) et al.45 As a preventive measure, Smit and Knox-Davies46
gene in a multilocus approach. O’Donnell et al.27 noted that suggested hot-water treatment and acid scarification for the
besides F. solani f. sp. cucurbitae race 2,28 N. vasinfecta is elimination of N. vasinfecta from tea seeds.
the only other representative of the F. solani species complex Temporini and VanEtten47 provided experimental evi-
which has been shown to be pathogenic both to plants and to dence for Neocosmospora sp. NRRL 22470 (termed by the
humans. authors as N. boniensis), that this species is capable of caus-
ing disease on pea (Pisum sativum). The experiment was
performed after the discovery of the homologues for the pea
54.1.2 Epidemiology and Pathogenesis
pathogenicity genes (PEP cluster) of Haematonectria hae-
Species of Neocosmospora are almost exclusively tropical or matococca mating population VI in strain NRRL 22470.
subtropical. N. vasinfecta var. vasinfecta has been isolated The PEP cluster involves the pda1 gene, the product of which
from different locations including South Carolina (United converts the pea phytoalexin pisatin to a less toxic compound
States), Guinea Bissau, Nigeria, Sierra Leone, Tanzania, by demethylation, and is therefore a presumed virulence fac-
Zaire, Guyana, India, Bangladesh, Pakistan, Hong Kong, tor. The PEP cluster shows a discontinuous phylogenetic dis-
and Australia, while N. vasinfecta var. africana is known tribution: it could not be found in other representatives of the
from Egypt, Ethiopia, Gambia, Guyana, Nigeria, Sierra F. solani species complex but was detected in the more dis-
Leone, Zambia, South Africa, Uzbhekistan, India, Pakistan, tantly related pea pathogen F. oxysporum f. sp. pisi, suggest-
Sri Lanka, Venezuela,15 and Argentina.29 N. boninensis, ing its possible translocation via horizontal gene transfer.47
N. parva, N. spinulosa, and N. tenuicristata are known Watanabe et al.48 isolated and characterized a soybean
only from their type isolates, which derived from Japan, saponin hydrolase from a soil-derived isolate of N. vasinfecta
Galapagos Islands, Brazil, and Japan, respectively, while N. var. vasinfecta, which is capable of degrading soybean sapo-
indica is known from two isolations from India. nin and generating soyasapogenol B. The gene encoding for
Neocosmospora species can most commonly be isolated the enzyme has also been cloned, sequenced, and success-
from soil, often from the rhizosphere of plants15; however, fully expressed in T. viride enabling further characterization.
they were also reported to occur in special habitats. The Due to their ability to form complex sterols in fungal mem-
type strain of N. tenuicristata has been isolated from marine branes and to cause a loss of membrane integrity, saponins
sludge.14 N. vasinfecta species could be recovered from hairs are supposed to take part in the protection of the producer
of rodents in Nigeria,30 and it is also among the common gen- plants from phytopathogenic fungi.49 Deglycosylated sapo-
era that can be isolated from the females and cysts of nema- nins show reduced antifungal activities; saponin hydrolases
todes.31–34 Due to its frequent association with nematodes, appear therefore to facilitate the virulence of plant pathogenic

fungi in host plants. As N. vasinfecta is capable of infecting metabolites might be among the possible virulence factors of
soybean, its saponin hydrolase may thus represent a possible N. vasinfecta as a human pathogen.
virulence factor. N. vasinfecta was also reported to be able to Neocosmospora species may carry transposable elements.
hydrolyse inulin, a polysaccharide contained in the roots of Close relatives of Fot1, a member of the pogo transposon
certain plants including sunflower.50 The inulinase (2,1-beta- family prevalent in the F. oxysporum complex were found
d-fructanohydrolase) hydrolyses inulin into pure fructose, in five species from the distant section Martiella, including
being an excellent alternative for the production of fructose a Neocosmospora sp. isolate from African soil.64 Two cop-
syrups. Inulin-hydrolyzing fungi might therefore be used in ies of the element, Neo1 and Neo2 were found in the isolate
biotechnological processes. showing 98% nucleotide identity with each other. As Fot1
Phytotoxic metabolites may also contribute to the viru- elements could not be detected in other sections that are more
lence of N. vasinfecta as a plant pathogen. In the 1970s, closely related to the F. oxysporum complex, the authors sug-
phytotoxic naphtharizine derivatives were reported from gested horizontal gene transfer as the possible explanation for
N. vasinfecta39,51 and supposed to be related to pathogenesis; the discontinuous distribution.
however, Roos52 found no correlation between their produc- Since the 1960s, N. vasinfecta has been used as a model
tion and virulence of the fungus. N. vasinfecta var. afri- organism for research on fungal transport and metabolism of
cana NHL2298, a strain isolated from soil at Johannesburg, inorganic ions including ammonia,65,66 bicarbonate,67 potas-
South Africa was found to produce an α-pyrone named sium,68,69 chloride,70–74 zinc,75–77 magnesium,78 cadmium,79
neovasinone, which accelerated the root elongation of let- aluminum,80 and mercury,81 as well as organic compounds
tuce seedlings,53 and the related metabolite neovasinin, including acetate,82 glucose,83 and polyols related with
which is phytotoxic against soybean.54,55 Neovasinin is a osmotic adjustment.84,85
fungal metabolite containing a unique bicyclic unit, 2H,5H-
pyrano(4,3-b)pyran-2-one. To explore the biosynthetic path-
way of neovasinin, a search was conducted for biogenetically
related metabolites. This resulted in the identification and The first report about the clinical involvement of the genus
structure determination of further Neocosmospora metabo- Neocosmospora dates back to 1993.86,87 It was the description
lites including neovasipyrones A and B, neovasifuranones of a case of leg granuloma (a localized mycotic cyst of soft
A and B56 as well as neovasipyridones A−F.57,58 Two phy- tissue in the leg) caused by N. vasinfecta in a renal trans-
totoxic metabolites with a benzofuranone ring, vasinfectins plant and dialysis patient from Senegal. The latency period
A and B—which are diastereomers of each other with the between infection and clinical manifestation was suggested
structure of (E)-2-(3-hydroxy-2,4-dimethyl-1-hexenyl)-2,7- to be several years with a low level of inflammation. A cir-
dimethyl-5-propanoyl-2H-benzo[b]furan-3-one—were also culatory source for the inoculum was suggested as there was
isolated from N. vasinfecta NHL2298.59 Vasinfectins A and no trauma that occurred at the infection site. Surgery and a
B were found to cause chlorosis at 0.3 μg/leaf on young soy- small dose of ketoconazole (truncated prematurely because
bean leaves in the leaf spot assay. Cutler et al.60 isolated the of a cyclosporin toxicity side effect) eliminated the infection.
plant growth regulatory metabolite radicicol—5-chloro-6- Five years later, Kac et al.88,89 reported a case of osteoarthri-
(7,8-epoxy-10-hydoxy-2-oxo-3,5-undecadienyl)-β-resorcylic tis due to Neocosmospora in the legs of a French parachut-
acid μ-lactone, also known as monorden—from the culture ist who suffered an open ankle dislocation in Senegal. The
filtrates of N. tenuicristata. Radicicol significantly inhibited authors initially reported the causal agent as N. africana,88
the growth of etiolated wheat coleoptiles, caused slight necro- but in the final paper they modified the diagnosis to N. vasin-
sis on 10-day-old corn plants, and also proved to moderately fecta89 according to the revision of the genus by Cannon and
inhibit Bacillus subtilis and B. cereus. Radicicol produced by Hawksworth.15 The fungus grew repeatedly from the wound
N. tenuicristata proved to be a cell differentiation modula- and from the progressing osteitis despite extensive cleaning
tor; it inhibited embryonic angiogenesis in a bioassay sys- measures and treatment with amphotericin B (AMB). The
tem involving chorioallantoic membranes of growing chick minimal inhibitory concentration of AMB toward the isolate
embryos.61 The abilities of radicicol to inhibit both the pro- was low; however, the parenteral antifungal therapy failed.
liferation of, and plasminogen activator production by vas- Both of the above mentioned patients were cured by radi-
cular endothelial cells were suggested to be involved in the cal surgical interventions that prevented dissemination of the
anti-angiogenic action. The authors concluded that radicicol disease, limiting it to the lower extremities. Because of the
might be a potential drug for treating different angiogenesis- poor susceptibility of the fungus, complete surgical excision
dependent diseases, such as solid tumors, psoriasis, rheuma- prompted by early diagnosis was the only means to influence
toid arthritis, and diabetic retinopathy.61 the clinical outcome.
N. vasinfecta var. vasinfecta, var. africana, and N. boni- In 2001, a disseminated N. vasinfecta infection had been
nensis were also shown to produce cyclosporins with antifun- reported by Cornely et al.90 in a 38-year-old patient from
gal activities against Alternaria kikuchiana.62,63 Cyclosporins Germany with acute nonlymphocytic leukemia who later on
are fungal metabolites with powerful immunosuppressive died of multiorgan failure. The initial clinical manifestations
properties, that can be applied for the prevention of rejec- included a 4-week history of fatigue, vertigo, and bone pain
tion in organ transplant recipients. On the other hand, these and subsequently the patient developed granulocytopenia

Neocosmosporas 453
with persistent fever and interstitial and patchy lung infil- of the Fusarium solani species complex, including a clinical
trates. Investigations also revealed hepatosplenomegaly and isolate of N. vasinfecta. Upon 48 h of incubation, the MICs
hypodensic lesions in the liver, suggesting possible hepato- were determined to be AMB: 8 μg/mL; 5FC: >64 μg/mL;
splenic involvement of fungal disease. The systemic fungal ITC: >8 μg/mL; VRC: >8 μg/mL; POS: >8 μg/mL; NAT:
infection was confirmed as the mold grew in swab cultures of 4 μg/mL; CAS: >16 μg/mL; MICA: >16 μg/mL; ANID:
a lateral purulent ulcerous lesion of the right foot, in sputum >16 μg/mL; and TRB: 1 μg/mL against the isolate.
samples, transtracheal aspirates, a second biopsy of the foot
lesion, and in blood cultures and the treatment with AMB 54.1.4 Diagnosis
and liposomal AMB failed.
Based on the information of the NCBI Nucleotide data- 54.1.4.1 Conventional Techniques
base, a N. vasinfecta infection occurred in a pediatric burn Although their anamorphs grow easily in routine culture
patient, which was the first registered case in the American media, the teleomorphs of Hypocreales are rarely found in
continent.91,92 A case of corneal ulcer due to N. vasinfecta culture. Neocosmospora is an exception from this point of
(initially diagnosed as Fusarium spp.) was reported by view, as it produces perithecia in culture enabling colony-
Manikandan et al.93 in an immunocompetent patient from and micromorphology-based identification. Samples can be
Aravind Eye Hospital, Coimbatore, South India. The patient cultivated on Sabouraud agar at 30°C and 37°C in ambient
being a farmer cultivating cotton and groundnut presented air; the fast-growing, white-to-pale-buff colonies develop
with pain, redness, and defective vision in his left eye of 5 without special requirements. The micromorphological fea-
days duration. Initially, Fusarium keratitis was diagnosed tures of the 3- to 4-day-old cultures resemble a Fusarium or
based on the fast growing, white colonies with cottony aerial Acremonium species. The fast growing fungal colonies on
mycelia on media and observation of the microscopic mor- PDA are flat, thin, and appear almost transparent. Microscopic
phology of hyaline, straight or slightly curved microconidia. observations reveal hyaline, elongated to cylindrical conidia
This diagnosis was later revised, as the isolate was identi- aggregated in slimy heads on conidiogenous cells developing
fied as N. vasinfecta based on the teleomorph morphology on undifferentiated hyphae. The size of conidia varies from
and molecular methods. Further, the recipient corneal button 5–10 μm in length to 2–3 μm in width and are mostly single
from therapeutic keratoplasty showed fungal growth after 3 celled or with one septum. Some conidia appear slightly
days, which was again identified as N. vasinfecta. Since the curved and intercalary chlamydospores are also observed.
publication of this case report, a second case (unpublished) Numerous ascomata (perithecia) form within 10–14 days that
of N. vasinfecta keratitis has also been diagnosed in the same give the colony a punctate appearance (incubated at light and
hospital, from a 77-year-old one eyed male patient who was dark conditions at 25°C). Supported by subculturing on oat-
known to be diabetic and asthmatic. The patient required meal agar with Lupinus sp., orange-brown to copper-colored
therapeutic keratoplasty. Furthermore, the isolates from fruiting bodies develop until day 8, which can be identified
the F. solani species complex studied by O’Donnell et al.27 as perithecial ascomata (200–300 μm diameter) after 14 days
included an additional clinical isolate of N. vasinfecta iso- of incubation.90 Ascomata are subspherical (300–400 μm in
lated from human eye in Louisiana. diameter), multilayered and smooth-walled, each with an
A low minimal inhibitory concentration of AMB was apical pore. Cylindrical asci, 90–110 × 10–12 μm in diam-
reported by Kac et al.89 against the isolated N. vasinfecta eter, are present inside the ascomata, each containing eight
strain. Cornely et al.90 reported the minimal inhibitory con- ascospores in a row. The ascospores are brownish, spheri-
centrations (MICs) of AMB and 5-flucytosine (5FC) as well cal to ellipsoidal, 10–15 to 8–12 μm, with thick roughened
as susceptibilities to fluconazole (FLC), itraconazole (ITC), walls and no germ pore present (Table 54.1; Figures 54.1
terbinafine (TRB), and voriconazole (VRC) for their case and 54.2).93
isolate as follows: AMB: >8 μg/mL, 5FC: >128 μg/mL, FLC:
>128 μg/mL, ITC: >2 μg/mL, VRC: 1 μg/mL, and TRB: 54.1.4.2 Molecular Techniques
0.125 μg/mL. The overall in vitro susceptibility test results As described above, N. vasinfecta can be identified by con-
confirmed the resistance of the isolate to AMB and FLC and ventional methods based on morphological characters.
nearly all of the few other antifungal agents available for sys- However, a long time is needed until formation of perithecial
temic therapy. In contrast, Manikandan et al.93 determined ascomata, and the micromorphological features of N. vasin-
that the keratitis isolate of N. vasinfecta was susceptible to fecta isolates may suggest Acremonium or Fusarium species
AMB which was in congruence with the success of the post- in the early days of incubation, which could lead to an inad-
operative AMB treatment. Nevertheless, they also reported equate diagnosis and inappropriate therapeutic regimen. This
that the strain proved to be resistant to natamycin (NAT), problem could be overcome by the application of fast and
ketoconazole, econazole, and clotrimazole at the highest reliable molecular techniques for identification.
concentration evaluated, with MICs of >1024, >32, >32, and Hue et al.94 designed a PCR primer pair that amplifies a
>32 μg/mL, respectively. O’Donnell et al.27 reported the anti- fragment from the rDNA of the main Fusarium species and
fungal susceptibilities of AMB, NAT, 5FC, ITC, VRC, TRB, N. vasinfecta, but not of 11 other medically important fungi.
posaconazole (POS), anidulafungin (ANID) caspofungin The method was optimized for blood and tissue samples. In
(CAS), and micafungin (MICA) against a panel of members the case of a suspected fungal infection, this technique is

(a) (b)
(c) 100 µm (d) 100 µm
FIGURE 54.1 (a) Culture of Neocosmospora vasinfecta grown on PDA at 25°C for 7 days. The colony appears flat, thin, and almost
transparent. Rings with punctate appearance due to the presence of numerous perithecia are apparent. (b) Culture of N. vasinfecta grown on
recipient corneal button (5% sheep blood agar) at 37°C for 7 days. (c) Perithecia development of N. vasinfecta. Bar = 100 μm. (d) Perithecial
ascomata of N. vasinfecta after 14 days on potato dextrose agar with lupine stem. Bar = 100 μm.
(a) 100 µm (b) 100 µm
(c) 100 µm (d) 10 µm
FIGURE 54.2 (a) Perithecia of Neocosmospora vasinfecta: orange-brown in color, subspherical (300–450 μm in diameter), smooth-walled
and each has an apical pore. (b) Perithecia contain cylindrical asci (80–100 × 11–15 μm in diameter). (c) Each ascus contains eight asco-
spores. Bar = 100 μm. (d) Ascospores of N. vasinfecta: single-celled, uniseriate, brownish, spherical to ellipsoidal, 10.0–15.5 × 7.5–12.0 μm
with roughened cell walls and no germ pore. Bar = 10 μm.

Neocosmosporas 455
applicable for the restriction of the pathogen to fusaria and and EF2 (5′-GGARGTACCAGTSATCATG-3′) amplifying a
their teleomorphs, but not for species specific identification. 716 bp product from tef1; as well as 5f2 (5′-GGGGWGAY
For the purposes of culture-based molecular identifica- CAGAAGAAGGC-3′) and 7cr (5′-CCCATRGCTTGYTTRC
tion of clinical N. vasinfecta isolates, sequence analysis of CCAT-3′), and 7cf (5′-ATGGGYAARCAAGCYATGGG-3′)
the ITS region inside the nuclear rDNA was performed both and 11ar (5′-GCRTGGATCTTRTCRTCSACC-3′) ampli-
by Cornely et al.90 and Manikandan et al.93 O’Donnell91 also fying 863 and 875 bp products from the rpb2 gene, respec-
used ITS along with a partial sequence of the 28S rRNA gene tively. PCR cycling program consists of 1 cycle of 94°C
and a fragment of the tef1 gene for the identification of a N. for 90 s and 40 cycles of 94°C for 30 s, 55°C for 90 s, and
vasinfecta isolate from a pediatric burn patient. Other possi- 68°C for 2 min, followed by 1 cycle of 68°C for 5 min and
bilities include the analysis of sequences from the 18S rRNA a 4°C soak.27 The PCR primers were utilized for sequenc-
gene—which was applied by Yanai95 for the identification of ing of the nuclear ribosomal gene product and rpb2, while
fungal isolates from arable peat soil as N. vasinfecta—and primers EF3 (5′-GTAAGGAGGASAAGACTCACC-3′) and
from the second largest subunit of the rpb2 gene.27 EF22T (5′-AGGAACCCTTACCGAGCTC-3′) were used for
sequencing of the tef1 fragment on an ABI 373 or ABI 377.
Cornely et al.90 performed PCR amplification and DNA
54.2 Methods
sequencing with the primers ITS2, ITS3, ITS4, and ITS5
54.2.1 Sample Preparation (5′-GGAAGTAAAAGTCGTAACAAGG-3′)97 on an ABI
377 automated sequencer (Applied Biosystems).
For DNA isolation, practically any protocol successfully Sequence analysis can be performed by BLASTN
applied for filamentous fungi could be sufficient also for similarity search at the website of the National Center for
N. vasinfecta. For the purposes of molecular identification of Biotechnology Information (http://www.ncbi.nlm.nih.gov/
the N. vasinfecta isolate from corneal ulcer,93 mycelia grown BLAST).98 GenBank accession numbers of some refer-
on yeast extract glucose medium (0.5 g yeast extract, 10 g glu- ence sequences of ITS, tef1 and 28S rRNA for N. vasin-
cose, 20 g agar in 1000 mL distilled water) at 25°C for 10 fecta are AY381138-AY381143, AY381144-AY381149, and
days are subjected to DNA isolation by the GeneElute™ Plant AY381150-AY381155, respectively.91 Reference sequences of
Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) the rpb2 gene (EU329497, EU329512, and EU329636)27 and
according to the manufacturers instructions. In the study of 18S rRNA (AB302197-AB302200, AB302205, AB302207,
Cornely et al.90 DNA was extracted by sonication in cetyl AB302208, AB302210, and AB302212)95 are also available
trimethyl ammonium bromide buffer (CTAB).96 for N. vasinfecta in the GenBank database.
54.2.2 Detection Procedures 54.3 C

Perspectives
The ITS region of the rRNA gene complex can be ampli-
fied by universal fungal primers. Subsequent analysis of Although little is known about N. vasinfecta as a pathogen
the nucleotide sequence helps confirm the identity of N. in humans, its clinical significance is gaining momentum as
vasinfecta.27,90,93,97 an emerging human fungal pathogen. In particular, human
Manikandan et al.93 used ITS1 (5′-TCCGTAG infections due to N. vasinfecta have caused serious pre-
GTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTG dicaments in the recent past and have been reported from
ATATGC-3′), and performed the PCR in 50 μL volumes con- various parts of the world highlighting organ specific or sys-
taining 5 μL 10× PCR buffer (ZenonBio, Szeged, Hungary), temic manifestations. The basic methods used to diagnose
200 μM each dNTP, 2.5 mM MgCl2, 0.5 μM each primer, infections caused by N. vasinfecta in humans have been
2.5 U of Taq DNA polymerase (ZenonBio, Szeged, Hungary), recognized to be complex and may also be inadequate. In
and 5 μL template DNA. PCR amplification was carried the early days of incubation, the micromorphological fea-
out in a T3 thermocycler (Biometra, Göttingen, Germany) tures of N. vasinfecta isolates may be misleading, suggest-
with 1 cycle of 94°C for 5 min, 30 cycles of 94°C for 1 min, ing Fusarium or Acremonium species, which in turn could
48°C for 1 min, and 72°C for 1 min, and a final elongation lead to an inadequate therapeutic regimen because of the
step at 72°C for 10 min. The PCR products were purified different AMB susceptibilities of these genera. This prob-
using GenElute™ MINUS ETBr SPIN COLUMNS (Sigma- lem could be overcome by the application of molecular
Aldrich),93 or by the Qiagen Gel Extraction Kit (Qiagen).90 techniques for fast and reliable identification. Furthermore,
The PCR products were sequenced using the ITS4 primer being aware of the varying ranges of MICs among different
with the dye deoxy terminator chemistry on an ABI 373A isolates of the species, each clinical isolate of N. vasinfecta
DNA sequencer (Applied Biosystems).93 should be examined for its minimal inhibitory concentra-
O’Donnell et al.27 employed the following primers in their tions to antifungals.
study: ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) N. vasinfecta is one of the two species within the F. solani
and NL4 (5v-GGTCCGTGTTTCAAGACGG-3′) amplifying species complex which have been shown to be pathogenic
a 986 bp product from the ITS region and domains D1 and D2 both to plants and to humans.27 Such organisms possess the
of 28S rRNA; EF1 (5′-ATGGGTAAGGARGACAAGAC-3′) potential to be developed to model organisms to investigate

whether common virulence factors are involved in plant and 24. Rossman, A.Y. et al., Genera of Bionectriaceae, Hypocreaceae
human pathogenesis. and Nectriaceae (Hypocreales, Ascomycetes), Stud. Mycol.,
42, 1, 1999.
25. O’Donnell, K., Molecular phylogeny of the Nectria haemato-
cocca–Fusarium solani species complex, Mycologia, 92, 919,
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55 Ochroconis
Ayako Sano and Kyoko Yarita
Contents
55.1 Introduction...................................................................................................................................................................... 459
55.1.1 Taxonomy and Morphology.................................................................................................................................. 459
55.1.3 Pathogenesis.......................................................................................................................................................... 460
55.1.4 Antifungal Susceptibilities................................................................................................................................... 460
55.1.5 Diagnosis.............................................................................................................................................................. 462
55.2 Methods............................................................................................................................................................................ 462
55.2.2.1 Species-Specific PCR............................................................................................................................ 463
55.2.2.2 Loop-Mediated Isothermal Amplification Method............................................................................... 463
55.3 Conclusion........................................................................................................................................................................ 465
References.................................................................................................................................................................................. 466
55.1 Introduction ribosomal RNA sequence [2]. As an independent species,

O. gallopava demonstrates less than 96% in sequence iden-
Ochroconis gallopava is a dematiaceous and thermo-toler- tity to the closest species O. calidifluminalis in this gene
ant fungal species that causes pulmonary, cerebral, and sys- region [3].
temic infections in humans and animals. In avian outbreaks, The colonies of O. gallopava are felty, flat, and brownish-
O. gallopava induces clinical symptoms that resemble those black, with a reddish-brown pigment exuding into the agar
of highly pathogenic avian flu. Therefore, correct identifica- [1]; however, the colors and textures may vary depending on
tion of O. gallopava is critical for the control and prevention culture media and duration of subculturing after isolation.
of this emerging zoonotic pathogen. Because of their obvious Colonies on Sabouraud dextrose agar (SDA) at 25°C, 37°C,
advantages, molecular tests have been increasingly applied and 42°C are uniformly floccose or felty and dark olive green
for rapid detection of O. gallopava. This chapter outlines on the surface and dark brown to reddish-brown pigment on
protocols based on polymerase chain reaction (PCR) and the reverse. Those on potato dextrose agar (PDA) are floc-
loop-mediated isothermal amplification (LAMP) for species- cose, dark olive green on the surface, and dark brown on the
specific determination of O. gallopava. reverse. Those on oat meal agar (OMA) are floccose, reddish-
brown to ashy green on the surface, and lacy on the reverse,
with a slight reddish-brown pigment. The fresh isolates of O.
55.1.1 Taxonomy and Morphology
gallopava from the nature are extremely floccose; however,
Ochroconis gallopava (W.B. Cooke) de Hoog 1983 is the they show the same appearance as the clinical isolates after
valid anamorphic name of a dematiaceous fungus whose several times of subculturing [4].
synonyms include Diplorhinotrichum gallopavum W.B. Hyphae are pale or yellowish brown to brown.
Cooke, 1964; Dactylaria gallopava (W.B. Cooke) Bhatt Conidiophores are mostly cylindrical to acicular, sometimes
and Kendrick, 1968; Ochroconis gallopava (W.B. Cooke) poorly differentiated. A few conidia attach at the tip of conid-
de Hoog, 1983; Dactylaria constricta (Abbott) Dixon and iophores. Conidia are two celled, subhyaline to pale brown,
Salkin var. gallopava (W.B. Cooke) Salkin and Dixon, 1987; smooth walled to verrucose, cylindrical to clavate, and con-
and Ochroconis gallopavum (W.B. Cooke) de Hoog, 1985 [1]. stricted at the septum. Typical conidia size of O. gallopava
Ochroconis gallopava is related closely to Ochroconis shown in the textbook ranged as 11–18 × 2.5–4.5 μm having
calidifluminalis, O. constricta, O. gamsii, O. humicola, O. wide apical cells [1]. According to the morphological studies
tshawytschae, Scolecobasidium terreum, S. cateniphorum, on O. gallopava by Dixon and Salkin [5], the conidia sizes
S. avellanea, S. verruculosum, S. verruculosum, Chalara of six strains of this fungal species range from 3.3 to 16.6 μm
spp., Fusicladium pini, F. rhodense, F. ramoconidii, F. in length and from 2.1 to 5.8 μm in width. The surface of the
amoenum, and S. tricladiatum based on nucleotide sequence conidia is covered with mucoid-like components that stain
analysis of the D1/D2 domain of the large-subunit (LSU) with alcian blue (Ohori et al., Personal communication).
459

55.1.2 Biology and Epidemiology According to Dixon et al. and Walsh et al., clinical and
animal isolates of O. gallopava are lethal to mice via intrave-
Tansey (1973) reported that O. gallopava is one of thermo- nous route [48,49]. Furthermore, through intratracheal inoc-
tolerant fungi [6]. The maximum growth temperature is ulation of O. gallopava conidia obtained from an outbreak
48°C–50°C. It shows excellent growth at 42°C as the same case in turkeys, Blalock and Derieux succeeded reproduction
as 35°C or 37°C, while slightly inhibited at 25°C [2,3]. of the disease caused by O. gallopava on turkey poultry [41].
According to Fukushiro et al. [7], the maximum growth tem- Based on experimental studies involving intravenous
perature of O. gallopava is 50°C, while those of the related infection for mice with O. gallopava conidia, the target organ
species such as O. constricta, O. humicola, and O. tshaw- might be the brain, although the fungal species also affected
ytschae are less than 37°C [1,5]. Therefore, thermo-tolerance the lung and other visceral organs such as liver, kidney, and
test may be important to differentiate O. gallopava from spleen [2,4]. The circulating movements shown by mice
related species forming clavate conidia. inoculated with conidia of O. gallopava pointed to the likely
Distribution of O. gallopava might be worldwide. The damage in the brains. The hemorrhage and marked lesions in
natural habitat of O. gallopava is thought to be places with the brains also supported the strong neurotropism [4].
very low pH and extreme temperatures such as in thermal These findings suggested that the central nervous sys-
soils [6,8–11], hot spring effluents [4,6,10], sewage from tem should be carefully examined in O. gallopava-infected
nuclear power plants [12,13], a pulp sample [2], and broiler- patients and animals. In fact, the cerebral involvements were
house litters [14,15]. also recorded in the human cases [2,16,17,19–23,25,33–35,
Up to the end of September 2009, more than 40 human and CBS database] followed by pulmonary disorders. Similar
cases have been reported [5,7,16–38]. The patients are located changes were also noted in the animal cases [14,15,25,39–47].
in the United States [2,5,16–18,20–23,25,28,30,31,36–38], In addition, O. gallopava isolates from hot springs had
South Africa [17], Australia [24–26], Canada [2,27,35], China the same ability to elicit cerebral symptoms from experi-
[32,33], New Zealand [2], the Netherlands and Germany mentally infected mice as clinical or animal isolates [4]. By
(referred from the database of CBS: Centraalbureau voor contrast, a clinical isolate reported by Ohori et al. [2] showed
Schimmelcultures, the Netherlands database), and Japan much lower virulence and no mouse died in spite of treatment
[2,7,34] (Table 55.1). The pathogen also causes outbreaks or with corticosteroid. The difference in virulence between
infections in birds and mammals [2,5,14,15,25,39–47]. The O. gallopava strains might also depend on histories of stor-
host animals include turkey [39,41], chick [14,15,25,40,42], age and/or condition of maintenance [50].
Japanese quail chick [43], gray-winged trumpeter [44], The fungal cells have the capability to overcome the host
snowy owl [45], cat [5,46], and dog [47] (Table 55.2). Animal defense mechanism in mice, although the degree of migra-
infections, especially in birds, have raised a serious problem tions of polymorphonuclear leukocytes and macrophages to
for differentiation from SARS and highly pathogenic avian mycelial cells in host tissues appear to depend on each fun-
influenza [2]. gal isolate. Fresh and wild strains of O. gallopava are highly
virulent even in healthy hosts. The fact that a wood pulp
worker without any underlying disease developed multiple
55.1.3 Pathogenesis
lung abscesses after repeated inhalation of O. gallopava in
The main clinical symptom of O. gallopava infection in composts provides evidence that constant exposure to a huge
humans is pneumonia [2,5,17,18,24–28,31,32,37,38, and number of O. gallopava spores may cause disease in healthy
CBS database]; however, some cases develop into sys- subjects [17,28].
temic infections [2,16,19,25,33–35], cerebral involvements
[2,17,20–23,25, and CGB database], and subcutaneous
55.1.4 Antifungal Susceptibilities
[7,30] and ocular lesions [29]. Many of the patients have
underlying diseases: organ transplantation as the highest Minimum inhibitory concentration (MIC) for O. gallopava
risk factor [2,18–17,30,31,33,37,38], hematological malig- is relatively low; MIC of amphotericin B ranges from 0.015
nancy [7,16,17,29,34], diabetes [16,21,24,25,30,31], AIDS to 2.0 μg/mL [20,21,29,30,31,33,51–53], that of flucytosine
[25,26,35], cancer [36], database of CBS: Centraalbureau ranges from 0.5 to 80.7 μg/mL [1,20,21,30,31], that of fluco-
voor Schimmelcultures, the Netherlands), immune dis- nazole ranges from 8 to 32 μg/mL [1,20,29,31], that of itra-
order [2,17], cardiovascular disorder [17], emphigus [32], conazole ranges from 0.02 to 0.5 μg/mL [20,21,29,30,31,33,
pneumoconiosis [2]. Two apparently healthy individuals, a 51–53], that of miconazole ranges from 0.5 to 2.0 μg/mL [52],
wood pulp worker [28] and a coal mine worker [17], also that of voriconazole ranges from 0.03 to 1.0 μg/mL [33,51–
became infected after frequent inhalation of or exposure to 53], that of ketoconazole ranges from 0.25 to 3.2 μg/mL
the pathogen (Table 55.1). [1,20], and that of terbinafine ranges from 0.03 to 0.06 μg/mL
On the other hand, infected birds and mammals showed [52]. These data suggest that amphotericin B, with or without
cerebral involvements predominantly (Table 55.2). There is a itraconazole, can be recommended as the first choice drug
possibility that the differences of body temperatures between against O. gallopava infection [20,24,28].
human and other animals influence the predominant symp- Our recent results on the antifungal susceptibility testing
toms of O. gallopava infection. of O. gallopava isolated from hot springs showed that the

Ochroconis 461
TABLE 55.1
Human Cases of Ochroconis gallopava Infection
Authors, Published Year, and Affected
Case Reference Number Country Sex Age Organ Remarksa Outcome
1 Dixon and Salkin, 1986 [5] USA ND ND Lung ND ND
2 Fukushiro et al., 1986 [7] Japan F 58 Subcutaneous Leukemia Survived
3 Terreni et al., 1990 [16] USA M 62 Systemic Leukemia, diabetic Died
4 Sides et al., 1991 [17] ND M ND Lung Immunocompromised ND
5 Sides et al., 1991 [17] South Africa ND ND Lung Coal mine worker ND
6 Sides et al., 1991 [17] South Africa ND ND Lung Coal mine worker ND
7 Sides et al., 1991 [17] USA ND ND Lung Immunocompromised Died
8 Sides et al., 1991 [17] USA ND ND Brain Immunocompromised ND
9 Sides et al., 1991 [17] USA M 47 Lung Cardiovascular disease ND
10 Sides et al., 1991 [17] USA M 60 Brain Lymphoma, nocardiosis Died
11 Mancini and McGinnis, 1992 [18] USA M 30 Lung Heart transplant recipient Survived
12 Prevost-Smith et al., 1993 [19] ND M 46 Systemic Heart transplant recipient Died
13 Vukmir et al., 1994 [20] USA M 68 Brain Liver transplant recipient Survived
14 Kralovic and Rhodes 1995 [21] USA M 63 Brain Liver transplant recipient, diabetic Died
15 Rossmann et al., 1996 [22] USA M 59 Brain Liver transplant recipient, nocardiosis Died
16 Bonham et al., 1996 [23] USA ND ND Brain Liver transplant recipient Survived
17 Jenney et al., 1998 [24] Australia M 58 Lung Heart transplant recipient, diabetic Survived
18 Horré and de Hoog, 1999 [25] UK ND ND Systemic AIDS ND
19 Horré and de Hoog, 1999 [25] Australia ND ND Systemic ND ND
20 Horré and de Hoog, 1999 [25] USA ND ND Brain Diabetes mellitus ND
21 Horré and de Hoog et al., 1999 [25,26] ND M 48 Lung HIV-positive transplant recipient ND
22 Horré and de Hoog et al., 1999 [25,26] Australia ND ND Lung ND ND
23 Horré and de Hoog, 1999 [25] USA ND ND Lung Transplant recipient ND
24 Burns et al., 2000, [27] Canada F 58 Lung Lung transplant recipient with Survived
pulmonary nodule
25 Odell et al., 2000 [28] USA M 38 Lung Wood pulp worker with pulmonary Survived
abscess
26 Bowyer et al., 2000 [29] UK M 69 Eye Chronic lymphocytic lymphoma Died
27 Mazur and Judson, 2001 [30] USA F 32 Subcutaneous Lung transplant recipient with diabetic Survived
28 Malani et al., 2001 [31] USA M 32 Lung Renal transplant recipient with Died
diabetic
29 Zhao et al., 2002 [32] China M 68 Lung Pemphigus Survived
30 Wang et al., 2003 [33] China M 13 Systemic Renal transplant recipient Died
31 Fukushima et al., 2005 [34] Japan F 66 Systemic Chronic lymphocytic lymphoma Died
32 Ohori et al., 2006 [2] USA M 54 Systemic Heart transplant recipient Died
33 Ohori et al., 2006 [2] Japan M 79 Lung Pneumoconiosis ND
34 Ohori et al., 2006 [2] Canada F 68 Lung ND ND
35 Ohori et al., 2006 [2] USA ND ND Lung ND ND
36 Ohori et al., 2006 [2] New Zealand M 83 Lung ND ND
37 Boggild et al., 2006 [35] Canada M 28 Systemic AIDS Died
40 Hollingsworth et al., 2007 [36] USA F 78 Lung Hypothyroidism, basal cell skin cancer Survived
41 Shoham et al., 2008 [37] USA F 64 Lung Renal transplant recipient Survived
42 Shoham et al., 2008 [37] USA M 60 Lung Renal transplant recipient Survived
43 Shoham et al., 2008 [37] USA M 50 Lung Liver transplant recipient Survived
44 Mayer and Bastani, 2009 [38] USA M 71 Lung Renal transplant recipient Died
38 Database of CBS Netherlands ND ND CSF Spondylodiscitis ND
39 Database of CBS Germany M 50 Lung Bronchial carcinoma Died
CBS, Centraalbureau voor Schimmelcultures, the Netherlands, CSF, cerebrospinal fluid, ND, no data.
Underlying disease or occupation.
a

TABLE 55.2
Animal Cases of Ochroconis gallopava Infection
Authors, Published Year, Affected
and Reference Number Country Animal Species Organ(s)
Georg et al., 1964 [39] USA Turkeya Brain
Connole, 1967 [40] Australia Chicka Brain
Blalock et al., 1973 [41] USA Turkeya Brain
Ranck et al., 1973 [42] USA Chicka Brain
Waldrip et al., 1974 [14] USA Chick, six casesa Brain
Randall and Owen, 1981 [15] UK Chick, two casesa Brain and lung
Shane et al., 1985 [43] USA Japanese quail chicksa Brain and lung
Dixon and Salkin, 1986 [5] USA Cat Lung
Karesh et al., 1987 [44] USA Gray-winged trumpeter chicks Brain
Salkin et al., 1990 [45] USA Snowy owl chick Brain
Padhye et al., 1994 [46] USA Cat Systemic
Horré and de Hoog, 1999 [25] India Chicka Brain
Ohori et al., 2006 [2] New Zealand Antipodean parakeet Lung
Singh et al., 2006 [47] USA Dog Systemic
a Epidemic outbreaks at poultry farms.
MIC values range from 0.25 to 1 μg/mL for amphotericin culture is positive or negative, it should be kept under obser-
B, 0.25–4 μg/mL for flucytosine, 0.25–1 μg/mL for itracon- vation up to 4 weeks. In our experience, it took 1 week to
azole, 0.5–2 μg/mL for miconazole, 1–2 μg/mL for voricon- recover from visceral organs of experimentally O. gallopava-
azole, 8–64 μg/mL for fluconazole, and ≤0.03–0.125 μg/mL inoculated mice [2,4].
for micafungin [3,54]. Furthermore, observations of mycological profiles may
take a few more weeks. When the isolate was dematiaceous
55.1.5 Diagnosis fungi at a glance of the colony, having excellent growth abil-
ity at 42°C and producing clavate and two-celled conidia, the
As opportunistic fungal infections in immunocompromised case was able to be suspected as fungal infection caused by
patients, aspergillosis, candidiasis, and/or cryptococcosis black fungi including O. gallopava.
are well recognized by their morbidity and mortality [38]. On the other hand, the hematological and blood chemistry
O. gallopava is listed as a causative agent for emerging data of the hosts may have been influenced by the underly-
fungal infection, causing fatal infections in immunocom- ing diseases in immunocompromised host. Chest x-ray and
promised hosts, especially patients with hematological computed tomography also help to detect pulmonary lesions.
malignancy [34]. On the other hand, frequent exposure to Therefore, rapid and accurate diagnostic methods except
fungal elements might cause the infection to humans with- for culturing and DNA sequencing are useful at clinical labo-
out immune disorder [28]. We should take into consideration ratories to differentiate O. gallopava infection from SARS
O. gallopava infection to the patients showing respiratory and highly pathogenic bird flu.
symptoms, systemic disorder, and/or intractable cutane-
ous massive lesions and to not only immunocompromised 55.2.1 Sample Preparation
patients but also individuals working at dusty environments.
Isolation of the causative agent is the gold standard for diag-
nosis. We strongly recommend trying isolation of the caus-
55.2 Methods ative fungal pathogen from clinical materials. Consistency of
Culture of the causative agent, O. gallopava, from clinical the findings of culture, cytology and/or histopathology, and
materials such as sputum, BAL (bronchoalveolar lavage), molecular biological data may bring a valid diagnosis.
and/or biopsy is the gold standard when the isolate corre- The sputum or BAL sample is directly spread on chloram-
sponds to cytological and histopathological findings contain- phenicol (100 mg/L)-added PDA plates at least in duplicate.
ing fungal components. However, it is impossible to diagnose When bacterial contaminations are expected, autoclaved-
O. gallopava infection on the basis of cytology or histopa- chloramphenicol (100 mg/L)-added saline is poured on the
thology alone. sputum or sediment of BAL at 10 times volume, is agitated
It takes a longer period until mycological identification. strongly, is stored at 35°C–37°C for at least 2 h, and is spread
Culture, isolation, and identification of the causative agent on the culture plate. Culture condition is recommended to
require at least a few weeks. To determine whether fungal use at 42°C and continue to observe up to 14 days. Then,

Ochroconis 463
suspicious colonies are picked up for morphological and geniculata, Curvularia lunata, Curvularia senegalen-
molecular biological studies. Using incubators adjusted at sis, Exophiala alcalophila, Exophiala dermatitidis, and
35°C and 42°C simultaneously might be a convenient way to Exophiala moniliae). The primer set was also tested on eight
isolate both O. gallopava and other fungal pathogen. clinically important fungal species: Aspergillus fumigatus,
While waiting for the fungal colony recovering from clini- Blastomyces dermatitidis, Candida albicans, Coccidioides
cal materials, some parts of molecular biological diagnosis posadasii, Cryptococcus neoformans, Histoplasma capsu-
may proceed simultaneously. latum, Penicillium marneffei, and Sporothrix schenckii for
DNA samples for molecular biological studies such as confirmation of the specificity [2].
species-specific PCR system and the LAMP method are pre- The sequences of the forward and reverse primers are
pared as follows: OgF3: 5′-AGG GAG TCT CGG GTT AAG GG-3′ posi-
DNA from purified fungal colonies is extracted with com- tion 119–138 th, and OgB3: 5′-CAT TCC CTT CGT CTT
mercial DNA extraction kits (i.e., DEXPAT ® kit [TaKaRa, TGT CC-3′ position 493–474 th of AB161059 consisting of
Ohtsu, Japan]). Although DEXPAT kit is designed for 615 th. The D1/D2 domain of the LSU ribosomal RNA gene
extracting DNA from paraffin-embedded tissue samples, we sequences of O. gallopava in the GenBank database was
routinely use it for the isolation of genes from fungal cultures. identical except for two wobble bases at 421st and 435th.
Our procedure for purified fungi is as follows: Approximately
Procedure
100 μL of fungal cells are placed in a sterilized microtube
(1.5 mL size); 0.5 mL of DEXPAT solution is then added, and
1. The PCR mixture (25 μL) contains 20–40 ng of
the mixture is homogenized with a plastic pestle. It is then
template DNA (2.5 μL), one Ready-To-Go™ PCR
incubated at 100°C for 10 min and centrifuged at 12,000 rpm
bead (Amersham Pharmacia), and 10 pmol each of
(13,201 × g) for 10 min. The supernatants are used as DNA
primers OgF3 and OgB3.
samples. The original procedure of DEXPAT kits for par-
2. The PCR mixture is subjected to an initial denatur-
affin-embedded tissue samples is as follows: Three sections
ation at 95°C for 4 min; 30 cycles of 94°C for 1 min,
of 10 μm-thick paraffin tissue are placed into a microtube
58°C for 90 s, and 72°C for 2 min; a final extension
(1.5 mL), and 0.5 mL of DEXPAT solution is added. After
at 72°C for 10 min.
boiling for 10 min, the tube is centrifuged at 12,000 rpm
(13,201 × g) for 10 min. The supernatant is then processed for
Note. The primer set also amplified the homologous part of
molecular biological studies. Biopsy tissue, blood, and BAL
O. calidifluminalis which is the most recently discovered
samples are fixed with ethanol at 70% final concentration at
Ochroconis sp. demonstrating closest phylogenetic and mor-
least for 10 min. Before fixation by the ethanol, the tissue
phological relationships to O. gallopava [3]. Furthermore,
and coagulated blood are ground out using a plastic homog-
the primer set may amplify other O. gallopava–related
enizer. The fixed sample is centrifuged, washed three times
species, such as Chalara sp., Scolecobasidium terreum,
with sterile water by centrifuging at 12,000 rpm (13,201 × g)
S. cateniphorum, and S. tricladiatum, although they have not
for 5 min, and 0.5 mL of DEXPAT solution is added. The
been confirmed.
mixture is incubated at 100°C for 10 min and centrifuged at
12,000 rpm for 10 min. The pus samples are collected with 55.2.2.2 Loop-Mediated Isothermal
sterile cotton buds. The cotton tip is cut off and placed in Amplification Method
a sterilized microtube (1.5 mL), and 0.5 mL of 70% etha-
Principle. LAMP method is a simple, rapid, specific, and
nol is added. After vortexing and centrifuging, the sample
cost-effective nucleic acid amplification method solely devel-
is washed three times with sterile water by centrifuging at
oped by Eiken Chemical Co., Ltd (http://loopamp.eiken.
12,000 rpm (13,201 × g) for 5 min, and 0.5 mL of DEXPAT
co.jp/e/lamp/index.html). It is characterized by the use of
solution is added. The cotton tip and the solution are incu-
four different primers specifically designed to recognize six
bated together at 100°C for 10 min and centrifuged at 12,000
distinct regions on the target gene, and the reaction process
rpm (13,201 × g) for 10 min.
proceeds at a constant temperature using strand displacement
reaction (Figure 55.1a) [55].
55.2.2 Detection Procedures Amplification and detection of genes can be completed in
a single step, by incubating the mixture of samples, primers,
55.2.2.1 Species-Specific PCR DNA polymerase with strand displacement activity and sub-
Principle. The species-specific primer set for O. gallopava strates at a constant temperature (around 65°C). It provides
was designed from the sequence of D1/D2 LSU rDNA of O. high-amplification efficiency, with DNA being amplified
gallopava (accession number AB161059 in GenBank) after 109–1010 times in 15–60 min. Because of its high specificity,
comparison with those from 14 dematiaceous fungal spe- the presence of amplified product can indicate the presence
cies (i.e., closely related species: O. constricta, O. gamsii, of the target gene.
O. humicola, and O. tshawytschae, and other dematiaceous LAMP method uses four primer sets: F (Forward)
fungal species: Alternaria alternata, Arthrobotrys javanica, 3, B (Backward) 3, FIP (Forward Inner Primer), and
Bipolaris spicifera, Cladophialophora carrionii, Curvularia BIP (Backward Inner Primer) selected from six distinct

BIP primer
5΄ B1c 3΄
F3 primer +
5΄ F3 3΄ 5΄ B2 3΄
Forward 5΄ F3 F2 F1 B1c B2c B3c 3΄
Complimentary 3΄ F3c F2c F1c B1 B2 B3 5΄

FIP primer
3΄ B3 5΄
5΄ F1c 3΄
+ B3 primer
5΄ F2 3΄
(a)
0gF3 primer End 0gFIP
5΄ F3 3΄ 5΄ F2 3΄ 5΄ F1 3΄
5΄ AGGGAGTCTCGGGTTAAGGG AGAGGGTGAGAGTCCCGT gccccttcgacgagtcgagt 3΄
3΄ tccctcagagcccaattccc tctcccactctcagggca CGGGGAAGCTGCTCAGCTCA 5΄

3΄ F3c 5΄ 3΄ F2c 5΄ 3΄ F1c 5΄
Start 0gFIP
0gF3 primer: 5΄–AGGGAGTCTCGGGTTAAGGG–3΄
0gFIP primer: 5΄–ACTCGACTCGTCGAAGGGGCAGAGGGTGAGAGTCCCGT–3΄
F1c F2
(b)
Start 0gBIP
5΄ B1c 3΄ 5΄ B2c 3΄ 5΄ B3c 3΄

5΄ ACTGGCCAGAGACCGATAGCG gcactttgaaaagagagtc ggacaaagacgaagggaatg 3΄
3΄ tgaccggtctctggctatcgc CGTGAAACTTTTCTCTCAGT CCTGTTTCTGCTTCCCTTAC 5΄

3΄ B1 5΄ 3΄ B2 5΄ 3΄ B3 5΄
End 0gBIP 0gB3 primer
0gB3 primer: 5΄–CATTCCCTTCGTCTTTGTCC–3΄
0gBIP primer: 5΄–ACTGGCCAGAGACCGATAGCGTGACTCTCTTTTCAAAGTGC–3΄
(c) B1c B2
FIGURE 55.1 (a) Design four types of primers based on the following six distinct regions of the target gene: The F3c, F2c, and F1c
regions at the 3′ side and the B1, B2, and B3 regions at the 5′ side. FIP: Forward inner primer consists of the F2 region (at the 3′ end) that is
complementary to the F2c region, and the same sequence as the F1c region at the 5′ end. F3 primer: Forward outer primer consists of the F3
region that is complementary to the F3c region. BIP: Backward inner primer consists of the B2 region (at the 3′ end) that is complementary
to the B2c region, and the same sequence as the B1c region at the 5′ end. B3 primer: Backward outer primer consists of the B3 region that
is complementary to the B3c region (http://loopamp.eiken.co.jp/e/lamp/primer.html). (b) Positions and sequences of the forward primers for
the species-specific LAMP for Ochroconis gallopava, and (c) those of the backward primers.
regions of the target gene (http://loopamp.eiken.co.jp/e/ Although PrimerExplore, special software to design
lamp/primer.html). The most important primer sets are LAMP primers, is available in the Web site (http://primerex-
F3 and B3. The primers should be selected from specific plorer.jp/e/), it seems to be useful as reference hints for base
genes or gene sequences based on species-specific PCR composition, GC contents, secondary structures, and Tm
and were confirmed after testing with intraspecies diver- value on designing primers based on our experience.
sity and a huge numbers of related pathogenic fungal spe- The target gene (e.g., DNA template) and the reagents
cies. Therefore, enormous numbers of trials and errors are are incubated at a constant temperature between 60°C and
required until the final primers are confirmed. Further, the 65°C (Figure 55.2a and b). The reaction steps were available
primers should completely differentiate the fungal genes at the Web site (http://loopamp.eiken.co.jp/e/lamp/principle.
from host ones. html).

Ochroconis 465
bps bps of 235th–216th and the forward sequence of 170th–187th.

The primer OgBIP (5′-ACT GGC CAG AGA CCG ATA
1000 1000 GCG TGA CTC TCT TTT CAA AGT GC-3′) is designed for
the forward sequence of 287th–307th and the complementary
500 500 sequence of 355th–336th in AB161059 (Figure 55.1b and c).
Procedure
1. One μL of fungal DNA template and 5 pmol each of

OgF3 and OgB3 and 40 pmol each of FIP and BIP
(a) M 1 2 3 4 5 6 7 8 9 10 11 12 13 M primers are mixed with 12.5 μL of a reaction mix-
ture in a kit (Loop AMP, Eiken, Tokyo, Japan) in a
bps bps
final volume of 25.0 μL.
2. The DNA mixtures are incubated at 63°C for 60 min
and 80°C for 2 min.
3. The amplified DNAs are detected by electropho-
resis in a 1.0% agarose gel stained with ethidium
1000 1000 bromide.
500 500 Note. Detection of the fungal gene from infected tissue sam-
ples by a LAMP method requires a longer incubation period
[56]. We recommend 120 min of incubation for detection of
(b) M 1 2 3 4 5 6 7 8 9 10 11 12 13 M
the gene from clinical samples [2].
The LAMP method for detection of species-specific gene
FIGURE 55.2 Images of the species-specific PCR and the of O. gallopava also amplified the closest species, O. calidi-
LAMP for Ochroconis gallopava. (a) Species-specific PCR for fluminalis, as the same band pattern as O. gallopava [3]. The
Ochroconis gallopava. M: Marker, 1–10: O. gallopava, 11: O. LAMP system may also amplify the partial gene of the D1/D2
gamsii, 12 and 13: O. tsawytschae. The related species such as domain of the LSU ribosomal RNA gene on some O. gal-
O. constricta, O. humicola, Alternaria alternata, Arthrobotrys
lopava-related fungal species: Chalara sp., Scolecobasidium
javanica, Bipolaris sp., Bipolaris specifera, Cladophialophora
terreum, S. cateniphorum, and S. tricladiatum. However, we
bantiana, C. carrionii, Curvularia geniculata, Cu. lunata var.
lunata, Cu. senegalensis, Exophiala alcalophiala, E. dermatiti- speculated that the band patterns might be different from
dis, E. jeanselmei, E. moniliae, E. spinifera, Fonsecaea pedro- those of O. gallopava based on our studies about genus
soi, Phialophora verrucosa, and Rhinocladiella atrovirens, were Paracoccidioides; P. lutzii [57], a closely related species to
negative. (b) Species-specific LAMP for Ochroconis gallopava. P. brasiliensis having 89% identity in the target gene, was
M: Marker, 1–10: O. gallopava, 11: O. gamsii, 12 and 13: O. tsaw- amplified as a different ladderlike band pattern from those
ytschae. The related species listed above were also negative. of P. brasiliensis by a LAMP method [58]. Nevertheless, the
pathogenicity of Ochroconis calidifluminalis is lower than
LAMP method is highly sensitive. We experienced many that of O. gallopava [3], and there has been no case report
faults of contamination of the genes. Once contamination of involving O. calidifluminalis. Moreover, the pathogenicities
the target gene occurs, all reactions become positive, even in a of other new species, Chalara sp., Scolecobasidium terreum,
negative control using distilled water as template. Therefore, S. cateniphorum, and S. tricladiatum, are also unknown
extremely careful procedures are required: reagents, pipettes, because they are environmental isolates with no case report.
plastic pipette tips, safety cabinet, and hands. The samples Therefore, molecular biological identification of unknown
should be handled separately from the reagents. The positive dematiaceous fungi and detection of O. gallopava and
control for the target gene should be done separately. Ochroconis sp. genes from clinical materials by the LAMP
This is one of the reasons why some fungal species com- may remain useful (Figure 55.2).
mon in normal human flora or in environments are not rec-
ommended for the LAMP method. Selection of the target
55.3 Conclusion
fungal species is also important. The fungal species should
be rare in laboratorial environment. O. gallopava is treated O. gallopava is a species of dematiaceous fungi and affects
as suitable for the LAMP method because it is one of the central nervous and respiratory systems in humans, ani-
rare species in clinical laboratory and an uncommon fungal mals, and birds. Outbreaks of O. gallopava infection in birds
species in our environments. The primer sequences of F3 resemble highly pathogenic bird flu and SARS. The pres-
and B3 are as the same as the species-specific primer sets ent O. gallopava species-specific primer sets for PCR and
for PCR: OgF3 and OgB3. The primer OgFIP (5′-ACT CGA LAMP methods may help differentiate O. gallopava infec-
CTC GTC GAA GGG GCA GAG GGT GAG AGT CCC tion from not only highly pathogenic bird flu and SARS but
GT-3′) is designed for the reverse complementary sequence also other fungal infections.

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56 Phaeoacremonium
László Galgóczy, Laura Kovács, Tamás Papp, and Csaba Vágvölgyi
Contents
56.1 Introduction...................................................................................................................................................................... 469
56.1.2.1 P. alvesii............................................................................................................................................... 470
56.1.2.2 P. amstelodamense............................................................................................................................... 470
56.1.2.3 P. griseorubrum................................................................................................................................... 471
56.1.2.4 P. krajdenii........................................................................................................................................... 471
56.1.2.5 P. parasiticum...................................................................................................................................... 471
56.1.2.6 P. rubrigenum....................................................................................................................................... 471
56.1.2.7 P. sphinctrophorum.............................................................................................................................. 471
56.1.2.8 P. tardicrescens.................................................................................................................................... 471
56.1.2.9 P. venezuelense..................................................................................................................................... 471
56.1.2.10 Unidentified Phaeoacremonium Species............................................................................................. 471
56.1.3 Diagnosis.............................................................................................................................................................. 473
56.1.3.1 Conventional Techniques..................................................................................................................... 473
56.1.3.2 Molecular Techniques.......................................................................................................................... 473
56.2 Methods............................................................................................................................................................................ 475
56.2.1.1 Media and Culturing Conditions.......................................................................................................... 475
56.2.1.2 Light Microscopy Sample Preparation................................................................................................. 475
56.2.1.3 Electron Microscopy Sample Preparation............................................................................................ 475
56.2.1.4 Genomic DNA Preparation.................................................................................................................. 475
56.2.2.1 Morphological Identification of Phaeoacremonium Species............................................................... 475
56.2.2.2 Molecular Identification of Phaeoacremonium Species...................................................................... 475
References.................................................................................................................................................................................. 478
56.1 Introduction 56.1.1 Classification, Morphology,

and Epidemiology
The hyphomycete genus Phaeoacremonium, the anamorph
of Togninia, includes ecologically, plant pathologically, and The genus Phaeoacremonium includes 30 species,3,4 but their
human medically important species.1 The majority of these number has been increasing continuously in consequence of
fungi have been isolated from infected woody host plants, the reidentification and the improved molecular techniques
especially from grapevines (Vitis vinifera L.) as causative of fungal taxonomy.
agents of Petri and Esca diseases,2 and also from larvae of This genus was described by Crous et al.5 in 1996 with
bark beetles.3 The incidence of human infections caused six species: P. parasiticum, P. aleophilum, P. angustius, and
by Phaeoacremonium spp. has increased during recent P. chlamydosporum isolated from grapevines and P. inflatipes
years especially among imunocompromised patients.2 and P. rubrigenum isolated from humans and plants. Later,
These data indicate that the diseases caused by different P. chlamydosporum was renamed Phaeomoniella chla-
Phaeoacremonium species could be more frequent in the mydospora and placed in a different genus.6 During the past
future. decade, further 25 species of the genus Phaeoacremonium
469

have been described: P. mortoniae,7 P. viticola,8 P. alvesii, both immunocompromised and immunocompetent patients
P. amstelodamense, P. australiense, P. griseorubrum, in various countries: P. alvesii, P. amstelodamense, P. gris-
P. krajdenii, P. scolyti, P. subulatum, P. tardicrescens, eorubrum, P. krajdenii, P. parasiticum, P. rubrigenum,
P. venezuelense,2 P. argentinense, P. austroafricanum, P. sphinctrophorum, P. tardicrescens, and P. venezuel-
P. novae-zealandiae, P. sphinctrophorum, P. theobroma- ense.1,2 From these nine species, only P. alvesii, P. krajdenii,
tis,1 P. pallidum, P. prunicolum, P. fuscum,9 P. croatiense, P. parasiticum, and P. venezuelense have been isolated from
P. hungaricum, P. sicilianum, P. tuscanum,10 P. cinereum, woody hosts.1 It is hypothesized by Mostert et al.2 that spe-
and P. hispanicum.4 cies causing plant diseases, which can grow at 40°C, could
The genus Phaeoacremonium contains the anamorphs of be human pathogens too. According to this assumption, the
Togninia species; they can be considered morphologically as following species can be expected to be detected from human
intermediate between Acremonium and Phialophora. The infections in the future: P. australiense, P. subulatum, and
distinctive morphological characters of Phaeoacremonium P. aleophilum.2
from Phialophora are its aculeate phialides and inconspicu- The suspected sources of Phaeoacremonium infections
ous, nonflaring collarettes, and of Acremonium is its pig- in humans are woody splinters, soil, and air. Presumably,
mented vegetative hyphae.5 General description of the genus the major sources of human infections are the woody splin-
extended with the new species derived from Mostert et al.1,2 ters, which cause phaeohyphomycotic cyst or mycetoma
Colonies on malt extract agar (MEA) medium are flat with if they are passed under the skin.1 The disease caused by
entire (nonragged) margins, moderately dense, predomi- Phaeoacremonium species is variable in their clinical
nantly felty, or woolly textured. Generally, they are medium- manifestation including subcutaneous infections, fungemia,
brown becoming paler toward the conidia-forming region; disseminated disease,12 endocarditis,13 eumycetoma,14 onych-
but orange-brown, gray-brown, and dark-brown colonies are mycosis,15 endophthalmitis,16 osteomyelitis,17 and acute or
also common. If the colonies are paler, their color may range chronic arthritis.18,19 The number of human cases caused by
from pale yellow to beige. Pink-colored colonies, from pale Phaeoacremonium species has been continuously increasing
to dark-pink, also occur. Mycelium is branched, and hyphae since 1990, especially among immunocompromised hosts.12
are septate and occur singly or bundled (4–27 hyphae).
Mycelia usually have warts in their surface, but their pres-
ence depends on the area of the colony, the age of the culture, 56.1.2 Clinical Features and Pathogenesis
and the medium used. Warts differ in their density and their 56.1.2.1 P. alvesii
size among different species. Additional mycelial ornamen-
Human infections caused by P. alvesii have been described
tation is a useful character for distinguishing taxa, it could be
in two case reports in the literature. In both cases, this fun-
smooth, verruculose, and verrucose. The mostly pale brown
gus caused subcutaneous infection in an immunocompetent
conidiospores could be long and branched, branched, or short
host.20,21 Isolates from these cases were originally identi-
and unbranched depending on the species. They differ in
fied as P. inflatipes20 and P aleophilum 21 based on their
their number of septum and their ornamentation. Three types
macro- and microscopic morphology only. Further inves-
of phialides were identified in the genus Phaeoacremonium,
tigations supplemented with molecular techniques revealed
and they differ in their size, form, and presence of the basal
that both fungi represent a new Phaeoacremonium species,
septum. They could be monophialidic or polyphialidic. The
which was named P. alvesii.2 The infected areas were sur-
aseptate and smooth-walled conidia are hyaline to subhya-
gically excised with21 or without20 antifungal treatment
line aggregating into round, slimy heads at the apices of
[itraconazole (ITRZ), 100 mg/day], which led to uncom-
phialides. There is size difference between the aerial and
plicated healing in both cases. Only one undescribed case
agar conidia. The aerial conidia vary in shape from oblong-
has been found in the literature, where this fungus was
ellipsoidal to obovate, cylindrical, allantoids, or reniform, in
isolated from synovial fluid of a patient with keratitis.2
contrast to agar conidia, which are relatively long and are
P. alvesii was proved to be sensitive to seven investigated
mostly allantoid or oblongellipsoidal. Conidia have homog-
antifungal agents with the following MICs: amphotericin
enous cellular contents when they are young, but they can
B (AMB) = 2 μg/mL, fluconazole (FCZ) = 8 μg/mL, ITRZ=
become two-guttulate after 7–14 days, especially on agar.
16 μg/mL, ketoconazole (KCZ) = 4 μg/mL, ravuconazole
Phaeoacremonium species are widely distributed
(RCZ) = 1 μg/mL, terbinafine (TBF) = 2 μg/mL, and vori-
throughout the world. P. parasiticum, P. aleophilum, and
conazole (VCZ) = 1 μg/mL.21
P. krajdenii are the most frequent of them. They have been
mostly isolated from a range of woody hosts as causative
agents of plant diseases, but P. parasiticum, P. aleophilum, 56.1.2.2 P. amstelodamense
and P. argentinense have been isolated from soil also.1 One P. amstelodamense human infection was deduced from
species, P. scolyti (originally identified as P. rubrigenum), only one case, in which the fungus was isolated from
has been isolated from bark beetles,2,11 and only one case elbow joint interior. It was originally misidentified as P.
has been described yet, where an animal infection in dog is inflatipes, but Mostert et al.2 reidentified it as a different,
caused by P. parasiticum.1 Nine Phaeoacremonium species new species and introduced it as P. amstelodamense into the
have been confirmed as human pathogens causing mycoses in Phaeoacremonium genus.

Phaeoacremonium 471
56.1.2.3 P. griseorubrum RCZ = 0.03–1 μg/mL, TBF = 2 μg/mL, and VCZ = 0.03–2 μg/

This species from human infection was originally described mL. It is suggested that surgical excision with the administra-
as P. rubrigneum from human infection by Matsui et al. in tion of AMB and an azole antifungal agent would be a suc-
1999.22 A further study revealed that it represents a new spe- cessful therapy for the treatment of P. parasiticum cutaneous
cies named P. griseorubrum.2 This species was isolated as infection.12
an opportunistic pathogen from a subcutaneous tumor of
an immunosuppressed patient.22 One undescribed case was 56.1.2.6 P. rubrigenum
reported by Mostert et al.2, when this species was isolated There is no documented case report in the literature when
from human blood. really P. rubrigenum caused human infection.2,22 Only two
cases are mentioned by Crous et al.5 and Mostert et al.2 when
56.1.2.4 P. krajdenii this fungus was isolated from human sources. One of them
The first published case where this fungus caused human was isolated from a patient with pneumonia,5 and the other
infection was ascribed to Phialophora repens,23 but it was was derived from infected eye.2
reidentified as P. krajdenii by Mostert et al.2 In this case,
the skin lesion treated successfully by excision of the nod- 56.1.2.7 P. sphinctrophorum
ule from the patient, who suffered from advanced leproma- Two misidentified Phialophora repens have been caused
tous leprosy.23 In the second case, the fungus, misidentified human infection with phaeohyphomycotic cyst and subcu-
as P. repens, was isolated from mycetoma on hand from a taneous cysts. Reidentification by morphological investiga-
patient with mild diabetes mellitus.24 The nodule was sur- tion completed with molecular techniques by Mostert et al.1
gically excised several times with administration of flucy- revealed that these isolates represent a new Phaeoacremonium
tosine (5FC).24 The MIC value for 5FC of this P. krajdenii species, P. sphinctrophorum.
isolate was 60 μg/mL.24 The last described case in the litera-
ture when P. krajdenii caused human infection was reported
56.1.2.8 P. tardicrescens
as pedal white grain eumycetoma in an immunocompetent
patient.14 It was treated with 400 mg/day oral dose of ITRZ Mostert et al.2 reidentified a P. inflatipes isolate derived from
with unknown outcome.14 Besides the above-mentioned ones, human source as P. tardicrescens, and introduced it as a new
there are six cases collected by Mostert et al.2 in which this species. Clinical features and pathogenesis of this fungus
fungus was identified as P. inflatipes originally in five of have not been revealed yet.
them. Two of them derived from unknown human source,
and the remaining caused cutaneous infection in foot. 56.1.2.9 P. venezuelense
Till date, this species has been detected only in three human
56.1.2.5 P. parasiticum cases causing cutaneous infections.1,2,43,44 Among them, one
P. parasiticum (formerly Phialophora parasitica5) is the most isolate was originally misidentified as Cephalosporium ser-
commonly isolated Phaeoacremonium species from humans; rae43. Surgical excision of the infected area was the suggested
it is able to cause infections in both immunocompetent and treatment of the infection in case of a patient with hepatic and
immunodeficient patients. Typical manifestation of the P. renal dysfunctions.44
parasiticum infection is the subcutaneous phaeohyphomyco-
sis.12,21,25–33 In some cases, this fungus could cause sepsis or 56.1.2.10 Unidentified Phaeoacremonium Species
was isolated from disseminated disease with fatal outcome One case was reported in the literature, when olec-
among immunocompromised host.12,13,34–37 Beyond these main ranon osteomyelitis was caused by an unidentified
manifestation forms, P. parasiticum was isolated from endo- Phaeoacremonium species in a patient who suffered trau-
phtalmitis,16 onychomycosis,15 arthritis,18,19 eumycetoma,38 matic olecranon bursitis.17 This disease was cured with sur-
and chronic granulomatous disease.39 Surgical excision with- gical bursal excision and antifungal therapy with ITRZ.17
out25,30 or with antifungal treatment, such as AMB+ITRZ or In another case report, P. inflatipes caused fungemia with
only FCZ, was successful therapy in P. parasiticum cutane- fatal outcome (nevertheless, AMB administration) in a
ous infections.27,29,31 Application of AMB+VCZ was reported child suffering from severe aplastic anemia.45 Identification
in endophthalmitis,16 topical administration of sulconazole in of this isolate as P. inflatipes remained doubtful because
onychomycosis,15 KCZ19 in arthritis, ITRZ+5FC in eumyce- homology of the partial rRNA gene sequences did not
toma,38 and AMB+VCZ with or without capsofungin in chronic exceed 95%. Furthermore, all isolates from human infec-
granulomatous disease.39 The MIC break- and MIC endpoints tions, which were originally determinated as P. infaltipes,
of the different drugs have not yet been established for this fun- proved to be misidentifications in the comparative work by
gus. The MIC values documented in the literature depend on Mostert et al.2 suggesting that P. inflatipes has not been iso-
the used susceptibility methods, media, and incubation times; lated from human infection yet.
these are the followings:12,21,40–42 AMB = 0.125–16 μg/mL, Tables 56.1 and 56.2 summarize the observed human
FCZ = 8 μg/mL, ITRZ = 0.03–32 μg/mL, KCZ = 2 μg/mL, cases and antifungal susceptibilities of Phaeoacremonium
miconazole = 2.5–10 μg/mL, posoconazole = 0.03–0.25 μg/mL, species.

TABLE 56.1
Reported Human Cases of Phaeoacremonium Infections
Age Surgical
Species (Year) Sex Underlying Disease Infection Site(s) AF Therapy Excision Outcome References
P. alvesii 83 F None Left foot None Y A (cure) [2,20]
19 M None Left ankle ITC Y A (cure [2,21]
P. amstelodamense n.d. n.d. n.d. Elbow joint int. n.d. n.d. n.d. [2]
P. griseorubrum 61 F Malignant rheumatoid Left foot FCZ Y A (failed) [2,22]
arthritis
n.d. n.d. n.d. Blood n.d. n.d. n.d. [2]
P. krajdenii n.d. M Lepromatous leprosy Scalp Prednisolon Y A (cure) [2,23]
63 M Mild diabetes mellitus Left hand 5FC Y A (cure) [2,24]
41 M None Right foot ITRZ Y n.d. [14]
31 M n.d. Foot n.d. n.d. n.d. [2]
n.d. F n.d. Foot n.d. n.d. n.d. [2]
n.d. F n.d. Foot n.d. n.d. n.d. [2]
n.d. n.d. n.d. n.d. n.d. n.d. n.d. [2]
n.d. n.d. n.d. n.d. n.d. n.d. n.d. [2]
P. parasiticum 45 M Renal transplant Skin, soft tissue None Y A (cure) [26]
41–44 M Renal transplant Leg, later also knee AMB, 5FC, KCZ, Y A (failed) [32–34]
ITR
45 M Liver transplant Disseminated AMB, ITR N D [34]
49 M None Knee AMB, 5FC N A (cure) [18]
30 F None Joint KCZ Y A (cure) [19]
61 M Renal transplant Leg FLC Y A (cure) [27]
92 F Unknown Disseminated AMB, TBF N D [35,36]
30 M Renal transplant Leg None Y A (cure) [25]
30 F None Foot ITR, 5FC Y A (cure) [38]
45 M Liver transplant Left hand, AMB, FCZ, ITR N D [13]
endocarditis
9–17 M Chronic granulomatous Lung CPF, ABLC, LAMB Y,N A (cure) [39]
disease VCZ, PSZ
55 M Renal transplant Left ankle, foot ITRZ, TBF, FCZ Y A (failed) [2,21]
30 F Aplastic anemia Disseminated ABLC N D [12]
40 M Cardiac transplant Hip, forearm ABLC, ITRZ Y D [12]
49 M Renal transplant Left foot AMB, ITRZ N A (cure) [29]
n.d. n.d. n.d. Eye, endophthalmitis AMB, VCZ N A (cure) [16]
n.d. M Kidney transplant Forefinger None Y A (cure) [30]
26 F None Forearm AMB, ITRZ Y A (cure) [31]
55 M None Onychomycosis SCZ Y A (cure) [15]
P. rubrigenum n.d. n.d. Pneumonia n.d. n.d. n.d. n.d. [5]
n.d. n.d. n.d. Eye n.d. n.d. n.d. [2]
P. n.d. n.d. n.d. Phaeohyphomycotic n.d. n.d. n.d. [1]
sphinctrophorum cyst
n.d. M n.d. Subcutaneous cyst n.d. n.d. n.d. [1]
P. tardicrescens n.d. n.d. n.d. n.d. n.d. n.d. n.d. [2]
P. venezuelense n.d. n.d. n.d. Foot n.d. n.d. n.d. [2,43]
28 M Bone marrow Right arm None Y n.d. [44]
transplant
n.d. n.d. n.d. Right ankle n.d. n.d. n.d. [2]
Unknown 54 F Myelodysplastic Elbow, osteomyelitis ITRZ Y A (cure) [17]
syndrome, IgA
deficiency
P. infaltipes? 2 M Aplastic anemia Disseminated AMB N D [45]
Abbreviations: M, male; F, female; AF, antifungal; 5FC, flucytosine; AMB, amphotericin B; ABLC, amphotericin B lipid complex; CPF, caspofungin;
FCZ, fluconazole; ITRZ, itraconazole; KCZ, ketoconazole; LAMB, liposomal amphotericin B; PSZ, posaconazole; SCZ, sulconazole; TBF, terbinafine;
VCZ, voriconazole; Y, yes; N, no; A, alive; D, dead; n.d., no data.

Phaeoacremonium 473
TABLE 56.2
Antifungal Susceptibilities of the Clinically Important Phaeoacremonium spp.
MIC (μg/mL)
Isolate AMB FCZ ITRZ KCZ MCZ PSZ RCZ TBF VCZ 5FC References
P. alvesii 2 8 16 4 n.d. n.d. 1 2 1 n.d. [21]
P. krajdenii n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 60 [24]
P. parasiticum 0.125–16 8 0.03–32 2 2.5–10 0.03–0.25 0.03–1 2 0.03–2 n.d. [12,21,40–42]
Abbreviations: AMB, amphotericin B, FCZ, fluconazole, ITRZ, itraconazole; KCZ, ketokonazole; PSZ, posoconazole; RCZ, ravuconazole; TBF,
terbinafine; VCZ, voriconazole; 5FC, flucytosine; n.d., no data.
56.1.3 Diagnosis
TABLE 56.3
Radial Growth of Clinically Important
Phaeoacremonium cases have been reported most frequently
Phaeoacremonium Species at Various Temperatures
as subcutaneous infections and usually involving phaeohy-
after 8 Days of Incubation at 25°C on MEA Medium by
phomycosis,1,2 which means the invasion of the human tissue
by fungi with dark, melanized cell wall. The symptoms of Mostert et al.
this disease are the dark phaeohyphomycotic cyst, a pain- Growth Temperatures (°C)
Radial
less or painful nodule, and a cavity filled with pus under the Species Minimum Optimum Maximum Growth (mm)
skin.1,2 There have been also other clinical manifestations
P. alvesii 15 30 37 9.5–11
of the Phaeoacremonium infections as described in Section
P. amstelodamense 15 30 40 11.5–12.5
56.1.2. P. griseorubrum 10 30 40 6–7.5
The histological traits of the Phaeoacremonium infec- P. krajdenii 15 30 37 9–14
tion are the mixed cell granuloma; the presence of septate, P. parasiticum 15 30 40 10.5–11.5
branched, and intertwined hyphal elements; and conidiog- P. rubrigenum 10 30 37 9.5–10
enous cells (phyalides) and conidia.14,20,22,28,44 P. sphinctrophorum 15 25–30 30–37 6–15
Identification of Phaeoacremonium species is tradition- P. tardicrescens 15 30 40 8–9
ally carried out by macro- and microscopic observation P. venezuelense 15 30 40 9–16
of the morphology and investigation of growth conditions.
The identification by macroscopic examination of the Source: Mostert, L. et al., Stud. Mycol., 54, 1, 2006.
Phaeoacremonium species is based on the colony color on
MEA medium, the radial growth on MEA, and the yellow
pigmentation on oatmeal agar (OA) and potato dextrose spacer regions (ITS1 and ITS2) of the ribosomal RNA gene
agar (PDA) medium.1,2 Key features of clinically important cluster; these regions are located between conserved genes
Phaeoacremonium species for their macromorphological encoding the 18S, 5.8S, and 28S rRNA.46 However, in case
identification are indicated in Tables 56.3 and 56.4 based of Phaeoacremonium, ITS region can be used only with lim-
on the studies of Mostert et al.1,2 Discriminating characters ited success for species-level identification because of the
for the microscopic identification are the mycelium texture, high level of homology and thus, alternative gene targets are
structure of conidiophore, length of conidiospore, diameters needed. Such alternatives may be the β-tubulin-, the actin-,
of warts, the predominant phialide type, and the predomi- or the calmodulin-encoding genes.1,2,46
nant Type II phialide shape.1,2 These characters are summa- Several methods are available for molecular identification
rized for clinically important species in Table 56.4. of Phaeoacremonium species, and they have been continu-
ously improved by new techniques. Restriction fragment length
56.1.3.2 Molecular Techniques polymorphisms (RFLP) patterns of the ITS region and the
Molecular techniques may help us to avoid the misidentifi- β-tubulin gene proved to be suitable to distinguish P. aleophi-
cation of the hypothesized species based on its macro- and lum, P. inflatipes, P. rubrigenum, and P. parasiticum.47,48 New
microscopic observation. The main advantage of the iden- species of Phaeoacremonium could successfully determined
tification by gene sequencing is that it does not require via- by DNA phylogenies inferred from ITS1 and ITS2, 5.8S rRNA,
ble organism or sporulation (molds), thus enabling a rapid β-tubulin, actin, and calmodulin gene sequences.2,7,8,49 For
diagnosis,46 This last mentioned reason is important in case rapid and clear species identification and detection, several
of Phaeoacremonium species because these fungi are slow polymerase chain reaction (PCR) primer sets and combinations
growing and their taxonomy is difficult.3 One of the most have been evaluated for ITS1 and ITS2, β-tubulin, and calmod-
frequently used targets for molecular identification of fungi ulin.1,2,12,44,50 Based on these studies, several Phaeoacremonium
including Phaeoacremonium is the internal transcribed species have been identified successfully from human

474
TABLE 56.4
Key Features for Macroscopic and Microscopic Identification of Clinically Important Phaeoacremonium Species by Mostert et al.
Yellow Maximum
Colony Color on Pigmentation on Mycelium Conidiophore Conidiophore Diameter of Predominant Predominant Type II
Species MEA OA and PDA Textures Structure Length (μm) Warts (μm) Phialide Type Phialide Shape
P. alvesii Medium pink or beige Variable Verruculose Mostly short and (14–)17–43(–50) av. 27 0.5 Type III Subcylindrical or navicular
unbranched
P. amstelodamense Beige to pale brown No Verruculose Mostly short and usually (15–)16–61(–90) av. 36 1 Type II Elongate-ampulliform,
unbranched constricted at the base
P. griseorubrum Dark pink No Verruculose Mostly short and (21–)23–70(–85) av. 38 1.5 Type II and III Elongate-ampulliform or
occasionally branched navicular
P. krajdenii Medium brown to dark No Verrucose Short and usually (16–)20–45(–76) av. 28 1 Type II Elongate-ampulliform,
brown unbranched attenuated at the base
P. parasiticum Brown with medium No Verrucose Mostly long and (24–)27–80(–130) av. 47 3 Type III Subcylindrical
brown center branched
P. rubrigenum Medium to purple pink No Verruculose Mostly short and (20–)23–51(–70) av. 34 1 Type III Elongate-ampulliform,
unbranched attenuated at the base
P. sphinctrophorum Brown to orange gray No Verrucose Mostly short and often (11–)13–39(–50) av. 23 No warts Type II Elongate-ampulliform

branched attenuated or constricted
at the base
P. tardicrescens Medium brown to No Verrucose Mostly short and (13–)16–52(–67) av. 31 0.5 Type I and III Subcylindrical to subulate
olivaceous-brown unbranched
P. venezuelense Beige to orange-brown No Verruculose Mostly short and (20–)28–48(–52) av. 31 1 Type III Subcylindrical or navicular
occasionally branched
Sources: Mostert, L. et al., Stud. Mycol., 54, 1, 2006; Mostert, L. et al., J. Clin. Microbiol., 43, 1752, 2005.
Note: Type of phialides: Type I, shortest, (2–)3–11(–17) μm, and have no basal septum, become polyphialidic in case of P. krajdenii; Type II, medium-sized, (5–)9–14(–16) μm, and are elongate-ampulliform or
navicular in shape, become polyphialidic in case of P. krajdenii; Type III, the longest, (10–)15–23(–34) μm, and are subcylindrical, navicular or subulate, monophialidic.
Phaeoacremonium 475
infections.1,2,12,14,15,44 The more rapid real-time PCR detection 2. Mount it in a specimen holder with a mixture of
protocols were evaluated for detection and identification based cryoblock and colloidal graphite.
on the differences between the ITS1 and ITS2 regions and the 3. Freeze the specimens in nitrogen slush (−212°C)
β-tubulin-encoding genes; however, this approach is not yet and transfer to the cryostage where a thin gold layer
available for all clinically important species.3,51 was splattered over the sample (75 s, 1.2 kV).
4. Transfer the sample to a cooled SEM stage chamber.
56.2 Methods 56.2.1.4 Genomic DNA Preparation

56.2.1 Sample Preparation Beyond the conventional DNA preparation methods,9,12,48,52
commercial DNA purification kits can be used for rapid sam-
For filamentous fungi, conventional DNA extraction methods ple preparation. Based on the literature, the following kits
are well suited to total genomic sample preparation.52 The are suited for this: FastDNA Kit (Bio101),1,50 DNeasy plant
disadvantages of these techniques are that they are slow and mini kit (QIAGEN),3 Maxwell® 16 Tissue DNA Purification
relatively high amount of samples are needed. In the studies Kit from Promega®,15 UltraClean™ Microbial DNA Kit
reviewed in the previous sections, commercial DNA purifi- (MO Bio).10
cation kits were used for rapid and safe sample preparation.
56.2.1.1 Media and Culturing Conditions 56.2.2 Detection Procedures

The following three media are preferred for morphologi-
cal identification of Phaeoacremonium species: MEA (5 g 56.2.2.1 Morphological Identification of
malt extract, 5 g yeast extract, 5 g glucose, 10 g KH2PO4) Phaeoacremonium Species
medium for macro- and microscopic morphology and for The steps of identification based on the morphological char-
measuring of the radial growth; OA (30 g of oat flakes, 1 g acters described by Mostert et al.1
of MgSO4 × 7H2O, 1.5 g of KH2PO4) and PDA (50% potato
Procedure
filtrate obtained by boiling 300 g diced potato in 500 mL
water and filtering, 20 g sucrose) medium for the detection of
1. Plate the strain onto MEA and incubate it at 25°C in
yellow pigmentation (all media are given for 1 liter and con-
the dark for 14 days.
tain 15 g agar).1,2,21 After incubation at 25°C for 5–14 days,
2. Cut mycelial plugs (2 mm diameter) from the mar-
Phaeoacremonium isolates grow well.1,2 The presence of
gin of the colony and transfer onto MEA, OA, and
near-UV light enhances the sporulation.1 Genomic DNA can
PDA media. Incubate the plates at 25°C and note
be extracted from mycelia grown either on solid or in liquid
the colony characters and pigment production after
malt extract medium after incubation at 25°C for 5–7 days.
8 and 16 days.
3. For determination of the cardinal growth tempera-
56.2.1.2 Light Microscopy Sample Preparation
tures, plate the plugs onto MEA in three replicates
Light microscopy sample preparation was describes by for each temperature and incubate the dishes in dark
Mostert et al.1 at temperatures ranging from 5°C to 40°C in 5°C
Procedure intervals, including 37°C to simulate human body
temperature.
1. Plate the strain onto MEA and incubate it at 25°C 4. To determine radial growth, measure the radius of
for 2–3 weeks in presence of near-UV light, which the colony, after incubation at 25°C for 8 days.
enhances the sporulation. 5. Use Tables 56.3 and 56.4 to identify the investigated
2. Collect aerial mycelium with fine needle 2–3 cm strain. In the microscopic observation, use Table
from the colony margin to avoid contact with the 56.4 for the identification of the investigated strain
agar, and mount in lactic acid on glass slide. based on its microscopic morphology.
3. For investigating the structures formed in and on
the agar surface, cut an agar block 3–5 mm from 56.2.2.2 Molecular Identification of
the margin of the colony with very little to no aerial Phaeoacremonium Species
mycelium, and mount it in lactic acid on glass slide. 56.2.2.2.1 Identification on Genus Level
56.2.1.3 Electron Microscopy Sample Preparation A nested PCR processed by Aroca and Raposo is appro-
priate to confirm that the investigated strain is actually a
Low-temperature scanning electron microscopy (SEM) pro-
Phaeoacremonium species.50 PCR amplification using primers
tocol derived from Mostert et al.1
ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′)53 and
Procedure ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)54 on DNA
extracts in the first reaction and then primers Pm1 (5′-CTC
1. Plate the strain onto small agar blocks (<25 mm2) of CAA ACC CTT TGT GAA CAT-3′, forward) and Pm2
MEA and incubate it at 25°C in the dark. (5′-CGA GCC CGC CAC TGA CTT-3′, reverse) on the product

of the first reaction results in a 415 bp fragments only in case of 56.2.2.2.2 Identification on Species Level
Phaeoacremonium species.50 Pm1 and Pm2 were designed in Using Species-Specific Primers
the ribosomal DNA ITS regions ITS1 and ITS2, respectively. Molecular identification of Phaeoacremonium species
Procedure including all clinically important taxa was carried out by
Mostert et al.1 using universal forward primer T1 (5′-AAC
1. Prepare the reaction mixture for the primary PCR in ATG CGT GAG ATT GTA AGT-3′)55 with specific reverse
a final volume of 25 μL containing 2 μL of 10× buf- primers developed in β-tubulin-encoding gene introns, and
fer, 0.2 μM (ITS1F and ITS4) primer, 2 mM MgCl2, a reverse primer ACT-783R (5′-TAC GAG TCC TTC TGG
0.2 mM deoxynucleoside triphosphates (dNTPs), CCC AT-3′)56 with specific forward primers both developed
0.75 U of Taq polymerase, 2.5 μL of BSA, and 1 μL in actin-encoding gene introns. The primers for the identi-
of DNA as template (approximately 10 ng of DNA). fication of all clinically important Phaeoacremonium spe-
2. Perform the first PCR in a thermocycler using the cies and the size of the amplified fragments are listed in
following reaction conditions: 2.5 min at 94°C; 35 Table 56.5.
cycles, with 1 cycle consisting of 15 s at 94°C, 30 s
at 53°C, and 90 s at 72°C, and a final extension step Procedure
at 72°C for 7 min.
3. Dilute (1:200) the PCR products from the first reac- 1. Prepare the reaction mixture containing 0.5 μL of
tion and use it in the secondary PCR. diluted sample (ranging from 10 to 100 ng/μL), 1×
4. Prepare the reaction mixture for the secondary PCR buffer, 2.5 pM of each primer, 200 μm of each
PCR in a final volume of 25 μL: 2.5 μL of 10× buf- of the dNTPs, 0.3 U of Taq DNA polymerase, and
fer, 0.5 μM (Pm1 and Pm2) primer, 4 mM MgCl2, 1.5 mM of MgCl2 and it made up to a final volume
0.8 mM dNTPs, and 1.25 U Taq polymerase. of 12.5 μL with sterile water.
5. Perform the second PCR in a thermocycler using 2. Perform the touchdown PCR in a thermocycler
the following reaction conditions: 5 min at 94°C; 30 using the following reaction conditions: 5 min at
cycles, with 1 cycle consisting of 30 s at 94°C, 30 s 94°C; 5 cycles, with 1 cycle consisting of 30 s at
at 57°C, and 50 s at 72°C, and a final extension step 94°C, 30 s at 66°C, and 60 s at 72°C; 5 cycles, with
at 72°C for 7 min. 1 cycle consisting of 30 s at 94°C, 30 s at 64°C, and
6. Visualize the PCR products after electrophoresis on 60 s at 72°C; and a final 25 cycles, with 1 cycle con-
1.5% agarose gels stained with ethidium bromide sisting 30 s at 94°C, 30 s at 62°C, and 60 s at 72°C,
and run in 1× Tris-borate-EDTA buffer. and a final extension step at 72°C for 6 min.
TABLE 56.5
Primers for the Identification of Clinically Important Phaeoacremonium Species
by Mostert et al.
Species-Specific Primer Fragment
Species Primer Pair (Pbr and/or Paf) Sequence (5′–3′) Size (bp)
P. alvesii T1 + Pbr5_1 ACG AGC TGA AGG TAA AAR GGA TC 548
ACT-783R + Paf2 GCC AAT CTG AGG CTA TGG AA 192
P. amstelodamense T1 + Pbr9 CGG TGA ACA TCA CGG GGG AG 456
P. griseorubrum T1 + Pbr9 CGG TGA ACA TCA CGG GGG AG 456
ACT-783R+Paf3 TCC GCC AAT TGA GGC TAC AA 194
P. krajdenii T1 + Pbr13 AGA TCG TTA GAC GTG TCC CG 272
P. parasiticum T1 + Pbr2_2 CGG TAG AGG TTT GGC GAC 446
P. rubrigenum T1 + Pbr5_1 ACG AGC TGA AGG TAA AAR GGA TC 548
ACT-783R + Paf1 GCC AAT CGA GGC TAT GGA G 191
P. sphinctrophorum T1 + Pbr2_1 AGC RCC TGT AGC TTT GCA G 611
P. tardicrescens T1 + Pbr1_2 TCC CGC TGA AGG AAA GGA AG 449
P. venezuelense T1 + Pbr3_1 ATC TCG AGA CAG AGC GGA TG 568
Source: Mostert, L. et al., Stud. Mycol., 54, 1, 2006.

Note: Pbr primers designed for introns of β-tubulin-encoding gene and Paf primers on introns of actin-
encoding gene. Primer Pbr5_1 not distinguishes P. rubrigenum and Pm. alvesii, while primer Pbr9 not
distinguishes Pm. griseorubrum and Pm. amstelodamense. Use additional species-specific primers
(developed in actin gene introns) in these cases. P. rubrigenum and P. alvesii are distinguished by the
ACT primers Paf1 and Paf2. A specific primer, Paf3, developed for P. griseorubrum. P. amstelodamense
gives a positive reaction with the primer Pbr9 and a negative one with Paf3.

Phaeoacremonium 477
3. Visualize the PCR products after electrophoresis on 4. Purify the PCR products with a commercial PCR
1% agarose gels stained with ethidium bromide and DNA purification kit.
run in 1× Tris-acetate-EDTA (TAE) buffer. 5. After the sequencing of the PCR product, com-
pare it with the β-tubulin sequences available from
56.2.2.2.3 Identification of Species Based on Partially GenBank using a BLAST search.
Amplified β-Tubulin Gene Sequences
Alternatively, Phaeoacremonium species can be identified The PCR product can be purified with commercial PCR DNA
by sequencing of the partial amplified β-tubulin-encoding purification kits, for example, GFXTM PCR DNA purifica-
gene, and its comparison with the β-tubulin sequences avail- tion kit (Pharmacia Biotech, Cerdanyola, Spain).58
able from genomic databases (GenBank, EMBL, and DDBJ) A Phaeoacremonium database containing all of the
using a Basic Local Alignment Search Tool (BLAST) search. known Phaeoacremonium species can be accessed from
Baddley et al.12 used T1 primer (5′-AAC ATG CGT GAG ATT the website of the CBS-KNAW Fungal Biodiversity Centre
GTA AGT-3′)55 with Bt2B primer (5′-ACC CTC AGT GTA (http://www.cbs.knaw.nl/databases/).
ACC CTT GGC-3′)57 based on the study of Mostert et al.2
Procedure 1 56.3 C
Perspectives
1. Prepare the reaction mixture containing 5 μL of
The incidence of fungal infections has increased dramati-
diluted sample, 1× PCR buffer 2.5 pM of each
cally over the past several years, especially among immu-
primer, 200 μM each of the dNTPs, 1.5 mM MgCl2,
nocompromised patient. Furthermore, more fungi have
and 0.5 U of Taq DNA polymerase; reaction mixture
emerged as opportunistic human pathogen.59 The data of the
is made up to a final volume of 25 μL with sterile
literature indicates that the human infections caused by dif-
water.
ferent Phaeoacremonium species have been more frequent in
the past two decades and this number could be increased in
lowing reaction conditions: 5 min at 96°C; 36 cycles,
the future.2 The treatment of Phaeoacremonium infections
with 1 cycle consisting of 30 s at 94°C, 30 s at 58°C,
is difficult because available data concerning the MIC
90 s at 72°C, and a final extension step at 72°C for
break- and endpoints of the generally used antifungal agents
7 min.
are very limited for the different species in the literature.
The suggested therapy is the surgical excision with admin-
1% agarose gels stained with ethidium bromide and
istration of AMB and extended-spectrum triazole antifungal
run in 1× TAE buffer.
agenst.12
4. Purify the PCR products with a commercial PCR
Taxonomy, detection, and identification of the members
of the Phaeoacremonium genus have been improved con-
5. After the sequencing of the PCR product, com-
tinuously; and numerous new species have been described
pare it with the β-tubulin sequences available from
and reidentified as new species by improving molecular
GenBank using a BLAST search.
techniques.1,2 Identification of Phaeoacremonium isolates by
morphological examination is well established and described
Guarro et al.44 used BT2-F (5′-GG(CT) AAC CA(AG)
by Mostert et al.1,2 The disadvantages of this technique are
AT(ATC) GGT GC(CT) GC(CT)-3′) and BT2-R (5′-ACC
that it is slow and sometimes lead to misidentification by an
CTC (AG)GT GTA GTG ACC CTT GGC-3′) primers based
inexperienced researcher. Application of different molecu-
on the study of Gilgado et al.58
lar methods allows fast and reliable identification. Detection
Procedure 2 of Phaeoacremonium spp. in genus level was evaluated by
Aroca and Raposo50 based on the differences of the ribosomal
1. Prepare the reaction mixture in the final volume of DNA ITS regions among different fungal genus. This nested
25 μL containing 20–60 ng of fungal DNA template, PCR method uses general fungal primers ITS1F and ITS4
10 mM Tris-HCl pH 8.3, 50 mM KCl, and 2.5 mM in the primary reaction and specific primers (Pm1 and Pm2)
MgCl2 (10× Perkin-Elmer buffer II plus MgCl2 solu- designed to the ribosomal DNA ITS regions ITS1 and ITS2
tion), 100 μM of each dNTPs, 1 μM of each primer, of some Phaeoacremonium species, respectively. Because
and 1.5 U of Taq DNA polymerase. of the limited applicability of the ITS region at the species
2. Perform the PCR in a thermocycler using the fol- level, other molecular markers were used to process a method
lowing reaction conditions: 5 min at 94°C; 35 cycles, for distinguishing the species46. A touchdown PCR method
with 1 cycle consisting of 30 s at 95°C, 1 min at appropriate to identify Phaeoacremonium isolates in species
60°C, 1 min at 72°C, and a final extension step at level was evaluated by Mostert et al.1 This method uses spe-
72°C for 7 min. cific primers designed to the different regions of the β-tubulin
3. Visualize the PCR products after electrophoresis on and the actin genes. More rapid real-time PCR detection pro-
1% agarose gels stained with ethidium bromide and tocols were evaluated using similar molecular markers for
run in 1× TAE buffer. detection and identification of Phaeoacremonium species,3,51

but these protocols have not been processed for all clinically 13. Heath, C.H. et al., Phaeoacremonium parasiticum infective
important species. endocarditis following liver transplantation. Clin. Infect. Dis.,
Species belonging to the Phaeoacremonium genus are 25, 1251, 1997.
14. Hemashettar, B.M. et al., Phaeoacremonium krajdenii, a
known principally as plant pathogenic fungi, but 9 (P. alvesii,
cause of white grain eumycetoma. J. Clin. Microbiol., 44,
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1143, 2002.

57 Phialemonium
Contents
57.1 Introduction...................................................................................................................................................................... 481
57.1.3 Diagnosis.............................................................................................................................................................. 482
57.2 Methods............................................................................................................................................................................ 483
57.3 Conclusions....................................................................................................................................................................... 484
References.................................................................................................................................................................................. 484
57.1 Introduction are often without a basal septum. Conidia are unicellular,
hyaline, and allantoid. On the other hand, P. obovatum pro-
57.1.1 Classification, Morphology, and Biology duces a distinctive pale green pigment in culture, in contrast
The genus Phialemonium consists of dematiaceous (i.e., to other medically important dematiaceous fungi which are
dark-walled) fungi belonging to the mitosporic Ascomycota olive green or brown to grey-black. P. obovatum conidia are
group, class Sordariomycetes, subphylum Pezizomycotina, obovoid or narrowest at the base.
phylum Ascomycota, kingdom Fungi. Currently, the genus Phialemonium species are saprophytic in nature and rep-
comprises two recognized species (Phialemonium curvatum resent a rare cause of invasive mold infections in humans,
and Phialemonium obovatum) and nine unassigned species. especially immunocompromised hosts and transplant recipi-
A former species in the genus Phialemonium dimorphospo- ents. Phialemonium spp. have the ability to sporulate within
rum is synonymized with P. curvatum [1]. Anamorphs of the the matrix of the agar medium, without the requirement of
genus are found in Cephalothecaceae. an air space above the hyphae, which is also noted in hya-
First created in 1983 by Gams and McGinnis to cover line filamentous molds such as Acremonium and Fusarium.
fungal isolates with morphological features intermediate The production of unicellular yeast-like forms or propagula-
between Acremonium (which is hyaline or nonpigmented) tion in vivo by Acremonium, Fusarium, and Phialemonium
and Phialophora (which is pigmented), Phialemonium enables their ready circulation in the bloodstream (resulting
appears pale in culture and the innate hyphal pigmenta- in fungemia) in comparison to hyphal structures of angio-
tion is observed in tissue only by histological stains with invasive fungi such as Aspergillus and Mucor. In addition,
specificity for melanin (e.g., the Fontana-Masson silver Phialemonium species are able to grow in culture at tempera-
stain) [2]. tures as high as 40°C, suggesting their potential for infection
Phialemonium colonies grow rapidly, reaching a diameter of the central nervous system.
of 25–30 mm in 7 days on Sabouraud dextrose agar (SDA).
Initially, colonies appear flat, moist, and white, and later
become yellowish, with yellow-green pigments diffusing
into media (in the case of P. obovatum). Conidiophores are Once thought to possess low pathogenic potential, the dema-
hyaline and resemble hyphae; intercalary and distinct septate tiaceous (or melanized) fungi have increasingly become com-
phialides (10–16 μm in length) without collarettes arise from mon causes of severe and disseminated phaeohyphomycosis,
hyphae; conidia (2–3 μm by 4–5 μm in size) are one-celled affecting cutaneous and subcutaneous tissues, the ocular
and hyaline, smooth walled, aggregating into slimy obovate region, frontal and maxillary sinuses, lungs, bones, and the
or allantoid balls [3]. nervous system [4]. The genus Phialemonium is considered
P. curvatum conidia are subglobose to ellipsoidal or as a phaeoid fungus, which may cause the lesions observed
curved. Phialoconidia formed in tapering phialides are in phaeohyphomycosis and hyalohyphomycosis [5–8].
located in short branches of the superficial and submersed Predisposing factors for fungal infections are antibiotic ther-
hyphae. These short prolongations are cylindrical and thin apy, post-transplant immunosuppressive therapy, and HIV
and lack a collarette. Adelophialides and some phialides infections. Another important risk factor for Phialemonium
481

infection is the use of arteriovenous (AV) grafts or fistulas apex. Three blood cultures and a graft tissue specimen cul-
for hemodialysis, as many infected patients had AV grafts ture grew P. curvatum. TEE showed a small, calcified veg-
or fistulas rather than catheters. It is possible that implanted etation on the anterior leaflet of the mitral valve that extended
synthetic grafts or surgically created fistulas may act as a into the left atrium, with mild mitral regurgitation. The third
nidus for subclinical Phialemonium infection, allowing case involved a 70-year-old woman undergoing long-term
small inocula to produce overt infection more readily in a hemodialysis. The patient had cerebrovascular accident,
graft or fistula than in a catheter. Prior to dialysis access, type II diabetes mellitus, and peripheral vascular disease. A
thorough skin antisepsis is required for grafts and fistulas. Phialemonium species grew on culture of a graft tissue speci-
Any suboptimal skin preparation may facilitate viable molds men. The fourth patient involved a 77-year-old man with type
on the skin to be inoculated into the bloodstream or the AV II diabetes mellitus and chronic renal failure requiring hemo-
graft. In addition, the use of prefilled syringes may also act dialysis. Ultrasonography of the AV graft revealed thrombo-
as the source of P. curvatum infection [9]. sis; histopathologic examination of resected graft material
P. curvatum infections have been reported in patients with showed inflammatory cells and numerous fungal elements,
bone marrow transplantation, and hemodialysis patients, who and culture of the AV graft and blood grew Phialemonium
developed overwhelming infection associated with funge- species [11].
mia and endocarditis [9–15]. A case of cutaneous infection P. obovatum has been linked to cases of endocarditis,
caused by P. curvatum was reported in a patient with multiple fungemia, and osteomyelitis [17–19]. A case of native valve
myeloma who underwent bone marrow transplantation. Two endocarditis caused by P. obovatum in a premature neonate
months after transplantation, the patient developed purplish was reported [20]. A 4.5-month-old infant with fatal P. obo-
and painful nodular lesions on the right ankle. Lesion mate- vatum infection of thermal burn wounds, viable tissue, and
rial contained septate hyphae and some apparently hyaline blood vessels is reported, with dissemination to the spleen.
dilated cells. On potato agar, an initially white colony later P. obovatum osteomyelitis has been observed in a previously
became chraceous with whitish borders forming moist and healthy 41-year-old man following discography. A case of P.
plane colonies. The colony became grayish on Sabouraud curvatum fungemia in a patient with chronic myelogenous
agar with sparse white mycelium. The fungus was identified leukemia was reported. The patient developed graft-ver-
as P. curvatum on the basis of its unicellular, hyaline, cylin- sus-host disease of the skin and liver with fever and severe
drical, and allantoid conidia, in addition to other characteris- diarrhea, 6 months after peripheral bone marrow transplan-
tic macroscopic and microscopic characteristics [16]. tation. Biopsies of the stomach, colon, and rectum showed
In the four cases of P. curvatum infections reported by granulomatous inflammation with marked crypt distortion,
Proia et al. [11], the first involved a 35-year-old man with simulating Crohn disease. A lung wedge biopsy of the lesion
chronic renal failure. The patient presented right ocular revealed septate branching hyphae (4–5 μm in diameter) with
pain, swelling, and vision loss, 1 year after cadaveric renal terminal globular structures (10 μm in diameter). Blood cul-
transplantation surgery and oral prednisone and cyclospo- tures grew a pure isolate of P. obovatum [21].
rine treatment through hemodialysis via AV fistula. Physical The in vitro susceptibility of Phialemonium indicated
inspection of the right eye revealed periorbital edema, a more consistent level of activity for voriconazole and
scleral injection with hemorrhagic foci, and vitreal clouding. posaconazole. Treatment with the new triazoles is associated
Gram staining of vitreal aspiration showed both hyphal and with improved survival [8]. Caspofungin, combined with
yeast-like forms. A transesophageal echocardiogram (TEE) amphotericin B deoxycholate may have an additive effect.
uncovered large vegetations on both mitral and aortic valves.
Primary blood cultures on BacT/ALERT aerobic and anaero-
57.1.3 Diagnosis
bic bottles (bioMérieux) at 30°C and 35°C became positive
after 4 days; subculture showed heavy growth of P. curva- Although Phialemonium is considered as a dematiaceous fun-
tum mold after 3 days. The patient underwent combined aor- gus, direct microscopic examination of the material obtained
tic and mitral valve replacement and right eye vitrectomy. from its lesions often shows hyaline septate hyphae and its
Gram staining of mitral valve tissue revealed septate hyphae, culture also appears pale. Since other phaeohyphomycotic
and cultures of mitral and aortic valve tissue specimens agents such as A. alternata, Bipolaris spicifera, Exophiala
grew P. curvatum. One day after the operation, the patient jeanselmei, or E. spinifera also appear hyaline, they may
became febrile and hypotensive and developed acute conges- complicate the diagnosis of Phialemonium infection. The
tive heart failure and disseminated intravascular coagulation differentiation of Phialemonium from other phaeohyphomy-
and then died. Postmortem examination revealed cardiomeg- cotic agents relies on the detection of the innate hyphal pig-
aly, marked left ventricular dilatation, splenomegaly, and a mentation in Phialemonium with the Fontana-Masson silver
large splenic abscess. A large “vegetation” had overgrown stain that binds specifically to melanin.
the prosthetic mitral valve and had invaded through the left Microscopically, the distinctive features of P. curvatum
ventricular wall. isolates include the presence of setose and cupulate sporo-
The second case concerned a 66-year-old man who had dochium, conidia formation from branched conidiophores,
undergone long-term hemodialysis after an AV graft. The conidium production on typical tapering adelophialide lack-
patient presented a soft systolic murmur over the cardiac ing a basal septum, and hyphae with lateral and intercalary

Phialemonium 483
phialidic conidiogenous cells, with typical clusters of conidia buffer 10%, followed by incubation for another 30 min at
aggregating in slimy heads [9]. By contrast, Phialemonium 65°C. One volume (∼700 μL) of chloroform-isoamyl alcohol
obovatum isolates show obovate straight conidia (3.5–5 by (vol/vol = 24/L) is added and mixed by inversion. After incu-
1–2 μm) borne on adelophialides (reduced phialides lacking bation for 30 min at 0°C (on ice water) and centrifugation at
a basal septum); and P. obovatum colonies on potato flakes 14,000 rpm at 4°C for 10 min, the top layer is transferred to
agar (at 25°C) demonstrate characteristic diffusible green a clean Eppendorf tube. The sample is added with 225 μL
pigment on reverse [20]. of 5 M NH4-acetate and incubated for at least 30 min (on ice
While in vitro culture enables more accurate morpho- water) and centrifuged. The supernatant is transferred to
logical characterization of Phialemonium spp., the process is a clean sterile Eppendorf tube and mixed with a 0.55 vol-
time-consuming and demands considerable technical exper- ume (∼510 μL) of ice-cold isopropanol. After centrifugation
tise. With the recent development and application of nucleic for 7 min at 14,000 rpm and 4°C (or room temperature), the
acid amplification and sequencing techniques, the precise supernatant is decanted. The pellet is washed with 70% ice-
and speedy identification of Phialemonium spp. has become cold ethanol two times and dried by using a vacuum dryer.
feasible. For example, PCR amplification of the internal tran- The powder is resuspended in 48.5 μL of Tris-EDTA buffer
scribed spacer (ITS) regions followed by nucleotide sequenc- with 1.5 μL of 10 mg of RNase/mL, incubated at 37°C for
ing contributed to the identification of P. curvatum from 15–30 min, and stored at −20°C until use [23].
patients [22]. Alternatively, Phialemonium isolates are grown on liquid
complete yeast medium, and DNA may be extracted using
the FastDNA Kit (Bio 101).
57.2 Methods
57.2.1 Sample Preparation 57.2.2 Detection Procedures
Clinical specimens suspected of dematiaceous fungi are
Proia et al. [11] utilized universal primers V9G (5′-T
treated with 10% KOH or stained with calcofluor white
TACGTCCCTGCCCTTTGTA-3′) and LR5 (5′-ATCC
and observed microscopically for septate hyphal elements.
TGAGGGAAACTTC-3′) to amplify the ITS region of
Fungal isolation is performed on sabourauol’s dextrose agar
nuclear ribosomal DNA followed by sequencing, leading to
(SDA) with chloramphenicol or potato dextrose agar (PDA)
the identification of Phialemonium isolates.
at 25°C and 35°C. Suspected Phialemonium isolates are sub-
cultured on PDA at 25°C. Preliminary identification is based Procedure
on macroscopic and microscopic features of the colonies.
After growth for 1–7 days. From day 1 to approx 4 is too 1. PCR mixture (50 μL) is composed of 25 pmol of
early to take mycelium, on PDA slants, approximately 1 cm2 each primer, 200 μmol of each dNTP, 1 U of Taq
of mycelium is collected by scraping the slant with a sterile polymerase (Super Taq; HT Biotechnology), 1×
stick in 1 mL of sterile H2O. The material is transferred to a standard PCR buffer, and 1 μL of genomic DNA
2 mL screw-cap tube, and the tube is centrifuged for 1 min extract.
at 6000 × g. If the mycelium does not pellet, the material is 2. Amplification is carried out with an initial 94°C for
contained with a pediatric blood serum filter. After removal 2 min; 35 cycles at 94°C for 35 s, 55°C for 50 s, and
of supernatant, the material is resuspended in 200 μL of IDI 72°C for 2 min; and a final 72°C for 6 min.
sample buffer and transferred to the lysis tube containing 3. After checking a portion of the PCR products
glass beads. Lysis tube is vortexed for 5 min, and the tube on agarose gel, the remaining PCR products are
is placed in a boiling water bath for 15 min. Following cen- purified with the GFX PCR DNA and Gel Band
trifugation for 5 min at 16,000 × g, the supernatant is stored at Purification Kit (Amersham Pharmacia).
−20°C until PCR is undertaken. 4. The PCR products are sequenced with the prim-
Alternatively, about 1 cm2 of fungal material is trans- ers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)
ferred to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)
mixture of silica gel and Celite (silica gel H, Merck 7736/ [White, et al. 1990] by means of the BigDye ter-
Kieselguhr Celite 545; Machery) and 300 μL of TES buf- minator cycle sequencing kit (Applied Biosystems).
fer [2 g Tris (hydroxymethyl)-aminomethane, 0.38 g Sequences are analyzed on an ABI Prism 3700
Na-ethylenediaminetetraacetic acid (EDTA), and 2 g sodium instrument (Applied Biosystems).
dodecyl sulfate in 80 mL of ultrapure water (pH 8)]. The fun- 5. The resulting sequences are aligned against pub-
gal material is ground with a micropestle for 1–2 min. The lished sequences using Clustal X, version 1.81.
volume is adjusted by adding 200 μL of TES buffer. After Phylogenetic relationships of the taxa are estimated
vigorous shaking and the addition of 10 μL of a 10 mg/mL from the aligned sequences by the maximum par-
concentration of proteinase K to the tube, the mixture is simony criterion, as implemented in PAUP, version
incubated at 65°C for 10 min. With the addition of 140 μL of 4.0b10. Heuristic searches are performed using
5 M NaCl solution, the mixture is combined with 1/10 vol- parsimony informative, unordered, and equally
ume (∼65 μL) of CTAB (cetyltrimethylammonium bromide) weighted characters; branch robustness is tested by

means of 1000 search replications, each on boot- 6. Guarro J et al. Phialemonium fungemia: Two documented
strapped data sets. Starting tree(s) are obtained via nosocomial cases. J Clin Microbiol. 1999;37:2493–2497.
stepwise, random sequence addition repeated 100 7. Huang J et al. Flow impeding fungal thrombus in the ascend-
ing aorta. Ann Thorac Surg. 2008;86(4):1373–1375.
times (repeated 10 times in bootstrap analyses).
8. Rivero M et al., Infections due to Phialemonium species: Case
A maximum number of 9000 trees (1000 trees in report and review. Med Mycol. 2009;47(7):766–774.
bootstrap analyses) are allowed [9]. 9. Strahilevitz J et al. An outbreak of Phialemonium infective
endocarditis linked to intracavernous penile injections for the
treatment of impotence. Clin Infect Dis. 2005;40:781–786.
57.3 Conclusions 10. Schonheyder HC et al. Late bioprosthetic valve endocarditis
caused by Phialemonium aff. curvatum and Streptococcus
The genus Phialemonium is a dematiaceous fungus (with sanguis: A case report. J Med Vet Mycol. 1996;34:209–214.
melanin in the cell walls and septa in the hyphae) that dem- 11. Proia LA et al. Phialemonium: An emerging mold pathogen
onstrates intermediate morphological features between the that caused 4 cases of hemodialysis—Associated endovascu-
hyaline or nonpigmented genus Acremonium and pigmented lar infection. Clin Infect Dis. 2004;39:373–379.
genus Phialophora. Because Phialemonium hyphal pig- 12. Zayit-Soudry S et al. Endogenous Phialemonium curvatum
endophthalmitis. Am J Ophthalmol. 2005;140(4):755–757.
mentation is only detectable in tissue with melanin-specific
13. Dan M et al. Phialemonium curvatum arthritis of the knee
histological stain such as the Fontana-Masson silver stain, following intra-articular injection of a corticosteroid. Med
it may be also confused with other phaeohyphomycotic Mycol. 2006;44(6):571–574.
agents including Alternaria alternata, Bipolaris spicifera, 14. Weinberger M et al. Isolated endogenous endophthalmitis due
Exophiala jeanselmei, and E. spinifera, which produce hya- to a sporodochial-forming Phialemonium curvatum acquired
line hyphae. through intracavernous autoinjections. Med Mycol. 2006
Along with an increasing occurrence of immunosup- May;44(3):253–259.
pression in human populations, invasive infectious diseases 15. Rao CY et al. Contaminated product water as the source
of Phialemonium curvatum bloodstream infection among
due to emerging pathogens such as Phialemonium species
patients undergoing hemodialysis. Infect Control Hosp
are reported with alarming frequency. Indeed, both species Epidemiol. 2009;30(9):840–847.
within the genus Phialemonium, P. obovatum, and P. cur- 16. Heins-Vaccari EM et al. Phialemonium curvatum infection
vatum, have been shown to cause endocarditis, fungemia, after bone marrow transplantation. Rev Inst Med Trop Sao
osteomyelitis, keratitis, and endophthalmitis, often with fatal Paulo. 2001;43(3):163–166.
outcome. Considering that many fungal organisms demon- 17. McGinnis MR, Gams W, Goodwin MN. Phialemonium obo-
strate varied sensitivity to antifungal drugs, it is necessary to vatum in a burned child. J Med Vet Mycol. 1986;24:51–55.
18. Magnon KC, Jalbert M, Padhye AA. Osteolytic phaeohypho-
correctly identify them to species level. As laboratory char-
mycosis caused by Phialemonium obovatum. Arch Pathol Lab
acterization of Phialemonium species based on their macro- Med. 1993;117:841–843.
scopic and microscopic features is lengthy and technically 19. Osherov A et al. Phialemonium curvatum prosthetic valve
demanding, molecular techniques such as PCR and sequenc- endocarditis with an unusual echocardiographic presentation.
ing have become an indispensible tool for their identification Echocardiography. 2006;23:6, 503–505.
and detection. 20. Gavin PJ et al. Fatal endocarditis caused by the dematiaceous
fungus Phialemonium obovatum: Case report and review of
the literature. J Clin Microbiol. 2002;40:2207–2212.
21. Scott RS, Sutton DA, Jagirdar J. Lung infection due to oppor-
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org/, accessed on August 1, 2010. and Crohn disease-like involvement of the gastrointestinal
2. Gams W, McGinnis MR. Phialemonium, a new anamorph tract. Ann Diagn Pathol. 2005;9(4):227–230.
genus intermediate between Phialophora and Acremonium. 22. Clark T et al. Outbreak of bloodstream infection with
Mycologia 1983;75:977–987. the mold Phialemonium among patients receiving dialy-
3. de Hoog GS et al. (eds.), Atlas of Clinical Fungi, 2nd sis at a hemodialysis unit. Infect Control Hosp Epidemiol.
edn. Utrecht, the Netherlands: Centraalbureau voor 2006;27:1164–1170.
Schimmelcultures, 2000. 23. Zeng JS et al. Spectrum of clinically relevant Exophiala species
4. Ajello L. Hyalohyphomycosis and phaeohyphomycosis: Two in the United States. J Clin Microbiol. 2007;45(11):3713–3720.
global disease entities of public health importance. Eur J 24. White TJ et al. Amplification and direct sequencing of fungal
Epidemiol. 1986;2:243–251. ribosomal RNA genes for phylogenetics, in PCR Protocols:
5. King D et al. A phaeohyphomycotic cyst and peritonitis A guide to methods and Applications, Innis, M.A., Gelfand,
caused by Phialemonium species and a reevaluation of its tax- D.H., Sninsky, J. and White, T.J., Eds., Academic Press Inc.,
onomy. J Clin Microbiol. 1993;31:1804–1810. San Diego, CA, 1990;315–322.

58 Pseudallescheria and Scedosporium
Ana Alastruey-Izquierdo, Maria Victoria Castelli, Leticia
Bernal-Martinez, and Juan Luis Rodríguez Tudela
Contents
58.1 Introduction...................................................................................................................................................................... 485
58.1.1 Taxonomy, Biology, and Morphology................................................................................................................... 485
58.1.1.1 Taxonomy............................................................................................................................................... 485
58.1.1.2 Biology................................................................................................................................................... 485
58.1.1.3 Morphology............................................................................................................................................ 486
58.1.3 Diagnosis.............................................................................................................................................................. 487
58.2 Methods............................................................................................................................................................................ 489
58.2.2.1 PCR Amplification of ITS1-ITS2 Regions............................................................................................ 489
58.2.2.2 β-Tubulin PCR....................................................................................................................................... 489
58.2.2.3 Real-Time PCR...................................................................................................................................... 490
References.................................................................................................................................................................................. 490
58.1 Introduction demonstrated that P. boydii/ S. apiospermum is a complex of

species. Scedosporium boydii is the anamorph of P. boydii,
Scedosporium is a ubiquitous filamentous fungus with a while S. apiospermum has no teleomorph described yet. In
worldwide distribution. Among this genus, there are two addition, some new species have been described by means of
medically important species: Scedosporium apiospermum sequencing part of the β-tubulin gene. The species currently
(the anamorph of Pseudallescheria boydii) and Scedosporium accepted in the P. boydii–S. apiospermum species complex
prolificans. Scedosporiosis is the term used to define the clin- are S. apiospermum, P. boydii, Scedosporium aurantiacum,
ical diseases caused by these species. The diseases caused by Pseudallescheria minutispora, Pseudallescheria angusta,
these fungi range from localized to disseminated infections Pseudallescheria ellipsoidea, Pseudallescheria fusoidea,
in immunocompetent and immunocompromised patients. and Scedosporium dehoogii. However, the latest molecular
The diagnosis of Scedosporiosis is difficult since clinical studies suggested that this complex could have more cryptic
features and histopathology are similar to those produced by species [6,7].
other common fungi [1,2]. The treatment of Scedosporiosis is S. prolificans was first isolated in 1974 from a greenhouse
also difficult because of the multiresistant nature of the genus. soil under the name Lomentospora prolificans [8]. The first
case, described in 1984, was a disseminated infection in an
58.1.1 Taxonomy, Biology, and Morphology immunocompromised host [9]. The mold causing the infec-
tion was named as Scedosporium inflatum. The identity of
58.1.1.1 Taxonomy these two taxa was recognized in 1991 by Gueho and de Hoog
P. boydii (Ascomycetes), the sexual state of S. apiospermum, [10] on molecular grounds, being renamed as Scedosporium
was first discovered in 1889 as a cause of a human otitis [3], prolificans. S. prolificans has no known sexual state.
while the anamorph state, called at that time Monosporium
apiospermum, was discovered in 1911 from a case of human 58.1.1.2 Biology
mycetoma [4]. In 1919, Castellani and Chalmers [5] renamed S. apiospermum is mainly found in temperate climates
the genus Monosporium as Scedosporium. and less frequently in the tropics. Although its natural
P. boydii has been described for many years as the sex- niche is unknown, it is always associated with environ-
ual state of S. apiospermum. Recent molecular studies have ments associated to human activity. It is frequently found
485

in manure-enriched or polluted environments as garden aggregates at the apex; they are ovoid to pyriform, smooth
soil, agricultural fields, sewer, or ditch mud and pollutes walled, olive to brown, 2–5 × 3–13 μm in size and having
pond bottoms [1]. Because of its ability to assimilate natu- a truncated base. Some isolates may produce round, thick-
ral gas and aromatic compounds, it has been suggested for walled solitary conidia arising directly from the hyphae
use in bioremediation [11]. S. prolificans is frequently found [1,12,13]. It has no sexual state described.
in warm, indoor soil, such as in potted plants as well as in
glasshouses [8].
58.1.1.3 Morphology In 1922, the ascomycete Allescheria boydii was isolated from
58.1.1.3.1 S. apiospermum/P. boydii a mycetoma patient in the United States. Since then, this mold
Macroscopy: Fast growing colonies, cottony to lanose, grey- has been involved in chronic subcutaneous infections before
ish-white becoming pale brown with a greyish black reverse. 1980 and systemic or disseminated infections in immuno-
The optimal temperature is 25°C although it can grow up to compromised patients in the following years. In 1982, Fisher
42°C. et al. [14] described the near-drowning syndrome caused by
Microscopy: Two asexual states are described for this S. apiospermum, and few years later Berenguer et al. [15]
species. The most prevalent is Scedosporium while the syn- reviewed its ability to cause brain infections. Although it had
anamorph Graphium is not frequently found in culture. The already been described as a pulmonary colonizer in 1955
cylindrical conidiogenous annellidic cells are produced ter- [16], it was not until 2000 when its importance as a colonizer
minally or laterally from the hyaline hyphae (Figure 58.1a). in cystic fibrosis patients was addressed [17].
Conidia are subhyaline to brown, smooth walled, and sub- Scedosporium apiospermum and Scedosporium prolifi-
spherical to elongate with a truncated base and measure cans are fungal opportunistic pathogens. They may cause
6–12 × 3.5–6.0 μm [12]. The Graphium synanamorph may be asymptomatic colonization or localized or disseminated
produced in a later stage in some colonies. It is characterized infection following trauma, surgery, and immunosuppres-
by erect synnemata (Figure 58.1b) with terminally conidiog- sion [18]. As stated above, infections by S. prolificans have
enous cells (smaller than the ones of the Scedosporium syn- been more recently described than those by S. apiospermum.
anamorph) producing broadly clavate conidia less pigmented Initially S. prolificans was found to cause bone and soft tis-
than the Scedosporium ones. sue infections in immunocompetent individuals. However,
The sexual state, Pseudallescheria, is favored by nutri- the importance of S. prolificans has increased in the last
tionally poor media such as cornmeal agar. It is homothal- years as it can cause disseminated infections, especially in
lic and produces yellow brown to black, closed fruit bodies neutropenic patients [19,20].
(cleistothecia) spherical and 140–200 μm in size. The cleisto- Scedosporiosis represents a broad spectrum of clinical
thecia contain ovate to subglobose (12–18 × 8–13 μm in size) diseases caused by these two species. Three clinical syn-
asci with eight lemon-shaped, smooth-walled, pale yellow dromes can be distinguished [1]:
to golden brown ascospores and 6–7 × 4.0–4.5 μm in size
[1,12,13]. 1. Asymptomatic or symptomatic colonization of cavi-
ties: These species can be colonizers of the lungs of
58.1.1.3.2 S. prolificans patients with pulmonary disorders, causing pulmo-
Macroscopy: Colonies grow moderately rapidly at 25°C on nary fungal balls in susceptible patients suffering
Sabouraud agar. They are expanding and flat and with a from sarcoidosis or tuberculosis, allergic broncho-
moist surface of olive grey to black color. Unlike S. apio- pulmonary pneumonia and pulmonary colonization
spermum, it is not able to grow in presence of cycloheximide. in cystic fibrosis patients. S. apiospermum is among
Microscopy: The conidiophores are short, characterized by the most common filamentous fungi colonizing the
having swollen flask-shaped bases and frequently showing lungs of cystic fibrosis patients. This occurrence of
a long annellated zone (Figure 58.1c). Conidia usually form S. apiospermum species complex in the lungs of
(a) (b) (c)
FIGURE 58.1 Microscopy of Scedosporium spp. (a) Scedosporium apiospermum, (b) Graphium synanamorph, (c) Scedosporium
prolificans.

Pseudallescheria and Scedosporium 487
these patients is remarkable since this species is species can make the classification of these fungi difficult
infrequently isolated from air. Regarding S. prolifi- [30]. Besides, conventional techniques are time consuming,
cans, regional differences have been found. Thus, the delay in treatment being the major disadvantage for this
this mold is, by far, more prevalent in Spain and mycosis.
Australia although it has been described in many
other countries as the United States and Belgium 58.1.3.1.1 Direct Microscopy
[19,21]. Sinuses, nasal septum, and external auditory The most common procedure employed to visualize fungal
conduct may also be colonized by these species con- structures in clinical samples is to macerate the sample
stituting the portal of entry for developing sinusitis in 20% KOH followed by fluorescence microscopy using
or external otitis [20]. Blankophor or Uvitek 2B. These stains are only useful to
2. Localized infections: These infections are usually detect fungal structures but they are not able to discrimi-
developed after traumatic inoculation of fungal nate between other fungi as Aspergillus, Fusarium, etc.
elements in healthy patients. Skin and soft tissue Direct exam can be useful for specific clinical pictures
infections, mycetoma; arthritis, osteomyelitis; lym- as mycetoma because grains of different etiologic agents
phocutaneous syndrome as well as eye infection; have specific characteristics that can help to identify the
and endophthalmitis have been described [22,23]. mold to genus level. For instances, grains of Scedosporium
3. Systemic invasive disease: Neutropenic patients, are white and large (1–2 mm), with a lobed surface, sur-
HIV patients, immunosuppressed recipients of solid rounded by a significant eosinophilic zone, which is not
organ, or stem cell transplant recipients are suscep- present in other grains from other agents of white myce-
tible to Scedosporium although these mycoses may toma [22].
be occasionally observed in immunocompetent
patients. S. apiospermum has caused infections in 58.1.3.1.2 Histopathology
victims of near drowning and evacuees of natural In tissue sections, these species appear as septated and
disasters, such as tsunamis and hurricanes [24]. branched hyphae and can be easily misidentified either as
Aspergillus, Fusarium, or black fungi. Scedosporium pres-
Most reports of S. prolificans infections have occurred in ents an irregular branching at acute angles and is dichoto-
Australia, Belgium, Spain, and the United States [19,21,23,25]. mous. Scedosporium spp. might also have terminal or
Although the causes for this highly localized prevalence are intercalary chlamydospores that can be confused with yeast.
not known yet, climatic conditions could be related [26]. Kaufman et al. [31] reported the use of a polyclonal fluo-
Invasive Scedosporium infections are characterized by rescent antibody that successfully identifies S. apiospermum
high mortality and poor response to antifungal agents. The in tissue sections. In addition, a technique based on hybrid-
majority of the available antifungal drugs show low in vitro ization in situ using DNA probes has been described to
activity against S. apiospermum and only some new triazoles improve these methods [32,33].
such as voriconazole and posaconazole seem to be active [1].
On the other hand, S. prolificans is a multi-resistant fungus, 58.1.3.1.3 Culture
tolerating virtually all systemically active antifungal agents Samples from respiratory tract, biopsies, or grains can be
including the new triazoles and echinocandins [1,27,28]. cultured. In cases of nonmycetoma infections, samples
from the respiratory tract and biopsy specimens from other
sites should be carefully handled to avoid contamination
58.1.3 Diagnosis
with other molds. These species grow well on routine fun-
Because of the usually fatal outcome of these infections, gal media such as Sabouraud glucose agar or malt extract
particularly in the immunocompromised patients, a correct agar (MEA). The incubation temperature is from 25°C to
and early diagnosis and identification of these fungi are nec- 35°C, some strains being able to grow at 42°C. The growth
essary [29]. response of S. apiospermum and S. prolificans is a useful
Regarding the European Organisation for Research feature for distinction between the two species as S. prolifi-
and Treatment of Cancer (EORTC) criteria, diagnosis of cans is not able to growth in conventional media containing
Scedosporium infection should be based on cytology and his- cycloheximide [34]. In addition, S. prolificans is frequently
topathology evidence in samples from normally sterile body recovered from blood cultures [23].
sites and isolation of the mold from cultures.
There are different diagnostic methods described for these 58.1.3.1.4 Susceptibility to Antifungals
species. However, the importance of the different available Scedosporium prolificans is a multiresistant mold [28].
diagnostic techniques may be determined by the type of However, some antifungals are active against S. apiosper-
infection [22]. mum species complex [28,35]. Voriconazole is the most active
antifungal against this species complex, whereas ampho-
58.1.3.1 Conventional Techniques tericin B and itraconazole showed a limited activity [28,35].
Culture and identification of the fungus is necessary to per- Posaconazole has also exhibited a good activity against
form a correct diagnosis, but other morphologically similar S. apiospermum complex (geometric mean: 1.17 mg/L;

unpublished observations). No clear differences regarding length of time required for diagnosis when classical micro-
antifungal activity have been detected among the different biological methods are used.
species of S. apiospermum complex so far [35]. Table 58.1 With the development of the polymerase chain reac-
displays the data of susceptibility in vitro of Scedosporium tion (PCR), it is possible to multiply a given DNA segment
clinical isolates. Table 58.1 includes data of 94 clinical strains quickly and identify the fungus, both from cultures and clini-
tested in the Spanish Mycology Reference Laboratory follow- cal samples. As a consequence, an early clinical diagnosis
ing the EUCAST (European Committee on Antimicrobial can be established.
Susceptibility Testing) reference procedure for susceptibility
testing of filamentous fungi. 58.1.3.2.1 Conventional PCR Assays
These assays have been developed to detect DNA from tis-
58.1.3.1.5 Serological Methods sues or cultured strains and are targeted toward the inter-
These methods like immunodiffusion tests are not commer- nal transcribed spacer (ITS) region of the ribosomal DNA
cially available yet [1,36]. An assay based on the detection gene cluster or to protein-coding genes such as β-tubulin
of a peptidorhamnomannan antigen for S. apiospermum is or calmodulin. Once amplification of the target region has
available [37]; however, cross reactivity with other fungal been performed, PCR-amplified segments must be identified.
species has been reported [38]. Several methods have been described:
Recently, Thornton et al. [24] have developed an immuno- Sequencing of the amplified segment, with a posterior
fluorescence and double-antibody sandwich enzyme-linked analysis of the sequence in a sequence database [6,7,35,39].
immunosorbent assays (DAS-ELISAs) to differentiate S. Use of probes [32,33,40] (1) Fluorescent dyes like SYBR
apiospermum from other infectious fungi and to track the green using a DNA intercalating dye that binds nonspecifi-
pathogen in environmental samples. The immunoassay uses cally to any double-stranded DNA generated during the PCR
an immunoglobulin M and IgG1 kappa-light chain monoclo- reaction. This method provides sensitive detection of DNA
nal antibodies (MAbs) specific to S. apiospermum, which but is not specific. Some instruments can perform a melt-
bind to an immunodominant carbohydrate epitope on an ing curve analysis to determine the melting temperature (Tm),
extracellular 120 kDa antigen present in the spore and hyphal which depends on the percentage of G+C content and length
cell walls of S. apiospermum and its sexual state, P. boydii. of the amplicon [41]. (2) Hybridization probes with sequence
The MAbs are specific and do not react with S. prolificans, homology to target DNA, which allows a specific detection.
Scedosporium dehoogii, or other clinically relevant fungi as
A. fumigatus, Candida albicans, Cryptococcus neoformans, 58.1.3.2.2 Microarrays
Fusarium solani, and Rhizopus oryzae. The specificity of A DNA microarray-based assay for the detection and identi-
this assay was confirmed by sequencing of the internally fication of 14 fungal pathogens in clinical samples including
transcribed spacer 1 rRNA-encoding region of environmen- S. apiospermum has been developed by Spiess et al. [42]. The
tal isolates. Unfortunately, there is not data regarding human assay combines multiplex PCR and consecutive DNA micro-
clinical samples array hybridization. PCR primers and probes were designed
from unique sequences of the 18S, 5.8S, and ITS1 regions of
58.1.3.2 Molecular Techniques the fungal rDNA genes. The assay has been tested in blood,
An increasing number of molecular techniques for the diag- bronchoalveolar lavage, and tissue samples from neutrope-
nosis of fungal infections have been developed in the last nic patients with proven, probable or possible invasive fungal
few years due to the growing prevalence of mycoses and the infection (IFI) or without IFI.
TABLE 58.1
Summary of Susceptibility Results In Vitro of Antifungal Agents Tested
against Scedosporium Speciesa
Species No. AMB ITC VRC POS TRB CPF MCF ANF
S. apiospermum 25 4.80 4.15 0.91 1.45 22.11 0.25 0.17 1.19
P. boydii 13 6.44 1.48 0.62 0.74 22.63 6.35 0.16 1.01
S. aurantiacum 5 10.6 9.19 1.15 1.74 24.25 0.69 0.69 0.02
S. dehoogii 3 16 2 1 1.41 32 11.31 0.03 1
P. ellipsoidea 6 6.96 1.14 0.5 0.75 21.11 0.71 0.02 0.02
S. prolificans 42 13.99 9.84 13.01 11.81 20.40 6.56 2.69 4
Notes: AMB, amphotericin B; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; TRB,
terbinafine; CPF, caspofungin; MCF, micafungin; ANF, anidulafungin.
a Minimal inhibitory concentration results of antifungals tested are displayed as geometric means
in mg/L.

58.1.3.2.3 Real-Time PCR 58.2.2 Detection Procedures

Recently, two real-time PCR (RT-PCR)-based assays using
58.2.2.1 PCR Amplification of ITS1-ITS2 Regions
Molecular Beacon probes targeting the ITS region of rDNA
for the specific and quantitative detection of S. prolificans There are several universal primers to amplify the ITS
and S. apiospermum have been described. DNA from dif- regions:
ferent samples as serum, blood, and lung tissues can be ana-
lyzed without post-amplification manipulations steps [29]. ITS-I-5.8S-ITS-II
The two assays were specific (100%) and had a good sensi- Primer Approximate
tivity and reproducibility when performed in Scedosporium [44] Sequence (5′–3′) Size (bp)
animal model of infection. In addition, these assays allow ITS 1 TCCGTAGGTGAACCTGCGG 600–630
a rapid identification of cultured strains suspected to be ITS 4 TCCTCCGCTTATTGATATGC
Scedosporium. ITS 3 GCATCGATGAAGAACGCAGC ITS 3 + 4 = 330
ITS 2 GCTGCGTTCTTCATCGATGC ITS 1 + 2 = 290
ITS 5 GGAAGTAAAAGTCGTAACAAGG ITS 5 + 4 = 645
58.2 Methods
58.2.1 Sample Preparation Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and
ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) can be used to
Fungal DNA extraction is the first step in the molecular diag- amplify the complete region ITS1-5.8S-ITS2 of these fungi.
nosis. There are several commercial and in-house methods The reaction mixtures contain 0.5 μM of each primer,
to perform this step. The extraction method described by 0.2 mM of each deoxynucleoside triphosphate, 1.5 mM
Holden et al. is described below: MgCl2, 2.5 U Taq DNA polymerase and 25 ng of DNA in a
DNA extraction from culture [43]: Subculture the iso- final volume of 50 μL of PCR buffer.
lated fungus in MEA (2% malt extract) or potato dextrose Cycling conditions include one initial cycle at 94°C for
agar (PDA) until visible growth at 30°C ± 2°C. Add distilled 2 min, followed by 35 cycles of 30 s at 94°C, 45 s at 56°C,
water to the subculture and scrape with a swab. Inoculate and 2 min at 72°C, with one final cycle of 5 min at 72°C. The
plastic Petri dish containing 3 mL GYEP (2% glucose, 0.3% reaction products must be analyzed in a 0.8% agarose gel.
yeast extract, 1% peptone) with 50–100 μL of the spore sus- The above conditions are examples, and each PCR should
pension obtained. Incubate until growth at 30°C ± 2°C. be optimized in individual laboratory.
Harvest mycelium with a plastic tip and put on a Whatman
paper. It is important to dry mycelium completely. Transfer 58.2.2.2 β-Tubulin PCR
dry mycelium to a 50 mL propylene tube with screw cap and
Two regions of the β-tubulin gene have been amplified to
add six glass beads (4 mm diameter). Introduce the tube into
identify these species:
liquid N2 for 30 s and vortex for 30 s. Repeat operation until
all mycelial powder is obtained. Add 0.8 mL of extraction
β-Tubulin
buffer (0.2 M Tris–HCl, 0.5 M NaCl, 10mM EDTA, SDS
1%) and 0.8 mL of phenol: chloroform: isoamilic alcohol Primer Sequence 5′–3′
(25:24:1) and shake gently. Transfer to an Eppendorf and BT2-F [6] GGYAACCARATHGGTGCYGCY
centrifuge at 12,000 rpm for 15 min at 4°C–8°C. Transfer BT2-R [6] ACCCTCRGTGTAGTGACCCTTGGC
supernatant to a new tube and add an equal volume of phe- TUB-F [45] CTGTCCAACCCCTCTTACGGCGACCTGAAC
nol: chloroform: isoamilic alcohol (25:24:1) and vortex TUB-R [45] ACCCTCACCAGTATACCAATGCAAGAAAGC
for 30 s. Centrifuge for 15 min at 12,000 rpm at 4°C–8°C.
Remove supernatant and extract with an equal volume of The reaction mixtures should be performed with 1 μM of
chloroform: isoamilic alcohol (24:1). Vortex for 30 s and each primer, 50 μM of each deoxynucleoside triphosphate,
centrifuge at 12000 rpm for 5 min at 4°C–8°C. Take super- 2 mM MgCl2, 2.5 U Taq DNA polymerase and 25 ng of DNA
natant and precipitate DNA with isopropanol. Centrifuge for in a final volume of 50 μL of PCR buffer.
15 min at 12,000 rpm at room temperature. Aspirate super- Cycling conditions include one initial cycle at 94°C for
natant and wash the pellet with 70% ethanol. Centrifuge 5 min, followed by 35 cycles of 30 s at 95°C, 1 min at 55°C
at 12000 rpm for 10 min at 4°C–8°C. Aspirate supernatant (TUB) or 60°C (BT2), and 1 min at 72°C, with one final cycle
and resuspend the pellet in 25–50 μL RNAase (1mg/mL). of 7 min at 72°C.
Incubate at 37°C for 1 h. The identity of Scedosporium can be verified through
DNA extraction from clinical samples: Different DNA sequencing analysis of the amplified segment. The PCR
extraction kits can be employed depending on the clini- product is first purified with one of the commercial kits avail-
cal sample. Examples of commercial kits available to able such as Chroma SPIN+TE-200; Chroma SPIN+TE-400;
extract DNA are Tissue: Wizard (Promega), QiAmpTissue Chroma SPIN+TE-1000; UltraClean™ (PCR Clean-up™
(Qiagen), etc.; Blood and other fluids (serum, bone marrow Kit, Mo Bio Laboratories Inc). For sequencing reactions, use
aspirate, bronchoalveolar lavage, etc.): “NucleoSpinBlood” commercial kits like BigDye Terminator cycle sequencing
(Macherey-Nagel), QiAmp (Qiagen). ready reaction: Applied Biosystems. Use 1 μM of the primers

and 3. μL of the purified PCR product in a final volume of identify fungi to the species level. However, the classical
10 μL. identification by means of observation of morphology is a
cumbersome and slow process. This situation has favored
58.2.2.3 Real-Time PCR the development of new methodologies based on sequencing
Two RT-PCR assays for the diagnosis of these species were of specific targets. Sequencing is now considered the gold
developed and validated for the classification of clinical standard to identify fungi to species level and its use has
strains and for the detection of DNA in clinical samples using allowed the identification of new cryptic species indistin-
a murine model of invasive infection [29]. PCR reactions were guishable by classical morphology examination. In the case
performed in the Chromo 4 System (MJ Research, Biorad). of Scedosporium apiospermum species complex, the clinical
The kit 2x SensiMix DNA (Quantace, Ecogen) was used as importance of these new species still needs to be addressed.
described by the manufacturer. The PCR reaction (20 μL) Most urgent need is the design and validation of new diag-
contained 0.5 μM of each of the primers, 4.5 mM MgCl2, nostic methodologies that able to detect the pathogen as
and 0.4 μM of the probe labeled with 5′ FAM and 3′ BHQ1 quick as possible in human clinical samples. To accomplish
(Sigma Genosys). Each reaction mixture contained 18 μL of this goal, two RT-PCR-based assays using molecular beacon
the master mix and a 2 μL aliquot of DNA from the extracted probes targeting the ITS region of rDNA for the specific and
sample (see above). The cycling conditions included a first quantitative detection of S. prolificans and S. apiospermum
step for preincubation (activation of the enzyme) and dena- have been developed. DNA from different samples as serum,
turation of the template DNA at 95°C for 10 min. Next steps blood, and lung tissues can be analyzed without post-amplifi-
included an amplification program of 40 cycles as follows: cation manipulation steps [29]. The two assays were specific
denaturation, 95°C, 15 s; annealing, 54°C for S. apiosper- (100%) and had a good sensitivity and reproducibility when
mum or 52°C for S. prolificans, 15 s; and extension, 72°C, performed in Scedosporium animal model of infection. In
5 s. Quantification standards were run in conjunction with addition, these assays allow a rapid identification of cultured
each set of samples. The amplicons generated were 132 bp strains suspected to be Scedosporium. Clinical studies must
for S. apiospermum and 176 bp for S. prolificans. PCR prod- be performed in order to know the diagnostic performance
ucts were subjected to electrophoresis in 2% agarose gels fol- of this RT-PCR methodology. In any case, molecular diag-
lowing the protocols of Sambrook [46] to confirm the PCR nostic techniques will be the gold standard for diagnosis of
results. Amplified fragments were sequenced (ABI Prism IFIs in the next future.
377 DNA sequencer, Applied Biosystem, Madrid, Spain)
and the obtained sequences were compared to S. prolificans References
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Scedosporium prolificans infection, a review of 162 cases. 40. Wedde M et al. PCR-based identification of clinically relevant
Med Mycol. 47(4), 359, 2009. Pseudallescheria/Scedosporium strains. Med Mycol. 36(2),
24. Thornton CR. Tracking the emerging human pathogen 61, 1998.
Pseudallescheria boydii by using highly specific monoclonal 41. Espy MJ et al. Real-time PCR in clinical microbiology:
antibodies. Clin Vac. Immunol. 16(5), 756, 2009. Applications for routine laboratory testing. Clin Microbiol
25. Maertens J et al. Disseminated infection by Scedosporium pro- Rev. 19(1), 165, 2006.
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Case report and review. Ann Hematol. 79(6), 340, 2000. cation of fungal pathogens in clinical samples from neutrope-
26. Ponton J et al. Emerging pathogens. Med Mycol. 38(Suppl 1), nic patients. J Clin Microbiol. 45(11), 3743, 2007.
225, 2000. 43. Holden DW. DNA mini prep method for Aspergillus fumigatus
27. Cuenca-Estrella M et al. Head-to-head comparison of the (and other filamentous fungi). In: B. Maresca, G.S. Kobayashi
activities of currently available antifungal agents against (eds.), Molecular Biology of Pathogenic Fungi, a Laboratory
3,378 Spanish clinical isolates of yeasts and filamentous Manual. New York: Telos Press, 1994, pp. 3–4.
fungi. Antimicrob Agents Chemother. 50(3), 917, 2006. 44. White TJ. Amplification and direct sequencing of fungal ribo-
28. Cuenca-Estrella M et al. Comparative in-vitro activity of somal RNA genes for phylogenetics. In: M. Innis (ed.), PCR
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against clinical isolates of Scedosporium prolificans and CA: Academic Press, 1990, pp. 315–322.
Scedosporium apiospermum. J Antimicrob Chemother. 43(1), 45. Cruse M et al. Cryptic species in Stachybotrys chartarum.
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30. Guarro J, Gene J. Acrophialophora fusispora misidentified
as Scedosporium prolificans. J Clin Microbiol. 40(9), 3544,
2002.

59 Sarcopodium
Contents
59.1 Introduction...................................................................................................................................................................... 493
59.1.3 Diagnosis.............................................................................................................................................................. 494
59.2 Methods............................................................................................................................................................................ 494
59.3 Conclusions....................................................................................................................................................................... 495
References.................................................................................................................................................................................. 495
59.1 Introduction superficial, solitary, gregarious or confluent, sessile, appla-

nate to cupulate, or pulvinate, subhyaline to dark brown,
59.1.1 Classification and Morphology setose, and up to 400 μm in diameter. Sterile hyphae (setae)
The genus Sarcopodium (obsolete synonyms: Tricholeconium, (65 × 1.5–2.5 μm) form a frill at the margin of the sporo-
Periolopsis, Actinostilbe, Kutilakesa, and Kutilakesopsis) dochium but are also interspersed with the conidiophores.
covers a group of plant-infecting fungi in the family They are single or form small fascicles of 3–5 hyphae
Nectriaceae, order Hypocreales, class Sordariomycetes, and are erect, unbranched or slightly branched toward
subphylum Pezizomycotina, phylum Ascomycota, king- the base, straight or flexuose, septate, subhyaline to dark
dom Fungi. Among the 12 recognized species in the genus, brown, smooth walled, thin to slightly thick walled, and
Sarcopodium araliae, Sarcopodium circinatum (the type spe- cylindrical, with obtuse apices. The conidiophores (up to
cies of the genus), Sarcopodium oculorum, and Sarcopodium 35 μm long) are well differentiated, straight or flexuose,
tortuosum are relatively common plant pathogens; and S. subhyaline to pale brown, and smooth walled. They are
oculorum, named after the infection site, has been shown to irregularly branched, and each branch usually bears a
cause human infection [1–3]. single terminal group of slightly appressed conidiogenous
On potato dextrose agar (PDA) at 25°C, S. oculorum colo- cells. The conidiogenous cells (8–13 μm × 1–1.8 μm) are
nies reach a diameter of 39–40 mm after 14 days. Initially, enteroblastic, monophialidic, terminal or lateral, hyaline
colonies are flat, mucoid, and cream but later become radi- to subhyaline, smooth walled, subcylindrical, and rarely
ally folded, brownish gray, and granulose due to the produc- intercalary with a cylindrical and lateral projection. These
tion of conidiomata. The colonies are grayish white toward intercalary conidiogenous cells are predominantly found
the periphery with sparse aerial mycelium; the reverse is on undifferentiated hyphae. The conidia are aggregated in
colorless to brown. On oatmeal agar (OA) and potato carrot cream slimy masses, which remain attached to the upper
agar (PCA) at 25°C, S. oculorum colonies grow more rapidly part of the conidioma and cover the entire surface. The
than on PDA, achieving a diameter of up to 48 and 45 mm, individual conidia (1.2–3 μm × 0.8–1.5 μm) are subhyaline,
respectively, after 14 days. Colonies are granulose at the cen- aseptate, smooth and thin walled, ellipsoid, and navicular
ter and smooth toward the periphery, with whitish, soft cot- or slightly allantoid. They reach up to 5 μm and are cylin-
tony aerial mycelium and brownish gray submerged hyphae; drical or allantoid when they emerge from undifferentiated
the reverse is brownish gray and paler toward the periphery. hyphae [3].
On PDA at 37°C, S. oculorum colonies measure 14–15 mm Although S. oculorum and S. circinatum have sessile
in diameter after 20 days. Colonies are elevated and cerebri- sporodochia, unbranched setae, and ellipsoid or cylindrical
form, with abundant sporulation, but no conidiomata devel- conidia, S. circinatum produces flexuous or circinate and
ops. S. oculorum does not grow at 40°C [3]. verrucose setae up to 6 μm wide and larger (7–10 μm × 2 μm)
S. oculorum typically produces two types of conidia: conidia that are never navicular or allantoid. S. oculorum is
one is derived from sporodochial conidiomata (cushion- differentiated from S. tortuosum by the latter’s orange slimy
like structures bearing numerous compact, short conid- conidial masses and branched setae. S. oculorum also resem-
iophores, which produce the conidial mass) and the other bles Myrothecium spp. and Stephanonectria keithii in the
is derived from undifferentiated hyphae. Sporodochia are order Hypocreales (Ascomycota). However, the sporodochial
493

sterile hyphae in Myrothecium are hyaline to subhyaline and acervular conidiomata (cup-shaped fruiting bodies) on natural
confined to the edge of the sporodochium, and in S. keithii substrates and turn sporodochial in vitro.
they are absent. Moreover, the conidial masses are green in Given that culture-based methods for fungal diagnosis
Myrothecium and brown in S. keithii [3]. require up to 3 weeks of incubation, PCR amplification and
sequencing analysis of rRNA intergenic spacer (ITS) regions
provide a rapid and accurate approach for S. oculorum iden-
tification [4–6].
Sarcopodium spp. are commonly present in plants, dead her-
baceous stems, and dead wood in many parts of the world,
but not in soil, air, or animals. 59.2 Methods
The only reported clinical case of Sarcopodium oculo-
rum infection in humans involved a 12-year-old Brazilian 59.2.1 Sample Preparation
boy with a vernal conjunctivitis [3]. The patient presented Sarcopodium specimens are grown on inhibitory mold agar,
with a pain in his right eye and felt the presence of a for- modified Sabouraud agars, or potato dextrose agar (PDA).
eign body. After a 5 month treatment for keratoconjunctivi- Oatmeal agar (OA) (30 g of oat flakes, 1 g of MgSO4 · 7H2O,
tis with specific antiallergic drugs, topical dexamethasone 1.5 g of KH2PO4, 15 g of agar, 1000 mL of tap water) and
and prednisone, the patient developed a corneal ulcer as a potato carrot agar (PCA) (20 g of potatoes, 20 g of carrot,
consequence of the allergic process, showing redness of the 18 g of agar, 1000 mL of tap water) may also be used.
eye with inflammatory infiltrate and a suspicion of infection. After growth for 4–7 days on PDA slants, lysates are
Direct examination of Gram-stained mounts of the deep cor- prepared from approximately 1 cm 2 of mycelia with IDI
neal scrapings revealed the presence of numerous septate and lysis kits (GeneOhm Sciences). Briefly, in a biological
branched hyphae and a few ellipsoidal conidia. Culture of safety cabinet, mycelia are collected by scraping the slant
the corneal scrapings on Sabouraud dextrose agar at 25°C, with a sterile stick in 1 mL of sterile, molecular-grade
30°C, and 37°C for 4–5 days, yielded several colonies of H 2O. The material is transferred to a 2 mL screw-cap
a single, darkly pigmented fungus, which was verified as tube. The tubes are centrifuged for 1 min at 6000 × g. If
Sarcopodium oculorum on the basis of its distinct morpho- the mycelium does not pellet, the material is contained
logical, biochemical, and molecular features. The infection with a pediatric blood serum filter (Porex Corp., Fairburn,
was resolved with natamycin and ketoconazole [3]. GA). After removal of supernatant, the material is resus-
pended in 200 μL of IDI sample buffer and transferred to
59.1.3 Diagnosis the lysis tube, which contains glass beads. Lysis tubes are
vortexed on the highest setting for 5 min and placed in a
The microscopic characteristics of Sarcopodium oculorum boiling water bath for 15 min and centrifuged for 5 min at
are examined under a light microscope upon preparation of 16,000 × g. The supernatant is stored at −20°C until ampli-
wet mounts with lactic acid. Photomicrographs may also be fication [5].
obtained by scanning electron microscopy. To differentiate
from other Sarcopodium species, a conidial suspension of
Sarcopodium oculorum may be inoculated onto sterilized 59.2.2 Detection Procedures
plant material since many plant pathogenic fungi show dif-
ferent morphological features on natural substrates compared
green DNA-binding dye and amplicon melting tempera-
to laboratory culture media.
ture analysis for fungal detection using pan-fungal primers
Sarcopodium is a hyphomycetous genus and is charac-
ITS1 forward (5′-TCCGTAGGTGAACCTGCGG-3′) and
terized by the presence of sporodochia with sterile, smooth
ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′) [7].
or ornamented, often coiled pale brown setae arising from
The identity of the fungi is verified by subsequent sequence
branched conidiophores and producing slimy conidia from
analysis.
phialides. The conidia are hyaline, aseptate, and fusiform to
ellipsoid or cylindrical. The presence of numerous, dark spo- Procedure
rodochia covered by a wet mass of cylindrical, hyaline conidia
may be confused with the typical conidiomata (pycnidia) of 1. PCR mixture is composed of 1× Lightcycler
Phoma or other similar coelomycetous fungi under a stereomi- FastStart DNA Master Hybridization Probes mix-
croscope. However, these structures are easily differentiated ture (Roche Applied Science) (containing deoxy-
at high magnification. The pycnidia are closed, spherical or nucleoside triphosphates, FastStart Taq DNA
obpyriform structures that are open only at the apical part by an polymerase, and 1 mM MgCl2, additional MgCl2 is
ostiole. Additionally, the pycnidia possess a pseudoparenchy- added to a final concentration of 4.6 mM), 0.4 μM
matous wall that is absent in the sporodochia. Conidiophores each of ITS1 forward and ITS4 reverse primers, 1×
are formed inside the pycnidia and line the internal cavity. SYBR green (Molecular Probes), and 3 μL tem-
Other coelomycetous fungi such as Colletotrichum, develop plate DNA.

Sarcopodium 495
2. Thermal cycling parameters include 95°C for 59.3 ConclusionS

76°C for 30 s; and a final extension at 72°C for 2 min. The genus Sarcopodium consists of 12 recognized fun-
3. The quality of the amplicon is determined using the gal species that are commonly found in plants, dead her-
derivative of the melt analysis curve (55°C–99°C, baceous stems, and dead wood worldwide. Only one case
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 of Sarcopodium oculorum vernal conjunctivitis has been
(Corbett Robotics, Inc). reported recently [3]. Because many fungal species such
4. The amplified product is purified for bidirec- as members of the Phoma and Colletotrichum genera may
tional sequencing using ExoSAP-IT (USB Corp). also cause keratomycosis, there is a need to correctly iden-
Five microliters of Big Dye Terminator Ready tify them for specific treatments. The fact that some of these
Reaction Mix v. 1.1 (Applied Biosystems) is fungi sometimes fail to sporulate in culture often delays the
added to 4 μL of each primer (0.8 pmol/μL) and results based on morphology and biochemistry. Use of PCR
3 μL of purified PCR product. Cycle sequencing and sequencing offers a rapid and precise means of deter-
is performed with a 9700 thermal cycler (ABI), mining the identity of Sarcopodium and the morphologically
using 25 cycles of 96°C for 10 s, 50°C for 5 s, and similar fungi.
60°C for 4 min. Sequencing reaction products are
passed through a Sephadex G-50 fine column to References
remove unincorporated dye terminators. Purified
1. Sutton, B. C. 1981. Sarcopodium and its synonyms. Trans Br
sequencing reaction products are run on an ABI
Mycol Soc. 76:97–102.
Prism 3100 Genetic Analyzer with a 50 cm capil- 2. Watanabe, T. 1993. Sarcopodium araliae sp. nov. on root of
lary array. Aralia elata from Japan. Mycologia. 85:520–526.
5. Sequences are analyzed with the SmartGene 3. Guarro, J. et al., Corneal ulcer caused by the new fun-
Integrated Database Network software version gal species Sarcopodium oculorum. J Clin Microbiol.
3.2.3 vr. SmartGene is a web-based software and 2002;40(8):3071–3075.
database system with reference sequences derived 4. Leaw, S. N. et al., Identification of medically important yeast
from the National Center for Biological Information species by sequence analysis of the internal transcribed spacer
regions. J Clin Microbiol. 2006;44(3):693–699.
5. Pounder, J. I. et al. Discovering potential pathogens among
fungi identified as nonsporulating molds. J Clin Microbiol.
Note. If real time PCR instrument is unavailable, stan- 2007;45(2):568–571.
dard PCR may be performed with primers ITS1 and ITS4, 6. Bagyalakshmi, R. et al. Newer emerging pathogens of ocu-
and the resulting amplicon is sequenced with the same lar non-sporulating molds (NSM) identified by polymerase
primers. Sequence-based identifications are defined by chain reaction (PCR)-based DNA sequencing technique tar-
percent identity: species, ≥99%; genus, 93%–99%; and geting internal transcribed spacer (ITS) region. Curr Eye Res.
2008;33(2):139–147.
inconclusive, ≤93%. For strains producing discrepant iden-
7. White, T. J. et al. 1990. Amplification and direct sequencing
tification between the methods based on phenotypic char- of fungal ribosomal RNA genes for phylogenetics. In M. A.
acteristics and ITS sequence analysis, the D1-D2 region of Innis, D. H. Gelfand, J. J. Snisky, and T. J. White (eds.), PCR
the large-subunit rRNA gene is amplified with primers NL1 Protocols: A Guide to Methods and Applications. Academic
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 Press, San Diego, CA, pp. 315–322.
species clarification (Leaw et al. 2006).

60 Sporothrix and Sporotrichosis
Conchita Toriello, María del Rocío Reyes-Montes,
Armando Pérez-Torres, and Amelia Pérez-Mejía
Contents
60.1 Introduction...................................................................................................................................................................... 497
60.1.1 Classification......................................................................................................................................................... 497
60.1.2 Epidemiology........................................................................................................................................................ 498
60.1.3 Morphology and Biology...................................................................................................................................... 498
60.1.5 Molecular Epidemiology...................................................................................................................................... 501
60.1.6 Laboratory Diagnosis of Sporotrichosis............................................................................................................... 503
60.2 Methods............................................................................................................................................................................ 505
60.2.2.1 RAPD-PCR Assay................................................................................................................................. 506
60.2.2.2 Identification of Sporothrix Species by Sequencing.............................................................................. 506
60.2.2.3 S. schenckii Detection by Nested PCR.................................................................................................. 506
References.................................................................................................................................................................................. 507
60.1 Introduction 60.1.1 Classification

Fungal species of the genus Sporothrix are ubiquitous fungi As no teleomorph of the genus Sporothrix has been
that include common soil saprobes on wood, peat moss, and found, S. schenckii was classified in the Deuteromycota,
organic litter, and also human, insect, and plant parasites. Hyphomycetes. However, following sequencing analyses of
Sporothrix is one of the anamorph genera associated with six gene regions: 18S rRNA, 28S rRNA, 5.8 rRNA, elonga-
the ascomycete genus Ophiostoma (former Ceratocystis), tion factor 1-α (EF1α), and two RNA polymerase II subunits
with species that are symbionts of bark beetles1,2 and also (RPB1 and RPB2), a recent milestone in fungal molecular
causal agents of sap stain in freshly cut wood.3 Ribosomal systematics is the estimated phylogeny of the Kingdom Fungi
DNA sequences have confirmed the phylogenetic relation of proposed by James et al.,13 leading to a higher-level classifica-
Sporothrix to this genus of the Ophiostomatales.4–6 These tion for all groups of Fungi.14 All previous work mentioned
two genera are largely distributed in nature and found in above, recognizing the close association of Ophiostoma/
identical biotopes, showing similar morphologies and physi- Sporothrix, is now demonstrated by the phylogenetic relation-
ological traits. The conidia of some Ophiostoma species are ship among 106 taxa from 12 orders in the Sordariomycetes
indistinguishable morphologically from those of Sporothrix based on four nuclear loci (nSSU and nLSU rDNA, TEF
schenckii, and they synthesize rhamnomannans, which also and RPB2).15 Therefore, today Sporothrix schenckii Hektoen
are the main surface antigens of S. schenckii7–9 and which & C.F. Perkins16 is classified as an anamorphic Ophio
are absent in most pathogenic fungi. Mariat10 proposed that stoma belonging to Ophiostomataceae, Ophiostomatales,
Ceratocystis stenoceras (now O. stenoceras) is the perfect Sordariomycetidae, Sordariomycetes, Ascomycota, and
stage of S. schenckii; however, although a clear relation exists Fungi.17 Other molecular investigations 6,12,15,18,19 showed a
between Sporothrix and Ophiostoma as mentioned above,4–6 high genetic variability in S. schenckii, pointing out diverse
analyses of restriction profiles of mDNA11, macro-restriction phylogenetic lineages and proposing S. schenckii as a com-
patterns with two enzymes,12 and DNA sequences of inter- plex of numerous cryptic species. In recent work,20 three new
nal transcribed spacer (ITS) regions of the ribosomal RNA species of Sporothrix of medical importance were described.
operon6 confirm the separation of both fungi. In all, 61 names of Sporothrix taxa are listed in the Index
497

TABLE 60.1
Species of the Sporothrix schenckii Complex
Species Morphologically Identified as Origin References
S. albicans S. schenckii Environmental Marimon et al.20
S. brasiliensis S. schenckii Clinical Marimon et al.20
S. globosa S. schenckii Clinical Marimon et al.20
S. mexicana S. schenckii Environmental Marimon et al.20
S. schenckii S. schenckii Clinical Hektoen and Perkins16
S. luriei S. schenckii var. luriei Clinical Marimon et al.21
fungorum by CABI Bioscience,17 including the new species penetrating the host. The mycelial morphotype shows hya-
of clinical relevance (Table 60.1). In the following sections, line, regularly septated, thin hyphae, 1–2 μm in diameter,
the names of Sporothrix spp. mentioned in the original pub- and oval, pyriform, or elongated conidia, 1.5–3 to 3–6 μm,
lications are used. which are born singly or in groups produced sympodially on
denticulate conidiogenous cells (sympodial conidia). Another
type of conidia produced is thick walled, dark brown, and
60.1.2 Epidemiology
usually borne individually on short denticles along the sides
S. schenckii has been largely known as the causative agent of hyphae.37 These conidia regarded as sessile conidia show
of sporotrichosis,22–24 a subcutaneous mycosis of human and different shapes among Sporothrix species of clinical rele-
other mammals, acquired generally by a traumatic contact vance.20 The yeast morphotype, easily obtained on different
with live or decayed vegetation, or any traumatism with a culture media and at 28°C and 37°C,38 shows yeast cells that
fungus-contaminated object or organic material, rarely are fusiform and ovoid of 2.5–5 to 3.5–6.5 μm, with single,
with insect bites,25 and recently, even with cat contact or double, or multiple budding. Yeast forms originate from the
scratches.26 In rare occasions, it might be acquired by spore sides and tips of the hyphae. When rarely observed in tis-
inhalation. This subcutaneous mycosis may be acute, well sue, yeast cells form “asteroid bodies” described as a cen-
delimitated, or chronic; but in recent times, the disease may tral yeast cell surrounded by an extracellular eosinophilic
develop as a very serious disseminated disease to osteoar- material forming spicules described 100 years ago.39 Yeast
ticular structures and viscera in immunosuppressed individu- cells are also observed in the host tissues as elongated yeasts
als, particularly with AIDS.27 This mycosis has been known of diverse forms, generally described as “cigar bodies.”
since the beginning of the twentieth century and mentioned S. schenckii requires thiamine, with the pyrimidine moiety
for the first time by Schenck in the United States in 1898. In being the effective structure.40 This fungus is able to grow
1900, Hektoen and Perkins16 described exhaustively the dis- over a wide pH range, mycelial morphotype from 3.0 to 12.5
ease. Nowadays, sporotrichosis is well known throughout the and the yeast morphotype from 2.4 to 9.5. Urease activity was
world; it is a mycosis rare in Europe but frequent in America, only observed in the mycelial morphotype.41 The key phe-
in tropical and subtropical climates, in some Asian countries, notypic features for recognizing five species of Sporothrix
and in Australia. The disease has been considered as occu- spp. of clinical relevance are the morphology of the sessile
pational due to the fact that infected patients are generally pigmented conidia, growth at 30°C, 35°C, and 37°C, and the
workers closely related to vegetation exposure where the assimilation of sucrose, raffinose, and ribitol.20
causal fungus has its ecological niche.28–31 One of the most The cell wall composition and polysaccharides of
famous epidemic of sporotrichosis occurred in 3000 gold S. schenckii have been widely reviewed previously9,42–44 show-
miners from South Africa infected with timbers on which ing mannose, glucose, and rhamnose constituents, this last
the fungus was growing.22 Nowadays, the largest epidemic of sugar very rare in other human pathogens. Rhamnomannans
this mycosis due to zoonotic transmission has been described with monorhamnosyl side chains predominate in conidia
in Rio de Janeiro, Brazil.26,32–35 Epidemiologic relevance of and yeast cells, whereas dirhamnosyl side chains in hyphae.9
results of feline sporotrichosis in Brazil shows that the isola- Peptido-rhamnomannans isolated from different culture
tion of the fungus from nails and oral cavity of cats indicates media and with diverse procedures are used in different
that transmission can occur through a scratch or bite, while immunological diagnostic tests and epidemiological studies.
isolation from the nasal fossae and cutaneous lesions postu- In particular, sporotrichin, a culture filtrate-derived peptide
lates the possibility of infection through secretions.33,35,36 polysaccharide,45,46 is especially efficient at detecting delayed-
type hypersensitivity and widely used in epidemiological
research.
60.1.3 Morphology and Biology
Melanin production of S. schenckii has been described
Sporothrix spp. are dimorphic fungi that exhibit a myce- on conidial47 and in yeast cells.48 The fungus ability of pro-
lial morphotype in nature and a yeast morphotype when ducing melanin over a broad pH range is advantageous for

Sporothrix and Sporotrichosis 499
its survival on environmental and pathogenic conditions.49 in vivo.48 Melanin is a virulence factor due to its presence on
S. schenckii produces melanin via the 1,8-dihydroxynaph- the cell wall during in vitro culture and during infection, and
thalene (DHN) pentaketide pathway on conidia, but not on may have a protective role for the fungus since melanin has a
hyphae.47 However, yeast cells can also produce melanin scavenger property of free radicals.47,48 Moreover, this patho-
in vitro and during mammalian infection.48 In recent work, genic attribute of S. schenckii could be related to impairment
Almeida-Paes et al.49 showed melanin particles derived from of oxidative mechanisms of phagocytic cells, mainly neutro-
hyphae grown in L-DOPA medium, providing evidence in phils and macrophages, as was observed in mongolian ger-
support of L-DOPA melanin synthesis distinct from the bils with experimental sporotrichosis.55
DHN processes in this fungus. This melanin production is An additional attribute for the pathogenic success of
variable among different strains of the fungus.49 Melanized S. schenckii through the ability to resist damage from reac-
S. schenckii strains appear to cause infection more easily tive oxygen species (ROS) elicited by host effector cells
than strains that produce low amounts of this pigment.50 could be the expression of catalases. An increased expres-
sion of catalase, at mRNA level, has been demonstrated dur-
ing the conversion to the yeast form of S. schenckii in vitro.
Moreover, it has been speculated that catalase may facilitate
S. schenckii is a pathogenic fungus because it causes host the phase transition. Since transcription of catalase mRNA
damage as a result from either direct fungal action or the host is rapidly upregulated after hydrogen peroxide challenge, it
immune response. However, defining microbial pathogenesis would be relevant in vivo to facilitate the intracellular sur-
in the context of host response or host damage permits the vival of S. schenckii by providing a nontoxic microenviron-
inclusion of many variables which affect the host–pathogen ment in the polymorphonuclear leukocyte and macrophage
relationship. Thus, virulence, as a relative property of the phagosomes.56 Neuraminidase may be another pathogenic
pathogen, is modulated by host susceptibility and resistance.51 factor in sporotrichosis because this enzyme can metabolize
Sporotrichosis, characterized by a wide range of cutaneous a number of host substrates as glycoproteins, lipoproteins,
and systemic clinical manifestations, is caused by S. schenckii, immunoglobulins, and fibrinogen.57
a fungus with an apparent regular pattern of virulence, which Recent works have been addressed to investigate the
draws attention to the immunological mechanisms of the host potential pathogenic attributes of some constitutive mol-
on the pathogenesis of the mycosis.52 Localized cutaneous ecules located at the cell wall of S. schenckii. For example,
and subcutaneous is the most common form of sporotrichosis a glycoprotein of 70 kDa (Gp70) from the cell wall of this
and is characterized by ulcerative lesions, associated regional fungus is involved in fungal adherence to dermal extracel-
lymphangitis, and lymphadenopathy. A distinctive clinical lular matrix, a process significantly reduced when yeast cells
finding is the presence of hard, spherical nodules along lym- are preincubated with anti-Gp70 serum in a concentration-
phatic vessels. Extracutaneous sporotrichosis can involve the dependent manner.58 Since adhesion to cell surfaces or extra-
lung, joints, bones, and other organs. The systemic form of cellular matrix molecules is the first step to infection and
sporotrichosis is usually associated to immunocompromised dissemination of pathogens in the host, it is relevant that yeast
patients.9 Interestingly, in these cases, primary skin lesions cells and conidia of S. schenckii can bind to type II collagen,
are scattered or often absent. It has been proposed that clini- fibronectin, and laminin; additionally, yeast cells can also
cal differences could be related to different virulence of S. bind to fibrininogen whereas conidia cannot bind to either
schenckii strains. However, virulence of strains isolated from fibrinogen or thrombospondin.59,60 Yeast cells of S. schenckii
nature is similar to the virulence of strains isolated from are able to adhere and invade an endothelial cells monolayer,
humans.53 a process modulated by IL-1β, TGFβ, and divalent cations,61
S. schenckii infection is acquired by a cutaneous micro- which could play a role in the dissemination of the fungus
traumatism and, probably also, by inhalation of the fungus. from the primary site of cutaneous infection to other organs.
Independently of the entrance route, the fungal cell wall is Cell walls of fungi contain multiple potential pathogenic
the first structure that interacts with cells and tissues of the attributes but are also highly immunogenic, and antibodies
host. The composition of S. schenckii cell walls has been to its molecules are produced during fungal infection. Thus,
extensively studied,9,44 but remains poorly understood with fungal cell walls have a dual status: like a Trojan horse and
regard to its role in infection, colonization, invasion, latency, like an Achilles heel. This analogy, once again, points out
and disease outcome. the relevance of host response in the outcome of fungus–host
The cell wall of S. schenckii contains biochemical com- interaction. An example of the above-mentioned is recent evi-
ponents that could act as virulence factors that can play a dence of antibody-mediated passive protection in a model of
central role in pathogen–host interaction eliciting a miscel- sporotrichosis obtained with a monoclonal antibody specific
laneous of pathogenic mechanisms. Thus, virulent strains of to a 70 kDa glycoprotein of S. schenckii cell wall, injected
S. schenckii form conidia with thick cell walls with irregu- before, during, and after experimental infection. The result
lar electrondense granules representing pigment particles on of passive protection was evaluated as a significant reduction
the outermost layer.54 This pigment has been characterized in the number of colony-forming units (CFUs) in the organs
as melanin using monoclonal antibodies against melanin to of mice.62 Interestingly, a 70 kDa glycoprotein of S. schenckii
detect it in conidia in vitro and in yeast cells in vitro and is now recognized as an adhesin for fibronectin, due to the

fact that the interaction or binding capacity of several strains Mycelial filaments are not usually observed in vivo; however,
of the fungus to fibronectin correlates with virulence, deter- its presence in tissue sections in human and murine sporotri-
mined by mortality rate, fungal burden, and histopathology.63 chosis has been reported.70–72
From the histopathological point of view, lesions of sporo- Histopathology of granulomatous skin lesions of sporotri-
trichosis are nonspecific. Severe infiltration of inflammatory chosis indicates a link between innate and adaptive immune
cells, consisting of abundant neutrophils, macrophages, and responses. Although neutrophils and macrophages are the
few plasma cells and lymphocytes, is frequently described main infiltrating cells, CD4-positive T cells and dendritic cells
in experimental and natural sporotrichosis. Typically, the (DCs) have also been identified in those lesions. These IFN-
fungus is often difficult to detect. However, in acute and gamma-producing T cells would eventually activate local
subacute experimental infection, it is easy to observe numer- macrophages to kill intracellular S. schenckii.73–75 However,
ous fungal cells that show morphological diversity but yeast activation of CD4-positive cells requires the participation
cells predominate, many inside of phagocytic cells at the of mature or activated DCs (after uptake and the process-
inoculated site and in regional lymph nodes (Figure 60.1). ing of fungal antigens), the most potent antigen-presenting
During a long time, the so-called asteroid bodies have been cells that play a key role in cell-mediated immunity, at least.
considered a hallmark of sporotrichosis, although they can In fact, activated DCs have been observed in the granuloma
occur in other systemic mycoses.22 The ultrastructure of of sporotrichosis.76 Recognition of fungal pathogens by
these structures reveals the presence of a central fungal cell, pattern-recognition receptors of DCs and macrophages77 is
surrounded by peripheral rays of an electrondense granular a necessary prerequisite to elicit several signaling pathways
layered material that resemble remnants of destroyed host associated to the activation of the innate immune system,
cells and adherent cell membrane fragments at the outermost releasing proinflammatory cytokines and modulating adap-
cell layer.64 Using hematoxylin–eosin stain, asteroid bodies, tive or acquired immunity.78
15–35 μm in diameter, are observed within abscesses and A recent study indicates that S. schenckii strains of the
consist of a central yeast cell surrounded by an extracellular cutaneous clinical form, but not those of visceral origin,
eosinophilic material forming spicules. Since asteroid bodies induce JNK, ERK, and p38 MAPK activation (intracel-
occur in lesion with extensive necrosis, it has been postulated lular signaling pathways), and IL-6 and TNF-α release in
that eosinophilic material results in part from precipitations DCs derived from monocytes.79 Since release of IL-10 was
of antigen–antibody complexes.65,66 The presence of asteroid not detected, it has been speculated that toll-like receptor-4
bodies appeared to be related to the type of lesion in view (TLR-4) but not TLR-2, expressed by DCs might be the
of the fact that it was observed in 17 of 32 primary lesions receptor to recognize S. schenckii strains of cutaneous ori-
whereas in 22 of 31 secondary lesions.65,67 It has also been gin and induce strong Th1 immune response. Interestingly,
observed in cytologic smears obtained by fine-needle aspira- S. schenckii strains of visceral origin induce Th2 response
tion biopsies.68 Other in vivo morphology of yeast cells is according to the significantly higher IL-4 production along
the termed cigar-shaped form, generally observed in large with the inability to induce strong Th1 response.79 Moreover,
and suppurative lesions69 and in experimental infections. DCs cocultured with S. schenckii strains of cutaneous origin
(a) (b)
(c) (d)
FIGURE 60.1 Subacute experimental infection by Sporothrix schenckii in mice. Lesions consist of abundant neutrophils, macrophages,
and some lymphoid cells. In experimental lesions, it is easy to observe fungal cells, mainly yeast cells, engulfed by phagocytic cells located
at inoculated site and in regional lymph nodes. Note the presence of great inflammatory infiltrate forming a granuloma located near to tunica
albuginea covering testis (a, b), and in mesenteric lymph node (c) after intraperitoneal inoculation of S. schenckii. Observe many foamy
macrophages with abundant fungal cells inside, distributed at the periphery of the granuloma (d). (a–c ×40; d: ×1000. PAS stain).

(yeast and conidia) exhibited a clearer morphological evi- Based on the RFLP patterns, 11 types were reported cor-
dence of activation with long and wider dendrites than when responding to S. schenckii, four types to C. stenoceras (=O.
they were cocultured with strains of visceral origin. However, stenoceras), four types to S. inflata, and seven types to
internalization of fungal cells of strains of cutaneous and vis- C. minor (=O. minor). Those isolates related to S. schenckii
ceral origin by DCs appeared comparable, regardless of the due to their phenotypical characteristics had different poly-
presenting forms (yeast or conidia).79 morphic patterns allowing discrimination from this species.
Takeda et al.83 divided 258 clinical isolates from four differ-
ent places in Japan into 10 types—phylogenetically related
60.1.5 Molecular Epidemiology
in two groups, group A and group B. Afterward, Cooper
Molecular taxonomy is becoming one of the main methods et al.,50 using the study done by Dixon et al.28 as a basis
for identifying fungal species. The introduction of these tech- and considering the phenotypical aspects from S. schenckii
niques is originating new and continuous taxonomic classifi- clinical isolates and those from nature from a sporotricho-
cations to help identify species that could only be classified sis epidemic in the United States in 1988, also used RFLP
by gender for the last few years. from mtDNA and total DNA methods. These investigators
In order to identify S. schenckii, traditionally research- selected representative isolates from each of the phenotypi-
ers used markers that could be phenotypically observed in cal groups (A–H) established by Dixon et al.28 and formed
the organisms. The phenotypical techniques are those that eight groups according to the polymorphic patterns revealed.
detect characteristics expressed by the microorganism. An important aspect of this study was the determination of
These markers have been useful for differentiating groups the similarity between the species isolated from patients
within a species, but of insufficient resolution for distinguish- with a nature group (group A) and differences with other
ing individuals in a population. However, genotypical tech- isolates from nature (B–H). This study suggests that the indi-
niques are more sensitive and specific since they involve the viduals within group A were possibly the etiological agents
direct DNA analysis of chromosomal or extra-chromosomal (S. schenckii) responsible for the epidemic. In addition,
genetic elements. These markers are a good system for exam- these results concur with those obtained by Dixon et al.28
ining, among other things, the diversity of levels between and who found that the microorganisms obtained from clinical
within species, population structures, and also allowing the cases were phenotypically identical to those of group I from
study of the origin and evolution of isolates. Currently, phe- nature. On the other hand, the study conducted by Cooper et
notypic and genotypic markers are used for studying patho- al.50 using RFLP showed greater discretion between strains
genic fungi, that is, the recent research that pointed out three than the one carried out by Dixon et al.28 who based their
new species of Sporothrix spp.20 studies on phenotypical characteristics. In another epidemio-
Classical phenotypic morphological, thermal tolerant, and logical study using RFLP, Hajjeh et al.84 analyzed clinical
biochemical markers are used for characterizing S. schenckii. and environmental isolates from a sporotrichosis outbreak in
In the molecular typification of Sporothrix, the majority of workers from three plant nurseries in Florida, and different
authors use identification methods based on genomic diges- from that found by Dixon et al.28 and Cooper et al.50 where
tion with restriction enzymes such as restriction fragment the clinical isolates had very similar patterns amongst them-
length polymorphisms (RFLPs), in addition to other tech- selves and different ones from the environmental isolates.
niques as amplified fragment length polymorphisms (AFLPs) These observations, as well as those on the phenotypical and
and randomly amplified polymorphic DNA (RAPD). On the genotypical characterization of clinical and environmental
other hand, polymerase chain reaction (PCR) techniques are isolates where the strains isolated from clinical cases dif-
more frequently used by amplifying specific genomic zones, fer from those isolated in nature, suggest diverse fungal sub-
usually ribosomic DNA (rDNA) or mitochondrial DNA types or varieties.
(mtDNA). Lastly, the amplification of small rDNA fragments Ishizaki et al.85–88 have conducted several studies using
is being used successfully (ITS) for identifying certain fun- mtDNA-RFLP for assessing the variability between iso-
gal species. lates from different geographical origins (America, Asia,
The first studies that were conducted for typifying and Oceania). Their results confirmed the identification of
S. schenckii strains were based on its phenotypical character- the clinical isolates studied such as S. schenckii previously
istics.22,28,54,80–82 Several investigators9,20,41 found morphologi- classified by its morphological characteristics. Similarly,
cal and physiological differences between clinical isolates they reported that the 24 types of S. schenckii by mtDNA-
and those from other sources. However, these characteristics RFLP formed two phylogenetic groups. Group A was com-
may be influenced by several variables, for which genotypi- posed by types 1–3, 11, 14–19, 22, and 23, while group B by
cal methods, using molecular biology techniques allowing for types 4–10, 12, 13, 20, 21, and 24. It was also reported that
more sensitive studies, complement phenotypical methods. group A strains were predominantly from South America
To date, a number of molecular epidemiological studies and Africa, while group B strains were from Australia and
have been carried out with S. schenckii (Table 60.2). One Asia. Complementing this study, Kawasaki et al.98 studied
of the first was conducted by Suzuki et al.11 who studied S. isolates from Brazil, Mexico, and Spain, using the same
schenckii isolates and related fungi such as Ceratocystis molecular method, and found that all of the isolates from
spp. (now Ophiostoma) and Graphium sp. by mtDNA RFLP. Spain were in group B (previously established)—different

TABLE 60.2
Different Genotypes within Sporothrix spp. of Clinical Relevance and Ophiostoma-Related Species
Molecular Typing Genotypes References
RFLP of mtDNA S. schenckii isolates from all the world as: 24 mtDNA types with two groups Suzuki et al.,11 Takeda et al.,83
A = South Africa, North, Central, and South America Ishizaki et al.,85–88 Lin et al.,89
B = Japan, Australia, China, Korea Mora-Cabrera et al.,90 and
Arenas et al.91
DNA ITS1-RFLP 204 S. schenckii isolates into four types correlated with geographical origins Watanabe et al.19
RFLP (HaeIII, MspI), whole cell DNA DNA patterns of clinical isolates identical to group I environmental isolates Cooper et al.50
Groups II–VI complex of related fungi: Sporothrix–Ophiostoma
RFLP hybridization of ApaI-digest 31 S. schenckii isolates (China), 15 individual patterns (DNA types A-O) Zhang et al.92
genomic DNA with a probe (rRNA) recognized
Types A–C = 51.61%
18S rDNA sequencing S. schenckii related phylogenetically to Ophiostoma Berbee and Taylor4
RAPD-PCR 44 S. schenckii isolates of Mexico, Guatemala, and Colombia: grouping Mesa-Arango et al.93
related to geographical origin. Separation of clinical and environmental
isolates of Mexico
DNA fingerprinting M13 88 S. schenckii isolates of Brazil (59 cat epidemic) and Spain Gutierrez Galhardo et al.94
rDNA ITS sequencing Nine subtypes not associated to clinical forms
DNA sequence of ITS including Clear separation of S. schenckii, O. stenoceras, and O. nigrocarpum species De Beer et al.6
5.8 rRNA Phylogenetically related S. schenckii appears as more than one species
Two groups: clinical and environmental
AFLP (EcoRI-AA, MseI-A) 32 S. schenckii isolates from Peru Neyra et al.95
Two populations (A, B) not related to substrate, pathology, or geography
Pulsed field gel electrophoresis (PFGE) Eight strains S. schenckii from Japan, three types—Six to eight Tateishi et al.96
chromosomes
CHEF Same PFGE pattern in S. schenckii-dominant strain isolated from clinical O’Reilly and Altman12
cases and environmental hay for 15 years in Western Australia
Sequence analysis of protein coding 60 S. schenckii isolates Marimon et al.18
loci for chitin synthase, β-tubulin, Six distinct putative phylogenetic species among complex
calmodulin
Calmodulin gene sequences 127 S.schenckii isolates (USA, Europe, Asia) Marimon et al.20
Pheno- and genetically separation. Three new species: S. brasiliensis,
S. globosa, and S. mexicana
S. albicans, S. inflata, and S. schenckii var. luriei, different from
S. schenckii, no longer considered as single species
Chitin synthase, β-tubulin, calmodulin Strain CBS 937.72 (=ATCC 18616), clinical isolate from South Africa Marimon et al.21
sequences New species: S. luriei
Partial sequences of calmodulin gene S. globosa in Central and South America Madrid et al.97y
from those from North and South America that were mainly were included in group A, where type 2 (13 isolates), type 3
in the group A, coincident with the results by Ishizaki (10 isolates), and type 28 (7 isolates) dominated. All of the
et al.85–87. Mora-Cabrera et al.90 analyzed isolates from isolates from India and Thailand were included in group B.
Mexico, Guatemala, and Colombia using mtDNA-RFLP and The 52 isolates from group A and 24 from group B corre-
reported six new types (type 25–30). Watanabe et al.19 used sponded to type I and type IV rDNA, respectively, reported
DNA ITS1-RFLP to study the phylogeny among the existing by Watanabe et al.19
S. schenckii types. They found four types: rDNA types I–III With another molecular typing method, Tateishi et al.96
corresponding to the mtDNA from group A (types 1, 2, 11, applied karyotype analysis to eight S. schenckii isolates and
14–19, 22, and 23) and type IV corresponding to the mtDNA divided them into three types. Sugita99 used single-strand
group B strains (types 4–10, 12, 13, 20, and 21) of Ishizaki et conformation polymorphism (SSCP) to study 20 S. schenckii
al.85–87 The authors mention that ITS-RFLP is a method with isolates previously classified by mtDNA-RFLP. These authors
a greater discriminatory power than mtDNA-RFLP. In a fur- used oligonucleotides designed from the region of a putative
ther study, Ishizaki et al.88 typified other isolates from India, gene that codes for a membrane transporting protein. The
Thailand, Mexico, Guatemala, Colombia, and Brazil using isolates were divided into three types corresponding to those
mtDNA-RFLP and ITS-RFLP, finding two new types (31 previously obtained in karyotype analysis.96
and 32). Type 30 previously reported by Mora-Cabrera et al.90 Reis et al.100 carried out a study using RAPD-PCR for iden-
was confirmed as type 3. Of the 48 isolates from Mexico, 41 tifying the genetic relationship among S. schenckii strains

from patients and cats involved in an outbreak of sporotri- isolates identified morphologically as S. schenckii, from
chosis in Brazil. Their results showed great genetic similarity Mexico, Guatemala, and Colombia, through partial
among the 15 cases studied. The DNA from a strain isolated gene sequences of CAL. The study revealed, apart from
from a patient showed a profile identical to the DNA isolated S. schenckii sensu stricto, S. globosa in Mexico, Central, and
from a cat. The polymorphic patterns obtained from the iso- South America for the first time.
lates from three other patients were very similar to patterns In regard to the sensitivity of Sporothrix to different anti-
obtained from the isolates of cats studied, suggesting a com- mycotics, Gutierrez Galhardo et al.94 investigated the phe-
mon source of infection and thought of as the mode of infec- notypes (sensitivity to antimycotics) and genotypes (DNA
tion. Lee et al.101 analyzed 10 S. schenckii clinical isolates, fingerprinting method with primer M13, sequencing of the
in addition to other fungi, using RAPD-PCR. The results ITS-rDNA region) of the isolates recovered from different
showed that each random primer amplified the characteristic clinical forms during an outbreak transmitted by cats. The
band patterns in DNA of 8 out of 10 isolates, but different minimum inhibitory concentration (MIC) values to ampho-
in two isolates, suggesting a different Sporothrix anamorph tericin B, itraconazole, posaconazole, ravuconazole, and ter-
from Korea. Mesa-Arango et al.93 analyzed the phenotypical binafine were low for the isolates, and no differences were
characteristics (conidia size and thermal tolerance) and geno- found between the different clinical forms of the disease.
types using RAPD of isolates from Mexico, Guatemala, and The phenotypical techniques revealed that the isolates were
Colombia. The isolates from Colombia showed a greater size associated among themselves, and the fingerprinting analy-
of conidia and were thermal sensitive in relation to the iso- sis showed that Rio de Janeiro epidemic strains were geneti-
lates from Mexico and Guatemala. Using RAPD, a great vari- cally related. Although nine subtypes were found, they were
ability was observed among the S. schenckii isolates, forming not associated with specific clinical forms. Similar results
four groups according to their geographical origin. No rela- were obtained with ITS sequence analysis, suggesting that
tionship was found among the polymorphic patterns and the the cases related to the outbreak had a common origin. In
clinical forms of the disease, although isolates from Mexico another antifungal susceptibility study of five species of
were separated into two groups: clinical and environmental. Sporothrix,102 significant differences were found among
Neyra et al.95 used AFLP for analyzing the genetic diver- these species, with S. brasiliensis showing the best response
sity of S. schenckii isolates of Peru and reference strains from to antifungals, and S. mexicana the worst response.
other countries. The isolates from Peru were divided into two
separate homogeneous groups. A high level of variability
60.1.6 Laboratory Diagnosis of Sporotrichosis
was seen among the two groups without finding a correlation
to the clinical form of sporotrichosis. The definitive diagnosis of sporotrichosis relies on isolating
Recently, based on the analysis of the ITS rDNA region, the organism from the site of infection in cutaneous sporotri-
it has been suggested that more than one species may exist in chosis. S. schenckii is relatively easy to grow when compared
S. schenckii.6 With the purpose of clarifying if the variability with other systemic fungi, such as Histoplasma capsulatum.
of this fungus is due to species divergence or intraspecific Within days to weeks, hyphal growth occurs. The morpho-
diversity, Marimon et al.18 determined the sequence analysis logical microscopic characteristic of the fungus allows pre-
of three protein coding loci [chitin synthase, β-tubulin, and sumptive identification of the organism as Sporothrix spp.,
calmodulin (CAL)] of 60 S. schenckii isolates from differ- but conversion to the yeast form should be attempted to firmly
ent geographical locations. The isolates formed three main establish the diagnosis of sporotrichosis.36 Although some
clades, one grouping all isolates from Europe, another with cases are benign, severe disease or unusual presentations can
only isolates from Brazil, and the third with isolates from occur in immunocompromised individuals such as human
other South American countries and Africa. The authors pro- immunodeficiency virus-infected patients and patients with
posed the existence of at least six phylogenetic species from chronic granulomatous disease.26,27,103 Cutaneous sporo-
different regions, confirming S. schenckii as a complex of trichosis lesions may be mistaken with other infections as
species. Later on, the same authors20 studied phenotypically tuberculosis, leishmaniasis, paracoccidioidomycosis, gum-
and genotypically (nuclear CAL gene) a total of 127 isolates, matous syphilis, and chromoblastomycosis.22,104,105 In such
differentiating S. albicans, S. inflata, and S. schenckii var. cases, the delay in the diagnosis of sporotrichosis can lead
luriei from S. schenckii, and proposing three new species: to fatality.106 These handicaps require more efficient methods
Sporothrix brasiliensis, S. globosa, and S. mexicana of clini- to detect accurately the pathogen in infected individuals, and
cal relevance (Table 60.2). They suggest the CAL gene as a recently molecular tests have been introduced to reveal small
good marker, and sessile pigmented conidia, growth at 30°C, amounts of genetic material of the fungus in clinical samples.
35°C, and 37°C, and the assimilation of sucrose, raffinose,
and ribitol as phenotypic features, for the recognition of these 60.1.6.1 Conventional Techniques
species. A later communication21 proposed S. schenckii var. Biological material can be swabbed or aspirated from cuta-
luriei as the species S. luriei, based on its phenotypic char- neous lesions, or biopsy material can be obtained from the
acteristics and a multilocus sequence analysis, to be different tissue lesion. In general, other samples such as sputum, syno-
to other species of the S. schenckii complex.20 Furthermore, vial fluid, cerebrospinal fluid (CSF), and, rarely, blood have
Madrid et al.97 studied another 32 clinical and environmental been reported to yield S. schenckii when cultured. The direct

examination of these samples is usually negative and when The techniques developed to detect antibody response in
positive, asteroid bodies are observed; the percentage of patients with mycoses have traditionally used crude extracts
positive samples increase significantly when the initial pus is with a large number of fungal fractions. While being rela-
discarded and samples are taken more deeply and examined tively easy to obtain, these extracts are not free of problems.
thoroughly.107 Generally, Sabouraud dextrose agar and/or The standardization of different batches is difficult, and
Sabouraud dextrose agar with antibiotics (the fungus is resis- the use of crude extracts may facilitate the cross-reactivity
tant to cicloheximide) are used for primary isolation, and the between antibodies from patients with different fungal and
culture is kept at 26°C–28°C. In general, a high percentage bacterial infections. However, in the last two decades, there
of cultures become positive and this is one of the most reli- has been an approach to develop more defined antigens.
able methods for diagnosis. Growth occurs within 3–5 days Several antigens have been obtained of the yeast phase of
and the colony morphology is variable. At first, small dirty S. schenckii, essentially carbohydrates and proteins. One
whitish, finely radiated humid-like colonies are observed. of the most outstanding and biological active antigen is the
Afterward, the colony becomes wrinkled and membranous, peptide-rhamnomannan fraction expressed in the cell wall
developing areas that become dark brown and black eventu- glycopeptide components.42 Using Concanavalin A (ConA),
ally. Coloration depends on strains and culture media, some three main glycoproteins of 84, 70, and 58 kDa were identi-
colonies remaining white and glabrous. Microscopically, fied in Sporothrix.110 Mendoza et al.111 reported exoantigens
the mycelia morphotype shows the characteristic sympodial in filtrate cultures of the mycelial morphotype of the fun-
and sessile conidia described elsewhere, depending on each gus, especially those of 90 and 50 kDa, which appear to be
species. species specific. They showed that the antigenic composition
Conversion of the mycelial to yeast form of S. schenckii of S. schenckii exoantigens of the mycelial morphotype did
is important for its identification and is easily performed in not cross-react with a single serum specimen of coccidioi-
different culture medium at 28°C or 37°C. To induce the domycosis, histoplasmosis, and paracoccidioidomycosis.111
transformation, conidia and mycelia are grown on brain S. schenckii exoantigens of the mycelial morphotype have
heart infusion agar at 37°C for 4–6 days. Other culture media also been used in immunodiffusion and immunoelectro-
are yeast peptone glucose agar and Rodríguez del Valle phoresis assays in which no cross-reactions were observed
medium38, both with a pH of 7.2–7.4 at 28°C for 48–72 h. when using chromoblastomycosis and leishmaniasis sera.112
Microscopically, yeast are fusiform and ovoid cells of 2.5–5 In other studies, an ELISA test was developed with a ConA-
to 3.5–6.5 μm, with single or multiple budding. binding fraction (SsCBF) of the peptido-rhamnomannan
fraction of S. schenckii that showed 90% sensitivity and 86%
60.1.6.1.1 Histopathology overall efficacy when tested against sera from patients with
Histopathology is another inexpensive and useful procedure lymphocutaneous, fixed cutaneous, disseminated cutaneous,
for this disease. When the biopsy sample is from a nodule, or multiple and extracutaneous forms of sporotrichosis and
the infiltrate is seen as a central and chronic suppurative serum samples from healthy controls and subjects with other
zone, with microabscesses containing polymorphonuclear diseases.113 Recently, Almeida-Paes et al.114 developed an
cells surrounded by lympho-histiocytic infiltrates. This zone assay (ELISA) for specific antibody detection in serum spec-
may contain asteroid bodies. The second zone is where an imens of patients with sporotrichosis. The assay used myce-
asteroid body is seen as a gemming cell with a radiating halo lial morphotype S. schenckii exoantigens and tested against
of eosinophils (Hoeppli-Splendore phenomenon). Around sera from patients with different clinical forms of sporotri-
the chronic suppurative zone is the medial or tuberculoid chosis. Potential cross-reactions were analyzed with heter-
zone constituted by epitheliod cells, some multinucleated ologous sera, as well as with sera from healthy controls. They
Langerhans giant cells and the third zone, or syphilloid area, found a sensitivity of 97% and a specificity of 89% in this
is comprised of plasmocytes, lymphocytes, fibroblasts, and assay. These results suggest ELISA as a sensitive diagnostic
vascular neoformations. This arrangement is not always tool for the serodiagnosis of sporotrichosis and can be used
found since the cellular elements are often mixed.108 It is also together with conventional methods of diagnosis, particularly
essential that multiple serial sections stained with periodic in those cases where cross-reactions or false-positive results
acid-Schiff (PAS) reagent be meticulously studied for the are experienced with serodiagnosis. Additionally, Almeida-
evidence of spores and hyphae of S. schenckii.109 Paes et al.115 demonstrated that antibodies produced during
S. schenckii infection are diverse, and that an exoantigen
60.1.6.1.2 Immunological Techniques ELISA (mycelial morphotype exoantigen of S. schenckii) for
The immunological diagnosis that includes serological and the detection of combinations of IgA, IgG, and IgM antibod-
skin tests may also be helpful for sporotrichosis diagnosis. ies is a highly sensitive and specific diagnostic procedure for
Agglutination and immunodiffusion tests are recommended sporotrichosis.
for the diagnosis and prognosis of sporotrichosis. The agglu- Intradermal skin tests with mycelial-derived sporotrichin
tinin and precipitin titers disappear with the resolution of have been widely used for the immunological detection of
the disease. Other serological tests include indirect immu- sporotrichosis since its first report by González-Ochoa.45
nofluorescence and enzyme-linked immunosorbent assay It is useful in epidemiological studies and as an auxiliary
(ELISA). method in atypical forms of the disease. An intradermal

injection (0.1 mL) is applied, and after 24–48 h, a 5 mm or Using the previously mentioned report of the nested PCR
more induration reaction is considered positive. Test positiv- assay of Hu et al.116 for S. schenckii detection, Xu et al.119
ity indicates an immunocontact with the specific S. schenckii assayed those probes in 38 S. schenckii DNAs (including the
fungus and is generally positive in 95% of the cases with 24 types of mtDNA described previously) from mice tissue
fungal exposure. infected by each fungal isolate and skin biopsies of patients
with sporotrichosis. In addition, two strains of Ceratocystis
60.1.6.2 Molecular Techniques minor and DNA from isolates from other 10 species of human
The definite diagnosis of sporotrichosis is achieved through pathogenic fungi were used. Amplification was observed in
the isolation of S schenckii in clinical samples and in some the 38 S. schenckii isolates, in infected mice tissue and in
cases complemented with immunodiagnostic procedures. human clinical skin biopsies, but not in C. minor DNA nor
However, both isolation and immunodiagnosis pose limita- in other fungal species. The detection limit for S. schenckii
tions, such as cultures of the biopsy specimens frequently DNA was 50 fg. The authors showed that nested PCR can
yielding negative results and the immune cross-reaction with identify S. schenckii from all the described types of mtDNA
other fungal species causing similar nosologies. Actually, and in isolates from different parts of the world. The nested
among diverse methods, the PCR is a viable option. PCR PCR assay with the18S rRNA gene sequence of Hu et al.116
is the most commonly used molecular test for diagnosis. It seems to be sensitive and specific and is another useful tool
amplifies fungal DNA from a small amount of the pathogen for a quick diagnosis of sporotrichosis.
from clinical samples and constitutes a fast and sensitive pro-
cedure for the diagnosis of sporotrichosis. PCR used for the
60.2 Methods
detection and identification of S. schenckii can be done in a
single step or in two sets (nested PCR). Isolates of Sporothrix spp. are stored on potato dextrose agar
Among different studies using PCR for S. schenckii at 4°C–7°C, in slant cultures submerged in mineral oil at
diagnosis, we will mention the most recent ones. Hu et al.116 room temperature and at 4°C, and conidial suspensions in
designed a nested PCR assay for the detection of S. schenckii 10% glycerol at 4°C liquid nitrogen. Isolates grow well after
with the use of an 18S rRNA gene sequence as a blank. 10 years storage in any of the methods. When needed, iso-
Different isolates were used for specificity and sensitivity, lates are grown on potato dextrose agar plates at 28°C for 5–7
including five clinical isolates, S. schenckii ATCC 10213 days. Two DNA extraction methods are given to suit different
strain, 10 clinical isolates of common fungi, three strains of laboratory facilities.
Mycobacterium spp. and Staphylococcus aureus, and nor-
mal human skin tissue samples. The amplification of the
fragment alone was done in six S. schenckii isolates dur-
ing the first PCR and in the nested version. The sequences Growth of S. schenckii. Transfer a single colony of
obtained were 100% identical to S. schenckii 18S rRNA gene mycelial-phase S. schenckii to Sabouraud’s dextrose broth
sequence. The nested PCR showed a sensitivity of 40 fg of with 1% yeast extract (SDY) and incubate at 28°C on a gyra-
S. schenckii DNA detecting 1 CFU of S. schenckii in tissue tory shaker at 120 rpm for 7 days to achieve sufficient cell
samples. Validation of the nested PCR assay in clinical iso- mass for DNA extraction. Harvest mycelial cells by centrifu-
lates and infected experimental mice showed a high sensitiv- gation and wash three times in sterile Milli-Q water.
ity and specificity, suggesting this assay as a quick diagnosis First DNA extraction method. Grind approximately 25 mg
with sufficient precision to be clinically useful for patients of washed mycelium with a mortar and pestle in liquid nitro-
with sporotrichosis. gen. Suspend in 500 μL of sterile lysis buffer (50 mM Tris–
In another study, Sandhu et al.117 described a probe based HCl, 150 mM NaCl, 100 mM EDTA) with 50 μL of 10%
on the 28S rRNA gene for detecting any pathogenic or sap- sodium dodecyl sulfate (SDS). Gently shake the tubes and
robic fungi during the first PCR, while in the second PCR, centrifuge at 18,300 × g for 5 min. Treat the supernatant with
oligonucleotides specific for S. schenckii were used for 5 μL of 10 mg of RNase (Qiagen Inc., Valencia, CA) per ml at
revealing the presence of this DNA–DNA hybridization in 37°C for 1 h and then subject to phenol–chloroform–isoamyl
clinical samples. The probe showed a high level of specific- alcohol (25:24:1) extraction by adding an equal volume of
ity when testing it with related pathogenic fungi and clinical this mixture. Precipitate nucleic acids by adding 10 μL of 3 M
samples. In order to obtain a rapid detection system, Kano NaOAc and two volumes of absolute ethanol. Wash the pel-
et al.118 designed a pair of specific oligonucleotides from let with 70% ethanol, dry, and resuspend in water. Quantify
the chitin synthase 1 gene for detecting S. schenckii in tis- DNA fluorometrically and check against standard DNA con-
sue samples. The S1, R1 and S2, R2 oligonucleotides did not centrations by agarose gel electrophoresis.
amplify the DNA from other fungal or bacterial pathogens, Second DNA extraction method. In this method, instead
nor human or feline cells. Using PCR with the S1, R1 and S2, of mycelia breakage by liquid nitrogen, a FastPrep® -24
R2 oligonucleotides, they detected 100 and 10 pg of genomic (Qbiogene, Inc, CA) equipment is used. Place approxi-
DNA of S. schenckii. Results point out this system as another mately 25 mg of mycelia into 2 mL microtubes contain-
useful molecular tool for the diagnosis of human and animal ing 0.2 g of glass beads (400–455 μm in diameter, Sigma
sporotrichosis. Chemical Co.), washed in hydrochloric acid and 500 μL

of sterile lysis buffer (50 mM Tris–HCl, 100 mM EDTA, Automated sequencing is done with an ABI dye termina-
3% SDS, and 1% mercaptoethanol). Stir microtubes in the tor cycle sequencing ready reaction kit and PCR primers in
FastPrep® -24 (Qbiogene) for two periods of 40 s at 6 m/s accordance with the recommendations of the manufacturer
speed with a 2 min interval in ice between stirrings. Gently (Applied Biosystems). Sequences are generated from both
shake the microtubes and then centrifuge at 18,300 × g for strands and edited and initially aligned with the Sequence
5 min. Treat the supernatant with 20 μL of 10 mg of RNase Navigator (v1.01; Applied Biosystems) software package, and
(Qiagen) per ml at 37°C for 1 h and then subject to phenol– the alignments optimized visually.
chloroform–isoamyl alcohol (25:24:1) extraction by adding
an equal volume of this mixture. Precipitate nucleic acids 60.2.2.3 S. schenckii Detection by Nested PCR
by adding 10 μL of 3 M sodium acetate and two volumes A nested PCR is carried out as described by Hu et al.116
of absolute ethanol. Wash the pellet with 70% ethanol, dry, DNA samples, from isolates and positive S. schenckii, are
and resuspend in water. Quantify DNA fluorometrically and processed as follows: Two sets of primers are used. The
check against standard DNA concentrations by agarose gel design of oligonucleotides used in this technique was based
electrophoresis. on the comparison of the sequence of 18S rRNA gene of
S. schenckii (accession no. M85053) and those of other fungi
60.2.2 Detection Procedures in the GenBank database (National Center for Biotechnology
Information, National Library of Medicine, Bethesda, MD).
60.2.2.1 RAPD-PCR Assay93
The outer primer set SS1, 5′-CTC GTT CGG CAC CTT
Perform a 25 μL reaction mixture that contains 10 ng of ACA CG-3′, and SS2, 5′-CGC TGC CAA AGC AAC GCG
the required DNA sample; 2.5 mM MgCl2; 200 μM each GG-3′, delimit a 305-nucleotide sequence of the gene. The
dATP, dTTP, dCTP, and dGTP (Applied Biosystems Inc., inner primers SS3, 5′-ACT CAC CAG GTC CAG ACA
Foster City, CA); 100 pmol of each primer [primers OPBG- CGA TG-3′, and SS4, 5′-CGC GGG CTA TTT AGC AGG
01, OPBG-14, and OPBG-19 (kit B; Operon Technologies TTA AG-3′, delimit a specific 152-nucleotide sequence.
Ltd.)]; sterile, distilled MilliQ water (Millipore) to make up DNA amplification is performed on a Perkin-Elmer Cetus
the final volume; and 1 U of Taq DNA polymerase (Applied DNA thermal cycler and the first PCR performed in a 50 μL
Biosystems). Controls, which contain all of the compo- reaction mixture containing 200 μM of dNTPs (Applied
nents listed above except DNA, are also set up and run with Biosystems), 1.5 mM MgCl2, 0.4 μM of each outer primer,
each set of reactions. Optimal amplification conditions for 1.5 U Taq DNA polymerase (Applied Biosystems), and 10 ng
S. schenckii RAPD analysis are 45 cycles of 1 min at 94°C of DNA. Cycling conditions are as follows: one cycle at 95°C
(denaturation), 1 min at 35°C (annealing), and 1 min at 72°C for 5 min; 40 cycles at 95°C for 1 min, 68°C for 1 min, and
(extension). Amplifications are carried out in a thermocycler 72°C for 1 min; and 1 cycle at 72°C for 10 min. The reaction
(Perkin-Elmer Cetus, Norwalk. CT). A final 7 min cycle at mixture of the nested PCR is identical, except that 3 μL of
72°C ensures full extension of all amplified products, and the first reaction product and the inner primer pair SS3 and
samples are maintained at 4°C. The polymorphic amplified SS4 are used.
patterns revealed by RAPD analysis-PCR assays are viewed, Amplification products are electrophoresed through 2%
and digital images of ethidium bromide (0.5 μg/mL)-stained agarose in Tris-borate-EDTA 0.5X buffer. Electrophoresis
agarose gels (2%) are captured with a documentation system is conducted at 90 V for 60 min. The 123 bp DNA Ladder
(GeneCam; Syngene, Cambridge, MA) and printed on a digi- (Invitrogen, Carlsbad, CA) is used as molecular markers. The
tal graphic printer (Sony Electronics Inc., Park Ridge, NJ). bands are visualized with a UV transilluminator after ethid-
RAPD analysis-PCR assays are repeated independently three ium bromide (0.5 μg/mL) staining. They are captured with
times with each primer with all isolates studied, with consis- a documentation system (GeneCam; Syngene, Cambridge,
tent gel patterns detected from one assay to another. MA) and printed with a thermal printer (Sony 650).
60.2.2.2 Identification of Sporothrix
Species by Sequencing
60.3 C
onclusions and
The CAL gene sequence is the most phylogenetically informa-
tive locus found for the recognition of these species.20 Marimon
Future Perspectives
et al.20 developed a PCR assay targeting this gene. CAL gene Despite the large impact of molecular techniques in the
is amplified by PCR: 20–60 ng of genomic DNA template in field of clinical mycology, they do not eliminate the routine
25 μL reaction. Reaction mixtures consist of 0.5–1 mM of use of classic techniques, and as recommended in clinical
each primer (degenerated primers CL1 and CL2A20), 1.0 U of practice management, both should be performed simultane-
AmpliTaq DNA polymerase (Perkin-Elmer), 10 mM Tris–HCl ously. Importantly, molecular methods have not been fully
(pH 8.3), 1.5 mM MgCl2, 50 mM KCl, and 0.2 mM deoxynu- validated by comparison with those used in routine identi-
cleotide triphosphates. The amplification program includes an fication of fungi and therefore are not employed in clinical
initial denaturation cycle at 94°C for 5 min, 35 cycles at 95°C laboratories as a reliable tool for the diagnosis of dissemi-
for 30 s, 60°C for 1 min, and 72°C for 1 min, and 1 cycle at nated sporotrichosis. These circumstances demand an urgent
72°C for 7 min. validation for routine use in diagnosis and in different types

of research. Although the advantages of molecular methods 8. Toriello, C. and Mariat, F., Etude comparée des polyosides
are unquestionable, its failures should be considered timely des champignons Ceratocystis stenoceras et Sporothrix
for purposes of future corrections and/or considerations to schenckii. Composition chimique et analyse immunologique,
Ann. Microbiol., 125A, 287, 1974.
improve implementation. Interestingly, physicians need a
9. Travassos, L.R. and Lloyd, K.O., Sporothrix schenckii and
better understanding of fungal infection to notify the dis- related species of Ceratocystis, Microbiol. Rev., 44, 683,
eases and take into account the epidemiological evidence 1980.
associated to the mycosis, but above all they should be 10. Mariat, F., Adaptation de Ceratocystis a la vie parasitaire chez
familiar with the use and limitations of each of the available l’animal. Etude de l’aquisition d’un pouvoir pathogéne com-
diagnostic tests for fungal diseases particularly for sporotri- parable a celui de Sporothrix schenckii, Sabouraudia, 9, 191,
chosis. It is evident that much work is still needed in order to 1971.
identify clearly species of the S. schenckii complex of clini- 11. Suzuki, K., Kawasaki, M., and Ishizaki, H., Analysis of
restriction profiles of mitochondrial DNA from Sporothrix
cal relevance especially in regard to their different antifun- schenckii and related fungi, Mycopathologia, 103, 147,
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Sporothrix species can now be added to a list of emerg- 12. O’Reilly, L.C. and Altman, S.A., Macrorestriction analysis of
ing pathogens, according to their capacity to cause disease clinical and environmental isolates of Sporothrix schenckii,
in otherwise healthy hosts as well as in immunocompro- J. Clin. Microbiol., 44, 7, 2006.
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14. Hibbett, S.D., A higher-level phylogenetic classification of
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17. CABI Bioscience, CBS, 2009, Index Fungorum. Available:
understood than are those of bacterial or parasite pathogens.
http://www.indexfungorum.org, accessed October 12, 2009.
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thus do not stimulate their study. The emerging pathogenic schenckii, J. Clin. Microbiol., 44, 3251, 2006.
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20. Marimon, R. et al., Sporothrix brasiliensis, S. globosa, and
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61 Stachybotrys
Contents
61.1 Introduction.......................................................................................................................................................................511
61.1.2.2 Pathogenesis........................................................................................................................................... 512
61.1.3 Diagnosis...............................................................................................................................................................513
61.2 Methods.............................................................................................................................................................................513
61.2.1.1 Trichothecene Collection and Analysis..................................................................................................513
61.3 Conclusions........................................................................................................................................................................514
References...................................................................................................................................................................................514
61.1 Introduction or pigmented and cylindrical in shape, with swollen upper

portions, forming clusters of 3–6. Conidia (4.5 × 9 μm) are
61.1.1 Classification, Morphology, and Biology oval, hyaline or pigmented, one-celled, and in clusters.
The genus Stachybotrys is an asexually reproducing, dema- First identified from a mold growing on domestic wallpa-
tiaceous fungus belonging to the mitosporic Hypocreales per in Prague in 1837, Stachybotrys is a member of the Fungi
group, order Hypocreales, class Sordariomycetes, subphy- Imperfecti known as “black mold” or “toxic black mold.”
lum Pezizomycotina, phylum Ascomycota, and kingdom The fungus is an inhabitant of soil and strata rich in cellu-
Fungi. The mitosporic Hypocreales group encompasses the lose (e.g., hay, straw, grain, hemp, plant debris, dead roots,
genera of Acremonium, Acrostalagmus, Cephalosporium, wood pulp, cotton, fabrics, paper, book bindery glue, plant
Chaetopsina, Cylindrocladiella, Escovopsis, Fusarium, fiber-processing plants, etc.). It has been isolated from con-
Gliocladiopsis, Gliocladium, Hobsonia, Illosporium, taminated grains, tobacco, insulator foams, indoor air, and
Myrothecium, Parasarcopodium, Polycephalomyces, water-damaged buildings. The fungus tolerates temperatures
Rotiferophthora, Sesquicillium, Solheimia, Stachybotrys, up to >60°C and survives over-winter. Stachybotrys spores
Stilbella, Trichothecium, Tubercularia, Ustilaginoideae, stay viable for years to decades, and conidia retain viability
Verticillium, and Xenocylindrocladium [1]. after passage through the gastrointestinal tract. However, the
In turn, the genus Stachybotrys is divided into 15 recog- organism is killed by composting of manure and by disinfec-
nized species: Stachybotrys bisbyi, Stachybotrys chartarum, tants [4].
Stachybotrys chlorohalonata, Stachybotrys cylindrospora, Stachybotrys chartarum is a known producer of tricho-
Stachybotrys dichroa, Stachybotrys echinata, Stachybotrys thecene mycotoxins and stachylysin (a hemolysin). The
elegans, Stachybotrys kampalensis, Stachybotrys longis- best characterized trichothecenes include satratoxins F, G,
pora, Stachybotrys microspora, Stachybotrys nephrospora, and H, roriden E, verrucarin J, and trichoverrols A and B,
Stachybotrys oenanthes, Stachybotrys parvispora, which share chemical formula of C25H34O6 or C26H38O6 and
Stachybotrys subsimplex, and Stachybotrys theobromae, in are tricyclic sesquiterpenes with a 12,13-epoxy-trichothec-
addition to 13 unassigned species. Two species, S. chartarum 9-ene ring. Stachybotrys species also produce spirolactams
(obsolete synonyms: S. alternans and S. atra) and S. echinata and spirolactones (related to anticomplement components),
(obsolete synonym: Memnionella echinata), are implicated phenylspirodrimanes (inhibitor of complement activation),
in human diseases [2,3]. cyclosporins, and endothelin receptor antagonists [5–7].
Stachybotrys spp. grow rapidly and mature in approxi- Trichothecenes modulate inflammatory reactions and
mately 4 days. Colonies are “cottony,” white initially and alter alveolar surfactant phospholipid concentrations, besides
turning black with age. Septate hyphae are hyaline first and being potent inhibitors of protein synthesis (e.g., scirpen-
become darkly pigmented later. Conidiophores are simple or triol, 15-acetoxyscirpendiol, diacetoxyscirpenol [DAS or
branched, bearing phialides at apices. Phialides are hyaline a nguidine], verucarin A, and T-2 toxin) and elongation or
511

termination (e.g., trichodermin, trichodermol, crotocol, Since then, other clinical manifestations have been associ-
trichothecolone, trichothecin, and verrucarol). Trichothecenes ated with exposure to Stachybotrys chartarum mycotoxins
are susceptible to destruction by alkali although resistant to and spores, ranging from (1) chronic fatigue or headaches,
sunlight, UV light, x-rays, heat (up to 120°C), and acids. (2) fever, (3) irritation to the eyes, mucous membranes of the
Some of the mycotoxins have been isolated from dust (e.g., mouth, nose, and throat, (4) sneezing, (5) rashes, (6) chronic
satratoxins, trichoverrols, verrucarol, verrucarins, and coughing, (7) nausea, (8) memory loss, (9) vomiting, (10)
trichoverrins) and grain (T-2 toxin, nivalenol, and deriva- bleeding in the lungs and nose, (11) hypersensitivity pneu-
tives of others). Humans may develop toxin-related disease monitis (HP), (12) allergic rhinitis, and (13) asthma exacer-
by ingestion of food products contaminated with the fungus bations. Occupants of mold-contaminated, water-damaged
toxins, exposure to mycotoxins in building (sick-building buildings often develop symptoms in the central nervous sys-
syndrome), and/or inhalation of propogules while undertak- tem and the immune system as well as pulmonary diseases,
ing experiments with the fungus [8]. allergy, and inflammatory reactions. Occupational stachy-
botrytoxicosis acquired by inhalation showed chest and upper
61.1.2 Clinical Features and Pathogenesis airway symptoms, fever, leucopenia, and dermatitis, which
started within 2–3 days of exposure and lasted for 3 weeks
61.1.2.1 Clinical Features [18]. In a recent investigation of 32 patients with symptoms
Stachybotrys may induce disease by (1) infection, (2) genera- attributed to mold exposures at work, 25 (88%) patients had
tion of a deleterious immune response, and (3) toxic-irritant S. chartarum as well as Aspergillus and Penicillium. 79%,
effects from the metabolites. Together with other molds (e.g., 70%, and 64% of the 32 patients presented with cough, short-
Aspergillus, Penicillium, Alternaria, and Cladosporium), ness of breath, and chest tightness, respectively, which per-
Stachybotrys may play a role in the development of sick- sisted more than 6 weeks in 91%, suggestive of sick-building
building syndrome [9–25]. syndrome. Thirty one percent of these patients had positive
Stachybotrys was first noted as a pathogen of horses in skin test to fungal extracts, suggesting IgE-mediated or other
Ukraine in the early 1930s. After the ingestion of hay con- nonimmune mechanisms could be the cause of their symp-
taminated with Stachybotrys, horses developed lip edema, toms [25,29].
stomatitis, oral necrosis, rhinitis, conjunctivitis, coagu-
lopathy, hemorrhage, and neurologic disorders (irritability, 61.1.2.2 Pathogenesis
gait disturbance, and blindness). So-called superinfections Contemporary construction methods that use cellulose sub-
occurred and deaths were observed. The disease syndrome strates (e.g., fiber board) favor the growth of cellulolytic
is referred to as stachybotrytoxicosis. In a rare “atypical” fungi such as S. chartarum. The fungus is shown to produce
or “shocking” form, the disease was primarily neurologi- trichothecene mycotoxins as mentioned above, although
cal and highly fatal, with areflexia (loss of sensorimotor several other fungi (e.g., Fusarium, Myrothecium verru-
reflexes), hyperesthesia (hypersensitivity to pain), hyperir- caria, Myrothecium roridum, Trichothecium, Trichoderma,
ritability, blindness, and stupor. Cattle were also affected to Cephalosporium, Verticimonosporum, and Cylindrocarpon)
a lesser extent, and younger animals fared better than older also synthesize these compounds. S. chartarum macrocy-
one. Similar diseases have been reported in other parts of the clic trichothecene mycotoxins (MTM) are dissociated read-
world. A case of sheep disease was described in the 1990s ily from the surface of the organism and are consequently
after animals consumed heavily contaminated grain cubes in spread in damp buildings. S. chartarum MTM remain toxic
South Africa. The affected animals displayed fever, listless- over extended periods of time, and individuals with exposure
ness, oral lesions, pancytopenia, hemorrhage, opportunistic to the fungus contain MTM in their sera [24]. High indoor
infections, and a significant mortality rate [4,26–28]. exposures to trichothecene mycotoxins are associated with
In areas of enzootic equine stachybotrytoxicosis, fodder infrequent ventilation or vacuuming of the building, pets,
handlers and others with close contact with musty straw (e.g., visible mold, and old carpets [18].
using straw for fuel or bedding) also developed a dermato- Trichothecenes are potent translational inhibitors and
logic and respiratory syndrome. Dermatologic symptoms stress kinase activators. Experimental exposure of tricho-
were dermatitis on the scrotum, medial thighs, axilla, the thecene mycotoxins in mice led to severe intra-alveolar,
hands, and other areas, which progressed from hyperemia bronchiolar, and interstitial inflammation. In experimental
to crusting exudates to necrosis, with subsequent resolu- Wistar rats, decreased viability of alveolar macrophages
tion. Some patients showed erosions on the oral and gingival and increased activity of the lysosomal enzyme cathepsin D
mucosa. Respiratory symptoms included catarrhal angina, in bronchoalveolar lavage cells after S. chartarum exome-
bloody rhinitis, cough, throat pain, chest tightness, and occa- tabolite exposure were noticeable [30]. S. chartarum metab-
sional fever. Some patients had transient leukocytopenia olites suppress red blood cells (RBC); decrease the total
(reduced white blood cell count) and hemorrhage (bleeding). RBC count, hemoglobin, and hematocrit; and increase total
S. chartarum (S. alternans) isolated from straw produced bronchoalveolar lavage fluid cell count (indicating inflam-
areal fructifications in experimental rabbits by a dermal tox- mation, lower alveolar macrophage counts, and increased
icity test and similar local and systemic responses on the skin granulocyte count related to the BALF cells) in these ani-
of volunteers [4]. mals [31].

Stachybotrys 513
Satratoxin-positive S. chartarum activates inflamma- high-volume air sampler (Thermo Electron Corporation).
some-associated caspase-1, which is needed for proteolytic Entrained solids are concentrated in a phosphate-buffered
processing of IL-1beta and IL-18, in human macrophages. saline (PBS) solution (pH 7.4) to a final volume of 10 mL
In addition, purified trichothecene mycotoxins, roridin A, [46].
verrucarin A, and T-2 toxin activate caspase-1 and strongly Following the collection, SpinCon samples (all 10 mL)
enhance LPS-dependent secretion of IL-1beta and IL-18. are filtered using Fisher 13 mm-diameter nylon syringe fil-
Satratoxin-positive S. chartarum and the trichothecenes it ters with a 0.45 μm pore size (Fisher Scientific). The filtered
produces also trigger the activation of caspase-3, which is fluid is transferred asceptically to 15 mL polypropylene coni-
an effector caspase of apoptosis. Thus, human macrophages cal centrifuge tubes, frozen at −80°C, and lyophilized using
sense trichothecene mycotoxins as a danger signal, which a VirTis Freezmobile (SP Industries). The dried samples
activates caspase-1 and further enables the secretion of are individually resuspended in 1 mL of pyrogen-free water
IL-1beta and IL-18 from the LPS-primed cells [32]. (25°C) for immediate testing.
Several enzymes from S. chartarum spores demonstrate Filters obtained from the Andersen PUF sampler are
proteolytic activity and are able to hydrolyze gelatin and transferred individually to 50 mL polypropylene centrifuge
collagen I and IV [33]. S. chartarum spore extracts induce tubes on-site. The filters are suspended in 40 mL of PBS,
high levels of IL-6, IL-8, and TNF-alpha in human tracheal vortexed vigorously for 60 s, removed from the tubes using
epithelial cells. This stimulation of cytokine production is sterile forceps, and then discarded. The PBS extracts are
abolished by a serine protease inhibitor Pefabloc. Thus, pro- filtered into new 50 mL tubes. These are frozen at −80°C,
teinases from S. chartarum spores significantly contribute to lyophilized, and resuspended in 1 mL pyrogen-free water for
lung inflammation and injury [34]. S. chartarum spores are immediate testing.
capable of inducing both apoptosis and necrosis in THP-1 Samples are analyzed for macrocyclic trichothecenes
cells, leading to cell death within 3–6 h. More specifically, using a QuantiTox kit for trichothecenes (EnviroLogix). This
S. chartarum spores increase the formation of reactive oxy- competitive ELISA kit incorporates trichothecene-specific
gen species (ROS) and oxidative DNA damage. S. chartarum antibodies immobilized in polystyrene microtiter wells and
trichothecenes T-2 toxin and satratoxin G are mainly respon- is highly specific for MTM of S. chartarum. To ensure that
sible for apoptosis [35]. the ELISA runs correctly, the macrocyclic trichothecene
roridin A is used at a concentration of 50 ng/mL in PBS as a
positive control for each set of tests. PBS alone is used as a
61.1.3 Diagnosis
negative control [39].
S. chartarum is a cellulose-decaying fungus that grows well Conidia and other airborne particulates are collected on
at room temperature and with humidity above 93%. Isolation glass microscope slides that have been coated with a thin
of S. chatarum requires special media with high concentra- layer of petroleum grease using a foam makeup applicator.
tion of cellulose and low concentration of sugar and nitro- For testing purposes, conidia are collected from plates that
gen to compete with Penicillium and Aspergillus. Growing have reached confluence (approximately 7–14 days), using
S. chatarum isolates on rice or potato dextrose agar results sterile cotton swabs. To collect the conidia, swabs are gently
in higher proteolytic activity of the spores than those grown rolled over the surface of the fungal growth. The cotton tips
on drywall. of the swabs are placed in 1 mL of sterile room-temperature
Thin-layer chromatography, high-performance liq- (25°C) PBS in 1.5 mL microcentrifuge tubes and vortexed for
uid chromatography (HPLC), tandem mass spectrometry approximately 1 min to remove conidia. The conidia are then
(MSMS), gas chromatography (GC), GC combined with counted using a hemacytometer and identified to the genus
mass spectrometry (GC-MS, GC-MSMS), and enzyme- level by a trained technician using an Olympus BH2-RFCA
linked immunosorbent assay (ELISA) have been applied to optical light microscope. For ELISA testing, the fungal spore
detect S. chartarum mycotoxins in mold-affected materials suspensions are centrifuged at 14,500 rpm for 1 min to pellet
inside buildings, in carpet dust from water-damaged build- the conidia. Care is taken not to disturb the conidium pellet,
ings, and in animal tissues [36–41]. Finally, various reports and only the top 80% of the supernatant is used for ELISA
have demonstrated the potential usefulness of molecular bio- testing. Each sample is run in duplicate wells on two separate
logical techniques for detection of molds in air and clinical occasions [39].
specimens [42–45].
61.2 Methods Pounder et al. [45] described a real-time PCR with SYBR
61.2.1 Sample Preparation green DNA-binding dye and amplicon-melting tempera-
ture analysis for fungal detection using pan-fungal primers
61.2.1.1 Trichothecene Collection and Analysis ITS1 forward (5′-TCCGTAGGTGAACCTGCGG-3′) and
Airborne trichothecene mycotoxins are collected using a ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′). The
SpinCon PAS 450-10 bioaerosol sampler (Sceptor Industries) identity of the fungi is verified by subsequent sequence
and an Andersen GPS-1 polyurethane foam (PUF) analysis.

Procedure in humans. Along with biochemical procedures, molecular

techniques have been developed to detect the fungus and its
1. The PCR mixture is composed of 1× Lightcycler mycotoxins [49].
FastStart DNA Master Hybridization Probes mixture
(Roche Applied Science) containing deoxynucleo- References
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33. Mason, C. D. et al., 2001. Effects of Stachybotrys chartarum 46. Pasanen, A.-L. et al., 1993. Laboratory experiments on mem-
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62 Trichoderma
László Kredics, Lóránt Hatvani, László Manczinger,
Csaba Vágvölgyi, and Zsuzsanna Antal
Contents
62.1 Introduction.......................................................................................................................................................................517
62.1.2 Epidemiology and Pathogenesis............................................................................................................................518
62.1.3.1 Trichoderma Infections in Patients Undergoing Peritoneal Dialysis..................................................... 520
62.1.3.2 Trichoderma Infections in Organ Transplant Recipients....................................................................... 520
62.1.3.3 Trichoderma Infections in Patients with Hematologic Malignancies.................................................... 521
62.1.3.4 Trichoderma Infections of the Respiratory Tract and Allergic Reactions............................................. 521
62.1.3.5 Occurrence of Trichoderma Associated with HIV Infection................................................................ 522
62.1.3.6 Miscellaneous Cases of Trichoderma Infections.................................................................................. 522
62.1.3.7 Antifungal Susceptibilities of Clinical Trichoderma Isolates............................................................... 522
62.1.4 Diagnosis.............................................................................................................................................................. 526
62.2 Methods............................................................................................................................................................................ 529
References.................................................................................................................................................................................. 530
62.1 Introduction introduction of sequence-based molecular methods10 and

multigene phylogenic approaches11 as well as the applica-
62.1.1 Classification, Morphology, and Biology tion of the genealogical concordance phylogenetic species
Trichoderma species are asexual, saprophytic filamentous recognition (GCPSR) concept12 enabled the revision of the
fungi with teleomorphs belonging to the genus Hypocrea morphology-based taxonomy in Trichoderma and the iden-
(Ascomycota, Pyrenomycetes, Hypocreales, Hypocreaceae). tification of new species. The number of described and con-
Comprehensive historical overviews about the develop- firmed, phylogenetically different Trichoderma species has
ments in the taxonomy of the genus Trichoderma were already reached 100.2
presented by Druzhinina et al.1,2 The genus was introduced Macroscopically, Trichoderma species can be generally
by Persoon in 1794 for a green fungus growing on fallen characterized by rapidly growing, initially smooth and trans-
branches and other substrates.3 Bisby4 suggested that the lucent or watery white colonies, later becoming floccose, with
genus Trichoderma consisted of a single species, T. viride. distinct tufts of conidiophores that frequently form concentric
Until 1969, most species were referred to as either T. viride ringlike zones, gradually changing their color from whitish
or T. koningii, depending upon whether they produced glo- green to light olive green at maturity. Microscopically,
bose or oval conidia, respectively. In 1969, Rifai5 revised conidia occur in balls at the apexes of flask-shaped phialides
the taxonomy of the genus Trichoderma and described that arise at angles from the conidiophores. The identifica-
nine “species aggregates” based on the types of branch- tion of Trichoderma strains at the species level by morpho-
ing systems of the conidiophores, the character of phialo- logical examinations is problematic. In order to aid the fast
spores, the phialide disposition, and conidial morphology. and reliable identification of isolates, the online resources
In the 1980s and early 1990s, Bissett6–9 dissected several of TrichOkey, a DNA-barcode system based on defined nucleo-
Rifai’s aggregate species into several defined taxa and pro- tide sequence differences in the ITS1 and ITS2 region,13,14 and
posed an infrageneric classification of Trichoderma, which TrichoBLAST, a multiloci database of phylogenetic markers
divides the genus into sections Trichoderma, Pachybasium, for Trichoderma and Hypocrea powered by sequence diagno-
Saturnisporum, Longibrachiatum, and Hypocreanum. The sis and similarity search tools15 have been developed.
517

Members of the genus Trichoderma take part in the infection was fatal in 17 out of the 36 cases (47.22%). Case stud-
decomposition of plant residues in the soil. Certain species, ies reported the involvement of eight species from the genus
including T. reesei are excellent producers of cellulolytic Trichoderma (T. longibrachiatum,33,38–40,43,45,46,48–50,53,57,59,60
enzymes and can therefore be applied for cellulose degra- T. citrinoviride,52 T. pseudokoningii36,42 and T. reesei61 from
dation in the biotechnological industry.16 Other Trichoderma section Longibrachiatum, T. harzianum32,44,54 from sec-
species (e.g., T. harzianum, T. virens, T. atroviride, T. asper- tion Pachybasium B, T. koningii,30,34 T. atroviride40 and T.
ellum) are well known as potential biocontrol agents with the viride29,31,41,51,55 from section Trichoderma) and a yet unde-
ability of antagonizing a series of plant pathogenic fungi.17 scribed Hypocreaceae sp. close to the genus Hypocrea/
Proposed mechanisms of antagonism include mycopara- Trichoderma56 in human infections.
sitism by the action of cell-wall degrading enzymes, anti- For the successful prevention of Trichoderma infections,
biosis by the production of antibiotics, and competition for the understanding of the potential sources of contamina-
space and nutrients through rhizosphere competence.18–21 tion and possible routes of infection is needed. Airborne
Facilitation of seed germination and growth of the plants via Trichoderma conidia were suggested to be possibly related
releasing important minerals and trace elements from soil to allergic diseases,71 and ambient air is also supposed to
and induction of the defense responses in plants are further be the likely source of infection for some cases of oppor-
beneficial features of these species. Trichoderma spp. gener- tunistic Trichoderma infections (e.g., sinusitis, pneumonia,
ally occur in soils, but certain Trichoderma species are also abscesses following surgery). Based on a review by Madsen
known from special habitats. During the last decades, repre- et al.72 Trichoderma species could be isolated from air and/
sentatives of the genus have been reported to be harmful as or dust samples of human environments including hospital
the causative agents of green mold, a disorder that results in wards, homes, air conditioners, farms, sawmills, herb pro-
substantial losses in the production of cultivated mushrooms cessing plants, carpentries, flats, roofs of houses, station-
including champignon (Agaricus bisporus) and oyster mush- ary cars, wood chip terminals, buildings heated by wood
room (Pleurotus ostreatus).22–24 Besides mushroom patho- chips, schools, combine harvesters, and storey buildings.
genic Trichoderma species, clinically relevant members of Trichoderma species were reported among the most fre-
the genus including T. longibrachiatum and T. harzianum quently occurring filamentous fungi in certain studies, e.g.,
were also isolated from compost and substrate samples used in the fungal microbiota of air conditioners in Brazilian
for mushroom cultivation.23 Trichoderma species have also intensive care units.73
been reported as pathogens of reptiles25,26 and, in increasing A series of studies suggest that water-related sites repre-
number of cases, as etiologic agents in human infections.27,28 sent potential sources of Trichoderma infections. Hageskal
Members of this genus were previously considered to be con- et al.74 investigated the diversity of mold species in Norwegian
taminants or harmless colonizers when isolated from human drinking water and found the occurrence of Trichoderma
clinical specimens, but nowadays they are recognized as species in all samples from all of the sampling points in
emerging opportunistic pathogens of humans, especially of surface water-derived water systems, the predominant fungi
immunocompromised hosts. being isolated (72 of 273 isolates, 26.4%). After A. fumigatus,
Trichoderma species were found to be the second most prev-
alent filamentous fungi cultured from samples from outlets,
62.1.2 Epidemiology and Pathogenesis
waste pipes, and shower heads in a bone marrow transplanta-
The number of clinical cases caused by Trichoderma species tion ward.75 In another study, Varo et al.76 collected 50 sam-
is increasing from year to year, which can be attributed to the ples from seven water distribution points of the water system
increasing number of immunocompromised patients as well as of a hemodialysis unit, and isolated a total of 116 filamentous
the increasing attention of microbiologists to these fungi that fungi, with Trichoderma isolates being the most frequent
were previously considered to be nonpathogenic. Altogether, (47 isolates, 40.5%). In a study by Enríquez-Matas et al.,77 a
59 cases of isolation of Trichoderma strains from clinical sam- fungal strain identified as T. viride was isolated in cultures
ples could be recovered from the literature and culture collec- of water samples from an ultrasonic humidifier installed
tion data. Case details available for 36 cases of Trichoderma in the home of a patient diagnosed with hypersensitivity
infections29–62 are summarized in Table 62.1. The first report pneumonitis. Precipitating immunoglobulin G antibodies to
about a Trichoderma infection described the inadvertent T. viride were detected in the patient’s serum by enzyme-
infusion of T. viride to an immunocompetent host with con- linked immunosorbent assay, suggesting that the hypersen-
taminated intravenous fluid,58 the patient remained well after sitivity pneumonitis might have been due to Trichoderma
24 h of amphotericin B treatment. Since this initial report, from the humidifier. Trichoderma species are abundant in
clinical isolation of Trichoderma species has been reported indoor environments of water-damaged buildings, where
from several countries including France,30,32,40,42,46,48,53,59–61,63 they are frequently among the dominating microfungi.78–81
Spain,34,44,51,64 United Kingdom,58 Netherlands,33 Belgium,41 Besides T. atroviride, T. viride and T. hamatum, the species
Italy,36 Slovenia,35 Austria,47,65,66 Hungary,67 Lithuania,52 detected in such environments include the clinically relevant
Turkey,37,54,68 Canada,57 United States,29,43,45,49,56 Brasil,69 species T. longibrachiatum, T. citrinoviride, and T. harzia-
Chile,38 India,70 Korea,39 Japan,50 and Mali.46 The prognosis num.79,80 The release of spores from wet wallpapered gyp-
for Trichoderma infection is usually poor: the outcome of the sum boards by nine species of typical indoor fungi including

Trichoderma 519
TABLE 62.1
Case Reports about Trichoderma Infections in the Literature
Age/Sex Clinical Diagnosis Source Etiology Therapy Outcome Reference
47/M CAPD peritonitis Peritoneal fluid, autopsy T. viride AMB Died [29]
63/F CAPD peritonitis Peritoneal fluid T. koningii Catheter removal, MCZ Cured [30]
44/M CAPD peritonitis Peritoneal fluid T. viride AMB Died [31]
82/M CAPD peritonitis Peritoneal fluid T. harzianum KTZ, 5FC Died [32]
48/M CAPD peritonitis Peritoneal fluid, autopsy T. longibrachiatum AMB Died [33]
41/M CAPD peritonitis Peritoneal fluid T. koningii FLZ, 5FC, AMB Died [34]
60/M CAPD peritonitis Peritoneal fluid Trichoderma sp. Catheter removal, KTZ Cured [35]
33/M APD peritonitis Peritoneal fluid T. pseudokoningii Catheter removal Cured [36]
40/M CAPD peritonitis Peritoneal fluid Trichoderma sp. Catheter removal, ITZ, Died [37]
AMB
13/F CAPD peritonitis Peritoneal fluid T. longibrachiatum FLZ, AMB, catheter Died [38]
removal
67/M CAPD peritonitis, Peritoneal fluid, aspirated fluid T. longibrachiatum AMB, Catheter removal Cured [39]
intraabdominal abscess
70/M Cholecystitis and peritonitis ND T. longibrachiatum VRZ, CSP Died [40]
44/F TX/abdominal Peritoneal fluid, haematoma T. viride Surgery, AMB, FLZ Died [41]
dissemination
45/F TX/lung and skin Bronchoalveolar lavage, skin T. pseudokoningii FLZ, AMB, ABLC, 5FC Died [42]
dissemination biopsy, lung, brain, heart,
stomach
29/F TX/acute invasive sinusitis Sinus debridement T. longibrachiatum Surgery, AMB, ITZ Cured [43]
68/M TX/disseminated infection Brain and lung abscesses, T. harzianum — Died [44]
autopsy
29/M TX/disseminated infection Lung, liver, intestinal wall, T. longibrachiatum AMB, ITZ, ABLC Died [45]
stool, autopsy
63/F TX/invasive infection Subcapsular hepatic collection T. longibrachiatum Surgical debridement Cured [46]
11/M TX/invasive infection Bronchoalveolar lavage, T. longibrachiatum ABLC Died [46]
pleural drains
62/M TX/disseminated infection Liver, gastrointestinal tract, Trichoderma sp. and VRZ Died [47]
lung, heart, kidneys and skin Absidia corymbifera
49/M TX/liver infection Liver biopsy T. atroviride FLZ Died [40]
17/F HM/brain abscess Brain biopsy, cerebral pus T. longibrachiatum Surgery, AMB, 5FC, Cured [48]
KTZ, ITZ
11/M HM/skin infection Skin biopsy T. longibrachiatum AMB, ABLC Cured [49]
66/F HM/necrotizing stomatitis Ulcerative mucogingiva T. longibrachiatum AMB, ITZ Died [50]
54/F HM/pulmonary infection Fine-needle aspiration from T. viride ABLC, VRZ, CSP Cured [51]
pulmonary consolidation
49/F HM/pneumonia Bronchoalveolar lavage T. citrinoviride AMB Cured [52]
16/M HM/invasive pulmonary Sputum, bronchoaspiration, T. longibrachiatum VRZ, CSP Cured [53]
infection bronchoalveolar lavage
9/M HM/invasive fungal infection Serum, sputum, skin lesions T. harzianum ABLC Died [54]
46/M Pulmonary mycetoma Sputum, lung biopsy T. viride Surgery Cured [55]
19/F Pulmonary fibrosis Lung biopsy Hypocreaceae sp. — Cured [56]
52/F Allergic fungal sinusitis Bilateral endoscopic antral T. longibrachiatum Sinus lavage, ITZ Cured [57]
lavage
26/F Fungemia by contaminated Blood Trichoderma sp. AMB Cured [58]
saline
58/M Fungemia Blood T. longibrachiatum AMB, VRZ, catheter Cured [59]
removal
12/M Otitis externa Ear discharge T. longibrachiatum Nystatin, polymyxin B Cured [60]
61/M Infection of cerebrospinal Cerebrospinal fluid, shunt T. reesei ABLC, VRZ, CSP Cured [61]
fluid device
66/M Endocarditis Aortic conduit Trichoderma sp. Surgery, antifungal drugs Cured [62]
Notes: M, male; F, female; APD, automated peritoneal dialysis; CAPD, chronic ambulatory peritoneal dialysis; 5FC, 5-fluorocytosine; AMB, amphotericin B;
ABLC, amphotericin B lipid complex; FLZ, fluconazole; ITZ, itraconazole; KTZ, ketoconazole; MCZ, miconazole; VRZ, voriconazole; CSP, caspo-
fungin; TX, transplant; HM, hematological malignancy; ND, no data available.

T. harzianum has been measured under controlled conditions protease activities proved to be common among the examined
by Kildesø et al.82 For T. harzianum, it appeared that many strains. Separation of trypsin- and chymotrypsin-like activi-
particles were released in agglomerates of conidia due to the ties by Sephadex G-100 column chromatography revealed
mucous layer covering the surface of the spores. Released that both systems are complex consisting of several isoen-
fragments, smaller than the conidia, were also found in the zymes. The pH-dependence of these two protease systems
case of T. harzianum. The authors suggested that these finer was also studied and the results suggested that the different
particles could be responsible for some of the adverse health isoenzymes have different optimal pH values.
effects seen in moldy buildings, especially, as the small par- Schmoll et al.88 applied the method of rapid subtrac-
ticles are much easier transported from the building envelope tion hybridization (RaSH) for the detection of T. longibra-
to the breathing zone. chiatum genes differentially expressed between a clinical
Trichoderma species have also been isolated from T. longibrachiatum isolate grown in confrontation with
food,83,84 which may explain positive stool culture results.45,67 bronchial epithelial cells, and the same strain grown in
The catheter may be a portal of entry for invasive42,49 and bronchial epithelial growth medium alone. Among the
peritoneal dialysis-related Trichoderma infections.29–39 genes found to be upregulated under conditions of simu-
Contaminated infusion products can also lead to dissemi- lated infection there was a cyanoviridin-N-homologue for
nated infections when infused intravenously.58 homologues of which an antiviral effect has been shown,
Antal et al.85 studied the potential virulence fac- a putative extracellular DNase for which bacterial homo-
tors of 9 saprophytic and 12 clinical T. longibrachiatum logues with virulence promoting function are known, a
strains—including strains from previously published case member of the protein family of mannosyltransferases,
reports34,42,43,45,49—in order to compare their capacity to cause which are required for virulence in Candida albicans as
infection in humans. All of the examined strains were able well as genes putatively involved in ATP/ADP transloca-
to grow at temperatures ranging from 10°C to 40°C with an tion and ferric iron uptake.
optimum at 30°C, and at pH values ranging from 2.0 to 9.0 at
25°C with an optimum at pH 4. Besides T. longibrachiatum,
other species belonging to the Longibrachiatum section of 62.1.3 Clinical Features
the genus (including H. orientalis) are also capable of grow- 62.1.3.1 Trichoderma Infections in Patients
ing at 37°C. Because growth at elevated temperatures is one
Undergoing Peritoneal Dialysis
of the virulence factors of fungi, it is not surprising that most
of the strains involved in Trichoderma infections belong to Twelve cases of Trichoderma peritonitis have been reported
the Longibrachiatum section of the genus. All of the clini- in details in the case of patients requiring peritoneal dialysis
cal strains were able to grow at physiological pH, which is due to end-stage renal failure. The reported species include
another prerequisite of growth in human hosts. Carbon and T. longibrachiatum,33,38–40 T. pseudokoningii,36 T. harzia-
nitrogen source utilization experiments revealed that all of num,32 T. koningii,30,34 T. viride29,31 as well as unidentified
the strains were able to utilize a series of basic amino acids Trichoderma species.35,37 A previous history of bacterial peri-
both as sole carbon and nitrogen sources. The growth of dif- tonitis was frequent. Reported symptoms included abdominal
ferent bacteria was inhibited by metabolites of the strains. pain and fever. The fungi were isolated from the peritoneal
Compounds produced by three clinical isolates also reduced fluid in all of these cases. Therapeutical interventions involved
the motility of boar spermatozoa, indicating their toxicity to an early catheter removal30,35–39 as well as the administra-
mammalian cells. Exposure to the fungal extracts of these tion of antifungals (amphotericin B, ketoconazole, flucon-
clinical isolates resulted in quenching of the yellow fluores- azole, miconazole, itraconazole, voriconazole, caspofungin,
cence of the midpiece of boar spermatozoa stained with JC-1/ 5-fluorocytosin). Only four of the patients survived.30,35,36,39
propidium iodide, suggesting that the dissipation of the mito- Further two peritonitis-related clinical isolates were depos-
chondrial membrane potential may be involved in the toxic ited at the University of Alberta Microfungus Collection and
effect. There were no significant differences in the examined Herbarium (UAMH): T. longibrachiatum UAMH 9515 from
features between T. longibrachiatum strains derived from the peritoneal effluent of a female and Hypocrea orientalis
clinical or soil samples.85 UAMH 9573 from a peritoneal catheter tip (deposited as
Evidences are available for the possible involvement T. citrinoviride).
of proteolytic enzymes in aspergillosis, coccidioidomyco-
sis, and sporotrichosis86; therefore, it can be supposed that 62.1.3.2 Trichoderma Infections in Organ
extracellular proteolytic enzymes may be involved in the Transplant Recipients
pathogenicity of Trichoderma strains as opportunistic patho- Immunocompromised solid-organ transplant recipients rep-
gens of humans. Kredics et al.87 screened supernatants from resent another important risk population of Trichoderma
induced liquid cultures of six clinical T. longibrachiatum infection. An infection of a perihepatic haematoma reported
isolates—including strains from previously published case to be due to T. viride occurred in a liver transplant recipi-
reports43,45,49—for proteolytic enzyme activities with 11 dif- ent.41 The causal agent persisted in the immunocompro-
ferent chromogenic p-nitroanilide substrates. The production mised host despite amphotericin B treatment and surgical
of trypsin-like, chymotrypsin-like, and chymoelastase-like removal of the infected haematoma; however, the patient

Trichoderma 521
died of unrelated complications. A fatal infection caused 62.1.3.3 Trichoderma Infections in Patients
by a strain identified as T. pseudokoningii was described with Hematologic Malignancies
after allogeneic bone marrow transplantation in a patient Hematologic malignancies are also among the diseases pre-
with acute erythroleukemia.42 The fungus was obtained disposing to Trichoderma infections. A brain abscess due to
from bronchoalveolar lavage and skin biopsy specimens T. longibrachiatum corresponding to a contiguous infection
at the site of insertion of an intravenous catheter. Autopsy from an ethmoidal invasive sinusitis in a leukemic patient
revealed a disseminated infection with hyphae present in with prolonged neutropenia was successfully treated with
the lungs, pretracheal abscesses, stomach, heart, and brain. extremely prolonged antifungal therapy including ampho-
The acute invasive sinusitis caused by T. longibrachiatum tericin B, 5-fluorocitozin, ketoconazole, and itraconazole as
in a patient 6 months after small bowel and liver transplan- well as with the neurosurgical resection of the abscess.48 A
tation could be successfully treated with endoscopic sinus pediatric patient with severe aplastic anemia and neutrope-
operations for debridement, amphotericin B irrigation of nia developed an invasive T. longibrachiatum skin infection,
the maxillary sinus, and oral itraconazole treatment for a which was successfully treated with intravenous amphoteri-
total of 6 months.43 A disseminated T. harzianum infection cin B therapy.49 A case of T. longibrachiatum stomatitis was
was diagnosed in the postmortem examination of a renal reported by Myoken et al.50 in a neutropenic patient with
transplant recipient, the fungus was recovered from lung malignant lymphoma. The infection rapidly disseminated
tissue microabscesses and mycotic brain lesions.44 The gas- from the oral mucosa to the lungs with a fatal outcome
trointestinal tract (GIT) was suggested as the possible portal despite the intensive antifungal therapy with amphotericin B
of entry in the case of a fatal, disseminated T. longibra- and itraconazole. De Miguel et al.51 reported a pulmonary
chiatum infection in an allogenic bone marrow transplant T. viride infection in an adult patient with acute myeloid
patient with acute lymphoblastic leukemia, as the causal leukemia. The fungus was isolated from pulmonary aspi-
agent could be recovered from stool surveillance cultures rate. The patient was treated unsuccessfully with liposomal
and a perirectal ulcer biopsy specimen.45 Chouaki et al.46 amphotericin B, which was then replaced with voriconazole
reported further two cases of invasive T. longibrachiatum and caspofungin, resulting in successful therapy. In another
infections in solid-organ transplant recipients. In the case case of pneumonia in an acute myeloid leukemia patient,
of a patient who received liver transplant due to hepatitis C T. citrinoviride was isolated from the bronchoalveolar
virus-induced cirrhosis, a sample from a subcapsular col- lavage.52 The infection was successfully cured with ampho-
lection related to suture threads and biopsy specimens of tericin B therapy. In a patient with B cell acute lymphoblastic
perilesional tissues yielded pure cultures of T. longibrachia- leukemia, Alanio et al.53 reported an invasive lung infection
tum. Concomitant surgical debridement and local adminis- by Trichoderma. The cultures of sputum, bronchoaspiration,
tration of povidone iodine resulted in total recovery. The and bronchoalveolar lavage fluid samples yielded T. longi-
other case of T. longibrachiatum infection was fatal in a brachiatum. The infection was successfully treated with a
cystic fibrosis patient who underwent pulmonary trans- combination of voriconazole and caspofungin. A fatal T. har-
plantation due to terminal respiratory failure. The fungus zianum infection was reported in another patient with acute
could be isolated from samples obtained from the transcu- lymphoblastic leukemia.54 The causal agent could be recov-
taneous tracheal puncture and bronchoalveolar lavage fluid. ered from samples of blood serum, skin lesions, sputum, and
Stelzmueller et al.47 diagnosed postmortem a disseminated throat of the patient. Further clinical isolates from patients
double-infection with a Trichoderma sp. and Absidia cor- with hematologic malignancies include T. longibrachiatum
ymbifera involving the liver, GIT, lung, heart, kidneys, and IP-92 0647 from a patient with acute leukemia,63 T. citrino-
skin in a liver transplant recipient who died from progres- viride IP-95 1151 from blood cultures of a patient in aplasia
sive graft-versus-host disease, sepsis, and multiorgan fail- associated with lymphoma,63 and two H. orientalis isolates:
ure. Ranque et al.40 reported the isolation of Trichoderma one from the blood culture of a 3-year-old child with acute
atroviride from a liver biopsy specimen of a liver transplant lymphoblastic leukemia and another one from the stool of a
recipient. Further clinical Trichoderma isolates from trans- 15-year-old child with non-Hodgkin lymphoma.67 These two
plant recipients were deposited at the Canadian Collection H. orientalis isolates derived from patients of the same pedi-
of Fungal Cultures (CCFC): T. pseudokoningii CCFC atrics clinic, thus an epidemiological connection could not
007753 and CCFC 007754 isolated from a bone marrow be ruled out.
transplant recipient and from a liver and bowel transplant
recipient, respectively; at the National Reference Center
of Mycoses and Antifungal Drugs at the Institute Pasteur 62.1.3.4 Trichoderma Infections of the Respiratory
(IP): IP-94 0958 isolated together with Aspergillus fumig- Tract and Allergic Reactions
atus from a patient who had a lung transplantation and T. viride was reported from sputum and lung biopsy speci-
IP-93 1282 isolated from a bronchoalveolar washing in a 2 mens of a patient with pulmonary mycetoma, which was
month bone marrow recipient63; and at the Centro Nacional cured by surgical resection.55 Druzhinina et al.56 reported
de Microbiologia, Immunologia y Virologia Sanitaria the isolation of an unknown Hypocreaceae species close to
(CNM-CM): T. longibrachiatum CNM-CM 1798 from the the genus Hypocrea/Trichoderma from the lung tissue of
blood culture of a patient with liver transplant. a patient with nonfatal pulmonary fibrosis, who improved

dramatically after receiving bronchodilators. The clinical T. citrinoviride but not to the other molds, suggesting that
and mycological investigations did not determine whether T. citrinoviride may play a role in the etiology of adult-onset
this fungus did indeed cause the infection or contribute to asthma or serve as an indicator of other causal factors. In
pulmonary fibrosis. Based on culture collection data, the another study, doctor-diagnosed asthma among school chil-
isolation of T. longibrachiatum is known from the spu- dren was also strongly associated with high IgG levels to
tum of a tuberculosis patient (CNM-CM 2277) and from a T. citrinoviride.97 In a lung epithelial cell assay, T. harzianum
lung of a man who died (T. longibrachiatum CBS 446.95), was shown to be an inflammogen.98 On the other hand, short-
while T. citrinoviride was isolated from a patient suffering term human exposure to T. harzianum did not result in more
from chronic bronchitis with fever and respiratory infec- clinical effects than placebo exposure.99
tion (CNM-CM 1792, deposited as T. viride, reidentified as
T. citrinoviride by Kredics et al., unpublished data). 62.1.3.5 Occurrence of Trichoderma
Tang et al.57 described a case of allergic fungal sinusitis Associated with HIV Infection
associated with T. longibrachiatum in a patient with a his- The clinical occurrence of Trichoderma may also be associ-
tory of atopy and asthma, the case was successfully man- ated with human immunodeficiency virus (HIV) infection,
aged with a combination of sinus lavage, oral corticosteroids, as suggested by the isolation of Trichoderma sp. from the
itraconazole, and allergen immunotherapy. Further, T. lon- cerebrospinal fluid of an AIDS patient,69 the isolation of
gibrachiatum strains were isolated from infected maxillar T. longibrachiatum from a HIV-positive patient (deposited
sinus (strain IP-94 151063) and from the sinus lavage sample in the American Type Culture Collection as strain ATCC
of a rhinosinusitis patient.67 Two strains of T. viride65 and a 208859100), and Trichoderma fungaemia in a patient with
strain of T. inhamatum 66 were reported from the nasal mucus pulmonary cancer, chemotherapy-induced neutropenia, and
of chronic rhinosinusitis (CRS) patients; however, a great HIV infection.59
number of other fungi were found to be associated with CRS
and it is not known which of them were responsible for the
62.1.3.6 Miscellaneous Cases of
eosinophilic reaction.
Trichoderma Infections
Trichoderma species may also be present as part of
the human microbiota: strains were isolated from sputum The involvement of Trichoderma species is also known from
(T. koningii UAMH 473 and UAMH 475), from sinus eth- two cases of mycotic keratitis,46,70 a case of onychomyco-
moidalis (T. pseudokoningii CBS 500.94), and from the nasal sis,68 a case of otitis externa,60 a case of infection of cere-
mucus (T. viride) of healthy patients.66 brospinal fluid shunt device,61 and a case of endocarditis.62
Some Trichoderma species have been found to be aller- Further known clinical Trichoderma isolates include T. lon-
genic.89 Vesper et al.90 have found that T. viride had signifi- gibrachiatum strain IP-96 0086 from a hematic wound of an
cantly higher concentrations in the water-damaged homes otherwise healthy patient,63 strain IP-93 1792 obtained from
of asthmatic children compared with control homes. The a cerebrospinal derivative catheter,63 IP-97 0711 from the
possible involvement of Trichoderma species in eosinophil liquid of a chylothorax,63 and T. longibrachiatum CNM-CM
activation has also been demonstrated: precipitating anti- 2171 from the foot skin of a premature infant with subcutane-
bodies against T. viride were detected in the sera of patients ous lesions.85
with acute eosinophilic pneumonia.91,92 Volatile organic
compounds (VOC) from a Trichoderma isolate identified 62.1.3.7 Antifungal Susceptibilities of
as T. viride were found to potentiate IgE-related histamine Clinical Trichoderma Isolates
release from human bronchoalveolar cells.93 The authors sug- Antifungal susceptibility testing of fungi involved in oppor-
gested that VOC of Trichoderma might cause adverse health tunistic infections is relevant for the choice of adequate
implications via mediator release, inflammation, and neural therapy, as the susceptibility of the organisms to antifun-
stimulation. Besides the visible growth of fungi, growth hid- gal agents is an important factor affecting the outcome of
den behind walls and ceilings might also represent a risk, as the infection. Data available about the susceptibilities of
VOC penetrate the polyethylene vapor barrier. T. longibra- clinical Trichoderma isolates are summarized in Table
chiatum has been reported to be one of the most prevalent 62.2. Different methods were applied for the determina-
molds in the manufacturing process of stoppers,94 although tion of the minimal inhibitory concentration (MIC) values
no evidence of allergic sensitization by T. longibrachiatum of the antifungal agents in the available studies.101–109 Table
in patients with asthma in the cork industry could be demon- 62.2 reflects that most of the clinical Trichoderma isolates
strated.95 Jaakkola et al.96 conducted a population-based inci- are resistant to fluconazole, 5-fluorocytosine, and ampho-
dent case–control study to assess the risk of asthma in relation tericin B, and susceptible or intermediate to itraconazole,
to specific IgG antibodies to eight dampness-related microbes ketoconazole, miconazole, and voriconazole; however, high
(Aspergillus fumigatus, A. versicolor, Cladosporium clado- MIC-levels of itraconazole44,50,60,85 and ketoconazole44,52,56
sporioides, Fusarium oxysporum, Sporobolomyces salmo- and voriconazole40 were also reported in certain cases.
nicolor, Stachybotrys chartarum, Streptomyces albus and Based on the available susceptibility data, voriconazole
T. citrinoviride). An increased risk of developing asthma might be an important drug in the treatment of Trichoderma
in adulthood was significantly related to IgG antibodies to infections.50,51,53,54,56,59,111–115

Trichoderma
TABLE 62.2
Antifungal Susceptibilities of Clinical Trichoderma Isolates (Minimum Inhibitory Concentrations in mg/L)
Isolate Method AMB 5FC FLZ ITZ KTZ MCZ PSZ VRZ RVZ TRB CSP AND AP CHG Reference
T. longibrachiatum Broth 2 64 0.5 0.5 2 [113]
microdilution104
UAMH 7955 Disk method *7 mm [111]
Etest102 1 >256 32 0.125 [85]
Macrobroth 1.16 >322.75 80 0.3 [43]
dilution101
T. longibrachiatum Broth 2.0 >256 16 1.0 [45]
micodilution103
UAMH 7956 Broth 2 64 0.5 0.125 2 [113]
microdilution104
Disk method *9 mm [111]
Etest102 2 64 16 0.25 [85]
microdilution104
Etest102 2 >256 16 0.5 [85]
T. longibrachiatum Both 2 >64 >64 2 [49]
macrodilution
ATCC 201044 Broth 2 128 1 0.25 4 [113]
microdilution104
Etest102 2 64 0.5 0.008 [85]
microdilution104
ATCC 208859 Etest102 2 >256 8 0.25 [85]
T. longibrachiatum Broth dilution 2.5 50 12.5 1.25 [48]
IP-93 1192 Disk method *34 mm [48]
T. longibrachiatum Broth dilution 5 [33]
T. longibrachiatum EUCAST107 0.5 >64 >64 >8 1 0.5 [53]
(MEC)
T. longibrachiatum EUCAST107 1 >64 2 4 0.5 0.5 [59]
T. longibrachiatum Broth 0.5 >32 >64 >32 0.5 [50]
microdilution
T. longibrachiatum Broth 2 128 1 0.25 8 [113]
microdilution104
IP 2110.92 Broth 0.09 >100 25 0.18 0.09 [42]
microdilution109
(continued)
523
524
Antifungal Susceptibilities of Clinical Trichoderma Isolates (Minimum Inhibitory Concentrations in mg/L)
Isolate Method AMB 5FC FLZ ITZ KTZ MCZ PSZ VRZ RVZ TRB CSP AND AP CHG Reference
(originally Etest
102 2 >256 16 1 [85]
identified as T.
pseudokoningii)42
microdilution104
CBS 446.95 Etest102 2 >256 2 0.5 [85]
microdilution104
CNM-CM 382 Broth 4.0 16.0 128.0 1.0 ≤0.25 [34]

microdilution108
(originally Disk method *8 mm [111]
identified as T. Etest102 8 >256 2 0.25 [85]
koningii)34
microdilution104
CNM-CM 1798 Etest102 0.016 >256 1 0.125 [85]
microdilution104
CNM-CM 2171 Etest102 2 >256 16 0.5 [85]
microdilution104
CNM-CM 2277 Etest102 2 >256 32 0.25 [85]
T. longibrachiatum Broth 0.5–2 >8 2 [114]
(5 isolates) microdilution103
Etest102 1.0–4 >8 [110]
T. longibrachiatum Broth dilution104 0.5–2 >8 >8 2 >8 [115]
(3 isolates)
Trichoderma
T. longibrachiatum NCCLS M-38P103 1–2 16 2 [46]
(2 isolates)
H. orientalis Broth 2 128 0.5 0.25 1 [113]
microdilution104
Etest102 2 64 32 0.25 [85]
T. citrinoviride NR >32 >32 >32 4 0.125 [52]
T. reesei Etest102 1.5 >32 1 [61]
T. harzianum Broth 2 1024 1 0.25 8 [113]
microdilution104
CBS 102174 Microdilution106 2 256 128 32 8 8 [44]
T. harzianum Broth >16 0.125 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 [54]
macrodilution104
T. atroviride EUCAST107 1 >64 >64 >8 8 0.5 [40]
T. viride Broth dilution 0.78 100 1.56 [29]
T. viride Broth 0.25 8 2 [51]
microdilution104
T. viride Broth dilution 3.1 >50 25 1.6 0.8 [41]
Trichoderma spp. Broth 0.06–2 [64]
(4 isolates) microdilution104
Trichoderma sp. Broth 1 >128 2 1 0.25 0.25 0.06 [112,116,117]
(1 isolate) microdilution105
Trichoderma sp. Broth 32 [118]
(1 isolate) microdilution104
0.125
(MEC)
Hypocreaceae sp. Etest102 0.125 >32 >256 16 >32 >32 1 >32 [56]
Notes: *, diameter of inhibition zone; NR, not reported; MEC, minimum effective concentration; Antifungal agents; 5FC, 5-fluorocytosine; AMB, amphotericin B; FLZ, fluconazole; ICZ, itraconazole; KTZ, keto-
conazole; MCZ, miconazole; VRZ, voriconazole; PSZ, posaconazole; RVZ, ravuconazole; TRB, terbinafine; CSP, caspofungin; AND, anidulafungin; CHG, chlorhexidine digluconate; AP, Akacid plus®;
ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures; CNM-CM, Centro Nacional de Microbiologia, Immunologia y Virologia Sanitaria; IFM, Medical Mycology
Research Center, Chiba University, Japan; NRCMA, French National Reference Centre for Mycoses and Antifungals; IP, Institut Pasteur; UAMH, University of Alberta Microfungus Collection and
Herbarium.
525
In the study of Marco et al.,116 posaconazole displayed The low susceptibility level of some clinical Trichoderma
good in vitro activity against a Trichoderma sp. isolate. isolates to antifungal drugs may cause difficulties in the ther-
Pfaller et al.117 investigated the in vitro activity of two echi- apy of infected patients. In cases of suspected or confirmed
nocandin derivatives, anidulafungin and caspofungin as Trichoderma infection, amphotericin B in combination with
well as itraconazole, amphotericin B, and 5-flucytosine itraconazole or ketoconazole,49 or the combination of caspo-
against 51 clinical isolates of filamentous fungi, including a fungin and voriconazole51,53 were suggested as adequate
Trichoderma strain. Anidulafungin was fourfold more active choices of treatment, the duration of which should be indi-
than caspofungin against Trichoderma. Kahn et al.118 have vidualized to each case according to the type and extent of
demonstrated that caspofungin inhibits β-d-1,3 glucan syn- the infection and underlying predisposing conditions.
thesis and reduces in vitro growth of a clinical Trichoderma
isolate. Terbinafine was also shown to have a very strong 62.1.4 Diagnosis
activity in vitro against clinical isolates of several species of
filamentous fungi, including Trichoderma spp.64 62.1.4.1 Conventional Techniques
The MICs of fluconazole and amphotericin B obtained The difficulties of definitive diagnosis of Trichoderma infec-
by the Etest and the agar dilution method for seven clinical tions were discussed in details by Chouaki et al.46 Despite the
Trichoderma strains were compared by Dóczi et al.119 The documented dissemination, culture of blood samples does
MICs of fluconazole obtained with the two methods were in not appear to be a valuable diagnostic tool, as the common
agreement, while higher MICs were obtained for amphoteri- isolation rate of fungi in hemocultures is low.120 Therefore,
cin B with the agar dilution method than with the Etest in the the diagnosis mostly relies on the microscopic visualization
case of six clinical isolates; however, most of these differ- of fungal elements (fine, septate hyaline hyphae, eventually
ences were within the ± 2 two-step dilutions. chlamydospores) in tissue samples obtained from accessible
Antal et al.85 used the Etest method modified for molds102 sites, along with the isolation of the fungus on culture. The
to reveal a wide-scale dataset about the antifungal suscep- diagnosis can be reinforced by the repeated isolation of the
tibilities of T. longibrachiatum, including 12 and 9 isolates same fungus from the same—or in the case of disseminated
from clinical and environmental samples, respectively. Data infections from different—sites. Differential diagnosis of
for the clinical isolates from this study are included in Table Trichoderma infections with aspergillosis and other hyalo-
62.2. The MIC values of the tested antifungal drugs were hyphomycoses is difficult because of the morphologically
found to be 0.016–8 μg/mL for amphotericin B, 64–256 μg/ similar hyphae. The highest level of certainty in diagnosing
mL for fluconazole, 0.5–32 μg/mL for itraconazole, and an invasive mycosis requires the establishment of the pres-
0.008–1 μg/mL for ketoconazole. All of the examined ence of fungi in tissue by biopsy or a needle aspirate; how-
T. longibrachiatum strains proved to be susceptible to keto- ever, such invasive techniques are not always possible due
conazole, resistant to fluconazole and with one exception, to the patients’ conditions.54 Therefore, the definition of an
also resistant to itraconazole. MIC values for amphotericin invasive fungal infection may have to rely on a combination
B were more variable among the strains: four of the soil iso- of less-specific clinical, laboratory and radiological data.121
lates were susceptible (MIC value < 1 μg/mL) and five of Methods for identifying molds including Trichoderma from
them were resistant (MIC value > 2 μg/mL), while one sus- histological specimens by the development of fluorescent
ceptible and 11 resistant strains could be found among the antibody conjugates and the use of molecular techniques may
clinical isolates. The same set of clinical isolates was tested also provide definitive diagnoses.44
by the broth microdilution method103 for their antifungal Altogether, eight species from the genus Trichoderma
susceptibilities to conventional antimycotics (amphotericin (T. longibrachiatum, T. citrinoviride, T. pseudokoningii,
B, fluconazole, voriconazole) and cationic antimicrobials T. reesei, T. harzianum, T. koningii, T. atroviride, T. viride)
(chlorhexidine digluconate and Akacid plus®) by Kratzer have been reported from clinical cases (Table 62.1). The ques-
et al.113 The data from this study for amphotericin B and flu- tion rises whether all of these species are really capable of
conazole (Table 62.2) are in good agreement with the Etest causing infections in humans as several clinical Trichoderma
results of Antal et al.85 The high in vitro activity of vori- isolates were identified by the use of culture-based morpho-
conazole against clinical and environmental Trichoderma logical characters only.34,38,42,44,51,52 The identification of
isolates suggests that it can be an appropriate choice in the Trichoderma strains is very difficult if only morphologi-
treatment of invasive Trichoderma infections. Evaluation of cal characters are considered and needs special expertise.
synergism of double antifungal combinations revealed no Summerbell122 provided the key features for morphological
interaction between convential antimycotics, some degree of distinction of clinically relevant Trichoderma species and
synergism was detected between azoles and cationic antimi- suggested to follow the key of Gams and Bissett123 for the
crobials, while the interaction of the two cationic antimicro- purposes of morphology-based identification.
bials was synergistic for each tested Trichoderma isolate.113 Four members of Trichoderma section Longibrachiatum:
The authors concluded that an earlier diagnosis could enable T. longibrachiatum, T. citrinoviride, T. pseudokoningii
the clinical application of cationic antimicrobials to support and T. reesei have been reported from clinical cases, with
the treatment of localized Trichoderma infections in the T. longibrachiatum being the most frequently occurring ethi-
future. ologic agent in the entire genus.3,43,45,48–50,53,57,59,60 Tang et al.57

Trichoderma 527
identified strain UAMH 10147, the causal agent of a case of morphological diagnosis was later confirmed by molecular
allergic fungal sinusitis as T. longibrachiatum based on fea- techniques.124,125 Kantarcioğlu et al.54 identified a clinical
tures agreeing with criteria described by Samuels et al.100: a fungal isolate as Trichoderma spp. phenotypically by com-
radius of 60 mm on potato dextrose agar (PDA) after 65 h at paring its morphological characteristics with the standard
35°C (radius of 55 mm at 30°C); a yellow diffusing pigment descriptions given by Summerbell,122 and performed molec-
absent at 35°C but present at 30°C; mostly solitary, cylin- ular identification, which revealed the identity of the isolate
drical phialides tapered at the neck; smooth and ellipsoidal as T. harzianum.
conidia measuring 3.5–5 × 2.5–3 μm; the common presence From section Trichoderma, data were published about
of chlamydospores appearing either terminal or oval to the clinical relevance of three species: T. koningii, T. atro-
globose or intercalary and cylindrical to barrel shaped, viride, and T. viride. Two clinical isolates from peritonitis
measuring 6–10 μm × 3–5 μm. This original diagnosis was were identified as T. koningii.30,34 Ragnaud et al.30 provided
confirmed by molecular techniques, similarly to other cases a morphological description that allows the identification of
of T. longibrachiatum infections.45,48,50,53,57,59,60 Among the the isolate at the genus level only. Campos-Herrero et al.34
two known clinical cases of T. citrinoviride, the study of identified their isolate based on its rapid growth, morphol-
Kuhls et al.63 mentioned a T. citrinoviride isolate from blood ogy on Sabouraud dextrose agar, a grass-green pigmentation
cultures, which has been identified by molecular techniques, and microscopic characteristics (branching, septate, and hya-
while Kviliute et al.52 have not provided any details of the line, clusters of single-celled conidia at the end of conidio-
identification in their case report. The occurrence of T. pseu- phores), and provided a microphotograph. Based on the three
dokoningii has been reported from two clinical cases.36,42 long terminal conidiophore branches with phialides borne
Rota et al.36 provided no details about the identification singly or in offset pairs and the ellipsoidal–oblong conidia
of their peritonitis isolate, while Gautheret et al.42 used an seen on this microphotograph, Summerbell122 recognized the
immunofluorescence assay besides morphology for the iden- isolate as a member of section Longibrachiatum. This was
tification of a Trichoderma isolate from a fatal infection in later confirmed by molecular techniques: the isolates turned
a bone marrow transplant recipient as T. pseudokoningii, out to be T. longibrachiatum.63,124,125 A neotypification of
the patient’s serum reacted strongly against T. pseudokon- T. koningii has been carried out by Lieckfeldt et al.128 who
ingii cultures fixed on slides with methanol. This diagnosis characterized this species with a maximum growth tempera-
could not be confirmed later by molecular techniques: the ture of approximately 33°C, which does not meet the criteria
isolate turned out to be T. longibrachiatum.63,124,125 Studies of an opportunistic human pathogen. The authors of the two
on section Longibrachiatum126,127 suggested that the species papers reporting the occurrence of T. koningii in peritonitis
T. pseudokoningii may be limited in distribution to Australia cases have likely applied the outdated T. koningii concept,
and New Zealand. Later, Samuels et al.100 have found some which included all species with ellipsoidal conidia. T. atro-
T. pseudokoningii isolates also in North America and Sri viride is known from a single clinical case only.40 Based on
Lanka and have suggested that the species is widely distrib- the morphological characterization, the isolate had conidio-
uted, but probably not as common as indicated by reports in phores with branching pattern at right angles; flask-shaped,
the literature. three or four verticillate phialides (8–10 μm × 2 μm) often
From Trichoderma section Pachybasium B, only T. har- curved in whorls of two, dark green, subglobose, short ellip-
zianum has been shown to be involved in a few cases of soidal, smooth-walled conidia (3.5 μm × 4 μm). The identity
human infections.32,44,54 The fungal isolate from brain and of this isolate has been confirmed by molecular techniques.
lung abscesses of a renal transplant recipient was identified Five studies reported the isolation of T. viride from clinical
as T. harzianum by Guarro et al.44 upon its morphology on cases.29,31,41,51,55 Summerbell122 evaluated the morphological
PDA (very quick growth, dense, and cottony colonies with description from three of these case reports29,41,55 and found
a yellowish green area at the center and white toward the that they do not support the identification of the isolates at the
periphery, the whole surface rapidly turning granular with species level as T. viride. De Miguel et al.51 reported T. viride
dark green masses because of the abundant production of as the ethiologic agent of a pulmonary infection on the basis
conidia in tufts, colorless colony reverse, production of a yel- of its macroscopic morphology on Sabouraud chlorampheni-
lowish exudate) and oatmeal agar (very quick growth, cot- col agar and microscopic characteristics based on Rifai5
tony, and whitish green colonies becoming olive green in (the types of branching systems of the conidiophores, the
tufted conidial areas, colorless reverse, no exudate), as well phialide disposition, and the character of the phialospores).
as microscopical characters (pyramidally branched conidio- To the best of our knowledge, this diagnosis has not been
phores with short branches near the apex, phialides usually tested by molecular techniques. Identification of clinical
in groups of 2–5, ampulliform or lageniform and mark- Trichoderma isolates as T. viride may be due to the fact that
edly constricted at the base, generally 4–7 × 3–3.5 μm, usu- the name T. viride was applied for a long period to a phyloge-
ally longer and slender near the apex of the conidiophore, netically diverse group of Trichoderma strains with rounded,
subglobose or short obovoid conidia of 2.5–3 × 2–2.5 μm, roughened conidia. Lieckfeldt et al.129 redefined this species
subhyaline to pale green and smooth walled, presence of based on isolates derived from the Hypocrea rufa teleomorph
subglobose or ellipsoidal, hyaline, smooth-walled chlamydo- and characterized it with a maximum growth temperature
spores up to 12 μm in diameter in old cultures). This careful below 35°C.

The above studies reflect that identification relying solely Druzhinina et al.132 performed a multilocus phylogenetic
on conventional methods may sometimes lead to erroneous analysis based on the ITS region of the ribosomal RNA gene
species identification; therefore, the application of biochemi- cluster and fragments of the translation elongation factor 1α
cal and molecular techniques is suggested to confirm the (tef1), calmodulin (cal1), and endochitinase (chit18-5) genes
species-level diagnosis of clinical Trichoderma isolates. with 15 clinical Trichoderma isolates (including strains from
Szekeres et al.125 performed studies on clinical previously published case reports34,42,43,45,49). The study also
Trichoderma isolates applying a biochemical method, iso- included 36 environmental isolates. In this study, 12 of the
enzyme analysis based on cellulose-acetate electrophoresis clinical isolates proved to be T. longibrachiatum while three
(involving glucose-6-phosphate dehydrogenase, glucose-6- strains were identified as Hypocrea orientalis, which was
phosphate isomerase, 6-phosphogluconate dehydrogenase, previously proposed as the teleomorph of T. longibrachia-
peptidases A, B and D, and phosphoglucomutase enzymes) tum.100 However, the concordance of gene genealogies rec-
according to Hebert and Beaton130 for the identification. The ognized T. longibrachiatum and H. orientalis to be closely
method was suggested as a cheap and useful alternative of related, but different phylogenetic species. The phylogenetic
molecular techniques for the quick identification of clinical analysis has supported the conclusion that H. orientalis is
T. longibrachiatum isolates. sexually recombining whereas strict clonality prevails in
T. longibrachiatum.132 This study broadened the phyloge-
62.1.4.2 Molecular Techniques netic span of human opportunists in the genus Trichoderma/
The appropriate identification of the fungal etiologic agents Hypocrea by identifying the sexually recombining teleo-
involved in opportunistic infections is crucial for the adequate morph species H. orientalis as a potential human opportun-
choice of specific therapeutic interventions. Certain molecu- ist, and confirmed that the clinical isolates do not belong to
lar techniques have the potential to reveal an exact diagnosis, a single population of T. longibrachiatum, but presumably
thus helping to overcome the difficulties of morphology-based every isolate of T. longibrachiatum or H. orientalis is capable
identification. In a number of case reports, the identity of the of causing opportunistic infections in humans.
clinical Trichoderma isolates was confirmed by molecular H. orientalis may not be the only sexually reproducing
techniques, like DNA-fingerprinting43,45,49 or the sequencing species of clinical importance within the genus Trichoderma/
of ribosomal DNA internal transcribed spacer (ITS) region Hypocrea: when a multigenic approach was used to identify
ITS1–5.8S–ITS2.40,45,46,48,50,53,54,57,59,60,63,67 Kuhls et al.63 used a clinical fungal isolate with Hypocrea-like teleomorph mor-
PCR-fingerprinting to identify six clinical Trichoderma phology,56 phylogenetic analysis of molecular markers (ITS1
isolates—including two strains from previously reported and ITS2 of the rRNA gene cluster, a large exon and a short
cases42,48—as T. longibrachiatum (five isolates) and T. citrino- intron of tef1, and a fragment of the RNA polymerase subunit
viride (one isolate). Sequencing experiments of the ITS-region B-encoding gene rpb2) showed that it belongs to an unknown
confirmed the identifications made on the basis of PCR- fungal species that occupies a unique taxonomic position
fingerprinting data. The results of this study have revealed that close to Trichoderma/Hypocrea.
the “T. pseudokoningii” strain from the study of Gautheret Altogether, from the 59 clinical Trichoderma isolates
et al.42 needs to be reidentified as T. longibrachiatum. known from the literature, a sequence-based molecular iden-
Besides isoenzyme analysis, Kredics et al.124 and Szekeres tification has been performed for 33 isolates. The identities
et al.125 used ITS sequence analysis to reidentify further clini- of 12 and 3 isolates were confirmed as T. longibrachiatum
cal Trichoderma isolates as T. longibrachiatum, including and H. orientalis, respectively by multigenic approaches.132
strain CNM-CM 382 from the case report of Campos-Herrero Further, 12 isolates were identified as T. longibrachiatum
et al.,34 which was originally identified as “T. koningii” by ITS sequence analysis46,48,50,53,57,59,60,63; however, as this
based on morphological characters. The genetic diversity method alone is not able to differentiate between T. longi-
of T. longibrachiatum as an emerging fungal pathogen was brachiatum and H. orientalis,13 it cannot be excluded that
also examined by the analysis of restriction fragment length some of these isolates belong to H. orientalis. Therefore, if
polymorphisms (RFLPs) of the mitochondrial DNA.131 The the causal agent is supposed to belong to one of these two
examined 17 isolates (9 strains from clinical sources includ- species, tef1 sequence analysis is suggested to be performed
ing strains from previously published case reports,34,42,43,45,49 in addition to ITS in order to reveal an exact molecular iden-
and 8 strains from environmental samples) exhibited 7 and tification at the species level. Further isolates have been
10 different mitochondrial DNA profiles with the restriction confirmed as T. citrinoviride (2 strains)63 (Kredics et al.,
enzymes BsuRI and Hin6I, respectively. The method had unpublished data), T. harzianum (2 strains),54,124 T. atroviride
a discriminatory power higher than that of ITS sequence (1 strain),40 and an unknown Hypocreaceae sp. close to the
analysis and was therefore suggested as a suitable tool for genus Hypocrea/Trichoderma (1 strain).56 These data clearly
epidemiological investigations. In these studies, clinical and indicate that—with the exception of a few cases—the clinical
environmental isolates did not form separate clusters on the isolates within the genus Trichoderma are restricted almost
resulting phylogenetic trees and dendrogram, suggesting that exclusively to section Longibrachiatum of the genus, sug-
not a specific population of T. longibrachiatum is responsible gesting that certain reports about the clinical occurrence of
for opportunistic infections in humans, but every environ- other Trichoderma species (e.g., T. viride, T. koningii) may
mental isolate has the capacity to cause infection. require revision.

Trichoderma 529
62.2 Methods core sequence 5′-GAGGGTGGCGGTTCT-3′ as primers,

a ccording to a previous study of the authors.136
62.2.1 Sample Preparation Sequence-based molecular techniques including the prim-
For DNA-based molecular techniques, isolation of DNA from ers and reaction conditions applied for the identification of
Trichoderma strains can be performed by any protocol that clinical Trichoderma isolates are summarized in Table
was successfully applied for filamentous fungi. Kuhls et al.63 62.3.137–142
used the method of Gruber et al.133 for DNA isolation. In the For amplification of the ITS region, most of the stud-
study of Antal et al.131 applying mtDNA RFLP, DNA sam- ies applied the combination of SR6R and LR146,50,60,63,132 as
ples were isolated from lyophilized mycelia by the method of primers, while other studies applied ITS1 and ITS453,67,124
Leach et al.134 Recent studies applied the GeneElute™ Plant or ITS5 and ITS4.54 Optimized protocols for amplification
Genomic DNA Miniprep Kit (Sigma–Aldrich) or the Qiagen of Trichoderma phylogenetic markers including ITS, tef1,
DNeasy Plant Mini Kit132 according to the manufacturers’ chi18-5, and cal1 for molecular identification are available
instructions. at the web page of the International Subcommission on
Trichoderma and Hypocrea taxonomy (http://www.isth.info).
Amplified PCR products can be purified by the GenElute™
62.2.2 Detection Procedures Minus EtBr Spin Columns (Sigma–Aldrich) or by the Qiagen
For mtDNA RFLP, the fast typing method of Varga et al.135 Gel Extraction Kit (Qiagen), and sequenced using the dye
was used by Antal et al.131 Total DNA samples were digested deoxy terminator chemistry on a DNA sequencer, e.g., ABI
with BsuRI (GG/CC) or Hin6I (G/CGC) restriction enzymes. 373 or 377 (Applied Biosystems). Sequencing of fragments
PCR-fingerprinting was carried out by Kuhls et al.63 with can be carried out with the aid of an appropriate service.
the oligonucleotides (GACA)4, (GTG)5 and the phage M13 For sequence analysis, results from BLASTN similarity
TABLE 62.3
Sequence-Based Molecular Techniques for the Identification of Clinical Trichoderma Isolates at the Species Level
Suggested Tool
Locus Primers PCR Conditions for Analysis Diagnostic Value
ITS SR6R: Initial denaturation: 1 min at 94°C TrichOkey Identification of clinical
5′-AAGTAGAAGTCGTAACAAGG-3′137 1 min at 94°C 2.013,14 Trichoderma isolates at the species
LR1: 5′-GGTTGGTTTCTTTTCCT-3′137 1 min at 50°C
1.5 min at 74°C
} 30 cycles level (not able to differentiate
between T. longibrachiatum and
Final extension: 7 min at 74°C11 H. orientalis)
ITS1: 5′-TCCGTAGGTGAACCTGCGG-3″137 Initial denaturation: 5 min at 95°C TrichOkey Identification of clinical
1.5 min at 94°C 2.013,14 Trichoderma isolates at the species
}
ITS4: 5′-TCCTCCGCTTATTGATA TGC-3′137
2 min at 55°C 35 cycles level (not able to differentiate
3 min at 72°C between T. longibrachiatum and
ITS5: Initial denaturation: 4 min at 94°C TrichOkey Identification of clinical
5′-GGAAGTAAAAGTCGTAACAAGG-3′137 2 min at 94°C 2.013,14 Trichoderma isolates at the species
ITS4: 5′-TCCTCCGCTTATTGATATGC-3′137 2 min at 55°C
2 min at 72°C
} 35 cycles level (not able to differentiate
between T. longibrachiatum and
tef1 EF1-728F: Initial denaturation: 1 min at 94°C TrichoBLAST15 Differentiation between
5′-CATCGAGAAGTTCGAGAAGG-3′140 1 min at 94°C T. longibrachiatum and
TEF1-LLErev: 5′
AACTTGCAGGCAATGTGG-3′141
1 min at 59°C
50 s at 74°C
} 30 cycles H. orientalis
Final extension: 7 min at 74°C23

chi18-5 Chit42-1a: Initial denaturation: 1 min at 94°C TrichoBLAST15 Differentiation between
5′-GCTYTCCATCGGTGGCTGGAC-3′11 1 min at 94°C T. longibrachiatum and
Chit42-2a:
5′-GGAGTTGGGGTAGCTCAGC-311
1 min at 62°C
1 min at 74°C

cal1 CAL-228F: Initial denaturation: 3 min at 94°C NCBI Differentiation between
5′-GAGTTCAAGGAGGCCTTCTCCC-3′140 30 s at 94°C BLAST143 T. longibrachiatum and
CAL-737R:
5′-CATCTTTCTGGCCATCATGG-3′140
30 s at 50°C
60 s at 72°C

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393, 2002. 128. Lieckfeldt, E. et al., Neotypification of Trichoderma koningii
112. Marco, F. et al., Antifungal activity of a new triazole, vori- and its Hypocrea koningii teleomorph, Can. J. Bot., 76, 1519,
conazole (UK-109,496), compared with three other antifun- 1998.
gal agents tested against clinical isolates of filamentous fungi, 129. Lieckfeldt, E. et al., A morphological and molecular perspec-
Med. Mycol., 36, 433, 1998. tive of Trichoderma viride: Is it one or two species? Appl.
113. Kratzer, C. et al., In vitro activity and synergism of amphoteri- Environ. Microbiol., 65, 2418, 1999.
cin B, azoles and cationic antimicrobials against the emerging 130. Hebert, P.D.N. and Beaton, M.J., Methodologies for Allozyme
pathogen Trichoderma spp., J. Antimicrob. Chemother., 58, Analysis Using Cellulose Acetate Electrophoresis. A Practical
1058, 2006. Handbook. Helena Laboratories, Beaumont, TX, 1993.
114. Espinel-Ingroff, A., In vitro fungicidal activities of voricon- 131. Antal, Z. et al., Intraspecific mitochondrial DNA polymor-
azole, itraconazole, and amphotericin B against opportunistic phism within the emerging filamentous fungal pathogen
moniliaceous and dematiaceous fungi, J. Clin. Microbiol., 39, Trichoderma longibrachiatum, J. Med. Microbiol., 55, 31,
954, 2001. 2006.
115. Espinel-Ingroff, A. et al., Optimal testing conditions for 132. Druzhinina, I.S. et al., Alternative reproductive strategies
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fungi, Mycopathologia, 141, 73, 1998. 1990.
117. Pfaller, M. et al., In vitro activity of two echinocandin deriva- 134. Leach, J. et al., Rapid miniprep of DNA from filamentous
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63 Verticillium
Malena P. Pantou and Milton A. Typas
Contents
63.1 Introduction...................................................................................................................................................................... 535
63.1.1.1 Classification.......................................................................................................................................... 535
63.1.1.2 Morphology............................................................................................................................................ 536
63.1.2 Biology, Epidemiology, and Genomics................................................................................................................. 537
63.1.2.1 Biology................................................................................................................................................... 537
63.1.2.2 Epidemiology......................................................................................................................................... 537
63.1.2.3 Genomics............................................................................................................................................... 539
63.1.4 Diagnosis.............................................................................................................................................................. 541
63.2 Methods............................................................................................................................................................................ 544
References.................................................................................................................................................................................. 546
63.1 Introduction and removed all members of Verticillium sect. Prostrata

into Hypocreales. Thus, the genera Lecanicillium and
63.1.1 Classification and Morphology Simplicillium were introduced to accommodate all entomog-
63.1.1.1 Classification enous and fungicolous verticillium-like anamorphic fungi,
Haptocillium for nematophagous species with adhesive
Fungi of the genus Verticillium are soilborne, mainly plant
conidia, and Pochonia comprising species that form dictyo-
pathogens that attack plants through the roots and are
chlamydospores and parasitize nematode cysts and eggs. The
responsible for Verticillium wilt diseases. The name of the
saprophyte V. tenerum (syn. V. luteo-album) was also reclas-
genus is derived from the so-called verticillate disposition
sified as Acrostalagmus luteo-albus.10 However, in spite of
of the conidiophore branches and phialides, i.e., the branch-
this first generous reclassification, the genus of Verticillium
ing of conidiophores in whorls at several levels (Figure 63.1e
was still a polyphyletic entity that required resolution into
and f). The mycelium of Verticillium spp. is hyaline, sim-
more natural units. Thus, molecular phylogenetic data
ple or branched, and septate. The conidia are ovoid to elon-
based on mitochondrial and nuclear ribosomal sequences
gate and are produced on long phialides positioned in a whorl
placed V. nigrescens, V. theobromae and Acrostalagmus
or spiral-like shape around the conidiophores (Figure 63.1a
luteo-albus close to Phyllachorales but clearly differen-
and b). With the exception of hyphal tip cells that are mul-
tiated them from the group of all other phytopathogenic
tinucleate, older hyphal cell compartments are mostly uni-
species of Verticillium.9 Consequently, V. nigrescens and
nucleate. All hyphal cells and conidia of Verticillium spp. are
V. theobromae were placed under the new generic names
almost exclusively haploid.
Gibellulopsis and Musicillium, respectively.11 Finally, the
Based on these general morphological features, the
placement of V. fungicola, an important mushroom parasite,
genus Verticillium for years became the depository of some
was also reconsidered according to molecular phylogenetic
200 described species, and, therefore, its taxonomy needed
evidence that showed the affinity of the latter to the genus of
urgently major revision, a task undertaken over the past years
Lecanicillium.9,12,13 Therefore, in current taxonomy, only five
mainly by Gams and coworkers, amidst the contribution of
closely related species, namely V. dahliae, V. longisporum,
some other groups.1–9 The revision resulted initially in retain-
V. albo-atrum, V. tricorpus and V. nubilum remain as mem-
ing only phytopathogens and saprophytes in Verticillium
bers of Verticillium s. str.
sensu stricto, viz. the “Nigrescentia” (Phyllachorales)
535

10 µm 10 µm 10 µm
(a) (b) (c)
10 µm 10 µm
50 µm
(d) (e) (f )
10 µm 10 µm 10 µm
10 µm
(g) (h) (i) (j)
(k) (l) (m) (n)
(o) (p) (q) (r)
FIGURE 63.1 Microscopic and macroscopic imaging of Verticillium species. (a) Conidia of V. dahliae and (b) V. longisporum. (c) Young
developing chlamydosporia of V. tricorpus. (d) A V. dahliae germling; the germinated conidium appears swollen due to osmotic entrance
of water beneath the cell wall, while the newly formed septae are prominent along the young hypha. (e, f) Typical verticillately branched
conidiophores; the abundant hyaline conidiophores are more or less erect, with a varying number of phialides arising at each node and a
darkened or hyaline base, depending on the species. (g, h, and i) Three successive developmental stages of microsclerotia, the characteristic
multicellular resting structures of V. dahliae. They are typically dark brown to blackish, torulose, or botryoidal, consisting of swollen almost
globular cells arising in the centre of the culture after 6–10 days of growth. (j) V. albo-atrum characteristic dark resting mycelium, produced
by this species as thickened and darkened hyphae instead of microsclerotia. (k, l, m, and n) Top and bottom plate views of 3-week-old darkly
pigmented and hyaline V. dahliae cultures respectively. (o and p) Intensity and patterns of pigmentation in Verticillium cultures. (q) Fluffy
mycelium, V. lecanii. (r) Sparse aerial mycelium, V. tenerum (now Acrostalagmus luteo-albus). Photos were taken with the use of a Zeiss
Axio Imager. A1Microscope, with in-build camera, using differential interference contrast. (Courtesy of I.A. Papaioannou.)
63.1.1.2 Morphology the fusion of two different haploid nuclei to form a heterozy-
V. dahliae and V. longisporum form microsclerotia, dense gous diploid nucleus. However, molecular phylogenetic data
aggregates of darkly pigmented, and thick-walled hyphal support its divergence from the parental species, while mor-
cells. Microsclerotia develop by swelling of the hyphae and phological features such as the increased length of conidia
septation of the cells that enlarge and produce lateral cells (Figure 63.1b), the shape of microsclerotia, and the DNA
(Figure 63.1 g through i). Subsequently, melanin is deposited content per nuclei further differentiate the species.9,15,16 The
in the cell walls and intercellular space.14 V. albo-atrum is dif- phytopathogen V. nubilum forms only chlamydospores, while
ferentiated from the above species by the absence of micro- V. tricorpus forms all three types of structures, microsclerotia,
sclerotia and the presence of dark resting mycelium (Figure resting mycelium, and chlamydospores, as well as a copious
63.1j). V. longisporum is considered by some scientists as a yellow orange pigment when grown on plates (Figure 63.1c).
variety of V. dahliae and by several others as a new species.15 The presence of dark hyphae connected to microsclerotia
It has been suggested that it may be a hybrid that originated along with the irregular shape of the latter is characteristic of
from V. dahliae and V. albo-atrum parental species, following V. tricorpus and discriminatory for the species.17

Verticillium 537
From the former members of the genus, V. nigrescens now also become colonized. At this stage, V. albo-atrum can
p roduces brown, intercalary or terminal chlamydospores and produce its conidia on infected plant tissues that may be dis-
presents rather short, hyaline conidiophores while V. theo- persed by air currents and start another cycle of disease.19 In
bromae has long, brown conidiophores and no chlamydo- V. dahliae infection, large amounts of microsclerotia are pro-
spores, but chains of moniloid cells that gradually darken duced, and with the decomposition of plant materials they are
(Table 63.1).11 V. theobromae mainly induces cigar-end released in the soil where they can survive for 10–15 years.
rots of bananas and is widespread only in the tropics, and The survival potential of the melanized, dark, resting myce-
V. luteo-album (now Acrostalagmus luteo-albus) can be eas- lium produced by V. albo-atrum is shorter. According to this
ily distinguished from the species of the sect. Nigrescentia life cycle, Verticillium diseases generally spread through the
by its brick-red colony color. Verticillium sect. Prostrata was use of contaminated equipment, the transfer of contaminated
introduced for species producing prostrate conidiophores soil, irrigation, and the use of infected seed or plant materials
that are often poorly differentiated from a fine, white, or such as rootstocks, bulbs, and tubers. V. albo-atrum, which
yellowish mycelium. A number of species produce dictyo- produces conidia on infected tissues, also spreads through
chlamydospores, but production varies amongst and within air currents. These spores cannot be detached by currents of
species and across a range of culture conditions. Conidia dry air, but are readily removed by a fine spray from an atom-
often aggregate on the tips of phialides in heads, but in a izer. It is probable that in nature wind-blown mist is effec-
few species they adhere in chains. Variation in morphologi- tive in detaching wettable spores from the conidiophores of
cal traits is accompanied by a considerably diverse host range Verticillium species.
for the species of the section and by the fact that some spe- The entomopathogenic fungi of the former V. lecanii
cies are known to produce differentiated erect conidiophores group are of particular interest since they display a com-
(e.g., V. suchlasporum, now Pochonia chlamydosporia). It is pletely different life cycle. They infect insects with their
therefore evident that as morphological boundaries are not conidia, which adhere on the insect and germinate on its sur-
always clear, molecular traits often need to be considered for face to give germ tubes, from which hyphae penetrate into
a final taxonomic assignment. V. lecanii, now placed under the tissues mechanically and enzymatically using an array of
the generic name Lecanicillium, is an important entomo- enzymes like chitinases, lipases, and proteinases. The fun-
pathogen of the sect. Prostrata that is characterized by the gus is dimorphic, and inside the host’s hemolymph (blood)
presence of white fluffy mycelium (Figure 63.1q) and the it grows as single yeast-like cells rather than in the form of
absence of resting structures and can be diversified from its mycelium. This switch in growth form may aid dispersion
close relative fungicolous species V. psalliotae based on the and colonization of the hemocoel, optimize nutrient acquisi-
formation of cylindrical versus falcate conidia.1 tion through the increase of the surface area, and disparate
the efforts of the host cellular immune system.20 Later, as
the host’s tissues are killed, the fungus reverts to the hyphal
63.1.2 Biology, Epidemiology, and Genomics form and grows throughout the softer portions of the cuticle,
covering the insect with a layer of millions of spores that
63.1.2.1 Biology
are dispersed through the air currents, while the insect itself
The formation of resting structures enables Verticillium acts as a successful carrier spreading conidia at other mem-
species to persist in the environment. Melanin appears to bers of its order. This is a critical step, as in the absence of
contribute to long-term survival of resting structures since resting structures the fungus persistence in soil is rather
it protects against parasitism, dehydration, toxic chemicals, limited. Nevertheless, it was shown that when the fungus is
and presumably against UV radiation.18 The life cycle of all used as a biological control agent, it may persist in soil for
Verticillium species is very similar and can be divided into a long periods of time and can infect new insects.21 In the case
dormant, a parasitic, and a saprophytic phase. In the dormant of V. chlamydosporium, a fungus that parasitizes nematode
phase, germination of fungal resting structures present in the cysts and produces dictyochlamydospores, it has been proven
soil is inhibited through mycostasis, as conidia and resting that it can proliferate in soil and survive in considerable num-
structures have no innate or constitutive dormancy. This stage bers for at least 3 months.22 Therefore, when used as a bio-
can be overcome by either increasing the available nutrient or logical control agent, pre-cropping applications of the fungus
decreasing (often diluting) the inhibitory agent. Hyphae that is suggested as it is estimated that the survival is long enough
grow out of the germinating resting structures can traverse a to kill nematode eggs and females that develop on roots of
limited distance, possibly directed by nutrient gradients, to spring-sown crops.
reach potential host plants. After crossing the endodermis of
the root, the fungus enters the vascular tissues where it can
form conidia, a process referred to as budding. The initial 63.1.2.2 Epidemiology
sporulation in the root is thought to account for the coloni- Airborne conidia of Verticillium have been detected at sev-
zation of stem vessels, which results in rapid accumulation eral sites above cultivated fields at various sites, in the atmo-
of fungal biomass a few days later. During tissue necrosis sphere of a London garden, in house dust in Edinburgh, and
or plant senescence, the fungus enters the saprophytic stage. even in sandstorm dust from Saudi Arabia.19,23,24 The iso-
Apart from the vascular tissues, shoots and roots of the plant lates were derived mainly from 7 and >7 μm particles, but

538
TABLE 63.1
Morphological Description of Verticillium Plant Pathogenic Species
V. dahliae V. albo-atrum V. longisporum V. tricorpus V. nubilum V. nigrescens V. theobromae
Myceliuma
Color Black Grayish black Black Golden black Brownish black Brownish black Grayish brown
Hyaline sectors + + + + + − −
Resting mycelium (μm) − 3–7 − 3.5–7 − − 2–3.5
Microsclerotia (μm) 32–135 × 15–45 − 37–150 × 15–45 25–85 × 20–50 − − −
Chlamydospores (μm) − − − 7.5–11 8.5–17 5.5–8 −
Conidia
Shape and color Hyaline, ellipsoidal Hyaline, ellipsoidal Hyaline, ellipsoidal Hyaline, ellipsoidal Hyaline, ellipsoidal Hyaline, ellipsoidal Hyaline, ellipsoidal
to irregular, to irregular, to irregular, to irregular, to irregular, to irregular, to irregular,
subcylindrical, subcylindrical, subcylindrical, subcylindrical, subcylindrical, subcylindrical, subcylindrical,

single-celled, rarely single-celled, single-celled, single-celled single-celled, single-celled, single-celled
one-septate rarely one-septate rarely one-septate occasionally occasionally
one-septate one-septate
Size (μm) 4–6 × 1.8–2 3.5–10.5 × 2–4 7.2–9 × 2–3 3.5–10 × 1.5–3.5 4–10 × 2.5–3.5 4–8.5 × 1.5–2.5 3–8 × 1.5–3
a Description of cultures growing on PDA at 20°C for 2–3 weeks.

Verticillium 539
they could be detected even on particles of 2–4 μm.25 The a fungus for a considerable period. Ultimately, however,
exposure at a waste composting facility was measured to c onditions at any site will become unsuitable for further
be 49 cfu/m3, which is not very elevated, while the average growth. The fungus must then reach other sites suitable for
exposure at domestic sites in the United States was estimated growth, or survive in a dormant state until favorable condi-
to be 314 cfu/m3, which is considered medium to high if it tions return. Spores have two major roles in the life of the
represents only one species.26,27 In a similar study, where fungus, dispersal to a new site and survival until favorable
accurate identification to the species level was performed, conditions return. Many fungi produce more than one kind of
V. lecanii was detected at 67% of the air samples taken dur- spore. It is thus possible, and common, for specialization to
ing harvesting and 35% of the farm workers given skin tests occur, with a single species producing some spore types that
produced positive reactions against the fungus.28 V. lecanii, are efficiently dispersed and others that have a high capacity
now Lecanicillium group, is an entomopathogenic fungus for dormant survival. Sporulation in fungi results in a com-
that is applied in greenhouse crops for the biological con- plexity and diversity of form that contrasts with the relative
trol of aphids and other pests. A wide variety of airborne simplicity and uniformity of structure found among vegeta-
particles including organic vapors of the outdoor environ- tive cells. It is for this reason that taxonomists, concerned
ment, farms, industries, and homes are implicated in rhinitis, with the classification of fungi, have devoted much attention
asthma, allergies, and a host of other upper respiratory tract to spores and sporophores.
infections. Respiratory symptoms and lung function impair-
ment are probably the most widely studied among organic 63.1.2.3 Genomics
dust-associated health effects. Fungi are well-known sources Due to the economic and biotechnological interest of certain
of allergens and also are sources of β-glucan, which causes species of the genus, most of the presently available genetic
nonallergic respiratory symptoms.29 It should be noted that and molecular information concerns two phytopathogens,
thorough experiments conducted to examine the potential namely V. dahliae and V. albo-atrum, and the entomopatho-
risk of workers exposed to fungal biological control agents gen V. lecanii. The two phytopathogenic species have been
used for crop spraying have shown that the doses needed to reported to cause Verticillium wilt to nearly 80 plant gen-
be inhaled in order to cause acute or chronic toxicity, or aller- era, including more than 410 plant species, and serious out-
genicity, ought to be several tens of thousand times higher breaks of the disease with almost complete yield losses in
than those applied in the field (http://www.rebeca-net.de; crops like cauliflower, cotton, and sunflower.34 On the other
http://www.rafbca.com). hand, members of the V. lecanii group have been extensively
Irrespective of older or more recent taxonomy, all exploited as bio-control agents against a number of insects
Verticillium species are mitosporic (i.e., absence of sexual during the past years and are currently available as commer-
reproduction and a known teleomorph), and the only proven cial products.35 Although the taxonomic and population stud-
way by which they can exchange genetic material is through ies on these fungi are constantly increasing, with the advent
the parasexual cycle.30 The cycle requires as its first step of molecular genetics the need for a better understanding of
the fusion of genetically different hyphae (hyphal anasto- the genetic properties of these fungi became more apparent.
mosis) and the formation of a heterokaryon. On rare occa- A first step toward this target was made with the study of
sions, nuclear fusion between haploid nuclei with different the chromosome numbers of various Verticillium species
genotypes may occur and heterozygous diploid nuclei are using pulsed field gel electrophoresis to be followed by the
formed. The diploid nuclei multiply by mitosis in the hetero- analysis of the complete mitochondrial genomes (mtDNAs)
karyon and mitotic crossing-over can take place, obviously of V. lecanii (syn. Lecanicillium muscarium), V. dahliae,
at reduced frequencies (approximately 1/500–1/100) com- and V. albo-atrum, obviously due to the pivotal role of mito-
pared with meiotic recombination.31 Diploid nuclei are very chondria in producing energy in cells, and the relatively
unstable and are finally reduced to the haploid condition, small size, high copy number, and fast evolutionary rates
at a relatively constant frequency of 10 −3 per nuclear divi- of mtDNAs.36 Several differences that could facilitate the
sion. During the progressive loss of chromosomes (a process genetic fingerprinting and discrimination of Verticillium spe-
called “haploidization”), they may go through different stages cies and/or isolates were detected, and these enhanced the
of aneuploidy and the resulting haploids may be of different notion that the “phytopathogenic” branch of Verticillium is
genotypes, depending on the number of genes involved in only distantly related to the entomopathogenic members of
heterozygocity.32,33 However, despite the proven great value the genus.37,38 Finally, in February 2008, the Fungal Genome
of the parasexual cycle in the genetic analysis of Verticillium, Initiative and the Broad Institute released the first sequence
its true significance in nature is questionable due to its rarity assembly for a V. dahliae strain (VdLs.17) genome and only
and the widespread occurrence of vegetative incompatibility, 5 months later, in July 2008, the first annotation of this
a phenomenon that seems to prevent heterokaryosis between genome along with the annotation of the genome of V. albo-
genetically loosely related strains. In laboratory conditions, atrum strain VaMs.102 was publically available (http://www.
the parasexual cycle is imposed by the application of appro- broadinstitute.org/annotation/genome/verticillium_dahliae/
priate selective conditions. Similarly, in nature, a suitable MultiHome.html). The two species were selected to have
substratum or a host may support the vegetative growth of distinct phenotypic variation and very different host range

so that the comparative analysis of their genomic variation weeks before the identification of the fungal infection he
would help expose the molecular mechanisms that underlie had been treated with intraperitoneal broad-spectrum anti-
pathogenicity, differentiation, and host-adapted virulence. biotic therapy for peritonitis caused by mixed enteric flora.
Thus, it was shown that the two genomes have fairly similar The fungus was isolated from the peritoneal fluid culture and
%GC content (V. dahliae: 55.85%, V. albo-atrum: 56.06%), from the tip of catheter and was identified based on macro-
they exhibit extended synteny in their chromosomes, and scopic and microscopic features. The macroscopic examina-
their total genome sizes differ by 1 Mb (V. dahliae: 33.83 Mb, tion of the plate cultures on Sabouraud’s dextrose agar (SDA)
V. albo-atrum: 32.83 Mb) and 314 genes (V. dahliae: 10,535, showed fast-growing white mycelial colonies that acquired a
V. albo-atrum: 10,221). The optimal V. dahliae’s genome yellowish-red color after a while. Microscopic observations
map that was generated with the use of restriction enzymes revealed branched, septate and erect conidiophores, and ver-
and light microscopy was correlated to the in silico restric- ticillate phialides that supported ball-shaped single-celled
tion maps of the genome assembly to produce eight linkage conidia (gloiosporae). Antimycotic therapy with fluconazole
groups. However, since the number of chromosomes in fungi and flucytosine was significantly effective only after the
may vary among isolates of the same species, and previous removal of the catheter and the discontinuance of the CAPD.
studies have indicated extensive chromosome number and/ A later report of Verticillium infection concerned a
or genome size variability in strains of V. dahliae but not 40-year-old male juvenile diabetic with a recent history of
V. albo-atrum, the possibility that other strains of V. dahliae a nephrectomy for renal cell carcinoma followed by radio-
may display different number of chromosomes or even pos- therapy and chemotherapy, factors that made him a highly
sess dispensable chromosomes like those reported in other susceptible candidate for mycotic infections.42 Chemotherapy
phytopathogenic fungi cannot be excluded.16,39,40 Considering along with interferon α-2a was administered to the patient
the ease with which forced heterozygous diploids and mitotic for a suspected pulmonary metastasis at the time that he
recombinants of V. dahliae and V. albo-atrum were produced developed a diffuse subcutaneous nodule at his left arm.
in the laboratory, it is very likely that the genome homology Fine-needle aspiration of the nodule and routine staining of
observed in V. dahliae, V. albo-atrum, and V. longisporum the smears with May-Grünwald-Giemsa and Papanikolaou
can facilitate the formation of hybrids in nature.33 stains scarcely revealed a few septate hyphae in the midst of
an acute inflammatory exudate. It was the inoculation of the
pus on culture plates that showed the growth of white cottony
colonies, while microscopic observation of a slide culture
Species of the genus Verticillium rarely cause infections in revealed elongated conidiophores arranged in whorls, septate
humans, but fungal infections have been increasing in inci- hyphae, and single-celled conidia. Subsequently the fungus
dence over the past decades due to an expanding number of was identified as a Verticillium spp., and fluconazole was
immunosuppressed patients such as those with hematologic given orally. The infection must have progressed through a
malignancies and transplants along with the prolonged use traumatic skin laceration, probably through the injection site
of broad-spectrum antibiotics. Treatment for Verticillium of insulin, and the authors conclude that, had it not diagnosed
infection is not standardized because of the rarity of human early enough, the lesion could have evolved to disseminated
infections and the lack of clinical therapeutic trials. Very few disease.
data are available on in vitro susceptibility tests. Based on the Similarly, a 6-year-old child, diagnosed as a case of acute
limited number of isolates, amphotericin B, ketoconazole, lymphoblastic leukaemia-L1, received chemotherapy and
itraconazole, and voriconazole appear active in vitro against during the second session of induction developed high-grade
Verticillium spp., with ketoconazole and voriconazole being fever.45 The child was initially treated with broad-spectrum
the most prominent solutions.41 Among few case reports ana- antibiotics but did not respond to the treatment. Blood cul-
lyzed below, Verticillium infection has been successfully tures on the 13th and 14th day of induction from two different
treated by systemic fluconazole, intravenous fluconazole and venepuncture sites revealed the growth of a mold after 48 h
subsequent oral flucytosine, topical amphotericin B and sys- of aerobic incubation from routine blood culture bottles. The
temic fluconazole, intravenous amphotericin B, caspofungin mold was morphologically identified to belong to the genus of
combined with oral voriconazole, a therapy that was dis- Verticillium, and since the fungus was also isolated from the
continued twice and replaced by liposomal amphotericin B central line tip, the patient must have contracted the infection
and itraconazole accordingly, and intravitreal and oral vori- through colonized central line. Antifungal treatment in the
conazole for surgical management of chronic postoperative form of amphotericin B was added to the treatment regimen,
fungal endophthalmitis.42–48 However, the optimal antifungal and the patient became afebrile after 48 h.
agent remains unresolved, and successful clinical outcome A case, where Verticillium was initially misidentified,
requires early diagnosis. was that of a 50-year-old male patient with an infiltrate in
One of the early reports of Verticillium infections was that his eye and no history of trauma.44 Clinical features sug-
of a 33-year-old farmer on continuous ambulatory peritoneal gested herpetic keratitis and was treated with antiviral
dialysis (CAPD) for end-stage renal failure due to chronic therapy, but his condition deteriorated. Subsequent micro-
pyelonephritis.43 The patient had been surgically treated in biologic investigation of corneal scraping revealed the pres-
neonatal age for intestinal and renal malformations, and 2 ence of septate hyphae that where morphologically identified

Verticillium 541
to belong to Verticillium spp., and appropriate therapy with corresponding regions of V. dahliae. Therefore, although
amphotericin B and fluconazole was administered. In a case initially recognized as a case report of Verticillium spp.
of postoperative fungal endophthalmitis where Verticillium infection, the taxonomic reform of the genus along with the
was recognized as the causative microorganism, the injec- molecular data acquired from the nuclear ribosomal regions
tion of vancomycin, ceftazidime, and amphotericin B did not of the fungus points to Lecanicillium, the entompathogenic
lead to the regression of inflammatory reaction, possibly due ex member of the genus.
to fungal resistance to amphotericin B.47 The treatment was
subsequently changed to oral and intravitreal voriconazole 63.1.4 Diagnosis
that was combined with vitrectomy with IOL removal and
en bloc capsulectomy which resulted in the resolution of the 63.1.4.1 Conventional Techniques
infection. The accurate identification of the etiological agent The diagnosis of Verticillium is mainly based on the micro-
is crucial as it designates the appropriate course of treatment. scopic observation of morphological features. Clinical
In a report that referred to cases of onychomycoses, the most material can be skin and nail scrapings, urine, sputum,
prevalent nail disease, the implementation of molecular tech- and bronchial washings, cerebrospinal fluid, pleural fluid
niques resolved the problem of fungal identification.49 PCR, and blood, tissue biopsies from various visceral organs, and
sequencing, and restriction fragment length polymorphism indwelling catheter tips. In general, direct microscopy and
(RFLP) of a part of the 28S nuclear rDNA region were car- culturing are performed on all specimens. Microscopy pro-
ried out in order to reach a final conclusion. According to vides vital information, often an immediate presumptive
the report, several cases of Penicillium and Alternaria spp., diagnosis, which is of particular importance in the immu-
which were identified based on morphological features of nosuppressed patient. Microscopy depending on the sample
the isolated fungi, proved later to represent contaminants usually consists of either (a) wet mounts in 10% KOH with
as molecular methods revealed the presence of other fila- Parker ink, or India ink, (b) smears for Gram, Giemsa, and
mentous fungi. Similarly, an isolate originally described as periodic acid-Schiff (PAS) staining, and (c) histopathol-
Fusarium was found to belong to the genus of Verticillium. ogy of tissue sections using hematoxylin and eosin, Gomori
While in the report the identity of the isolate is not specified methenamine silver (GMS), and PAS. Special care should be
to the species level, the sequence, based on which the classi- placed on the selection of appropriate stain as Das et al.42
fication was made, corresponds to Lecanicillium muscarium describe that smears of fine-needle aspirate from the fungal
(former V. lecanii). nodule that were routinely stained with May–Grünwald–
Molecular techniques were also used to identify the infec- Giemsa (MGG) were able to demonstrate very few hyphae
tious agent at a different occasion. A 40-year-old female in the midst of an acute inflammatory exudate. These hyphae
patient with promyelotic leukemia presented multiple could have easily been missed, and the true diffusion of the
hypoechoic hepatosplenic nodules detected by abdominal fungal hyphae was revealed only after GMS, PAS, and Gram
sonography, 57 days after the third course of consolidation staining. However, even though histopathology offers the
chemotherapy.46 Initially, she was treated with oral flucon- most rapid confirmatory diagnosis, it is not always possible
azole for presumed hepatosplenic candidiasis, as the most to obtain suitable biopsy material or to make a specific iden-
common cause of hepatosplenic abscesses in patients with tification of the fungus.
leukemia treated with intensive chemotherapy is Candida This is especially true for hyalohyphomycosis that
spp.50 But fever and right upper quadrant pain developed 20 Verticillium causes, since the presence of hyaline myce-
days later and consequently the therapy was replaced by con- lium can be attributed to a wide variety of causative agents
ventional amphotericin B. Abdominal computed tomography like Acremonium, Beauveria, Fusarium, Geotrichum,
(CT) demonstrated multiple hypodense lesions in liver and Paecilomyces, Penicillium, and Scopulariopsis. Therefore,
spleen that progressed in size despite the applied therapy. culture preparation of the specimen is a prerequisite for
Histopathologic examination of a liver biopsy demonstrated Verticillium identification. The fungus can grow on a wide
granulomatous inflammation, and few broad septate hyphae variety of culturing media such as potato dextrose agar
were detected after Gomori’s methenamine silver staining. (PDA), SDA, and Czapek Dox agar. The addition of inhibi-
However, bacterial and fungal cultures of liver tissue were tory substances that would eliminate contamination from
negative probably due to the previous prolonged exposure of bacterial growth (e.g., chloramphenicol and gentamycin) can
the patient to antifungal reagents. Fungal identification was delay the growth of the fungus, while the presence of cyclo-
achieved after DNA extraction from the paraffin-embedded heximide inhibits it. Generally, it is important to use media
specimen from the liver biopsy, PCR amplification, and with and without inhibitory agents, and specimens from nor-
sequencing of the ITS1, ITS2, and D1–D2 nuclear ribo- mally sterile sites should be preferably inoculated to media
somal regions. The acquired sequence for D1–D2 region without inhibitory substances. Although culture character-
displayed high degree of identity (98.8%) against an ento- istics such as surface texture, topography, and pigmentation
mopathogenic Verticillium strain that is no longer a member are variable (Figure 63.1 k through r) and are therefore the
the genus. This discrepancy was furthermore reflected in the least reliable criteria for identification, they can be proven
low percentages of sequence identity that the ITS1 (33.8%) useful. Colonies of Verticillium are fast to moderate grow-
and ITS2 (49.5%) regions displayed when compared to the ing, white to pale yellow in color at first, becoming pinkish

brown, red, green or yellow with a colorless, yellow, black, the mitochondrial small rRNA gene were able to differenti-
or reddish brown reverse. Some of the Verticillium species ate Verticillium spp. from all other ascomycetes tested on the
can be identified by the production of resting structures (after basis of amplicon size and DNA hybridization patterns when
1–2 weeks of incubation), macroscopically visible as a dark- the amplicon was used as probe.53 Similarly, RAPD analy-
ening of the cultures (Figure 63.1 k–l, o–p). Examination of sis of Verticillium isolates provided clearly discriminatory
darkened cultures under a low power microscope, either in banding patters at the species and subspecies level that could
situ or squashed onto a slide, should reveal the nature of the occasionally be associated with geographic origin and abil-
resting structures (e.g., microsclerotia and dark resting myce- ity to form heterokaryons—vegetative compatibility group,
lium, Figure 63.1i–j). For the rest of the species that do not VCG.54,55 Although RAPDs are technically fast, simple, and
form resting structures, the observation of conidiophores and very sensitive, as they can detect even a single nucleotide sub-
conidia is indicative of their taxonomic status (Table 63.1). stitution in the priming region of the used primers by per-
Conidiophores are usually well differentiated, verticillately mitting or preventing the production of a characteristic band,
branched over most of their length, bearing whorls of slen- they also have some disadvantages. The major drawback is
der awl-shaped divergent phialides. Conidia are hyaline or reproducibility as RAPDs are sensitive to the vagaries of the
brightly colored, mostly one-celled, and are usually borne testing procedure. Additionally, they are very susceptible to
in slimy heads (glioconidia, Figure 63.1a–b). An online aid contamination by nontarget DNA and can only be performed
for the identification to the genus level of Verticillium against reliably on DNA from axenic cultures. Like RAPD markers,
medically important conidial molds is currently available amplified fragment length polymorphism (AFLP) markers
at: http://www.mycology.adelaide.edu.au/Fungal_Jungle/ are amplified with the use of arbitrary sequences, but with
Identification_Keys.html. greater reproducibility and fidelity. AFLP analysis compris-
ing 349 loci showed that the closest relative to V. longisporum
63.1.4.2 Molecular Techniques was V. dahliae.56 Fluorescent AFLP analysis also demon-
The use of morphologic criteria such as colony pigmentation strated that isolates of V. dahliae within a VCG subgroup
during growth on specific media and microscopic features are molecular similar, suggesting that the species is a highly
has been useful for distinguishing Verticillium against other structured, clonal pathogen.57
similar genera. However, using morphologic criteria as the RAPD and AFLP analyses due to their high discrimina-
sole source for fungus identification does not always result tory potential have been mainly focusing on revealing molec-
in safe conclusions, as the phenotypic traits of the genus are ular differences at the subspecies level. An effort to develop
quite uniform and confusing for the untrained eye, and col- a rapid molecular method for the detection and identification
ony pigmentation can vary significantly depending on culture of species that caused vascular wilt to tomato resulted in
conditions (Figure 63.1 k through r). Hence, there is consid- the design of a DNA array.58 Based on the reverse dot blot
erable interest in developing molecular markers/approaches technology, immobilized oligonucleotide detectors hybrid-
that would easily and accurately identify species and strains ized with a labeled PCR product corresponding to the fun-
of this pathogenic fungus. Although molecular markers are gal ITS ribosomal regions were detected in a sample. The
definitive and more stable than phenotypic observations, it hybridization pattern could reveal the presence of colonizing
should be noted that there is no ideal molecular marker for pathogens and subsequently could identify them as members
every organism or every purpose. Thus, some markers may of the species V. albo-atrum, V. dahliae, and Fusarium oxy-
be good for the discrimination of species or higher taxonomic sporum. The advantage of this technique was that it allowed
groups, whereas others are better for distinguishing individ- the simultaneous detection of multiple microorganisms, a
ual isolates or strains. Nevertheless, the irreversible character trait very useful when handling potentially complex samples.
of molecular markers that makes them such a valuable tool The extension of this preliminary effort will be the design
for fungal species identification underlines their validity for of an array that would allow the identification of all fun-
their application in numerous pathogenic conditions. gal species present in a multiplex sample, a task presently
In this direction, most of the studies on molecular markers undertaken for all lower eukaryotes by Dr. G. Anderson
of Verticillium regard the genus in the context of its phyto- (Berkeley University, California) who has already developed
pathogenic and/or entomopathogenic potential and are focus- the PhyloChip based on 16S sequences, capable of detecting
ing on the discrimination of representative species from other 9000 different operational taxonomic units of prokaryotes
microorganisms that share the same ecological habitats and (i.e., taxa, genera, families, orders) in one experiment.
have similar taxonomic characteristics. From several molec- The most successful tools applied so far for species iden-
ular methods tested for species and/or isolate identification tification in Verticillium are the nuclear rRNA gene complex
within the genus, RFLP and random amplified polymorphic repeat and the mitochondrial DNA (mtDNA). Both mole-
DNA (RAPD) analyses are perhaps the most popular probably cules are multicopied and seem to have a complementary role
due to the easiness to perform them in the laboratory. RFLPs in the reconstruction of fungal phylogeny. The rRNA gene
have been used to reveal inter- and intra-species relationships, complex repeat (V. dahliae sequence total length 7216 bp;
indicating that molecular subgrouping of isolates has a loose AF 104926) is undoubtedly the most popular because it con-
association with hosts and geographic origins.51,52 In addition, tains both highly conserved and universally present coding
PCR-RFLP using primers NMS1/NMS2 that amplify part of genes (18S or SSU, 5.8S, and 28S or LSU), as well as variable

Verticillium 543
regions like the internal transcribed spacers ITS1 and ITS2, identification since a single gene does not always faithfully
and the non-transcribed intergenic spacer (IGS) region, all represent the history of the entire genome containing it,
of which can be used for phylogenetic comparisons at many and comparisons may mislead and give wrong conclusions
taxonomic levels. Since the 28S rRNA gene was shown in about the relationship of the fungus with the members of
many other fungi to frequently harbor intron sequences, both the same species or even genus. In the case of Verticillium,
its 5′and 3′ends were used by several research groups as tar- these precautions appear to be very important since it has
gets for species identification in the genus but with moderate been demonstrated that morphological classification alone or
success.59 On the contrary, a unique 839-bp group-I intron in combination with sequence data from one gene led to the
was located in the 18S rRNA gene of all V. longisporum misclassification of isolates of V. albo-atrum strains, which
strains examined and was found inserted after a highly con- later, with additional molecular data, proved to be closely
served insertion position that is common to most ascomy- related to V. fungicola and V. psalliotae, species that are
cetes. Since this intron was absent from the corresponding placed currently under different generic names.64
18S regions of all the other phytopathogenic and entomo- The other molecule that can be used either as an alternative
pathogenic Verticillium species that were also tested in this or a complementary to rRNA gene complexes for the recon-
study, it can be considered as a specific identity marker for struction of fungal phylogeny is mtDNA. mtDNA evolves
V. longisporum.60 5–10 times faster than the nuclear DNA, has a high AT con-
Unlike the conserved 18S and 28S rRNA genes, IGS tent, relative small size, and a rather conserved gene content
region has been proven highly variable for Verticillium and compared to a considerably flexible gene order. Studies on
extremely informative since it permitted isolate grouping at Verticillium have shown that several mitochondrial genes
the subspecies level.59,61 However, due to its large size, the can be used for the identification of different species with
interference of its secondary structures in obtaining good an emphasis given to the NADH dehydrogenase subunit 1
PCR products, and the rarity of corresponding sequences in gene (nad1), the cytochrome oxidase subunit III gene (cox3),
data banks, it is less used than the other parts of the rRNA and the cytochrome b (cob) gene.8,9 However, none of these
repeat. Thus, the most popular locus for DNA-based myco- groups have evaluated so far the discriminatory value of the
logical studies at the subgeneric level, and hence for species mitochondrial cytochrome C oxidase subunit I (cox1), a gene
identification, remains the ITS region. This roughly 550 bp that consists of a newly developed molecular marker for fun-
segment combines the advantage of resolution at various gal taxonomy and identification.65 The complete mitochon-
scales (ITS1: rapidly evolving, 5.8S: very conserved, ITS2: drial genome of V. dahliae revealed a gene order that seems
moderately rapid to rapid), with the ease of amplification to be common amongst the phytopathogens of the genus but
of a multicopy region into a readily obtainable product. is not shared by the clade of Prostrata, e.g., V. lecannii (syn.
The large number of fungi for which ITS sequences have Lecanicillium muscarium).37,38 In this context, the amplifi-
been generated in data banks further increase the useful- cation of the intergenic region nad3-nad1 was characteris-
ness of this region for purposes of comparison. Using the tic of the Verticillium phytopathogens only, while amlpicon
basic local alignment search tool (BLAST), it is now easy size alone could differentiate V. nigrescens isolates from
to compare sequences of any unidentified fungus with those all other species.37,38 A second PCR product correspond-
deposited previously in GenBank and predict or identify its ing to the nad1-nad4 intergenic region, which was present
species. However, it is always important to bear in mind in all Verticillium species (Prostrata and Nigrescentia), was
the fact that some of ITS sequences in GenBank have been capable to resolve all discrepancies and identify strains at the
deposited without a strict quality control as far as species species level. Finally, a specific for V. longisporum primer,
identification is concerned, and consequently there are ITS VlonspF combined with Vdnad4R could clearly and unam-
sequences that have been deposited under erroneous taxo- biguously discriminate V. longisporum strains from V. dahl-
nomic names. iae and V. albo-atrum (Table 63.2).38
The usefulness of ITS for the identification of Verticillium Apart from the efforts for molecular identification and
species has been demonstrated in several population studies differentiation of Verticillium species, some work has also
with the fungus.2,8–9 However, several parameters must be been done on the molecular quantification of the presence
taken into consideration before using it as a putative fungal of these fungi on different plants. This was achieved with
barcode. First of all, a large number of co-specific specimens the use of real-time PCR technique (RT-PCR or qPCR), a
from as many populations and geographical regions as pos- method by which the quantity of V. dahliae and V. albo-
sible, as well as the erection of a tailored, closed-submission atrum DNA was measured in the stems and roots of resistant
database for the purpose, will be required.62 Secondly, the and susceptible olive tree genotypes, potato cultivars, and
intra-specific and often intra-isolate variations that have been alfalfa plants in order to monitor pathogen colonization and
recorded in several fungal groups indicate that heterogeneity estimate the resistance and tolerance to Verticillium wilt.66,67
among rRNA repeats because of its multicopy nature creates Although most of the current studies are focusing on the
complications for direct sequencing of ITS-PCR products quantification of the fungus in its natural inhabitant, which is
and makes it difficult to accurately define taxonomic groups in plant specimens for the phytopathogens, it is assumed that
at the species level.63 Finally, it should be stressed that no only minor adjustments will be necessary in order to achieve
single gene can be used as the “gold standard” of fungal similar results in routine clinical specimens.

TABLE 63.2
Proposed Primer Pairs for the Identification of Verticillium Species
Annealing
Primer Pair Region Sequences 5′–3′ Temperature (°C) Amplicon Size (Species) References
18S-ITS1/28S- ITS1-5.8S- GTCCCTGCCCTTTGTA/ 50 710 bp (V. dahliae) [9]
ITS2 ITS2 CCTGGTGGTTTCTTTTCC 750 bp (V. lecanii)
RPB1-Af/RPB1-Cr rpb1 GARTGYCCDGGDCAYTTYGG/ 45 755 bp (V. dahliae) http://faculty.
CCNGCDATNTCRTTRTCCATRTA washington.
edu/benhall
NMS1/NMS2 rns CAGCAGTGAGGAATATTGGTCAATG/ 45 458 bp (Verticillium spp.) [53]
GCGGATCATCGAATTAAATAACAT
18SVDF/18SVDR 18S GCGAAACTGCGAATGGCT/ 60 1.65 kb (V. dahliae) [60]
GTAATGATCCCTCCGCTG 2.5 kb (V. longisporum)
VDAL6/VDAL7 IGS GCTGTGCGTTGCAAGGCA/ 63 1.6 kb (V. dahliae) [59]
GAGCCATTCGCAGTTTCG
cox3A/cox3B cox3 TGATTTAGAGATSTAATATCAGAAG/ 48 363 bp (Verticillium spp.) [9]
CCGTGGAAACCTGTSCCAAAATA
nad1A/nad1B nad1 ATGGCSAGTATGCAAAGAAGA/ 50 479 bp (Verticillium spp.) [37]
GCATGTTCTGTCATAAASCCACTAAC
VlonspF/Vdnad4R nad1-nad4 AACAATTAATTTATAATAAAGTTA/ 48 500 bp (V. longisporum only) [38]
intergenic TAAAATTTTAAATTTTCTTTTATTG
SVert1513for/ GGTGACGCAGCAGACGAGCGGGACCGTG/ 72 1513 bp (V. albo-atrum only) [67]
SVert1513rev GCACTTATAGTTGTTACCCCTGCGTCACC
DB19/DB22 CGGTGACATAATACTGAGAG/ 54 539 or 523 bp (V. dahliae [57]
GACGATGCGGATTGAACGAA only)
Nad3a/atp9R nad3-atp9 ATTTGAATGTGGTTTTCAT/ 45 450 bp (L. muscarium)a [69]
intergenic GAGAATAATTGGTTTTTTAATG 600 bp (L. lomgisporum)a
a Former V. lecanii species.
63.2 Methods disruption of cell walls with beads produced the highest
yields with Aspergillus hyphae, a model filamentous fungal
63.2.1 Sample Preparation pathogen.68 At present, there are no publications to evaluate
The choice of the clinical specimen to be analyzed and diag- Verticillium DNA extraction from different clinical speci-
nose an invasive infection is a key question in medical micro- mens with different methods, but the authors presume that
biology. For diagnosis of an invasive fungal infection, the ideal all methods suitable for Fusarium are also applicable to
material must be easily assessable and free of fungal DNA in Verticillium, since the cell wall and life cycle of the two fungi
a healthy individual so that the distinction between invasive are rather similar. In the two reports where molecular evalu-
infection and colonization can be made easily. Moreover, the ation of the presence of Verticillium was carried out, DNA
clinical material should be taken from a body site affected by extraction was performed in one occasion from paraffin-
the infection. Blood should be collected using the appropriate embedded tissue specimen without stating the protocol that
anticoagulants as heparin is a potential inhibitor for the DNA was followed and in the other occasion from nail scrapings
polymerase, and tissue samples treated with formalin should and fragment after nail dissolvement and with the use of a
be washed thoroughly with sterile saline as formalin is also commercially available kit.46,49
an inhibitor of DNA polymerase. In general, DNA extraction The precautions and preconditions, which are encoun-
should aim to reduce the amount of the extracted human DNA tered due to the complexity and variety of clinical specimens
and at the same time to minimize the losses of the target fun- that are directly processed, can be overcome when fungal
gal DNA, should also eliminate potential DNA polymerase DNA isolation is performed from starting material grown on
inhibitors and avoid contamination with airborne spores. plate/liquid cultures used for the identification of the fungus.
For filamentous fungi and in consequence for Verticillium, Even though this method is time consuming since it requires
an extra step for cell wall disruption is necessary. Since the an extra step for the growth of the fungus on artificial
cell wall of Verticillium is somewhat resistant to enzymatic medium, it is less prone to produce false-negative results due
treatment approaches (e.g., zymolyase), mechanical disrup- to extraction difficulties. According to the proposed proto-
tion is recommended. In a comparison of six DNA extraction col, 100 mg of fungal scrapings is collected from the culture
methods for the recovery of fungal DNA from bronchoal- of Verticillium on an agar plate and placed in an Eppendorf
veolar lavage (BAL), those employing mechanical agitation/ tube. After an overnight incubation at −20°C, 500 μL of the

Verticillium 545
extraction buffer (200 mM Tris–HCl pH 8.5, 250 mM NaCl, gene, the translation elongation factor tef1, and the ribosomal
25 mM EDTA, 0.5% SDS) is added to the freeze mycelium polymerase B2 rpb2 for molecular markers, and as data on
and lysis is achieved through the use of a micropestle. A cen- different genomic loci accumulate, the regions that are used as
trifugation step of 30 min at maximum speed (14,000 rpm) fungal barcodes maybe altered.
is carried out in order to discard the cellular debris. The For the amplification of the ITS1-5.8S-ITS2 region
supernatant is quickly transferred to a new Eppendorf tube (~450 bp), the suggested primers (Table 63.2) can be used and
containing 20 μL of RNAse A (20 mg/mL), and the mixture the produced amplicons will comprise, except from the entire
is incubated at 37°C for 30 min. The solution is extracted at ITS1-5.8S-ITS2, partial sequences of the flanking 18S and
least twice with one volume of phenol:chloroform:isoamyl 28S regions as well. Amplification reaction should be per-
alcohol (25:24:1) and centrifuged for 10 min at maximum formed in a total volume of 50 μL, so that adequate quantity is
speed. DNA is precipitated with the addition to the aqueous produced for subsequent sequencing reactions. When tested
phase of 0.54 volumes (250 μL) of isopropanol and the incu- in our laboratory, each reaction included 5 μL reaction buf-
bation of the mixture at −20°C for at least 1 h. Following a fer (10 mM Tris–HCl pH 9.0 at 25°C, 1.5 mM MgCl2, 50 mM
centrifugation step of 15 min at maximum speed, the pellet is KCl, 0.1% Triton X-100), 100 μM of each dNTP, 40 pmol
rinsed with 70% ethanol and centrifuged once again at maxi- of each primer, 2 U of Thermophilic Taq DNA polymerase
mum speed for 15 min. The supernatant is discarded with (5.0 U/μL; Promega), 50 ng of template DNA, and sterile
extra care to remove all residual ethanol, without disturbing ultra pure water. Amplification is carried out in a PTC-200
the pellet. The pellet is air dried and resuspended in 100 μL Gradient Peltier Thermal Cycler (MJ Research) programmed
TE-buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0) or as follows: initial denaturation at 94°C for 3 min; 35 cycles of
sterile distilled water. DNA quality can be checked visually denaturation at 94°C for 1 min; annealing at 50°C for 1 min;
after electrophoresis on a 0.7% agarose gel in 1× TAE buffer extension at 74°C for 2 min; and final extension at 74°C for
(Trisacetate-EDTA), while DNA quantity can be estimated 5 min. PCR products are analyzed on a 0.7% agarose gel in
with a spectrophotometer by measuring the optical density 1× TAE buffer. A 1 kb DNA ladder (Invitrogen, Paisley) is
at 260 nm. included as DNA size marker, and clear single bands cor-
responding to an approximate size of 700 bp are purified as
63.2.2 Detection Procedures mentioned below for the subsequent sequencing reactions.
For the use of all other primer pairs, the annealing tempera-
For the detection of Verticillium, PCR amplification and ture should be adjusted accordingly.
sequencing of at least two independent gene regions is proposed. Sequencing reactions can be carried out in a variety of
The first should be the traditionally examined ITS1-5.8S-ITS2 available automated platforms according to the manufactur-
region which will provide a generic sequence that can be com- er’s instructions, but all PCR products prior to sequencing
pared with all corresponding sequences in databases and place should be purified from unbound dNTPs. This purification
the microorganism under inspection within, and rarely close step can be performed with various commercially available
to, the genus Verticillium. As a rule, the nucleotide sequence spin columns (e.g., QIAquick PCR Purification Kit, Qiagen,
obtained should group V. dahliae, V. albo-atrum, and V. lon- Nucleospin Extract, Macherey-Nagel). Sequencing of the
gisporum together and clearly separate them from all the other PCR products must be carried out in both directions, and
sensu stricto Verticillium and former V. lecanii members. As DNA sequences from these reactions must be aligned in order
a second molecule, and depending on the accuracy that a sci- to remove the regions corresponding to the primers used at
entist wants to achieve, any of the primers proposed in Table the amplification step and clarify all discrepancies between
63.2 can give additional information for genus, species, or them. The consensus sequence can subsequently be tested for
even subspecies identification. For example, the nuclear ribo- DNA similarity with BLAST against other available fungal
somal 18S subunit and the mitochondrial small ribosomal rns sequences deposited in GenBank. In the case of ITS or rpb1,
genes due to their relatively conserved character facilitate the other databases especially dedicated to the purpose of fungal
recognition of taxa at the level of genus. Similarly, the nuclear species identification can be used for sequence comparison
rpb1 and the mitochondrial nad1 and cox3 genes, alone or in like the one publically offered by the Assembling the Fungal
combination with the above gene sequences, are capable of Tree of Life (AFTOL) organization. According to the hits
distinguishing between closely related species. On the other recorded from this search, a positive identification of the fun-
hand, the IGS and the mitochondrial intergenic regions that gus can be achieved, sometimes to the species level.
are known to display an increased heterogeneity, provide
sequences that enable us to identify strains/isolates of a par- 63.3 C
ticular species. In addition, when searching specifically for
Perspectives
one of the species V. dahliae, V. albo-atrum, and V. longis-
porum, the use of the species-specific primers described in The ability of fungi to cause human disease appears to be an
Table 63.2 permits the immediate screening of the sample for accidental phenomenon. With the exception of a few derma-
their presence through a single PCR reaction and without the tophytes, pathogenicity among the fungi is not necessary for
necessity of the extra sequencing step. These propositions do the maintenance or dissemination of the species. In general,
not disqualify the use of different genes, such as the β-tubulin the development of human mycoses is related primarily to the

immunological status of the host and environmental exposure, 3. Zare, R. and Gams, W., A revision of Verticillium section
rather than to the infecting organism. Verticillium is an “oppor- Prostrata. IV. The genera Lecanicillium and Simplicillium
tunistic” fungus which causes infections almost exclusively in gen. nov., Nova Hedwigia, 73, 1, 2001.
4. Zare, R. and Gams, W., A revision of Verticillium section
debilitated patients whose normal defense mechanisms are
Prostrata. VI. The genus Haptocillium, Nova Hedwigia, 73,
impaired. Diagnosing infections remains a problem in the 271, 2001.
management of fungal diseases, particularly in the immuno- 5. Zare, R., Gams, W., and Evans, H.C., A revision of Verticillium
compromised host. Signs and symptoms are not specific, colo- section Prostrata. V. The genus Pochonia, with notes on
nization is difficult to distinguish from invasive disease, blood Rotiferophthora, Nova Hedwigia, 73, 51, 2001.
cultures are commonly negative, and patients are often unable 6. Sung, G. H. et al., A revision of Verticillium sect. Prostrata.
to undergo invasive diagnostic procedures. In order to improve II. Phylogenetic analyses of SSU and LSU nuclear rDNA
the survival for these infections, an early diagnosis is required. sequences from anamorphs and teleomorphs of the
Clavicipitaceae, Nova Hedwigia, 72, 311, 2001.
Conventional microbiological, histological, and radiologi- 7. Zare, R. and Gams, W., A monograph of Verticillium section
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Saccharomycotina and Taphrinomycotina

64 Candida
P. Lewis White, Michael D. Perry, and Rosemary A. Barnes
Contents
64.1 Introduction.......................................................................................................................................................................551
64.1.1 Taxonomy, Morphology, Biology, and Genomics..................................................................................................551
64.1.1.1 Taxonomy................................................................................................................................................551
64.1.1.2 Morphology, Biology, and Genomics.................................................................................................... 552
64.1.2 Disease Incidence, Mortality, and Epidemiology................................................................................................. 554
64.1.4 Diagnosis.............................................................................................................................................................. 558
64.2 Methods............................................................................................................................................................................ 563
64.2.1.1 DNA Extraction from Candida Cultures............................................................................................... 563
64.2.1.2 DNA Extraction from Clinical Specimens............................................................................................ 563
64.3 Future Perspectives and Conclusions............................................................................................................................... 564
References.................................................................................................................................................................................. 565
64.1 Introduction is estimated at 10%–49% for invasive disease. The cost to

health care administrators is substantial, and in the United
Candida remains the most common cause of fungal disease States it has been estimated at $1.7 billion per annum.3
worldwide with infections ranging from superficial candido- Molecular diagnostics have the potential to overcome the
sis of mucosal membranes requiring “over-the-counter” phar- limitations of conventional practice. Many of the published
maceutical intervention to life-threatening invasive disease protocols provide excellent analytical performance with
necessitating extensive medical, even surgical treatment. An some clinical validity but suffer from a lack of standardiza-
improved awareness of invasive disease and an increasing sus- tion with limited evaluation of clinical utility. Most current
ceptible population are countered by the availability of effec- Candida specific assays have excellent sensitivity and nega-
tive prophylaxis and limited techniques for accurate diagnosis. tive predictive value, making them a useful screening tool
The incidence of disease is, at best, described as steady but but diagnostic utility is affected by the prevalence of dis-
varies geographically and is influenced by general hospital ease and the impact of antifungal prophylaxis. Assays solely
practice. Indeed, comparison of incidence between countries targeting Candida cannot exclude diseases caused by other
is difficult. For countries without surveillance programs or yeasts or molds, and a diagnostic role reliant on assay speci-
the capacity to diagnose the disease using conventional tech- ficity to confirm candidal disease may be preferred. The use
niques, the incidence may be greatly underestimated. of molecular diagnostics designed to test for invasive disease
Early diagnosis of invasive disease can improve mortal- could be applied to superficial disease, although it is hard to
ity rates,1 but conventional microbiology techniques can be justify this in health economic terms and it cannot distin-
slow, lacking both sensitivity and specificity. Many cases are guish between disease and colonization.
treated empirically or treated preemptively on the basis of the The chapter will cover the topic of Candida and candido-
presence of risk factors. Unfortunately, these risk factors are sis focusing mainly on invasive disease although many of the
common amongst critically ill patients within the intensive aspects will apply to superficial disease.
care setting. Unless additional clinical interpretation guide-
lines are applied, both empirical and preemptive strategies 64.1.1 Taxonomy, Morphology, Biology,
may result in unnecessary prescribing of antifungal therapy at
and Genomics
great expense and drug-related toxicity. Crude mortality rates
for invasive disease range from 20% to 75% depending on 64.1.1.1 Taxonomy
patient population, type of disease, and the species involved.2 The genus Candida comprises approximately 200 spe-
Attributable mortality has not been accurately assessed but cies although only approximately 10% of species have
551

been associated with human disease.2 Candida species are Budding may be multilateral but growth of yeast cells is usu-
eukaryotic organisms of the Kingdom Fungi belonging to ally polarized and directed by polarisomes. The formation of
the phylum Ascomycota, subphylum Saccharomycotina, chains of connected but separated buds is not uncommon.7
class Saccharomycetes, and order Saccharomycetales and Candida species prefer aerobic growth conditions and
are members of the mitosporic Saccharomycetales family. although they will grow in the presence of elevated CO2,
They are opportunistic human pathogens and commensals strict anaerobic conditions may inhibit growth.11 Growth
of mucosal membranes and skin with most infections arising ranges are typically between 20°C and 37°C and the pH
from an endogenous source.4 Changes within the host allow range 2.5–7.5. Growth at 37°C is a prerequisite for patho-
the organism to cause opportunistic infection although the genic strains and, although it appears that 37°C is the optimal
yeast is not entirely passive during the infective process and growth temperature, many of the more pathogenic species
possesses various virulence and immunomodulatory factors.5 can grow at 40°C (Table 64.1).6,12 Growth rates vary depend-
ing on conditions with optimal doubling times of less than
64.1.1.2 Morphology, Biology, and Genomics 1 h for C. albicans.11 Growth below 33°C leads to individual
The unicellular organism survives by obtaining nutrients yeast cell formation via polar budding, whereas growth at
from both living and dead organic matter. Most Candida higher temperatures in the presence of inducers and nutrients
species are described as dimorphic, but are better described at neutral pH usually confers germ-tube formation leading to
pleiomorphic, producing true budding round, oval, ellip- hyphal structures.11
soid or elongate yeast cells (blastoconidia), elongated, con- Another interesting concept of candidal growth is the for-
nected yet separated pseudohyphae, true hyphal structures, mation of biofilms, often associated with the use of medical
chlamydospores, and opaque mating cells. Cellular structure devices such as venous catheters or mechanical ventilators.
depends on environmental conditions and transition between Through the upregulation of adherence factors, the yeast
morphological states is rapid in response to environmental adheres to the surface of the device or other cells and forms
pressure. The ability to change between hyphal and yeast a basal layer from which hyphal forms propagate to form an
forms may play an important role in virulence through tissue upper layer that secretes an extracellular matrix.13 Biofilms
invasion, colonization/dissemination, and biofilm formation. are particularly resistant to antifungal therapy postulated to
One species C. glabrata rarely produces pseudohyphae or be a result of limited drug penetration, although it has been
hyphal structures and was previously classified in the genus shown that drug diffusion through candidal biofilms is not
Torulopsis. It is still debated whether it should be classified limited and over production of drug efflux pumps may have
in the genus Candida. an important role, particularly in early biofilm production.13
Colonial appearance is characteristically creamy/white It is likely that biofilm resistance is a composite response reli-
and noted by the absence of pigmentation. Colonies are gen- ant on more than efflux pumps and removal of the infected
erally smooth and wet looking although under certain growth implanted device is recommended.13
conditions, a fringed border representing pseudohyphae or When exposed to unfavorable environmental conditions,
hyphal growth can be seen. Microscopically, C. albicans C. ablicans forms chlamydospores. These large, thick-walled
blastoconidia are typically 2–7 × 3–8 μm in size but dimen- cells are high in lipid and carbohydrate content, are three- to
sions vary between species. C. glabrata produces smaller fourfold the size of typical blastospores and form at the end
balstoconidia (3.4 × 2.0 μm). C. kefyr, C. krusei, and C. lipo- of branched filamentous suspensor cells when oxygen, light,
lytica may produce elongated blastoconidia up to 15 μm in temperature, and nutrients are limited.7,8 It is proposed that
length.6 Table 64.1 summarizes the phenotypic characteris- these stable structures are long-term resting cells although
tics of eight common species of pathogenic Candida. extended viability compared to active states has not been
C. albicans possesses the ability to reversibly switch col- demonstrated.7 Chlamydospores are not thought to have a
ony surface appearance from creamy/white to opaque. The role in pathogenicity.8
“white cells” form the distinctive smooth creamy domed As the candidal cell wall is in primary contact with the
colonies consisting of budding yeasts whereas the “opaque surrounding environment, it is critical to cell survival irre-
cells” have pitted cell walls resulting in flatter colonies of spective of pathogenicity or commensalism. Initially, the
grey appearance consisting of rough elongate cells possibly candidal cell wall was perceived to have a purely inert role
with mating projections.7 providing structural rigidity and cellular protection.5 This
The “opaque cells” are the mating-competent form of simplistic view has been replaced with the widely accepted
C. albicans.8 A stimulus is not always necessary for C. albi- role of a dynamic structure responding to changing envi-
cans cells to switch but pH, oxygen concentration, low inten- ronmental pressures and serving dual function; defensively
sity treatment with UV, mating pheromones, and a limitation as an anatomical barrier, and by incorporating molecules
of zinc in growth medium affect the frequency of “phenotypic (mainly proteins) essential for interaction with the envi-
switching,” which has been proposed to enhance pathogen- ronment including virulence factors, it has the potential
esis of the organism.7–10 Despite the ability to mate sexually to act in a pathogenic manner. It is comprised mainly of
candidal reproduction is almost entirely asexual by budding, carbohydrate (80%–90%), with limited protein and lipid,
as most of the cells are not homozygous at the “cell type con- forming a complex multilayered barrier of mannoprotein
trolling” MTL locus as the region is highly polymorphic.7 (30%–40%), β-glucan (50%–60%) and chitin (0.6%–3%).5,14

Candida 553
TABLE 64.1
Morphological and Physiological Characteristics of the Most Common Candida Species
Species
C. C. C. C.
Attribute albicans C. glabrata C. tropicalis parapsilosisa C. kefyr dubliniensis C. krusei guilliermondii
Teleomorph Not known Not known Not known Not known Kluyveromyces Not known Issatchenkia Pichia
marxianus orientalis guilliermondii
Colony color White/ White/ White/cream White/cream White/cream White/cream White/ White/cream
cream cream cream/gray
Colony Smooth/ Smooth/ Smooth/wet Smooth/wet Smooth/ dull Smooth/wet Smooth/flat/ Smooth/wetc
appearance wetb wet (dull/ rough) or dull/rough dull
Blastoconidia Sub/ Round to Sub/spherical Spherical to Elongated or Sub/ Elongated or Sub/spherical
shape spherical ellipsoid ovoid ovoid spherical ovoid
Blastoconidia size 2–7 × 3–8 3.4 × 2.0 3–5.5 × 4–9 2–3.5 × 3–4.5 3–6.5 × 5.5–16 2–7 × 3–8 2–5.5 × 4–15 2–4 × 3–6.5
(μm)
Growth at 37°C + + + + + + + Variable
Germ-tube + − − − − + − −
Fermentation
Glucose + + + + + + + +
Galactose Variable − + Variable +/slow Variable − Variable
Sucrose − (slow) − Variable −/slow + − − +
Maltose + − + −/slow − + − −
Lactose − − − − Variable − − −
Trehalose Variable Variable +/slow −/slow − Variable − +
Assimilation
Glucose + + + + + + + +
Galactose + − + + Slow + − +
l-Sorbose Variable − Variable +/slow Variable − − Variable
Sucrose Variable − Variable + + + − +
Maltose + − + + − + − +
Cellobiose − − +/slow − Variable − − Variable
Trehalose + (slow) − + + −/weak + − +
Lactose − − − − Variable − − −
Melibiose − − − − − Variable − Variable
Raffinose − − − − + − − +
Melezitose Variable − Variable + − + − Variable
Soluble starch + − + − − + − −
d-Xylose + − + + Slow Variable − +
l-Arabinose Variable − − + Variable − − Variable
d-Arabinose Variable − − − − − − Variable
d-Ribose − (slow) − − (slow) Variable Variable − − +
l-Rhamnose − − − − − − − Variable
d-Glucosamine Variable − Variable Variable − − + +
NAD-glucosamine + − + + − + + +
Glycerol Variable +/slow Variable + Slow +/slow + +
Erythritol − − − − − − − −
Ribitol Variable − +/slow +/slow Slow +/slow − +
Galactitol − − − − − − − Variable
d-Mannitol + − + + Variable + − Variable
d-Glucitol − (slow) − + + Variable + − Variable
αMD-glucoside Variable − Variable + − +/slow − Variable
d-Gluconate − (slow) − Variable +/slow − −/slow − Variable
dl-Lactate + − Variable − + + + Variable
Myo-inositol + − − − − − − −
(continued)


Morphological and Physiological Characteristics of the Most Common Candida Species
Species
C. C. C. C.
Attribute albicans C. glabrata C. tropicalis parapsilosisa C. kefyr dubliniensis C. krusei guililermondii
2KD-gluconate + Variable + + − + − +
d-Glucuronate − − − − − − − −
Nitrate − − − − − − − −
Urease − − − − − − − −
0.1% + − + − + + Variable Variable
Cyclohexamide
Source: Data summarized from Ellis, D. et al., Description of Medical Fungi, 2nd edn., Nexus Print Solutions, Adelaide, Australia, 2007.
Key: (+), Positive; (−), Negative; Variable: Result is strain dependent; Slow: May be slow in developing a positive result; +/slow: Positive, although some
strains may be slow in developing a positive result; + (slow): Positive, although occasional strains may be slow in developing a positive result; −/slow:
Negative, although some strains may be slow in developing a positive result; − (slow): Negative, although occasional strains may be slow in developing
a positive result; −/weak: Negative, although some strains may develop a weak positive result.
a Candida parapsilosis complex contains three species (C. parapsilosis, C. orthopsilosis, and C. metapsilosis) that are phenotypically indistinguishable.
b Note the possibility of the opaque mating cells that may be dull and rough and the possibility of a rough margin due to pseudohyphal or hyphal growth.
c Occasionally dull and rough.
β-Glucan/chitin are essential structural polysaccharides secreted lipase genes, there appear to be genes permit-
and form the rigid framework to which mannoproteins are ting increased oxidative metabolism/respiration and genes
linked, providing the cell surface characteristics.14 Overall, responsible for morphological restructuring, environmental
cell-wall composition is similar regardless of the candidal monitoring, and response.15
cell structure (yeast, pseudohyphae, or hyphal), although the Five hundred and sixty seven genes have been deemed
amount of chitin in hyphal structures is threefold that of its essential with an estimated 1400 genes necessary for
yeast counterpart.5 growth.18 The essential rDNA genes have shown varying
The complete genome sequence of C. albicans was pub- copy numbers between both strains and species with some
lished in 2004.15 C. albicans possesses a diploid genome strains of C. albicans possessing over 200 copies, making
spanning 14,851 kb comprising eight pairs of chromosomes them ideal targets for molecular detection.20 Comparison of
(R, 1–7), of which chromosomes R and 1 are the larg- the genomes of C. albicans and its closest relative C. dublini-
est (>3000 kb) and chromosomes 6 and 7 are the smallest ensis revealed 168 species-specific genes possibly indicative
(≅1000 kb).15 Significant natural heterozygosity is demon- of recent evolutionary divergence and provides a useful model
strated between strains such that no two homologous chro- for investigating the increased virulence of C. albicans.21
mosomes are identical in DNA sequence.15 Heterozygosity
is not distributed evenly throughout the genome and is most 64.1.2 Disease Incidence, Mortality,
evident on chromosomes 5 and 6.15 There is no known hap-
and Epidemiology
loid state and attempts to confirm meiosis have yet to be suc-
cessful, a major hurdle to completing the sexual cycle.15 It has The true burden of candidal disease is not possible to quan-
been proposed that mating can occur by combining two dip- tify as surveillance of superficial diseases such as genital
loid genomes to produce tetraploid organisms that randomly candidosis (thrush) is not performed routinely. However, the
lose chromosomes to become diploid with indiscriminate serious nature of invasive disease has led to the development
recombination of the eight chromosomes.16 The genome con- of fungal registries in developed countries although these are
tains more than 6000 open reading frames (ORF) possibly often incomplete and confined to documentation of candi-
coding for large proteins (>100 amino acids).15 Comparison demia only. Pfaller and Diekema in their extensive review
of the C. albicans genome with that of Saccharomyces cere- of Candida epidemiology predict nosocomial infection
visiae, Schizosaccharomyces pombe, and the human genome rates of between 2.5% and 10% resulting in 7,000–28,000
revealed that almost 50% of the ORF found matches in all cases of candidemia and 2,800–11,200 deaths in the United
three genomes and approximately 15% had further matches States per annum.2 In the United states, candidemias are the
in both the yeast genomes.15 Genes specific to C. albicans fourth leading cause of nosocomial bloodstream infection
could explain its heightened pathogenicity compared to with a median incidence rate for all types of invasive candidal
S. cerevisiae.15,17–19 In addition to large gene families related disease of 23.5 cases per 100,000 population over the years
to pathogenesis including agglutinin like sequence genes 1996–2003.2 Outside of the United States, incidence rates
(ALS), iron transport, secreted aspartyl proteinase, and are much lower and probably reflect differences in patient

Candida 555
TABLE 64.2
Incidence of Invasive Candidal Disease
Country Incidence Rate/105
[Reference] Range Population Patient Group No. Sites Most Common Agent
Norway [91] 1991–2003 1.9–3.3 All 22 C. albicans (70%)
United Statesa [23] 1992–1993 8 All 87 C. albicans (53%)
United Statesb [22] 1998–2000 10 All 47 C. albicans (45%)
United Statesc [92] 1998–2001 6.0 All 17 C. albicans (57%)
United Statesd [2] 1996–2003 22–29 All — C. albicans
Iceland [93] 1980–1999 1.4–4.9 All 16 C. albicans (64.6%)
Iceland [94] 1991–2006 3.7–5.8 All 16 C. albicans (54.2%–70.6%)
Finland [95] 1995–1999 1.9 All — C. albicans (70%)
Canada [96] 1999–2004 2.8 All 4 C. albicans (51.2%)
Spaine [29] 1997–1999 3.5 All 19 C. albicans (Approx. 44%)
Spain [97] 2002–2003 4.3 All 14 C. albicans (51%)
Denmark [98] 2003–2004 11.0 All 34 C. albicans (63%)
Denmark [99] 2004–2006 10.4 All 41 C. albicans (53.8%–66.1%)
U.K.f [100] 2002–2005 2.3–3.1 All — C. albicans (51.2%–55.1%)
Scotland [101] 2005–2006 4.8 All — C. albicans (50%)
England [102] 2006 3.3 All — C. albicans (53%)
a San-Francisco and Atlanta.

b Connecticut State and Baltimore.
c Iowa State.
d The U.S. National Hospital Discharge Survey results.
e ECMM Survey.
f Excludes Scotland.
demographics and medical practice, particularly those within has been superseded by invasive mold infections and the
the intensive care setting (Table 64.2). In recent years, the distribution of species causing invasive candidal disease is
global incidence of disease is best described as steady but changing.2,24 Generally, five species of Candida (C. albi-
varies with geographical location, patient age, ethnicity, cans, C. glabrata, C. krusei, C. parapsilosis, and C. tropi-
hospital practice, and underlying disorder. The incidence is calis) cause more than 90% of all candidemias (Table 64.3).
greatest at the extremes of the age spectrum with the very Candida albicans remains the most common cause of both
young (<1 year old) and elderly (>65 years old) more prone to superficial and invasive candidal disease although global
disease.2 An association with ethnicity has also been estab- data from the ARTEMIS DISK Surveillance program
lished in two U.S. studies where the incidence of disease was showed a 10% reduction in the incidence of C. albicans
greater in all age groups in the black compared to the white disease between 1997 and 2003, coinciding with increases
population.22,23 Links between incidence and the ICU are mainly in the incidence of C. tropicalis and C. parapsilosis.2
well established and relate to the use of clinical procedures In a recent study of data collated from 2019 patients from 23
and the disorders encountered within this setting. A range of U.S. centers involved in the prospective antifungal therapy
medical conditions and surgical procedures places patients (PATH) alliance C. albicans remained the most common
at varying degrees of risk of invasive candidal disease and single cause of invasive candidal disease (45.6%) but was
many will require additional medical interventions such as outweighed by the overall incidence of non-albicans disease
venous catheters, mechanical ventilation, total parenteral (54.4%).25 Generally, C. glabrata is the second most common
nutrition, prolonged antibacterial or steroid use, all of which species although in neonatal units and outbreak situations,
are additional independent risk factors. Patients undergoing C. parapsilosis is more prevalent, associated with skin, in
major abdominal surgery and solid-organ transplantation or particular hand colonization of health care workers and abil-
suffering from pancreatitis, premature birth, HIV infection, ity to form biofilms on catheters. C. glabrata is particularly
hematological malignancy, bone marrow transplantation, GI prevalent in North America and to a lesser extent in Europe,
tract rupture, or solid tumors and burns may all suffer from whereas in the Middle East and South America, C. tropi-
invasive candidal disease. The geographical incidence of calis and C. parapsilosis are more frequently encountered
invasive candidal infections is described in Table 64.2. (Table 64.3). The impact of fluconazole prophylaxis is dem-
Globally invasive candidal disease remains the most com- onstrated in a single-center study evaluating candidemia over
mon cause of invasive fungal disease (IFD) but in specific a 10-year period where C. albicans (13.5%) was demoted to
patient groups, i.e., hematology, invasive candidal disease the fourth most common cause of invasive candidal disease,

556
TABLE 64.3
The Global Epidemiology of Invasive Candidal Disease
North North England Asia/ Latin Saudi
Species (%) Global Global America America Europe and Wales England Scotland France Spain Denmark Denmark Pacific Australia America Brazil Arabia
C. albicans 73.3 62.3 51 45.6 56 60.1 53 52.0 57.0 51 63 59.8 56 62 50 40.9 53
C. glabrata 11.0 12.0 22 26.0 14 9.4 18 22.7 16.7 9 20 20.5 10 17 7 4.9 7
C. tropicalis 4.6 7.5 7 8.1 7 3.8 3 2.0 4.9 10 4 4.6 14 9 20 20.9 19
C parapsilosis 4.2 7.3 14 15.7 13 10.6 11 11.7 7.5 23 4 4.0 16 12 16 20.5 16
C krusei 1.7 2.7 2 2.5 2 1.5 1 1.0 5.2 4 3 4.1 2 13 2 1.1 2
C. guilliermondii 0.5 0.8 <1 0.3 1 0.6 1 3.3 — — — — <1 — 4 2.4 1
C. lusitaniae 0.5 0.6 <1 0.8 1 0.3 1 2.0 — — — — <1 — <1 — 1
C. pelliculosa — — — — 0.02 — — — — — — — — — 6.2 —
C. dubliniensis — — — 0.3 — — — 3.0 — — — 2.6 — — — — —
C. pseudotropicalis — — — — — — — — — — — — — —

Other — >2 — — 0.68 — 2.3 — 3 3 1.9 — — — 2.6
Candida spp. 3.9 4.9 — 0.5 — 13 11 — 3.6 — 4 2.6 — 4 —
Year 1997/1998 2003 2001/2004 2004/2008 1997– 1990/1999 2006 2005/2006 2005/2006 2002/2003 2003/2004 2004/6 2001/4 2002 2001/4 2003/4 1996–
1999 2004
Reference [2] [2] [103] [25] [29] [104] [102] [101] [105] [97] [98] [99] [103] [27] [103] [106] [107]
Candida 557
behind C. glabrata (30.6%), C. krusei (24.2%), and C. parap- The pathogenicity of Candida varies between species.
silosis (13.9%) in hematology patients when compared to the C. albicans is the most potent followed by C. tropicalis.31
previous 5 years.26 The prophylactic use of fluconazole was Sixty-four gene families have been positively identified in
determined as a risk factor for C. glabrata and C. krusei can- highly pathogenic Candida species and are associated with
didemia.26,27 This supports the results of the PATH alliance the cell wall and cell morphology, including hyphal and bio-
where previous azole use was associated with increased inci- film functions.32 To become a human pathogen, an organism
dence of C. krusei candidemia.25 However, this observation must be able to grow at 37°C and the ability to grow at higher
is not universal and long-term azole prophylaxis in one sur- temperatures (40°C) may contribute to virulence, particularly
gical ICU did not result in an increased incidence of azole- as invasive candidal infections result in fever.12 C. albicans,
resistant species.28 C. dubliniensis, C. famata, C. glabrata, C. inconspicua,
Changing epidemiology may impact mortality rates and C. kefyr, C. krusei, C. lusitaniae, and C. tropicalis all have
in both the PATH alliance and the European Confederation the capacity to grow at 40°C.6
of Medical Mycology (ECMM) survey, crude mortality rates Following infection, it is paramount that the interaction
were greater for disease caused by C. krusei (52.9%–55.3%) with the host is maintained and this is achieved through the
and lower for disease caused by C. parapsilosis (23.7%– production of adhesins (Als1p, Hwp1p, Int1p, and Mnt1p).16
25.9%).25,29 The mean crude mortality for all species and that Adherence to human buccal cells appears to be greater for
for C. albicans in the PATH alliance and ECMM survey was C. albicans than C. parapsilosis.33 C. albicans possesses
35.2%–38.5% and 35.9%–37.9%, respectively.25,29 It was also eight ALS family genes coding for cell well glycoprpoteins
noted that C. krusei disease was associated with patients with (adhesins) that are adhesive and/or promote flocculation and
neutropenia and corticosteroid use.25 Rather than attribut- filamentation and the ability to adhere to different cell types,
ing this increased mortality entirely on a putative enhanced extracellular matrices, or foreign bodies such as endotracheal
virulence of C. krusei, the patient comorbidities and poten- tubes or catheters.31,34 Adhesins are situated at the cell surface
tial delay in the initiation of appropriate treatment are more as is the case for any hydrolytic enzymes or molecules used
likely to be implicated. to avoid host defences. Proteolytic enzymes secreted by the
fungi can hydrolyze host membranes to not only enhance tis-
sue damage and the ease of invasion but to improve the bind-
ing efficiency between pathogen and host.16,31 Maintaining an
Candida species can cause both superficial and invasive optimally functioning cell wall is essential and gene dele-
disease and for most cases, the source of the infection is tions affecting this have resulted in attenuated virulence in
endogenous. Superficial disease can be oropharyngeal, vivo.35
genital, cutaneous, and paronychial. In certain patients with Secreted aspartyl proteinases (SAPs) are well established
congenital immunological or endocrinological disorders, as C. albicans virulence factors and are only found in the
recurrent disease occurs and results in chronic mucocutane- two most pathogenic species C. albicans and C. tropicalis.31
ous candidosis. Oral candidosis has several major clinical Produced by a family of 10 genes, SAPs function by destroy-
presentations: pseudomembranous candidosis where raised ing tissue and host defenses or by digesting proteins for nutri-
white removable lesions/plaques can develop in debilitated ents. They have an important role in both colonization and
patients or those with HIV disease; erythematous candidosis infection, and it is proposed that they are required for inva-
presents as a flat red lesion on the palate and tongue resulting sion.36 Recent research indicates that C. albicans metabolites
in tenderness and swelling in patients on antibacterial agents (possibly SAP and ALS) cause actin rearrangement in mac-
or long-term corticosteroid use; and hyperplastic candidosis rophages significantly reducing their ability to phagocytose
produces asymptomatic fixed lesions that occur on the inside both C. albicans and C. glabrata.37
of the cheeks of smokers or patients with oral trauma or den- Within host tissue, Candida will take on a mix of mor-
tal neglect and the lesions are dysplastic and considered pre- phologies. The formation of germ-tubes in human serum at
malignant. Genital candidosis usually presents with itching, body temperature indicates that this may be the preferred
redness, and burning with occasional discharge, fissuring, morphology for C. albicans within human tissue, although
and the presence of plaques. varying forms are found in vivo, demonstrating the effect
Invasive disease can cause a variety of clinical manifesta- of environmental pressures and the capacity the organism
tions with translocation from the GI tract or skin resulting has to adapt and invade different niches.38 During infection,
in candidemia and hematogenous dissemination to a variety hyphal structures are more invasive than yeasts and can pen-
of organs in the critically ill population. Symptoms are non- etrate deeper into endothelial tissue, expanding the area of
specific including a refractory fever and macronodular rash. tissue damage and allowing increased scope for yeast prolif-
Symptoms representative of specific manifestations (e.g., eration.38 However, it cannot be definitively proven that one
peritonitis, endocarditis, meningitis) are no different to bac- form of candidal cell is more virulent than the other.31
terial disease. For a concise but informative review of all the Candida cell surface molecules can trigger innate host
clinical presentations of both superficial and invasive candi- responses via the toll-like receptors and other recognition
dal diseases, see the book Fungal Infection; Diagnosis and molecules.39 Certain mannoproteins can trigger a Th1 cyto-
Management.30 kine response, a reaction to acute candidemia resulting in

sepsis. Conversely, exposure to other mannoproteins, dectin, of C. albicans, although C. dubliniensis also possesses this
and glucans may result in a Th2 cytokine response, a reaction ability.45 Another useful test to distinguish C. albicans and
to persistent fungal infection. The application of this Th1/ C. dubliniensis from other Candida species is the production
Th2 paradigm to human infection is far from clear and recent of large, thick-walled chlamydospores when grown under a
recognition of the role of regulatory T cells (T-reg) and Th17 cover slip (oxygen limited) on corn-meal agar. It has been
responses further complicates the picture. Recent evidence noted that C. albicans strains usually produce only one ter-
suggests Candida can modulate and evade host immunity minal chlamydospore whereas C. dubliniensis produces
and use immunomodulatory molecules that enable them to two to three, generally on shorter pseudohyphae.8 However,
promote protective tolerance leading to colonization and this is not truly definitive and relies on the discretion of the
persistence.40 mycologist, a more accurate test being the production of
chlamydospores by C. dubliniensis and not C. albicans when
64.1.4 Diagnosis grown on Staib agar.7
An alternative approach not reliant on culture is the
64.1.4.1 Conventional Techniques serological detection of candidal cell wall antigens and/
The conventional diagnosis of candidal disease is based on or anti-Candida antibodies. The main targets are mannan
the combination of clinical features with microbiological, and β-d-Glucan, with mannan being specific for Candida
radiological, and histopathological evidence. The ability to species and β-d-glucan being found in a range of differ-
culture the organism can be important but in anatomical ent fungi. For improved sensitivity, the mannan antigen
sites where Candida is commensal, it is beneficial to show test can be combined with anti-mannan antibody test and
evidence of an invasive process. Microscopy presents typi- a sensitivity/specificity of 80%/93% has been achieved.46
cal yeast cells, pseudo or true hyphae remembering that only The usefulness of the antibody test in certain immunosup-
C. albicans produces true hyphae and that C. glabrata only pressed populations incapable of raising a sufficient immune
produces yeast cells in tissue.30 The isolation of Candida response must be questioned. All the above tests benefit
from sterile sites, including deep fluids/tissue biopsies from quality control achieved by the availability of stan-
(excluding GI tract) and blood is significant and lytic blood dardized commercial kits and as such are included in dis-
culture methods should be performed in all suspected cases ease-defining criteria despite variable clinical performance.
of invasive disease and will provide detection rates between Both latex agglutination (LA) and ELISA kits are available
50% and 80%, depending on patient population and under- although the former has a reduced sensitivity (25% LA vs.
lying candidal disease.30 Culture from oral, genital, GI and 75% ELISA).47 Detection of mannanaemia and anti-mannan
respiratory tract must be balanced against the possibility of it antibodies has shown varied performance between candi-
arising as a result of colonization. dal species ranging from 100% sensitivity for C. albicans to
If Candida is successfully isolated from a patient, then 40% for C. parapsilosis.48
all isolates from clinically significant sites should be identi- In a recent comparison of nonculture diagnostics in
fied to a species level to determine antifungal susceptibil- patients with candidemia, the following sensitivities were
ity.41 Classically, Candida species are indentified on the basis generated 88%, 47%, 41%, 47%, and 75% for PCR, β-d-
of their assimilation and fermentation patterns, as described glucan, mannan ELISA, anti-mannan antibody detection,
in Table 64.1 and many commercial kits are available for and combined mannan-antibody, respectively.49 For a more
this purpose (API 20C and API Candida, BioMerieux; expansive review of fungal cell wall immunodiagnostics, see
AUXACOLOR, Biorad). Both the AUXACOLOR and API the recent article by Kedzierska et al.50
Candida systems correctly identified >90% of clinical yeast The difficulty in definitively diagnosing invasive candi-
isolates when comparatively evaluated.42 Alternatively, the dal disease results in many patients receiving unnecessary
most common species of Candida can be identified using empirical antifungal therapy. Without a “gold-standard”
chromogenic agar where usually glucosaminide derivatives diagnostic test, diagnosis is often based on a combined strat-
are metabolized by Candida to produce colonial colors that egy of risk factors, microbiology, and clinical symptoms.
vary according to species or group of species.43 Varying disease definitions have been described in the lit-
As C. albicans remains the most common cause of both erature and makes comparison between studies difficult.
superficial and invasive disease, the identification of this spe- To overcome these problems, consensus definitions for IFD
cies takes precedence in most clinical laboratories and tests using host, clinical, and microbiological criteria have been
have been developed to distinguish between C. albicans and agreed.51 Whilst these encompass Candida and certain
most other species. The phenomenon of “phenotypic switch- molds, they were designed for clinical and epidemiological
ing” has been used as a laboratory technique to differenti- research purposes and were not intended for routine clinical
ate C. albicans from non-albicans species. When grown on practice. Initially, covering patients with cancer and hemato-
heated blood agar in an atmosphere of 5% CO2, C. albicans logical malignancy, the revised definitions were extended to
colonies grow with an irregular margin that distinguishes patients with hereditary immunodeficiencies, connective tis-
them from other Candida species.44 In addition, the formation sue disorders, and solid-organ transplant patients. However,
of germ tubes within 3 h when incubated in serum at 37°C is critically ill patients on the ICU and surgical patients are not
a commonly used laboratory test for determining the identity covered by these criteria, which concentrate on candidemia,

Candida 559
histologically proven disease, and chronic disseminated techniques.58,59 This may be due to the uneven distribution of
candiodosis. As a consequence, the majority of the invasive pathogens within clinical specimens (especially tissue biop-
candidal burden will not fall within the remit of these con- sies), the transient nature of candidemias, and/or the lack of
sensus definitions. consensus regarding optimal specimen type and subsequent
Several published studies have investigated the predictive extraction procedures and platforms.
value of risk factors for diagnosis of invasive fungal infec- Choices of extraction method are influenced by speci-
tion in non-neutropenic, critically ill adult patients. Candida men type, which is in turn dictated by the pathology of the
colonization is an independent risk factor for infection and disease. The vast majority of papers have described ampli-
precedes invasive disease in most cases. More than 50% of fication procedures utilizing primer sequences that target
ICU patients will become colonized during hospitalization conserved multi-copy genes to heighten assay sensitivity and
and distinguishing infection from colonization may be dif- specificity. Frequently, these primers flank less-conserved
ficult. Risk of infection increases with the number of sites regions of the genome to allow for species differentiation by
colonized and is dependent on the colonizing species.52 various detection methods. Assays have been developed for
Further studies in surgical ICU patients have confirmed this direct detection from clinical specimens and for identifica-
trend and recommend the use of a Candida colonization tion of cultured isolates. The majority of publications focus
index to assess the degree of colonization. A ratio of ≥0.5 on samples for the diagnosis and/or preemptive monitoring
calculated from the number of noncontiguous sites colonized of patients at risk of invasive candidiasis (IC). Expedited
with the same strain over the number of sites sampled, has identification from clinical specimens and cultured isolates
been shown to have a good positive predictive value.53 Some allow for more timely and appropriate treatment of infected
studies have attempted to develop or evaluate a risk score for patients, a reduction in the cost and morbidity associated
invasive fungal infection or an algorithm for use of antifun- with inappropriate treatment of those without disease.
gal drugs (either prophylactically or preemptively) in non-
neutropenic, critically ill adult patients.54,55 Many of these 64.1.4.2.1 Specimen Type
scoring systems have been developed and evaluated within The most useful specimen for diagnosing candidemia should
the same data set. Given the geographical variation in inci- be blood. As already discussed, blood culture is not 100%
dence, scoring systems will identify different proportions of successful although its limited positivity confirms the
patients. Sensitivity will vary accordingly. presence of viable organism within the peripheral circula-
tion. Possible routes of entry of the organism into the blood
64.1.4.2 Molecular Techniques stream are highlighted in Figure 64.1 with the three main
With the increasing population of patients at risk and the sources of Candida being endogenous, hospital acquired,
emergence of resistant species causing these infections, or via a traumatic disruption to the anatomical barriers. The
the need for rapid and accurate diagnostics is imperative. most pertinent blood fraction is open to debate and may dif-
Molecular techniques have the potential to offer optimum fer according to disease presentation. Most investigations
accuracy of detection of these pathogens with as yet unparal- use whole blood although serum and plasma testing have
leled speed, although the economic necessity of batch testing been shown to be a suitable alternative (Table 64.4). Testing
may compromise diagnostic efficiency. whole blood requires large volumes for optimal detection of
Publications on molecular detection and identification of the low fungal burden but also increases the opportunity for
Candida species are many and varied as highlighted in Table the presence of inhibitors. The consequent laborious extrac-
64.4. Clinical sensitivity and specificity are difficult to com- tion procedures and the protocol dependent inefficiency to
pare between studies of varying design and sensitivity will extract “free DNA” are further weaknesses. Smaller volumes
be influenced by the specimen type and the consequent DNA and simpler extraction procedures make serum and plasma
extraction protocol. Furthermore, the evaluation of theoreti- testing attractive. However, loss of cell-associated frag-
cally more sensitive molecular methods by comparison to ments/organism and the presence of DNAses within blood
“reference” conventional techniques is somewhat of a mis- may compromise utility. In addition, the interpretation of
nomer. Nonetheless, three publications highlighted in Table DNAemia is still poorly understood.
64.4 reported clinical sensitivity and specificity results for Other clinical specimens utilized include various respi-
Candida specific PCR and sensitivity ranged from 90.9% to ratory secretions,60,61 fresh and paraffin-embedded tissue
100% with specificity 97% or greater.47,56,57 Between these specimens and biopsies,62,63 ocular samples,64 urine,65 and
three studies, both hematology and critical-care patients have nail clippings.66 The significance of detection of Candida in
been studied and both serum and whole blood have been suc- non-sterile anatomical sites, particularly respiratory, genital,
cessfully tested confirming the presence of both a free DNA and GI tract must be balanced against the detection of com-
and cell-associated DNA source and a potential clinical ben- mensal organisms and it is difficult to assign significance
efit in varying patient populations. without other evidence of an invasive process, e.g., histopath-
Limit and range of detection differ widely although ologically. The majority of publications report pan-fungal
most tests reportedly detect clinically relevant levels of assays that include Candida species within their detection
Candida (10 0 to 101 cfu/mL). Despite this, some authors have capability although with decreased sensitivity compared to
reported discordant results compared to traditional culture the assays utilizing blood specimens (Table 64.4).

TABLE 64.4
Molecular Techniques Used to Detect Candida Species from Clinical Specimens
Predominant Limit of
Sample Type Detection Candida Species Detected Target Region Detection Method Reference
Serum 0.22–2.3 copies/ Calb, Cdub, Cglab, Cpara, Ckrus, 18S rRNA and ITS Real-time PCR—hydrolysis [57]
mL Ctrop
Serum 1 organism/mL Clab, Cglab, Cpara, Ctrop ITS Semi-nested PCR and AGE [70]
Whole blood 1 cfu/mL Calb, Cdub, Cglab, Ckef, Ckrus, 18S rRNA PCR and Southern blot [71]
Cpara, Ctrop
Whole blood 1–5 cfu/mL Calb, Cdub, Cglab, Ckef, Ckrus, 18S rRNA Real-time PCR—biprobe [47]
Cpara, Ctrop
Whole blood 1–5 cfu/mL Calb 18S rRNA Real-time PCR—hybridization [88]
Whole blood and 1–10 cfu/mL Calb, Cglab, Ckrus, Clus, Ctrop 18S rRNA NASBA-enzymatic, [82]
plasma bead-based, and membrane-
based detection
Whole blood 1–100 cells/mL Calb, Cglab ITS region PCR-DEIA [75]
Whole blood 2 cfu/mL Calb, Cdub, Cglab, Cguil, 18S rRNA Real-time PCR—hybridization [108]
Ckef, Ckrus, Clus, Cnorv,
Cpara, Ctrop
Whole blood 2 cfu/mL Calb, Ckrus, Cpara, Ctrop ITS region PCR-ELISA [74]
Whole blood 3 cfu/mL Calb, Cdub, Cglab, Cguil, 18S rRNA Real-time PCR—biprobe [59]
Ckef, Ckrus, Clus, Cnorv,
Cpara, Ctrop
Whole blood 4 cfu/mL Pan-fungal 18S rRNA PCR and Southern blot [72]
Whole blood 4–5 cfu/mL Pan-fungal 18S rRNA PCR and AGE [69]
Whole blood 5 cfu/mL Calb 18SrRNA PCR-ELISA [73]
Whole blood 5 cfu/mL Calb, pan-fungal ITS region Real-time PCR—hydrolysis [56]
Whole blood 10 cfu/mL Pan-fungal 18S rRNA Real-time [109]
PCR—hybridization
Whole blood/biopsy <10 organisms/ Calb, Cdub, Cglab, Cguil, Ckef, 28S rRNA RQ-PCR [110]
reaction Ckrus, Clus, Cpara, Ctrop
Whole blood and 50 copies Calb, Cdub, Cfam, Cglab, Cguil, RNase P RNA Real-time PCR—hydrolysis [111]
plasma Ckrus, Cpara, Ctrop
Whole blood 200 fg DNA Calb, Cglab, Cguil, Ckef, Ckrus, P450 LDMG PCR-REA [112]
Cpara, Ctrop
Respiratory Samples 5–10 cfu/mL Calb, Cdub, Cglab, Ckrus, Cpara, ITS region Real-time PCR—biprobe [60]
Ctrop
Respiratory Samples 25 cfu/mL Pan-fungal 28S rRNA Real-time PCR—hydrolysis [61]
Tissues 2–20 cfu Pan-fungal ITS region PCR-MLH [62]
Tissues — Pan-fungal ITS region PCR-sequence [63]
Nail clippings 100–1000 plasmid Calb, Cdub, Cglab, Cguil, Ckrus, 28S rRNA Nested PCR and AGE [66]
copies Cpara, Ctrop
Ocular samples 1 fg DNA Pan-fungal ITS region Semi-nested PCR and AGE [64]
Urine 1–50 cfu/mL Calb CaHSP70 PCR and dot-blot [65]
Blood culture 1 cell/2 μL sample Calb, Cglab, Ckrus, Cpara, Ctrop ITS 2 region PCR and EPF [113]
Blood culture media 2 cfu/mL Calb, Cdub, Cglab, Cguil, Ckrus, ITS region Multiplex PCR and AGE [68]
Clus, Cpara, Ctrop
Blood culture media 10 cfu/mL Calb, Cdub, Cglab, Cguil, Ckrus, ITS region MT-PCR [58]
Cpara, Ctrop
Blood culture 10 cfu/mL Panfungal and Calb, Cdub, Cfam, 18S, ITS and 28S Semi-nested PCR-xMAP [79]
Cglab, Cguil, Cinc, Ckef, Ckrus, rRNA regions
Clipo, Clus, Cmem, Cnorv,
Cpara, Cparar, Cpel, Csak, Ctrop,
Cuti, Cval, Czey
Blood culture 100 cfu/mL Calb, Cglab, Ckrus, Cpara, Ctrop 18S rRNA and ITS Nested and real-time [114]
region PCR—hydrolysis

Candida 561

Molecular Techniques Used to Detect Candida Species from Clinical Specimens
Predominant Limit of
Sample Type Detection Candida Species Detected Target Region Detection Method Reference
Fungal isolates — Calb Numerous regions LSplex PCR and microarray [78]
(77 different primer
pairs employed)
Fungal isolates — Yeasts including: Calb, Cdub, ITS region PCR and ACE [76]
Ckrus, Clamb, Cpara, Ctrop
Fungal isolates — Pan-fungal 18S rRNA fTEFAP [80]
Fungal isolates — Calb, Cglab, Ckef, Ckrus, Cpara, ITS region Real-time PCR—hydrolysis [89]
Ctrop
PCR, conventional PCR; CaHSP70, C. albicans heat shock protein 70; AGE, agarose gel electrophoresis; EPF, end-point fluorescence; ACE, automated capil-
lary electrophoresis; fTEFAP, fungal tag-encoded FLX amplicon pyrosequencing; LSplex, large-scale multiplex; MT-PCR, multiplex-tandem PCR;
MLH, multiplex liquid hybridization; xMAP, Luminex xMAP technology; NASBA, nucleic acid sequence-based amplification; EIA, enzyme immuno-
sorbent assay; DEIA, DNA-enzyme immunosorbent assay; RQ-PCR, real-time quantitative PCR; REA, restriction enzyme analysis; P450 LDMG,
cytochrome P-450 lanosterol-14α-demethylase gene.
Abbreviations: Calb, C. albicans; Cdub, C. dubliniensis; Cfam, C. famata; Cglab, C. glabrata; Cguil, C. guilliermondii; Cinc, C. inconspicua; Ckef, C. kefyr;
Ckrus, C. krusei; Clamb, C. lambica; Clipo, C. lipolytica; Clus, C. lusitaniae; Cmem, C. membranifaciens; Cnorv, C. norvegenesis; Cpara, C. parapsi-
losis; Cparar, C. pararugosa; Cpel, C. pelliculosa; Csak, C. sake; Ctrop, C. tropicalis; Cuti, C. utilis.; Cval, C. valida; Czey, C. zeylanoides.
Endogenous source
Mucosal or GI tract
Colonisation/commensal
Nosocomial source
skin, contaminated
Host unbalance/debilitation equipment, care worker
Commensal to pathogen Induced Clinical Catheter; TPN.

immunosupression intervention cytotoxic therapy
Superficial
Tissue invasion
disease
Invasive disease Anatomical

Haematogenous disruption
dissemination
Angioinvasion
Traumatic source
Blood skin, GI tract,
environmental
Wholeblood Clot Serum/plasma
Possible specimens for PCR*
*Tissue may also be used
FIGURE 64.1 Candidal disease, sources, disease progression, and samples for molecular testing.
The identification of Candida species from culture using organism multiplying within the blood culture bottle and if a
molecular techniques may be useful, particularly for pecu- specimen with insufficient inoculum or nonviable organisms
liar species that are difficult to identify using classical tech- is introduced into a blood culture system, the concentration
niques. The application of pyrosequencing looks particularly of target will be diluted 10-fold to below the detection limit
promising.67 These assays utilize straightforward extraction of PCR testing. In this instance, it would have been easier to
procedures and provide adequate performance when testing test the original blood directly.
cultures. Improved turnaround time in relation to conven-
tional identification is reported, as well as accurate identifi- 64.1.4.2.2 Detection Technologies
cation of polyfungal infections.68 The process is limited by its A wide range of real-time (RT) and end-point detection
reliance on suboptimal conventional culture detection. Direct assays have been employed for the diagnosis of Candida dis-
molecular testing of blood cultures will be reliant on the ease and are represented in Table 64.4. Conventional PCR

followed by end-point detection techniques such as agarose technologies are also increasingly common within molecular
gel electrophoresis (AGE),64,68–70 blotting,65,71,72 enzyme laboratories although adapting assays for fast kinetics is not
immunoassay (EIA),73–75 multiplex liquid hybridization always straightforward.
(MLH),62 and automated capillary electrophoresis (ACE)76 Nested/semi-nested assays may be required in order to
have demonstrated suitable analytical sensitivity for clini- achieve clinically significant sensitivity, thereby increasing
cal diagnostics. However, these techniques are often time- the turnaround times and cost. Indeed, the experience of the
consuming, with amplification time ranging from 2 to 3 h and authors, supported by many of the papers published for RT
time for detection of at least 1 h. They also require facilities, molecular diagnosis of fungal disease, shows that to achieve
equipment, and expertise with limited availability in most the required analytical sensitivity for clinical diagnostics,
diagnostic facilities. Insufficient specificity and sensitivity for more than 35 cycles of amplification is frequently required.
species-level identification may also be a drawback with the For most cases, this only leaves a small window of oppor-
most basic AGE analysis and additional post-amplification tunity for accurate detection as beyond 40 cycles, many RT
processing required. All of these assays require the open- assays give spurious non-reproducible results due to a com-
ing of reactions vessels subsequent to amplification. This bination of Poisson’s law and probe degradation/nonspecific
increases the risk of false-positive results through amplicon interactions between the oligonucleotides. Nested or semi-
contamination, especially for nested protocols, which may be nested systems have been used to overcome this problem, but
incorporated to improve sensitivity. the necessity to handle PCR products leads to contamination
RT technologies have become popular, partially due to problems and more frequent false positivity, compromising
increased rapidity, sensitivity, and specificity in comparison the specificity/positive predictive value of a diagnostic assay
to many end-point detection systems. These protocols often when compared to RT systems. For any PCR test, it is impor-
require no more than 1 h to perform the same number of cycles tant to interpret results correctly within the clinical context,
of amplification that would take 2–3 h on a conventional ther- placing greater significance on multiple positive results than
mal block. The incorporation of specific detection probes a single non-reproducible PCR positive result.
within the amplification mix also allows for greater specific- Multiplex PCR reactions where two or more primer pairs
ity than most conventional PCR detection systems and allows are used to increase the detection range while maintain-
visualization of results as amplification occurs. The compat- ing analytical specificity are technically demanding, but
ibility of RT platforms with diagnostic facilities, especially in allow for the detection of a larger number of targets within
terms of their foot-print and closed-tube nature, has further a single test. Multiplex PCRs have been developed in tan-
enhanced their popularity by reducing and controlling the dem with both RT and conventional detection techniques as
contamination risk. Batch testing remains a financial impera- well as more recently with microarray analysis,77,78 Luminex
tive in clinical molecular diagnostics although the develop- Technology,79 and rapid sequencing techniques.67,80 A reduc-
ment of platforms that circumvent batch testing requirements tion in assay sensitivity is often seen when reactions are
may in the future facilitate on-demand even bedside testing. multiplexed, leading some researchers to develop nested/
RT detection is limited by the number of channels, usu- semi-nested procedures despite the potential drawbacks
ally no more than four, available to detect reporter dyes and already discussed.79 Currently, no publications are available
by the nature of the reaction kinetics employed. When only that demonstrate the application of microarray analysis or
genus-specific detection is required, RT detection is relatively rapid sequence detection directly to clinical specimens, prob-
straightforward. Where a species-specific assay is sought to ably a result of a reduced analytical threshold inherent with
differentiate the main pathogenic species or identify poten- these protocols as they now exist.
tially resistant species (C. glabrata or C. krusei), it becomes Candidal RNA has also been targeted via NASBA and
necessary to devise a more complex detection grouping in reverse transcriptase-PCR (RT-PCR).81,82 Both RT-PCR and
order to leave capacity for an internal control reporter. This NASBA are end point analysis system suffering similar limita-
is essential if false-negative results are to be avoided. If a tions to conventional PCR. To our knowledge, no RT-NASBA
RT pan-fungal approach is desired in addition to Candida methods, utilizing beacon technology, have been described.
detection, the similarity of conserved regions to the human However, reverse transcriptase PCR has been combined with
genome and differences in less-conserved regions amongst RT detection but only after the synthesis of cDNA.81,83 It is
clinically significant yeasts and molds makes differentiation possible to perform the reverse transcription and RT-PCR in
challenging using a single RT assay. a single tube and this should be performed to avoid the prob-
A vast array of RT platforms have been used for Candida lems mentioned.
detection, including bi-probes, hydrolysis (TaqMan) probes, There are a host of assays suitable for the accurate and
and hybridization (FRET) probes (Table 64.4). The choice rapid detection and/or identification of Candida species
of probe can be limited by platform and the choice of plat- from clinical specimens and cultured isolates giving inter-
form is most often dictated by existing technologies within ested health professionals from varied facilities the oppor-
laboratories although cost, familiarity with, and diversity of tunity to validate molecular assays for their particular needs
application of new equipment and user preference are also (Table 64.4). The choice of assay should depend on the range
important. Plate-based platforms are becoming popular as of detection required (Candida/resistance species/panfun-
they allow for larger batches to be processed at one time. Fast gal) dictated by the patient group of concern. The optimum

Candida 563
specimen is still debated. Users should ensure that extraction and EDTA anticoagulated should only be used, as heparin
procedures are fit for purpose for the chosen sample type can inhibit PCR processes and sodium citrate has been asso-
(Figure 64.1). ciated with fungal contamination, albeit Aspergillus. The
DNA extraction process is laborious and involves red cell
64.2 Methods lysis, white cell lysis, fungal lysis, followed by DNA purifica-
tion and precipitation and is described as follows:
1. Add 10 mL of red cell lysis buffer to 3 mL blood in
64.2.1.1 DNA Extraction from Candida Cultures a sterile 15 mL tube (If processing fresh non-frozen
1. Harvest 1 mL of overnight liquid culture by centrif- blood shake at room temperature for 5 min).
ugation at 5000 × g for 5 min, remove supernatant, 2. Centrifuge at 3000 × g for 10 min in a sealed rotor
and resuspend in lyticase buffer. Alternatively, sim- centrifuge. Decant the supernatant, leaving the pel-
ply scrape several colonies from an agar plate and let and/or the remaining 0.5 mL of fluid, whichever
resuspend in lyticase buffer. is the greater volume.
2. Extract DNA from Candida isolates using recombi- 3. Repeat steps 1 and 2, then proceed to step 4.
nant lyticase (Sigma-Aldrich) and the QiaAmp tis- 4. Resuspend the pellet in 1 mL white cell lysis buffer.
sue kit (Qiagen) as described previously.84 5. Incubate tubes at 65°C for 30–45 min.
For a cheaper alternative to recombinant lyticase, 6. Centrifuge at 7500 × g in a sealed centrifuge for
simply vortex the harvested Candida culture with 10 min.
Roche MagNA Lyser Green Beads for 30 s. Pulse 7. Discard the supernatant being careful not to disturb
centrifuge and wash with 200 μL of molecular the pellet (that may not be visible). Add 30 μL of the
grade water. Transfer washings to the Qiamp tissue Roche MagNA lyser Green Beads and vortex vigor-
kit and continue as directed by the manufacturer’s ously for 30 s.
protocol. 8. Pulse centrifuge, wash beads with molecular grade
Note: (1) If enhanced sensitivity is required when water, transfer washings to QIAamp tissue kit and
precipitating DNA with ethanol or isopropanol, follow manufacturer’s protocol.
incubate the solution on ice for 30 min to increase
the yield. Secondly, after the elution of the DNA, Red Cell Lysis buffer (RCLB): 10 mM Tris pH 7.6, 5 mM
YM-100 microconcentrators (Millipore) can be MgCl2, 10 mM NaCl. Sterilize by autoclaving and store at
used to increase the DNA concentration, although room temperature.
this increases the opportunity for contamination. White Cell Lysis buffer (WCLB): 10 mM Tris, 50 mM
(2) All reagents are filter-sterilized through 0.2 μm NaCl, 0.2% SDS, 10 mM EDTA. Make up and filter steril-
filters before use. (3) After the fungal cell lysis, it is ize and store at room temperature. Make up proteinase K at
possible to replace QiaAmp tissue kit with Roche 20 mg/mL and freeze aliquots of 100 μL at −80°C. On the
High Pure Template DNA spin columns or even use day of extraction, thaw aliquots and add contents to 10 mL of
automated extractors such as the Qiagen EZ1 (EZ1 rest of buffer to give 200 μg/mL and use immediately.
tissue kit) or Roche MagNA Pure LC (Total NA kit). Note: An interesting concept permitting the targeting of
3. For a quick and simple candidal DNA extraction, both the free DNA and cell-associated DNA from a whole
simply harvest cells as above, remove supernatant, blood sample is to separate the plasma and blood cell frac-
and resuspend pellet in 60 μL of Microlysis plus buf- tions by centrifugation, then remove the plasma and save.
fer (Microzone) and incubate at 65°C for 15–30 min, Process the cellular material as described above (steps 1–8),
then 95°C for 5 min. The extract is now ready for then re-introduce the plasma when the commercial DNA
PCR amplification. purification and precipitation is performed.85
Note: The use of bead-beating after cell harvesting followed 64.2.1.2.2 Serum or Plasma
by Microlysis plus extraction may improve DNA yield. The use of serum or plasma only permits the detection of
free circulating DNA as any cell-associated DNA is lost dur-
64.2.1.2 DNA Extraction from Clinical Specimens ing sample processing. When the specimen is centrifuged to
64.2.1.2.1 Whole Blood remove the human blood cells, candidal blastospores, phago-
In principle, when using whole blood, both free DNA and cell- cytosed, or viable will also be sedimented whereas the free
associated DNA can be targeted, although in practice, free DNA will be retained in the plasma or serum fraction. Free
DNA is lost during the processing procedure. Cell-associated DNA may be released from the fungi by the action of the
DNA sources include both the viable organism as would be host immune response, autolysis due to nutrient limitation,
the case in a candidemia and organism rendered nonviable by cellular damage arising from the interstitial pressures expe-
the action of the host immune system. This nonviable target rienced during tissue invasion, or affect of antifungal thera-
should provide molecular testing with an improved sensitivity pies, most of which target the fungal cell wall or membrane.
compared to culture. Optimal volumes for testing are 2–4 mL The larger the sample volume, the increased opportunity

for detection of DNAemia and typically 1 mL volumes are 5′-FAM ATC TTT TTG ATG CGT ACT GGA CCC TG—
used, but volumes are often limited by the system used and BHQ1-3′, C. glabrata probe, 5′-JOE GGC TAA CCC CAA
many automated systems only permit the use of volumes up GTC CTT GTG GCT T—BHQ1-3′ and C. krusei 5′-ROX
to 0.5 mL. DNA within samples can be concentrated by using TAC CTA TGG TAA GCA CTG TTG CGG C—BHQ2-3′)
YM-100 microconcentrators (Millipore). DNA extraction that bind to a Candida-specific sequence within the PCR
from serum or plasma can be performed using most com- amplicon.
mercial manual or automated kits, but detection thresholds
Procedure
should be determined and contamination excluded prior to
routine diagnostic use.
1. Prepare PCR mixture (20 μL) consisting of 2.0 μL
Note: It is not necessary to perform bead-beating or lyti-
Light-Cycler FastStart DNA Master hybprobe
case digestion when using serum.
master-mix (Roche) [containing dNTPs, Faststart
64.2.1.2.3 Tissue Taq DNA polymerase, 10 mM MgCl2], 2.0 μL each
of L18F and L18R primers (final concentration
The essential step when testing tissue specimens is the effi-
750 nM), 2.0 μL Candida probe (final concentration
cient digestion of the tissue to allow access to the invading
400 nM), 2.4 μL of 25 mM MgCl2, up to 9.6 μL tem-
fungi. Both paraffin/formalin embedded and fresh tissue
plate DNA, and molecular grade water as required.
have been used and from a technical perspective, it is easier
Note: The probe can be monoplex or multiplex.
to use fresh tissue as this does not require additional pro-
2. Conduct PCR amplification on a Roche Light-
cessing to remove the fixative. Conversely, if a specimen has
Cycler, Corbett Rotorgene, or Applied Biosystems
been shown to contain fungal elements by histopathological
TaqMan as follows: 1 cycle of 95°C for 10 min; 50
investigation, then testing the same specimen excludes the
cycles of 95°C for 15 s, 62°C for 30 s with a single
possibility of false negativity arising from testing a different
acquisition to the required channels (FAM-generic,
biopsy or part of the biopsy that does not contain the invasive
JOE-glabrata and ROX-krusei), 1 cycle 40°C
agent. Tissue should be digested with a combination of SDS/
for 30 s.
proteinase K and may require overnight incubation. After
complete tissue lysis, the fungal cell is targeted by bead-
Note: The generic assay has been optimized for Roche Light-
beating or lyticase before completing the process using one
Cycler and may require further optimization for other plat-
of the previously mentioned commercial methods.
forms. The multiplex assay has only been performed on the
Corbett Rotorgene and reaction volumes can be adjusted to
64.2.2 Detection Procedures maintain the same concentrations but allow a 15 μL DNA
input in a 50 μL reaction volume. Different fluorophores
Providing the PCR assay meets the detection requirements
are required for multiplex use on the Roche Light-Cycler.
of the user any PCR amplification system can be used to test
The Candida assay specifically recognizes C. albicans,
any clinical specimen or culture providing the DNA extrac-
C. kefyr, C. krusei, C. dubliniensis, C. tropicalis, C. parap-
tion is suited to purpose and is capable of processing the
silosis, and C. glabrata but not C. guilliermondii, C. lipo-
specimen type.
lytica, C. famata, A. fumigatus, A. niger, S. cerevisiae, or
Below a detailed quantitative RT-PCR system is described.
Cryptococcus neoformans. Although all the species above
It is based on a qualitative bi-probe assay (Sybr Green with
produce an amplicon with primers L18F and L18R, only C.
Cy-5 probe) designed to detect, but not differentiate the main
albicans, C. kefyr, C. krusei, C. dubliniensis, C. tropicalis,
pathogenic species of Candida (i.e., C. albicans, C. dublini-
C. parapsilosis, and C. glabrata produce a probe signal. The
ensis, C. glabrata, C. kefyr, C. krusei, C. parapsilosis, and
assay has a sensitivity of 5 C. albicans cfu per mL of spiked
C. tropicalis) using the Idaho Technologies Light-Cycler sys-
whole blood.86
tem.86 Due to sequence variations, the original assay was found
For a hybridization (FRET) probe detection system, see
to have a variable detection limit when testing certain species,
the manuscript of Loeffler et al.88 For a hydrolysis (TaqMan)
particularly reduced for C. glabrata.87 Furthermore, the devel-
probe detection system capable of further differentiation of
opment of new RT technologies lead to the modification of the
Candida species, see the manuscripts of McMullan et al. and
assay for use on more widely used improved platforms (e.g.,
Guiver et al.57,89
Roche Light-Cycler, Applied BioSystems TaqMan, or Corbett
RotorGene) and was multiplexed to allow the differentiation
of C. glabrata and C. krusei. This assay uses the same pan- 64.3 Future Perspectives
fungal primers (L18F, 5′-CTCGTAGTTGAACCTTGG-3′;
and Conclusions
L18R, 5′-GCCTGCTTTGAACACTCT-3′) that target two
conserved regions encompassing a variable region within the The advantages of molecular diagnostics are well known.
18S rRNA gene, resulting in the production of a 140 bp ampli- One of the main disadvantages of molecular detection of
con that corresponds to the nucleotide positions of 620–760 in any specific pathogen has been that molecular assays are
the 18S rRNA gene of C. albicans. The amplified product is not catch-all tests. For diseases such as IFD, with a low
then detected by hydrolysis probes (Generic Candida probe, prevalence in a population, the ability to exclude multiple

Candida 565
pathogens in a single reaction is beneficial. The increasing detection systems and the technology described above will
ability to efficiently perform multiplex amplification and certainly have a future, possibly syndromic, role. It is only a
detection or rapidly sequence even large sequences of any matter of time before ways of increasing sensitivity of detec-
given genome hold the potential to provide more compre- tion are found and these technologies can also be applied in
hensive pan-genomic/syndromic approaches to molecular diagnosing candidal, indeed broad-range IFD directly from
diagnosis in the future. This is demonstrated by recent publi- clinical specimens.
cations on Luminex technology,79 MT-PCR,58 Microarray,77,78
and Pyrosequencer80 based detection and/or identification of
fungal pathogens, including Candida species. References
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65 Debaryomyces
María J. Andrade, Mar Rodríguez, Elena Bermúdez, Félix
Núñez, Miguel A. Asensio, and Juan J. Córdoba
Contents
65.1 Introduction...................................................................................................................................................................... 569
65.1.2 Biology, Pathogenesis, and Clinical Features....................................................................................................... 570
65.1.3 Diagnosis.............................................................................................................................................................. 571
65.1.3.1 Physiological and Morphological Analysis............................................................................................ 572
65.1.3.2 Molecular Analysis................................................................................................................................ 572
65.2 Methods............................................................................................................................................................................ 573
65.2.1 Reagents and Equipment...................................................................................................................................... 573
65.2.2.1 Yeast Isolates.......................................................................................................................................... 573
65.2.2.2 DNA Extraction..................................................................................................................................... 573
65.2.3.1 RFLP Analysis of the IGS rDNA Region.............................................................................................. 574
65.2.3.2 RFLP Analysis of Mitochondrial DNA................................................................................................. 575
65.2.3.3 RAPD-PCR............................................................................................................................................ 576
65.3 Conclusions....................................................................................................................................................................... 578
References.................................................................................................................................................................................. 578
65.1 Introduction 65.1.1 Classification and Morphology

Species of the ascomycetous genus Debaryomyces are Genus Debaryomyces was established by Klocker16 with the
among the most common yeasts isolated from many natu- single species of D. globosus that was classified later in the
ral habitats such as air, soil, pollen, tree exudates, plants, genus Saccharomyces.17 Currently 19 species are included:
fruits, insects, feces, gut of vertebrates, and seawater.1 D. carsonii, D. castellii, D. coudertii, D. etchelsii, D. hanse-
This genus is generally recognized by the production of nii, D. maramus, D. melissophilus, D. nepalensis, D. occiden-
persistent asci by mother cell-bud conjugation and the talis, D. polymorphus, D. pseudopolymorphus, D. robertsiae,
formation of warty ascospores, usually one or two per D. udenii, D. vanrijiae, D. yamadae,18 D. prosopidis,19
ascus.2 Debaryomyces hansenii is the most important D. mycophilus,20 D. singareniensis,21 and D. vietnamensis.22
species of the genus and is characterized by its cryo- and The yeasts of this genus show spherical cells, and pseu-
halo-tolerance.3,4 This species and its anamorph Candida domycelium is absent, primitive, or occasionally well
famata have been isolated from a large diversity of natural developed. All species are perfect and haploid and have a
sources including fruit, air, water, and soil but most fre- vegetative reproduction by multilateral budding.17 The sexual
quently from a wide variety of foods, mainly those with reproduction proceeds via heterogamous conjugation of two
low water activity.5–8 cells of different form or size, generally mother and bud,
D. hansenii is considered normally as a non-pathogenic although the isogamous conjugation also occurs.17 The con-
yeast9 and has rarely been isolated from humans. However, jugation commonly leads to a diplophase followed by meiosis
some clinical cases have been reported with D. hansenii or and ascospore formation.23 One to two spherical, globular,
C. famata, generally in patients with immunological disor- ovoidal or lenticular smoothy, or warty spores are usually
ders, including bone infection,10 alveolitis,11 septicaemia,12 formed per ascus, but in some species up to four spores could
retinopathy,13 ocular endophthalmitis,14 or central nervous be present.17 Debaryomyces species are distinguished from
system infection.15 other ascomycetous yeast genera by the special internal ultra-
Classification, pathogenic relevance, and diagnosis of structure of the spore’s wall, consisting of a dark outer layer
Debaryomyces are discussed in this chapter. and two light inner layers separated by a dark line.17
569

The nuclear base composition of the Debaryomyces spe- and Candida guilliermondii belong to a monophyletic clade
cies is 37 mol% G+C or higher.24 The karyotype analysis containing organisms that translate CTG as serine instead of
reveals a high degree of polymorphism.25 leucine, suggesting they are sister taxa.36
The most common species of this genus, D. hansenii,
grows as spherical to short, oval cells, single, in pairs or
65.1.2 Biology, Pathogenesis, and Clinical Features
in short chains. As well as other species of the genus, the
pseudomycelium is absent, primitive, or exceptionally well Debaryomyces species are osmotolerant and can grow in
developed.17 Nevertheless, a yeast-to-mycelium dimor- media containing up to 4 M NaCl.37 Species of this genus are
phism has been described in continuous fermentation, with characterized physiologically by their inability to assimilate
the yeast morphology being reversibly affected by the dis- nitrate, as well as their weak or nonexistent fermentation
solved O2: low aeration leads to hyphae formation and the capacities,17 and chemotaxonomically by their expression of
increase in O2 concentration resulted in recuperation of the coenzyme Q-9.38 The type species of this genus, D. hansenii
oval shape.26 After conjugation, usually one or seldom two and its anamorph C. famata, is a cryotolerant, marine yeast,
spherical spores with a warty wall are formed per ascus.17 which can grow with salinity levels up to 24%.39 These and
The wartiness is not always distinct under light microscope, others characteristics that are used for testing strains of
and in scanning micrographs it appears as small blunt protu- D. hansenii are summarized in Table 65.1.
berances or as small ridges.27 The presence of spores gives D. hansenii is able to grow at 10% NaCl or 5% glucose,
the culture a brown color. and these characteristics are used to discriminate D. han-
D. hansenii is a highly heterogeneous species, with senii from other ascomycetous yeasts. On the other hand,
remarkable phenotypic differences between strains, such D. hansenii is one of the lipid-accumulating, “oleaginous”
as variations in their ability to metabolize various carbon yeasts and can accumulate lipids to concentrations up to
sources, the expression of different lipase and protease 70% of their dry biomass,40 and their metabolism is clearly
activities, and their diverse optimal growth conditions.28,29 dominated by pathways that contribute to lipid metabolism.
Therefore, a number of synonymous species and varieties D. hansenii can be cultivated in media with up to 25% NaCl
was recorded due to the variability in morphological, physi- or 18% glycerol.41 It is isolated from environments with
ological, and biochemical criteria commonly employed high salt concentrations, such as seawater or several types
in yeast classification.17 This heterogeneity in taxonomic of food. In fact, moderate NaCl concentrations improve the
characteristics has resulted in the description of varieties growth of D. hansenii cells. The positive effect of NaCl on
linked to D. hansenii. According to the current taxonomy, D. hansenii growth is even more evident in the presence
two varieties of D. hansenii are distinguished, D. hanse- of several stress conditions, such as high temperature 42 and
nii var. fabryi and D. hansenii var. hansenii, with different low or high pH levels in the media. D. hansenii is reported
properties, which may be discriminated by their maximum to grow optimally at 20°C–25°C, which might be a conse-
growth temperatures, the sequence divergences of their 26S quence of its natural occurrence in habitats such as seawa-
rRNA genes, and differences in the electrophoretic mobility ter. D. hansenii can grow at 5°C and even below 0°C.43 At
of their glucose-6-phosphate dehydrogenase.30–32 However, 10°C, this yeast is able to grow at pH 4.0–6.0 when aw is up
according to genomic studies, it has been proposed that to 0.99. D. hansenii can grow at aw values as low as 0.65.44,45
the varieties hansenii and fabryi should be considered as Although at 37°C selective growth of D. subglobosus
two genetically distinct species.33 In this sense, a polypha- belonging to the complex D. hansenii2 has been reported,
sic approach study re-examining 65 strains of D. hanse- it is difficult to use this temperature as the only criterion to
nii, including isolates from varieties fabryi and hansenii, differentiate Debaryomyces and specially those of complex
concluded that three distinct species should be considered D. hansenii.
within the D. hansenii group: D. hansenii, D. fabryi, and Debaryomyces spp. have been rarely isolated from
D. subglobosus.2 These three taxa may be differentiated humans, but they are considered opportunistic pathogens.
by polymerase chain reaction (PCR) fingerprints, DNA There are diverse reports about sanitary problems caused
reassociations, and phenotypic characterization, mainly in especially for C. famata, though some few ones refer to D.
maximum growth temperatures. Additionally, the diver- hansenii. Wong et al.10 reported some infections caused by
gence in intron sequences between the D. hansenii vari- Debaryomyces species. Torulopsis candida (C. famata) was
eties (D. hansenii var. hansenii, D. hansenii var. fabryi, isolated from a patient with chronic skin lesions on the hands
C. famata var. flareri, and D. tyrocola) unambiguously and feet. D. hansenii was also found in one case of bone
distinguished the groups as four separate taxa: D. hanse- infection that a 23-year-old woman suffered during 4 years.
nii, D. tyrocola, D. fabryi, and the new taxon D. macquar- Several clinical samples were identified as D. hansenii (and
iensis.34 In addition, this gene genealogy-based approach its anamorph C. famata) in superficial infections.46 D. hanse-
revealed genetic exchanges between these populations, indi- nii was also responsible for a persistent candidemia observed
cating an unexpected genetic diversity within this part of in a patient heavily treated with various fungicides.12 A
the genus Debaryomyces.34 65-year-old female was diagnosed with extrinsic allergic
D. hansenii and its anamorph C. famata are phylogeneti- alveolitis resulting from exposure to inhaled organic dusts,
cally related to Candida albicans.35 In addition, D. hansenii with D. hansenii being the dominant species in indoor air

Debaryomyces 571
TABLE 65.1
Characteristics of Complex Group Debaryomyces hansenii
Possible Results for
Characteristics Complex Group D. hansenii
Fermentation of:
• Galactose, glucose, maltose, melibiose, raffinose v
• Lactose −
Assimilation of substrates:
• l-Arabinose, cellobiose, ethanol, galactose, d-glucitol, d-gluconate, 2-keto-d-gluconate, glucose, α-methyl-d- +
glucoside, glycerol, lactose, maltose, d-mannitol, raffinose, ribitol, d-ribose, salicin, soluble starch, succinic acid,
sucrose, trehalose, d-xylose
• d-Arabinose, citric acid, erythritol, galacitol, 5-keto-d-gluconate, d-glucosamine, N-acetyl-d-glucosamine, inulin, v
dl-lactate, hexadecan, melezitose, melibiose, l-rhamnose, l-sorbose
• Inositol, methanol, saccharate −
Splitting of arbutin +
Assimilation of nitrate −
Assimilation of nitrite v
Assimilation of creatine +
Growth in vitamin-free medium −
Growth on 50% (w/w) glucose-yeast extract agar +
Growth at 37°C v
G+C (mol%) 38.2–38.6
Shape of the ascospores Spherical
Wall of the spores Warty
Number of spores per ascus 1, 2
Formation of pseudomycelium Absent or very primitive
Sources: Adapted from Kreger-Van Rij, N.J.W., The Yeast, a Taxonomic Study, 3rd edn., Elsevier Science Publishers, Amsterdam, the Netherlands, 1984;
Nakase, T. et al., The Yeasts, a Taxonomic Study, 4th edn., Kurtzman, C.P. and Fell, J.W., Eds., Elsevier Science Publishers, Amsterdam, the
Netherlands, 1998; Del Bove, M. et al., Food Microbiol., 26, 453, 2009; Córdoba, J.J. et al., Molecular Detection of Foodborne Pathogens, Liu,
D. Ed., CRC Press, Taylor & Francis Group, Boca Raton, FL, 2009.
+, positive reaction; −, negative reaction; v, variable reaction.
samplings.11 A 66-year-old man stem cell transplant recipient artery bypass and in a patient with continuous ambulatory
had no response to therapy with amphotericin B and voricon- peritoneal dialysis.58,59 Thus, medical importance of D. han-
azole and died after a septic shock. Positive blood cultures senii and its anamorph C. famata may rely on the susceptibil-
have demonstrated persistent candidemia due to D. hansenii ity of immunocompromised patients and on its resistance to
(C. famata).12 the treatments applied to different pathologies.
Different species of Candida are part of the natural
microbiota and, thus, are regarded as commensal organisms
65.1.3 Diagnosis
in humans. C. famata was thought to be non-pathogenic for
humans. However, this yeast was isolated in combination Since some Debaryomyces spp. can act as opportunistic
with other Candida spp. from a relevant number of clinical pathogens, accurate and sensitive methods for detection in
cases, including ocular endophthalmitis,14 acute zonal occult clinical and environmental samples are needed. For diag-
outer retinopathy,13,47 and central nervous system infection.15 nosis purposes, a complete characterization of yeasts at
In addition, C. famata is not rarely implicated in human species and strain level is required for most clinical cases.
fungemia.48–51 The occurrence of C. famata in patients with Different physiological and morphological methods have
problems of the oral cavity is also documented.52 New treat- been traditionally used in taxonomic differentiation of
ments of hospitalized patients seem to have favored the Debaryomyces.1,38,60,61 However, several studies have shown
emergence of C. famata as a pathogen or as responsible of that traditional identification methods based on phenotypic
nosocomial infections, but they may be underreported.53 properties of yeasts (morphological, biochemical, and physi-
In this sense, C. famata has been associated with catheter- ological tests) are laborious, lack discriminatory power, and
related bloodstream infection54 and rarely with other infec- misidentification occurs frequently.62 In addition, identifica-
tions,55–57 generally in immunocompromised patients. tion on the basis of morphological properties generates the
C. famata was implicated in a complication after a coronary so-called double binomial nomenclature, with one name for

the vegetative state (anamorph) and another for the sexual not cover species of genus Debaryomyces, including Yeast
state (teleomorph). This is the case of C. famata/D. hanse- Identification System API 20C (BioMérieux), the Uni-Yeast-
nii and more recently C. flareri/D. fabryi.34 Furthermore, Yek system (Remel), the Minitek system (BBL),60,66,67 Yeast
morphological methods generally produce ambiguities and Identification Panel (Baxter-MicroScan), and MicroScan
inaccuracies in the results, because the morphological and Rapid Yeast Identification.68,69 API ID32C system allows the
physiological characteristics are strongly influenced by identification of three species of the genus Debaryomyces:
growing conditions. D. hansenii, D. marama, and D. polymorphus.70 Similarly,
Progress in the molecular biology in the last decade the Vitek Yeast Biochemical card (BioMérieux) allows the
has opened up possibilities of characterizing yeasts at the identification of D. hansenii strains.71,72
genomic level. The sequencing of the genes coding for 18S Additionally, several methods have been developed for
and 26S ribosomal RNA (rRNA), as well as internal tran- automatic identification of yeasts on the basis of biochemical
scribed spacer (ITS) and intergenic spacer (IGS) region, has tests, such as the highlighting system Vitek 2® (BioMérieux)
brought about many changes in the identification and clas- with colorimetric and fluorimetric VITEK 2 yeast cards73,74
sification of yeasts at species and strain level.22,63 In addition, and Biolog YT Microplate® (AES Laboratories).
techniques based on random amplified polymorphic DNA Furthermore, selective and differential chromogenic
(RAPD-PCR) and restriction fragment length polymorphism solid media have been developed for the detection of
(RFLP) have been already recognized as reliable tools for Debaryomyces spp., although they have been used only with
the rapid identification of yeasts.64 We here review the main food samples.75,76
physiological and morphological methods, as well as the
molecular techniques. 65.1.3.2 Molecular Analysis
Molecular diagnostic methods possess several advantages
65.1.3.1 Physiological and Morphological Analysis over conventional methods based on phenotypic character-
Conventional identification of Debaryomyces spp. is based istics, such as higher reliability and sensitivity. They are also
on physiological, morphological, and biochemical charac- potentially more accurate, reproducible, and rapid.71 These
teristics. Morphological and sexual characteristics are not characteristics could serve as an important prerequisite for
useful for identifying Debaryomyces at the genus and spe- the timely onset of antifungal therapy. In addition, molecu-
cies level, because ambiguous identification may be obtained lar analysis is not influenced by environmental conditions
due to strain variability. The physiological tests used for of yeasts cells, because the nucleotide sequence of the DNA
identifying purposes are associated with the utilization of does not change during growth.
carbon and nitrogen sources, growth factor requirements, Nowadays, different molecular methods are available for
growth at elevated temperatures and on media of high sugar both qualitative and quantitative yeasts detection, as well as
or sodium chloride content, formation of typical character- for species identification, although most of them focus on
istic metabolites, and susceptibility to antibiotics. The uti- detection of Candida spp.
lization of these tests for Debaryomyces characterization Molecular identification can be carried out by nucleic
requires considerable experience and skill for evaluating acid hybridization or amplification. Methods of nucleic acid
the specific tests. Furthermore, great difficulties for the dif- hybridization use a DNA probe that has a sequence comple-
ferentiation at the species level in this genus could be found, mentary to the target sequence. 18S rRNA-targeted oligo-
since many biochemical and physiological tests show the nucleotide probes have been designed for rapid and reliable
same results for different species. In this sense, the classical identification of yeasts, such as the genus Debaryomyces
growth temperature test at 37°C to differentiate D. hanse- and the species D. hansenii.77,78 However, such probes do
nii of D. fabryi used alone confuses several strains of these not allow a full identification of all different species of
species.22 Debaryomyces.
The main biochemical tests are electrophoresis of pro- Other molecular methods are based on nucleic acid
teins, coenzyme Q analysis, allozyme analysis, and ultra- amplification. The most popular method of amplification is
structure and chemical composition (polysaccharides, fatty the PCR technique. Several conventional PCR procedures
acids) of the cell wall.17 Missoni et al.65 found the evalua- have been developed for the identification of D. hansenii.22,79
tion of cell fatty acids by gas chromatography very useful in Different variations of this technique (PCR-RFLP, RAPD,
the routine diagnosis and epidemiological monitoring of the real-time PCR, or quantitative PCR [Q-PCR], nucleic acid
infection due to Candida spp. (including C. famata). These sequence-based amplification [NASBA], etc.) are also
techniques are not always stable or reproducible because used to differentiate species of the genus Debaryomyces.
they depend on the physiological status of yeast strains. For PCR methods based on four housekeeping genes (ACT1,
example, fermentation of sugars is not very accurate because TUB2, RPL31, and RPL33) have been recently employed
the slow release of CO2 is not so immediate to be trapped in to the delineation of species among strains assigned to
a Durham tube. D. hansenii.34
Many commercial methods based on the above morpho- One of the problems associated with using PCR for micro-
logical and physiological characteristics have been developed bial investigation is the possibility of detecting naked DNA
for the identification of yeasts, although most of them do derived from dead and degrading yeast cells instead of live

Debaryomyces 573
yeasts, an outcome that may be unintended. An alternative analysis appears as one of the most suitable methods to
to prevent naked DNA detection by PCR consists of using differentiate yeast strains. Querol et al.91 developed a new
NASBA system that selectively amplifies RNA. This method mtDNA restriction analysis method based on the extrac-
has been used with yeasts of genus Candida.80 However, this tion of total yeast DNA and the use of GC-rich restriction
is not a big problem for clinical specimens, since the pres- endonucleases that recognize a high number of sites in the
ence of dead cells may be of clinical significance. yeast nuclear DNA, but few sites in the mtDNA. This tech-
The RAPD-PCR is a variation of the PCR useful for the nique has successfully been used to characterize strains
characterization of yeasts based on amplification of genomic of genus Debaryomyces 81,83,84,92,93 and clinical isolates of
DNA with a single short primer. Due to low-temperature Candida.94
hybridization, primer joins unspecific sites throughout the Other method used for the characterization of
genome, allowing amplification of DNA fragments of differ- Debaryomyces is pulsed-field gel electrophoresis (PFGE).
ent length. The use of RAPD-PCR permits the generation of With this technique, a restriction enzyme digests the complete
fingerprints which are specific for species and even strains. genome, and large DNA molecules are resolved by continu-
The RAPD-PCR technology has been used for the correct ous reorientation of the electric field during gel electropho-
identification of several species of Debaryomyces with dif- resis, determining chromosome length polymorphism (CLP).
ferent primers, mainly derived from micro- and mini-satel- This pattern is specific for yeast species, due to genetic and
lite DNA.62,79,81–85 These micro- and mini-satellite primers evolutionary phenomena that take place in the chromosomes
amplify the genomic DNA of these highly polymorphic (insertions, deletions, and translocations). This method has
regions, thus providing resolution among yeast strains. Using been reported as a useful tool in the differentiation of species
RAPD-PCR, Prillinger et al.33 concluded that D. hansenii and strain level of Debaryomyces.29
and D. fabryi are distinct entities and reinstated the varieties Overall, the most reliable, simple, and fast methods to
at species level. differentiate Debaryomyces spp. at strain level rely first on
One of the most promising PCR techniques in the detec- RFLP analysis of mtDNA and then on RAPD-PCR.83
tion of microorganisms is Q-PCR. Several Q-PCR assays
have been developed for detecting and enumerating C. famata 65.2 Methods
in fungal infections.47,86
Other molecular methods are based on the determination In this chapter, two methods for the differentiation of
of the RFLP. This procedure allows the differentiation of Debaryomyces spp. are proposed: single RFLP analysis of
organisms by analyzing patterns of rupture that are gener- the IGS rDNA region and a combined method that includes
ated in a specific site of the genome when it is cut by restric- mtDNA restriction analysis and then RAPD-PCR.95 For
tion enzymes. Then, gel electrophoresis displays a pattern of these methods, total yeast DNA from presumptive clinical
polymorphic bands corresponding to the fragments of differ- yeasts isolates should be first isolated.
ent sizes that are generated on the cut of each endonuclease.
This fragment length polymorphism appears because organ- 65.2.1 Reagents and Equipment
isms from different species and even strains differ in the
distance of cleavage sites for each restriction enzyme. The Reagent and equipment for RFLP of IGS and mtDNA and
similarity of the generated patterns allows for correlations RAPD-PCR analyses are listed in Table 65.2.
between species and strains.
The PCR-RFLP is a useful method for identification of 65.2.2 Sample Preparation
some yeast genus, using the restriction analysis of differ-
ent regions of rRNA/DNA genes (ITS-5.8S rDNA, 18S 65.2.2.1 Yeast Isolates
rDNA, etc.). This technique includes two steps: amplifica- Clinical specimens collected from patients are cultured on
tion of the rDNA region and digestion of the PCR product. Sabouraud dextrose agar or yeast peptone glucose (YPG)
Therefore, this method requires a longer time frame than agar (1% w/v yeast extract; 2% w/v peptone; 2% w/v glucose;
current PCR methods that achieve identification and typing 1.7% agar) aerobically at 27°C for 48–72 h. The obtained iso-
without further treatment of the PCR products. The results of lates can be subcultured in cryotubes containing Sabouraud
PCR-RFLP depend on the region of genome amplified and dextrose broth or YPG broth (1% w/v yeast extract; 2% w/v
the enzymes used. For example, PCR-RFLP of ITS1-5.8S peptone; 2% w/v glucose) and stored with 40% (v/v) glycerol
rDNA-ITS2 and 18S rDNA regions has been reported as a at −80°C.
good method for the differentiation of Candida species,87–89
but it does not allow to distinguish Debaryomyces spp.81 65.2.2.2 DNA Extraction
However, the PCR-RFLP of the IGS of rDNA is proposed as Genomic DNA is extracted from yeasts grown in YPG broth
a valuable technique for discriminating species of the genera (Figure 65.1). Yeast cells, taken from a single colony, are
Debaryomyces and Candida.22,88,90 cultured in 50 mL conical tubes containing 10 mL of YPG
Another technique relied on RFLP is the mitochondrial broth at 25°C for 48 h to reach an appropriate cell density
DNA (mtDNA) restriction analysis. Among all the molecu- and placed inclined in an orbital shaker under constant agi-
lar techniques described in literature, mtDNA restriction tation at 200 rpm to obtain large amounts of mtDNA. Yeast

TABLE 65.2
Culture Media, Reagents, and Equipment Required for Yeast DNA Extraction, RFLP Analysis of IGS and
Mitochondrial DNA, and RAPD-PCR
Culture Media and Reagents
Sabouraud dextrose agar Ethylenediaminetetraacetic acid (EDTA) disodium salt, RNase
dihydrate
Sabouraud dextrose broth Lyticase Sterile deionized water
Bacteriological agar Tris-(hydroxymethyl)-aminomethane PCR reagents (see Table 65.3)
Yeast extract Hydrochloric acid (HCl) 35% Restriction enzymes and 10× buffer (see Table 65.3)
d-Glucose anhydrous, extra pure Sodium dodecyl sulfate (SDS), molecular biology grade DNA molecular markers
Bacteriological peptone Potassium acetate, extra pure Bromophenol blue, indicator
Glycerol Isopropanol Ethidium bromide
d-Sorbitol extra pure Ethanol absolute
Equipment
Laminar flow cabinet Water bath Microwave oven
Stomacher lab blender Centrifuge for 50 mL conical tubes UV transilluminator
Incubator Centrifuge for 2 and 0.5 mL microtubes Thermal cycler and PCR tubes
Orbital shaker 1 mL, 200, 50, 5, 1, and 0.5 μL pipettes Horizontal electrophoresis unit with the appropriate gel
casting tray and combs
Vortex 5 mL cryotubes
Freezer Spectrophotometer
cells are harvested from pure cultures by centrifugation at respectively. Pure DNA should have a ratio value between
4000 rpm for 5 min; resuspended in 500 μL of 1 M sorbitol, 1.8 and 2.0.
0.1 M ethylenediaminetetraacetic acid (EDTA) pH 7.5; and
transferred to 2 mL sterile microcentrifuge tubes. At this 65.2.3 Detection Procedures
stage, 40 μL of a lyticase solution (20 mg/mL) is added to
digest yeast cell wall and obtain spheroplasts. After 45 min 65.2.3.1 RFLP Analysis of the IGS rDNA Region
incubation at 37°C in a water bath with occasional shaking, The IGS region of the rDNA, which is located between
the suspension is centrifuged at 13,000 rpm for 1 min and the 26S and 18S genes, is amplified by PCR using the primers
supernatant is discarded. The pellet is resuspended in 500 μL CNL12 (5′ CTGAACGCCTCTAAGTCAG 3′) and CNS1
of a solution containing 50 mM Tris–HCl, 20 mM EDTA pH (5′ GAGACAAGCATATGACTACTG 3′).96 Amplification
7.4 to release cellular DNA from spheroplasts. Next, 50 μL reaction mixture is prepared on ice in volumes of 50 μL
of 10% (w/v) sodium dodecyl sulfate (SDS) is added, and the containing the reagents and concentrations summarized in
mixture is incubated at 65°C for 10 min in a water bath. To Table 65.3. The thermal cycling parameters are summarized
remove proteins, 200 μL of 5 M potassium acetate is added, in Table 65.3. After the program ends, amplification prod-
and the mixture is shaken and stored on ice for 15 min. Next, ucts are cooled in the thermal cycler at 4°C until removed,
the mixture is centrifuged at 13,000 rpm for 5 min and 500 μL and then kept at −20°C until required. A negative control,
of the resulting aqueous phase (top layer) is transferred to a which consists of an equal volume of water replacing the
new sterile microtube together with an equal volume of ice- DNA template, can be included. PCR-amplified DNA frag-
cold isopropanol and left 5 min at room temperature. After ments are analyzed in 1% (w/v) agarose gels at 80 V.90,97
the precipitated nucleic acids are centrifuged at 13,000 rpm Aliquots of 10 μL of PCR products are individually
for 10 min, the supernatant is decanted and the pellet is fur- digested with the restriction endonucleases HapII, HhaI, and
ther washed with 1 mL of ice-cold 70% (v/v) ethanol and cen- MboI at 37°C overnight90,97 (Table 65.3). Restriction frag-
trifuged at 13,000 rpm for 1 min. Ethanol is aspirated with a ments are separated by electrophoresis on 2.5% (w/v) agarose
pipette and the pellet is dried at 37°C for 45 min. Dried DNA gels at 60 V98 (Figure 65.2).
is suspended in 50 μL of TE buffer (10 mM Tris–HCl, 1 mM A DNA molecular marker can be incorporated into the
EDTA pH 8.0) and 1 μL of RNase (20 mg/mL) is added to gel to estimate the size of both amplification and restric-
digest the contaminating RNA. Then, the solution is incu- tion products. Gels are stained with an ethidium bromide
bated for 30 min at 37°C in a water bath and immediately solution (0.5 μg/mL) and visualized under UV light. The
placed at −20°C. size of the amplified IGS rDNA region of D. hansenii is of
To be used for PCR, concentration and purity of the about 2800 bp.90 Restriction patterns of tested clinical yeasts
extracted DNA have to be determined by the optical isolates are compared with those of reference strains for
density (OD) at 260 nm and the ratio OD 260/280 nm, identification.

Debaryomyces 575
Clinical specimen
YPG agar
Yeast cells
10 mL YPG broth
Incubation
(25ºC, 200 rpm, 48 h)
Centrifugation
Supernatant
(4000 rpm, 5 min)
500 µL 1 M sorbitol, 0.1 M EDTA, pH 7.5
Resuspension 40 µL lyticase 20 mg/mL
Cell wall digestion
(37ºC, 45 min)
Spheroplasts
Centrifugation
(13,000 rpm, 1 min) Supernatant
500 µL 50 mM Tris–HCl, 20 mM EDTA, pH 7.4
Resuspension
Free DNA
50 µL 10% SDS
Protein resolubilization
(65ºC, 10 min)
200 µL 5 M KOAc
Protein precipitation
(0ºC, 15 min)
Centrifugation
Bottom layer
(13,000 rpm, 5 min)
500 µL Isopropanol, 5 min
DNA precipitation
(5 min)
Centrifugation
Supernatant
(13,000 rpm, 10 min)
1 mL 70% Ethanol
Washing
Centrifugation
(13,000 rpm, 1 min) Supernatant
Drying
(37ºC, 45 min) 50 µL TE buffer
Dissolution
1 µL RNase 20 mg/mL
RNA digestion
(37ºC, 30 min)
Isolated DNA
Figure 65.1 Diagram of DNA extraction from Debaryomyces spp.
65.2.3.2 RFLP Analysis of Mitochondrial DNA on horizontal 0.8% (w/v) agarose gels at 100 V (Figure 65.3).
mtDNA restriction analysis involves the digestion of A DNA molecular marker can be incorporated into the
total DNA with restriction enzymes selective for GC-rich gel as a standard for the calculation of the restriction frag-
sequences, such as HaeIII, which results in an over-diges- ment size. Gels are stained and visualized as described in
tion of the nuclear DNA to render specific bands from Section 65.2.3.1. Restriction profiles of tested clinical yeasts
mtDNA. Thus, this methodology allows detecting mtDNA isolates are compared with those of reference strains for
RFLPs without first separating the mtDNA from nuclear characterization.
DNA. To reduce DNA digestion time, a microwave oven can
Digestion mixture is performed on ice in a volume of be used.99,100 The digestion mixture is placed into a water
15 μL, containing the reagents and amounts summarized in bath and heated three times at maximum level of the
Table 65.3. After the incubation overnight at 37°C in a water microwave (1250 W) for 20 s each, giving a spin between
bath, restriction fragments are electrophoretically separated each time.

Table 65.3
PCR and RFLP Reagents and PCR Conditions for Identification and Characterization of Debaryomyces spp.
IGS PCRa mtDNA RFLPb RAPD-PCRb
Reagents Volume Reagents
(Concentration) Volume (μL) Reagents (μL) (Concentration) Volume (μL)
Mg2+-free reaction HaeIII (20 U/μL) 1 Mg2+-free reaction
buffer buffer
(10 mM Tris–HCl pH 5 10× buffer 1.5 (10 mM Tris-HCl pH 5
8.8, 8.8,
50 mM KCl, 0.1% DNA 5 50 mM KCl, 0.1%
Triton X-100) Triton X-100)
MgCl2 (50 mM) 3 Sterile deionized 7.5 MgCl2 (50 mM) 3
water
PCR nucleotide mix 1 PCR nucleotide mix 1
(10 mM) (10 mM)
CNL12 (100 ng/μL) 1 (GACA)4 (100 ng/μL) 2
CNS1 (100 ng/μL) 1 DNA (10 ng/μL) 10
DNA (10 ng/μL) 10 Taq DNA polymerase 0.5
(2 U/μL)
Taq DNA polymerase 0.5 Sterile deionized 28.5
(2 U/μL) water
Sterile deionized water 28.5
PCR stagesa PCR programa PCR stages PCR program

Initial denaturation 94°C 85 s Denaturation 94°C, 1 min
Denaturation 95°C, 35 s Annealing 36°C, 1 min 30 cycles
Annealing 58°C, 55 s 35 cycles Extension 55°C, 5 min
Extension 72°C, 2 min Final extension 55°C, 5 min
Final extension 72°C, 10 min
IGS RFLPa
Reagents Volume (μL)

HhaI, MboI, or HapII 0.1
(10 U/μL)
10× buffer 4
IGS-PCR 10
Sterile deionized water 25.9
a Adapted from Quirós, M., Debaryomyces hansenii: Estudio de su capacidad para deteriorar alimentos y desarrollo de métodos para su detección rápida,
Doctoral thesis, UCM, Madrid, Spain, 2005; Romero, P. et al., FEMS Yeast Res., 5, 455, 2005; Quirós, M. et al., Anton. Leeuwen., 90, 211, 2006.
b Adapted from Andrade, M.J. et al., Int. J. Food Microbiol., 107, 48, 2006; Córdoba, J.J. et al., Molecular Detection of Foodborne Pathogens, Liu, D. Ed.,
CRC Press, Taylor & Francis Group, Boca Raton, FL, 2009.
65.2.3.3 RAPD-PCR Table 65.3 for a final volume of 50 μL. The reaction is car-
RAPD-PCR is based on the amplification of genomic DNA ried out in a thermal cycler under the amplification condi-
using a single primer with an arbitrary nucleotide sequence. tions summarized in Table 65.3.
Consequently, this method does not depend on prior knowl- RAPD-PCR products are separated by 1% (w/v) aga-
edge of the DNA template sequence from the assayed yeasts. rose gel electrophoresis at 100 V (Figure 65.3). A molecular
RAPD-PCR with primers derived from simple repetitive weight DNA standard can be incorporated into the gel to esti-
sequences, such as (GACA)4, has been applied for genetic mate the size of the amplification products. Gels are stained
characterization of D. hansenii.83,84 and visualized as described in Section 65.2.3.1. Patterns of
The amplification reaction mixture must be prepared on tested clinical yeast isolates are compared with those of ref-
ice, using the reagents and concentrations summarized in erence strains for characterization.

Debaryomyces 577
Isolated DNA
Primers
CNL12, CNS1
Reaction buffer
(MgCl2, nucleotide mix,Taq polymerase)
Amplification
(see Table 65.3)
Molecular marker, loading buffer

IGS-PCR products
Restriction enzyme
Electrophoresis gel
(1% agarose in TAE buffer) HapII, HhaI, MboI
–
Digestion buffer
Electrophoresis +
(80 V) Water bath
0.5 µg/mL ethidium bromide (37ºC, overnight)
Molecular marker,
Visualization loading buffer
(UV)
Digested PCR products
Electrophoresis gel
(2.5% agarose in TAE buffer)
–
+ Electrophoresis
(60 V)
0.5 µg/mL ethidium bromide
Visualization
(UV)
Figure 65.2 Diagram of IGS-RFLP for analysis of Debaryomyces spp.
Isolated DNA
Restriction enzyme Primer
HaeIII (GACA)4
Digestion buffer Reaction buffer

(MgCl2, nucleotide mix,Taq polymerase)
Water bath Microwave oven

Amplification
(37ºC, overnight) (1250 W, 3 × 20 s)
(see Table 65.3)
Molecular marker, loading buffer

Digested DNA RAPD-PCR products
(0.8% agarose in TAE buffer) Electrophoresis gel (1% agarose in TAE buffer)
– –
+ Electrophoresis +
(100 V)
0.5 µg/mL ethidium
bromide
Visualization
(UV)
Figure 65.3 Diagram of mitochondrial DNA restriction and RAPD-PCR analyses for Debaryomyces spp.

65.3 Conclusions 6. Cocolin, L. et al. Study of the ecology of fresh sausages and
characterization of populations of lactic acid bacteria by
Yeasts of genus Debaryomyces are among the most common molecular methods. Appl. Environ. Microbiol., 70, 1883, 2004.
yeasts isolated from many natural habitats. These yeasts 7. Fadda, M.E. et al. Occurrence and characterization of yeasts
are considered normally as non-pathogenic. However, some isolated from artisanal Fiore Sardo cheese. Int. J. Food
species of this genus have been occasionally isolated from Microbiol., 95, 51, 2004.
human diseases such as bone infection, retinopathy, allergic 8. Simoncini, N. et al. Dynamics and characterization of yeasts
during ripening of typical Italian dry-cured ham. Food
alveolitis, or septicaemia in immunocompromised patients. Microbiol., 24, 577, 2007.
Nineteen different species have been included in the genus 9. Warren, N.G. and Hazen, K.C. Candida, Cryptococcus, and
Debaryomyces. Most of them are osmotolerant and can grow other yeasts of medical importance. In: Manual of Clinical
in media containing up to 4 M NaCl. Species of this genus Microbiology, 7th edn., Murray, P.R., Ed. ASM Press,
are characterized physiologically by their inability to assimi- Washington, DC, 1998.
late nitrate, as well as their weak or nonexistent fermentation 10. Wong, B. et al. Bone infection caused by Debaryomyces han-
capacities. senii in a normal host: A case report. J. Clin. Microbiol., 16,
545, 1982.
Since some Debaryomyces spp. can act as opportunistic
11. Yamamoto, Y. et al. Extrinsic allergic alveolitis induced by
pathogens, it is necessary that accurate and sensitive methods the yeast Debaryomyces hansenii. Eur. Respir. J., 20, 1351,
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complete characterization of the yeast at species and strain Debaryomyces hansenii (teleomorph Candida famata) and
level is required. Traditional identification methods, based Scopulariopsis brevicaulis in a stem cell transplant patient
on phenotypic properties of yeasts, are laborious, lack dis- receiving liposomal amphotericin B and caspofungin for sus-
pected Aspergillosis. Infection, 33, 397, 2005.
criminatory power, and misidentification occurs frequently.
13. Carrasco, L. et al. Isolation of Candida famata from a patient
Progress in the molecular biology has opened up possibilities with acute zonal occult outer retinopathy. J. Clin. Microbiol.,
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IGS rDNA region or a combined method that includes first candida (Candida famata) endophthalmitis simulating
mtDNA restriction analysis and then RAPD-PCR have been Propionibacterium acnes syndrome. Arch. Ophthalmol., 109,
proposed to characterize Debaryomyces spp. These methods 1718, 1991.
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and Science (grants AGL2004-03291, AGL2007-64639, and Publishers, Amsterdam, the Netherlands, 1998.
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66 Geotrichum
Silvia D’Arezzo, Paolo Visca, and Corrado Girmenia
Contents
66.1 Introduction...................................................................................................................................................................... 581
66.1.2 Epidemiology........................................................................................................................................................ 582
66.1.3 Clinical Features, Pathogenesis, and Therapy...................................................................................................... 583
66.1.4 Diagnosis.............................................................................................................................................................. 583
66.2 Methods............................................................................................................................................................................ 586
66.2.2.1 Identification of Geotrichum by PCR Amplification of the D2 Region................................................ 587
66.2.2.2 RFLP Analysis....................................................................................................................................... 587
66.2.2.3 Automated Fluorescent Capillary Electrophoresis................................................................................ 587
66.2.2.4 DNA Sequence Analysis........................................................................................................................ 587
References.................................................................................................................................................................................. 588
66.1 Introduction carbohydrate assimilation patterns and other biochemical

properties.
66.1.1 Classification and Morphology De Hoog et al. [3] considered in a taxonomic revision all
Geotrichum is a filamentous yeast-like fungus found world- the arthroconidial ascomycetous yeast-like genera together
wide in soil, water, air, and sewage, as well as in plants, combining studies of morphology, physiology, molar gua-
cereals, and dairy products [1–3]. It is also found in normal nine + cytosine percentage (mol% G + C), and DNA–DNA
human flora and is isolated from sputum and feces. The homology. In particular, the revision considered Geotrichum
genus Geotrichum includes 18 species, some of which can be and its teleomorphs Galactomyces and Dipodascus, and
pathogenic to humans [4–7]. The classification of arthroco- four species were recognized in Geotrichum, two in
nidial yeast-like strains has long been problematic, and a cru- Galactomyces, and 13 in Dipodascus. More recent phylo-
cial issue of fungal taxonomists has been the differentiation genetic studies of hyphal ascomycetous yeasts based on the
between the genus Trichosporon and the genus Geotrichum determinations of the sequence divergence of the large-sub-
[1,2]. Various studies demonstrated that the type species of unit (25S–28S) ribosomal RNA (rRNA) gene concluded that
Trichosporon was of basidiomycetous affinity, and, on the Galactomyces, Geotrichum, and Dipodascus are related to
basis of differences in carbohydrate composition of the cell the ascomycetous yeasts and should be placed in the order
walls, a separation of the basidiomycetous and ascomycetous Saccharomycetales [12–16].
arthroconidial species as Trichosporon and Geotrichum spe- Based on more recent taxonomic revisions [4–7], the
cies, respectively, was proposed [8–11]. genus Geotrichum is composed of 18 species. Of these,
In clinical laboratory, Geotrichum capitatum and six species including G. candidum have a teleomorph in
Geotrichum candidum, the two most frequently encountered Galactomyces, six species including G. capitatum have a
Geotrichum species in humans, and Trichosporon spp. are teleomorph in Dipodascus, and six species have an unknown
generally indistinguishable from one another on the basis teleomorph.
of colony morphology alone. G. candidum produces only Hereafter, we will focus on the two species G. candidum
arthroconidia; both G. capitatum and Trichosporon spp. pro- and G. capitatum, because of their involvement in superficial
duce arthroconidia as well as blastoconidia. Only G. capi- or deep infections in humans. A schematic taxonomic clas-
tatum can also produce anelloconidia, which unfortunately sification of these two pathogens is shown in Figure 66.1.
may be misidentified as arthroconidia or blastoconidia. G. capitatum was originally known as Trichosporon
Therefore, these species are usually differentiated by their capitatum and classified among the basidiomycetes. Later,
581

the stools but do not colonize the digestive tract. Although

Phylum: Ascomycota
many workers are exposed to large quantities of G. candi-
Class: Hemiascomycetess dum spores (e.g., cheesemakers and workers in starters fac-
Order: Saccharomycetales
tories), no professional disease implicating G. candidum has
been reported to date. The pathogenicity of G. candidum in
Teleomorphic
Family
Anamorphic
Dipodascaceae Candidaceae
Galactomyces Genus Geotrichum humans has not been defined [25]. Infections are very rare
state
state
Galactomyces Species Geotrichum and may be superficial and deep invasive. Intestinal coloni-
candidus candidum zation by G. candidum may occur when mucosal immunity
is disturbed and may be the source of dissemination in cases
(a) of severe immunodeficiency. The cases of G. candidum ill-
ness reported in literature may be overestimated because
Phylum: Ascomycota of the possibility of misidentification with other pathogens
Class: Hemiascomycetes such as G. capitatum or Trichosporon spp., particularly in
Order: Saccharomycetales older publications before the recent taxonomic classification
of arthroconidial fungi, or the difficult etiological attribu-
Teleomorphic
Family
Anamorphic
Dipodascaceae Candidaceae
tion of some mixed infections in which multiple bacteria and
Dipodascus Genus Geotrichum
state
state
fungi are involved.
Dipodascus Species Geotrichum
capitatus capitatum G. capitatum has been isolated from environmental
sources such as woods and poultry feces and is widely dis-
(b)
tributed in nature as a soil saprobe. More frequently than
G. candidum, G. capitatum is part of the normal flora
Figure 66.1 Present nomenclature of Galactomyces candidus/ of human skin and may be isolated from sputum and the
Geotrichum candidum (a) and Dipodascus capitatus/Geotrichum digestive tract of healthy people. As with other opportunis-
capitatum (b). tic yeasts, the number of infections due to this species has
increased during the past two decades, as a consequence of
the rise of hosts presenting factors that predispose them to
however, in light of its cell wall structure and septal pores and fungal infections such as cytotoxic chemotherapy, neutrope-
its tendency to produce numerous arthroconidia and few blas- nia, broad-spectrum antibiotic treatment, steroids, and inva-
toconidia, it was considered to be an ascomycete. The appro- sive catheterization [26–28].
priateness of this classification was further supported by the A literature review published in 2005 [28] demonstrated
discovery of its sexual form (teleomorph), Dipodascus capita- that patients with hematological malignancies are by far the
tus [3,17,18], and it was thus assigned to the genus Geotrichum. most common victims of G. capitatum infections. Patients
The subsequent discovery of its ability to produce anelloco- of this type accounted for 92% of reported invasive G.
nidia, as well as arthroconidia and blastoconidia, led Salkin to capitatum infections, and acute leukemia, acute myeloid
reclassify G. capitatum as the single species of a new genus: leukemia in particular, was the underlying disease most
Blastoschizomyces capitatus [19]. However, from a taxonomic frequently associated with all these infections. Most of the
point of view, the designation G. capitatum is still regarded as infected patients had been treated with conventional cyto-
the correct anamorphic name [20,21]. toxic chemotherapy, and very few had received blood stem
cell transplants. The infections usually occurred during a
period of profound neutropenia (neutrophil count, less than
66.1.2 Epidemiology
100/mm 3).
Members of the arthroconidial yeast genera Dipodascus and Although acute leukemia is the major underlying condi-
Galactomyces and their Geotrichum anamorphs are iso- tion in G. capitatum infections, its incidence seems to be
lated mainly from plant materials and to a lesser extent from low even in this group. On the basis of a retrospective study
human and other animal specimens. of Italian cases, the incidence rates for G. capitatum infec-
G. candidum is a ubiquitous fungus found in a wide tions among patients with acute leukemia were only five
range of habitats such as plant tissues, silage, soil, milk, air, cases per 1000 adult patients with acute leukemia (or 0.5%).
and water. It is also a component of the natural flora of the The geographic distribution of G. capitatum infections
digestive tract in humans and many other mammals [22,23]. is by no means homogeneous, and there is a significantly
It is found naturally in cheese and fermented milk, but in higher frequency of G. capitatum infections in Europe
France it has also been added as a ripening agent for at least (87% of reported cases) than in the United States (5%) [28].
30 years. It is reported by the International Dairy Federation Furthermore, 87% of the European cases occurred in Italy,
and European Food and Feed Culture Association as a Spain, and France. Similar pictures emerge from the data
microorganism with a documented history of use in dairy collected during the ARTEMIS DISK Surveillance Study,
products [24]. Sources of exposure are ingestion, inhala- a prospective study conducted in more than 30 countries to
tion, and contact [25]. Cheese consumption proved to be a identify global trends in the susceptibility of yeast patho-
major exposure source. Usually, the strains survive up to gens to fluconazole and voriconazole [29]. Isolates collected

Geotrichum 583
between June 1997 and December 2002 included 40 G. cap- The optimal therapy for geotrichosis has yet to be identi-
itatum strains of whom 32 (80%) came from Europe, and fied. Most of data are available for G. capitatum, whereas the
over half of the European isolates were recovered in Italy paucity of clinical and microbiological data reported to date
[28,29]. This finding seems to suggest that climatic factors does not allow to define specific indications for G. candidum.
might play a selective role in the epidemiology of G. capita- However, it could be hypothesized that there is a similar
tum infections. antifungal susceptibility profile for both Geotrichum spe-
cies. Conventional amphotericin B, alone or associated with
other antifungal agents, has been the drug most frequently
66.1.3 Clinical Features, Pathogenesis, and Therapy
employed in first-line therapy of G. capitatum infections. The
The clinical features of geotrichosis frequently resemble those low number of cases treated with alternative antifungal regi-
of superficial and invasive candidiasis, although there are some mens does not allow any comparative evaluation of the effi-
significant differences. Both G. candidum and G. capitatum cacy of these strategies. Several investigators have proposed
are recognized as a cause of superficial, mucosal and skin dual-drug therapy with amphotericin B and flucytosine as
infections not only in immunocompromised patients but also a valid option, but there is no evidence in the literature that
in healthy subjects, whereas deep invasive infections have this (or any other) drug combination was more effective than
been reported almost exclusively for G. capitatum in patients single-drug regimens. As for the newer antifungal drugs, it is
with underlying hematological diseases. currently impossible to make any predictions of their clinical
Invasive infections by G. candidum are mainly pulmo- efficacy. The literature contains reports of only a few cases in
nary or bronchopulmonary but also cutaneous and dis- which voriconazole or caspofungin was used as initial or sal-
seminated. The first cases of bloodstream and invasive vage antifungal therapy [27,46,47]. However, in vitro suscepti-
disseminated infection by this pathogen have been reported bility findings can be a useful guide in selecting an antifungal
in 1952 and 1965, respectively [30–32]. Invasive infections regimen for geotrichosis. Amphotericin B and voriconazole
may be localized to the skin and soft tissues, for example, are very active drugs against this yeast whereas a variable
when a traumatism enables fungal inoculation [33], but dis- susceptibility to flucytosine, fluconazole, and itraconazole has
seminated geotrichosis has been rarely reported in some been observed [27,48]. Importantly, echinocandins are intrin-
patients immunosuppressed by HIV infection or chemo- sically not active against Geotrichum species [28].
therapy [34–42].
The first case of G. capitatum infection was reported in 66.1.4 Diagnosis
1965 [43]. The fungus is isolated from blood in over 70%
of invasive infections, and approximately two-thirds of the 66.1.4.1 Conventional Techniques
reported cases of fungemia are associated with clinically or Early diagnosis of deep-seated Geotrichum species infection
microbiologically documented invasive tissue localization; in is difficult to establish because clinical symptoms are not
a very small number of cases, a central venous catheter is specific and frequently resemble those of invasive candidosis.
the portal of entry of the infection [28]. These clinical fea- Blood cultures are the “gold standard” for diagnosis of inva-
tures are in contrast with those observed in cancer patients sive G. capitatum infections, although they usually take sev-
with candidemia, as shown in a large prospective European eral days to show detectable growth of yeast cells and remain
study on candidemia in cancer patients in which only 10% of negative in about 30% of cases. As anticipated, the isolation
patients had a clinically or microbiologically histologically of Geotrichum species from respiratory tract is easy to obtain
documented organ involvement and a correlation of funge- but of ambiguous significance considering the possibility of a
mia with a central venous catheter was demonstrated in 31% simple colonization.
of cases [44]. The EORTC/IFICG and NIAID/MSG defini- Noncultural methods could be useful for the improve-
tions provide no specific indications on the significance of ment of laboratory diagnosis, but clinical data on G. capi-
G. capitatum recovery from respiratory-tract specimens [45]. tatum antigens or DNA detection are not available to date.
Since the microorganism is the potential component of the Consequently, the microbiological diagnosis of geotrichosis
normal microbial flora of the human digestive and respira- continues to be represented by conventional cultural methods
tory tracts, it is difficult to distinguish between colonization of blood and, less frequently, of other specimens.
and infection. However, several experiences seem to support Recently, false-positive reactivity in the Platelia
the view that the clinical significance of isolation of G. capi- Aspergillus assay, a sandwich enzyme-linked immunosor-
tatum from sputum in neutropenic patients seems to be com- bent assay (ELISA) assay for the detection of the Aspergillus
parable with that of molds and Cryptococcus neoformans, galactomannan antigen, has been observed in a series of
and for this reason, the recovery of G. capitatum from respi- hematologic patients with invasive G. capitatum infection
ratory tract specimens of patients with clinically documented [49,50]. In vitro studies showed that G. capitatum exoanti-
pneumonia may be indicative of probable pulmonary geotri- gens may be cross-reactive with the Aspergillus galacto-
chosis [28]. mannan assay. This cross-reactivity may be related to the
The outcome of G. capitatum invasive infections appears presence of galactomannans demonstrated in the cell wall
to be worse than that associated with invasive Candida infec- of Geotrichum spp. and their teleomorphs Dipodascus and
tions, with a mortality rate of about 60% of cases [28]. Galactomyces [51]. This unexpected reactivity in the Platelia

Aspergillus test could be considered a useful diagnostic and real-time monitoring of invasive infection by Geotrichum spp.
prognostic tool in the management of Geotrichum infections. is hampered by the lack of noncultural markers [28,49,50].
A positive galactomannan assay result for a patient with an
infective clinical picture suggestive of Candida infection 66.1.4.2.1 Detection and Identification
could indicate the possibility of a G. capitatum infection, On this premise, the application of polymerase chain reac-
considering also that galactomannan serum detection may tion (PCR) technology to molecular diagnostics holds great
precede by several days the documentation of the infection promise for the early identification of Geotrichum spp. For
by conventional cultures. rapid detection of Geotrichum spp. in clinical samples, target
gene sequences and practicable primers have been combined
66.1.4.2 Molecular Techniques with effective DNA extraction methods (Table 66.1). PCR
Early detection of infectious fungi has a significant impact assays may be applied to different clinical samples, including
on the clinical management of many fungal diseases. From respiratory specimens, blood fractions (serum, plasma, and
a clinical perspective, early and accurate diagnosis of patho- whole blood), urine, and other sterile body fluids [53–55].
genic fungi is crucial to contain the infection and minimize DNA targets used in molecular diagnostic tests for
empirical antifungal therapy, which is the primary cause of Geotrichum spp. include the group of genes encoding the
the emergence of antifungal resistance [52]. All these issues nuclear rRNAs (Figure 66.2a) that are present in multiple
apply to Geotrichum infections. copies and repeated in tandem. In yeasts, the 5.8S, 18S,
Molecular methods have solved many problems of myco- and 25S–28S rRNA genes (or ribosomal DNAs [rDNAs])
logical diagnosis by providing species-level identification of are transcribed as a 35S–40S precursor encompassing both
fungi through sequence-based analysis of suitable taxonomic internal and external transcribed spacers (ITS and ETS,
markers, yielding a more accurate identification and short- respectively), which are spliced out of the transcript [56]. The
ening of the time required for microbiological confirmation conserved regions alternate with divergent domains (D1 and
of life-threatening fungal infections. Moreover, DNA-based D2) and highly variable regions (ITS). Between each cluster,
techniques have become essential tools to trace the epide- there is an intergenic spacer (IGS), while a 5S rRNA gene has
miology of many fungal diseases. In general, nucleic acid- a variable position and transcription direction, depending on
based methods are (i) less subjective than microscopy- or the fungal group (Figure 66.2a). The ribosomal conserved
culture-based methods, (ii) unaffected by the growth condi- sequences can be used to screen for yeast presence, while
tions of the fungus, (iii) capable of discriminating between the variable ITS and D1/D2 sequences provide species-level
phenotypically undistinguishable species, and (iv) more rapid identification [55,57,58].
than traditional methods. Because of their high specificity Many in-house PCR assays have been developed for the
and sensitivity, molecular assays have become increasingly identification of Geotrichum spp. PCR with fungal primers
adopted by clinical laboratories to complement the informa- designed on the conserved sequences of the 5.8S and 25S–
tion gained from conventional methods. 28S rDNA results in amplification of the species-specific
In the large majority of invasive infections, Geotrichum ITS2 regions, which are variable in amplicon length [59].
spp. is isolated from blood. However, blood cultures usu- The variability in length of the ITS2 region is utilized to
ally take several days to show detectable growth, and in the make specific diagnosis of pathogenic fungal isolates from
remaining cases of deep-seated infection without fungemia, blood and tissues, including G. candidum. Fluorescently
the diagnostic approach may be very difficult. Moreover, labeled amplicons can be rapidly and accurately sized
Table 66.1
DNA-Based Assays for Identification of Geotrichum spp.
DNA Target Primer Sequence (5′–3′) Detection Method Reference
18S rRNA and ITS1 NS1 GTAGTCATATGCTGTCTC PCR + restriction analysis Senses-Ergul et al. [60]
ITS2 GCTGCGTTCTTCATCGATGC
5.8S and 28S rRNA ITS41 TCCTCCGCTTATTGATAGC PCR + fluorescent capillary electrophoresis Turenne et al. [59]
ITS86 GTGAATCATCGAATCTTTGAAC
ITS1 and ITS2 ITS1 TCCGTAGGTGAACCTGCGG PCR + sequencing White et al. [74]
ITS1 and ITS2 ITS4 TCCTCCGCTTATTGATATGC PCR + restriction analysis Florez et al. [61]
ITS5 GGAAGTAAAAGTCGTAACAAGG
D1/D2 NL1 GCATATCAATAAGCGGAGGAAAAG PCR + sequencing Ersoz et al. [53]
NL4 GGTCCGTGTTTCAAGACGG
D2 U1 GTGAAATTGTTGAAAGGGAA PCR (±sequencing) Putignani et al. [55]
U2 GACTCCTTGGTCCGTGTT

Geotrichum 585
a
a
r
l
l
M
Ca
Cp
Gc
Ck
Cg
5΄-ETS ITS1 ITS2 D1 D2 3΄-ETS IGS1 IGS2
18S (SSU) 5.8S 25/28S (LSU) 5S
(a)
Candida spp. (~260 bp)

Geotrichum spp. (~125 bp)
(b)
Cal 1- GTGAAATTGTTGAAAGGGAA GGGCTTGAGATCAGACTTGGTATTTTGCATGTTGC -- TCTCTCGGGGGC---- GGCCGCTGCGGTTTACCGGGCC
Cpa 1- GTGAAATTGTTGAAAGGGAA GGGCTTGAGATCAGACTTGGTATTTTGTATGTTAC -- TCTCTCGGGGGT---- GGCCTCTACAGTTTACCGGGCC
Cgl 1- GTGAAATTGTTGAAAGGGAA GGGCATTTGATCAGACATGGTGTTTTGCGCCCCTTG - CCTCTCGTGGGCTTGGGACTCTCGCAGCTCACTGGGCC
Ckr 1- GTGAAATTGTTGAAAGGGAA GGGTATTGCGCCCGACATGG- GGATTGCGCACCGCTGCCTCTCGTGGGC-- GGCGCTCTGGGCTTTCCCTGGGCC
Gca 1- GTGAAATTGTTGGAGGGGAAGG------------------------------------------------------ -------------------
Gcl 1- GTGAAATTGTTGGAGGGGAAGG---------------------------------------------------------- ---------------
************ * ******** * * *** *** *** ****** *** * * * *****
Cal AGCATCGGTTTGGAGCGGCAGGATAATGG- CGGAGGAATGTGGCACGGCTTCT---- GCTGTGTGTTATAGCCTCTGAC - GATACTGCCAGCCTAGA

Cpa AGCATCAGTTTG- AGCGGTAGGATAAGTG- CAAAGAAATGTGGCACTGCTTCG---- GTAGTGTGTTATAGTCTTTGTC - GATACTGCCAGCTTAGA
Cgl AGCATCGGTTTT - GGCGGCCGGAAAAAAC- CTAGGGAATGTGGCTCTGCGCCTCGGTGTAGAGTGTTATAGCC CTGGGG- AATACGGCCAGTCGGGA
Ckr AGCATCGGTTCT - TGCTGCAGGAGAAGGGGTTCTGGAACGTGGCTCTTC---------- GGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGA
Gca ------------------------------------------------------------------------------------- CGATGGTAGGAA
Gcl -------------------- ----------------------------------------------------------------- CGATGGTAGGAA
* * * * * * * * * * * * *** ** * ** ***** * * * ***** ** ** * * * * * * * *
Cal CCGAGGACTGCGGT--- TTTTACCTAGGATGTTGGCA TAATGATCTTAAGTCGCCCGTCTT -------- ---- GAAACACGGACCAAGGAGTC- 258

Cpa CTGAGGACTGCGGC--- TTCGGCCTAGGATGTTGGCATAATGATCTTAAGTCGCCCGTCTT ------------ GAAACACGGACCAAGGAGTC- 257
Cgl CCGAGGACTGCGATACTTGTTATCTAGGATGCTGGCATAATGGTTATATGCCGCCCGTCTT ------------ GAAACACGGACCAAGGAGTC- 269
Ckr CCGAGGACTGCGGCCGTGTAGGTCACGGATGCTGGCAGAACGGCGCAACACCGCCCGTCTT------------ GAAACATGGACCAAGGAGTC- 259
Gca TAAGAGGCTGCGGT-- TTGAAATAATTGTTTTTCGGGCCACGGTCTCCTGA - GCCTGCTTTCGCACCC GTCTTGAAACACGGACCAAGGAGTC- 124
Gcl TAAGAGGCTGCGGT-- TTGAAATAAATGTTTTTCGGGCCACGGTCTCCTGA -GCCTGCTTTCGCACNNNNNNNGAAACACGGACCAAGGAGTC- 124
* ***** ** * * * * ** * * * * * * ****** *************
(c)
Figure 66.2 (a) Physical organization of the fungal rRNA gene cluster. All cluster components are included in the representation. SSU,
small subunit (18S rRNA gene); LSU, large subunit (25S–28S rRNA gene); D1 and D2 regions are the highly divergent regions of the LSU
rRNA gene, shown as black boxes. ITS1, ITS2, and ETS1, ETS2 represent intergenic and extragenic spacers, as shown as dark gray boxes,
respectively. Intergenic spacers (IGS1 and IGS2), between each cluster are nontranscribed regions which separate rRNA clusters from one
another along the chromosome. (b) Agarose gel electropherogram of PCR amplicons generated with primers defining the D2 region of the
25S–28S rRNA gene. Differences between Candida and Geotrichum spp. are shown by the different size of the amplicon (ca. 260 and
125 bp, respectively). (c) Alignment of the D2 region of the 25S–28S rRNA genes of C. albicans, C. parapsilosis, C. glabrata, C. krusei,
G. capitatum, and G. clavatum. The 5′- and 3′-DNA regions matching the forward and reverse primer sequences are in bold. The boxed
region denotes a deletion on the D2 region of Geotrichum spp. 25S–28S rRNA genes leading to smaller sized (124 bp) amplicon. Conserved
positions are marked with an asterisk below the alignment. Abbreviations: Cal, Candida albicans; Cgl, Candida glabrata; Ckr, Candida
krusei; Cpa, Candida parapsilosis; Gca, G. capitatum; Gcl, G. clavatum.
with an automated capillary electrophoresis system [59] amplicon size of pathogenic Geotrichum spp. is shorter than
(Table 66.1). PCR of the ITS1 and ITS2 regions and direct that obtained for other pathogenic yeast isolates (ca. 120 bp
sequence analysis of the amplicons, coupled with classical vs. a mean size of ca. 260 bp; Figures 66.2b and c) [55].
detection methods, proved effective for identification of G. To the best of our knowledge, there are no commercial sys-
capitatum as the etiologic agent of superficial geotrichosis tems available capable of identifying G. capitatus, including
in an old patient [54]. During a mini epidemic of G. capita- those based on rRNA gene sequence analysis (MicroSeq ID
tum involving three cases, PCR and sequencing analysis of Fungal gene library, Applied Biosystems, version 2.0). Since
the D1/D2 region was performed to ensure identification of G. capitatum reference sequence has not yet been included
this organism using the primer pair NL1 and NL4 (Table in the commercial MicroSeq ID Fungal gene library, D2
66.1). In the same study, randomly amplified polymorphic sequencing and interrogation of free-access databases (e.g.,
DNA (RAPD) analysis was applied to allow discrimina- the GenBank at: http://www.ncbi.nlm.nih.gov/) is advisable
tion among isolates [53]. More recently, the hypervariable for identification of this species [55].
D2 region was found to contain significant information for Another common approach for Geotrichum species dif-
confident identification of Geotrichum isolates at the spe- ferentiation relies upon differences in restriction fragment
cies level [55]. G. capitatum can be easily differentiated length polymorphism (RFLP) profiles obtained with differ-
from other yeast species, most prominently Candida spp., ent restriction enzymes. Restriction enzyme digestion of the
by simple visual inspection of the D2 amplicon size fol- ITS1-18S rRNA amplicons [60] and ITS1–5.8S–ITS2 prod-
lowing agarose gel electrophoresis, and identification can ucts [61] has successfully been applied to identification of
be confirmed by amplicon sequencing (Table 66.1). The D2 G. candidum (Table 66.1).

New advanced molecular approaches based on matrix- (microsatellites) have been used to amplify the regions located
assisted laser description ionization time of flight-mass between two microsatellites in a large number of eukaryotic
spectrometry (MALDI-TOF) are currently under setting microorganisms [68]. Used in combination, RAPD-PCR
for the rapid detection and identification of fungi in clini- and RAM-PCR techniques permit G. candidum strains to
cal microbiology laboratories. A recent study demonstrates be unambiguous differentiated by using three RAPD probes
that MALDI-TOF can be used as a fast technique for reliable and one RAM probe [66,67]. Different from RAPD-PCR, the
identification of Geotrichum spp. [62]. With state-of-the art RAM-PCR involves large primers and an annealing tempera-
instruments and user-friendly software, mass spectrometry ture at 50°C, leading to higher hybridization specificity [66,67].
specialists are not needed and laboratory technicians are able The RAM-PCR method therefore seems to be suitable for the
to run the system after a few days of training. Investment and purpose of inter-laboratory analysis. Nevertheless, obtaining
maintenance of the instrument are balanced by low consum- reproducible fingerprints requires standardized experimen-
able costs [62]. MALDI-TOF appears to be a very promising tal conditions, such as using the same thermal cycler, cycling
technique, which could open a new horizon for the fast and conditions, and concentrations of reagents (template DNA,
reliable diagnosis of fungal infections. PCR buffer, dNTPs, and Taq DNA polymerase).
It is foreseeable that improvement in DNA sequenc- To determine the size of the chromosomal DNA mol-
ing technology together with the development of free user- ecules from 13 strains of G. candidum differing in habitat
friendly sequence analysis software and validated yeast and morphotype, pulsed field gel electrophoresis (PFGE)
databases will facilitate transfer of genotypic identification has been used without previous macrorestriction analy-
to the clinical laboratory routine. Thus, molecular-based sis [66,67]. The strains investigated had five to eight chro-
Geotrichum spp. identification is ready to be used not only mosomes, 0.6–4.5 Mb in size. PFGE profiles, defined as
by specialized research facilities but also in the clinical diag- electrophoretic karyotypes, showed a high degree of poly-
nostic laboratory. morphism, indicating considerable variability between
strains. Notably, genome size and the presence of large chro-
66.1.4.2.2 Typing mosomes appeared correlate with the morphotype [66,67].
Among the many potential applications for strain typing are G. candidum strains with a mold-like or intermediate mor-
outbreak analysis and environmental monitoring, patient photype tended to have larger genomes than strains with a
monitoring and treatment follow-up, local and global epi- yeast-like morphotype.
demiology, database construction, strain identification, and
many more. Typing of isolates helps to define prevention
measures and improved guidelines for patient management 66.2 Methods
due to a greater understanding of the potential sources of
infections.
It has been suggested that a number of Geotrichum spp. Specimen handling and preparation have a significant
cases have a nosocomial origin, emanating from a com- impact on the performance of molecular diagnostic tests
mon source within the hospital environment [17]. Typing of for fungal detection. The sample preparation method should
G. capitatum isolates by molecular methods made it pos- release intracellular DNA from the fungal cell wall, con-
sible to demonstrate [17,63] or exclude [64] clonal correlation centrate DNA targets that may be present in very small
among clinical isolates in the nosocomial setting, supporting amounts, and eliminate contaminants, potential inhibitors,
the value of molecular typing in investigation of epidemio- and other extraneous materials without degrading the DNA.
logical correlates of G. capitatum. The availability of an easy-to-perform DNA extraction
With the development of molecular typing, it has become procedure, providing pure DNA devoid of PCR inhibitors,
possible to demonstrate inter- or intraspecific DNA polymor- would be ideal for any PCR-based diagnostic test. There are
phism in fungi despite a lack of information about the target a multitude of nucleic acid extraction techniques [69]. The
sequences. One PCR-based method independent of whole preferable method represents a compromise between effi-
genome knowledge is RAPD, which has long been applied to ciency, purification yields, and transferability to the labora-
prokaryotic genomes and successfully extended to eukaryotic tory routine. DNA may be extracted using in-house methods,
microorganisms, including G. candidum [17,25,53,61,65]. commercial kits, and automated commercial techniques.
However, RAPD analysis exhibits a low level of reproducibil- Mechanical destruction with glass beads and freeze-thaw
ity because of the low-stringency conditions used in the PCR, steps with liquid nitrogen or a heat-alkali treatment have
and these conditions lead to mismatched pairings. Therefore, successfully been applied [69,70]. DNA extraction following
the patterns may be complex and hardly comparable between enzymatic digestion of the fungal wall is another effective
laboratories [66,67]. method. However, the use of toxic chemicals such as phe-
RFLP analysis of whole-cell DNA has also been applied nol–chloroform mixtures further limits the use of in-house
to differentiate clinical isolates of G. capitatum [63,64]. methods in the clinical routine [71]. The use of commer-
More recently, using the random amplified microsatellites cial kits (e.g., QIAmp Tissue, Qiagen; GeneReleaser,
technique (RAM or inter-single sequence repeats, [ISSR]), BioVentures; Puregene D 6000, Gentra; Dynabeads DNA
the specific oligonucleotides of single sequence repeats DIRECT, Dynal; and DNAzol, Molecular Research Center)

Geotrichum 587
shortens the extraction procedure, but the efficiency of Note. PCR analysis of the hypervariable D2 region of the
extraction of fungal DNA can vary considerably between fungal 25S–28S rDNA provides clear differentiation between
commercial kits [69,70]. Automated commercial techniques Geotrichum and Candida based on the D2 amplicon size, and
(e.g., MagNA Pure LC; Roche Diagnostics) are better suited identification can be confirmed by amplicon sequencing. The
for routine clinical laboratories. High-speed cell disrup- D2 amplicon size of pathogenic Geotrichum spp. is shorter
tion (HSCD) incorporating chaotropic reagents and lysing than that obtained for other pathogenic yeast isolates (ca. 120
matrices provides rapid disruption of cells and high yields bp vs. a mean size of ca. 260 bp [Figures 66.2b and c]) [55].
of DNA [72].
A simple thermolytic procedure for G. capitatum DNA 66.2.2.2 RFLP Analysis
extraction has been used as routine protocol in the clinical Procedure
laboratory for direct PCR amplification of the D2 region
[55]. Sample contamination should also be considered 1. Prepare a PCR mixture for the amplification of
as major problem in molecular diagnosis because of the ITS1 or ITS1 and ITS2 regions and perform the
extremely high sensitivity of all nucleic acid amplification PCR amplification in a thermocycler using prim-
techniques. Fungal spores, such as conidia from Aspergillus ers and PCR conditions described by Senses-Ergul
spp. and other molds, might be present in the air. Thus, air- et al. [60] and Florez et al. [61].
borne spore inoculation during the DNA extraction process 2. Prepare an RFLP mixture containing purified
could potentially lead to false-positive results, especially in PCR amplicons, 1× corresponding restriction buf-
pan-fungal assays [73]. However, the risk of contamination fer (dependent on the enzyme used and the manu-
is not higher in fungal PCR assays than in other diagnostic facturer) and restriction enzymes HaeIII and MspI
PCRs if general precautions are taken. In order to control [60], or AluI, HaeIII, HhaI, HinfI, RsaI, and Sau3AI
naturally arising DNA from airborne sources (e.g., fungal [61]. Incubate overnight at the temperature indicated
spores), negative controls should be included during each by the manufacturer.
DNA extraction procedure. Negative controls consist of 3. Seperate the fragments on 3% agarose gels, achieve
sterile water or blood from healthy individuals and should visualization by staining with ethidium bromide
be subjected to all preparation steps in parallel with the (0.5 μg/mL), and photograph the results under UV
extracted samples [72]. light [60].
66.2.2.3 Automated Fluorescent

66.2.2 Detection Procedures Capillary Electrophoresis
66.2.2.1 Identification of Geotrichum by PCR To detect fluorescently tagged amplicons by an automated
Amplification of the D2 Region capillary electrophoresis sequencer, a capillary is filled
with a replaceable liquid polymer for high resolution under
Procedure
denaturing conditions. The sample containing fluorescent
The following procedure is adapted from Ref. [55]. PCR product is injected into the capillary and is subject to
constant voltage and temperature (60°C). The fluorescently
1. A single fungal colony >1 mm in diameter is picked tagged fragments of DNA resulting from amplification by
with a micropipette tip, suspended in 20 μL of ster- fluorescent primers are detected by a laser beam and auto-
ile doubly distilled water, and thermolyzed for 5 min matically analyzed by the instrument [58]. Up to hundreds
at 95°C. of samples can be loaded depending on the instrument, and
2. Prepare a PCR mixture (50 μL) containing 2 μL of each run is completed within 30 min. Results can be viewed
thermolysate, 750 mM Tris–HCl pH 8.8, 200 mM as soon as each sample has been processed. The instrument
(NH4)2SO2, 0.1% Tween 20 (v/v), 1.5 mM MgCl2, detects up to four various fluorescent “colors,” allowing for
0.2 mM of each dNTP, 25 pmol primer U1 (5′-GTGA the inclusion of an internal standard with each run. Accurate
AATTGTTGAAAGGGAA-3′) and 25 pmol primer fragment size analysis is based on the electrophoretic mobil-
U2 (5′-GACTCCTTGGTCCGTGTT-3′), and 0.7 unit ity of the sample relative to the internal standard [58].
of Taq DNA polymerase (Red Hot DNA polymerase,
Abgene, Epsom, United Kingdom) [55]. 66.2.2.4 DNA Sequence Analysis
3. Perform the PCR amplification in a thermocycler A variety of automated sequencers are available on the mar-
using a cycling program consisting of a denaturation ket, but their use requires specialized personnel with direct
step at 95°C for 3 min; 35 cycles of 94°C for 45 se and indirect costs often unaffordable by clinical laboratories.
50°C for 45 s, and 72°C for 1 min; and a final exten- However, many companies offer sequencing services at rea-
sion step at 72°C for 7 min [55]. sonable price and provide fast (internet-based) reporting of
4. Separate the PCR products on a 2% agarose gel and sequencing data.
stain with ethidium bromide (0.5 μg/mL). The pres- Sequence-based identification is achieved by a simple
ence of a single product of expected size is inter- process involving free internet-based tools for sequence
preted as evidence of successful amplification. quality assessment and database interrogation at GenBank

(http://www.ncbi.nlm.nih.gov/) or EMBL (http://www.ebi. will be unavoidable steps. Lastly, the clinical meaning of a
ac.uk/embl/), using BLASTN (http://www.ncbi.nlm.nih.gov/ positive PCR test should be interpreted in the light of our
BLAST/) and FASTA homology search algorithms (http:// knowledge on the clearance kinetics of fungal DNA in the
www.ebi.ac.uk/fasta33/nucleotide.html). infected patient; this would help in distinguishing between
colonized individuals and infected patients.
66.3 C
onclusions and
References
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67 Kluyveromyces
Dongyou Liu
Contents
67.1 Introduction...................................................................................................................................................................... 591
67.1.3 Diagnosis.............................................................................................................................................................. 592
67.2 Methods............................................................................................................................................................................ 592
67.2.2.1 Real-Time PCR Amplification and Sequencing Analysis of ITS Regions............................................ 592
67.2.2.2 PCR and Sequencing Analysis of ITS1 and ITS2................................................................................. 592
67.3 Conclusion........................................................................................................................................................................ 593
References.................................................................................................................................................................................. 593
67.1 Introduction Kluyveromyces phaffii group encompassing Kluyveromyces

blattae, Kluyveromyces phaffii, and Kluyveromyces yar-
67.1.1 Classification and Morphology rowii; (2) the Kluyveromyces marxianus group consisting
The genus Kluyveromyces is an ascomycetous yeast belonging of Kluyveromyces aestuarii, Kluyveromyces dobzhanskii,
to the family Saccharomycetaceae, class Saccharomycetes, Kluyveromyces lactis, K. marxianus, and Kluyveromyces wick-
subphylum Saccharomycotina, phylum Ascomycota, and erhamii; (3) the Kluyveromyces thermotolerans group includ-
kingdom Fungi. The family Saccharomycetaceae consists of ing Kluyveromyces thermotolerans, Kluyveromyces waltii,
28 genera: Ascobotryozyma, Citeromyces, Debaryomyces, and Saccharomyces kluyveri; and (4) the Saccharomyces
Dekkera, Eremothecium, Issatchenkia, Kazachstania, cerevisiae group containing the remaining Kluyveromyces
Kluyveromyces, Kodamaea, Komagataella, Kregervanrija, species and the reference Saccharomyces species (sensu lato
Kuraishia, Lachancea, Lindnera, Lodderomyces, and sensu stricto) and Candida glabrata [8].
Nakaseomyces, Nakazawaea, Naumovia, Pachysolen, Pichia, Morphologically, Kluyveromyces marxianus (anamorph:
Saccharomyces, Saturnispora, Tetrapisispora, Torulaspora, Candida kefyr) may be similar to other Candida spp. On
Vanderwaltozyma, Williopsis, Zygosaccharomyces, Sabouraud dextrose agar at 25°C, Kluyveromyces marxianus
and Zygowilliopsis as well as some unclassified colonies are cream to brown, glossy, and smooth. On corn-
Saccharomycetaceae [1]. meal agar at 25°C for 72 h, the fungus forms highly branched
In turn, the genus Kluyveromyces covers 16 recognized pseudohyphae. Under the microscope, K. marxianus shows
species: Kluyveromyces aestuarii, Kluyveromyces blat- evanescent asci containing 2–4 cresent-shaped spores [9].
tae, Kluyveromyces dobzhanskii, Kluyveromyces hubei-
ensis, Kluyveromyces lactis, Kluyveromyces lodderae,
Kluyveromyces marxianus, Kluyveromyces nonfermentans,
Kluyveromyces phaffii, Kluyveromyces piceae, Kluyveromyces Kluyveromyces marxianus is used commercially to produce
siamensis, Kluyveromyces sinensis, Kluyveromyces thermo- the lactase enzyme. In addition, it is also produced as a nutri-
tolerans, Kluyveromyces waltii, Kluyveromyces wickerhamii, tional yeast and bonding agent for fodder and pet food, and as
and Kluyveromyces yarrowii, in addition to 20 unassigned a source of ribonucleic acid in pharmaceuticals.
species [1–4]. Of these, Kluyveromyces marxianus (synonyms: The first documented case of Kluyveromyces marxianus
Kluyveromyces cicerisporus and Kluyveromyces fragilis; ana- (Kluyveromyces fragilis) involved an immunosuppressed car-
morph: Candida kefyr or Candida pseudotropicalis) has been diac transplant patient with pulmonary manifestations. The
implicated in burn wounds and blood and vaginal infections K. marxianus isolates were sensitive to 5-fluorocytosine and
in humans [5–7]. miconazole as well as amphotericin B. The patient responded to
Based on the analysis of mitochondrial cytochrome-c oxi- amphotericin B treatment and recovered without sequelae [20].
dase II gene sequences, the genus Kluyveromyces appears as a K. marxianus has also been found to cause vaginitis [5,9,10].
polyphyletic taxon that consists of the four main groups: (1) the A K. marxianus strain was isolated from a 33 yo woman with
591

clear signs of vaginitis, leucorrea, pruritis and vulvovaginal 67.2.2 Detection Procedures
erythema [9]. In addition, seven K. marxianus (C. kefyr) iso-
lates were identified from women showing thick curdy vaginal 67.2.2.1 Real-Time PCR Amplification and
discharge, vulvovaginal itching and discomfort [10]. Sequencing Analysis of ITS Regions
67.1.3 Diagnosis green DNA-binding dye and amplicon-melting temperature
Pathogenic yeasts are responsible for bulk of human clini- forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
cal cases of invasive mycosis that often produces indistin- reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
guishable symptoms and results in significant morbidity and of the fungi is verified by subsequent sequencing analysis.
mortality, especially in high-risk/immunocompromised indi-
viduals. Despite their diversity and taxonomical complexity, Procedure
yeasts are renowned for their apparent lack of characteristic
morphological features, contributing to the difficulty in the 1. PCR mixture is composed of 1 × Lightcycler
identification and diagnosis of disease-causing yeasts includ- FastStart DNA Master Hybridization Probes mixture
ing Kluyveromyces on the basis of macroscopic, microscopic, (Roche Applied Science) containing deoxynucleo-
and biochemical examination [11]. side triphosphates, FastStart Taq DNA polymerase,
Molecular techniques such as PCR amplification and and 1 mM MgCl2 (additional MgCl2 is added to a
sequencing analysis offer a rapid, sensitive, and specific final concentration of 4.6 mM), 0.4 μM each of ITS1
alternative to conventional methods for yeast speciation. By forward and ITS4 reverse primers, 1× SYBR green
exploiting the nucleotide differences at the small-subunit (Molecular Probes), and 3 μL template DNA.
(18S) rRNA gene, the D1/D2 domains of the large-subunit 2. Thermal cycling parameters include 95°C for
(28S) rRNA gene, and the internal transcribed spacer (ITS) 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s,
regions, accurate determination of yeasts of interest can be and 76°C for 30 s; and a final extension at 72°C for
achieved within a day instead of weeks that is required by 2 min.
biochemical testing [12–16]. 3. The quality of the amplicon is determined using the
45 s hold at 55°C, and 5 s/°C) using the RotorGene
67.2 Methods
3000 (Corbett Robotics, Inc).
67.2.1 Sample Preparation 4. The amplified product is purified for bidirectional
DNA can be extracted from yeast culture by the glass bead microliters of Big Dye Terminator Ready Reaction
lysis method. A loopful of culture from malt yeast extract Mix v. 1.1 (Applied Biosystems) is added to 4 μL of
agar is transferred to 200 μL lysis buffer (2% Triton X-100, each primer (0.8 pmol/μL) and 3 μL of purified PCR
1% SDS, 10 mM NaCl, 10 mM Tris/HCl pH 8.0, and 1 mM product. Cycle sequencing is performed with a 9700
EDTA) in a 1.5 mL tube, and 200 μL phenol/chloroform/isothermal cycler (ABI), using 25 cycles of 96°C for
amyl alcohol (25:24:1) is added. Cells are lysed by vortexing 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing
for 4 min in the presence of 20 mg glass beads. The aqueous reaction products are passed through a Sephadex
layer is treated once more with phenol/chloroform/isoamyl G-50 fine column to remove unincorporated dye ter-
alcohol, and the DNA is precipitated with chilled 2-propa- minators. Purified sequencing reaction products are
nol. The pellet is washed with 70% ethanol and finally resus- run on an ABI Prism 3100 Genetic Analyzer with a
pended in 50 μL TE buffer (10 mM Tris/HCl pH 8.0 and 50 cm capillary array.
1 mM EDTA) [17]. 5. Sequences are analyzed with the SmartGene
Alternatively, DNA may be prepared from yeast cultures Integrated Database Network software version
after 2 days of incubation on Sabouraud agar using Whatman 3.2.3 vr. SmartGene is a web-based software and
FTA filter paper technology. Briefly, yeast aqueous suspen- database system with reference sequences derived
sions (200 μL) are applied directly to Whatman FTA micro- from the National Center for Biological Information
cards, which are then placed, open, in a Panasonic microwave (NCBI) GenBank repository.
(800 W) while still damp and subjected to two cycles of 30 s
on full power, with a pause of at least 30 s between each Note. Sequence-based identifications are defined by percent
cycle. Punches (2 mm in diameter) are then removed from identity: species, ≥99%; genus, 93%–99%; and inconclusive,
dried FTA filters by using a Harris micropunch and washed ≤93%.
twice for 1 min each with 100 μL of Whatman FTA wash
reagent, followed by two washes for 1 min each with 100 μL
of TE buffer (10 mM Tris–HCl pH 7.5 and 0.1 mM EDTA). 67.2.2.2 P
CR and Sequencing Analysis
Washed filters are then dried for 5 min at 55°C on a dry heat of ITS1 and ITS2
block, and PCR mixes are added directly to the washed and Leaw et al. [15] utilized fungus-specific universal
dried FTA filter punches [16,18]. primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)

Kluyveromyces 593
and ITS2 (5′-GCATCGATGAAGAACGCAGC-3′) to 67.3 Conclusion

amplify the ITS1 region and universal primers ITS3
The genus Kluyveromyces is a member of the ascomycetous
(5′-GCATCGATGAAGAACGCAGC-3′) and ITS4
yeast family Saccharomycetaceae that comprises at least
(5′-GCATATCAATAAGCGGAGGA-3′) to amplify the
28 genera. Among the 16 recognized species and 20 unas-
ITS2 region. Subsequent sequencing analysis of the result-
signed species in the genus Kluyveromyces, Kluyveromyces
ing amplicons facilitated identification of a range of fungal
marxianus (synonyms: Kluyveromyces cicerisporus and
pathogens.
Kluyveromyces fragilis; anamorph: Candida kefyr or Candida
Procedure pseudotropicalis) has been occasionally involved in human
burn wound and blood and vaginal infections [20]. Given
1. PCR mixture (50 μL) is made up of 10 mM Tris– their close morphological and biochemical similarity to other
HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM Candida spp., identification of Kluyveromyces spp. using con-
each of deoxynucleoside triphosphates, 1.2 U of Taq ventional techniques is difficult, if not impossible. Application
DNA polymerase, 0.4 μM (each) of the ITS1 region of molecular techniques such as PCR amplification and
primers (ITS1/ITS2) or the ITS2 region primers sequencing analysis is essential for correct and efficient deter-
(ITS3/ITS4), and 2 μL (1–5 ng) of DNA template. A mination of Kluyveromyces from other related yeasts.
negative control is included in each run by replac-
ing the template DNA with sterile water in the PCR
mixture. References
2. Amplification is conducted with an initial 94°C for 1. The UniProt Consortium. Available at http://www.uniprot.
3 min; 30 cycles of 94°C, 60°C, and 72°C for 1 min org/, accessed on August 1, 2010.
each; and a final 72°C for 3 min. 2. Nagahama T et al. Kluyveromyces nonfermentans sp. nov.,
3. After checking the PCR products on 1.5% aga- a new yeast species isolated from the deep sea. Int J Syst
rose gel, the amplicons are purified using a com- Bacteriol. 1999;49:1899–1905.
3. Lachance MA. Current status of Kluyveromyces systematics.
mercial PCR cleanup kit. The DNA fragments are
FEMS Yeast Res. 2007;7:642–645.
sequenced using an ABI Prism 377 automated DNA 4. Am-In S, Yongmanitchai W, Limtong S. Kluyveromyces sia-
sequencer (Applied Biosystems) with a BigDye mensis sp. nov., an ascomycetous yeast isolated from water in
Terminator cycle sequencing kit (version 3.1; a mangrove forest in Ranong Province, Thailand. FEMS Yeast
Applied Biosystems). All amplicons are sequenced Res. 2008;8(5):823–828.
on both strands using primers ITS1 and ITS2 for 5. Corpus K et al. Candida kefyr, an uncommon but emerging
the ITS1 region and primers ITS3 and ITS4 for the fungal pathogen: Report of two cases. Pharmacotherapy
ITS2 region. 2004;24:1084–1088.
6. Gupta N et al. Epidemiology and molecular typing of
4. After sequencing, portions of the 18S, 5.8S, and Candida isolates from burn patients. Mycopathologia
26S rRNA gene sequences of the PCR products 2004;158:397–405.
are removed to obtain the exact ITS1 and ITS2 7. Miranda TT et al. Diversity and frequency of yeasts from
sequences. the dorsum of the tongue and necrotic root canals asso-
5. For all yeasts, the sequences of the 3′ ends of the ciated with primary apical periodontitis. Int Endod J.
18S and 5.8S rRNA genes are GCGGAAGGA 2009;42(9):839–844.
TCATTA and GTTTGAGCGTCATTT, respec- 8. Belloch C et al. Phylogeny of the genus Kluyveromyces
inferred from the mitochondrial cytochrome-c oxidase II
tively, and the sequences of the 5′ ends of the 5.8S
gene. Int J Syst Evol Microbiol. 2000;50:405–416.
and 26S rRNA genes are AAACTTTCAACAA and 9. García-Martos P et al. Sexual forms of yeasts in clinical sam-
GACCTCAAATCAG, respectively. ples. Mycopathologia 1996;136(2):67–70.
6. The ITS1 or ITS2 sequence is compared with 10. Erdem H et al. Identification of yeasts in public hospital pri-
those available at the GenBank databases using the mary care patients with or without clinical vaginitis. Aust N Z
BLAST sequence analysis tool (http://www.ncbi. J Obstet Gynaecol. 2003;43:312–316.
nlm.nih.gov/BLAST/) (blastn) with default settings 11. Hospenthal DR et al. Presumptive identification of Candida
species other than C. albicans, C. krusei, and C. tropicalis
except that sequences are not filtered for low com-
with the chromogenic medium CHROMagar Candida. Ann
plexity. Species identification is determined from Clin Microbiol Antimicrob. 2006;5:1.
the lowest expected value of the BLAST output. 12. Rimek D et al. Identification of contaminating fun-
gal DNA sequences in Zymolyase. J Clin Microbiol.
Note. For strains producing discrepant identification 1999;37(3):830–831.
between the methods based on phenotypic characteris- 13. Chen Y-C et al. Identification of medically important yeasts
using PCR-based detection of DNA sequence polymorphisms
in the internal transcribed spacer region 2 of the rRNA genes.
large-subunit rRNA gene is amplified with primers NL1 J Clin Microbiol. 2000;38:2302–2310.
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 14. Chen Y-C et al. Polymorphic internal transcribed spacer
(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for region 1 DNA sequences identify medically important yeasts.
species clarification. J Clin Microbiol. 2001;39:4042–4051.

15. Leaw SN et al. Identification of medically important yeast 18. Borman AM et al. An improved protocol for the prepa-
species by sequence analysis of the internal transcribed spacer ration of total genomic DNA from isolates of yeast and
regions. J Clin Microbiol. 2006;44(3):693–699. mould using Whatman FTA filter papers. Mycopathologia
16. Linton CJ et al. Molecular identification of unusual pathogenic 2010;169:445–449.
yeast isolates by large-ribosomal-subunit gene sequencing: 2 19. Pounder JI et al. Discovering potential pathogens among
years of experience at the United kingdom mycology refer- fungi identified as nonsporulating molds. J Clin Microbiol.
ence laboratory. J Clin Microbiol. 2007;45(4):1152–1158. 2007;45(2):568–571.
17. Bhardwaj S et al. PCR-based identification and strain typ- 20. Lutwick LI, Phaff HJ, Stevens DA. Kluyveromyces fragilis
ing of Pichia anomala using the ribosomal intergenic spacer as an opportunistic fungal pathogen in man. Sabouraudia
region IGS1. J Med Microbiol. 2007;56:185–189. 1980;18(1):69–73.

68 Pichia and Kodamaea
Dongyou Liu
Contents
68.1 Introduction...................................................................................................................................................................... 595
68.1.2.1 Pichia anomala...................................................................................................................................... 596
68.1.2.2 Pichia fabianii....................................................................................................................................... 596
68.1.2.3 Pichia farinosa...................................................................................................................................... 596
68.1.2.4 Kodamaea ohmeri................................................................................................................................. 597
68.2 Methods............................................................................................................................................................................ 598
68.2.2.1 PCR Identification of Pichia anomala.................................................................................................. 598
68.2.2.2 PCR Identification of Pichia guilliermondii (Candida guilliermondii)................................................ 598
68.2.2.4 Sequencing Analysis of ITS1 and ITS2................................................................................................. 599
68.3 Conclusion........................................................................................................................................................................ 600
References.................................................................................................................................................................................. 600
68.1 Introduction guilliermondii (anamorph: Candida guilliermondii var. guil-

liermondii), and Pichia norvegensis (anamorph: Candida
68.1.1 Classification and Morphology norvegensis) are of medical importance.
The genera Pichia and Kodamaea are ascomycetous The genus Kodamaea, also classified in the family
yeasts belonging to the family Saccharomycetaceae, class Saccharomycetaceae, contains five recognized species:
Saccharomycetes, subphylum Saccharomycotina, phylum Kodamaea anthophila, Kodamaea kakaduensis, Kodamaea
Ascomycota, kingdom Fungi. The family Saccharomycetaceae laetipori, Kodamaea nitidulidarum, and Kodamaea ohmeri
covers 28 genera: Ascobotryozyma, Citeromyces, and five unassigned species (i.e., Kodamaea sp. BG-3,
Debaryomyces, Dekkera, Eremothecium, Issatchenkia, Kodamaea sp. I11, Kodamaea sp. NRRL Y27634, Kodamaea
Kazachstania, Kluyveromyces, Kodamaea, Komagataella, sp. YS 85, Kodamaea sp. YS9). Of these, Kodamaea ohmeri
Kregervanrija, Kuraishia, Lachancea, Lindnera, (obsolete synonyms: Pichia ohmeri and Yamadazyma ohmeri;
Lodderomyces, Nakaseomyces, Nakazawaea, Naumovia, anamorph: Candida guilliermondii var. membranaefaciens),
Pachysolen, Pichia, Saccharomyces, Saturnispora, a yeast commonly used in food industry for fermentation in
Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, pickles, rinds, and fruits, is implicated in human infections
Zygosaccharomyces, and Zygowilliopsis, in addition to some [2,3].
unclassified Saccharomycetaceae [1]. Morphologically, Pichia and Kodamaea spp. resem-
The genus Pichia (obsolete synonyms: Hansenula, ble Candida spp. Being ascomycetous yeasts, Pichia and
Hyphopichia, and Yamadazyma) is an ascospore-producing Kodamaea often produce asci each containing one to four
teleomorph taxon consisting of > 100 recognized species hat-shaped or globose ascospores on acetate ascospore agar,
and more than 150 unassigned species. The anamorphs of Gorodkowa medium, V-8 medium, or 2% glucose malt agar.
the genus Pichia are found in the genus Candida, and both The ascospores of Pichia and Kodamaea are stained red with
Pichia and Candida form ascospores and share consider- Kinyoun stain, while the cells other than ascospores take the
able sequence similarity in the 28S rRNA gene. Among the counterstain [4].
large number of species within the Pichia genus, Pichia ano- P. anomala is identified on the basis of its inability to pro-
mala (obsolete synonym: Hansenula anomala; anamorph: duce germ tubes and urease, a standard sugar assimilation pat-
Candida pelliculosa), Pichia fabianii, Pichia farinosa (syn- tern, and the production of one to four hat-shaped ascospores.
onym: Pichia miso; anamorph: Candida cacaoi), Pichia Kodamaea (Pichia) ohmeri colonies show a color change from
595

pink to blue on CHROMagar Candida medium on which few January 2004), 1046 children were admitted to the pediatric
other yeast species share such color change [5,6]. ICU, and 17 (1.6%) of them developed P. anomala funge-
mia. The median age of infected children was 1.1 years, and
the overall mortality rate was 41.2% (7/17). On multivariate
analysis, the presence of a central venous catheter was identi-
Members of the Pichia and Kodamaea genera are free-living fied as a significant factor for this outbreak. Molecular stud-
ascosporogenous yeasts that constitute part of the normal ies showed that the outbreak was caused by a single strain,
or transient flora of the human throat and alimentary tract which was absent on health-care workers’ hands or in the
[7]. Generally considered as contaminants, some Pichia and environment.
Kodamaea spp. are now recognized as clinically significant Kalenic et al. [16] described an outbreak of Pichia
opportunistic pathogens in immunocompromised patients or (Hansenula) anomala infections in eight adult patients
individuals with other predisposing factors (e.g., prematurity, treated at a surgical ICU during a 5 month period. Among the
low birth weight, long duration of hospital stay, and existence risk factors analyzed for this outbreak (i.e., blood alkalosis,
of prosthetic valve). The clinical diseases caused by Pichia reduced urea, duration of hospitalization, bacteremia and col-
and Kodamaea spp. encompass urinary tract infection, peri- onization with Pseudomonas aeruginosa, and an APACHE
tonitis, enteritis, prosthetic valve endocarditis, nosocomial II score >17 during bacteremia or fungemia), only the dura-
fungemia, disseminated infection, etc. [8,9]. tion of blood alkalosis was significant in case patients.
Recently, Park et al. [17] reported a case of Pichia ano-
68.1.2.1 Pichia anomala mala-associated keratitis of the eye in a 50-year-old woman
Pichia anomala (formerly Hansenula anomala) has been with systemic lupus erythematous and Stevens-Johnson syn-
identified as an important opportunistic fungal pathogen drome. The patient presented with ocular pain, and culture
causing nosocomial fungemia, interstitial lung disease, endo- of corneal scrapings yielded P. anomala. Following topical
carditis, enteritis, and keratitis in children and adults [10–13]. amphotericin B, natamycin, and systemic inidazole treat-
Chakrabarti et al. [12] documented a Pichia anomala- ments, the patient showed a full recovery.
related outbreak of nosocomial fungemia involving 379 neo- In vitro susceptibility study of 58 isolates of Pichia ano-
nates and children (4.2% admissions) in the hospital pediatric mala to five antifungal drugs using two broth microdilution
wards over a period of 23 months (April 1996 to February methods clinical and laboratory standards institute (CLSI)
1998). Based on a culture prevalence survey, 14 (28%) out and European committer on antimicrobial susceptibility
of the 50 consecutive premature neonates were colonized testing (EUCAST) showed that Pichia anomala is generally
with P. anomala at the umbilicus, mouth, rectum, and groin sensitive to fluconazole, voriconazole, amphotericin B, and
sites on the date of delivery and 10 of these colonized neo- caspofungin, but less susceptibile to itraconazole [18].
nates later developed P. anomala fungemia. Neonates with
documented sepsis responded well to treatment with fluco- 68.1.2.2 Pichia fabianii
nazole or amphotericin B, or itraconazole. Significant risk Bhally et al. [19] documented Pichia fabianii as the cause
factors associated with P. anomala fungemia in premature of an infection in a 5-week-old female twin delivered at 25
neonates included lower gestational age, a very low birth and 37 weeks. The patient presented with respiratory distress
weight (<1500 g), and a longer duration of hospital stay. Upon syndrome and necrotizing enterocolitis, and was febrile,
analysis by multilocus enzyme electrophoresis, P. anomala thrombocytopenic, and requiring minimal ventilatory sup-
outbreak isolates (from both patients and health-care work- port. Blood cultures yielded a yeast, which was identified as
ers’ hands) appeared identical, suggesting that carriage on P. fabianii by sequencing a subunit of D2 of the large subunit
the hands of health-care personnel was likely to be respon- (LSU) rRNA gene. The organism was susceptible in vitro to
sible for dissemination of the fungus. The outbreak was only amphotericin B, fluconazole, and 5-fluorocytosine, and the
controlled after institution of a health education campaign patient responded to amphotericin B and removal of vascular
to improve hand-washing practices and after introduction of catheter. Hamal et al. [20] also isolated Pichia fabianii from
a nystatin-fluconazole prophylaxis to all premature neonates the blood of a patient with aortic valve endocarditis. The iso-
and high-risk infants. lates were initially identified biochemically as Candida pel-
In a separate study, Bakir et al. [14] reported nosocomial liculosa but confirmed subsequently as P. fabianii by direct
port catheter infection due to P. anomala in three children sequencing of the ITS2 region of rRNA gene.
receiving cancer chemotherapy, a preterm infant with blood-
stream infection, and an infant with severe combined immu- 68.1.2.3 Pichia farinosa
nodeficiency. All P. anomala isolates were susceptible to Pichia farinosa (synonym: Pichia miso) is a halotolerant
amphotericin B and fluconazole, and patients responded well yeast with widespread distribution and has been isolated
to amphotericin B treatment. The common clinical feature in from various sources such as miso soup and giraffe dung.
these patients was the presence of prior antimicrobial therapy. The fungus is characterized by the production of a salt-
Pasqualotto et al. [15] also reported an outbreak of Pichia mediated killer toxin that kills yeasts of several genera,
anomala fungemia occurring in a pediatric intensive care including Saccharomyces cerevisiae [21]. Adler et al. [22]
unit (ICU). During the study period (from October 2002 to reported a case of Pichia farinosa bloodstream infection in

Pichia and Kodamaea 597
a 13-year-old boy with anaplastic large-cell lymphoma, who the 18S rRNA gene, the D1/D2 domains of the 26S rRNA
had a Broviac catheter inserted into the right jugular vein. The gene and the ITS regions. The isolate was resistant to flucon-
patient presented with fever (40°C), vomiting, and local red- azole but sensitive to amphotericin B deoxylate. After treat-
ness and swelling along the catheter tunnel. Cultures of blood ment with amphotericin B deoxylate, the fever and cellulitis
specimens on Sabouraud dextrose agar grew yeast that was inflammation subsided and the patient was discharged in a
identified as Candida boidinii by using the API 20 C AUX stable condition.
identification system, which lacks the species P. farinosa in Taj-Aldeen et al. [36] reported the first case of Kodamaea
its database (bioMérieux), and as Pichia farinosa by using (Pichia) ohmeri fungemia in a premature neonate with low
the ID 32 C identification system (bioMérieux). Sequencing birth weight (680 g). During the first week, the baby devel-
analyses of PCR-amplified fragments covering internal tran- oped clinical necrotizing enterocoli, and a blood culture
scribed spacer 1 (ITS1), the 5.8S rRNA gene, and ITS2 as taken on the 13th day of life showed a yeast-like organism
well as the ribosomal LSU D1/D2 variable regions revealed using the Bactec automated culturing system (BD Diagnostic
the identity of the fungus as P. farinosa. The isolate was sen- Systems) and pediatric bottle. The organism failed to pro-
sitive to amphotericin B and caspofungin and less sensitive to duce ascospores on Sabouraud dextrose agar and malt extract
fluconazole. After initiation with amphotericin B treatment agar +2.5% glucose even with a prolonged incubation period
and removal of the Broviac catheter, the patient became afe- (14 days), and was tentatively identified as K. ohmeri with
brile after 2 days, and neutropenia was resolved. The patient Vitek II and API ID 32C (bioMérieux). Sequence analysis of
was discharged with a course of oral fluconazole for 2 weeks the 5.8S rRNA gene confirmed its identity as K. ohmeri. The
and no evidence of recurrence or complications was noted in organism was susceptible to amphotericin B and flucytosine,
subsequent checkup. but dose dependently susceptible to itraconazole and flucon-
azole. The isolate failed to produce ascospores on Sabouraud
68.1.2.4 Kodamaea ohmeri dextrose agar and malt extract agar + 2.5% glucose even
Kodamaea (Pichia) ohmeri is an ascosporogenous yeast with a prolonged incubation period (14 days). The patient
commonly used in food products. The fungus has been impli- responded to amphotericin B/liposomal amphotericin B and
cated in fungemia, endocarditis, fungiurea, peritonitis, and fluconazole treatment.
wound infection in immunocompromised individuals and the De Barros et al. [37] also described a case of fungemia
elderly [6,23–35]. caused by Kodamaea ohmeri in a 3-year-old female patient,
Shin et al. [30] reported a case of fungemia and phle- who had previously received antimicrobial therapy due to a
bitis due to Pichia ohmeri in a 59-year-old hospitalized peritoneal infection and nosocomial pneumonia, and had a
patient, who had previously received intensive antimicro- central venous catheter implanted. K. ohmeri was isolated
bial therapy for a ventriculoperitoneal shunt infection and from blood and the tip of the catheter 48 h after its implan-
subsequent nosocomial pneumonia. The patient developed tation. The yeast was identified by standard microbiological
intermittent fever and suffered from tender swelling of the methods and sequence analysis of the D1/D2 domains and
right leg due to peripheral phlebitis at the site of insertion the ITS1 + 2 spacer regions of the ribosomal DNA. The yeast
of a peripheral venous catheter (which had already been was susceptible to amphotericin B, and liposomal AmB was
removed at the onset of fever). Cultures of samples from the used successfully to resolve the infection.
insertion site as well as blood yielded P. ohmeri. Following
a 14 day amphotericin B therapy, the fungemia and phlebitis
were cleared.
Han et al. [31] also described two cases of Kodamaea Due to the increasing involvement of Pichia spp. in human
ohmeri-associated infections in patients with preexisting infections, there is a need to develop rapid methods for the
conditions. The first one involved a 14-year-old boy who identification of these organisms at the species and subspe-
was neutropenic following chemotherapy for leukemia. The cies level. The availability of strain typing information is
infection was cured by catheter removal and the use of flu- also critical for epidemiological tracking of the outbreaks,
conazole. The second case concerned a 74-year-old man detection of cross-transmission of nosocomial pathogens,
who had undergone surgeries for a subcutaneous tumor. The and determination of the source and virulence of infection
patient developed polymicrobic cellulitis and succumbed to strains.
the complications despite surgical debridement and antibiotic Phenotypic techniques are useful for the identification of
therapy. Kodamaea ohmeri isolates were identified correctly Kodamaea (Pichia) ohmeri, including the formation of char-
by biochemical tests and were susceptible to amphotericin acteristic pink-blue colonies on CHROMagar media [5,6] and
B and dose dependently susceptible to itraconazole and demonstration of distinct biochemical profiles on commer-
fluconazole. cial yeast identification systems (e.g., API or Vitek) [38,39].
Yang et al. [35] reported a case of fungemia due to K. However, these methods failed to give conclusive results for
ohmeri in a 71-year-old man with cellulitis. The patient pre- Pichia anomala (formerly Hansenula anomala), Pichia fabi-
sented with leg edema, fever, and change of consciousness. anii, and Pichia farinosa on a number of occasions [20,22].
Blood culture yielded a yeast colony, which was identified PCR amplification and sequencing analysis of the 18S rRNA
as K. ohmeri by Vitek YBC card, API 20C, sequencing of gene, the D1/D2 domains of the 28S rRNA gene, and the ITS

regions have proven crucial for their correct determination 1% SDS, 10 mM NaCl, 10 mM Tris/HCl pH 8.0, 1 mM
[3,40–46]. EDTA) in a 1.5 mL tube and 200 μL phenol/chloroform/iso-
Pulsed-field gel electrophoresis (PFGE) karyotyping and amyl alcohol (25:24:1) is added. Cells are lysed by vortexing
restriction endonuclease analysis of genomic NotI-digested for 4 min in the presence of 20 mg glass beads. The aqueous
DNA (REAG-N) are also valuable for the identification of layer is treated once more with phenol/chloroform/isoamyl
Pichia and Kodamaea stains [6]. In addition, while the ITS alcohol and the DNA is precipitated with chilled 2-propanol.
harbors limited sequence variation to be useful for Pichia The pellet is washed with 70% ethanol and finally resus-
anomala typing, the intergenic spacer 1 (IGS1) region (which pended in 50 μL TE buffer (10 mM Tris/HCl pH 8.0, 1 mM
varies from 1213 to 1231 bp in length) of the rRNA is inter- EDTA) [49].
spersed with repeats and has more variation than the ITS
regions, providing a more discriminatory tool in the typing of 68.2.2 Detection Procedures
P. anomala strains [47–49]. By using a pair of specific prim-
ers corresponding to the 18S rRNA gene sequence, Pichia 68.2.2.1 PCR Identification of Pichia anomala
guilliermondii (anamorph: Candia guilliermondii) can be Bhardwaj et al. [49] designed several primers (IGF1, IGF2,
reliably differentiated from other members of the Candia IGR1, IGR2, and 5SR1) from the conserved region of rRNA
famata complex [8]. gene intergenic spacer region 1 (IGS1) for specific identifica-
tion of Pichia anomala (Table 68.1).
68.2 Methods Procedure
68.2.1 Sample Preparation 1. PCR mixture (50 μL) is made up of 50 ng genomic

Clinical samples (e.g., tracheal secretion, skin scraping, cer- DNA, 25 pmol each of primer pair (IGF1/IGR1,
vical swab, urine, blood) are cultivated on Sabouraud agar IGF1/IGR2, IGF2/IGR1, IGF1/5SR1, or IGF2/5SR1
(SGC 2; bioMérieux) for 5 days at 30°C. Yeast isolates are in separate tubes), 200 μM each dNTP, 2.5 mM
identified using traditional morphology and the ID 32C MgCl2, and 2 U Taq polymerase.
and VITEK 2 ID-YST biochemical identification methods 2. The mixture is subjected to an initial denaturation
(bioMérieux). at 94°C for 5 min, followed by 30 cycles of 94°C for
DNA from clinical samples can be prepared with a 1 min, 55°C for 1 min, and 72°C for 1 min, with a
QIAamp DNA Blood Kit (Qiagen). Viscous tracheal secre- final extension at 72°C for 10 min.
tion samples are diluted with an equal volume of sputasol 3. The resulting amplicons are separated by electro-
(Oxoid) and liquefied at 37°C for 1 h. Thereafter, 400 μL of phoresis in a 1% agarose gel, stained with ethidium
the sample is transferred to a tube containing lysing matrix bromide (0.5 µg/mL), and visualized under UV light.
E and mechanically disrupted in a RiboLyser. Subsequently,
200 μL of the sample is incubated with 200 μL buffer AL Note. The expected products from different combinations of
at 56°C for 10 min prior to DNA extraction. Valvular tissue primers (IGF1/IGR1, IGF1/IGR2, IGF2/IGR1, IGF1/5SR1,
(20 mg) is treated with 180 μL buffer ATL and 20 μL pro- and IGF2/5SR1) are of 1.1, 0.6, 0.7, 1.25, and 0.75 kb, respec-
teinase K (30 U; Sigma-Aldrich) at 56°C until the tissue is tively. They are highly specific for Pichia anomala.
completely lysed, followed by incubation with 200 μL buffer
68.2.2.2 P CR Identification of Pichia guilliermondii
AL at 70°C for 10 min prior to DNA extraction. Skin and nail
(Candida guilliermondii)
scraping samples are suspended in 200 μL sterile distilled
water and transferred into a tube with lysing matrix E. After Yamamura et al. [8] developed a specific PCR assay for Pichia
mechanical disruption, 180 μL of buffer ATL and 20 μL of guilliermondii (Candida guilliermondii) using primers
proteinase K are added. Samples are incubated at 56°C over- CUGF (5′-CGGGGAGGTAGTGACAATAC-3′) and CUGR
night, followed by incubation with 200 μL of buffer AL at (5′-CAAACACCACAAGGGCGAAT-3′) derived from 18S
70°C for 10 min. The remaining steps are undertaken accord- rRNA gene. Use of these primers facilitates amplification
ing to the manufacturer’s instructions. DNA is eluted from
the QIAamp column with 50 μL sterile distilled water [50].
TABLE 68.1
Cervical swabs are rinsed in lysis buffer (Digene Hybrid
Capture 2 CT/GC DNA kit; Digene). Total DNA is extracted PCR Primers for Species-Specific
from 200 μL cervical swab lysis solution, 200 μL ethylenedi- Identification of Pichia anomala
aminetetraacetic acid (EDTA)-anticoagulated blood, 400 μL Primer Sequence (5′ → 3′)
serum, and 400 μL urine using the NucliSens easyMAG (bio- IGF1 AGTATACTGGCTAACAGAAGTTGGCTA
Mérieux) automated DNA extraction system. DNA is eluted IGF2 GGAATCGTGACCAAAAAATGGGAAAT
in 55 μL easyMAG elution buffer [50]. IGR1 ATAATTTGGCCCAAATCTATTCTCCCA
DNA can be purified from yeast culture by the glass bead IGR2 CTAACGGGGGGAGGATCCAGTATAAAA
lysis method. A loopful of culture from malt yeast extract 5SR1 CACCGTTTCCGTTCCGATC
agar is transferred to 200 μL lysis buffer (2% Triton X-100,

of a ca. 250 bp product from DNA of P. guilliermondii (C. G-50 fine column to remove unincorporated dye ter-
guilliermondii), not that of related taxa including Pichia ano- minators. Purified sequencing reaction products are
mala, Pichia ohmeri, Candida albicans, Candida parapsilo- run on an ABI Prism 3100 Genetic Analyzer with a
sis, Candida tropicalis, Candida krusei, Candida glabrata, 50 cm capillary array.
Candida kefyr, Candida utilis, Candida dubliniensis, and 5. Sequences are analyzed with the SmartGene
Candida auris. Integrated Database Network software version
Procedure
1. PCR mixture (100 μL) is made up of 100 μM dNTPs,
1× PCR buffer (GE Healthcare), 2.5 U of DNA Taq
polymerase (GE Healthcare), 30 pmol each of prim-
Note. Sequence-based identifications are defined by percent
ers CUGF and CUGR, and 3–20 ng DNA.
identity: species, ≥99%; genus, 93%–99%; and inconclusive,
2. Amplification is performed with an initial 94°C for 5
≤93%.
min, 25 cycles of 95°C for 1 min, 60°C for 15 s, and
72°C for 15 s, followed by a final 72°C for 10 min.
3. PCR products are separated by electrophoresis on 68.2.2.4 Sequencing Analysis of ITS1 and ITS2
a 1.2% agarose gel, stained with ethidium bromide, Leaw et al. [45] utilized fungus-specific universal prim-
and visualized under ultraviolet light. ers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and
ITS2 (5′-GCATCGATGAAGAACGCAGC-3′) to
Note. A ca. 250 bp product is obtained only from DNA of amplify the ITS1 region, and universal primers ITS3
P. guilliermondii (C. guilliermondii), but not that of other (5′-GCATCGATGAAGAACGCAGC-3′) and ITS4
fungal taxa. (5′-GCATATCAATAAGCGGAGGA-3′) to amplify the
ITS2 region. Subsequent sequencing analysis of the result-
68.2.2.3 Sequencing Analysis of ITS Regions ing amplicons facilitated identification of a range of fungal
Pounder et al. [51] described a real-time PCR with SYBR pathogens.
Procedure
1. PCR mixture (50 μL) is made up of 10 mM Tris–
HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM
each of deoxynucleoside triphosphates, 1.2 U of Taq
Procedure DNA polymerase, 0.4 μM (each) of the ITS1 region
primers (ITS1/ITS2) or the ITS2 region primers
1. PCR mixture is composed of 1× Lightcycler FastStart (ITS3/ITS4), 2 μL (1–5 ng) of DNA template. A
DNA Master Hybridization Probes mixture (Roche negative control is included in each run by replac-
Applied Science) (containing deoxynucleoside tri- ing the template DNA with sterile water in the PCR
phosphates, FastStart Taq DNA polymerase, and mixture.
1 mM MgCl2 (additional MgCl2 is added to a final 2. Amplification is conducted with an initial 94°C for
concentration of 4.6 mM), 0.4 μM each of ITS1 for- 3 min; 30 cycles at 94°C, 60°C, and 72°C for 1 min
ward and ITS4 reverse primers, 1× SYBR green each; and a final 72°C for 3 min.
(Molecular Probes), and 3 μL template DNA. 3. After checking the PCR products on 1.5% aga-
2. Thermal cycling parameters include 95°C for rose gel, the amplicons are purified using a com-
10 min; 50 cycles at 95°C for 5 s, 60°C for 20 s, and mercial PCR cleanup kit. The DNA fragments are
76°C for 30 s; and a final extension at 72°C for 2 min. sequenced using an ABI Prism 377 automated DNA
3. The quality of the amplicon is determined by using sequencer (Applied Biosystems) with a BigDye
the derivative of the melt analysis curve (55°C–99°C, Terminator cycle sequencing kit (version 3.1;
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 Applied Biosystems). All amplicons are sequenced
(Corbett Robotics, Inc.). on both strands using primers ITS1 and ITS2 for
4. The amplified product is purified for bidirectional the ITS1 region and primers ITS3 and ITS4 for the
sequencing using ExoSAP-IT (USB Corp). Five ITS2 region.
microliters of Big Dye Terminator Ready Reaction 4. After sequencing, portions of the 18S, 5.8S, and
Mix v. 1.1 (Applied Biosystems) is added to 4 μL of 26S rRNA gene sequences of the PCR products are
each primer (0.8 pmol/μL) and 3 μL of purified PCR removed to obtain the exact ITS1 and ITS2 sequences.
product. Cycle sequencing is performed with a 9700 5. For all yeasts, the sequences of the 3′ ends of the
thermal cycler (ABI), using 25 cycles of 96°C for 18S and 5.8S rRNA genes are GCGGAAGGA
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing TCATTA and GTTTGAGCGTCATTT, respec-
reaction products are passed through a Sephadex tively, and the sequences of the 5′ ends of the 5.8S

and 26S rRNA genes are AAACTTTCAACAA and 4. Garcia-Martos, P. et al., 1996. Sexual forms of yeasts in clini-
GACCTCAAATCAG, respectively. cal samples. Mycopathologia 136, 67.
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in the paediatric intensive care unit: Case reports and review
those available at the GenBank databases using the
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dren: From asymptomatic colonization to tissue invasion.
Pediatr Infect Dis J 10, 400–402.
Note. The ITS2 region, located between the 5.8S and 28S sub- 8. Yamamura, M. et al., 2009. Polymerase chain reaction assay
units in the RNA gene, provides a more specific target than for specific identification of Candida guilliermondii (Pichia
the ITS1 for determination of K. ohmeri [6]. For strains pro- guilliermondii). J Infect Chemother 15(4), 214–218.
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ers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and mala—An outbreak in a cancer hospital. Mycoses 40, 193–196.
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12. Chakrabarti, A. et al., 2001. Outbreak of Pichia anomala
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38. Latouche, G.N. et al., 1997. Comparison of use of phenotypic 50. Vollmer, T. et al., 2008. Evaluation of novel broad-range
and genotypic characteristics for identification of species of real-time PCR assay for rapid detection of human pathogenic
the anamorph genus Candida and related teleomorph yeast fungi in various clinical specimens. J Clin Microbiol 46,
species. J Clin Microbiol 35, 3171–3180. 1919–1912.
39. Buchaille, L. et al., 1998. Evaluation of six commercial sys- 51. Pounder, J.I. et al., 2007. Discovering potential pathogens
tems for identification of medically important yeasts. Eur J among fungi identified as nonsporulating molds. J Clin
Clin Microbiol Infect Dis 17, 479–488. Microbiol 45, 568–571.

69 Pneumocystis
Steve M. Taylor and Steven R. Meshnick
Contents
69.1 Introduction...................................................................................................................................................................... 603
69.1.3 Diagnosis.............................................................................................................................................................. 605
69.2 Methods............................................................................................................................................................................ 607
Acknowledgments.......................................................................................................................................................................612
References...................................................................................................................................................................................612
69.1 Introduction of Pneumocystis as a trypanosome in 1909 quickly yielded to

revision by Delanoe and Delanoe in 1912, but it remained
The fungus now known as Pneumocystis jirovecii that infects a protozoan through much of the twentieth century. Several
humans entered modern medicine as a trypanosomal infec- factors contributed to this misclassification, including the
tion of guinea pigs reported by Carlos Chagas in 1909.1 The inability to culture the organism in vitro; therapeutic success
eminent parasitologist’s misclassification was soon corrected, with antiprotozoal, but not antifungal, antimicrobials; and
and it was rechristened in deference both to its distinct para- limited interest in the organism due to its clinical rarity.
sitic morphology and to early investigator Dr. Antonio Carini In the 1980s, its newfound clinical importance and
as Pneumocystis carinii.2 It retained this name through the advances in DNA technology fueled interest, and molecu-
balance of the twentieth century but assumed varied medi- lar phylogenetic analyses definitively demonstrated that
cal guises, first as a rare cause of sporadic respiratory infec- P. jirovecii is a fungus, though an atypical one.8 It lacks ergos-
tion, then as a significant cause of pneumonia (Pneumocystis terol in its cell wall—possessing instead an unusual abun-
carinii-pneumonia [PcP]) in undernourished children in dance of cholesterol—rendering it impervious to the polyene
Europe in the wake of World War II.3 Subsequently, severely antifungal agents that target ergosterol synthesis, and the cell
immunosuppressed children and adults receiving cytotoxic wall lacks the rigidity and impermeability of most yeasts.
chemotherapy became the main victims of this persistent yet Additionally, sequencing of Pneumocystis isolates from dif-
uncommon pathogen.4 In 1981, clusters of PcP in injection- ferent vertebrate species demonstrated that Pneumocystis
drug users and men who have sex with men in the United strains are species-specific, and it is unclear that either cross-
States presaged the recognition of the acquired immunodefi- infections or genetic commingling occur between strains.9
ciency syndrome (AIDS),5 and in the early years of the AIDS Genotyping these host species-specific isolates con-
epidemic PcP was thrust center stage as a common and devas- firmed significant sequence divergence of key loci, com-
tating AIDS-defining opportunistic infection.6 With aggres- plicating Pneumocystis nomenclature with a proliferation
sive chemoprophylaxis in high-risk patients and the effective of host species-specific designations. In 2002, human-
treatment of human immunodeficiency virus (HIV) infec- derived Pneumocystis was officially renamed P. jirovecii
tion in the developed world, the overall incidence of PcP has (pronounced “yee-row-vet-zee”), acknowledging both its
declined significantly, though it remains an important cause of genetically distinct heritage and the contributions of Czech
opportunistic pneumonia in patients with immunodeficiency parasitologist Otto Jirovec, who first described the fungus in
due to therapeutic immunosuppression, congenital disorders, human disease.10 P. carinii was retained to describe one of
or inadequately treated or undiagnosed HIV infection.7 two strains that infect rats, though, to avoid confusion, the
abbreviation PcP continues to refer to pneumonia caused by
P. jirovecii (Pneumocystis pneumonia).
Since its initial recognition as a human pathogen, P. jirovecii The P. jirovecii life cycle is incompletely understood, as
has changed both names and kingdoms. Chagas’ classification attempts to reproducibly cultivate the organism in vitro have
603

failed.11 Because of this and the difficulty of generalizing has been described. Since the widespread application of
observations from models of infected animals, insight into its HIV therapy and thus the prevention of severe immunocom-
biology and life cycle has been intuited from the microscopic promise, the incidence of PcP has declined significantly
examination of human lung specimens and by analogy to life among HIV-positive patients. Among HIV-negative patients,
cycles of other ascomyceteous fungi.12 Both major forms of the incidence varies according to type and duration of
the organism—trophic and cystic—comprise the life cycle, immunosuppression.15
which is propagated by both asexual and sexual reproduc- Mortality from PcP remains substantial, and has not sig-
tion. Trophic forms exist in clusters with avidity for Giemsa nificantly improved among HIV-infected patients despite
staining and can reproduce by binary fission. Alternatively, the advent of antiretroviral therapy in the mid-1990s.16,17
these haploid trophic forms can conjugate to form diploid Recent reviews of HIV-infected patients in San Francisco16
cyst forms, whose thick cell walls are readily identified by and in London17 document mortality rates of approximately
methenamine silver and toluidine blue-O stains. The clinical 10% in the 2000s, with death associated with advanced
pathological consequence is that in human lung infections, age, medical comorbidity, and subsequent episodes of PcP.
the haploid trophic forms typically predominate and com- Among HIV-negative patients, mortality is nearly 40%, in
prise up to 90% of the microscopically visible life forms. accordance with its more rapid and fulminant presentation
(see above).18
Extrapulmonary infection with Pneumocystis has been
reported, but its clinical importance is unknown.19 Autopsy
P. jirovecii causes pneumonia with atypical clinical and series of HIV-infected patients have identified P. jirovecii
radiographic features.13 Classic symptoms in adults include histologically and molecularly in the reticuloendothelial, gas-
fever, shortness of breath, and a nonproductive cough, though trointestinal, renal, and hematopoetic systems; these lesions
these are neither specific nor sensitive for the pathogen. In are typically associated with patients receiving either inhaled
patients infected with HIV, the onset of symptoms is subacute or no prophylaxis.20
or insidious, whereas in patients with immunosuppression Pneumocystis enters the body via inhalation and infects
due to cancer, transplantation, or cytotoxic chemotherapy, alveoli.12 Organisms have been identified in a wide variety of
the clinical course is often more rapid.14 In children, mal- environmental reservoirs, though it is unclear whether new
nourishment predisposes to infection that begins with poor infections are acquired from these sources or from human-to-
feeding and gradually progresses to respiratory distress and human spread. Serologic epidemiology suggests widespread
cyanosis. An abnormal lung exam is frequent, but hypoxemia exposure to the organism in humans, usually within the first
is almost uniformly present. Radiographically, pneumonia several years of life, consistent with the peak incidence of
due to P. jirovecii is notoriously varied and nonspecific. Its clinical infection in HIV-infected infants.21 The natural his-
appearance ranges from classic lobar pneumonia to diffuse tory of infection after acquisition is unclear. Colonization
interstitial infiltrates to, in early disease, normal radiogra- with P. jirovecii, defined as the presence of organisms in the
phy, and nearly every radiographic phenomenon has been absence of clinical infection, is common in adults with HIV
reported (see Figure 69.1). or non-HIV-related immune suppression and chronic lung
The overwhelming risk factor for disease is immunocom- disease, although notably minimal in health, immunocom-
promise, usually due to either advanced HIV infection or petent adults.22 The implications of colonization studies are
the administration of cytotoxic chemotherapy or high-dose twofold: minimal colonization among health adults suggests
corticosteroids; in particular, an association between the that lifelong latent infection is unlikely in most adults, and
tapering phase of corticosteroids and the onset of infection colonized individuals may serve as a reservoir for transmis-
sion to susceptible individuals. Clinical infection may result
from either activation of these latent infections due to immu-
nosuppression or from the acquisition of new organisms,
though evidence from molecular genotyping studies suggests
the latter.10
The development of clinical disease following infection is
typically contingent upon an attenuation of host immunity.
Impairment of either humoral or cellular immunity has been
associated with development of pneumocystosis, though the
mechanisms of this are complex and poorly understood. In
normal lungs, alveolar macrophages primarily affect clear-
ance of organisms after infection, and this activity is impaired
in patients with AIDS or cancer.12 Additionally, macrophage
Figure 69.1 Radiographic appearance of P. jirovecii-pneumonia. activation by P. jirovecii cell-surface antigen β-glucan stimu-
Left: a standard anterior/posterior chest x-ray shows bilateral perihi- lates release of cytokines including tumor necrosis factor and
lar reticular opacities. Right: computer tomography of the chest can interleukin-8, which serve to augment organism clearance by
show multilobar ground-glass opacities. recruiting additional inflammatory cells. This downstream

Pneumocystis 605
macrophage activity is highly dependent upon the presence

of upstream T cells that are primarily of the CD4+ lineage, as
evidenced both by clearance of infection in T-cell deficient,
macrophage-replete mice reconstituted with CD4+ cells and,
more simply, by the close association between infection risk
and CD4+ cell level in patients with AIDS.
Pathologically, progressive diminution of host immunity
allows proliferation of organisms resident in alveoli.13 The
histologic picture is typically one of a foamy, intra-alveolar
eosinophilic infiltrate; more advanced cases often progress
to hyaline membranization with interstitial fibrosis, which
is pathologically similar to changes that characterize the
adult respiratory distress syndrome (ARDS). These patho-
logic changes in the lung result not only from direct effects
of the organism but also the proinflammatory response, a
paradoxical phenomenon given that clinical infection virtu-
ally requires advanced immune suppression. Nevertheless, Figure 69.2 P. jirovecii detected in respiratory specimens by
experimental evidence in rat models of pneumonia and clini- colorimetric staining. (a) Wright-Giemsa preparations do not stain
cal experience in AIDS patients with PcP both suggest that the cyst walls but do stain the intracystic bodies (10×), (b) hema-
exuberant or dysregulated natural and adaptive immunity in toxylin and eosin staining typically reveals a foamy, intra-alveolar
the lung contribute to pathophysiology.20 infiltrate, with trophic forms failing to stain (40×), (c) the Gomori
methenamine silver stain readily stains cysts (40×), and (d) tolu-
idine blue-O stains cysts (40×).
69.1.3 Diagnosis
The diagnosis of PcP is supported by clinical, radiographic, step in the diagnostic evaluation; its sensitivity is widely vari-
and serum chemistry data, but the gold standard test remains able, though at some experienced centers approaches 80%. In
the staining and microscopic examination of respiratory contrast, neither oropharyngeal wash nor expectorated spu-
specimens.23 As above, the clinical and radiographic findings tum reliably demonstrates organisms detectable by staining
are nonspecific and substantially overlap with those of lower in patients with PcP.20
respiratory tract infections due to other pathogens. Serum Organisms are detected microscopically by either colo-
testing can support the diagnosis, as an elevated serum lac- rimetric staining or immunofluorescence (see Figure 69.2).
tate dehydrogenase (LDH), though nonspecific, is frequently Staining is more widely employed, and a variety of stains
considered supportive of the diagnosis. Additionally, serum reliably identify the different life forms of P. jirovecii. The
levels of S-adenosyl-methionine (AdoMet), an enzyme essen- cyst-stage-specific stains toluidine blue-O and methena-
tial for fungal metabolism, may offer a means to diagnose mine silver will stain only fungi and thus fail to identify the
PcP nonmicrobiologically,24 though this is controversial.25 more-abundant trophic forms. Giemsa and Diff-Quik stain
More recently, serum (1→3) β-d-glucan, a major component all stages of P. jirovecii and thus address this shortcoming,
of the cyst wall of P. jirovecii, has been advocated as a sensi- but will also stain yeast and other extracellular debris and
tive serum marker of PcP, though it has not yet been widely thus complicate examination. Some laboratories employ a
employed in clinical practice.26 staining algorithm incorporating a rapid screening exami-
nation followed by a more-intensive preparation for defini-
69.1.3.1 Conventional Techniques tive diagnosis. In tissue specimens, methenamine silver and
Securing the diagnosis traditionally requires the demonstra- toluidine blue-O commonly diagnose cysts, and standard
tion of organisms in respiratory specimens.20 A variety of hematoxylin–eosin often reveals a foamy, intra-alveolar
specimen types has been investigated, and the diagnostic eosinophilic infiltrate that is characteristic of PcP.
yield is dependent on the quality of specimen and its route As an alternative to colorimetric straining, immunofluo-
of collection. Historically, open lung biopsy was necessary rescent staining also allows for rapid microscopic detection,
to obtain an appropriate specimen for analysis, but this though it requires specialized equipment. On induced spu-
has been supplanted by fiber-optic bronchoscopy and less- tum, it may be more sensitive and specific than colorimetric
invasive collection techniques. Sputa induced by the inhala- staining,28 but these advantages are largely abrogated when
tion of saline mist can be examined as an initial diagnostic bronchoalveolar lavage (BAL) fluid is examined.29
step, but the highest diagnostic yield is provided by fluid and
tissue obtained during bronchoscopy. The examination of 69.1.3.2 Molecular Techniques
biopsies or fluid gathered at bronchoscopy reputedly have a Because of the inability to reliably cultivate the organism
sensitivity over 90% for the diagnosis, though the need for for definitive microbiologic diagnosis, molecular diagnos-
invasive specimen collection limits their clinical utility.27 tic assays have generated significant interest for the detec-
Because of this, induced sputa can be examined as the first tion of Pneumocystis. Since the first description of diagnosis

by DNA amplification in 1990,30 subsequent reports have tract in the absence of clinical pneumonia has been termed
promulgated protocols with a wide variety of gene targets, “carriage,” “colonization,” and “subclinical infection,” and
detection chemistries, specimen inputs, and operating char- molecular assays have generated evidence of significant
acteristics. Despite this variety, a common finding of most carriage of P. jirovecii in children, HIV-infected adults,
assays when compared with conventional methods is a high non-HIV-infected immunosuppressed adults, health-care
sensitivity for organisms but a diminished specificity that workers, and adults with chronic lung diseases (reviewed by
likely reflects the detection of organisms in a carrier, rather Morris et al.22). Because the clinical significance of this car-
than infecting, state. rier state is unclear, the detection of P. jirovecii by molecular
The most widely investigated protocols target sequences assays in asymptomatic individuals complicates their use in
of the rRNA of P. jirovecii, including the mitochondrial large diagnosis of clinical disease.
subunit (mtLSUrRNA),30 the internal transcribed spacers Nevertheless, the specificity of these assays has been eval-
(ITS),31 or the 18S subunit.32 In order to shorten testing turn- uated both microbiologically and clinically. Most assays have
around time and minimize the risk of contamination by post- tested negative on panels of non-P. jirovecii pulmonary patho-
amplification processing, assay chemistry has evolved from gens either in pure culture or in clinical respiratory speci-
nested PCR with Southern blotting or agarose gel electro- mens, indicating 100% microbiologic specificity.45,46,48,49,57
phoresis to real-time amplification with detection by fluores- However, a subsequent comparison of assays found cross-
cence resonance energy transfer (FRET) or TaqMan probes reactivity with S. cervisiae, C. albicans, or C. neoformans
or by SYBR green fluorescence.33–38 These real-time PCR for assays targeting the 18S rRNA and thymidylate syn-
technologies have been employed by protocols to generate thase genes.49 Because clinical specificity is bedeviled by
a quantitative estimate of organism burden and thus poten- the detection of P. jirovecii DNA in clinically noninfected
tially distinguish between asymptomatic P. jirovecii carriage patients, specificities have been reported between 86% and
and clinically significant infection. Additionally, reverse 100%, depending on the assay.30,32,44,50,52 Specificity may be
transcriptase (RT)-PCR protocols have been described that improved by quantifying organism burden, and, to this end,
detect P. jirovecii mRNA in an effort to identify only viable several assays have been adapted to exploit real-time detec-
organisms from clinical or environmental samples.39,40 For tion chemistry. Assays targeting MSG have been modified to
amplicon detection, protocols employing enzyme immunoas- include amplicon detection with FRET probes, and though
says41 or solution hybridization enzyme-linked assays42 have the application of a post hoc cutoff improved the specificity
generally been supplanted by protocols exploiting real-time to detect clinical disease to nearly 100% in respiratory speci-
technology for detection. mens58 and oral washes,59 a predictable consequence was a
Notably, one report describes the development and appli- slight decrease in clinical sensitivity. In contrast, improve-
cation to clinical samples of a loop-mediated isothermal ments in specificity without significant diminution of sensi-
amplification (LAMP) protocol to detect P. jirovecii,43 which tivity in real-time assays have been achieved with TaqMan
may reduce the cost of testing compared with real-time assays targeting both HSP7060 (sensitivity 95% and specific-
technologies. ity 96%) and dihydroopteroate synthase (dhps)35 (sensitivity
94%, specificity 96%).
69.1.3.2.1 Operating Characteristics of PCR Assays
Compared with conventional diagnosis of PcP by staining 69.1.3.2.2 Comparisons between Assays
or clinical factors, many molecular assays for P. jirovecii Despite the promulgation and promotion of diverse pro-
performed on respiratory specimens have a sensitivity that tocols, few investigations have assessed the agreement
approaches 100%. When performed on induced sputum or between assays. Fischer et al.57 compared the sensitivities
BAL, sensitivity of 100% has been reported for assays tar- of the mtLSUrRNA and MSG assays on serial dilutions of
geting the ITS,44 18S rRNA,32 5S rRNA,45 thymidylate syn- clinical samples and found the MSG assay was 10-fold more
thase,46 and major surface glycoprotein (MSG).47,48 When sensitive than the mtLSUrRNA. Flori et al.61 found nearly
first described, the assay targeting the mtLSUrRNA reported identical diagnostic accuracy between assays targeting the
sensitivity of 87%–91%,30,49 but subsequent modifications mtLSUrRNA and MSG, though the incorporation of a quan-
incorporating a nested50,51 or touchdown52 amplification have titative cutoff in the MSG real-time protocol allowed for an
improved sensitivity to 100%. A notable exception to the rule improvement in specificity for clinical disease. Linssen et al.62
of the high sensitivity of molecular assays is that for P. jirovecii found high agreement between different microbiology labo-
dihydrofolate reductase (dhfr) promulgated by Schluger ratories performing real-time and conventional PCR assays.
et al.53 due to its reported sensitivity of 17% in respiratory Two studies have expressly compared the diagnostic accu-
specimens54 and the possibility that it may amplify a nonspe- racy of panels of published protocols. One study compared
cific target55; the clinical sensitivity of a subsequent nested the operating characteristics of six common P. jirovecii
assay targeting dhfr has not been rigorously evaluated.56 molecular assays published by 1995 on stain-positive and
The high sensitivity of these assays complicates defini- -negative BAL specimens.49 The sensitivity of assays target-
tive assessment of their specificity because they frequently ing the ITS and the 18S rRNA was 100%, but assays were
detect P. jirovecii in the absence of compelling clinical evi- less sensitive when targeting the mtLSUrRNA (87%), thy-
dence of PcP. This presence of P. jirovecii in the respiratory midylate synthase (60%), 5S rRNA (33%), and dhfr (23%).

Pneumocystis 607
Differences in sensitivity were attributed to differences in this end, real-time assays have been advocated as offering
both gene copy number and amplification conditions, as the greater diagnostic utility in this population.74
ITS and 18S assays were the only nested assays. All assays
returned negative results on the subset of BALs with both
negative stains for P. jirovecii and positive results for other 69.2 Methods
pulmonary pathogens, indicating microbiologic specificities
of 100%.
A similar comparison was repeated in 2007,63 compar- Genomic DNA (gDNA) extraction from respiratory speci-
ing protocols targeting ITS,49 dhps (nested),64 dhps (single),65 mens (oral washes, induced sputa, or BAL fluid) may require
dhfr,56 MSG (heminested),47 mtLSUrRNA (nested),66 18S preextraction preparation. Specimens may be frozen prior
rRNA,67 and 5S rRNA (real-time).48 The greatest sensitivi- to extraction. Mucous-containing BAL or sputum samples
ties, when compared with methenamine silver staining, were may require the addition of an equal volume of 0.9% NaCl
achieved by the mtLSUrRNA nested assay (100%), the real- to resuspend and subsequent pelleting by centrifugation,61 or
time assay targeting 5S rRNA (88%), and the heminested the addition of lysis buffers,72 dithiothreitol,46,51 or N-acetyl-
MSG assay (71%). The study did not report a comprehen- l-cysteine,36 though these latter additions may necessitate
sive comparison of the assays’ clinical or microbiological amplification controls to ensure the absence of introduced
specificities. PCR inhibitors. Oral wash specimens are obtained by vigor-
ous rinsing of the oral cavity for 10–30 s with 50 mL of sterile
69.1.3.2.3 Nonpulmonary Specimens saline,48 centrifugation at 2800 × g for 10 min, and resuspen-
When applied to less-invasive specimens such as blood sion of the pellet in 1 mL of supernatant. Preparation of oral
and oral washes, data are mixed on the diagnostic utility of wash, sputum, or BAL fluid for gDNA extraction can be
PCR-based assays for P. jirovecii. In patients with PcP, most accomplished by centrifuging 2 mL of specimen at 3000 × g
assays have failed to detect P. jirovecii DNA in blood,32,68,69 for 15 min, digestion of the pellet with proteinase K at 60°C
or returned positive results in PcP-negative control patients.69 for 2 h, and boiling for 10 min to deactivate the enzyme.52
In contrast, one report found that detection in blood of the However, BAL fluid is frequently input directly into com-
P. jirovecii rRNA ITS was highly sensitive and specific for mercial extraction protocols without processing.60
PcP,54 though this has not been repeated in other cohorts. Paraffin-embedded lung tissue samples may also be used
Oral wash specimens have been investigated as a nonin- as a source of gDNA for amplification, after treatment of sec-
vasive method of diagnosis, and several reports have docu- tions with xylene and ethanol and digestion with proteinase
mented sensitivities as high as 88%–91%,52,57,59 while other K.75 Additionally, stained or unstained smears of BAL fluid
reports found the assays much less sensitive.70 The exami- or lung biopsies have successfully yielded DNA for amplifica-
nation of multiple oral washes increases the sensitivity but tion.76 After soaking slides in methanol or in ethanol and water
decreases specificity, though this diminution of specificity to remove Giemsa or silver stains, slides should be scraped
may be mitigated by the use of an RT-PCR assay.40 with a sterile scalpel into sterile water, with the scraped con-
tents then transferred to a glass-fiber filter disk and processed
69.1.3.2.4 Non-HIV-Infected Patients for DNA extraction after proteinase K digestion. Despite this,
Several of the P. jirovecii molecular assays are less robust in stained or unstained slides generally yield less gDNA than
non-HIV-infected patients.71 Two reports compare the utility BAL that has been frozen or stored in ethanol.35
in different patient groups of nested PCR protocols target- For all specimens, phenol–chloroform, Chelex, or com-
ing the P. jirovecii mtLSUrRNA in the respiratory specimens mercial extraction kits are appropriate extraction alternatives.
of patients with pneumonia. The assay accurately predicted Commercial extraction kits employed include the Wizard
clinically proven PcP in HIV-infected patients but was poorly Genomic DNA Purification Kit (Promega), MagnaPure LC
predictive in leukemia and lymphoma patients.72 Similarly, Isolation Kit (Applied Biosystems), QiaAMP DNA Mini Kit
the positive predictive value of an mtLSUrRNA nested assay (Qiagen),62 Gene Clean II (BIO 101),72 QIAamp Tissue Kit
was >97% in HIV patients but <50% in transplant patients, (Qiagen),73 DNeasy Tissue Kit (Qiagen),51 and NucliSENS
patients with malignancies, or non-HIV-infected immuno- (bioMerieux).43
suppressed patients.73 Other reports in non-HIV infected Few studies compare the success of extraction protocols.
immunosuppressed patients describe “false-positive” (i.e., Huggett et al.60 employed a real-time PCR assay targeting
PCR-positive patients without other compelling clinical evi- HSP70 on gDNA extracted from BAL fluid by the QIAamp
dence of PcP) rates as high as 82%50 and quantitative trends UltraSens Virus Kit or the DNeasy Tissue Kit, with the
toward lower organism burdens (compared with HIV-infected QIAamp system isolating greater amounts of DNA as evi-
patients).58 It is unclear if these findings in non-HIV-infected denced by increased gene copy number when assayed. Huang
patients reflect asymptomatic carriage, incipient clinical et al.47 report a comparison of DNA Extraction Reagent
infection, or a biologic difference in virulence between the (Perkin Elmer) and phenol–chloroform extraction from oral
lungs of HIV- and non-HIV-infected patients. Nevertheless, washes, sputa, and BAL fluid, and found no difference in
greater assay specificity for clinical disease is a priority in detection rates when assaying with two protocols targeting
immunocompromised non-HIV-infected patients, and, to MSG and mtLSUrRNA.

Though their diagnostic utility is largely unproven, serum infectious threat to patients with drug-resistant HIV, those
specimens may be processed along much the same lines, with poor adherence and access to antiretroviral therapy or
with the addition of proteinase K to serum (4:1), incubation PcP prophylaxis, and those in receipt of organ transplan-
at 56°C for 2 h, boiling for 10 min, and proceeding with phe- tation or other immunosuppressive drugs.7 Additionally,
nol–chloroform extraction on an aliquot of the remainder.54 though its incidence and contribution to opportunistic
infection in patients with AIDS in developing countries
remains unclear, these patients may represent a large at-
risk population. For these reasons, the importance of
Protocols employing PCR, RT-PCR, and real-time PCR for P. jirovecii abides.
detection target a variety of genes and incorporate many At this time, numerous well-characterized assays achieve
modifications; see Table 69.1 for specifics of the most current accurate detection of P. jirovecii in clinical samples, though
and widespread protocols. The original protocol targeting there are none that are commercially available, and protocol
the mtLSUrRNA30 has been updated for improved sensitiv- specifics vary widely. Sample preparation and DNA extrac-
ity by either adding a nested amplification or maintaining tion have been achieved by a wide variety of commercial and
a single round of amplification with a touchdown protocol. conventional protocols, and assays have targeted more than
Both Wakefield66 and Weig et al.72 amplify mtLSUrRNA in 10 different genes and employed conventional PCR, real-time
two rounds of amplification and electrophoresce products PCR with fluorescent probes and SYBR green, RT-PCR, and
on an agarose gel, with positive samples demonstrating the LAMP.
successful amplification of a 260 or 193 bp product, respec- For the clinical microbiologist, what role should molecular
tively. Helweg-Larsen et al.,52 however, maintain the original assays play in the diagnosis of P. jirovecii infection? In AIDS
single round of amplification of mtLSUrRNA but incorpo- patients with PcP, the added benefit of molecular detection of
rate a touchdown step in the cycling conditions, with positive P. jirovecii remains unclear because conventional staining is
amplification indicated by the presence of a 346 bp band after widely available and accurate. In immunosuppressed patients
agarose gel electrophoresis. without AIDS, the high reported rates of false-positive PCR
Assays targeting the ITS have been employed not only for results can contribute to diagnostic confusion. Recent incor-
detection but for typing P. jirovecii strains. From the origi- poration of real-time technologies may add luster to the clini-
nal protocol,31 the second round of amplification has been cal application of these assays by offering timelier testing in
altered77–79 to yield products suitable for molecular diversity AIDS patients and allowing quantitation of organism burden
and phylogenetic analysis, though the validity of this practice to differentiate asymptomatic carriage from clinical infec-
has been questioned.80 Similarly, assays targeting dhfr and tion. Additionally, these assays may provide a basis for the
dhps have variable sensitivity35,49 and have generally been inclusion of P. jirovecii as a target in multiplex respiratory
employed for genotyping rather than detection. Mutations in pathogen detection panels designed for immunocompro-
both dhfr64 and dhps56 have been detected in isolates from mised hosts.
patients after exposure to antifolate medications, and have Compared with conventional methods, molecular assays
thus been of interest as possible mediators of P. jirovecii drug have several unique advantages: they more reliably detect
resistance.81 P. jirovecii carriage in noninvasive specimens collected for
The assay developed by Huang et al.47 targeting the MSG epidemiologic investigations; they can generate data to allow
has been infrequently employed as a conventional PCR strain typing for molecular phylogeny studies; and they allow
assay but frequently reported upon after adaptation for real- genotyping to detect described or prospective molecular cor-
time PCR with a touchdown amplification and real-time relates of clinical drug resistance. These tools exploit the
FRET probes.48,57–59,61,62 SYBR green detection has been benefits of molecular technologies to expand our understand-
employed by two assays that target the mtLSUrRNA82 and ing of P. jirovecii’s basic biology, clinical pathogenesis, and
the 5S rRNA, but the potential for nonspecific amplifica- response to antimicrobial therapy.
tion of targets of other pathogens likely limits their appli- Though molecular tools will refine our understanding
cation to clinical samples. Of late, TaqMan probes, or 5′ of P. jirovecii in HIV-infected and immunosuppressed
nuclease probes, have been employed by assays targeting patients in the developed world, perhaps their most prom-
P. jirovecii β-tubulin,34 dhps,35 dhfr and 5.8S rDNA,38 and ising application is in better characterizing the biology and
PjHSP70.60 Such assays, like the other FRET assays, have burden of P. jirovecii in the developing world. With over
allowed for estimates of organism burden and assay-specific 20 million people living with HIV in sub-Saharan Africa
cutoff values to distinguish P. jirovecii carriage from clini- (compared with 1.4 million in North America), careful
cal disease.35,48,60 investigation is needed to characterize both their individ-
ual risk of infection and the epidemiology and spread of
69.3 C
onclusions and strains within populations. Properly adapted and applied,
molecular assays can both inform our biological under-
Future Perspectives
standing of P. jirovecii as well as optimize the clinical
The advent of antiretroviral therapy has dramatically care of patients both infected and at risk for infection with
reduced the incidence of PcP, but it remains a significant P. jirovecii.

Pneumocystis
Table 69.1
Molecular Protocols for P. jirovecii Detection Employing PCR, RT-PCR, and Real-Time PCRa
Target Amplification Conditions
(Approximate (after Denaturation/
Reference bp Length) Primers (5′–3′) Reaction Mixture Activation) Detection Output
PCR
Kitada et al., 5S rRNA 5S sense: AGTTACGGCCATACCTCAGA 50 pmol each primer (94°C for 1.5 min, 55°C for Polyacrylamide or agarose gel electrophoresis
199145 (120 bp) 5S antisense: AAAGCTACAGCACGTCGTAT 1.5 mM MgCl2 2.5 min, 72°C for
Template/reaction 1.9 min) × 25 or 40
vol: 10/100 μL
Schluger et al., dhfr (NR) Sense: CTGCAAAATCCTTGGATCAT 1 μM each primer (92°C for 1 min, 55°C for Southern blotting: probe
199153 Antisense: CTTTAGTACCAACCCAAGAT 1.5 mM MgCl2 1 min, 72°C for 1 min) × 45 GATAGAATTATGGCTACAATA
Template/reaction
vol: 5/95 μL
Lipschik et al., 18S rRNA 340a: CCAGATTAGCTTTTGCTGATCGCGGG 50 pmol each primer (94°C for 1 min, 50°C for Southern blotting; phosphorus-32 labeled probe
199232 Round 1 (378 bp) 708: ACTTTCCAGTAATAGGCTTATCG Template/reaction 1 min, 72°C for 2 min) × 36, 372a: AAGGAAAATGAACTTGCTGGCTCT
Round 2 (312 bp) JK1: TGTTGGCATGAAGCCAATGGAA vol: 5/50 μL 72°C × 10 min
JK2: CAATAACCCATCACCAGTCCGAAG
Olsson et al., Thymidylate PC1: ATTTATGGGTTTCAATGG 0.4 μM each primer (94°C for 30 s, 50°C for 25 s, Agarose gel electrophoresis
199346 synthase PC2: GTTCCCTTTAATATTGC 3 mM MgCl2 70°C for 1 min) × 40, 70°C
(403 bp) Template/reaction for 5 min
vol: 2/50 μL
Lu et al., 199431 ITS 1724F: AAGTTGATCAAATTTGGTC 0.2 μM each primer (94°C for 1 min, 47°C for Agarose gel electrophoresis, cloning, sequencing
Round 1 (693 bp) ITS2R: CTCGGACGAGGATCCTCGCC Template/reaction 1 min, 72°C for 2 min) × 35,
Round 2 (550 bp) ITS1F: CGTAGGTGAACCTGCGGAAGGATC vol: NR/100 μL 72°C for 10 min
ITS2R1: GTTCAGCGGGTGATCCTGCCTG Then, (94°C for 1 min, 58°C
for 1 min, 72°C for
2 min) × 35, 72°C × 5 min
Wakefield, mtLSUrRNA pAZ102E: GATGGCTGTTTCCAAGCCCA 1 μM each primer (94°C for 1.5 min, 55°C for Cloning and sequencing
199666 Round 1 (340 bp) pAZ102H: GTGTACGTTGCAAAGTACTC 3 mM MgCl2 1.5 min, 72°C for 2 min) × 40
Round 2 (260 bp) pAZ102X: GTGAAATACAAATCGGACTAGG Template/reaction Then, (94°C for 1.5 min,
pAZ102Y: TCACTTAATATTAATTGGGGAGC vol: NR/NR 55°C for 1.5 min, 72°C for
2 min) × 10, (94°C for
1.5 min, 63°C for 1.5 min,
72°C for 2 min) × 30
Lane et al., Dhps F1: CCTGGTATTAAACCAGTTTTGCC 0.4 μM each primer (92°C for 30 s, 52°C for 30 s, Agarose gel electrophoresis
199764 Round 1: B45: Template/reaction 72°C for 60 s) × 35, 72°C for
Round 2: 335 bp CAATTTAATAAATTTCTTTCCAAATAGCATC vol: 5/100 μL 5 min
AHUM: Then, (92°C for 30 s, 55°C
GCGCCTACACATATTATGGCCATTTTAAATC for 30 s, 72°C for 60 s) × 35,
BN: GGAACTTTCAACTTGGCAACCAC 72°C for 5 min
(continued)
609
610
Molecular Protocols for P. jirovecii Detection Employing PCR, RT-PCR, and Real-Time PCRa
Target Amplification Conditions
(Approximate (after Denaturation/
Reference bp Length) Primers (5′–3′) Reaction Mixture Activation) Detection Output
Weig et al., mtLSUrRNA pAZ102E: GATGGCTGTTTCCAAGCCCA 25 pmol each primer (94°C for 1 min, 55°C for Agarose gel electrophoresis
199772 Round 1 (340 bp) pAZ102H: GTGTACGTTGCAAAGTACTC 3 μL of 25 mM MgCl2 1 min, 72°C for 90 s) × 35,
Round 2 (193 bp) pLE1: TCGGACTAGGATATAGCTGG Template/reaction vol: 72°C for 1 min
pLE2: CCCTTTCGACTATCTACC 10/50 μL (first round) Then, (94°C for 1 min, 58°C
1/50 μL (second for 1 min, 72°C for
round) 90 s) × 30, 72°C for 10 min
Helweg-Larsen mtLSUrRNA pAZ102E: GATGGCTGTTTCCAAGCCCA 0.4 mM each primer (94°C for 15 s, 72 → 62°C Agarose gel electrophoresis
et al., 199852 (346 bp) pAZ102H: GTGTACGTTGCAAAGTACTC Template/reaction for 30 s (decrement of 1°C
vol: NR/100 μL cycles 1–10), 72°C for
15 s) × 50, 72°C for 5 min
Huang et al., MSG (249 bp) JKK14/15: 1 μM each primer (95°C for 1 min, 65°C for Southern blotting, Probe JKK16:
199947 GAATGCAAATCYTTACAGACAACA 3 mM MgCl2 1 min, 72°C for 1 min) × 43, TGCAAACCAACCAAGTGTACGACAGG
JKK17: AAATCATGAACGAAATAACCATTGC Template/reaction 72°C for 10 min
vol: NR/50 μL
RT-PCR
Maher et al., Phsb1 (425 bp) Phsb1-161C: TGTTAAAAAAGACATGAAAATG Reverse transcription: (94°C for 1 min, 50°C for Agarose gel electrophoresis
200139 Phsb1-566nc: CAGCAGTGGCTTTAACTGAA Phsb1-566nc at 4.3 μM 1 min, 72°C for 1 min) × 40
Real-time PCR
Kaiser et al., mtLSUrRNA pAZ102E: GATGGCTGTTTCCAAGCCCA 0.6 mM each primer (95°C for 5 s, 56°C for 10 s, SYBR green

200182 pAZ102H: GTGTACGTTGCAAAGTACTC 5 mM MgCl2 72°C for 20 s) × 45, then
Template/reaction dissociation step
vol: 2/20 μL
Palladino et al., 5S rRNA 5S sense: AGTTACGGCCATACCTCAGA 0.5 μM each primer (95°C for 15 s, 55°C for 10 s, SYBR green
200137 5S antisense: AAAGCTACAGCACGTCGTAT 72°C for 10 s) × 38, then
dissociation step
Larsen et al., MSG JKK14/15: 1 μM each primer (95°C for 5 s, 65 → FRET probes: PCMSGFRET1U:
200248 GAATGCAAATCYTTACAGACAACA 5 mM MgCl2 50°C × 10 s [decrement of CAAAAATAACAYTSACATCAACRAGGCG-
JKK17: AAATCATGAACGAAATAACCATTGC Template/reaction 1°C for cycles 2–6, then 2°C fluorescein
vol: 5/20 μL for steps 7–11], 72°C for PCMSGFRET1D: Red
0.2 μM FRET probe 20 s) × 46 640-TGCAAACCAACCAAGTGTACGACAGG
PCMIM1U:
GATATCGTCCATTCCGACAGCATC-fluorescein
PCMIM1D: Red
705-CCAGTCACTATGGCGTGCTGCTAG
Pneumocystis
Brancart et al., β-tubulin 1186F: GATCCGAGACATGGTCGCTATT 0.3 μM each primer (95°C for 15 s, 60°C for TaqMan probe: 1212T:
200534 1257R: TTCAACCTCCTTCATGGAAACAG 0.66 μM probe 1 min) × 45 TGTTGCAGCGATTTTCCGCGGTA
Template/reaction
vol: 5/25 μL
Alvarez- dhps DHPS_F: GCTTGGTCCAAGTCGCAAAA 0.9 mM each primer (95°C for 15 s, 60°C for TAMRA probe: VIC-ATTTACAGGGTGTCTTACA
Martinez et al., DHPS_R: CAGCAGTGCCCCAAATCC 0.25 μM probe 60 s) × 45 GGTGATGTTATGCCAA
200635 Template/reaction
vol: 2/25 μL
Arcenas et al., Cdc2 Fwd: AGGTAGGAGAAGGTAAGAAA 0.5 μM each primer (95°C for 10 s, 55°C for 15 s, FRET probes:
200636 Rev: GCTGTGCTTGGAACCC 0.4 μM Red-640 probe 72°C for 15 s) × 45 GATCTTGAAAATGGCACAATAGTAG-
0.2 μM fluorescein fluorescein
probe Red-640-
Template/reaction TTAAAAAAATCCGGCTAGAAGCAGAAG
vol: 5/20 μL
Bandt and 5.8S rRNA 5.8S_F: TGGCTCTCGCGTCGATG 10 pmol each primer (95°C for 15 s, 61°C for 5 s, TAMRA probe: 5.8S_PR:
Monecke, 5.8S_R: TTCGATGATTCACTAAATTCTGCAA 4 pmol probe 72°C for 5 s) × 55 FAM-
200738 Template/reaction AGAACGTGGCAAAATGCGATAAGTAGTGTGA
vol: 5/20 μL
Bandt and dhfr Dhfr2.F: GGCTGATCAAAGAAGCATGGATA 10 pmol each primer (95°C for 15 s, 61°C for 5 s, TAMRA probe: Dhfr2_PR:
Monecke, Dhfr2_R: 4 pmol probe 72°C for 5 s) × 55 FAM-
200738 CGGCATAGACATATTCGATACTTGTT Template/reaction TGCGTGAAACAGATACATGGAGCTCTACCC
vol: 5/20 μL
Huggett et al., PjHSP70a Fwd: CGTCTTGTAAACCACTTCATTGC 0.3 μM each primer (95°C for 10 s, 72°C for 20 s, BHQ1 probe: FAM-AAGAAAGATCTTTCAGGG
200860 Rev: AGTCCGTTTAGCACGCTCAC 0.075 μM probe 72°C for 30 s) × 45
Template/reaction
vol: NR/12.5 μL
a NR, not reported; mtLSUrRNA, large subunit of mitochondrial rRNA; ITS, internal transcribed spacer; MSG, major surface glycoprotein; dhfr, dihydrofolate reductase gene; dhps, dihydroopteroate synthase gene;
PjHSP70, P. jirovecii heat-shock protein 70 gene; FRET, fluorescence resonance energy transfer; FAM and VIC, fluorescent dyes; consultation of the referenced original manuscript is recommended before appli-
cation of any assays.
611
Acknowledgments 20. Mandell, G., Bennett, J., and Dolin, R., Principles and
Practice of Infectious Diseases, Churchill Livingstone, New
We thank Robert Miller, Joe Kovacs, Peter Walzer, C. Ben York, 2004.
Beard, and Laurence Huang for their helpful reviews of 21. Vargas, S.L. et al., Search for primary infection by
a draft of this chapter. We thank Melissa Miller and Nacy Pneumocystis carinii in a cohort of normal, healthy infants.
Henshaw for the images of P. jirovecii in stained clinical Clin Infect Dis 32, 855–861 (2001).
22. Morris, A., et al., Epidemiology and clinical significance of
specimens.
Pneumocystis colonization. J Infect Dis 197, 10–17 (2008).
23. Kovacs, J.A. and Masur, H., Evolving health effects of
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70 Saccharomyces
Franca Rossi and Sandra Torriani
Contents
70.1 Introduction.......................................................................................................................................................................615
70.1.1.1 Classification...........................................................................................................................................615
70.1.1.2 Morphology.............................................................................................................................................616
70.1.1.3 Biology....................................................................................................................................................616
70.1.2 Clinical Features, Pathogenesis, and Epidemiology..............................................................................................617
70.1.2.1 Clinical Features.....................................................................................................................................617
70.1.2.2 Pathogenesis............................................................................................................................................618
70.1.2.3 Epidemiology..........................................................................................................................................619
70.1.3 Laboratory Diagnosis............................................................................................................................................619
70.2 Methods............................................................................................................................................................................ 621
70.2.1.1 Sample Handling................................................................................................................................... 621
70.2.1.2 Extraction of DNA from Colonies or Pure Cultures............................................................................. 621
70.2.1.3 Extraction of Total DNA from Human Fecal Samples.......................................................................... 621
References.................................................................................................................................................................................. 624
70.1 Introduction S. cerevisiae is used in the food industry worldwide as a

principal agent of fermentation in naturally leavened bakery
70.1.1 Classification, Morphology, and Biology products, alcoholic beverages like wines, beers, fermented
70.1.1.1 Classification juices, and distillates. Strains of S. cerevisiae, thanks to
their high level of resistance to ethanol, anaerobic condi-
Saccharomyces cerevisiae belongs to the fungal phy-
tions, low pH, and osmotic stress, are essentially the only
lum Ascomycota, lineage Hemiascomycetes, subphylum
organisms remaining alive at the end of wine fermentation.
Saccharomycotina, order Saccharomycetales.1,2 The latter is
This yeast can be found in nature on various plants and in
composed of three clades among which the Saccharomyces
the soil. In studies aimed at defining the source from which
complex, constituted by the genera Saccharomyces and
it contaminates grapes in the vineyards and the winery
Kluyveromyces, is the most densely sequenced. The phylog-
environment, it has been isolated from the bodies of insects,
eny of this group was defined by multi-gene sequence com-
most frequently honey bees, but also wasps and Drosophila.
parison1 and by comparative hybridization of genomic DNA
These are supposed to be the carriers that transfer the yeast
on S. cerevisiae cDNA microarrays.3 S. cerevisiae and the
to the interior of damaged fruits. In a phylogenetic study
sibling species S. bayanus, S. cariocanus, S. kudriavzevii,
based on diversity at 5 loci in a sequence of 7 kb, 184 poly-
S. mikatae, S. paradoxus, and S. pastorianus, which is con-
morphic sites were identified; nucleotide variation in 81
sidered a hybrid of S. cerevisiae and S. bayanus, constitute
isolates originating from natural and artificial fermenta-
the Saccharomyces sensu stricto clade. Adaptation of the
tions, tree exudates, and immunocompromised patients
Saccharomyces sensu stricto species to low-oxygen environ-
from Europe, Africa, Southeast Asia, and North America
ments was driven by the duplication of genes involved in the
supported the hypothesis that “domesticated” strains are
“fermentative lifestyle,” alcohol dehydrogenase (ADH) and
derived from natural populations. The phylogenetic tree
pyruvate decarboxylase (PDC) encoding alcohol dehydroge-
suggested that lineages at the root were derived from tree
nase and pyruvate decarboxylase, respectively, following a
exudates in North America and Africa. The comparison of
whole genome duplication event that occurred in a common
oak-associated and vineyard-associated yeasts suggested
ancestor of the subgroup “Saccharomyces complex.”4
615

that both habitat and geographical distance have a role in solid medium, many S. cerevisiae strains are dimorphic and
determining genetic diversity.5 can form complex colonies or mats constituted by multicel-
S. cerevisiae has been isolated from humans and shown lular pseudohyphae by modifying their budding pattern from
to be present in the digestive tract, vagina, skin, and oro- multi- to unipolar. This process, observed both in haploid and
pharynx of healthy hosts; however, it has not yet been clearly diploid cells during nitrogen starvation and in presence of
demonstrated whether this is a true commensal species or carbon sources other than glucose,15 is known as “pseudofila-
its colonization is transient and related to food consumption. mentation” or pseudohyphal growth (PHG).
Due to high concentration of the B vitamins, minerals, Other phenotypes include the formation of biofilms16 and
and cofactors, this yeast is used as a nutritional supple- complex fluffy-structured colonies with cells connected by
ment. The S. cerevisiae variant “S. boulardii” (S. cerevisiae extracellular glycosylated matrix material.17 When in suspen-
Hansen CBS 59266,7 patent strain held in the American Type sion, some industrial strains can form specialized biofilms
Culture Collection [ATCC]), first isolated from litchi fruit in called flors at the interface liquid–air, which are necessary
Indochina and once considered a separate species, is used for particular wine-making processes.18 The ability to floc-
worldwide as a biotherapeutic agent. S. boulardii has been culate is also a strain-dependent character.14,19
used in Europe as a probiotic preparation for oral adminis- For the different morphologies observed within the
tration under different commercial names (Ultra-Levure, S. cerevisiae species, like PHG, substrate invasion, flor for-
Precosa®, Codex, etc.). S. boulardii is also used as a feed mation, flocculation, and biofilms on plastic surfaces, the
additive, an example is Levucell SB®, and as a food supple- adhesin Flo11p plays a key role. This is a 196 kDa manno-
ment. This yeast is highly effective for the treatment of sev- protein that functions as target for adhesion on the yeast cell
eral types of acute diarrhea and colitis by exerting multiple wall.18 It is also implicated in cell adherence to surfaces by
beneficial activities like the inactivation of enteropathogenic conferring hydrophobicity.16
bacterial toxins by proteolytic enzymes, trophic effects on Pseudohyphal filaments consist of elongated undetached
the small intestine mucosa mediated by the release of poly- cells that result in a colony morphology presenting a cen-
amines in the endoluminal compartment, improvement of tral body from which numerous branched spokes depart.
the absorption of glucose by secretion of high amounts of The elongated cells form filaments in the agar beneath and,
sucrases, enhancement of antibody response specific for bac- in some strains, beyond the colony perimeter. McCusker
terial pathogens, and attenuation of inflammatory symptoms et al.20 observed that many colonies of clinical and non-
by different mechanisms.8,9 clinical strains grown on casein formed pseudohyphae (Psh+
Clinical strains and commercial strains used in the food phenotype); however, pseudohyphal formation was seen
industry could not be discriminated sharply by genetic, phe- more frequently and tended to be more extensive (longer
notypic, or metabolic profiling because of the existence of pseudohyphal chains and more pseudohyphal chains ema-
food-associated strains that share high similarity with rep- nating from a colony) for clinical isolates. A similar obser-
resentatives of the other group and the high diversity within vation was reported in a study carried out with 118 clinical
the two groups.10–12 and industrial S. cerevisiae strains. The exceptions among
The entire genomes of three S. cerevisiae strains, i.e., the industrial strains were S. boulardii, two baker’s yeasts,
those of the laboratory strain S288c originating from a rot- and one wine strain, Fermivin Crio.21 A screening of 1026
ting fig,13 of the clinical strain YJM789,14 and of the wine strains isolated from Italian vineyards showed that roughly
strain RM11-1a (http://www.yeastgenome.org), are available half were able to show invasive filamentous growth when
to date. starved for nitrogen. Also, 2.5% of the strains had a filigreed
(“rough”) or fluffy colony phenotype. Furthermore, several
70.1.1.2 Morphology strains also showed differences in the treatments that induce
Cells of S. cerevisiae are spheroidal, ovoidal, or elongate. modification of growth morphology, indicating the presence
Pseudohyphae may be formed but true hyphae are not proof genetic variation in response to stimuli that can produce
duced. This yeast can stably propagate by budding as haploid filamentous growth.17
or diploid cells. Vegetative cell division characteristically
occurs by axial or bipolar budding in which a daughter cell 70.1.1.3 Biology
is initiated as an outgrowth from the mother cell. After cell Life cycle, cell structure assembly, metabolic pathways, gene
separation a scar is left in the budding region on the mother function, and expression regulation of S. cerevisiae are the
cell surface. Haploid cells mate in response to pheromones. most well known among eukaryotes since this organism con-
Diploid cells undergo meiosis and sporulation in condition stituted for decades a model system for its relatively small and
of complete starvation, i.e., absence of fermentable carbon compact genome, comprising 16 chromosomes of sizes 200–
and nitrogen sources. Asci are unconjugated, persistent, and 2200 kb in the haploid stage, and ease of genetic transfor-
contain one–four spherical to ovoidal ascospores. mation and manipulation. Knowledge about the physiology
The variety of morphological phenotypes is a consequence of this yeast has increased after whole genome sequenc-
of adaptive responses to environmental signals. While it is ing and annotation for S. cerevisiae S288c. In this strain,
mainly known as a unicellular species with ellipsoid cells that about 6000 genes are present of which 3.8% have introns.13
divide by multilateral budding and form smooth colonies on Chromosomes contain movable retrotransposons, subdivided

Saccharomyces 617
in the types Ty1–Ty5. These are 5.3–5.7 kb in size and are isolates are able to switch mating type; these are referred to
constituted by retroviral DNA flanked by ∼330 bp terminal as homothallic strains. The wild-type allele of the HO gene
repeats long terminal repeats (LTRs) or δ sequences. The Ty for homothallism encodes a site-specific endonuclease that
transposable elements are involved in insertion/deletion, or promotes mating type switching from a to α or vice versa.
“indel,” events. The clinical strain sequenced, S. cerevisiae HO is transcribed only in the mother cell, so that when a
YJM789, is the haploid isoform of YJM145, a yeast isolated homothallic spore divides, the mother cell switches mating
from the lung of an AIDS patient with pneumonia, which type and immediately fuses with the newly produced daughter
causes death in complement-deficient mice19 and differs in cell.26 A positive correlation exists between growth rate and
phenotype from S288c in being flocculant, displaying colony the transcription of genes of both cytosolic and mitochondrial
morphology switching, and growing at high temperatures.20 translation machinery. Genes related to functions associated
Its genome sequence is studied for correlating genetic traits with oxidative energy metabolism, especially those involving
to the ability of causing infections. Several open reading peroxisomes, i.e., fatty acid metabolism, oxygen metabolism,
frames (ORFs) are found to be unique to YJM789, some of and autophagy, are negatively correlated with growth rate.
which might have been acquired through horizontal trans- The hypothesis is that it serves to protect the cell from the
fer. From genome comparisons between S. cerevisiae S288c longer exposure to oxidative metabolism necessary to produce
and this strain, it emerged that single nucleotide polymor- enough energy at lower growth rates.27 In S. cerevisiae, PHG
phism (SNPs) are the primary cause of phenotypic vari- is regulated by at least two signaling pathways: (1) the nutri-
ability. The remaining variability is generated by insertion/ ent-sensing cyclic AMP–protein kinase A (PKA) pathway and
deletions and consequent difference in gene copy number, (2) the mitogen-activated protein kinase (MAPK) pathway.
with the subtelomeric regions rich in redundant sequences Recently, based on DNA-microarray analysis, a model of yeast
as preferential locations for nonhomologous recombination.14 PHG regulation by autophagy was also derived that illustrates
Moreover, several genes encoding cell surface proteins (such how this recycling process plays a critical role in response of
as TIR1, HSP150, FIT1, AGA1, MNN4, and FLO10), bear- PHG to nitrogen starvation in a gradual manner by mitigating
ing intragenic tandem repeats, have different frame repeat the effects of low nitrogen availability.28 Pseudohyphal dif-
numbers between S288c and YJM789. Their variation in tan- ferentiation in S. cerevisiae is regulated by the cell surface G
dem repeats may generate functional cell surface variability, protein-coupled receptor (GPCR) GPR1 that receives extra-
that contributes to adaptation to the environment and pos- cellular signal from glucose by the Ras signaling system and
sible host immune evasion.22 While strain S288c has 50 Ty by the MEP2 encoded ammonium permease. Ras1/2, partially
elements of all classes, YJM789 genome contains only 17 Ty redundant small GTP-binding proteins, activates the MAPK
elements, all of which are Ty1, Ty2, and Ty5.14 pathway to cause pseudohyphal differentiation.29 The gene
Other nucleic acid types are mitochondrial DNA (mtDNA), MEP2, encoding an ammonium transporter, has been impli-
which encodes components of the mitochondrial translational cated in transducing the signal in response to low ammonium
machinery, approximately 15% of the mitochondrial proteins levels and is subject to nitrogen catabolite repression (NCR).
and the 2 μm autoreplicative circle plasmids. Almost all A strain defective in MEP2 is unable to form pseudohyphae.
S. cerevisiae strains contain three families of viral dsRNA Recent studies suggest that MEP2 plays a role in pseudohy-
that confer the capacity to produce a killer toxin active against phal differentiation through the reuptake of secreted ammo-
other yeasts and are transmitted when packaged in viral par- nium under nitrogen starvation conditions.30
ticles. Saccharomyces cerevisiae also contains a 20S circular The regulation of the gene FLO11, encoding Flo11p,
single-stranded RNA that acts as an independent replicon and appears to be critically dependent on the relative levels of glu-
is inherited as a non-Mendelian genetic element. cose and ammonium.29 One known example of genetic poly-
Due to the gene asset permitting efficient growth in anaer- morphism accounting for a different capability of PHG is the
obic conditions, S. cerevisiae has the ability to consume glu- FLO8 gene, a transcription factor required for pseudohyphal
cose preferentially via fermentation rather than respiration, formation, which in strain S288c contains an inactivating
to grow in total absence of oxygen and to be induced to form amber mutation, yet S. cerevisiae YJM145 forms abundant
respiratory-deficient mitochondrial mutants (“petites”).23 pseudohyphae20 and its FLO8 ORF has no amber mutation.
Fermentation is such a rapid process in S. cerevisiae that opti- Another example of inactivating polymorphism with a conse-
mization of glycolysis allows synthesizing a similar number quence on morphology found in the S288c involves the AMN1
of ATP molecules per second as in the aerobic metabolism.24 gene. The same gene in S. cerevisiae YJM789 contains an
S. cerevisiae tolerates high levels of ethanol, which may be SNP as that confers a more clumpy phenotype to this strain.14
utilized as a source of energy once that glucose is depleted.25
The life cycle of S. cerevisiae alternates between diplo-
phase and haplophase. Clonal reproduction is predominant. 70.1.2 Clinical Features, Pathogenesis,
In heterothallic strains, haploid cells are of two mating types, and Epidemiology
a and α. Mating of a and α cells results in a/α diploids that
undergo meiosis upon starvation. The four haploid spores 70.1.2.1 Clinical Features
resulting from meiosis are contained within the wall of the Though it is commonly perceived as a harmless microorgan-
mother cell (the ascus). The spores of about 70% of natural ism given its thousand-year association with fermented food

and beverage production, numerous case reports of this yeast (fluffy yellow exudates) and esophagitis (yellow-white plaques
exist in the medical literature. Local infections caused by on an erythematous background) can be present in both con-
S. cerevisiae may occur as a consequence of mechanical fac- ditions, and is even indistinguishable from infections due
tors in mildly immunocompromised or immunocompetent to other microorganisms that cause endocarditis.35 Fever is
persons.31,32 Saccharomyces cerevisiae is responsible of vagi- not always present. Histological documentation of deep-site
nitis in a minority of cases with yeast etiology.33 Medical lit- involvement demonstrated the association of S. cerevisiae
erature reports described cases of vaginal infections directly with necrosis and granulomatous reaction.38
caused by S. cerevisiae strains used in domestic or profes- In two studies of in vivo pathogenesis in a mouse model,
sional bread-making.34 different isolates of S. cerevisiae displayed a continuum of
Serious gastrointestinal or systemic infections have been virulence in mice, defined as the capacity for the organism to
observed in immunosuppressed patients. The portal of entry proliferate in the brain and/or persist for an extended period
for invasive infections is mainly digestive: a case described of time.39 After intravenous inoculation, most clinical strains
in an immunocompetent patient was due to the ingestion of tested were able to proliferate principally in the brain, but
health food containing viable yeasts. Symptoms were recur- also in kidneys, spleen, liver, and lungs, whereas nonclini-
rent fever, malaise with nausea, and night sweats. Yeasts cal isolates were readily cleared. Histological inspection of
were isolated from bone marrow and urine specimens. The brains infected with a virulent clinical isolate and an aviru-
patient recovered when he stopped ingesting health food con- lent strain showed that the former was more able to invade
taining yeasts.35 In some instances, the S. cerevisiae strains the parenchyma while the latter was mostly localized in the
responsible for infection were winery-resident strains or bak- capillaries.19
ery strains.31,34 The ability of S. cerevisiae strains to cause infection
S. cerevisiae is currently considered an emerging patho- depends on the possession of fungal properties frequently
gen in part as an effect of the refinement of yeast identifica- associated with pathogenesis, i.e., the ability to grow at high
tion methods, but also for new patient predisposing factors temperatures (38°C–42°C), the ability to invade host cells,
as a consequence of the increase of the population immuno- morphological versatility, and the ability to produce and
compromised for underlying chronic or debilitating diseases, secrete degradative enzymes (proteinases and phospholi-
increased use of immunosuppressive drugs or broad-spec- pases), which could play a role in blocking capillaries, a fun-
trum antibiotics, as well as parenteral nutrition, use of intra- gal characteristic associated with animal mortality.40
vascular catheters, and, finally, the increasing numbers of The ability to grow at 37°C showed no significant asso-
HIV-infected individuals. Infections with lethal outcome ciation with virulence, instead growth at 39°C was signifi-
were observed in patients with critical conditions such as cantly associated with virulence. The capacity to grow at
AIDS, neoplastic and metabolic diseases, intestinal surgery. 42°C is tightly associated with virulence39 and was found
Invasive Saccharomyces infections showed a significantly almost exclusively in clinical strains: In a screening of 118
increased incidence since the 1990s35 and a recent 15 month S. cerevisiae strains, the ability to grow at 42°C was found
survey of fungal bloodstream infections in 54 Belgian hospi- mainly in clinical isolates and in very few industrial strains,
tals reported a threefold increase in the frequency of S. cere- namely, S. boulardii, the wine strain Uvaferm 71B, and two
visiae.36 Of 21 isolates, 19 gave the profile of S. boulardii. baker’s yeasts.21 The high-temperature growth phenotype of
In one patient with multiple yeast infections, S. cerevisiae YJM789, in particular, has been dissected to an SNP resolu-
was associated with C. albicans. Concomitant isolation of tion for several local regions of divergence.14 A gene impli-
S. cerevisiae and a second microorganism, mainly entero- cated in the capacity to grow at 42°C is SSD1, which encodes
bacteria, was reported in some cases of fungemia. In single a protein able to affect various cellular processes including
organ infections, the main sites were the lungs and the heart maintenance of cell integrity, and seems to play an impor-
valves.35 From these evidences, it is clear that S. cerevisiae tant role in the virulence of Saccharomyces organisms. The
should be considered as an opportunistic pathogen of low encoded protein, Ssd1, regulates the biological activity of
virulence rather than a nonpathogenic yeast. Hsp104. This chaperone mediates protein disaggregation
after heat stress, a process that permits the recovery of cell
70.1.2.2 Pathogenesis viability responding to signals from cellular integrity path-
Human infections caused by S. cerevisiae occurred with a ways. The possible involvement of this protein in cell prop-
broad range of clinical manifestations, from fungemia, pneu- erties bound to pathogenesis is indicated by evidence that
monia, and deeply invasive dissemination to localized infec- deletion of its genetic determinant SSD1 led to a significant
tions in specific organs or body districts. Virulent S. cerevisiae increase in virulence for both clinical and plant isolates, as
possess the capability to colonize and persist in various nor- revealed in a mouse model of invasive infection, likely for
mally sterile body sites (e.g., the lung and blood) and, conse- the alteration in composition and cell wall architecture that
quently, is able to survive and disseminate in vivo. However, overstimulates the pro-inflammatory response.41
it has been observed that clinical and nonclinical strains are Regarding the production of extracellular lytic enzymes
poorly capable of adhering to intestinal cell monolayers.37 possibly implied in virulence, it was found that generally the
Invasive Saccharomyces infection is clinically indistinguish- potentially virulent strains produce higher amounts of phos-
able from invasive candidiasis, notably because chorioretinitis pholipases compared to industrial strains.21

Saccharomyces 619
A microbial virulence-related trait that promotes host patients because they are supplied exclusively with sterile
c olonization and invasion is the ability to survive oxidative food, and isolates from patients concurrently hospitalized
stress, exerted by reactive oxygen species (ROS), a compo- in the same unit were identical. Hospital-acquired trans-
nent of mammalian host defense. Among 103 S. cerevisiae mission of this yeast and possible systemic fungal infec-
strains, isolated from clinical and other sources and tested tion is an important cause of death of these patients since
for survival oxidative stress exerted by tert-butyl hydroper- they receive cytotoxic therapy and preventive antifungal
oxide, a stable organic analog of H2O2, clinical isolates, some treatment that can select Saccharomyces strains resistant
soil isolates, and strains from insect guts exhibited the high- to antifungal drugs like amphotericin B and fluconazole.
est mean resistance to ROS expressed as survival rates.42 The ability of the yeasts to persistently colonize multiple
The genetic diversity among the clinical isolates determined body sites and contamination via hands of health care pro-
using five genetic loci suggested multiple origins of ROS viders can explain why in a study regarding colonization
resistance. In a pathogenesis assay with Caenorhabditis ele- of hematological patients S. cerevisiae increased markedly
gans, an established model host, S. cerevisiae infected the during the follow-up; it was detected 1–30 weeks after ini-
nematodes causing disease and death.43 Data indicated that tiation of the cytotoxic chemotherapy in different patients
the host produced reactive oxygen species (ROS) in response and only in one case the patient was already colonized at
to fungal infection. Yeast deletion mutants of genes sod1 and admission.47 In a similar investigation, contamination from
yap1, could not withstand ROS and failed to cause disease, food was considered possible since Saccharomyces strains
thus, indicating that these genes can be considered virulence were absent in the hospital environment and on the hands
markers. or swabs from the personnel.48 Saccharomyces organisms
S. cerevisiae can give rise to the initial steps of bio- were most frequently isolated from blood, either exclusively
film formation by adhering on plastic materials.16 This can or with involvement of other organ(s). The rates of favor-
explain why use of intravascular or other types of catheters able outcome among immunocompromised and immuno-
was among the causes of increase of S. cerevisiae infec- competent patients did not differ significantly, indicating a
tions in nosocomial environments. In support of the possible low virulence of this species.35
implication of biofilm formation in infection transmission via Epidemiologic and clinical differences between S. cere-
catheters is the prompt resolution of symptoms when these visiae and S. boulardii invasive infections were revealed.
were removed44 and the persistence of the infection when Systemic infections like fungemia and septicemia caused
these were left in place.45,46 by S. boulardii resulted from oral administration both in
immunocompromised and immunocompetent patients, thus
70.1.2.3 Epidemiology indicating the ability of this organism to cross the intestinal
For the establishment of the epidemiological trend, viru- mucosa.49,50 S. boulardii was exclusively isolated from blood.
lence levels, and mechanisms, 92 documented cases of Predisposition factors for S. boulardii infections included
proven invasive S. cerevisiae infection satisfying the defi- intensive care unit hospitalization, presence of an indwelling
nitions of the Invasive Fungal Infection Cooperative Group catheter, and intestinal disease. The lack of organ involve-
of the European Organization for Research and Treatment ment and the better prognosis could be due to the relatively
of Cancer-Mycosis Study Group of the National Institute of low virulence of S. boulardii39 or the lower frequency of
Allergy and Infectious Diseases were taken into account. immunocompromise in the group of patients with S. boular-
These definitions imply fungemia, isolation from normally dii infection. Though the level of virulence of S. boulardii is
sterile fluids (e.g., pleural and synovial fluids), and deep- modest, it has been reported to vary among different batches
site infections either proven by histological examination and is therefore not predictable.6 By applying Affimetrix
for sites susceptible to colonization (lungs, peritoneum, oligonucleotide chips designed on the whole S. cerevisiae
esophagus). Fifteen cases were diagnosed before 1990 and sequenced genome, a distinctive genetic feature of S. bou-
76 after 1990.35 All patients had at least one condition facil- lardii with implications for pathogenicity was found, i.e., a
itating the development of invasive fungal infection; the higher copy number for genes of the cAMP pathway involved
most frequent were an intravenous catheter and a previous in PHG.7 Other overrepresented genes are involved in protein
receipt of antibiotic therapy. S. boulardii was considered synthesis and stress response. Epidemiological data available
to be the etiological agent in 37 cases (40.2%). Molecular led the EFSA (European Food Safety Authority) to recom-
identification was performed in 23 cases, mainly by means mend that S. boulardii should certainly be contraindicated
of restriction enzyme analysis of mtDNA. Among the 37 for patients of fragile health, as well as for patients with a
patients with presumptive S. boulardii infection, five did central venous catheter in place.51
not take this probiotic at the time of diagnosis. However,
the similarity of the genotypic profile between S. boular-
dii isolates from patients and from probiotic preparations 70.1.3 Laboratory Diagnosis
strongly suggested nosocomial acquisition, with catheters
being a likely portal of entry because of possible con- 70.1.3.1 Conventional Techniques
tamination through hand transmission. This explanation is When not required for epidemiological studies, the identifica-
given for cases of S. cerevisiae infection in hematological tion of the yeasts in daily practice is not carried out in clinical

microbiology laboratories; the only routine procedure can Different genetic fingerprinting methods have been
be microscopic observation of the organisms from positive used to compare clinical isolates and strains from other
cultures. Specimen categories include blood and other body sources. Initially, karyotyping by pulsed field gel elec-
fluids, feces, and swabs from different sites (throat, vagina, trophoresis (PFGE), restriction length polymorphisms of
or external body sites). Cases of unfavorable outcome require whole genome or mtDNA were applied for strain discrimi-
culturing of internal organs like lungs, liver, and kidneys nation.58 A study regarding DNA typing of 60 clinical and
in order to individuate the site of infection and the cause of nonclinical isolates of S. cerevisiae reported that EcoRI
death. Cultures of catheter tips are necessary, when contami- DNA digestion profiles of clinical isolates were very het-
nation is suspected to derive from such devices, to make the erogeneous, exhibiting little clonality. However, it was pos-
right decision about their removal. In some outbreaks, the sible to subgroup these isolates into two clusters, A and B,
hospital environment was also sampled in order to individu- on the basis of the presence or absence of a 3.0 kb band on
ate the source of infective Saccharomyces. This was done by agarose gel electrophoresis of EcoRI-digested DNA and a
directly touching Petri plates with finger tips of the involved statistical association of virulence with the group A DNA
personnel and sweep plating surfaces and clothes.48 For iden- type was found.58
tification of the species, the first step is direct seeding of sam- Different S. cerevisiae-specific PCR-based genetic profil-
ples onto selective media such as Sabouraud dextrose agar ing techniques were developed and applied to the distinction
(SDA) supplemented with chloramphenicol and incubation of clinical strains. Among these techniques, amplification
for 24–48 h. Isolated colonies are inoculated into miniatur- of microsatellites from different loci was found to be highly
ized identification trays available from different manufac- reproducible, able to discriminate single strains according
turers. The most frequently used are API 32C and API 20C to the guidelines proposed by the European Study Group on
AUX test strips or Vitek 2 ID-Yst cards (bioMèrieux, Marcy Epidemiological Markers.35 Microsatellites are short sequence
l’Etoile, France). Results are acquired in 48 h and identifi- repeats (SSR), usually less than 10 nt and highly polymorphic
cation is based on carbohydrate fermentation profiles. Some in length, found in many ORFs and in noncoding regulatory
systems concomitantly provide data on susceptibility to anti- regions. All the S. cerevisiae strains, including S. boulardii,
fungal drugs. Another conventional detection system is a dif- gave 100% positive PCR results with primers flanking all
ferential plating medium called chromogenic Candida agar microsatellites tested by Hennequin et al.59 Limitedly to the
(CHROMagar Company, Paris, France) that has been proven loci considered, the method was proven to be specific for
to allow the growth and distinction of many yeast species, S. cerevisiae since isolates belonging to the closely related
including S. cerevisiae.52 species S. pastorianus, S. paradoxus, and S. bayanus failed
to be reproducibly amplified.59 Microsatellite amplification
70.1.3.2 Molecular Techniques profiles were exploited for strain clustering and different loci
The choice of molecular techniques for identifying yeasts were used to this aim.12,59,60 Amplification products of sizes
involved in infections offers advantages like faster diagnosis in the range 110–370 bp are obtained that are separated on
and unequivocal identification. Moreover, after identification, polyacrylamide or agarose gels depending on size and differ-
the same nucleic acid samples extracted from pure cultures ence in length.59,60
can be used for strain typing by methods enabling the defini- In the first study on clinical strains, it was found that some
tion of the infection source. Three species-specific PCR tests of them had microsatellite repeat profiles similar to bakery
are described in literature for the culture-independent detec- strains. Moreover, this method provided a distinction of
tion of S. cerevisiae: two have been applied to wine and one S. boulardii based on the size of the amplification product
to feces.53–55 All these tests were designed for quantification obtained for the microsatellite trinucleotide repeat sequence
by real-time PCR, however, the specific primer sets can be (CAG)9,59,61 concomitantly confirming its affiliation to the
used for identification by conventional PCR. S. cerevisiae species.
Particularly noteworthy for the diagnosis of fungal infec- Before the assessment of this technique, multiple enzy-
tions is the APEX (arrayed primer extension) microarray sup- matic restrictions of mtDNA10 were required for a definitive
port projected for one-step detection of common pathogenic identification since the lack of univocal phenotypic traits
fungi from clinical specimens based on their ITS1 and ITS2 did not permit to identify S. boulardii in a routine man-
sequences, in which a specific probe for S. cerevisiae was ner. Another widely used PCR fingerprinting procedure for
included.56 Authors stated that identification by this method S. cerevisiae is determination of inter-δ sequence amplifi-
is unambiguous, sensitive, and reproducible. cation profile.10 The amplification products are separated
When DNA from pure cultures is available, a rapid method by electrophoresis on agarose gel. Also with this method,
for the identification of S. cerevisiae as well as of other yeast de Llanos et al.10 found that a S. cerevisiae isolate from an
species is PCR amplification of the ITS1-5.8S-ITS2 rDNA infection case was highly similar to two commercial bread-
internal transcribed spacer region, followed by digestion making strains. The two molecular types were also found
with frequent cut restriction endonucleases CfoI, HaeIII, and similar to several clinical isolates from feces, pharynx, and
HinfI,57 and by separation on a 3% agarose gel. Sequencing vagina previously characterized.
of the domains D1 and D2 located at the 5′ end of gene 26S Finally, another PCR-based fingerprinting method pro-
is another reliable identification method.1 posed is based on amplification of minisatellite repeats,

Saccharomyces 621
very commonly present inside the ORFs of cell wall protein a direct detection method specific for S. cerevisiae has been
genes in S. cerevisiae and other yeasts.62 These repeats are experimented to date.
recombinogenic and can be a source of genetic variability
among strains. This typing method was developed to exploit 70.2.1.2 Extraction of DNA from
minisatellite variability for the distinction of different strains Colonies or Pure Cultures
isolated from wine. The minisatellite regions selected in the For preparing pure cultures of the isolated clinical S. cere-
sequences available for the reference strain S. cerevisiae visiae strains, single well-isolated colonies that look round,
S288c had to satisfy the following criteria in order to allow convex, cream colored, and smooth or slightly wrinkled are
amplification from different strains and detection on agarose picked from plates and transferred directly in tubes with lysis
gel: at least 80% identity between repeats and at least 20 bp solution or in broth for subculturing. In this second case, the
repeat length, respectively. Moreover, the test was restricted colony is allowed to develop for 48 h at 30°C. Part of the cul-
to nonenzymatic proteins supposed to be more variable since ture will be used to prepare a frozen stock of the isolate by
their sequence variation would not cause a lethal loss of func- transferring 1 mL of it in a sterile cryovial, adding ca. 200 μL
tion. The genes chosen were AGA1 coding for the anchoring of sterile glycerol, mixing, and storing at −80°C. DNA suit-
subunit of α-agglutinin; DAN4, member of the PAU pro- able for typing by a PCR-based method from a pure culture of
teins of unknown function; HSP150, a cell wall–bound heat S. cerevisiae can be derived from the protocol of Hennequin
shock protein; and the major cell wall glycoprotein SED1. et al.59 applied prior to microsatellite amplification. The pro-
Amplification products ranged in size between 1000 and cedure includes the following steps:
1600 bp and were separated on agarose gel. In Table 70.1 all
the mostly adopted methods to type S. cerevisiae are listed 1. Harvest yeast cells from 3 mL of culture by centrif-
and briefly described. ugation at 10,000 × g for 5 min.
2. Remove the supernatant and wash the cell pel-
70.2 Methods let with 1 mL of TE buffer; centrifuge the cells at
10,000 × g for 5 min.
70.2.1 Sample Preparation 3. Resuspend the cell pellet in 200 μL of lysis buffer;
incubate for 1 h at 37°C.
70.2.1.1 Sample Handling
4. Add 200 μL of lysis solution; incubate at 65°C for
In clinical laboratories, sample types for the isolation and 30 min; keep on ice for 5 min.
identification of yeasts are blood and other body fluids or 5. Add 100 μL of 5 M potassium acetate; keep at 4°C
lavages, swabs from different sites, and tissue specimens. for 45 min; centrifuge for 5 min at 10,000 × g.
Currently, blood samples are first inoculated in 6. Transfer the supernatant in a clean tube; precipitate
BACTEC™ Plus Aerobic/F and Anaerobic/F bottles and DNA by addition of 30 μL of ammonium acetate
then are monitored using the BACTEC 9240 system (Becton solution plus three volumes of 100% ethanol; cen-
Dickinson) or similar systems. Yeast-positive cultures identi- trifuge for 10 min at 10,000 × g.
fied by Gram’s stain are streaked on isolation media or can 7. Remove the supernatant and add 1 mL of 70% etha-
be used for nucleic acid extraction and identification by spe- nol; centrifuge for 2 min at 10,000 × g.
cies-specific molecular methods. Direct streaking onto Petri 8. Carefully remove the ethanol by gentle aspiration;
dishes is a common practice also for the analysis of swabs, dry in open tubes under a fume cabinet until all
lavages, and tissue specimens. Frequently used media for residual ethanol evaporates.
yeast isolation are SDA agar (casein hydrolyzate 5 g/L, tis- 9. Resuspend in 50 μL of TE buffer; store at 4°C for
sue hydrolyzate 5 g/L, dextrose 40 g/L, agar 15 g/L, pH 5.6) up to 6 months.
plus 4 g/L chloramphenicol; YEPD (yeast extract 10 g/L,
peptone 20 g/L, glucose 20 g/L, agar 15 g/L); and Wallerstein
Laboratory (WL) nutrient agar (Oxoid Ltd.) both supple- 70.2.1.3 Extraction of Total DNA from
mented with 0.1 g/L chloramphenicol or 15 μg/L biphenyl to Human Fecal Samples
inhibit bacteria and molds, respectively. Solid samples like Application of direct detection of S. cerevisiae in clinical
feces or dough are homogenized in ninefold volume of ster- samples by PCR would expedite the diagnosis of infec-
ile tryptone solution (casein tryptone 1 g/L, NaCl 9 g/L) in tions due to this yeast. In a study aimed at estimating
Stomacher bags. If not analyzed soon all sample types must the efficiency of different methods for extracting a high
be stored at −80°C in sterile plastic bags or tubes before inoc- amount of good quality DNA from fungal species, it was
ulation or direct DNA extraction. found that best results are obtained by combining enzy-
All the identification methods described in the following matic treatment with mechanical disruption by mortar
part of this section are based on PCR amplification. Both and pestle grinding of material frozen in liquid nitrogen.63
direct detection in a clinical or food sample and identifica- This method was applied later and allowed high sen-
tion of an organism in pure culture will be reported. Sample sitivity in the PCR-based detection of S. cerevisiae and
types considered are pure cultures and feces. The latter was Saccharomyces sensu stricto DNA from feces.54 In the
chosen because it is the only type of clinical sample in which reported experiments, fecal samples were frozen at −80°C

TABLE 70.1
Molecular Methods Developed for the Discrimination of S. cerevisiae Isolates at the Species or Strain Level
Method Procedural Details Discrimination Level Disadvantages
Karyotyping Purification of total DNA by extraction from cells Single strain Time-consuming
immobilized in agarose plugs Delicate and expensive equipment
Electrophoretic separation in a minimum of 23 h by
PFGE
Profile analysis
EcoRI RFLP Total DNA extraction from pure cultures Strain clusters High amount of unfragmented DNA needed
Digestion for 90 min with EcoRI endonuclease Time-consuming
21 h run on 1% agarose gel No distinction of the restriction profile group
Profile analysis B from S. paradoxus
mtDNA restriction Total DNA extraction from pure cultures Single strain Relatively high amount of unfragmented
Digestion overnight with HinfI or RsaI DNA needed for digestion (∼0.5 μg)
endonucleases Time-consuming
Run on 1% agarose gel
Profile analysis
ITS1-5.8S-ITS2 region DNA extraction from pure cultures Species None
amplification and PCR with primers flanking the ITS1-5.8S-ITS2
restriction region
Restriction with endonucleases CfoI, HaeIII, and
HinfI
Separation on 3% agarose gel
Amplification of δ DNA extraction from pure cultures Single strain Unstable patterns with some primer sets
sequences PCR with primers targeted to transposon δ sites Not all strains produce amplification
Separation on 1.5% agarose gel products
Profile comparison
Amplification of DNA extraction or cell disruption by freeze–thaw Single strain Need of separation on polyacrylamide gels
microsatellite sequences treatment from pure cultures in a sequencing apparatus in the original
PCR with primers flanking microsatellite sequences method or labeling with different
of different genetic loci fluorochromes due to the range of
Separation on polyacrylamide gels containing dimension of the amplification products
formamide or on agarose gel obtained for some genetic loci
Profile comparison
Amplification of the DNA extraction or cell disruption by heat treatment Single strain Need to combine the profiles obtained for
minisatellites in cell from pure cultures amplicons from different loci with four
wall protein genes PCR amplification with a primer set targeted on the different primer pairs
sequences flanking the minisatellite regions of cell
wall protein genes
Separation on agarose gel
Profile comparison
Real-time PCR targeted DNA extraction by mechanical cell disruption Culture-independent To date experimented only on feces and wine
on rRNA genes and ITS Amplification in a normal PCR reaction followed by detection and
or on S. cerevisiae- conventional electrophoresis or in a real-time PCR identification of
specific random apparatus for quantification using SYBR Green as S. cerevisiae
amplification products a fluorescent dye
in sterile plastic bags before DNA extraction. The phases 4. Transfer the sample in a 2 mL centrifuge tube; cen-
of the method are as follows: trifuge at 10,000 × g for 10 min at 4°C.
5. Carefully transfer the supernatant in a new tube.
1. Thaw the fecal sample; transfer about 1 g of it in a 6. Add 500 μL isopropanol and 150 μL of 3 M
laboratory mortar previously well cleaned and ster- sodium acetate solution pH 4.8; mix for 5 min
ilized by keeping overnight at 200°C in a stove. and keep for additional 5 min at room tempera-
2. Add 500 μL of extraction buffer; immediately ture; centrifuge at 10,000 × g for 10 min at room
freeze by liquid nitrogen insufflations. temperature.
3. Grind for 2 min; add 500 μL of phenol–chloroform– 7. Carefully discard the supernatant and add 500 μL
isoamyl alcohol mixture and rotary mix at room of 70% ethanol to the pellet; centrifuge for 2 min at
temperature for 10 min. 10,000 × g.

Saccharomyces 623
8. Remove as much ethanol as possible by gentle aspi- 5. Stain the gels in 0.5 μg/mL ethidium bromide aque-
ration; dry in the open tubes under a fume cabinet ous solution.
until all residual ethanol evaporates. 6. Estimate the sizes of PCR amplicons by comparison
9. Resuspend in 100 μL of TE buffer containing DNase against a DNA length standard (e.g., 100 bp ladder,
free RNase A; store at 4°C allowing RNA digestion Invitrogen). Examples of profiles are reported in
overnight. Figure 70.1.
For specific PCR detection, the method developed by

Chang et al.54 is represented here. The oligonucleotides
Detection from pure cultures can be carried out either SCDF (5′-AGGAGTGCGGTTCTTTG-3′) and SCDR
by the ITS1-5.8S-ITS2 rDNA internal transcribed spacer (5′-TACTTACCGAGGCAAGCTACA-3′) targeting the D1/
region amplification/RFLP (restriction fragment length D2 region of 26S rRNA gene and amplifying a 310 bp DNA
polymorphism) method recommended by Esteve-Zarzoso fragment from S. cerevisiae are used. Amplification is done
et al.57 for the identification of several yeast species or by using DyNAmo™ HS SYBR® qPCR kit (FINNZYMES)
S. cerevisiae-specific PCR tests.53–55 In addition, the iden- master mix, 10 pmol of each primer, and 1 μL template. The
tification of S. boulardii can be carried out by specific cycling program comprises initial denaturation at 95°C for
amplification of the (CAG)9 microsatellite repeat at locus 15 min; 45 cycles of denaturation at 94°C for 20 s, annealing
YLR177w.59,61 The former method is worth being carried at 58°C for 30 s, and extension at 72°C for 45 s; and a final
out because when a yeast contaminant does not belong to extension at 72°C for 5 min. Amplification is performed in an
the species S. cerevisiae it anyhow allows the true specie OPTICON™2 DNA Engine, MJ Research.
to be determined. The S. cerevisiae-specific PCR tests are Finally, the S. boulardii-specific microsatellite ampli-
useful in culture-independent detection, too. The PCR test fication test with primers Boul3 (5′-CTTAAACAAC
specific for S. boulardii, when applied on pure cultures, can AGCTCCCAAA-3′) and Boul4 (5′-ATTTCTGATGC
elucidate the epidemiology of infection cases caused by this GCTGATTCAT-3′)61 generates a PCR product of 130 bp.
particular strain. The reaction mixture is composed of 1.5 mM MgCl2, 200 μM
PCR/RFLP of the ITS1-5.8S-ITS2 rDNA internal tran- of each dNTP, 0.2 μM of each primer, and 1 U of Taq DNA
scribed spacer region comprises amplification with oligo- polymerase. The cycling program included initial denatur-
nucleotides ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) ation at 94°C for 4 min; 28 cycles of 94°C for 30 s, 56°C for
and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′); digestion 45 s, 72°C for 30 s; and a final extension at 72°C for 10 min.61
with frequent cut restriction endonucleases CfoI, HaeIII, and PCR fragments are denatured (94°C for 4 min), separated
HinfI; and separation on a 3% agarose gel. Species identifica-
tion is achieved by comparison with molecular weight tables
M 1 2 3 4 5 6 7 M
reporting the profiles obtained for each yeast species. For this
purpose, primers delimiting a region of the rRNA coding
region comprising the 5.8S rRNA gene and the two internal
transcribed spacers ITS1 and ITS2 are used. S. cerevisiae CfoI
shows an 880 bp amplicon that gives bands of 385, 365, and
130 bp with CfoI; 320, 230, 180, and 150 bp with HaeIII; and
365 bp and 155 bp with HinfI.
1. Prepare PCR mixture containing 0.5 μM of each

primer, 10 μM deoxynucleotides, 1.5 mM MgCl 2
and 1× PCR buffer, sterile double-distilled water up HaeIII
to the desired volume and 1 μL of DNA sample in a
30 μL reaction.
2. Run a cycling program consisting of initial denatur-
ation at 95°C for 5 min; 35 cycles of denaturing at
94°C for 1 min, annealing at 55 ± 5°C for 2 min, and
extension at 72°C for 2 min; and a final extension at
72°C for 10 min. Hinf I
3. Digest the PCR products (10 μL or approximately
0.5–1.0 μg) without further purification with 10 U
(1 μL) of the restriction endonucleases CfoI, HaeIII,
and HinfI at 37°C for 15 min.
4. Separate the PCR products and their restriction Figure 70.1 ITS1-5.8S-ITS2 PCR/RFLP profiles of seven
fragments on 1.4% and 3% agarose gels, respec- S. cerevisiae isolates from grapes (numbers 1–7). M; 1 kb Plus DNA
tively, in 1× TAE buffer. Ladder (Invitrogen).

on 7.5 M urea-12.5% polyacrylamide gels in 0.5× TBE buf- transporter MEP2 gene that show higher expression during
fer (5.4 g/L Tris base, 2.75 g/L boric acid, 2.92 g/L EDTA pseudofilamentation,29 the gene PLB1 encoding a phospho-
pH 8.1), and then stained with ethidium bromide solution as lipase,64 and genes SOD1 and YAP143 are possible candi-
above. Length polymorphisms are evaluated by comparison dates. Research in the direction of finding suitable target
with the migration of a reference strain, with a slightly dif- genes can be validly aided by the development of DNA
ferent size of the amplicon, e.g., 133 bp from S. cerevisiae microarray supports constructed on the basis of available
S288c.59 S. cerevisiae genome sequences. Other targets to be inves-
tigated with respect to their contribution to virulence and
to the capacity to evade host immune defenses are genes
70.3 C
onclusions and encoding cell surface proteins. Since this property can
Future Perspectives depend on the number of minisatellite repeats found in their
The introduction of molecular diagnostics, with the conse- ORFs,22 an amplification strategy proposed by Marinangeli
quent increase in rapidity, specificity, and ease of execution, et al.62 could allow linking the profiles found for some of
has not only expedited the identification and diagnosis of those genes to virulence. At present, this kind of molecular
S. cerevisiae infections, but also revealed the true dimen- method has not yet been developed. The current testing for
sion of the risk related to the diffusion and hazardousness the safety of food starter strains still relies on phenotypic
of this opportunistic pathogen. Reviewing the infection evaluation of characters strongly associated to pathogenic-
cases attributable to S. cerevisiae through 2005,35 it is clear ity like growth capacity at 42°C, PHG, and phospholipase
that the improvement of diagnostic procedures has played activity.21
a key role in tracing a veritable outline of the incidence Given that little or no information is available to the public
trend. about the hazardousness of the bakery yeast, correct instruc-
PCR-based species-specific identification is now widely tions, especially directed to food and beverage operators,
adopted in research and clinical laboratories to supplant the could prevent the onset of many infectious cases and favor
current procedures based on rather equivocal physiological the development of good hygienic practices that minimize
identification keys. As the methods proposed for S. cerevi- the environmental diffusion of the yeast and its delivery via
siae detection/identification have been designed for culture- food to infection-predisposed individuals.
independent quantitative analysis with the double advantage
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Part II
Bastidiomycota

71 Coprinopsis and Hormographiella
Contents
71.1 Introduction...................................................................................................................................................................... 629
71.1.3 Diagnosis.............................................................................................................................................................. 630
71.2 Methods............................................................................................................................................................................ 631
71.2.2.1 Sequencing Analysis of the ITS 2 Region............................................................................................. 631
71.2.2.2 PCR-RFLP Analysis of SSU and ITS 2 Regions.................................................................................. 631
71.3 Conclusions....................................................................................................................................................................... 631
References.................................................................................................................................................................................. 632
71.1 Introduction 71.1.2 Clinical Features and Pathogenesis

71.1.1 Classification and Morphology Coprinopsis cinerea is observed growing on lawns.
Hormographiella aspergillata has been isolated from
The genus Coprinopsis is a basidiomycete belonging human lung tissue, bronchoalveolar lavage fluid, brain tis-
to the family Psathyrellaceae, order Agaricales, class sue, cerebrospinal fluid, and eye and skin scrapings, and was
Homobasidiomycetes, phylum Basidiomycota, kingdom responsible for pneumonia, lung abscess, endocarditis, and
Fungi. The family Psathyrellaceae currently consists of five keratomycosis in immunocompetent and immunosuppressed
genera: Coprinellus, Coprinopsis, Parasola, Psathyrella, individuals [6–9]. The fungus is responsible for keratomyco-
and Psathyrellaceae incertae sedis [1]. In turn, the genus sis in dogs [5].
Coprinopsis covers 36 recognized species and 11 unas- Nenoff et al. [8] described a fatal case of pulmonary
signed species. Of these, Coprinopsis cinerea (inky cap infection with Hormographiella aspergillata in a 40-year-
fungus) (obsolete synonym: Coprinus cinereus; anamorph: old woman, who underwent autologous bone marrow trans-
Hormographiella aspergillata) is a mushroom species, plantation for acute lymphoblastic leukemia. A Coprinopsis
whose involvement in human disease process is through its (Coprinus) sp. was isolated repeatedly from bronchial
anamorph H. aspergillata. secretions and bronchoalveolar lavage. Histopathological
The anamorphic genus Hormographiella (class Asco examination of the lung tissue demonstrated septate hyphae
mycetes, phylum Ascomycota) is composed of four mold characteristic of Aspergillus and Coprinopsis sp.
species: H. aspergillata, H. candelabra, H. candelabrata, Verweij et al. [9] documented another fatal case of
and H. verticillata [2]. The anamorph of Coprinopsis cinerea pulmonary infection due to H. aspergillata involving a
(formerly Coprinus cinereus), H. aspergillata is associated 24-year-old man receiving intensive cytotoxic treatment
occasionally with diseases in humans and animals [3]. for acute lymphoblastic leukemia. The patient became neu-
Hormographiella aspergillata shows moderate growth tropenic with fever and headache and complained of right-
on mycological media (e.g., Sabouraud glucose agar, potato sided pleuritic chest pain. Chest radiographs showed focal
dextrose agar, and chocolate agar). Colonies measure 3 cm pulmonary infiltrates in the right lower lobe, progressing
in 10 days at 30°C and are dense, velvety with cottony tufts to the left upper and lower lobes of the lung. The patient
and irregular margin, white to slightly cream, and pale on was treated with amphotericin B and then itraconazole.
reverse. Hyphae are hyaline and septate; conidiophores However, the patient died from respiratory failure. A nec-
are simple and well-differentiated. Arising from the end rotizing bronchopneumonia was noted at autopsy. Periodic
of conidiophores either in clusters or in irregular groups, acid-Schiff and Gomori methenamine silver stains revealed
arthroconidia are catenate, hyaline, smooth-walled, single- a large mass of hyphae in the cavity of the lung abscess.
celled, and rectangular or cylindrical with truncate ends, but The hyphae, measuring between 1.9 and 4.6 μm in diam-
with terminal cells rounded at the tip [4,5]. eter, were septate, showing irregularly shaped swellings
629

and branching at acute angles or dichotomously. Cultures allogeneic HLA-matched bone marrow transplant and later
of the left and right lungs grown for 2 days yielded white developed odynophagia and persistent febrile neutropenia. A
to cream, cottony and dense fungal colonies, while cul- computed tomography (CT) scan of the lungs revealed pul-
tures were not obtained from other organs. Microscopic monary nodules. The patient had white plaques involving
examination of the fungus showed hyaline, septate hyphae, the soft palate and pharynx. A smear from a throat culture
macronematous conidiophores with no clamp connections, showed hyphal elements and conidiophores, and a biopsy
and straight or curved, thin-walled conidia accumulating of the palate lesion revealed submucosa and mucosa infil-
around the conidiophore. As the isolate displayed undi- trated with hyphal forms with sparse septation, rare branch-
agnostic arthroconidial shape and failed to sporulate, its ing, and chlamydoconidia, which was confirmed by ITS 2
identity was confirmed as H. aspergillata (C. cinereus) on sequencing as Rhizomucor variabilis. Following treatment
the basis of small subunit (SSU) and internal transcribed with posaconazole and caspofungin, the palate lesion had
spacer (ITS) restriction fragment length polymorphism decreased in size, with improved symptoms. However, the
(RFLP) patterns. patient had persistent severe refractory pancytopenia and
There was also a report of a lung abscess due to H. asper- developed an altered mental status and a generalized seizure.
gillata (anamorph. Coprinus cinereus) in a patient with non- A CT scan of the brain showed multiple hypodense lesions
Hodgkin’s lymphoma [10]. In addition, Lagrou et al. [11] of the cerebral hemispheres and cerebellum and a repeat CT
described the involvement of Hormographiella aspergil- scan of the lungs uncovered cavitating lesions in the right
lata in lung tissues of a 34-year-old man, who underwent an upper lobe/right middle lobe. A modified therapy consisting
uncomplicated allogeneic peripheral blood stem cell trans- of liposomal amphotericin (Ambisome) and caspofungin was
plantation from a human leukocyte antigen (HLA)-identical initiated. The patient developed high fever and new small
sibling for acute myeloid leukemia. The patient presented with erythematous skin papule on the right knee and the left arm.
serious capillary leak syndrome, hepatic and renal failure, Cultures of the skin biopsy yielded a white mold, which was
progressive encephalopathy, neutropenic fever, bilateral pul- identified as Hormographiella aspergillata. The patient died
monary infiltrates, dyspnea, and cough. Microscopic exami- from respiratory failure 2 weeks after the appearance of the
nation of bronchoalveolar fluid (BAL) samples with Grocott initial skin lesion.
staining showed Aspergillus-like molds, and cultures of the Hormographiella spp. are considered sensitive to some
BAL samples yielded fast-growing, white to slightly cream- azoles such as miconazole but resistant to fluconazole or itra-
colored dense fungal colonies. Clusters of rectangular, cylin- conazole; they are variably sensitive to amphotericin B and
drical arthroconidia with truncate ends were observed but are resistant to flucytosine [3].
with terminal cells rounded at the tip produced from conidio-
phores. The patient died from respiratory failure and refrac-
71.1.3 Diagnosis
tory septic shock. At autopsy, multiple intraparenchymal and
subpleural hemorrhagic nodules were found in both lungs. Infections caused by Hormographiella aspergillata are
Hematoxylin and eosin staining of the lung tissues confirmed reported increasingly, especially in patients with acute leu-
the presence of septated hyphae. The fungus was identified kemia or other underlying diseases. It is important to identify
as Coprinus cinereus by amplification and sequencing of the Hormographiella from other molds as this fungus displays
ITS 2 region. It is interesting that the teleomorph was identi- varied sensitivity to antifungal drugs.
fied here rather than the anamorph, and further verification Filamentous basidiomycetes are notably difficult to
may be required. identify because they do not always show key diagnostic
Conen et al. [12] also documented three cases of invasive features and tend to only form undifferentiated arthroco-
pulmonary infections with Hormographiella aspergillata in nidia. For example, some Hormographiella isolates fail
patients undergoing treatment for acute leukemia. The fun- to sporulate, and others do not display diagnostic clamp
gus was detected in BAL, and despite systemic antifungal connections or arthroconidial shape. Other common fungi
therapy with voriconazole and amphotericin B, all three forming arthroconidia include Geotrichum candidum,
patients died. Greer et al. [13] presented a case of H. aspergil- Trichosporon species, Geomyces pannorum, Malbranchea
lata-related endocarditis involving a 77-year-old female with species, Arthrographis kalrae, and Scytalidium species.
a bioprosthesis for symptomatic mitral valve stenosis. The Arthrographis, Geomyces, and Hormographiella also pro-
patient developed an unusual clot-like mass on the atrial side, duce conidiophores. However, the Hormographiella conid-
due to extensive colonization by a thick filamentous fungus iophore is clearly broader than the conidiogenous hyphae,
with true hyphae, pseudohyphae, and yeast forms. The fun- which is sometimes useful [2].
gus was identified as Hormographiella aspergillata, which is Definitive determinations of Hormographiella are only
perhaps surprising as yeast forms are not associated with the possible with molecular techniques using RFLP patterns of
fungus in question. PCR-amplified ITSs (~600 bases) and SSU rRNA (~1700
Abuali et al. [4] described the isolation of Hormographiella bases). Alternatively, sequencing analysis of ITS 2 and the
aspergillata from a skin lesion of a 14-year-old female patient D1/D2 variable domains (~600 bases) at the 5′ end of the
with acute myelogenous leukemia. The patient underwent an large subunit rRNA gene (D1/D2) is required [14–16].

Coprinopsis and Hormographiella 631
71.2 Methods (5′-TCCTCCGCTTATTGATATGC-3′) to amplify a 350 bp

fragment of the ITS 2 region, interspacing the 5.8S rRNA
71.2.1 Sample Preparation and 28S rRNA of fungal ribosomal RNA genes for identifica-
Clinical specimens (e.g., BAL fluid) are first examined by tion of Hormographiella.
microscopy for mycotic elements with KOH and other stains. Procedure
Tissue samples (e.g., skin, lung, liver, spleen, and kidney tis-
sues) are fixed in 10% formalin, embedded in paraffin, sec- 1. The PCR mixture (30 μL) is composed of 12 pmol of
tioned at 5 μm, and stained with periodic acid-Schiff Gomori each primer, 0.25 mM each of dATP, dCTP, dGTP,
methenamine silver with eosin counterstain or hematoxylin and dTTP, 5 mM MgCl2, 1× reaction buffer, 2.5 U
and eosin. Taq DNA polymerase, and 3 μL paraffin extract.
Portions of the samples are inoculated on mycological 2. Amplification is performed with an initial 94°C for
agars (e.g., Sabouraud dextrose agar, Mycosel, inhibitory 3 min; 35 cycles of 94°C, 55°C, and 72°C for 30 s
mold agar, chocolate agar) and incubated at 30°C or 37°C in each; and a final 72°C for 7 min.
ambient air. Tissue samples are homogenized (Stomacher 80 3. PCR products are analyzed on a 1.5% agarose gel
Lab-Blender) and streaked onto mycologic agars. Production stained with ethidium bromide. Fragments are
of conidia may be stimulated by subcultivation on Takashio purified with the high pure PCR purification kit
medium (Sabouraud agar with 0.2% glucose). The isolate may (Roche Diagnostics) and 50 ng of purified PCR
be transferred to a potato dextrose agar plate and overlaid product is sequenced with the BigDye v.3.1 termi-
with sterile coverslips and incubated. The mold is examined nator sequencing kit (Applied Biosystems) using the
by placing the coverslip onto a glass slide with a lactophenol same primers used for PCR. Reactions are run on
cotton blue stain. an ABI 3130xl (Applied Biosystems) and sequences
For DNA extraction, approximately 1 cm2 of fungal mate- are edited using Sequencher 4.6 (GeneCodes, MI).
rial is transferred to a 2 mL Eppendorf tube containing a 2:1 Sequences proofread in both directions are com-
(wt/wt) mixture of silica gel and Celite (silica gel H, Merck pared against GenBank using BLAST.
7736/Kieselguhr Celite 545; Machery) and 300 μL of TES
buffer [2 g Tris (hydroxymethyl)-aminomethane, 0.38 g Note. The primer combination ITS 3/ITS 4, covering the
Na-EDTA, and 2 g sodium dodecyl sulfate in 80 mL of ultra- ITS 2 region, amplified a product of about 350 bp from
pure water (pH 8)]. The fungal material is ground with a the clinical sample. Sequence analysis of the fragment
micropestle for 1–2 min. The volume is adjusted by adding gave 343 bp of sequence information after trimming of the
200 μL of TES buffer. After vigorous shaking and the addi- primer sequences. Comparison against GenBank showed
tion of 10 μL of a 10 mg/mL concentration of proteinase K to a 99.7% identity (one mismatch over the 343 bp) to the
the tube, the mixture is incubated at 65°C for 10 min. With corresponding sequence of C. cinerea, the anamorph of
the addition of 140 μL of 5 M NaCl solution, the mixture is which is H. aspergillata (see also Lagrou et al. [11] above).
combined with 1/10 volume (~65 μL) of cetyltrimethylammo- Interestingly, other primer combinations covering ITS 1 or
nium bromide (CTAB) buffer 10%, followed by incubation parts of 18S rRNA gene did not result in any amplification
for 30 min at 65°C. One volume (~700 μL) of chloroform–iso- products in this case.
amyl alcohol (vol/vol = 24/L) is added and mixed by inver-
sion. After incubation for 30 min at 0°C (in iced water) and 71.2.2.2 P CR-RFLP Analysis of SSU
centrifugation at 14,000 rpm at 4°C for 10 min, the top layer and ITS 2 Regions
is transferred to a clean Eppendorf tube. To the sample is
Verweij et al. [9] described the use of primers NS1 and NS24
added 225 μL of 5 M NH4-acetate and it is incubated for at
(for amplification of SSU) and primers ITS 1 and ITS 4
least 30 min (in iced water) and centrifuged. The supernatant
(for amplification of ITS 1 and 2, including 5.8S ribosomal
is transferred to a clean sterile Eppendorf tube and mixed
RNA [rRNA]) for identification of Coprinopsis cinerea and
with a 0.55 volume (~510 μL) of ice-cold isopropanol. After
other molds. Amplicons are digested with the restriction
centrifugation for 7 min at 14,000 rpm at 4°C (or room tem-
enzymes HinfI, HaeIII, RsaI, and DdeI and electrophoresed
perature), the supernatant is decanted. The pellet is washed
on 1.4% agarose gels. Patterns are compared by using the
twice with ice-cold 70% ethanol and dried by using a vacuum
Image Master (Pharmacia) software package for evaluation
dryer. The powder is resuspended in 48.5 μL of Tris-EDTA
of molecular weights. The fungus had different patterns to
buffer with 1.5 μL of 10 mg of RNase/mL, incubated at 37°C
the other fungi analyzed.
for 15–30 min, and stored at −20°C until use [17].
71.2.2 Detection Procedures 71.3 Conclusions

71.2.2.1 Sequencing Analysis of the ITS 2 Region Hormographiella aspergillata is the anamorph of
Rampazzo et al. [5] utilized universal primers ITS 3 Coprinopsis cinereus (Coprinus cinereus), which is a basidio-
(5′-GCATCGATGAAGAACGCAGC-3′) and ITS 4 mycete with a typical mushroom form normally occurring in

compost and sewage [2]. H. aspergillata has been associated 7. Bartz-Schmidt, K. U. et al. (1996). Chronic basidiomycetous
with human infections such as pneumonia, endocarditism, endophthalmitis after extracapsular cataract extraction and
and endophthalmitis. Due to the difficulty of identification intracapsular lens implantation. Graefe’s Arch. Clin. Exp.
Ophthalmol., 234, 591–593.
on the basis of macroscopic and microscopic features, fila-
8. Nenoff, P. et al. (1997). Rare fatal simultaneous mould infec-
mentous basidiomycetes such as Hormographiella species tion of the lung caused by Aspergillus flavus and the basid-
are poorly recognized in the clinical laboratory. Application iomycete Coprinus sp. in a leukemic patient. J. Med. Vet.
of PCR and sequencing analysis of the ITS2 and other gene Mycol., 35, 65–69.
regions permits rapid and accurate identification of H. asper- 9. Verweij, P. E. et al. (1997). Fatal pulmonary infection caused
gillata, shortening the time needed for its diagnosis from by the basidiomycete Hormographiella aspergillata. J. Clin.
as much as 4–6 weeks to a day, and hence facilitating early Microbiol., 35, 2675–2678.
antifungal therapy. 10. Surmont, I. et al. (2002). A pulmonary infection caused by
Coprinus cinereus (Hormographiella aspergillata) diagnosed
after a neutropenic episode. Med. Mycol., 40, 217–219.
11. Lagrou, K. et al. (2005). Fatal pulmonary infection in a leu-
References kaemic patient caused by Hormographiella aspergillata.
1. The UniProt Consortium. Available at http://www.uniprot. J. Med. Microbiol., 54, 685–688.
org/, accessed on August 1, 2010. 12. Conen, A. et al. (2010). Hormographiella aspergillata: An
2. Guarro, J. et al. (1992). Hormographiella, a new genus emerging mould in acute leukaemia patients? Clin. Microbiol.
of hyphomycetes from clinical sources. Mycotaxon, 45, Infect., Published Online: May 18, 2010.
179–190. 13. Greer, E.L. et al. (2008). Truffle’s revenge: A pig-eating fun-
3. Gené, J. et al. (1996). Molecular characterization, related- gus. Cardiovasc. Pathol., 17(5), 342–343.
ness and antifungal susceptibility of the basidiomycetous 14. De Baere, T. et al. (2002). Identification of cultured isolates of
Hormographiella species and Coprinus cinereus from clinically important yeast species using fluorescent fragment
clinical and environmental sources. Anton Leeuwen, 70(1), length analysis of the amplified internally transcribed rRNA
49–57. spacer 2 region (ITS2). BMC Microbiol., 2, 21.
4. Abuali, M. M. et al. (2009). Rhizomucor variabilis var. reg- 15. Scorzetti G. et al. (2002). Systematics of basidiomycetous
ularior and Hormographiella aspergillata infections in a yeasts: A comparison of large subunit D1/D2 and internal
leukemic bone marrow transplant recipient with refractory transcribed spacer rDNA regions. FEMS Yeast Res., 2(4),
neutropenia. J. Clin. Microbiol., 47(12), 4176–4179. Erratum 495–517.
in: J. Clin. Microbiol., 2010;48:1018. 16. Romanelli, A. M. et al. (2010). Sequence-based identification
5. Rampazzo, A. et al. (2009). Hormographiella aspergillata of filamentous basidiomycetous fungi from clinical speci-
keratomycosis in a dog. Vet. Ophthalmol., 12, 43–47. mens: A cautionary note. J. Clin. Microbiol., 48(3), 741–752.
6. Speller, D. E. and MacIver, A. G. (1971). Endocarditis caused 17. Zeng, J.S. et al. (2007). Spectrum of clinically relevant
by a Coprinus species: A fungus of the toadstool group. Exophiala species in the United States. J. Clin. Microbiol.,
J. Med. Microbiol., 4, 370–374. 45(11), 3713–3720.

72 Cryptococcus
Massimo Cogliati, Anna Maria Tortorano, and Maria Anna Viviani
Contents
72.1 Introduction...................................................................................................................................................................... 633
72.1.3 Diagnosis.............................................................................................................................................................. 635
72.2 Methods............................................................................................................................................................................ 637
72.2.2.1 PCR Identification.................................................................................................................................. 638
72.2.2.2 Molecular Typing................................................................................................................................... 639
72.3 Conclusion........................................................................................................................................................................ 639
References.................................................................................................................................................................................. 640
72.1 Introduction Furthermore, same-sex reproduction may occur between

two different α strains [5].
72.1.1 Classification, Morphology, and Biology F. neoformans, C. neoformans teleomorph, produces
The yeasts of the genus Cryptococcus are spheroidal, ovoi- spheroidal, oblong, finely roughened basidiospores, while
dal, or elongate in shape, and reproduce mainly by multilat- F. bacillispora, C. gattii teleomorph, produces smooth bacil-
eral budding. Key characteristics of the genus are the ability lary basidiospores [2,3]. In addition, these two species differ
to assimilate inositol as sole carbon source, produce urease, in the ability to hydrolyze glycine and in the resistance to
react with diazonium blue B, and the lack of fermentative canavanine. In presence of C. gattii, the canavanine-glycine-
ability. Most of the isolates have a glucoronoxylomannan bromothymol (CGB) agar turns blue within 2–5 days, while
(GXM) capsule that differs in size according to the strain or no reaction occurs in presence of C. neoformans. C. neo-
to environmental factors [1]. formans has an EDTA-resistant urease activity and does not
At least 40 Cryptococcus species have been found in a assimilate d-proline in comparison with C. gattii [6].
wide variety of environmental locations. Among clinical The major environmental source of C. neoformans is
isolates, C. neoformans and C. gattii are the most frequent avian, mainly pigeon, droppings due to the ability of this
causes of disease, although other species such as C. albidus, species to utilize creatinine as a source of nitrogen by a cre-
C. laurentii, C. uniguttulatus, C. humiculus, C. curvatus, atinine deiminase that is repressed by the overproduction of
and C. luteolus have been reported to cause infection occa- ammonia. On the contrary, the absence of the enzyme repres-
sionally [1]. sion in C. gattii causes overproduction of ammonia, strong
Recognized as separate species, C. neoformans and alkalinization of the substrate, and inhibition of the yeast
C. gattii belong to two distinct monophyletic lineages and growth in avian droppings [6]. Recently, pigeon guano has
exhibit morphological differences during sexual reproduction. been shown to support the growth of both species, but C. gat-
More than 35 years ago, Kwon-Chung [2,3] described tii cannot sexually reproduce. Therefore, pigeon guano does
the teleomorph, Filobasidiella, belonging to the order not represent a niche for this species [7].
Tremellales and phylum Basidiomycota. Conjugation C. neoformans and C. gattii have been isolated from soil
between mating type (MAT) α and a cells produces dikary- and trees, mainly from decaying wood in the hollows. The
otic hyphae with clamp connections. A basidium is formed at colonization of decaying wood and tree hollows by both
the tip of the hypha. After karyogamy and meiosis, four mei- C. neoformans and C. gattii is enhanced by the produc-
otic nuclei are formed that produce, by mitosis, four chains tion of a laccase enzyme with phenoloxidase activity, which
of uninucleated basidiospores released from the basidium. degrades lignin [6].
MAT α and, more rarely, MAT a cells alone are also able On the basis of the specific structure of GXM, the main
to produce basidiospores by monokaryotic fruiting [4]. capsule component, C. neoformans, is classified into three
633

serotypes (A, D, and AD), and C. gattii in two serotypes maturation and activation of human dendritic cells [25].
(B and C). Serotype A corresponds to C. neoformans var. Another important virulence factor is the melanin pro-
grubii and serotype D to C. neoformans var. neofomans duced from polymerization of phenolic compounds, such as
[6,8]. GXM structure alterations may occur at low rate deter- cathecolamins, by the fungal laccase. The melanin is depos-
mining the colony phenotypic switching phenomenon [9]. ited in the cell wall conferring the brown color to the yeast
Cryptococcus colony may switch from mucoid to smooth cells. Melanization protects the yeast from oxidative damage
to wrinkled phenotype or vice versa. Mucoid and wrinkled by scavenging host-produced antioxidants [26] and reduces
phenotypes are reported to be more virulent than the smooth the fungal susceptibility to amphotericin B and caspofungin
ones in mouse models [10]. [27]. Melanin is essential for extrapulmonary dissemination
C. neoformans is ubiquitous, whereas C. gattii, isolated as it allows survival of the yeasts inside the alveolar macro-
firstly from Eucalyptus camaldulensis and from several tree phages and their transport into the lymph nodes and then into
species in Australia and in other tropical and subtropical the bloodstream. The existence in the brain of melanin pre-
regions, has recently been isolated from different native tree cursors (l-dopamine and epinephrine) has been hypothesized
species on Vancouver Island, British Columbia, Canada, and to explain the neurotropism of cryptococci. Other hypotheses
from the surrounding soil and air [11]. The specific route of about the fungus neurotropism concern the presence in the
the recent introduction of C. gattii to the Pacific Northwest brain of specific receptors that attract the yeast or the yeast
has not yet been established [11]. protection in the central nervous system (CNS) from the host
Cryptococcus MAT distribution is a further enigma that immune responses [8]. The production of capsule and melanin
still remains to be solved. MAT α population represents the was shown to be linked to the expression of several genes con-
majority of the isolates, whereas MAT a cells continue to tained in the MAT locus. MAT α cells were shown in animal
survive in specific niches. In addition, the MAT a locus is model to be more virulent than a cells [28,29] and to be more
conserved in hybrid diploid strains, which act as reservoir of efficient than a cells in crossing the blood–brain barrier dur-
this allele. A possible explanation for this distribution bias is ing coinfection [30]. In addition, the Vancouver hypervirulent
the ability of α cells to reproduce by both monokaryotic fruit- clone of C. gattii was proven to descend from two α MAT
ing and same-sex mating [5]. Genotyping of several C. neo- parents [15]. Other enzymes, namely, protease, phospholi-
formans and C gattii isolates by PCR fingerprinting [12,13], pase, and urease, are also considered virulence factors.
amplified fragment length polymorphism (AFLP) [14], and Cryptococcal infection is acquired by inhalation from an
multilocus sequence typing (MLST) [15–17] showed that environmental source of the infectious propagules (basidio-
the prevalent genotype in the world is VNI (var. grubii) and spores or poorly or no capsulated dried yeast cells of less
that VNIV genotype (var. neoformans) is present not only in than 3 μm in diameter) that can reach the alveoli where they
Europe [18] but also in North [19] and South Americas [12], rehydrate and acquire the polysaccharide capsule [6,8]. The
India [20], and Japan [21]. Interestingly, in these regions, a pulmonary infection is typically asymptomatic. The yeasts
consistent number of VNIII strains were identified that were can be either cleaned by macrophages or remain as a dor-
shown to be diploid hybrids originated from an intervarietal mant latent form within a granuloma. When host immunity is
mating between VNI and VNIV strains [22,23]. Intracluster compromised, the dormant yeasts can reactivate, proliferate
VNI diploids were also observed among Botswana and North inside the macrophages, and be released into the extracellular
Carolina isolates [24]. Four molecular types (VGI, VGII, environment. The released yeasts can enter other phagocytes
VGIII, and VGIV), not corresponding to serotypes B or C, and dissemination occurs.
were also identified in C. gattii species and a specific C. gat- The host immune response to cryptococcal infection is the
tii genotype, VGIIa, was shown to be responsible of the out- result of a complex interplay between cellular and humoral
break in Canada [15]. immunity. Innate immunity acts early and is activated by
specific recognition of molecular structures of cryptococ-
cal cells and of secreted products. In the lung, cryptococci
first interact with alveolar macrophages and dendritic cells
Several factors contribute to the virulence of C. neoformans that participate in fungal recognition, phagocytosis, antigen
and C. gattii [25]. The ability to grow at 37°C is essential presentation, and activation of the host response. Humoral
for survival in the human host. The polysaccharide capsule, response with production of a specific set of antibodies
composed of GXM (more than 90%) and galactoxyloman- can play an important role in the elimination or control of
nan, protects the yeast cell from phagocytosis and interferes cryptococci in the body [31,32]. However, cellular-mediated
with host immunity. As a facultative intracellular pathogen, immunity remains the critical component for protection
the yeast releases the capsular polysaccharide intracellularly, against cryptococci [33]. For instance, effector cells against
and the accumulation of the polysaccharide in the cytoplas- the yeast include CD4+ and CD8+ lymphocytes, NK cells,
mic vesicles results in macrophage dysfunction and lysis. In and activated professional phagocytes that produce granulo-
addition, the polysaccharide represses the migration of neu- matous inflammation. The granuloma formation is the result
trophils, interferes with cytokine secretion, inhibits T-cell of TH1-response mediated by IL12. A shift toward a TH2-
proliferation, induces macrophage apoptosis, and delays response probably antagonizes the clearance of the yeast

Cryptococcus 635
from host tissue. The TH17 lymphocytes have recently been 72.1.3 Diagnosis
r ecognized as cause of the exaggerated immune response in
AIDS patients with active cryptococcosis treated with highly 72.1.3.1 Conventional Techniques
active antiretroviral therapy (HAART). The conventional diagnosis of cryptococcosis is based on the
Impairment of the host’s defences against cryptococci can microscopic demonstration of the fungus, its growth in cul-
lead to dissemination of the infection, most likely by migra- ture, and on the capsular antigen detection [6,39].
tion of macrophages with ingested fungal cells from the The India ink examination of the CSF is a rapid test for
lung to the draining lymph nodes and via the bloodstream the diagnosis of cryptococcal meningitis. The yeast cells are
to cause systemic infection involving many body sites and visualized within a clear halo (the capsule) against the dark
organs, especially the brain. Headache, fever, lethargy, nau- background of the India ink and they can be easily differ-
sea and vomiting, memory loss, personality changes, stupor, entiated from leukocytes or fat droplets by the presence of
and coma are the signs and symptoms of meningoencepha- some budding cells. The sensitivity of the test depends on
litis observed in the majority of the patients. In patients with the fungal burden and the volume of the examined sample,
AIDS, symptoms usually appear late in the course of the and it may be improved by examining the pellet from the
disease when the fungal burden in the brain is high. In con- centrifuged CSF.
trast, in non-AIDS patients, the onset is insidious and symp- Cryptococci are difficult to observe in routine hematoxy-
toms may last months and years before diagnosis is made. lin–eosin histological preparations of bioptic samples. By
At lumbar puncture, the opening pressure may be markedly Gomori methenamine silver and periodic acid Schiff stains,
elevated, especially in patients with AIDS due to the high fungal cells are recognized by their oval shape and narrow-
fungal burden [34]. Cerebrospinal fluid (CSF) cell counts are based budding. However, the Mayer’s mucicarmine stain is
low (≤20/mmc) in AIDS-associated cryptococcosis and up needed to reveal the polysaccharide capsule. The presence
to 150–200/mmc in non-AIDS patients. CSF glucose level of the capsule allows the differentiation of cryptococcal cells
is low (<40 mg/dL) and protein level is elevated (>45 mg/dL) from the yeast forms of the dimorphic fungi Blastomyces
mainly in non-AIDS patients [35]. dermatitidis and Histoplasma capsulatum.
Cryptococcal infections caused by var. grubii and var. Cryptococci are not fastidious organisms and can be cul-
neoformans occur worldwide, mainly in the immunocom- tured from biological samples on standard fungal media in
promised host. The medical importance of cryptococcosis aerobic conditions. Antibiotics, such as chloramphenicol or
increased dramatically with the AIDS epidemic. Patients gentamycin, should be added to the media to reduce bacte-
with HIV infection are at risk of cryptococcosis late in the rial contamination. On the contrary, cycloheximide should
course of the viral infection when the CD4 lymphocytes not be added as it inhibits most of the isolates. Cultures
counts are less than 100 cells/mm3. Despite the widespread are incubated at 30°C–35°C, avoiding temperature ≥37°C.
use of HAART, the global burden of HIV-associated cryp- Yeast colonies usually develop in 2–5 days. Nevertheless,
tococcosis approximates 1 million cases annually. The mor- incubation up to 3–4 weeks is recommended, mainly if the
bidity and mortality of this fungal disease are still extremely patient is receiving or has received an antifungal treatment.
high in developing countries where access to HAART and to Cryptococci grow on agar media as white to cream-colored
healthcare is limited [36]. In medically developed countries, colonies that may turn to brown after prolonged incubation.
most of the cases occur in patients with newly diagnosed HIV The mucoid appearance of the colony is related to the size of
infection and in HIV-negative patients receiving high-dose the capsule that varies with strain and medium composition.
corticosteroids, or monoclonal antibodies (e.g., alemtuzumab Cryptococcal antigen assays that measure in the body flu-
and infliximab) or other immunosuppressive agents for their ids the polysaccharide released from the yeast have proven to
underlying conditions. be a valuable diagnostic test, more sensitive than India ink
Unlike C. neoformans, C. gattii causes disease also in examination and culture. The slide agglutination using latex
otherwise healthy immunocompetent individuals. In these particles (LA) coated with polyclonal antibodies or with anti-
subjects, the most common clinical manifestations are cryp- GXM monoclonal antibodies is widely used. The specificity
tococcoma of the lung and mass lesions in the brain with and sensitivity of the test performed on serum samples have
long-term neurological sequelae [37]. been improved by pretreatment of the sample with pronase.
Infection due to C. gattii has most often been reported in A titer of at least 1:4 is suggestive of cryptococcal infection
tropical and subtropical regions; however, since 1999, cases and a titre ≥1:8 is indicative of active infection. Antigen titer
have been identified in domestic and wild animal popula- is much higher in AIDS patients than in HIV-negative indi-
tion and in residents in, and travelers to Vancouver Island viduals. The CSF antigen titer has been shown to be a prog-
and lower mainland British Columbia, Canada. The annual nostic factor as high CSF antigen titers correlate with failing
incidence rate (5.8 and 25.1 cases per million on mainland host defence and poor outcome [34].
British Columbia and on Vancouver Island, respectively) is The apparent subjectivity of reading and grading the LA
the highest worldwide [38]. In recent years, several human reaction is overcome by the more recently developed enzyme
and animal cases of C. gattii disease have been recognized in immunoassay (EIA) that measures the major component of
Pacific Northwest regions of the United States [38]. the capsular polysaccharide, the GXM. However, the format

of the kit, designed for the simultaneous testing of multiple Recently, a nested PCR of ITS regions was compared with
samples, makes it inappropriate for its use in emergency. conventional diagnostic techniques by Saha et al. [45] who
Polysaccharide antigen screening in serum of HIV- investigated 359 samples from 52 patients with cryptococ-
positive patients with CD4 cell count <100 cells/μL living in cosis and 30 negative controls. The results indicated that the
areas with a high incidence of cryptococcal disease should sensitivity and specificity of PCR, EIA, and LA were compa-
be performed before the initiation of antiretroviral therapy in rable using urine, CSF, and serum for the diagnosis of cryp-
order to diagnose and treat subclinical infections before it is tococcosis, both before and after the initiation of treatment.
unmasked by the immune reconstitution [40]. A reverse cross-blot hybridization assay, based on ERG11
A positive serum or CSF antigen titer, even in absence of sequence, was applied by Posteraro et al. [46] to detect impor-
the isolation in culture or microscopic evidence of the fun- tant medical yeasts including C. neoformans in clinical sam-
gus, should be taken into serious consideration. ples. The results agreed with those of culture and phenotyping
The measurement of anticryptococcal antibodies is not of for the two CSF positive samples from patients with crypto-
consistent clinical benefit for the diagnosis of active infec- coccosis. The authors concluded that the PCR assay is a rapid
tion, while the antibody detection may have a prognostic method (7 h) compared to conventional identification (4 days).
value in AIDS patients during recovery from active infec- The usefulness of molecular methods in the diagnosis of
tion, when the antigen titer declines. cryptococcosis was in particular demonstrated by Abbott
Even if European Organization for Research and et al. [47], who employed in situ hybridization (ISH) utiliz-
Treatment of Cancer/Mycoses Study Group (EORTC/MSG) ing oligonucleotide probes directed against fungal ribosomal
definitions state that a positive result of CSF India ink or of RNA to detect C. neoformans and other dimorphic fungi in
serum or CSF LA is sufficient to establish a diagnosis of paraffin-embedded tissue sections. The ISH confirmed the
proven cryptococcosis [41], an extended diagnostic workup diagnosis of the five cases, two of which were culture-proven
is strongly recommended including culture of blood, CSF, cryptococcosis. ISH should be limited to cases with a posi-
urine, bronchoalveolar lavage in case of lung involvement, tive histological diagnosis for yeast or dimorphic fungi. The
and biopsy sample of skin lesions. method offers a valuable tool in the rapid identification of
fungal infections, especially in circumstances when tissue
72.1.3.2 Molecular Techniques cultures are an afterthought.
Molecular techniques offer an excellent alternative to con- Real-time PCR for C. neoformans identification in
ventional methods for an early diagnosis of most important clinical samples was applied in two different studies. Lau et al.
fungal diseases as they are rapid, can detect low fungal load, [48] set up a multiplex PCR, with amplicons being detected
and can be used for small size samples. However, molecular via analysis of the melting curve in a real-time thermocycler.
methods for the diagnosis of cryptococcosis have poorly been The method was able to detect and identify simultaneously
developed because of the limited demand of these tests due to 12 different fungal pathogens including C. gattii and C. neo-
the high yield of polysaccharide antigen detection tests. formans. Seventy blood specimens previously identified by
Prariyachatigul et al. [42] first applied PCR in the detec- conventional methods were analyzed. Molecular identifica-
tion and identification of C. neoformans in 27 CSF samples, tion was 100% in agreement with culture-based identification
20 of which from patients having or suspected to have cryp- including three cryptococcal cases.
tococcal meningitis. Sensitivity of PCR for direct detection Veron et al. [49] reported a TaqMan• real-time PCR pro-
of C. neoformans in CSF was high compared to the CSF cul- tocol for diagnosing cryptococcosis. DNA extracted from 101
ture (17 positive out of 20 proven or highly suspected cases various biological samples were investigated and the seven
vs. 11/20), whereas it was low compared with LA (20/20) samples culture-positive for C. neoformans or C. gattii also
and India ink examinations (19/20). The authors suggested tested positive by real-time PCR. No false negatives were
that these results were due to the ability of LA and India found among the 94 clinical samples that were Cryptococcus
ink to detect cells or antigens, no matter whether the yeasts culture-negative.
were dead or alive, while the reduced sensitivity of PCR was Although molecular biology is still scarcely applied in the
attributed to the rapid degradation of DNA. routine diagnosis of cryptococcosis due to the lack of stan-
Rappelli et al. [43] developed a nested-PCR assay to dardized, easy to use procedures in clinical laboratory, it has
detect C. neoformans-specific internal transcribed sequence greatly contributed to the progress of epidemiological studies
1 (ITS1) sequence in CSF. They obtained positive reactions on cryptococcosis where important progresses toward stan-
with all 21 clinical samples from patients previously diag- dardization of the genotyping method have been made.
nosed as having cryptococcal meningitis by conventional Molecular techniques as PCR fingerprinting [12,13], AFLP
techniques, and negatives reactions with all 19 negative [14], and MLST [15–17] have been employed to genotype a
controls. large number of clinical and environmental Cryptococcus
Paschoal et al. [44] examined a total of 72 CSF samples, strains isolated from different part of the world.
obtained from patients with and without AIDS, by specific Recently, Meyer et al. [50] reached a consensus in the
PCR of ITS1–5.8S–ITS2 regions. The results demonstrated establishment of a standard nomenclature for the main
that PCR test had the highest sensitivity rate, superior to cul- genotyping clusters: VNI and VNII (var. grubii), VIII
ture (85.7%) and India ink test (76.8%). (AD-hybrids), VNIV (var. neoformans), and VGI, VGII,

Cryptococcus 637
VGIII, and VGIV (var. gattii). The authors also described 2. Add 20 μL of proteinase K (10 mg/mL) mix by
a reference MLST protocol that includes seven housekeep- vortexing, and incubate at 56°C until the tissue
ing genes (URA5, PLB1, LAC1, SOD1, CAP59, IGS1, and is completely lysed. To disperse the sample,
GPD1). At present, gene sequences obtained with the above vortex the microcentrifuge tube occasionally
protocol can be recorded in a dynamic database (Multi Locus during incubation or place it in a shaking water
Sequence Typing) at http://www.mlst.net. bath or on a rocking platform.
3. Briefly spin to remove drops from the inside of
72.2 Methods the microcentrifuge tube lid.
4. Add 25 μL of 250 mM NaOH, 25 μL sodium
72.2.1 Sample Preparation dodecyl sulphate (SDS) 5%, and incubate at
95°C for 10 min.
1. DNA extraction from serum, CSF, and urine (Saha
5. Neutralize with HCl.
et al., 2009) [45]
6. Add 200 μL AL buffer (Qiagen) to the sample,
Procedure mix by pulse-vortexing for 15 s, and incubate at
1. Boil the sample for 5 min. 70°C for 10 min. Briefly spin to remove drops
2. Centrifuge at 1000 × g for 15 min. from the inside of the microcentrifuge tube lid.
3. Discard supernatant to obtain 200 μL pellet. 7. Add 200 μL ethanol (96%–100%) to the sample,
4. Add 500 μL of guanidine thiocyanate (6M), and mix by pulse-vortexing for 15 s. After mix-
500 μL of phenol saturated in Tris(hydroxymethyl) ing, briefly spin to remove drops from the inside
aminomethane (Tris), and mix. of the microcentrifuge tube lid.
5. Boil for 15 min. 8. Carefully apply the mixture to the QIAamp spin
6. Centrifuge at 1000 × g for 15 min. column (Qiagen) in a 2 mL collection tube and
7. Discard supernatant to obtain 200 μL pellet. centrifuge at 6000 × g for 1 min. Place the spin
8. Add 180 μL of ATL buffer (Qiagen), 20 μL of column in a clean 2 mL collection tube and dis-
proteinase K (20 mg/mL), and mix. card the tube containing the filtrate.
9. Incubate at 56°C for 30 min. 9. Add 500 μL AW1 buffer (Qiagen) and centri-
10. Briefly spin to remove drops from the inside of fuge at 6000 × g for 1 min. Place the spin col-
the microcentrifuge tube lid. umn in a clean 2 mL collection tube and discard
11. Add 200 μL ethanol (96%–100%) to the sample the collection tube containing the filtrate.
and mix again by pulse-vortexing for 15 s. After 10. Add 500 μL AW2 buffer (Qiagen) and centri-
mixing, briefly spin to remove drops from the fuge at full speed for 3 min.
inside of the microcentrifuge tube lid. 11. Place the spin column in a clean 1.5 mL micro-
12. Carefully apply the mixture to the QIAamp spin centrifuge tube and discard the collection tube
column (Qiagen), in a 2 mL collection tube, and containing the filtrate.
centrifuge at 6000 × g for 1 min. Place the spin 12. Add 200 μL AE buffer (Qiagen) or DNA-free
column in a clean 2 mL collection tube and dis- distilled water.
card the tube containing the filtrate. 13. Incubate at room temperature for 5 min and then
13. Add 500 μL AW1 buffer (Qiagen) and centri- centrifuge at 6000 × g for 1 min.
fuge at 6000 × g for 1 min. Place the spin col- 14. Store the elution at −20°C.
umn in a clean 2 mL collection tube and discard
3. DNA extraction from cultures (Viviani et al.,
the collection tube containing the filtrate.
1997) [13]
14. Add 500 μL AW2 buffer (Qiagen) and centri-
fuge at full speed for 3 min. Procedure
15. Place the spin column in a clean 1.5 mL micro- 1. Culture Cryptococcus isolate on two Sabouraud
centrifuge tube and discard the collection tube dextrose agar slants for 48 h at 35°C.
containing the filtrate. 2. Gently wash with 2 mL of sterile distilled water
16. Add 50 μL of warm AE buffer (Qiagen). the agar surface of the slants.
17. Incubate at room temperature for 5 min and then 3. Transfer the yeast suspension in a 2 mL micro-
centrifuge at 6000 × g for 1 min. centrifuge tube.
18. Store the elution at −20°C. 4. Count the cell concentration using a Bürker
hemocytometer and dilute the suspension to 108
2. DNA extraction from tissue samples (Hendolin
yeasts/mL.
et al., 2000) [51]
5. Centrifuge 2 mL of the 108/mL yeast suspension
Procedure at 1300 × g for 5 min.
1. Cut tissue samples in 5–25 mg pieces, place 6. Discard supernatant and suspend the pellet
them in a 1.5 mL microcentrifuge tube, and add in 1 mL of protoplasting solution: 20 mg lys-
180 μL of ATL buffer (Qiagen). ing enzymes [Sigma-Aldrich] in 1 mL sodium

citrate–sorbitol [SCS] (2 mL of 1 M sodium CneoRev (5′-GGTAATCACCTTCCCACTA

citrate [pH 5.8] and 98 mL of 1 M sorbitol). ACACAT-3′)
7. Seal the microcentrifuge tube with parafilm and • 0.25 μM of CneoProbe (5′-FAM-ACGTCG
incubate in a water bath at 37°C for 1 h. GCTCGCC-MGB-3′) (MGB: minor groove
8. Mix 10 μL of 2% SDS (in DNAase-free dis- binder, Applied Biosystems)
tilled water) and 10 μL of cell suspension on a • 3 μL TaqMan Exogenus Internal Positive
slide and check protoplast formation at phase- Control (IPC, Applied Biosystems)
contrast light microscopy. If protoplasts do not • 5 μL of DNA template
reach about the 90% of the cells, incubate the 2. Run real-time PCR on an ABI Prism 7300
suspension at 37°C for a longer time. (Applied Biosystems)
9. Centrifuge at 1300 × g for 5 min and discard the
2. Nested-PCR identification (Bialek et al., 2002) [52]
supernatant.
10. Suspend the pellet in 600 μL of lysing buf- Procedure
fer (8% dodecyltrimethyl-ammonium bromide 1. First-round PCR amplification mixture in a
[DTAB, Sigma-Aldrich], 1.5 M NaCl, 100 mM final 50 μL volume:
Tris [pH 8.6], 50 mM EDTA [Sigma-Aldrich]). • 10 mM Tris–HCl (pH 8.3), 50 mM KCl, and
11. Incubate in a water bath at 68°C for 30 min. 2.5 mM MgCl2 (10× buffer II plus MgCl2
12. Add 900 μL of cold chloroform and mix by solution, Applied Biosystems)
inverting the tube. • 100 μM of each deoxynucleoside triphosphate
13. Centrifuge at 11,600 × g for 10 min. • 1 μM of each primer, Fungus I (5′-GTTAAA
14. Transfer 350 μL of the upper aqueous phase in a AAGCTCGTAGTTG-3′) and Fungus II
1.5 mL microcentrifuge tube. (5′-TCCCTAGTCGGCATAGTTTA-3′)
15. Add 100 μL of CTAB (5% hexadecyltrimethyl- • 1.5 U of AmpliTaq DNA polymerase
ammonium bromide [CTAB, Sigma-Aldrich] in (Applied Biosystems)
0.4 M NaCl) and gently mix. • 10 μL of template DNA
16. Add 700 μL of DNase-free distilled water and 2. Thermal cycling conditions:
mix by inverting the tube. • Initial denaturation at 94°C for 5 min
17. Centrifuge at 8000 × g for 2 min and discard the • 35 cycles at 94°C for 30 s, 50°C for 30 s, and
supernatant. 72°C for 1 min
18. Add 150 μL of 1.2 M NaCl and gently mix till • Final extension at 72°C for 5 min
the pellet dissolve. 3. Nested-PCR amplification mixture in a final
19. Add 500 μL of ice-cold 99%–100% ethanol and 50 μL volume:
mix by inverting the tube. • 10 mM Tris–HCl (pH 8.3), 50 mM KCl, and
20. Centrifuge at 8000 × g for 10 min and discard 2.5 mM MgCl2 (10× buffer II plus MgCl2
the supernatant. solution, Applied Biosystems)
21. Add 500 μL of 70% ethanol and mix by invert- • 50 μM of each deoxynucleoside triphosphate
ing the tube. • 1 μM of each primer, Cryp I (5′-TCC TCA
22. Centrifuge at 8000 × g for 10 min and discard CGGAGTGCACTGTCTTG-3′) and Cryp II
the supernatant. (5′-CAGTTGTTGGTCTTCCGTCAATCT
23. Spin dry. A-3′)
24. Add 30–50 μL of DNA-free distilled water and • 1.5 U of AmpliTaq DNA polymerase
gently mix till the pellet dissolve. (Applied Biosystems)
25. To store the DNA for long time, add 3–5 μL of • 1 μL of the first-round amplification reaction
Tris EDTA 10 × solution (10 mM Tris PH 7.4, mixture
1 mM EDTA) 4. Thermal cycling conditions (two-step PCR):
26. Store at −20°C. • Initial denaturation at 94°C for 5 min
• 30 cycles at 94°C for 30 s and 72°C for 1 min
72.2.2 Detection Procedures • Final extension at 72°C for 5 min
72.2.2.1 PCR Identification 3. 18S rDNA fragment amplification and hybridization
1. Real-time PCR identification (Veron et al., 2009) [49] (Prariyachatigul et al., 1996) [42]
Procedure Procedure
1. Amplification mixture in a final 25 μL volume: 1. PCR amplification mixture in a final 50 μL
• 1× TaqMan Gene Expression Master Mix volume:
(Applied Biosystems) • 10 mM Tris–HCl (pH 8.3), 50 mM KCl (10×
• 0.9 μM of each primer, CneoFrw buffer II solution, Applied Biosystems)
(5′-GCCGCGACCTGCAAAG-3′) and • 2.5 mM MgCl2

Cryptococcus 639
• 200 μM of each deoxynucleoside triphosphate • 2.5 U of AmpliTaq DNA polymerase

• 25 pmol of each primer, CPL1 (5′-GGAGGTAG (Applied Biosystems)
TGACAATAAATA-3′) and CPR4 • 25 ng of genomic DNA
(5′-TGCTAATGTATTCGGGCGATT-3′) 2. Thermal cycling conditions:
• 1 U of AmpliTaq DNA polymerase (Applied • 35 cycles at 94°C for 20 s, 50°C for 1 min,
Biosystems) and 72°C for 20 s
• 10 μL of genomic DNA • Final extension at 72°C for 6 min
2. Thermal cycling conditions: 3. Reference strains:
• Initial denaturation at 94°C for 5 min ATCC MYA-4564 (WM148, serotype A, VNI),
• 50 cycles at 94°C for 45 s, 62°C for 45 s, and ATCC MYA-4565 (WM626, serotype A,
72°C for 45 s VNII), ATCC MYA-4566 (WM628, serotype
• Final extension at 72°C for 5 min AD, VNIII), ATCC MYA-4567 (WM629, sero-
3. Hybridization type D, VNIV), ATCC MYA-4560 (WM179,
• Transfer DNA from the gel to a nylon mem- serotype B, VGI), ATCC MYA-4561 (WM178,
brane Hybond-N+ (Amersham Biosciences) serotype B, VGII), ATCC MYA-4562 (WM161,
by capillary transfer serotype B, VGIII), and ATCC MYA-4563
• Hybridize the membrane with the species- (WM779, serotype C, VGIV).
specific probe INSR4 (5′-AGGCGGTCC
TCCTCACGGAGTGC-3′) labeled with 2. Multilocus sequence typing (Meyer et al., 2009) [50]
a colorimetric detection system (Dig Procedure
Oligonucleotide 3′-end labeling and detec- 1. Amplification mixture in a final 50 μL volume:
tion system, Boehringer, Mannheim) • 10 mM Tris–HCl (pH 8.3), 50 mM KCl (10×
• Wash the membrane according to manufac- buffer solution, Applied Biosystems)
turer’s instructions and develop it for color • 3 mM MgCl2
4. Internal transcribed sequence 1 (ITS1 rDNA frag- • 200 μM of each deoxynucleoside triphosphate
ment amplification (White et al., 1990, modified) [53] • 20 pmol of forward and reverse primers
• 2.5 U of AmpliTaq DNA polymerase
Procedure (Applied Biosystems)
1. Amplification mixture in a final 50 μL volume: • 100 ng of genomic DNA
• 10 mM Tris–HCl (pH 8.3), 50 mM KCl (10× 2. Primer sequences and thermal cycling condi-
buffer solution, Applied Biosystems) tions are reported in Table 72.1.
• 3 mM MgCl2
• 200 μM of each deoxynucleoside triphosphate
72.3 Conclusion
• 50 pmol of each primer, CN4 (5′-ATCACCT
TCCCACTAACACATT-3′) and CN5 (5′- Molecular diagnosis of cryptococcal disease is far from being
GAAGGGCATGCCTGTTTGAGAG-3′) a standardized procedure in clinical laboratory where con-
• 2.5 U of AmpliTaq DNA polymerase ventional techniques continue to represent the gold standard.
(Applied Biosystems) Antigen detection, despite the rare occurrence of false nega-
• 100 ng of genomic DNA tive (due to poorly or noncapsulated yeasts) or false positive
2. Thermal cycling conditions: results (due to cross reactions with Trichosporon infection),
• Initial denaturation at 94°C for 3 min remains a valid diagnostic tool, especially in emergency as it
• 25 cycles at 94°C for 30 s, 62°C for 30 s, and provides diagnosis in less than 1 h.
72°C for 1 min Molecular techniques could provide some advantages
• Final extension at 72°C for 10 min compared to conventional methods as DNA detection has
high target specificity and may be able to couple detection
72.2.2.2 Molecular Typing and identification simultaneously. This specificity is reached
also by culture method that, however, requires longer time
1. PCR fingerprinting (Meyer et al., 2003) [12]
for fungal growth and identification.
Procedure Molecular techniques might be particularly useful for the
1. Amplification mixture in a final 50 μL volume: analysis of biological samples from nonsterile sites, where
• 10 mM Tris–HCl (pH 8.3), 50 mM KCl, and high contamination by Candida may mask the growth of
1.5 mM MgCl2 (10× buffer plus MgCl2 solu- cryptococci in culture medium. Among molecular-biology
tion, Applied Biosystems) techniques, ISH has proven to be a valuable diagnostic adjunct
• 3 mM magnesium acetate to positive histology as it provides a rapid identification of the
• 200 μM of each deoxynucleoside triphosphate fungus in the tissue when culture is an afterthought.
• 30 ng of M13 (5′-GAGGGTGGCGGTTCT-3′) A technical problem of molecular methods is represented
primer by difficulties in the DNA extraction due to Cryptococcus

Table 72.1
Multilocus Sequence Typing Scheme
Gene Locus and Primer Sequence (5′-3′) Sequence
(Reference) PCR Cycling Conditions Start Sequence End Sequence Length (bp)
rRNA intergenic spacer 1 (IGS1) 94°C 3 min—35 cycles: TAAGCCCTTGTTAA AGATTTATTG 723
IGS1F ATCCTTTGCAGACGACTTGA 94°C 30 s, 60°C 30 s,
IGS1R GTGATCAGTGCATTGCATGA [16] 72°C 1 min—72°C 10 min
Glyceraldehyde-3-phosphate dehydrogenase (GPD1) 94°C 3 min—35 cycles: GGTTTCGGTACGG GACCCTGCCAA 543
GPD1F CCACCGAACCCTTCTAGGATA 94°C 45 s, 63°C 1 min,
GPD1R CTTCTTGGCACCTCCCTTGAG [15] 72°C 2 min—72°C 10 min
Laccase (LAC1) 94°C 3 min—30 cycles: GTAAGTATCAGCT CAAGCTAAACA 469
LAC1F AACATGTTCCCTGGGCCTGTG 94°C 30 s, 58°C 30 s,
LAC1R ATGAGAATTGAATCGCCTTGT [15] 72°C 1 min—72°C 10 min
Phospholipase B (PLB1) 94°C 3 min, 30 cycles: TGTTACTTGGATT CTGGAACATCG 532
PLB1F CTTCAGGCGGAGAGAGGTTT 94°C 45 s, 61°C 45 s,
PLB1R GATTTGGCGTTGGTTTCAGT [16] 72°C 1 min—72°C 5 min
Orotidine monophosphate pyrophosphorylase (URA5) 94°C 3 min—35 cycles: TTTTCGGCAACTCT TGGAAAGCTC 601
URA5F ATGTCCTCCCAAGCCCTCGAC 94°C 45 s, 63°C 1 min,
URA5R TTAAGACCTCTGAACACCGTACTC [12] 72°C 2 min—72°C 5 min
Capsule-associated protein (CAP59) 94°C 3 min—35 cycles: ACGGTACGCGCC GAGACAGAATG 559
CAP59F CTCTACGTCGAGCAAGTCAAG 94°C 30 s, 56°C 30 s,
CAP59R TCCGCTGCACAAGTGATACCC [15] 72°C 1 min—72°C 5 min
Cu, Zn superoxyde dismutase (SOD1) 94°C 3 min—35 cycles: CCACGTGCTCGCA CCTGTCAATGC 700
SOD1CNF AAGCCTCTCATCCATATCTT 94°C 30 s, 52°C 30 s,
SOD1CNR TTCAACCACGAATATGTA 72°C 1 min 30 s—72°C
SOD1CGF GATCCTCACGCCATTACG 5 min
SOD1CGR GAATGATGCGCTTAGTTGGA [54]
intracellular growth or to the presence of a large polysaccha- 8. Lin, X. and Heitman, J., The biology of the Cryptococcus
ride capsule. So far, molecular techniques have yet to super- neoformans species complex. Ann. Rev. Microbiol., 60, 69,
sede conventional methods for diagnosis of Cryptococcus 2006.
9. Goldman, D.L. et al., Phenotypic switching in the human
infections. On the contrary, molecular techniques have
pathogenic fungus Cryptococcus neoformans is associ-
been applied successfully for advances in the knowledge of ated with changes in virulence and pulmonary inflamma-
Cryptococcus ecology, epidemiology, and pathogenesis. tory response in rodents. Proc. Natl. Acad. Sci. U.S.A., 95,
14967, 1998.
10. Jain, N. and Fries, B.C., Phenotypic switching of Cryptococcus
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24. Lin, X. et al., Diploids in the Cryptococcus neoformans sero- Microbiol., 36, 3438, 1998.
type A population homozygous for the alpha mating type 44. Paschoal, R.C. et al., Neurocryptococcosis: Diagnosis by
originate via unisexual mating. PLOS Pathog., 5, 1, 2009. PCR method. Rev. Inst. Med. Trop. Sao Paulo, 46, 203, 2004.
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73 Malassezia
Takashi Sugita, Mami Tajima, Hisae Tsubuku, Mayumi Miyamoto, Enshi Zhang,
Ryoji Tsuboi, Masako Takashima, Yoshio Ishibashi, and Akemi Nishikawa
Contents
73.1 Introduction...................................................................................................................................................................... 643
73.1.1 Taxonomy............................................................................................................................................................. 643
73.1.2 Epidemiology........................................................................................................................................................ 643
73.1.3 Immunological Aspects........................................................................................................................................ 645
73.1.4 Diagnosis.............................................................................................................................................................. 645
73.2 Method.............................................................................................................................................................................. 647
73.2.2.1 Identification by PCR and Sequencing.................................................................................................. 647
73.2.2.2 Nested PCR Detection........................................................................................................................... 647
73.2.2.3 Real-Time PCR Quantitation................................................................................................................. 649
73.3 Conclusion........................................................................................................................................................................ 649
References.................................................................................................................................................................................. 650
73.1 Introduction the microorganism in 1889. Independently, Sabouraud estab-

lished the new genus Pityrosporum in 1904. For approxi-
The genus Malassezia colonizes only on the skin of human mately 100 years, many taxonomists debated whether
or nonhuman animals and does not exist in the environment Malassezia and Pityrosporum were synonymous, and
[1,2]. With the exception of one species, Malassezia pachy- now Pityrosporum is treated as a synonym of Malassezia.
dermatis, these microorganisms require lipids for growth. Nevertheless, the name Pityrosporum is often used in
The fatty acid biosynthesis pathway of Malassezia was elu- papers and books, although this use is not in accordance
cidated when the entire genome was determined in 2007 [3]. with fungal nomenclature rules. The greatest influence on
The fatty acid biosynthesis pathway of Malassezia differs the taxonomy of the genus Malassezia was the discovery
from that of typical fungi and is actually similar to that of of the heterogenicity of M. furfur. This microorganism was
animals. Consequently, Malassezia prefer sebum-rich areas, once considered to be the common causative agent of sev-
such as the scalp and face. Although Malassezia is a major eral skin diseases; however, in 1996, it was determined that
component of the cutaneous fungal microbiota of humans, M. furfur consisted of five distinct species [1]. The reclas-
these microorganisms are associated with several skin dis- sification of M. furfur has contributed to the elucidation of
eases. Malassezia are responsible for the development or the causative agents of Malassezia-skin related diseases
exacerbation of pityriasis versicolor, seborrheic dermati- (see Section 73.1.2). Of the known Malassezia species, four
tis, folliculitis, and atopic dermatitis [4–6]. Relationships have affinity for animals (M. caprae, M. equina, M. nana,
between Malassezia and psoriasis or acne vulgaris have and M. pachydermatis), whereas the remaining nine species
also been suggested. colonize human skin (M. dermatis, M. furfur, M. globosa,
M. japonica, M. obtusa, M. restricta, M. slooffiae, M. sym-
podialis, and M. yamatoensis).
73.1.1 Taxonomy
The taxonomy of anamorphic basidiomycetous yeast in the
73.1.2 Epidemiology
genus Malassezia dates to the nineteenth century [7]. In
1853, Robin isolated a culture from a patient with pityria- Although the clinical conditions pityriasis versicolor, sebor-
sis versicolor and named the species Microsporum furfur. rheic dermatitis, atopic dermatitis, and psoriasis are quite dif-
Subsequently, the new genus Malassezia was established for ferent, the cutaneous Malassezia microbiota in patients with
643

Table 73.1
Detection of Malassezia DNA in Patients with Malassezia-Related Skin Diseases and Healthy Subjects
Detection of Malassezia DNA (%)
Number
of M. M. M. M. M. M. M. M. M.
Subject Patients globosa restricta slooffiae dermatis sympodialis japonica yamatoensis obtusa furfur
Seborrhoeic dermatitis [9] 31 100 100 39 39 36 13 10 10 7
Pityriasis versicolor (lesional 49 100 100 8 25 35 6 4 4 10]
region) [10]
Atopic dermatitis (lesional 36 100 100 31 31 58 33 14 28 33
region) [9]
Healthy subject [9] 30 100 100 17 30 37 10 7 10 27
Note: M. globosa and M. restricta DNA were detected by a real-time PCR (unpublished data).
these skin diseases is almost identical [8–12]. Pityriasis versi- M. restricta is twice that of M. globosa in atopic dermatitis
color develops on the trunk and upper arms of people in their and psoriasis. Therefore, the Malassezia microbiota is identi-
20s and 30s, occurring more often during the summer than cal qualitatively but significantly different quantitatively in
the winter, whereas seborrheic dermatitis develops in areas each skin disease.
of the body that are rich in sebaceous glands, such as the Malassezia colonizes the sebaceous areas of the skin.
face, scalp, and upper trunk. Atopic dermatitis is a chronic Therefore, given the dependency of sebaceous gland activity
disease involving cycles of remission and deterioration and on age and gender, the Malassezia microbiota of healthy indi-
is characterized by hypersensitivity to dry skin. Psoriasis is a viduals should be assessed at every age and in both genders.
multifactorial skin disease with a strong genetic component, The latest large-scale study examined 770 healthy Japanese
including several genes encoding proteins involved in epider- aged 0–82 years [13]. In males, the total amount of Malassezia
mal differentiation and immune, inflammatory, and pathogen DNA remained essentially constant between 0 and 9 years of
responses. Of the nine human-associated Malassezia species, age and then increased markedly up to 16–18 years of age
both M. globosa and M. restricta are commonly detected (Figure 73.2). In females, the amount of Malassezia DNA
from almost all patients, whereas the other species are found increased until 10–12 years of age, decreased until 19–22
in fewer than 60% of cases (Table 73.1). Quantitative analysis years of age, and then increased again until 30–39 years of
gives additional interesting results. Although the Malassezia age, before finally decreasing gradually with age beyond 39
microbiota is similar regardless of skin disease, the ratio years. M. globosa and M. restricta accounted for more than
of M. globosa to M. restricta differs according to disease 70% of the total Malassezia DNA in both males and females
(Figure 73.1). In seborrheic dermatitis, M. restricta predomi- at all ages.
nates over M. globosa, whereas M. globosa is the predomi-
nant species in pityriasis versicolor. The colonization level of
107
100
80
Number of plasmid copy
106
Distribution (%)
60
40
105
20
0 104
PV SD AD PS
0–3
4–6
7–9
10–12
13–15
16–18
19–22
23–29
30–39
40–49
50–59
60–69
70–79
80–
FIGURE 73.1 Proportions of M. globosa and M. restricta in Age (years)

patients with Malassezia-related skin diseases. Closed squares,
M. globosa; open squares, M. restricta; PV, pityriasis versicolor; FIGURE 73.2 Changes in the amounts of Malassezia DNA with
SD, seborrheic dermatitis; AD, atopic dermatitis; PS, psoriasis. age. Closed circles, males; open circles, females.

Malassezia 645
73.1.3 Immunological Aspects 73.1.4 Diagnosis

Specific IgE antibody against several cutaneous microor- 73.1.4.1 Conventional Techniques
ganisms, including Malassezia, is produced in patient’s In the clinical laboratory, conventional identification meth-
serum as patients with atopic dermatitis lack ceramide bar- ods for yeast identification are based on biochemical char-
rier function. No anti-Malassezia IgE antibody is detected acteristics. Two commercial kits, API 20C Aux™ and ID
in serum from healthy subjects. Numerous Malassezia aller- 32C API™ (bioMérieux), are the most popular for yeast
gens (Mala f 2–4, Mala s 1, and Mala s 5–13) have been identification. These kits can identify 47 and 69 yeast spe-
identified and characterized [14–16]. Recently, MGp42, cies, respectively, within 48 h. Unfortunately, as Malassezia
a new major M. globosa allergen, was identified and may requires a lipid for growth, these kits cannot be applied for
be a cleavage product of intact HSP70 [17]. The precise Malassezia identification. An identification method combin-
mechanism by which Malassezia colonization induces ing (i) growth on Sabouraud dextrose agar (SDA); (ii) the
IgE antibody production and the inflammatory cascades growth temperature on modified Dixon agar; (iii) the uti-
that lead to atopic dermatitis remain unclear. The produc- lization of Tween 20, 40, 60, and 80; (iv) the hydrolysis of
tion of IgE antibodies has been implicated in the Th2-type esculin; (v) the catalase reaction; and (vi) the utilization of
immune response [18–20]. Keratinocytes produce a range polyethoxylated castor oil has been proposed (Table 73.2)
of proinflammatory and immune cytokines in response to [27]. This method has been useful for discovering new
microorganisms and skin damage [21,22]. A recent study Malassezia species. In addition, the accuracy of identifi-
demonstrated that keratinocytes play a critical role in the cation is improved significantly by using CHROMagar™
pathogenesis of atopic dermatitis by secreting several Malassezia medium (Kanto Chemical, Tokyo, Japan) [28].
Th2-type cytokines [23]. Cytokine secretion profiles using M. sympodialis, M. globosa, M. dermatis, and M. pachy-
antibody array analysis have revealed that M. globosa and dermatis produce a precipitate after incubation at 32°C for
M. restricta induce the secretion of distinct Th2-type cyto- 4 days on CHROMagar Malassezia medium. Pale purple
kines from human keratinocytes: M. globosa induces IL-5, and pink colors are developed. Of 370 Malassezia strains
IL-10, and IL-13 secretion, and M. restricta induces IL-4 belonging to nine species, 363 strains (98.1%) were identi-
secretion. These observations suggest a possible relation- fied accurately using the characteristics listed in Table 73.2.
ship between Malassezia colonization and the increased
IgE production in atopic dermatitis. Another important 73.1.4.2 Molecular Techniques
connection between Malassezia colonization and atopic As yeast species have few distinct morphological charac-
dermatitis is the increased secretion of granulocyte-macro- teristics, molecular techniques are a powerful tool for spe-
phage colony-stimulating factor (GM-CSF) and cutaneous cies identification. The most broadly utilized identification
T-cell-attracting chemokine (CTACK) from keratinocytes method is rRNA gene sequence analysis. Figure 73.3 shows
[23]. M. globosa is capable of inducing GM-CSF secretion the rRNA gene locus in M. globosa; it is approximately 8000
from keratinocytes. GM-CSF contributes primarily to the bp long. Within this, the internal transcribed spacer (ITS)
maintenance of the chronic inflammatory process in atopic region and D1/D2 26S rRNA gene have been used widely for
dermatitis by enhancing the antigen-presenting capacity fungal identification. IGS analysis is also preferable for strain
of Langerhans cells (LCs) and dendritic cells (DCs) [24]. typing or for use as an epidemiological tool, but is limited to
M. restricta induces the secretion of CTACK from kerati- Malassezia [29–31], Trichosporon [32–36] (see Chapter 77
nocytes. CTACK selectively attracts cutaneous lymphocyte on the genus Trichosporon), and Cryptococcus [37,38].
antigen-positive memory T cells to the inflamed sites [25]. A
recent study revealed that CTACK is upregulated in atopic 73.1.4.2.1 Culture-Dependent Methods
dermatitis patients [26]. Based on these findings, the follow- The PCR primers used to amplify the ITS region, includ-
ing is a possible mechanism by which Malassezia species ing the 5.8S rRNA gene, D1/D2 26S rRNA gene, and IGS
induces an IgE-immune response in patients with atopic 1 region, are listed in Table 73.3. Primers ITS1, ITS4, NL1,
dermatitis: (1) skin barrier dysfunction facilitates skin pen- and NL4 amplify the ITS region and D1/D2 rRNA gene of
etration by colonizing Malassezia, allowing the interaction all fungi, and DNA sequencing is performed with the same
of Malassezia with epidermal cells including LCs, DCs, primers. Primers 26BF and GBR are used to amplify the
and keratinocytes; and (2) local antigen-presenting cells IGS1 region of Malassezia species, but cannot be applied to
(APCs) such as LCs and DCs subsequently process and M. sympodialis and M. dermatis [29]. Identification criteria
present Malassezia antigens to induce immune responses. (% DNA similarity) should be considered when a strain is
This could be augmented by keratinocyte-derived GM-CSF. identified using rRNA gene sequencing. Generally, strains
Malassezia-stimulated keratinocytes produce Th2 cyto- with greater than 99% DNA sequence similarity in both the
kines, including IL-4 and IL-13, which in turn stimulate B ITS region and D1/D2 26S rRNA gene are considered to be
cells to undergo IgE class switching and the production of conspecifics (see Chapter 77 on the genus Trichosporon).
Malassezia-specific IgE. In addition, keratinocyte-derived The length of the D1/D2 26S rRNA gene is identical across
IL-5 may attract and activate eosinophils locally in atopic all species, and the DNA sequence similarity within a spe-
dermatitis lesions. cies exceeds 99% for all Malassezia species (Table 73.4).

646
Table 73.2
Typical Phenotypic Features in Nine Human-Associated Malassezia Species
Growth on mDixon at Utilization of
Precipitate
Growth Color on Production 10% 0.5% 0.5% 0.1% Polyethoxylated Catalase
Species on SDA 32°C 37°C 40°C CHROM on CHROM Tween 20 Tween 40 Tween 60 Tween 80 Esculin Castor Oil Reaction
M. pachydermatis + + + + Pale pink + + + + + ± ± +
M. sympodialis − + + + Pale pink + − + + + + − +
M. globosa − + ± or − − Purple + − − − − − − +
M. dermatis − + + + Pale pink + + + + + − − +
M. furfur − + + + Pale pink − + + + + − + +
M. slooffiae − + + + Pale pink − ± or + + + − − − +
M. obtusa − + ± or + − Pink − − − − − + − +

M. restricta − + + − Pink − − − − − − − −
M. japonica − + + − Pink − − ± + − + − +
Source: Referred from a paper of Kaneko, T. et al. J. Clin. Microbiol., 45, 3737, 2007.
Notes: +, positive; −, negative.
Malassezia 647
1765 252 156 290 3390 444 118 1738 73.2 Method
18S 26S 18S
ITS1 ITS2 IGS1 IGS2
5.8S 5S In vitro culture: As mentioned above, Malassezia species,
FIGURE 73.3 Schematic representation of the rRNA gene locus
with the exception of M. pachydermatis, require a lipid for
in Malassezia. ITS, internal transcribed spacer; IGS, intergenic growth. Therefore, lipids such as olive oil, glycerin, or Tween
spacer region. The length of a subunit or region is from M. globosa are added to the medium. Dixon media or Leeming and
CBS 7966. Notman agar (LNA) are widely used to culture Malassezia.
A small amount of pure culture (approximately one loop) is
prepared.
By contrast, the length of the ITS region differs in each spe- DNA extraction from scale samples: Sample scale from
cies, ranging from 161 to 266 bp long, and both M. globosa a subject is obtained using an appropriate method, such as
and M. restricta show intraspecies sequence diversity. The swabbing with a cotton applicator, scraping with a scalpel,
length of the M. globosa and M. restricta ITS1 region ranges stripping with tape, or washing with appropriate buffer.
from 237 to 266 and from 210 to 261 bp, respectively. The Stripping with tape may be best because this method is sim-
former species shows 88% sequence within-species similar- ple and does not stress the subject [8]. A transparent dressing
ity, and the latter shows 73% similarity. Of the 13 Malassezia (e.g., OpSite [3 × 7 cm; Smith and Nephew Medical, Hull,
species, M. caprae, M. dermatis, M. equina, M. japonica, United Kingdom]) is applied to the subject’s skin. Then, the
M. obtusa, M. slooffiae, and M. yamatoensis do not show OpSite dressing is removed and placed in 1.5 mL of lysing
intraspecies ITS diversity. Many Malassezia species can be solution (100 mM Tris–HCl pH 8.0, 30 mM EDTA pH 8.0,
identified using only the D1/D2 rRNA gene sequence, except and 0.5% sodium dodecyl sulfate) and incubated for 15 min
for M. sympodialis and the phylogenetically closely related at 100°C. The OpSite dressing is removed from the tube, and
species M. caprae, M. dermatis, and M. equina. M. caprae the suspension extracted with phenol–chloroform–isoamyl
and M. equina were separated from M. sympodialis sensu alcohol (25:24:1, vol/vol/vol). Subsequently, the samples
lato based on phylogenetic analysis, but they also differ phe- are extracted with chloroform–isoamyl alcohol (24:1, vol/
notypically. The D1/D2 rRNA gene sequences of these four vol), and the DNA is precipitated with 2-propanol using
species have greater than 98%–99% sequence similarity and Ethatimate (Nippon Gene, Toyama, Japan) as a precipitation
they share 82%–93% and 88%–95% sequence similarity in activator. The DNA pellet is resuspended in 50 μL of 1× TE
the ITS1 and ITS2 regions, respectively (Table 73.5). DNA (10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0). An unused
sequencing of both the ITS regions and the D1/D2 rRNA OpSite dressing is used as a negative control.
gene sequence is recommended when isolates may belong to
M. sympodialis and related species. Malassezia species can
be identified using IGS sequences, but analysis of the region 73.2.2 Detection Procedures
is preferred when used as an epidemiological tool.
73.2.2.1 Identification by PCR and Sequencing
In addition to rRNA gene sequencing, other molecular iden-
tification methods have been introduced, including random DNA sequence analysis of the ITS region or D1/D2 26S
amplification of polymorphic DNA (RAPD) [39,40], ampli- rRNA in the rRNA gene is the most reliable and accurate
fied fragment length polymorphism (AFLP) [40], restriction identification method. These regions are amplified by PCR
fragment length polymorphism (RFLP) analysis [41,42], ter- with primers ITS1 and NL4, as shown in Table 73.3. PCR
minal fragment length polymorphism (tFLP) [43], denaturing is performed with an initial 3 min denaturation at 94°C, fol-
gradient gel electrophoresis (DGGE) [40], and single strand lowed by 30 cycles of 30 s at 94°C, 30 s at 57°C, and 1.5 min
conformation polymorphism (SSCP) analysis [44]. at 72°C, and a final 10 min extension at 72°C. The PCR
products are sequenced with the primers ITS1 and ITS4
73.1.4.2.2 Culture-Independent Methods or NL1 and NL4, and the resulting DNA sequence is sub-
One member of the genus Malassezia, M. restricta, is often jected to a blast search (http://www.ncbi.nlm.nih.gov/blast/
absent from cultured samples because its growth on plates Blast.cgi). A species is defined as organisms sharing >99%
is slower than that of related species. All of the Malassezia identity in their D1/D2 26S rRNA gene. For the ITS region,
species in a sample must be recovered from the medium the sequence similarity shown in Table 73.2 is employed to
to obtain an accurate quantitative analysis. Unfortunately, define each species.
no optimal growth medium for all Malassezia species is
available. Culture-dependent methods can yield significant 73.2.2.2 Nested PCR Detection
numbers of viable cells, but have limited ability to produce Nested PCR conducted using the two primer sets shown in
accurate analytical results. Therefore, methods that do not Table 73.3 can be applied for detection of the nine human-
require an incubation period (i.e., molecular-based, culture- associated Malassezia species [10]. Extracted DNA (3 μL)
independent methods) have been developed to analyze the from each sample is added to 37 μL of PCR master mix-
distribution of the Malassezia microbiota. Each species is ture, which consists of 4 μL of 10× PCR buffer (100 mM
detected using nested PCR or real-time PCR [8–13,45–48]. Tris–HCl pH 8.3, 500 mM KCl, 15 mM MgCl2), 3.2 μL of

Table 73.3
The PCR Primers Used to Detect Malassezia DNA
Region or subunit Primer Sequence (5′–3′)
Primers for identifying Malassezia species

ITS region ITS1 (forward) TCCGTAGGTGAACCTGCGG
ITS4 (reverse) TCCTCCGCTTATTGATATGC
IGS region 26BF (forward) GCTTTCGAGTGCATACCACAC
GBR (reverse) GGAAATAGGATGAGAGAAAC
D1/D2 26S rRNA gene NL1 (forward) GCATATCAATAAGCGGAGGAAAAG
NL4 (reverse) GGTCCGTGTTTCAAGACGG
Target species Primer Sequence (5′–3′)
Primers for detecting DNA of each Malassezia species by nested PCR

First PCR for six Malassezia speciesa Forward ATCCTTTGCAGACGACTTGA
Reverse TGCTTAACTTCGCAGATCGG
First PCR for three Malassezia speciesb Forward ACCTGCAGAAGGATCATTAGTGA
Reverse TCCTCCGCTTATTGATATG
Second PCR for each Malassezia species

M. dermatis Forward CGCACCTTGCGCTCCATGGT
Reverse AGCCTGGTTTCCCAGGCAGCGG
M. furfur Forward TGTGTACCATAGGCACCCAC
Reverse CACGGTGATAAAGGGATGCA
M. globosa Forward TCGAGTGCATACCACACTCGAG
Reverse TACGGTGCTTTCACGGTTCT
M. japonica Forward CGTATGTGGATCTTATCCTAT
Reverse TGACTAGTGTCGTAGGCACGGTA
M. obtusa Forward CATGGTCCATCTCCCACACA
Reverse AGAGAGTGCGTGGCGCATGGT
M. restricta Forward CGACCTAGTCGACTACATCCTACT
Reverse TTCGGAGATACAAGCCTCCAT
M. slooffiae Forward ACGCACGCTAACACAACGTG
Reverse TGTGCGATTCGAAGCGCACA
M. sympodialis Forward CGCACCTTGCGCTCCATGGT
Reverse GGTACAATCCCCAGGCAGCAA
M. yamatoensis Forward CGATCAAACTTCTCTGTGTCCAG
Reverse TGTGTGGGAGGTAGAAGAGGCA
Target species Primer and probe Sequence (5′–3′)
Primers and TaqMan probe for detecting Malassezia DNA by real-time PCR
All Malassezia species Mala-F (forward) CTAAATATCGGGGAGAGACCGA
Mala-R (reverse) GTACTTTTAACTCTCTTTCCAAAGTGCTT
Mala-MGB (probe) FAM-TTCATCTTTCCCTCACGGTAC-MGB
M. globosa M. glob-F (forward) GGCCAAGCGCGCTCT
M. glob-R (reverse) CCACAACCAAATGCTCTCCTACAG
M. glob-MGB (probe) FAM-ATCATCAGGCATAGCATG-MGB
M. restricta M. rest-F (forward) GGCGGCCAAGCAGTGTTT
M. rest-R (reverse) AACCAAACATTCCTCCTTTAGGTGA
M. rest-MGB (probe) FAM-TTCTCCTGGCATGGCAT-MGB
a M. furfur, M. globosa, M. japonica, M. obtusa, M. restricta, M. yamatoensis.

b M. dermatis, M. sympodialis, M. slooffiae.

Malassezia 649
Table 73.4
Summary of the ITS1, ITS2, and D1/D2 LSU rRNA Gene Sequences
ITS1 ITS2
D1/D2 26S rRNA gene
Species Length (bp) Similarity (%) Length (bp) Similarity (%) Similarity (%)
Malassezia caprae 164 100 231 100 100
Malassezia dermatis 161 100 233 100 100
Malassezia equina 162–164 >99 232 100 100
Malassezia furfur 209–210 >95 370 100 >99
Malassezia globosa 237–266 >88 278–290 >91 >99
Malassezia japonica 208 100 337 100 100
Malassezia nana 182–186 >97 241–242 >98 >99
Malassezia obtusa 213 100 367 100 100
Malassezia pachydermatis 186–187 >94 328–344 >94 >99
Malassezia restricta 210–261 >73 276 >99 >99
Malassezia sloffiae 195–196 >99 313–316 >99 100
Malassezia sympodialis 162–163 100 234–235 100 100
Malassezia yamatoensis 176 100 298 100 100
Table 73.5
DNA Sequence Similarity of the Phylogenetically Close Species
M. sympodialis, M. caprae, M. dermatis, and M. equina
Subunit or Spacer Region Species M. caprae M. dermatis M. equina
D/D2 26S rRNA gene M. dermatis 98
M. equina 99 99
M. sympodialis 99 99 99
ITS1 M. dermatis 93
M. equina 82 85
ITS2 M. dermatis 95
M. equina 91 91
200 μM deoxynucleoside triphosphates (an equimolar mix- 10 min, and 40 cycles of 95°C for 15 s, and 60°C for 1 min.
ture of dATP, dCTP, dGTP, and dTTP), 15 pmol of each A standard curve is plotted using the cycle threshold val-
primer, and 0.5 U of Ex Taq DNA polymerase (Takara). In ues obtained by amplifying successive 10-fold dilutions of
the nested PCR step, 1 μL of the first amplification product a known concentration of plasmid DNA (from 101 to 109
is added to a new reaction mixture with the same composi- copies). The assay can detect 101 copies. The second method
tion as the first. detected the nine human-associated Malassezia species
using SYBR GREEN [48]
73.2.2.3 Real-Time PCR Quantitation
A real-time PCR assay can also quantify Malassezia DNA
73.3 Conclusion
from scale samples without the need to culture isolates. Two Malassezia furfur has long been considered as the major
methods have been introduced. One detects all Malassezia causative agent of all Malassezia-related skin diseases. The
species, especially the two major components, M. globosa species heterogenicity of this microorganism is elucidated,
and M. restricta, with TaqMan probes [43]. First 25 μL of and analysis of the Malassezia microbiota has progressed
TaqMan Universal PCR master mix (Applied Biosystems), rapidly. Molecular-based approaches have contributed
200 nM each of the forward and reverse primers, 250 nM greatly to this research. The culture of Malassezia is difficult
TaqMan probe (see Table 73.2), 3 μL of DNA, and Milli-Q compared to that of other pathogenic fungi, making direct
water in a total volume of 50 μL are placed in each well Malassezia DNA detection a valuable tool. Molecular-based
of a 96-well plate. Amplification and detection are per- methods for detecting and identifying Malassezia will con-
formed with the cycle parameters 50°C for 2 min, 95°C for tinue to be mainstream.

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74 Rhodotorula
Diego Libkind
Contents
74.1 Introduction...................................................................................................................................................................... 653
74.1.1 Taxonomy............................................................................................................................................................. 653
74.1.2 Environmental Distribution.................................................................................................................................. 654
74.1.4 Diagnosis.............................................................................................................................................................. 656
74.1.4.1 Conventional Identification.................................................................................................................... 656
74.1.4.2 Molecular Identification......................................................................................................................... 656
74.2 Methods............................................................................................................................................................................ 659
74.2.1.1 Sample Collection and Culture.............................................................................................................. 659
74.2.1.2 DNA Extraction..................................................................................................................................... 659
74.2.2 Identification Procedures...................................................................................................................................... 659
74.2.2.1 Micro/Minisatellite Primed PCR.......................................................................................................... 659
74.2.2.2 Species-Specific PCR............................................................................................................................ 660
74.2.2.3 Restriction of the Amplified 5.8S-ITS Region of the Ribosomal rRNA Operon.................................. 660
74.2.2.4 rDNA Sequencing Analysis................................................................................................................... 660
References.................................................................................................................................................................................. 662
74.1 Introduction stage: Sporidiobolus), which has been found, in a few occa-
sions, involved in human fungemia or has been isolated from
When dealing with the isolation of fungi from clinical clinical specimens.3–8
samples, in a few cases, yeast colonies with a characteris-
tic salmon-pink to coral-red color appear. A classic pro-
cedure of many medical mycologists is to assign such
74.1.1 Taxonomy
isolates to the genus Rhodotorula on the basis of this sole
phenotypic attribute. Today, thanks to the improvement of The genus Rhodotorula is polyphyletic and has 47 dif-
molecular biology techniques (especially DNA sequenc- ferent species, constituting a very heterogeneous taxon.
ing) and molecular phylogenetics, it is well known that Most of Rhodotorula species belong to the subphylum
yeasts producing pigmented colonies (also called pigmented Pucciniomycotina, either to the class Microbotryomycetes
or red yeasts) may belong to several basidiomycetous gen- (29 species) or to the class Cystobasidiomycetes (15 spe-
era other than Rhodotorula. Examples of these genera are cies). The remaining three species are classified in the
Rhodosporidium (teleomorphic stage of many Rhodotorula subphylum Ustilaginomycotina and do not produce carot-
species), Sporobolomyces, Sporidiobolus, Dioszegia, enoid pigments. R. glutinis is the type species of the genus
Cystofilobasidium, Xanthophyllomyces, and Cryptococcus. Rhodotorula and belongs to the order Sporidiobolales (Class
The distinctive pink-to-red coloration of their colonies is the Microbotryomycetes), together with the abundant yeast R.
result of the intracellular production and accumulation of mucilaginosa (previously known as R. rubra). A third clini-
carotenoid compounds. The composition of carotenoid pig- cally important Rhodotorula species is R. minuta, which,
ments may vary qualitatively in red yeasts depending on the unlike R. glutinis and R. mucilaginosa, is ascribed to the
species. The most important carotenoids found in yeasts are order Cystobasidiales (Class Cystobasidiomycetes). The case
torularhodin, torulene, and β-carotene.1,2 of R. glutinis is peculiar. Until recently, it was regarded as
However, among yeast with current medical relevance, the a ubiquitous yeast, and at least 370 collection strains were
pigmented species known to be most frequently involved in considered to belong to this species.9 The classical circum-
human mycoses in fact belong to Rhodotorula. There are no scription of R. glutinis was based mostly on a few physi-
records of clinical isolates for almost any other pigmented ological and cultural properties. However, several studies
genera. The only exception is Sporobolomyces (and its sexual have reported the physiological and genetic heterogeneity of
653

R. glutinis.10–14 More recently, Sampaio et al.15 and Gadanho in this chapter: R. mucilaginosa, R. glutinis complex, and
and Sampaio16 reevaluated the circumscription of this species R. minuta.
using a large set of isolates that had been previously identi-
fied by phenotypic criteria as R. glutinis and found that most
74.1.2 Environmental Distribution
of the isolates did not actually belong to this species. The
most common were anamorph strains of Rhodosporidium Rhodotorula species can be commonly isolated from natural
babjevae, but other species of Rhodotorula (R. dairenensis, environments (e.g., soil, water, phylloplane, etc.). A notable
R. graminis, and Rhodotorula spp.) and Rhodosporidium case is that of R. mucilaginosa, which is one possibly the
(Rh. diobovatum, Rh. sphaerocarpum) had also been mis- most ubiquitous basidiomycetous yeast species. Besides a
identified as R. glutinis. It has been also shown that from probable association with humans, Rhodotorula mucilagi-
a total of 45 isolates, only 4 had been properly assigned to nosa is found in a wide range of natural habitats including
R. glutinis.16 The species with the closest phenetic and phy- living or decomposing plant constituents and soil,17–21 and
logenetic proximity to R. glutinis are R. graminis and Rh. various aquatic environments such as freshwater lakes,22
babjevae. Therefore, it is likely that many of the isolates of estuaries,23 coastal waters,24 and open ocean and deep-sea
R. glutinis from humans, which have been reported, may environments.25,26 Moreover, R. mucilaginosa is also present
actually have been confused with other yeast species. For the in extreme environments like hyper-acidic waters,27,28 ura-
case of medically important yeasts, a reliable identification is nium leachate,29 and cold environments30–32 among others.
needed for the selection of the appropriate antifungal. The R. glutinis complex is also worldwide in distribution
From the above, it becomes clear that the accurate identi- and has been isolated from a variety of substrates. Known
fication of R. glutinis and closely related species is only pos- sources include air, freshwaters, marine waters, terrestrial
sible by the use of molecular techniques. In order to avoid environments (soil, phylloplane, flowers, etc.), food and bev-
confusions, hereinafter the term R. glutinis complex will be erages, animals, and humans.9
used to refer to R. glutinis and closely related Rhodotorula In comparison to R. mucilaginosa and R. glutinis com-
and Rhodosporidium species often confused with R. glutinis. plex, R. minuta has been less frequently isolated in natural
The R. glutinis complex is hence a group of species, and its environments. This species was detected in air, seawater
characteristics and properties correspond to that of the sum (including deep-sea environments),25 and freshwater.22
of species included in it. Concerning the presence of these Rhodotorula spe-
Figure 74.1 provides a schematic representation of the cies in food, R. mucilaginosa is the species with the largest
phylogenetic placement of Rhodotorula and Rhodosporidium frequency of appearance and the widest range of food and
species within the phylum Basidiomycota, as well as the beverages from which it has been detected. Several studies
main phenotypic characteristics of the species discussed reported the isolation of R. mucilaginosa from peanuts, apple
Subphylum
Basidiomycota
Cryptococcus Agaricomycotina + +
Pseudozyma/Ustilago Ustilaginomycotina + –
Rhodotorula/Rhodosporidium – –
Pucciniomycotina
INOSITOL
PAC
(A)
– R. mucilaginosa
R. mucilaginosa Glucuronic acid

and
R. minuta mycosporines
–
+ R. minuta
Rhodotorula
Nitrate
rhodosporidium
Gallic acid Saccharic acid
+
R. glutinis – –
+
R. glutinis
complex
Rh. kratochvilovae –
Rh. diobovatum + +
Rh. babjevae – +
(B)
Figure 74.1 Schematic representation of the phylogenetic placement of Rhodotorula (R., asexual) and Rhodosporidium (Rh., sexual)
within the phylum Basidiomycota (A) and the main phenotypic characteristics of the species discussed in this chapter (B)—R. glutinis
complex, R. minuta, and R. mucilaginosa. Most species in Cryptococcus form white colonies (open circle) and a minority of species
form pink colonies (gray circle). Colony colors are similar in Rhodotorula, but their proportions are symmetrical. The members of the
Ustilaginomycotina do not form pink colonies. The ability to utilize inositol as sole carbon source and to produce amyloid compounds
(PAC) allows the discrimination of the yeasts belonging to the three main lineages of the Basidiomycota. The most relevant Rhodotorula
and Rhodosporidium species can be distinguished based on the ability to utilize nitrate as sole nitrogen source, the capacity to utilize gluc-
uronic, gallic, and saccharic acids, and on the production of mycosporines. (From Libkind, D. et al., Syst. Appl. Microbiol., 28, 749, 2005.)

Rhodotorula 655
cider,33 cherries,34 fresh fruits,35–37 fruit juice,38 cheese,38–40 reviewed 128 literature cases (up to 2006) of Rhodotorula-
sausages,41 edible mollusks,42 and crustaceans,43 among oth- associated infections and found that 79% of the cases were
ers. The remaining two Rhodotorula species are less fre- fungemia, 7% eye infections, and 5% peritonitis related with
quent, and normally when they are present, R. mucilaginosa continuous ambulatory peritoneal dialysis. Furthermore, in
is also present. However, a few exceptions are known; as an 87% of the cases, the patient was immunosuppressed or had
example Callon et al.44 reported the isolation of R. glutinis cancer.
complex and R. minuta as the only representatives of pig- R. mucilaginosa is the species responsible for the major-
mented yeasts from goat milk. ity of these medical cases (∼70%) followed by R. glutinis
Rhodotorula species can be found in association with complex (∼10%) and R. minuta (∼6%). Reports in which
humans, having been isolated from various types of human Rhodotorula isolates could not be properly identified to the
specimens like feces, skin, and sputum, forming part of the species level correspond to the remaining percentage of the
normal flora.45 These yeasts are generally considered com- cases.
mensal saprobes,46–49 occurring most frequently in the envi- It should be noted that in many of these cases, sufficient
ronment than in human tissues. It is important to know that evidence supporting the involvement of the yeast isolate in
Rhodotorula spp. have been found to be one of the most fre- the pathogenic process is lacking. In some cases, the basis
quent isolated microorganism from nurses and nonnursing for considering Rhodotorula yeasts as pathogenic is based
hospital personnel hands.50–51 Moreover, many patients stud- on the resolution of fever accompanied by the disappearance
ied on the very day of hospital admission have been shown of the yeast from blood cultures without evidence of clear-
to be carriers of such yeasts.52 Rhodotorula yeasts possess ance of an infectious focus. Such evidence is suggestive that
a strong affinity for plastic, having been recovered from the blood isolate was the etiologic agent of fever, but it is not
various medical equipments like hemodialysis machines and definitive.56
fiber-optic bronchoscopes,53,54 and several environmental With respect to other pathogenic yeasts, like those of the
sources including shower curtains, bathtub grouts, and tooth- genus Candida, the incidence of Rhodotorula infections
brushes.55 A direct consequence of such broad exposure to is quite low. Early epidemiological studies in the United
these yeasts is that, in humans with depressed immunologi- States and Europe showed that the incidence of Rhodotorula
cal systems, Rhodotorula spp. can occasionally behave as infections was between 0.5% and 2.3% of total fungemia
opportunistic pathogens causing a variety of systemic infec- cases.63,99,100 Moreover, Lunardi et al.58 reported that in a 3
tions. As a result, patients may suffer increased morbidity year surveillance of the occurrence of Rhodotorulosis in a
and mortality due to colonization, allergy, and invasive infec- Brazilian tertiary care hospital, the overall incidence of
tion from such yeasts. such infections was 0.056 episodes per 1000 hospital admis-
sions. In a more recent work, Pfaller et al. (2009)101 reported
the incidence of non-Candida yeast clinical isolates from
all over the world in a period of ca. 10 years and showed
Rhodotorula species that are regarded as emerging yeast that Rhodotorula represented only 4.1%. Of a total of 462
pathogens are R. mucilaginosa (synonym R. rubra),56,57 which Rhodotorula isolates studied, R. mucilaginosa was the most
seems to be frequently associated with human infections, and frequent, with 64 isolates, followed by R. glutinis complex
the less frequent R. glutinis complex and R. minuta.58 Such (37) and R. minuta (1). The remaining strains (76%) could
species have been implicated in a broad spectrum of funge- not be identified to the species level using conventional iden-
mia and other malignancies mostly in immunocompromised tifications methods. The incidence of yeast-related mycoses
hosts; however, a few cases of Rhodotorulosis in inmuno- in a Portuguese oncology hospital during a 6-year period was
competent patients have been reported.59–61 recently reported, and R. mucilaginosa was the only pig-
To the best of our knowledge, one of the first cases of mented yeast detected representing just 1.9% of the cases.61
fungemia caused by Rhodotorula spp. was reported in It should be noted that the infections caused by unusual
1960 for a patient with endocarditis.62 A dramatic increase yeasts like Rhodotorula spp. could be under-reported because
in the number of cases of Rhodotorulosis was observed in of the difficulties in accurate diagnosis and a tendency of
the following years. Factors predisposing such opportunistic attributing isolates to specimen contamination.102
infections include underlying immunosuppression, the use Nowadays, Rhodotorula spp. are still rare in the diagnos-
of broad-spectrum antibiotics and/or narrow-spectrum anti- tic mycological laboratory.103 However, an increase in the
fungals, and the presence of foreign devices such as central frequency of invasive mycoses due to these opportunistic can
venous catheters.55,63 However, the use of indwelling devices be anticipated, considering the ever-increasing application
is by far the major risk factor for infection with these oppor- of newer technologies and therapies in the medical practice,
tunistic yeasts.64–66 Rhodotorula spp. have been incriminated which result in longer and more severe immunosuppression
in cases of ocular infections,67–71 meningitis,72–77 peritio- of nosocomial patients. Furthermore, given the Rhodotorula
nitis,78,79 endocarditis,80 ventriculitis,81 cancer-associated spp. intrinsic resistance to the widely used triazole and echi-
infections,59,57,82 infections related to central venous cath- nocandin antifungal agents, patients receiving such drugs,
eters and other indwelling devices,47,49,53,57,66,74,83–86 and vari- which actually select for Rhodotorula spp.,59 will be more
ous others types of infections.87–97 Tuon and Costa (2008)98 susceptible to the development of Rhodotorula fungemia.68,104

Even though Rhodotorula species appear to be less not be considered appropriate for treatment of such cases
virulent than more common yeast pathogens like Candida due to Rhodotorula spp. high resistance to these kind of
and Cryptococcus neoformans, for example, fatal cases in drugs.8,84,115,116,125
which Rhodotorula spp. was suspected to be the etiologi-
cal agent have indeed been reported. In the literature, the 74.1.4 Diagnosis
overall mortality rate of Rhodotorula fungemia of reported
cases is around 15%,58,85,98,105,106 although a few authors con- 74.1.4.1 Conventional Identification
sider it higher.64,107–109 Rhodotorula-related sepsis syndrome Conventional identification of yeast species and strains is
and other life-threatening complications have also been based on morphological, physiological, and biochemical
reported.47,65,110 characteristics. In order to reliably identify yeast to the spe-
The generally low pathogenicity of Rhodotorula spp. is cies level, it is necessary to conduct 50–100 phenotypic tests.
probably related to its reduced ability to grow at 37°C, an attri- This procedure is laborious, time-consuming (it usually takes
bute typically enhancing the virulence of pathogenic strains.111 3 weeks or more), and only an experienced person can obtain
Several clinical and environmental isolates of R. glutinis com- proper interpretation of the test results.126 Furthermore,
plex have been reported to grow at 37°C112; however, it should sometimes ambiguous identifications may be obtained due to
be noted that the species Rh. toruloides, often misidentified as intra-specific physiological variability of the strains.
R. glutinis, and hence included in the R. glutinis complex, can For the identification of mycoses-causing yeasts, as is the
grow at 37°C.9 Other explanations for the low pathogenicity of case of some Rhodotorula species, a reliable identification is
Rhodotorula probably derive from the scarce ability of these required for epidemiological investigations and for the selec-
yeasts to secrete degradative enzymes such as proteinase113 tion of an appropriate antifungal treatment.
and phospholipases,114 which may be associated with patho- Conventional identification of yeast isolates in the clinical
genic processes caused by opportunistic yeasts. laboratory is based on one of several commercially available
Rhodotorula spp. infections have been successfully treated biochemical and enzymatic panels. However, some disad-
with catheter removal alone (for indwelling device-associated vantages of these identification kits include the existence of
cases), antifungal therapy, and with a combination of these two limited databases122,123 and frequent misidentification.85,127–136
approaches.58,65,115,116 Antifungal treatment of Rhodotorula Because of the ever-expanding spectrum of fungal patho-
infections typically involve the use of amphotericin B, given gens (including yeasts), the field of medical mycology has
that Rhodotorula spp. was shown to be highly susceptible become an extremely challenging study of infections caused
to such compound in vitro71,115 and that it has been proven by opportunistic fungi. Given that the identification of such a
to be effective for clearing various types of Rhodotorula wide and taxonomically diverse array of fungal pathogens by
fungeamia.65,81,117–119 Liposomal amphotericin B should be also conventional methods is often difficult and sometimes incon-
considered because it is less nephrotoxic than conventional clusive,137 molecular biology techniques are being incorpo-
amphotericin B deoxycholate while maintaining antifungal rated in clinical laboratories to provide more accurate and
activity against Rhodotorula spp.120 More than a few cases prompt diagnosis.
have been reported in which treatment with this antifungal
worked successfully for curing various types of Rhodotorula 74.1.4.2 Molecular Identification
infections.53,90,104,121 Alternative antifungals for the treatment Molecular diagnostic methods possess several advantages
of Rhodotorulosis include Flucytosine73,118 showing excel- over conventional methods based on phenotypic characteris-
lent in vitro activity84,122 and the promising new triazoles: tics, such as higher reliability, reproducibility, and through-
Ravuconazole115–116 and Isavuconazole.123 Of worth noting is put. Due to their high specificity and sensitivity, these
the evidence provided by the work of Diekema et al.115 who molecular methods should be used routinely in the clinical
tested the antifungic susceptibility of 64 clinical Rhodotorula laboratories as a complement of the information provided by
isolates including R. mucilaginosa (24 isolates), R. glutinis conventional methods and particularly, to help in the diagno-
complex (29 isolates), R. minuta (5 isolates) as well as other sis of dubious cases.
non-identified Rhodotorula species (6 isolates), finding no sig- A variety of molecular approaches for genotyping and
nificant differences among them in the activities of any of the identification of yeast isolates have been described. These
tested agents. These results suggest that the frequent infection- methods include species-specific DNA primers,131,138–140 mul-
causing Rhodotorula species behave similarly toward most tiplex PCR,141,142 real-time PCR assays,142,143 PCR fingerprint-
antifungal treatments. Interestingly, Silici124 showed that six ing (i.e., random amplification of polymorphic DNA [RAPD],
different honey bee propolis were highly effective in inhibit- micro/minisatellite primed-PCR [MSP-PCR]),15,16,22,24,144
ing a strain o Rhodotorula spp. displaying very low minimum PCR-enzyme immunoassays,145 single-strand conformation
inhibitory concentration (MIC) values (≤0.01 μg/mL). Such polymorphism (SSCP) analysis of ribosomal DNA (rDNA),146
results are promising and should be corroborated with a larger restriction fragment length polymorphism (RFLP) analysis
set of isolates in order to evaluate propolis as an alternative of 5.8S-internal transcribed spacer (ITS) rDNA,147–149 electro-
antifungal agent for Rhodotorula mycoses. phoretic karyotyping by pulsed-field gel electrophoresis,150–152
Narrow-spectrum azoles (e.g., fluconazole) and echi- denaturing/temperature gradient gel electrophoresis (DGGE/
nocandins (e.g., caspofungin) type antifungics should TGGE),153–158 amplified fragment length polymorphism,155,159

Rhodotorula 657
flow cytometry or Luminex®,160–163 DNA microarray,163–165 up of motifs of 15–30 pb arranged in tandem. Such regions
and DNA sequencing.166,167 However, not all these techniques are highly polymorphic and thus provide sufficient resolu-
can be readily applied in the average clinical laboratory due tion for the discrimination among yeast species. However,
to their complexity, high costs, and/or limitations in the array the discrimination power of each MSP-PCR primer dif-
of yeast species that can detect. Several of such methods have fers, and it is related to the phylogenetic position of the taxa
been only developed for usual pathogenic yeasts (Candida under study. After PCR reaction, MSP-PCR DNA banding
spp., Malassezia spp., Cryptococcus neoformans) and are patterns are obtained by agarose gels electrophoresis. These
not yet applicable for less common opportunistic yeasts like are species specific, allowing the unambiguous identification
Rhodotorula spp. For a list of available molecular techniques of most yeast species tested so far by comparison with pro-
for yeast identification, as well as their advantages and disad- file databases or reference strains. Often, small variations in
vantages, see Table 74.1. minor bands may provide differentiation up to strain level,
PCR-based techniques in general provide the required which is useful for epidemiological studies.144,168 Several
simplicity, specificity, and sensitivity to identify most fungal studies have successfully employed the MSP-PCR method
species in a short time period. Most of these methods do for rapid genotyping of large sets of collection strains and/
not directly provide identification, unless results are com- or environmental isolates which included Rhodotorula spe-
pared with databases or tests are run together with refer- cies.15,16,22,28,32 In such cases, the species R. mucilaginosa and
ence strains (i.e., PCR fingerprinting, multiplex PCR, ITS R. minuta were easily identified by direct comparison with
sequence length polymorphism, and SSCP-ITS). A PCR reference strains. Libkind et al.144 studied almost 100 envi-
fingerprinting method that proved to be very useful for the ronmental isolates of R. mucilaginosa and found that more
rapid and simple genotyping of Rhodotorula isolates is the than 90% of these shared similar MSP-PCR profiles as the
MSP-PCR technique.16,22,144 It consists in the PCR amplifica- type strain using either M13 or (GTG)5 primers, thus allow-
tion of genomic DNA using synthetic oligonucleotides that ing prompt identification. On the other hand, studies within
detect micro/minisatellite DNA. Microsatellite DNA con- the R. glutinis complex have demonstrated that this closely
sists of sequence repeated motifs of ca. 2–10 bp arranged related group of species shares similar MSP-PCR banding
in tandem at various loci, while minisatellite DNA is made patterns with primers like M13. The discrimination among
Table 74.1
Molecular Detection Techniques for Yeasts with Special Reference to Rhodotorula spp. and Their Characteristics
Method Characteristics References
PFGE Laborious, time consuming, lack of databases, needs reference strains, requires [150–152]
specific equipment
PCR fingerprinting Fast, simple, needs reference strains, suitable primers may vary with yeast group
MSP-PCR High repetitiveness, high discriminatory power [16,144]
RAPD Low repetitiveness [155,184]
Species-specific primers/multiplex PCR Fast, simple, needs specific primersb [131,138–142,185]
Sequence length polymorphism (ITS1 or ITS2) Fast, simple, limited databases [129,130]
SSCP-ITS Fast, simple, needs reference strains, limited to known species [144,186]
RFLP-ITS Simple, specificity depends on yeast group, limited database [147—157]
PCR–T/DGGE Complicated, needs experience, low repetitiveness, useful for culture-independent [153–158]
detection
D1/D2 rDNA sequence Highly conserved, more complete database, provides accurate identifications [166,171]
ITS rDNA sequence More variable, less complete database. Useful for analyses of closely related [133,167,172]
species
Other gene sequences (IGS, 18S, cytochrome b) Limited databases [187–189,192]
Real-time PCR Sensitive, fast, needs specific probes,b and equipment [142,143,182]
FISHa Simple, fast, needs specific probes.b Useful only if cells are in high numbers and [183,190]
metabolically active
Pyrosequencing® Low cost, limited to short DNA fragments [191]
DNA microarray High-throughput, needs specific DNA probes,b specific equipment [144,155–157]
Flow cytometry, Luminex®a High-throughput, needs specific DNA probes,b specific equipment [160–162]
Notes: PFGE, pulsed-field gel electrophoresis; MSP-PCR, micro/minisatellite primed-PCR; RAPD, random amplification of polymorphic DNA; SSCP, sin-
gle-strand conformational polymorphism; RFLP, restriction fragment length polymorphism; T/DGGE, temperature/denaturing gradient gel electro-
phoresis; FISH, fluorescent in situ hybridization.
a Techniques that to the best of our knowledge have not been yet tested or developed for Rhodotorula spp. identification.
b Implies that such technique is limited to known species.

these species can be obtained with the alternative MSP-PCR emergent pathogens would not be detected with these tech-
primer (GAC)5.16 niques until they are incorporated into each database.
To the best of our knowledge, the MSP-PCR technique has Currently, the molecular technique most commonly used
not been yet tested for clinical isolates of Rhodotorula, how- for accurate identification of yeasts is gene sequencing,
ever; Latouche et al.168 applied this technique for Candida because it offers a rapid and robust method for recogniz-
clinical strains and found it fast, reproducible, and more cost- ing species and resolution is not limited to closely related
effective than available biochemical approaches. taxa. Yeast species identification based on DNA sequencing
An alternative PCR method that can be used to directly have emphasized either in coding (D1/D2 variable domains
identify Rhodotorula spp. (as well as others fungal patho- of the large subunit rDNA) or in noncoding (ITS) regions
gens) involves the use of species-specific primers.138,139 The of the rDNA. As a result, largely complete databases of D1/
technique employs three primers in a PCR amplification of D2168 and ITS167 sequences are available for molecular clas-
a ∼600 bp DNA segment, consisting of two external univer- sification and identification of yeasts, including all known
sal (upstream and downstream) primers and one internal Rhodotorula species.
species-specific primer. Species identification requires the The value of the D1/D2 region of the 26S rDNA for iden-
formation of species-specific rDNA nucleotide amplicon tifying clinically relevant yeast isolates by DNA sequencing
which is significantly smaller than the nontarget segment. has been clearly demonstrated.171 Sequence analysis of this
Multicopy genes such as those of ribosomal origin (either region provides enough resolution for the correct discrimi-
the D1/D2 region of the large rDNA subunit or the noncod- nation of R. mucilaginosa and R. minuta, given that both
ing ITS regions of the rRNA-encoding gene) are preferred species show low genetic heterogeneity in such region.144
by many researchers to achieve well-defined results.138,139 Contrarily, for R. glutinis, a single base mismatch differ-
However, because they may generate false-positive results, entiates it from the closely related species R. graminis and
other researchers have looked at single-copy genes of R. babjevae.16 Thus, D1/D2 sequence analysis may be useful
high specificity.169 Specific rDNA signature nucleotides or for the identification of R. glutinis complex, but does not pro-
sequences of the target yeast species are used for the devel- vide enough resolution for the discrimination of the various
opment of the specific oligonucleotide primers. A worth not- related species included in this group. As an example, Leaw
ing disadvantage of this detection technique is that primers et al.172 were not able to differentiate R. glutinis from Rh.
are designed based on sequences available in the GenBank babjevae and R. graminis using D1/D2 sequence analysis.
database, which may not represent the whole nucleotide A higher discriminatory level may be achieved when
variability for each species. This is particularly important using the more variable ITS region in rDNA sequenc-
for Rhodotorula spp. given that R. mucilaginosa144,152 and ing studies. This sequence is equally suitable than the D1/
R. glutinis complex16 are genetically heterogeneous species. D2 region for accurate identification of both R. minuta and
Thus, it can be anticipated that false-negative results could R. mucilaginosa. Furthermore, it has been demonstrated
be obtained using species-specific primers for the molecular that even sequencing of either the ITS1 region alone or the
diagnosis of Rhodotorula species. Furthermore, to the best of ITS2 region alone may be enough for the correct identifica-
our knowledge, currently there is only one available specific tion of these two yeast species.172 For the particular case of
primer for Rhodotorula spp., which was designed to detect R. mucilaginosa, strain variability can also be detected with
R. mucilaginosa.139 ITS sequencing,144 which is interesting for epidemiological
Other techniques have been developed for rapid yeast geno- investigations. Again, difficulties may appear when trying to
typing that also involve PCR (Table 74.1), one of which is the discriminate species of the R. glutinis complex by sequenc-
RFLP of the 5.8S-ITS rDNA sequence (RFLP-ITS). RFLP- ing the ITS region. Gadanho and Sampaio,16 using the com-
ITS consists of a PCR amplification of the ITS fragment plete ITS region, determined that R. glutinis and R. graminis
and subsequent digestion with three restriction enzymes.148 differed in one mismatch and three indels, whereas three
The length of the ITS fragment together with the lengths of mismatches differentiated R. glutinis from R. babjevae.
the fragments resulting from the enzymatic restriction are Studies testing the usefulness of ITS1 or ITS2 sequences
used for species identification employing existing RFLP-ITS alone for yeast identification found that a strain suspected to
databases (i.e., http://www.yeast-id.com). However, it may be be R. glutinis (strain BCRC 20576) could not be differenti-
impossible to differentiate very closely related species using ated from Rh. babjevae using such regions.172 However, in
this method, given that sequence differences occurring out- such case, the criterion used to assign the strain BCRC 20576
side the restriction sites cannot be detected by RFLP-ITS to R. glutinis is not provided and thus is probable that this
analysis, and thus, fragments with identical sizes do not nec- strain did never actually belong to R. glutinis.
essarily have identical sequences.170 Furthermore, RFLP-ITS A key factor determining the correct identification of the
database is still quite limited for basidiomycetous yeast spe- sequenced yeast isolate using the BLAST tool of the GenBank
cies, particularly for Rhodotorula spp. is the sequences used for comparison. Unfortunately, today
A major drawback of PCR-based techniques, in general, the GenBank database includes many rDNA sequences
is the fact that their diagnostic capabilities are limited to the that have been wrongly submitted or have been submitted
respective existing databases, which commonly only include with inappropriate species designations. Hence, inexpe-
species considered pathogenic. Thus, new species becoming rienced clinical mycologists might be leaded to incorrect

Rhodotorula 659
identifications. When using rDNA sequencing for yeast iden- 2. Transfer two full loopfulls of the biomass into a
tification, one should be aware of this fact and select for com- 1.5 tubes containing 500 μL of lyzing buffer and
parison purposes only trustworthy sequence submissions, the equivalent to 200 μL of 425–600 μm glass
preferably those corresponding to type strains. Additional beads.
hints for a correct use of GenBank database while identify- 3. Vortex vigorously for 3 min, incubate at 65°C for
ing Rhodotorula yeasts are given in Section 74.2.2.4. 1 h, and repeat vortexing (3 min).
4. Centrifuge suspensions for 15 min at 10,000 rpm
and preferably at low temperatures (5°C).
74.2 Methods 5. Collect supernatant and transfer to new tube; store
There are several molecular methods that allow the accurate at −20°C for up to 10–12 months.
differentiation and detection of Rhodotorula spp. species. In 6. For PCR studies, dilute 1:600 in double-distilled
this section, appropriate molecular techniques for identifica- water in order to obtain a DNA solution ranging
tion and genotyping of Rhodotorula isolates from clinical from 5 to 50 ng/mL; add 5 μL to the PCR mix and
samples are recommended. store at −20°C for up to 4–6 months.
Alternatively, several commercial kits for yeast genomic

74.2.1 Sample Preparation DNA extraction are available.
74.2.1.1 Sample Collection and Culture
The sample collection and analysis procedures vary for each 74.2.2 Identification Procedures
material drawn. With the exception of blood or swab sam-
ples which are used to inoculate appropriate liquid medium 74.2.2.1 Micro/Minisatellite Primed PCR
for yeast growth, other specimens are aseptically collected, The MSP-PCR typing method is a valuable tool for myco-
homogenized, diluted, and submitted to plating procedures. logical laboratories due to its rapidity, high output, and
In the clinical laboratory, the isolation of yeasts is commonly high strain discrimination potential. The following are the
performed on Sabouraud dextrose agar (10 g/L mycologi- suggested MSP-PCR primers for each medically relevant
cal peptone, 40 g/L dextrose, pH 5.6) containing 100 mg/L Rhodotorula species:
chloramphenicol, though Rhodotorula species may be more R. mucilaginosa: the core sequence of the phage M13
effectively grown in YM agar (3 g/L yeast extract, 3 g/L malt (5′-GAG GGT GGC GGT TCT-3′) or the synthetic oligo-
extract, 5 g/L mycological peptone, 10 g/L dextrose, pH 5). nucleotide (GTG)5 provide enough species level resolution
It should be pointed out that the Pagano Levin medium was R. minuta: same as R. mucilaginosa
not suitable for the detection of R. mucilaginosa in various R. glutinis complex: M13 primer allows the detection of
human specimens.173 R. glutinis species complex, though discrimination of species
The characterization of the yeast isolate by genetic meth- within this group or strains may be achieved with the (GAC)5
ods should be performed only after the possibility that the primer
strain under study is a saprophytic contaminant has been
Procedure
discarded.
74.2.1.2 DNA Extraction 1. Prepare a PCR mixture containing 0.8 μM each

primer, 2 mM of each deoxynucleotide, 3.5 mM
Firstly, for DNA extraction, pure cultures of the Rhodotorula
MgCl2, 1 U of Taq DNA polymerase and 1× PCR
isolate must be obtained. For this, single colonies should be
buffer, sterile double-distilled water up to the
picked from the isolation plates and streaked on the surface
desired volume, and 5 μL of the DNA sample (1:600
of yeast-specific agar plates. After growth, a well isolated
dilution) for a 25 μL reaction.
colony is transferred to fresh culture medium either in tubes
2. The cycling program consists of initial denatur-
or in plates. At the moment of DNA extraction, the yeast is
ation at 95°C for 5 min; 40 cycles of denaturing at
inoculated in the surface of fresh agar medium plates and
94°C for 45 s, annealing at 50°C–55°C for 1 min and
incubated for 48–72 h at room temperature (20°C–25°C).
extension at 72°C for 1 min; and a final extension at
A rapid and simple DNA extraction protocol suitable for
72°C for 6 min.
any of the above-mentioned PCR-based methods has been
3. Run gel electrophoresis on 1.4% agarose gel, 0.5×
thoroughly described by Sampaio et al.15 Here we provide
TBE buffer; at 90 V for 3.5 h. Include a molecular
a shorter and equally effective method for extraction of
size marker on each gel, a mixture of DNA cleaved
genomic DNA from a pure culture of Rhodotorula yeasts.
with HindIII and ΦX174 DNA cleaved with HaeIII
Procedure is recommended.
4. Stain the gel in 0.5 μg/mL ethidium bromide aque-
1. Grow the Rhodotorula spp. isolate for 24–48 h in ous solution.
regular yeast agar plates (i.e., YM) at room tempera- 5. Compare obtained MSP-PCR profiles with database
ture (20°C–25°C). or reference strains.

74.2.2.2 Species-Specific PCR corresponding to 50 μL of final volume). After

Species-specific oligonucleotide primers may be designed for heating the mixture containing the cell material
PCR identification of known Rhodotorula species or group for 15 min at 95°C, 1 U of Taq DNA polymerase is
of species. The procedure uses standard PCR components added in 25 μL double-distilled water.
and protocols (see Section 74.2.3.1) including DNA from the 2. The cycling program consists of initial denaturation
test species and three primers: two universal external limit- at 95°C for 5 min; 40 cycles of denaturing at 94°C
ing primers (either universal D1/D2 region or ITS primers) for 1 min, annealing at 55°C for 2 min, and exten-
and a species-specific internal primer. Species identifica- sion at 72°C for 2 min; and a final extension at 72°C
tion requires the amplification of a species-specific rDNA for 10 min.
nucleotide segment that is significantly smaller than the non- 3. Digest the PCR products (15 μL or approximately
target segment. Primers to be employed for the detection of 0.5–1.0 μg) without further purification with 1 U/μL
R. mucilaginosa with species-specific PCR primers139 are of the restriction endonucleases CfoI, HaeIII, and
External universal primers: NL1 (forward): 5′-GCA TAT HinfI at 37°C overnight.
CAA TAA GCG GAG GAA AAG-3′; and NL4 (reverse): 4. Separate the PCR products and their restriction
5′-GGT CCG TGT TTC AAG ACG G-3′; fragments on 1.4% and 3% agarose gels, respec-
Internal specific primer (forward) of the D1/D2 region: tively, in 0.5× TBE buffer.
5′-TCA GAC TTG CTT GCC GAG CAA TCG-3′.139 5. Stain the gels in 0.5 μg/mL ethidium bromide aque-
ous solution.
74.2.2.3 Restriction of the Amplified 5.8S-ITS 6. Estimate the sizes of PCR amplicons by comparison
Region of the Ribosomal rRNA Operon against a DNA length standard (e.g., 1000–100 bp
ladder).
A frequently used method for rapid identification of yeasts
consists of PCR amplification of the ITS1–5.8S–ITS2 rDNA 74.2.2.4 rDNA Sequencing Analysis
ITS region, followed by digestion with restriction endonucle-
The rRNA genes are the preferred target for DNA sequenc-
ases and by separation by agarose gel elecrophoresis.35,147,148
ing. The main reasons are the availability of conserved and
Studies using the RFLP-ITS method for the identification of
highly variable regions within the rRNA operon and its pres-
Rhodotorula yeasts are scarce, comprising only a few reports
ence in multiple copies in fungal cells allowing high sensi-
of wine 174,175 and orange-juice spoilage yeasts.35
tivity, as well as accurate molecular identification for most
The RFLP-ITS profiles to date known corresponding to
yeasts.171,172
Rhodotorula spp. are the following: R. mucilaginosa strains
The 5′-end of the 26S rDNA D1/D2 domain (D1/D2) and
typically show bands of the following molecular weights: ITS
the 5.8S-ITS region of the ribosomal rRNA gene (ITS) are
amplicon: ∼600 bp, CfoI: 320 + 240 + 80, HaeIII: 425 + 215,
the most frequently sequenced regions for yeast identifica-
and HinfI: 340 + 225 + 75,174,175 whereas R. minuta has ITS
tion. Hence, large databases are available for non-taxono-
amplicons of 660 bp and CfoI: 300 + 300, HaeIII: 400 + 215,
mist to quickly and accurately identify most known yeast
HinfI: 345 + 215.174 R. glutinis complex, on the other hand,
species, as well as recognize new species, by sequencing
generates ITS amplicons of approximately 640–660 bp and
approximately 600 nucleotides and doing a BLAST search
restriction fragments of variable sizes with all three enzymes
in GenBank.
(see the database http://www.yeast-id.com). Unfortunately,
In general, strains differing in no more than three nucle-
some strains of R. glutinis complex share similar RFLP-ITS
otide differences (0%–0.5%) are considered conspecific,
profiles with R. mucilaginosa. The only differences observed
while strains showing six or more noncontiguous substitu-
are seen in the fragments produced with HaeIII, which gen-
tions (1%) are separate species.176 Strains with intermediate
erally are 425 + 215 bp for R. mucilaginosa and 430 + 210
nucleotide substitutions are also likely to be separate species,
for R. glutinis complex.35,148 The protocol for the RFLP-ITS
though further molecular and conventional characterization
technique is described in the following. The amplification/
is needed for reassurance. On the other hand, the ITS regions
digestion procedure is applicable also directly from fresh
ITS1 and ITS2, which are separated by the 5.8S gene of
yeast colonies for rapid identification (see following text).
rDNA, are also highly substituted and often used for species
Procedure identification, but for many species, ITS sequences provide
no greater resolution than that obtained from 26S domains
1. Prepare PCR mixture containing 0.5 μM each D1/D2.177,178
primer, 10 μM deoxynucleotides, 1.5 mM MgCl2 List of universal primers for rDNA sequencing:
and 1× PCR buffer, sterile double-distilled water up
to the desired volume, and 5 μL of the DNA sample • D1/D2 region: NL1 (forward): 5′-GCA TAT CAA
(conveniently diluted) for a 25 μL reaction. For the TAA GCG GAG GAA AAG-3′ and NL4 (reverse):
alternative method of direct amplification from a 5′-GGT CCG TGT TTC AAG ACG G-3′ primers.
single colony, the cell material (picked with sterile • ITS region: ITS1 (forward): (5′-TCC GTA GGT
tip or needle) is added to a reaction mixture of 25 μL GAA CCT GCG G-3′) and ITS4 (reverse) (5′-TCC
(though mix components are at a concentration TCC GCT TAT TGA TAT GC-3′) primers.

Rhodotorula 661
• ITS1 region: ITS1 (forward): (5′-TCC GTA GGT 3. The presence of PCR products must be confirmed
GAA CCT GCG G-3′) and ITS2 (reverse) (5′-GCT by 1% gel electrophoresis.
GCG TTC TTC ATC GAT GC-3′) primers. 4. Purification of the PCR product should be carried
• ITS2 region: ITS3 (forward) (5′-GCA TCG ATG out using DNA purification kit following manufac-
AAG AAC GCA GC-3′) and ITS4 (reverse) (5′-TCC ture’s indications.
TCC GCT TAT TGA TAT GC-3′) primers. 5. Sequence PCR product using one or both of the prim-
ers either by sending samples to a DNA Sequencing
Procedure Service or, if applicable, using your own Sequencer
equipment following the manufacture’s protocol.
1. Prepare PCR mixture containing 0.5 μM each 6. Visually correct sequences for nucleotide ambigui-
primer (see following text), 10 μM deoxynucleo- ties or errors and compare to GenBank nucleotide
tides, 1.5 mM MgCl2 and 1× PCR buffer, sterile database by using the BLASTN program (NCBI,
double-distilled water up to the desired volume, and Bethesda, MD).179
5 μL of the DNA sample (diluted, see above DNA
extraction protocol) for a 25 μL reaction. For the Type strains of medically relevant Rhodotorula species and
alternative method of direct amplification from a their GenBank accession numbers for identification purposes
single colony, the cell material (picked with ster- are shown in Table 74.2.
ile tip or needle) is added to a reaction mixture of
25 μL (though mix components are at a concentra- 74.3 C
onclusions and
tion corresponding to 50 μL of final volume). After
Future Perspectives
heating of the mixture containing the cell material
for 15 min at 95°C, 1 U of Taq DNA polymerase is In spite of the large contact that humans have with
added in 25 μL double-distilled water. Rhodotorula yeasts as a consequence of the broad distribu-
2. The cycling program consists of initial denaturation tion in natural and artificial environments of these microor-
at 95°C for 5 min; 40 cycles of denaturing at 94°C ganisms, a relatively low incidence of Rhodotorulosis cases
for 1 min, annealing at 55°C for 2 min, and exten- have been reported. This suggests that Rhodotorula infec-
sion at 72°C for 2 min; and a final extension at 72°C tions are opportunistic and that the pathogenicity of these
for 10 min. yeasts is limited by factors that are currently unknown and
Table 74.2
The Type or Reference Strains for Medically Relevant
Rhodotorula and Other Pigmented Yeast Species
Species Type Strain D1/D2e ITSe
R. mucilaginosa CBS 316T AF070432 AF444541
R. glutinis CBS 20T AF070430 AF444539, AF387775
R. dairenensis CBS 4406T AF070429 AF444501
Rh. sphaerocarpum CBS 5939T AF070432 AF444499
Rh. toruloides CBS 14a AF207884 AB049028
Rh. kratochvilovae CBS 7436T AF071436 AF444520
Rh. diobovatum CBS 6085T AF070421 AF387782, AF444502
Rh. babjevae CBS 7808T AF070420 AF444542
R. graminis CBS 2826T AF070431 AF444505
R. minuta CBS 319T AF189945 AF190011
Other
Sporidiobolus johnsoniib CBS 5470T AF070435 EF592109
Sporidiobolus salmonicolorc CBS 490T AF070439 AF387784
Sporidiobolus metaroseusd CBS 7687T EU003461 EU003482
Sporidiobolus pararoseus CBS 491T AF189977 AY015429
Note: R.: Rhodotorula, Rh.: Rhodosporidium.

a Not the type strain but a widely used reference strain.
b Formerly Sporobolomyces holsaticus.
c Formerly Sporobolomyces salmonicolor.
d Formerly Sporobolomyces roseus.
e Genbank accession number.

need investigation. Nevertheless, it is clear that Rhodotorula based on real-time PCR,142,180,182 DNA microarrays,136,163,181
yeasts, in particular the species referred to in this chapter, fluorescent in situ hybridization (FISH),183 and Luminex®160
must be considered medically relevant given that inmu- technologies. Recently, Hsiue et al.136 tested a new oligonu-
nocompromised patients are susceptible of developing cleotide array system targeting the ITS1 or ITS2 regions163
fungeamia when exposed to such yeasts. to directly detect fungal pathogens from positive blood
Future investigations must also focus on the factors that cultures. This array system includes probes for R. muci-
favor infection by Rhodotorula yeasts, and on the search laginosa, R. glutinis, and R. minuta and was able to detect
for phenotypic and genotypic characteristics that may help efficiently R. mucilaginosa from blood cultures.136 It remains
discriminate potential pathogenic strains from those that are to be determined if this technique can be used directly from
not. In this task, molecular genotyping tools will play a sig- human specimens.
nificant role by allowing the rapid screening of clinical and However, to the best of our knowledge, the rest of the
environmental isolates for the detection of molecular traits techniques mentioned above have not been yet tested for the
proper of more virulent strains. The increasing importance of detection of Rhodotorula spp. directly from human speci-
Rhodotorula yeasts as infective agents makes of the possibil- mens. Thus, future investigations and developments in this
ity to distinguish between potentially pathogenic and harm- area should take account of Rhodotorula species due to their
less strains on the basis of genetic traits a pressing need. increasing importance as opportunistic mycoses-producing
Molecular techniques for the detection, identification, yeasts.
and typing of potentially pathogenic strains of Rhodotorula
must provide rapid and accurate identification and discrim- References
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of Rhodotorula, a requisite for such molecular technique 1. Libkind, D. and van Broock, M.R., Biomass and carotenoid
is that it must grant precise species differentiation within pigments production by Patagonian native yeasts, World J.
Microbiol. Biotechnol., 22, 687, 2006.
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2. Buzzini, P. et al., Carotenoid profiles of yeasts belonging to
mine which of these species can actually cause disease and the genera Rhodotorula, Rhodosporidium, Sporobolomyces
in the hypothetical case that all did, which has the higher and Sporidiobolus, Can. J. Microbiol., 53, 1024, 2007.
incidence of infections. Such epidemiological investiga- 3. Bergman, A.G. and Kauffman, C.A., Dermatitis due to
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pathogens, thus allowing the assessment of their antifungal Sporobolomyces infection in an AIDS patient, J. Infect. Dis.,
164, 623, 1991.
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The implementation of molecular biology techniques tis in an AIDS patient: Pathogen or passenger? AIDS, 8, 387,
in the clinical laboratory for the routine detection of 1994.
Rhodotorula spp. is vital for a prompt and precise diagnosis, 6. Morrow, J.D., Prosthetic cranioplasty infection due to
and the subsequent application of the appropriate antifungal Sporobolomyces, J. Tenn. Med. Assoc., 87, 466, 1994.
treatment. Current conventional methodologies based on 7. Sharma, V., Shankar, J., and Kotamarthi, V., Endogeneous
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often leading to inconclusive identifications or even to incor- Eye, 20, 945, 2006.
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gradient gel electrophoresis to the study of yeast diversity in 170. Villa-Carvajal, M., Querol, A., and Belloch, C., Identification
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253, 2004. transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal
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in the extreme environments of the Iberian Pyrite Belt: A 171. Linton, C.J., Molecular identification of unusual pathogenic
comparison between universal and fungi-specific primer sets, yeast isolates by large ribosomal subunit gene sequencing: 2
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Microbiol. Ecol., 57, 139, 2006. ence laboratory, J. Clin. Microbiol., 45, 1152, 2007.
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denaturing gradient gel electrophoresis (DGGE), FEMS Yeast 173. Silva, J.O. et al., Performance of selective and differential
Res., 1, 79, 2001. media in the primary isolation of yeasts from different bio-
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158. Prakitchaiwattana, C.J., Fleet, G.H., and Heard, G.M., 175. Sabate, J. et al., Isolation and identification of yeasts associ-
Application and evaluation of denaturing gradient gel electro- ated with vineyard and winery by RFLP analysis of ribosomal
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Yeast Res., 4, 865, 2004. 2002.
159. Gupta, A.K. et al., Identification and typing of Malassezia 176. Kurtzman, C. and Fell, J.W., Yeast systematics and phylog-
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75 Schizophyllum
Sophie Cassaing, Marie-Denise Linas, and Antoine Berry
Contents
75.1 Introduction...................................................................................................................................................................... 669
75.2 Methods............................................................................................................................................................................ 673
75.2.1.1 Pretreatment........................................................................................................................................... 673
75.2.1.2 DNA Extraction..................................................................................................................................... 673
75.2.2.1 S. commune-Specific PCR..................................................................................................................... 674
75.2.2.2 Tm Determination after Real-Time Panfungal PCR............................................................................. 674
75.2.2.3 Sequencing............................................................................................................................................. 674
75.3 Conclusion........................................................................................................................................................................ 675
References.................................................................................................................................................................................. 675
75.1 Introduction a protrusion (clamp) from the apical cell. The other nucleus
migrates and divides synchronously near the clamp. Septa
Schizophyllum is a wood-decaying fungus and S. commune are formed across the mitotic spindle, one at the basis of the
is the single species up to now involved in human diseases. clamp and one between apical and subapical cells. Thus, one
S. commune is found on every continent except Antarctica nucleus remains entrapped into the clamp. Then the clamp
where the wood substrate is lacking. Although it is encoun- and the subapical cell fuse and the clamp nucleus is released
tered throughout the year, it is more abundant during the cold toward the subapical cell, restoring the dikaryotic state of the
raining season in tropical countries.5 subapical cell.11,12 As a characteristic of basidiomycete fungi,
S. commune was discovered in 1930 by Hans Kniep. the dikaryotic phase can be prolonged over several years or
The main role of S. commune is to recycle carbon from rot- even indefinitely.
ten wood, but it has also been described as a fruit orchards Only the dikaryon is competent to form fruit body and
pathogen or protruding through the plastic film on baled to sporulate. Fruit body appears as a split-gill fungus, mac-
grass silage.1 S. commune is considered edible in some coun- roscopically visible on dying trees, and occurs single or in
tries such as Malaysia, China,2 Thailand,3,4 Nigeria,5 Congo, small colonies. It is 3–7 cm wide with a fan-shaped cap. The
Cameroon,6 Peru, and Southwest Mexico.7 S. commune is rich hymenium underneath the fruit body is formed of gills where
in fibers, proteins, and vitamins such as thiamine and ribo- numerous basidia are arranged in palisade, each bearing four
flavin. In addition, some compounds of this mushroom have basidiospores on erect sterigmata that are released into the
been shown to possess an immunomodulatory activity and environment.13 Fruiting bodies are also macroscopically vis-
potent macrophages/T-cell-mediated antitumor effect.4,8–10 ible in culture when the mycelium is dikaryotic.
Mating, fertilizing, fruiting, and meiosis are determined in
Schizophyllum by two unlinked genetic compatibility factors
named A and B, each one consisting of two closely linked
S. commune is a filamentous heterothallic basidiomycete loci α and β. Aα is estimated to have 9 different specificities
from the Agaricales order and the Schizophyllaceae family since Aβ is estimated to have 32 specificities. Bα and Bβ are
(http://www.cbs.knaw.nl). The life cycle of S. commune is both estimated to have nine specificities. As a consequence,
made up of two phases: the unmated homokaryon and the more than 28,000 mating types are encountered in the sexual
mated dikaryon. Sexual reproduction involves the formation progeny.11,14–16
of mycelium special structures named clamp connections: Both A and B gene functions are required for dikaryotic
upon cell division, one of the nuclei migrates and divides into growth. Sexual reproduction occurs only if the two haploid
669

hyphae are compatible. The condition for a fully compatibil- or after hematoxylin–eosin, Grocott–Gomori stain, or lacto-
ity is a difference in specificity at Aα or Aβ and a differ- phenol Cotton blue stain. Histologically, mucinous material
ence at Bα or Bβ.11,15,17–20 The B mating-type genes encode with eosinophils and Charcot-Leyden crystals may be visu-
G-protein-coupled receptors and their ligands that are small alized in pulmonary or sinusal specimens when an allergic
lipopeptide pheromones. These pheromone–receptor com- process is involved.
plexes regulate nuclear exchange and migration.17,19,21–25 The
A mating-type genes encode DNA-binding regulatory pro- 75.1.3.1.2 Culture
teins involved in controlling synchronous nuclear division The fungus is easy to culture on most of standard media
with clamp connection development.26 used in clinical laboratories, such as Sabouraud dextrose
(with or without gentamicin/chloramphenicol) and potato
dextrose agars incubated between 25°C and 37°C, even until
75.1.2 Clinical Features and Pathogenesis 43°C.5,16,31,32 The colony reaches a diameter of several centi-
Despite its large distribution and the common presence of meters in a week. The optimal temperature is not well speci-
environmental basidiospores liable to be inhaled through fied: the rate of growth is described slightly faster at 43°C
the nasal route, S. commune is rarely reported as a cause by Sigler et al.31 when the optimum growth temperature for
of infection in humans. Firstly regarded as a nonpathogenic Nigerian strains is 25°C at pH 5.5.5 Media such as potato dex-
fungus, it is now emerging as a cause of mycosis. It is respon- trose, 2% malt extract, and phytone-yeast agars can be used
sible for disease in both immunocompetent and immunosup- for subcultures. S. commune is susceptible to cycloheximide
pressed people such as patients with diabetes or in patients at a concentration of 0.4 mg/mL.16
with human immunodeficiency virus infection. Infections
include local or disseminated disease and many clinical pre- 75.1.3.1.2.1 Macroscopic Colony Aspect The myce-
sentations can be confused with Aspergillus infections. In lium presents as a woolly, rather dense, white mold. Colonies
1950, Kligman reported S. commune as a possible agent of have a rapid growth rate up to 50–60 mm after 7 days. Zones
onychomycosis,27 and in 1994, Kamei was the first to identify of condensation of the mycelium as masses of hyphae can
the fungus in the human respiratory tract.28 More recently, appear in 1- or 2-week-old cultures. The reverse is pale
although the number of reports of S. commune mycosis is cream-colored. Culture plates or tubes often release a char-
increasing, the review of the English-language medical lit- acteristic strong disagreeable odor. Under conditions of light
erature reports no more than 50 cases (Table 75.1). Diseases (alternative dark and light),33 and if the genotype is formed
mostly consist of lung disorders and infections of the sinuses. from fully compatible homokaryons, dikaryotic mycelia can
Pulmonary diseases (48%) include allergic bronchopulmo- produce characteristic fan-shaped or shell-like basidiocarps
nary mycosis, bronchial mucoid impaction, a case of chronic (Figure 75.1). These fruiting bodies are provided with lon-
eosinophilic pneumonia, a case of fungus ball, and a case gitudinally split gills and are visible to the naked eye after
of bilateral nodules. Chronic or allergic sinusitis represents a culture of 1 week to several months. They are up to 2 cm
43% of reports with a clear predominance in women (81%). in height and in diameter. The formation of fruiting bodies
S. commune sinusitis concerns healthy patients as well as is easier at 25°C compared to 37°C. Sometimes, tube-like
patients with diabetes or HIV infection. When infection structures of a few millimeter in height can appear with a
occurred in AIDS individuals, the CD4 lymphocyte count central aperture. These elements likely correspond to aborted
was 150/mm3 and 1/mm3.29,30 In most of the reported cases, fruiting bodies (infertile) and are the result of two homokary-
sinusitis caused by S. commune is unilateral (94%). Maxillary ons partially compatible, differing only at the A locus or only
sinus is more often affected (38%), but all sinuses are con- at the B locus.16,17,31,33–36
cerned (Table 75.2). Several sinuses are involved in 31% of
cases. The diagnosis remains difficult because the clinical 75.1.3.1.2.2 Microscopy Characteristics As observed in
presentation and histopathological findings can be suggestive biological samples, hyphae observed in culture are 1.5–3 μm
of aspergillar sinusitis. wide. They can present with discreet rounded swellings (cre-
nate hyphae). Dikaryotic isolates are easier to identify because
they present with clamp connections that are involved in cell
75.1.3 Laboratory Diagnosis division and constitute an essential marker for the recogni-
tion of a basidiomycete. The presence of spicules (also named
tubercles or pegs) is also highly representative of S. com-
75.1.3.1.1 Microscopic Examination of Biological mune, but monokaryotic isolates may fail to produce these
Samples and Histopathology structures.16,31,35 If the mycelium is formed from incompatible
Mycological examination of biological specimen (e.g., nasal or partially compatible homokaryons, if it fails to produce
flushing, sinus content, bronchoalveolar fluid (BAF), and tis- clamps, or if it is overlooked, the identification becomes more
sue biopsy) consists of the search for hyaline, septae hyphae, difficult and these isolates are inclined to be labeled “Mycelia
varying in width from 1.5 to 5 μm. Hyphae can branch at sterilia.”35,37 Moreover, clamps and spicules can be lost upon
acute or right angles but without dichotomous fork.16,31,32 subcultures.35 In contrast, some isolates present clamps only
These fungal elements are visualized after KOH preparation in subcultures and after several months.16

Schizophyllum 671
TABLE 75.1
S. commune Human Infections Reported in the English Literature between 1950 and 2009
Biological Sample Clinical Feature Age Sex Underlying Disease Reference
Toe nails Onychomycosis 33 M — [27]
CSF Atypical meningitis 24 M — [42]
Palate Palate ulceration 4 months M Dehydration [43]
Sinus content Sinusitis 30 F None [44]
Sinus content Sinusitis 75 F Obesity, diabetes [44]
Sinus content Sinusitis 35 F — [45]
Sinus content Sinusitis 62 F HIV, diabetes [29]
Sinus content Sinusitis 42 M HIV [30]
BAL fluid Allergic bronchopulmonary 57 F Healthy [28]
mycosis
Lingular brushing
Lobectomy specimen Lung fungus ball 53 F Tuberculosis [31]
Diabetes
Sinus content Sinusitis 35 M None [46]
Brain biopsy Brain abscess after 58 M Hypertension [32]
Lung biopsy pulmonary infection Coronary artery disease
Bronchial plugs Bronchial mucoid impaction 67 F Previous tuberculosis [47]
Sputum
Bronchial plugs Bronchial mucoid impaction 72 F — [48 cited by 3]
— Allergic bronchopulmonary 53 F — [49 cited by 3]
mycosis
— Allergic bronchopulmonary 44 F — [50 cited by 3]
mycosis
Nasal discharge Sinusitis 36 F None [35]
Sinus content
Sinus content Sinusitis 62 F Diabetes [16]
Bronchial plugs Bronchial mucoid impaction 51 F Bronchiectasis [51]
Lobectomy specimen Pulmonary mucous 74 M Tuberculosis [39]
consolidative lesion
N/A Bronchial mucoid impaction 51 F — [52 cited by 3]
Nostrils flushing Sinusitis 60 F — [37]
Nostrils flushing Sinusitis 51 F — [37]
N/A Allergic bronchopulmonary 79 F — [53 cited by 3]
mycosis
BAL fluid Chronic eosinophilic 74 F Diabetes [54]
pneumonia
Sputum Allergic bronchopulmonary 27 F Bronchial asthma [55 cited by 3]
mycosis
Sputum Allergic bronchopulmonary 33 F Bronchial asthma [55 cited by 3]
mycosis
Sinus content Sinusitis 47 M Diabetes [56]
Renal failure
Cyst content Bronchogenous cyst 56 M None [58]
Bronchial plugs Bronchial mucoid impaction 54 F None [3]
Sputum
Bronchial aspirate Bronchopneumonia 59 M Hodgkin’s disease [36]
Lung biopsy Bilateral pulmonary nodules 56 F Cardiac transplantation [60]
Sinus content Sinusitis 57 F Asthma, myasthenia, [40]
rheumatoid arthritis
Corticosteroids

TABLE 75.2
Sinusitis Caused by Schizophyllum commune
Localization Treatment Outcome Reference
Maxillary Surgery — [44]
Maxillary Surgery Sinus pain for a year [44]
Maxillary Surgery Favorable [45]
Maxillary Surgery Probable cure [29]
Antifungal treatment Death due to lymphoma
Maxillary Drainage Possible recovery [30]
Antifungal treatment Death due to meningoencephalitis
Maxillary, ethmoidal, and frontal Surgery — [46]
Corticosteroids
Maxillary Surgery Favorable [35]
Antifungal treatment
Maxillary and sphenoidal Surgery Favorable [16]
Maxillary and ethmoidal Surgery and antifungal treatment Favorable [37]
Ethmoidal and sphenoidal Antifungal and antihitaminal treatment Favorable [37]
Ethmoidal and sphenoidal Surgery Favorable [37]
Ethmoidal and sphenoidal Surgery — [37]
Sphenoidal Surgery and antifungal treatment Favorable [56]
Ethmoidal Surgery Favorable [57]
Ethmoidal and sphenoidal Surgery — [59]
Bilateral maxillary, ethmoidal, and sphenoidal Surgery and antifungal treatment Favorable [40]
dextrose agar plate beside a plug of a known single-basidio-

spore isolate (e.g., UAMH 7793, 7694, and 7695). A micro-
scopic examination of the mycelium from the contact zone
is performed after 7–10 days of coculture to display the
presence of clamp connections demonstrating a process of
dikaryotization. Compatibility for dikaryotization between
mycelia derived from different fruiting bodies reaches almost
100%.16,31,39
75.1.3.1.3.3 Serological Tests Some authors report

the detection of serum antibodies (IgG, IgM, and IgA) to
S. commune cytosol or extracellular culture antigen to con-
firm a sensitization to the fungus using the method described
by Kamei et al.28 In case of an allergic pathology, high levels
FIGURE 75.1 S. commune on malt extract agar after 7 days at of serum IgE are also observed.40
25°C showing fan-shaped fruiting bodies.
Unfortunately, more than half of S. commune clinical iso- Excepted when isolates consist in typical dikaryotic myce-
lates are monokaryotic and then, without clamp connections. lium, conventional culture methods often lack of char-
Thus, the identification has to be suspected by the woolly exten- acteristic clues to identify S. commune with certitude.
sive culture, the unpleasant odor, and the presence of spicules. Moreover, cultures need sometimes to be prolonged for
several weeks to visualize clamp connections or fruiting
75.1.3.1.3 Additional Studies bodies and dikaryotization assays need the possession of
75.1.3.1.3.1 Tolerance to Benomyl Tolerance to beno- monokaryotic strains derived from a single basidiospore.
myl is tested by assessing growth rates on Sabouraud dex- So certitude diagnosis often calls to molecular methods.
trose agar with and without benomyl at concentrations of 2 The sequence enclosed between the highly conserved small
and 10 μg/mL.38 (18S) and large (28S) ribosomal subunit genes, and par-
ticularly the one and two internal transcribed spacer (ITS)
75.1.3.1.3.2 Dikaryotization Assay To confirm the apti- regions is a valuable target for identification of S. commune,
tude to dikaryotization of an isolate, a plug of a supposed using analysis of the restriction fragment length polymor-
monokaryotic S. commune strain is placed onto a potato phism (RFLP) or nucleotide sequencing.13,37,41 Although

Schizophyllum 673
Buzina et al.37 showed a reliable identification of S. com- 75.2 Methods

mune using EcoRI and AvaI for RFLP, sequencing, if it
is possible, appears the most accurate method to identify 75.2.1 Sample Preparation
this species. Using ITS2 and ITS3 as sequencing primers, 75.2.1.1 Pretreatment
a similarity of 99.4%–100% (of 660 bp) has been shown
Some specimens such as sputum or BAF if viscous need a
between the nucleotide sequences of 10 strains obtained
first treatment at room temperature with a mucolytic agent
either from clinical samples or from fungal collections.37
(v/v) as Digest-Eur® (Eurobio). After vortex, wait until com-
If morphological criteria are sparse, sequencing presents
plete fluidization. Then centrifuge 5 mL of fluidized sputum,
the advantage to allow identification of most fungal spe-
BAF, or other biological fluid at 900 × g for 20 min and pre-
cies using the same set of universal fungal primers (Tables
serve a 500 μL pellet.
75.3 and 75.4). In contrast, if there is a strong suspicion
of S. commune based on cultural criteria, a S. commune-
focused PCR using the specific set of primers scom1 and 75.2.1.2 DNA Extraction
scom237 (Table 75.5) is a faster and cheaper method to con- All clinical samples (surgical specimens such as sinus or tissue
firm identification. biopsies, biological fluids) and cultures need to be disrupted
TABLE 75.3
Sets of Panfungal Primers Previously Described for the Identification of S. commune by Nucleotide
Sequencing (English Literature)
Initial PCR Primers Sequencing Primers Sequencing Target Final Product Length (bp) Reference
ITS5 ITS2 Partial 18S rDNA–partial 28S 660 [37]
ITS4 ITS3 rDNA [61]
NS1 V9G V9 variable domain of the 18S 450 [41]
ITS4 5.8G rDNA–central region of the 5.8S rDNA [61]
ITS1 ITS1 Partial 18S rDNA–partial 28S 590–630 [61,62]
ITS4 ITS4 rDNA [60]
Unnamed 1 Unnamed 1 18S rDNA 500 [63]
Unnamed 2 Unnamed 2
TABLE 75.4
Sequences of Panfungal Primers for the Identification of S. commune
Panfungal Primers Sequences (5′–3′) Reference
ITS1 TCCGTAGGTGAACCTGCGG [61]
ITS2 GCTGCGTTCTTCATCGATGC [61]
ITS3 GCATCGATGAAGAACGCAGC [61]
ITS4 TCCTCCGCTTATTGATATGC [61]
ITS5 GGAAGTAAAAGTCGTAACAAGG [61]
NS1 GTAGTCATATGCTTGTCTC [41]
V9G TTACGTCCCTGCCCTTTGTA [41]
5.8G AATGTGCGTTCAAAGATTCG [41]
Unnamed 1 ATTGGAGGGCAAGTCTGGTG [63]
Unnamed 2 CCGATCCCTAGTCGGCATAG [63]
MITS2A GATGAAGAACGCAGCGAAAT Cassaing, S. et al., Unpublished data
MITS2B ATGCTTAAGTTCAGCGGGTA Cassaing, S. et al., Unpublished data
TABLE 75.5
Sequences of S. commune Specific Primers
Target PCR Primers (5′–3′) Product Length (bp) Reference
Part of ITS region flanking scom1 GTTGACTACGTCTACCTCAC 305 [37]
the 5.8SrDNA scom2r GTTAGGCTCCAGCAGACCTC

before DNA extraction. Several physical (e.g., liquid nitrogen, SYBR Green 10× (Roche Applied Science), 0.4 μM
solid beads) or enzymatic (e.g., lysozyme 10 mg/mL in 10 mM of each panfungal primers MITS2A and MITS2B
Tris–HCl, pH 8.0, or lyticase 0.5 mg/mL) lysis methods are (Table 75.4), additional MgCl2 to reach a final con-
used. The combination of solid beads contained in a screw centration of 3.5 mM, 0.25 U of UNG (Fermentas
cap microtube and lysis buffer allows both the splitting and Life Sciences), and 2.5 μL of DNA.
homogenization of tissues and the break up of hyphal struc- 2. Thermocycling and detection are performed in glass
tures without the projection of microparticles. Commercial kits capillaries on a LightCycler instrument (Roche
are available, including tubes filled with beads alone or associ- Applied Science). An initial preheating step of 8 min
ated with a lysis buffer (e.g., IDI lysis kit, GeneOhm Sciences; at 95°C is used to activate the DNA polymerase and
MagNA Lyser Green Beads®, Roche Applied Science). inactivate UNG. Then, a touchdown procedure fol-
lows, consisting of a first amplification of 5 cycles (15 s
1. In a biological safety cabinet, place the sample (a frag- at 95°C, 10 s at 70°C, and 19 s at 72°C), then a second
ment of biopsy, 500 μL of pelleted fluidized BAF or amplification of 11 cycles (15 s at 95°C, 10 s at tem-
sputum or 0.5 cm2 of mycelium) in a microtube filled peratures decreasing from 70°C to 60°C with 1°C dec-
with ceramic beads (MagNA Lyser Green Beads, remental steps, and 19 s at 72°C, and, finally, a third
Roche Applied Science) and 500 μL of BLB (MagNA amplification of 22 cycles (15 s at 95°C, 10 s at 60°C,
Pure Bacteria Lysis Buffer, Roche Applied Science). and 19 s at 72°C). A total of 38 cycles is performed.
2. Insert the tubes into the rotor of the MagNa Lyser 3. Cycling is followed by a melting curve analysis. The
instrument (Roche Applied Science) and secure it expected Tm of the PCR product for S. commune is
with the retention plate. 86°C ± 0.3.
3. Cell disruption (fast oscillating) is performed twice
for 35 s at 7000 rpm. 75.2.2.3 Sequencing
4. After a brief centrifugation to pellet the cell debris, If the PCR is performed on a clinical specimen DNA or a
place the tube for 20 min at room temperature prior nonsporulating mold without any morphological orientation,
to DNA extraction. a panfungal PCR using universal fungal primers followed by
nucleotide sequencing will be more informative. The proto-
Proceed to the DNA extraction using, for example, the
col below details the procedure targeting the ITS1 region.
high pure PCR Template Preparation Kit® (Roche Applied
Science): Initial conventional PCR
1. Transfer 200 μL of supernatant for DNA extraction 1. Prepare a mixture (final volume 50 μL) for conven-
into a microtube. tional PCR with 0.5 μL Taq polymerase (Ampli
2. Extract DNA following the manufacturer’s Taq Gold® DNA polymerase, Applied Biosystems),
instructions. 0.4 μM of each primer (ITS1 and ITS2), 0.2 mM
3. Store DNA at +4°C until amplification and sequenc- of each dNTPs (MP Biomedicals), 2 mM MgCl2
ing or at −20°C if analysis is deferred. (Applied Biosystems), and 10 μL of DNA.
2. Run the cycling program on a thermocycler (e.g.,
75.2.2 Detection Procedures Veriti, Applied Systems): 1 cycle at 96°C for 10 min,
then 35 cycles with 96°C for 1 min, 58°C for 45 s,
75.2.2.1 S. commune-Specific PCR
72°C for 1 min, and then 72°C for 10 min.
Buzina et al. published specific S. commune primers37 (see 3. Perform an electrophoresis of the PCR product
Table 75.5) suitable for a targeted diagnosis, for example, in together with a DNA molecular weight (e.g., pUC
the case of a previous clinical history with S. commune. In Mix Marker 8, Fermentas) using a 2% agarose gel
others cases, this fungus is not frequent enough to use spe- containing 0.5 μg/mL ethidium bromide.
cific PCR as a first diagnostic test. 4. Visualize the PCR product under UV light box. A
75.2.2.2 Tm Determination after 228 bp band is expected.
Real-Time Panfungal PCR
PCR sequencing
If S. commune is suspected with a woolly white mycelium,
a panfungal real-time PCR with the analysis of the PCR 1. Perform two PCR sequencing reactions (10 μL) con-
product, Tm (melting temperature) is a good and rapid com- taining 1 μL of the previous PCR product, 2 μL of
plementary test for the identification of this mold. The pro- primer ITS1 or ITS2 (1 pmol/μL), 2 μL of Big Dye
tocol below details a PCR targeting the ITS2 region. Terminator v1.1 (Applied Biosystems), and 5 μL of
Procedure water.
2. Cycle sequencing is performed as follows: a first
1. Prepare a panfungal Sybrgreen PCR mixture (10 μL) step at 96°C for 1 min and then 25 cycles as follows:
with 1 μL of LightCycler® FastStart DNA Master 96°C for 10 s, 50°C for 5 s, and 60°C for 75 s.

Schizophyllum 675
3. PCR products are passed through a Performa® DTR 10 Miyazaki, K. et al., Activated (HLA-DR+) T-lymphocyte
gel filtration cartridge (Edge Bio) to remove unin- subsets in cervical carcinoma and effects of radiotherapy and
corporated dye terminators, excess of dNTPs, and immunotherapy with sizofiran on cell-mediated immunity and
survival, Gynecol. Oncol., 56, 412, 1995.
salts.
11 Casselton, L.A. and Olesnicky, N.S., Molecular genetics of
mating recognition in basidiomycete fungi, Microbiol. Mol.
Sequencing Biol. Rev., 62, 55, 1998.
12 Schubert, D. et al., Ras GTPase-activating protein gap1 of
1. Purified sequencing products are read on an ABI the homobasidiomycete Schizophyllum commune regulates
Prism 3100 Avant Genetic Analyzer® (Applied hyphal growth orientation and sexual development, Eukaryot.
Biosystems) according to the manufacturer’s Cell, 5, 683, 2006.
13 de Hoog, G.S., Filamentous basidiomycetes, Atlas of Clinical
instructions.
Fungi, 2nd edn., p. 242, Universitat Rovira i Virgili, Reus,
2. The BLASTn algorithm (National Center for 2000.
Biotechnology Information) is used for the analysis 14 Raper, J.R., Boyd, D.H., and Raper, C.A., Primary and sec-
of sequences and species identification (e.g., http:// ondary mutations at the incompatibility loci in Schizophyllum,
www.ncbi.nlm.nih.gov/BLAST). Proc. Natl. Acad. Sci. U.S.A., 53, 1324, 1965.
15 Fowler, T.J. et al., Crossing the boundary between the Balpha
and Bbeta mating-type loci in Schizophyllum commune,
75.3 Conclusion Fungal Genet. Biol., 41, 89, 2004.
S. commune is described as a growing emergent pathogen, 16 Sigler, L. et al., Maxillary sinusitis caused by medusoid form
of Schizophyllum commune, J. Clin. Microbiol., 37, 3395,
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1999.
review suggests that this fungus has now to be included 17 Vaillancourt, L.J. et al., Multiple genes encoding pheromones
in the differential diagnosis of Aspergillus in pulmonary and a pheromone receptor define the B beta 1 mating-type
and sinuses diseases. Because of the frequent poorness specificity in Schizophyllum commune, Genetics, 146, 541,
of the primary culture and the difficulties of the identifi- 1997.
cation based on morphological features, the laboratory 18 Raper, C.A. and Raper, J.R., Mutations modifying sexual
diagnosis is still laborious and frequency of S. commune morphogenesis in Schizophyllum, Genetics, 54, 1151, 1966.
infections is likely underestimated. There is no doubt that 19 Wendland, J. et al., The mating-type locus B alpha 1 of
Schizophyllum commune contains a pheromone receptor
molecular techniques including nucleotide sequencing are gene and putative pheromone genes, EMBO J., 14, 5271,
become essential for the identification of this filamentous 1995.
basidiomycete. 20 Wang, C.S. and Raper, J.R., Isozyme patterns and sexual mor-
phogenesis in Schizophyllum, Proc. Natl. Acad. Sci. U.S.A.,
66, 882, 1970.
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76 Sporobolomyces
Dongyou Liu
Contents
76.1 Introduction...................................................................................................................................................................... 677
76.2 Methods............................................................................................................................................................................ 678
76.3 Conclusion........................................................................................................................................................................ 678
References.................................................................................................................................................................................. 679
76.1 Introduction face, and incubating for an extended period at 25°C. Colonies
on the initially uninoculated plate form a mirror image of the
76.1.1 Classification and Morphology colonies that are on the initially inoculated plate.
The genus Sporobolomyces is a basidiomycetous yeast
classified in the mitosporic Sporidiobolales group,
order Sporidiobolales, class Microbotryomycetes, phy-
lum Basidiomycota, kingdom Fungi. The mitosporic Sporobolomyces spp. are phylloplane yeasts that have wide-
Sporidiobolales group comprises two genera: Rhodotorula spread distribution in environments and occur in animals and
and Sporobolomyces; and the genus Sporobolomyces cur- humans. Several Sporobolomyces spp. (e.g., S. salmonicolor,
rently contains >20 recognized species as well as >30 S. roseus, and S. holsaticus) have been implicated in human
unassigned species, of which Sporobolomyces roseus and infections, inducing dermatitis, cerebral infection, lymph-
Sporobolomyces salmonicolor are involved in human infec- adenitis, fungemia, and allergic alveolitis, notable in immu-
tions [1–14]. The teleomorphs of the genus Sporobolomyces nosuppressed individuals, as a consequence of advanced
are found in the genus Sporidiobolus [15]. medical intervention [16–23].
Sporobolomyces is a ballistoconidium-forming yeast A case of Sporobolomyces-related dermatitis involved a
that is present in environments (e.g., soil, air, water), plants, 30-year-old woman with a pruritic skin lesion. A yeast-like
animals, and humans [2]. Sporobolomyces species (e.g., S. organism, Sporobolomyces holsaticus, was isolated from
salmonicolor, S. roseus, and S. holsaticus) may cause derma- the lesion [18]. Additionally, Sporobolomyces species was
titis, cerebral infection, lymphadenitis, fungemia, and aller- responsible for extrinsic allergic alveolitis in a 28-year-old
gic alveolitis, particularly in immunosuppressed patients. horseback rider with exposure to a horse barn. The patient
Sporobolomyces colonies grow rapidly and mature in 5 presented with fever 4 h after exposure and precipitins posi-
days. The optimal growth temperature is 25°C–30°C, with tive against Sporobolomyces but negative against other fungi
some isolates growing poorly at 35°C–37°C. Colonies are and horses. Sporobolomyces was isolated from straw in the
smooth, often wrinkled, and glistening with bright red to barn and cessation of exposure to this barn (but contin-
orange color, resembling Rhodotorula spp. ued exposure to horses) had contributed to improvement in
Sporobolomyces produces oval to elongated yeast-like clinical condition [17]. Similarly, Sporobolomyces salmo-
cells, pseudohyphae, true hyphae, and one-celled, usually nicolor was shown to be a likely cause of allergic disorders
reniform (kidney-shaped) ballistoconidia (2–12 × 3–35 μm). in 14 employees working in a hospital building with severe,
Blastoconidia are forcibly discharged from denticles repeated, and enduring water and mold damage. The patients
located on ovoid to elongate vegetative cells and function presented cough, asthma, alveolitis, and rhinitis [22].
to produce satellite colonies. The shot-off (or discharge) of The common antifungal drugs amphotericin B (AMB)
Sporobolomyces ballistoconidia can be observed by inoculat- and fluconazole (FLC) may be useful for treatment of
ing one plate, taping it on top of an uninoculated plate, face to Sporobolomyces infections [24,25].
677

76.1.3 Laboratory Diagnosis a Harris micropunch and washed twice for 1 min each with
100 μL of Whatman FTA wash reagent, followed by two
Observation of characteristic macroscopic and micro- washes for 1 min each with 100 μL of TE buffer (10 mM Tris–
scopic features in clinical samples and cultured isolates has HCl pH 7.5, 0.1 mM EDTA). Washed filters are then dried for
formed the basis of conventional laboratory identification of 5 min at 55°C on a dry heat block, and PCR mixes are added
Sporobolomyces and other yeasts. To aid the microscopic directly to the washed and dried FTA filter punches [30].
detection of fungal elements in clinical specimens and tis-
sue biopsy, several stains (e.g., KOH, lactophenol, calcofluor
white, PAS, Gomori methenamine silver) may be utilized. 76.2.2 Detection Procedures
Further characterization of Sporobolomyces is conducted Bai et al. [8] utilized primers ITS1 (5′-GTCGTAACAA
with cultured isolates using a range of biochemical tests such GGTTTCCGTAGGTG-3′) and NL4 (5′-GGTCCGTGTTT
as carbon and nitrogen compound assimilation patterns and CAAGACGG-3′) to amplify a DNA fragment covering the
vitamin requirement tests [8]. Application of commercial ITS region and 25S rRNA D1/D2 domain for identification of
yeast identification systems (e.g., API and VITEK) facilitates Sporobolomyces and other yeasts.
easy biochemical differentiation of yeasts.
Considering that detection of characteristic morphologi- Procedure
cal features requires specialized skills, in vitro development
of reproductive structures of yeasts is time-consuming, and 1. PCR mixture (100 μL) is made up of 200 μM con-
biochemical tests seldom provide species-specific deter- centrations of each deoxynucleoside triphosphate,
mination, molecular techniques have been increasingly 250 nM concentrations of the appropriate prim-
employed for improved detection and identification of yeasts ers, 2 U of HotStarTaq polymerase (QIAGEN), and
including Sporobolomyces. By targeting the conserved 18S, either 5 μL of extracted genomic DNA or a single
5.8S, and 28S nuclear rRNA genes, the phylogenetic rela- filter punch.
tionships among yeasts can be accurately assessed [26,27]. 2. Amplification is conducted with initial enzyme acti-
Furthermore, PCR amplification and sequencing analysis of vation at 94°C for 15 min, followed by 40 cycles at
the more rapidly evolved internal transcribed spacer 1 and 94°C for 30 s, 52°C for 1 min, and 72°C for 2 min.
2 regions (ITS1 and ITS2, respectively) allow rapid species 3. After checking a fraction of total amplification
differentiation within a genus [28–31]. products in 1.2% agarose gels run for 45 min at
120 V in Tris-borate buffer, the remainder of the
PCR products are adjusted to final concentrations
76.2 Methods of 10% (wt/vol) PEG 8,000 and 10 mM MgCl2
and then centrifuged for 10 min at 12,000 rpm in a
bench-top centrifuge. The resulting DNA pellets are
Clinical samples are first examined by microscopy with the washed in 75% ethanol, air dried, resuspended in
use of fungal stains; portions of the samples are inoculated sterile water.
on Sabouraud dextrose agar (SDA), potato dextrose agar 4. The nucleotide sequences of the PCR products
(PDA), and other culture media and incubated at 30°C and are determined on an ABI PRISM 377 DNA
35°C for a few days [25]. sequencer using the ABI BigDye cycle sequenc-
For DNA extraction, 2 day old yeast colonies from SDA ing kit with the forward primers ITS1 and NL1
plate are resuspended in 100 μL of lysis buffer (100 mM (5′-GCATATCAATAAGCGGAGGAAAAG-3′)
Tris, 30 mM EDTA, 0.5% [wt/vol] sodium dodecyl sulfate, and the reverse primers ITS4 (5′-TCCTCCGCTTA
pH 7.5), vortexed briefly and incubated at 100°C for 15 min. TTGATATGC-3′) and NL4. Electrophoresis was
Following addition of 100 μL of 2.5 M potassium acetate, carried out.
the suspension is incubated on ice for 1 h and centrifuged at 5. The sequences of the ITS regions or 26S rDNA D1/
14,000 × g for 5 min. The supernatant is transferred to a new D2 domains of the testing strains and the reference
tube, an equal volume of isopropanol is added, and the tube sequences from GenBank are aligned with the pro-
is centrifuged for 5 min. After decanting the supernatant, gram clustal x and adjusted manually. Phylogenetic
500 μL of 100% ethanol is added, and the tube is centrifuged trees are constructed from the evolutionary distance
for 20 min. The supernatant is decanted, and the extracted data calculated from Kimura’s two-parameter model
DNA is air dried and resuspended in 100 μL of sterile water using the neighbor-joining method. Bootstrap anal-
and stored at −20°C [28]. yses are performed on 1000 random resamplings.
Alternatively, yeast aqueous suspensions (200 μL) are
applied directly to Whatman FTA microcards, which are then
76.3 Conclusion
placed, open, in a Panasonic microwave (800 W) while still
damp and subjected to two cycles of 30 s on full power, with Sporobolomyces spp. are ballistoconidium-forming yeasts
a pause of at least 30 s between each cycle. Punches (2 mm in with widespread distribution. Several Sporobolomyces spp.
diameter) are then removed from dried FTA filters by using (e.g., S. salmonicolor, S. roseus, and S. holsaticus) have been

Sporobolomyces 679
implicated in human diseases such as dermatitis, allergic the Sporidiobolales isolated from aquatic environments
alveolitis, and disseminated infections in both immunocom- in Patagonia, Argentina. Int. J. Syst. Evol. Microbiol.
petent and immunosuppressed individuals [32]. While it is 2005;55:503–509.
14 Satoh K, Makimura K. Sporobolomyces koalae sp. nov.,
possible to identify Sporobolomyces and other yeasts through
a basidiomycetous yeast isolated from nasal smears of
macroscopic and microscopic examination as well as bio- Queensland koalas kept in a Japanese zoological park. Int. J.
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77 Trichosporon
Takashi Sugita, Reiko Ikeda, Akemi Nishikawa, Masako Takashima,
Nanthawan Mekha, Natteewan Poonwan, Ayse Kalkanci, and Semra Kustimur
Contents
77.1 Introduction...................................................................................................................................................................... 681
77.1.1 Taxonomy, Morphology, and Epidemiology......................................................................................................... 681
77.1.1.1 Taxonomy............................................................................................................................................... 681
77.1.1.2 Morphology............................................................................................................................................ 681
77.1.1.3 Epidemiology......................................................................................................................................... 681
77.1.3 Diagnosis.............................................................................................................................................................. 683
77.2 Methods............................................................................................................................................................................ 684
77.2.2 Detection Preparation........................................................................................................................................... 684
77.2.2.1 Identification by PCR and Sequencing.................................................................................................. 684
77.2.2 2 Real-Time PCR Detection..................................................................................................................... 684
77.3 Conclusion........................................................................................................................................................................ 685
References.................................................................................................................................................................................. 685
77.1 Introduction ubiquinones; species belonging to the Cutaneum clade have

CoQ-10, whereas species belonging to the other clades have
77.1.1 Taxonomy, Morphology, and Epidemiology CoQ-9 [1].
Of the basidiomycetous yeasts, Trichosporon is the second
largest genus worldwide, following the genus Cryptococcus. 77.1.1.2 Morphology
One-third of Trichosporon species are associated with Morphologically, Trichosporon species are characterized by
infection or allergy. In recent years, the number of patients the production of arthroconidia. Abundant arthroconidia are
with breakthrough Trichosporon infections after receiving observed when the microorganism is grown in corn meal
candin-derived antifungal agents has increased. The progno- agar. The colonies are cream colored, moist or dry, and with
sis in patients with disseminated trichosporonosis is poor and or without a white farinose covering. True hyphae are pres-
mortality is high. Thus, early, rapid detection of the pathogen ent and lateral, clavate, or globose blastoconidia may also
is important in the diagnosis and treatment of trichosporono- be present. Hyphal septa have, as far as is known, dolipores,
sis. This chapter describes a molecular detection method for which may be with or without vesicular or tubular parenthe-
Trichosporon. somes. With India ink (bo-ku-ju in Japanese) staining, a thin
capsule can also be observed surrounding the cells [2].
77.1.1.1 Taxonomy
Taxonomically, the basidiomycetous yeast in the genus 77.1.1.3 Epidemiology
Trichosporon belongs to the order Trichosporonales, class These microorganisms cause superficial and deep-seated
Tremellomycetes, and subphylum Agaricomycotina. Of the infections (Table 77.1). Of the genus Trichosporon, approxi-
basidiomycetous yeasts, the genus Trichosporon with 39 mately one-third of the species have been isolated from
species is the second largest genus worldwide, following the clinical specimens. Historically, Trichosporon species have
genus Cryptococcus. There are also many candidate species, long been known to be the causative agents of white piedra.
and Trichosporon will likely expand to 50 species within Two species are most often associated with this infection:
a few years. The genus is further divided into five clades: T. ovoides is responsible for white piedra of the head, while
Cutaneum, Ovoides, Brassicae, Gracile, and Porosum. These T. inkin is limited to the genital area [2]. Other species are
clades correlate well with some other taxonomic characteris- often isolated from patients with superficial infections, such
tics. Species of this genus possess CoQ-9 or CoQ-10 as major as T. asahii, T. asteroids, and T. cutaneum [2–4]. However,
681

TABLE 77.1
Trichosporon Species Isolated from Clinical Specimens
Infection Deep-seated infection T. asahii, T. asteroides, T. debeurmannianum, T. inkin, T. japonicum, T. loubieri, T. mucoides
Superficial infection T. ovoides (capital white piedra), T. inkin (white piedra of the genital area), T. asahii,
T. asteroides, T. cutaneum
Allergy Summer-type hypersensitivity T. dermatis, T. asahii, T. asteroides, T. coremiiforme, T. faecale, T. japonicum, T. ovoides,
pneumonitis T. domesticum, T. montevideense
Source: Sugita, T., The Yeasts, A Taxonomic Study, 5th edn., Boekhout, T., Fell. J.W., Kurtzman. C.P. (eds.), Elsevier, Amsterdam, the
Netherlands, 2011, pp. 2015–2061.
1787 123 157 175 3380 485 118 1610
18S 26S 18S
ITS1 ITS2 IGS1 IGS2

5.8S 5S
FIGURE 77.1 Schematic representation of the rRNA locus in Trichosporon. ITS, internal transcribed spacer; IGS, intergenic
spacer region.
white piedra is limited to Africa at present and other superfi- The major genotype obtained from infectious specimens is
cial infections due to Trichosporon species are rare. type 1, while that from the homes of SHP patients is type
Deep-seated trichosporonosis is an important fungal infec- 3. Random amplification of polymorphic DNA analysis also
tion clinically. The first case was reported in a brain abscess suggests that T. asahii strains obtained from clinical speci-
patient in 1970 in the United Kingdom [5]. Since then, the mens are genetically different from those obtained from the
number of cases has increased, primarily in immunocom- homes of SHP patients [23].
promised patients. For about 5 years, deep-seated tricho-
sporonosis has been recognized as a breakthrough infection
after treatment with candin-derived antifungal agents, such
as micafungin [6–9]. Although several species have been As mentioned above, Trichosporon species cause superficial
isolated from clinical specimens, T. asahii is the causative infection, although the incidence of this is low. Deep-seated
agent in the vast majority of cases [2,3]. This microorgan- trichosporonosis can be life threatening, with high mortal-
ism has nine genotypes in the intergenic spacer region (IGS), ity. Populations at risk are people with human immunode-
which is located between the 26S and 5S rRNA genes and ficiency virus (HIV) infection, bone marrow and organ
the population of genotypes differs in different countries
[10–12] (Figures 77.1 and 77.2). The genotype distribution of
100
isolates in Japan and Turkey is similar: approximately 80%
are genotype 1. The major genotypes of isolates from the Japan
USA
United States are types 3 and 5, while the major Thai isolates Thailand
80
are types 1 and 3. There is a geographic substructure among Turkey
T. asahii clinical isolates. T. asteroids [13], T. debeurman-

nianum [14], T. inkin [15,16], T. japonicum [17], T. loubieri
Distribution (%)
60
[18,19], and T. mucoides [20] are also occasionally isolated
from clinical specimens.
Trichosporon species not only cause infections but also
40
allergies. The microorganism triggers summer-type hyper-
sensitivity pneumonitis (SHP) [21], which follows the devel-
opment of type 3 or 4 allergy with the repeated inhalation of
20
Trichosporon arthroconidia, which often contaminate home
environments during the summer. In Japan, the summer is
hot, humid, and rainy. Such conditions favor the growth of
0
Trichosporon species. Although several Trichosporon spe- 1 2 3 4 5 6 7 8 9
cies may be distributed in the homes of patients with SHP,
Genotype
T. dermatis (41%) and T. asahii (51%) are the major causes
[22]. Interestingly, the genotype distribution of T. asahii in FIGURE 77.2 Genotype distribution of Trichosporon asahii clin-
patients with infection and SHP is significantly different. ical isolates from Japan, the United States, Thailand, and Turkey.

Trichosporon 683
transplant recipients, and patients with profound neutropenia at two positions in the D1/D2 26S rRNA gene. The nucleo-
[24–26]. The most recent epidemiological survey was con- tide differences in the ITS region and D1/D2 26S rRNA gene
ducted by a Japanese group who retrospectively analyzed among T. asahii, T. coremiiforme, and T. faecale are only
33 cases of Trichosporon fungemia in patients with hema- one or two base pairs. The DNA sequence of the IGS1 region
tologic malignancies, treated between 1992 and 2007 [27]. is more diverse, compared with ITS or D1/D2 26S rRNA
Trichosporon fungemia occurred as a breakthrough infection gene, indicating that IGS1 analysis can readily and reliably
during antifungal therapy in 30 patients (91%), 18 of whom differentiate between such closely related species [12]. The
were receiving micafungin. Of the 33 cases, 25 (76%) died 99% similarity in ITS sequences observed between two spe-
of the infection. This morality rate is similar to other reports cies corresponds to approximately 55%–95% IGS1 sequence
[28,29]. The resolution of infection was associated with neu- similarity. The 98% ITS sequence similarity in another pair-
trophil recovery, the absence of hyperglycemia, and therapy wise comparison corresponds to approximately 45%–55%
including azoles. Survival was significantly longer in patients IGS1 sequence similarity (Figure 77.3). Approximately 20%
receiving antifungal therapy containing azoles than in those difference has been observed in the IGS1 region among
who did not receive azoles. T. asahii, T. coremiiforme, and T. faecale and a 7% differ-
Trichosporon species also cause SHP with the repeated ence between T. domesticum and T. montevideense. Using
inhalation of their spores [21]. Patients develop cough, chills, the primers shown in Table 77.2, from 195 to 719 bp of the
headache, fever, and difficulty breathing as acute symptoms. IGS1 region can be amplified.
These symptoms disappear on eliminating the causative anti- Thus, IGS1 sequencing analysis is a reliable method
gen, Trichosporon, from colonizing places. In chronic SHP, for identifying Trichosporon species. A high-throughput
the patient’s lungs become fibrotic. As anti-Trichosporon-
specific IgG antibody is produced in the patient’s serum,
detection of this antibody can be used to diagnose SHP. 100
77.1.3 Diagnosis 99
% ITS DNA sequence similarity

The conventional identification method for yeast, including 98
Trichosporon species, is based on biochemical characteris-
tics. Two commercial kits, API 20C Aux™ and ID 32C API™
(bioMérieux), are the most popular for yeast identification. 97
These kits can identify 47 and 69 yeast species, respectively,
within 48 h. However, only three species (T. inkin, T. asahii,
T. mucoides) in the genus Trichosporon can be identified 96
using these kits. Morphologically, Trichosporon is character-
ized by the production of arthroconidia, which is a key char-
95
acteristic at the genus level.
30 40 50 60 70 80 90 100
% IGS 1 DNA sequence similarity
77.1.3.2.1 Culture-Dependent Methods FIGURE 77.3 Sequence similarities between the IGS1 and ITS
As yeast species have few unique morphological character- regions. Data are derived from Trichosporon DNA sequences.
istics, molecular techniques are a powerful tool for species
identification. The most broadly used identification method
is rRNA gene sequence analysis. Figure 77.1 shows the
rRNA locus in Trichosporon, which is approximately 7800 TABLE 77.2
bp long. Of this, the internal transcribed spacer (ITS) region PCR Primers and Probes Used to Detect Trichosporon
and D1/D2 26S rRNA gene have been widely used for fun- Species
gal identification. The most important part of rRNA gene
Primer and
sequence analysis is to establish identification criteria. When Probe Sequence (5′–3′)
there is less than 1% difference in the ITS region or D1/D2
Trichosporon 26SF, ATCCTTTGCAGACGACTTGA
26S rRNA gene between two strains, they are conspecifics
species forward
[30,31], although some Trichosporon species do not conform TR5R, AGCTTGACTTCGCAGATCGG
to this standard. No difference in the ITS region is observed reverse
between T. laibachii and T. multisporum, although these Trichosporon Forward AGTATAAGGGATCAAAAATGAGAGCCA
species have seven differences (1.1%) in their D1/D2 26S asahii Reverse CCTCTGAGGCCTTGCTCCTG
rRNA gene. T. montevideense and T. domesticum also share TaqMan TGATGGCCTTGGTTGAGA
identical nucleotide sequences in the ITS region but differ

suspension array system of uniplex and multiplex D1/D2 26S 77.2.2 Detection Preparation
rRNA, ITS, and IGS probes is a rapid, accurate method to
identify species of Trichosporon [32]. 77.2.2.1 Identification by PCR and Sequencing
DNA sequence analysis of the IGS1 region in the rRNA gene
77.1.3.2.2 Culture-Independent Methods is the most reliable and accurate identification method. The
The Trichosporon species of the greatest clinical importance IGS1 region is amplified by PCR with primers 26SF and
is T. asahii because infection due to T. asahii can be life TR5R (see Table 77.2). PCR is performed with an initial 3 min
threatening and its prognosis is poor. Thus, the early detec- denaturation at 94°C, followed by 30 cycles of 30 s at 94°C,
tion and accurate identification of the causative agent are 30 s at 57°C, and 1 min at 72°C, and a final 10 min exten-
necessary to provide appropriate treatment. As a first-gener- sion at 72°C. The PCR products are sequenced with primers
ation method, a direct detection method was developed for 26SF and TR5R. The IGS1 sequences range in length from
T. asahii DNA from blood [33–37]. This polymerase chain 195 to 719 bp, and the resulting DNA sequence is searched
reaction (PCR) assay could detect T. asahii DNA from the using BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).
polysaccharide antigen-negative samples, suggesting that A species is defined as a group of organisms sharing >95%
PCR had greater sensitivity than antigen detection methods identity in their IGS1 region.
(Table 77.3) [34]. Quantification of the pathogen in clini-
cal samples is also of great clinical significance. Three sets 77.2.2 2 Real-Time PCR Detection
of oligonucleotide primers and TaqMan probe pairs were First, 100 μL of serum is mixed with an equal volume of
designed for detecting the major causative agent of tricho- lysis buffer (100 mM KCl, 20 mM Tris–HCl pH 8.3, 5 mM
sporonosis, T. asahii [38]. The sequences were designed MgCl2, 0.2 mg/mL gelatin, and 0.9% Tween 20 solution) and
from the IGS1 region of the rRNA gene (Table 77.2). A real- proteinase K is added to the mixture. The mixture is incu-
time PCR assay can detect as few as 10 copies of T. asahii bated for 60 min at 55°C and then for 10 min at 95°C to inac-
DNA. For 21 blood samples, between 1.8 × 103 and 2.1 × 104 tivate the proteinase K. The sample is extracted twice with
copies of T. asahii DNA were detected. Of these, 16 were phenol–chloroform–isoamyl alcohol and precipitated with
positive for polysaccharide antigen using the latex aggluti- ethanol. Ethatimate™ (Nippon Gene, Toyama, Japan) is used
nation test with titers of 1:4–1:64. For the five samples that as a precipitation activator. For real-time PCR [38], 25 μL of
were negative by the latex agglutination test, 1.8 × 103 to a TaqMan Universal PCR master mix (Applied Biosystems),
4.9 × 103 copies of T. asahii DNA were detected. There was 200 nM each of the forward and reverse primers, 250 nM
a good correlation between the number of DNA copies and TaqMan probe (see Table 77.2), 3 μL of each DNA sample,
the titer of polysaccharide antigen (r 2 = 0.910). Real-time and Milli-Q water in a total volume of 50 μL are placed in
PCR can be used to diagnose deep-seated trichosporono- each well of a 96 well plate. Amplification and detection are
sis due to T. asahii. A practical example is described in performed with the cycle parameters 50°C for 2 min, 95°C
Section 77.2.2. for 10 min, and 40 cycles of 95°C for 15 s, and 60°C for 1 min.
A standard curve is plotted using the cycle threshold values
obtained by amplifying successive 10-fold dilutions of a
77.2 Methods known concentration of plasmid DNA (from 101 to 109 cop-
ies). A practical example is shown in Table 77.4 [39].
A 16-year-old boy received an allogeneic bone marrow
DNA is prepared from a small amount of a pure culture transplant from a human leukocyte antigen (HLA)-identical
(approximately one loop) using any DNA extraction method. unrelated donor for T cell acute lymphoblastic leukemia. On
TABLE 77.3
Blood Culture, Nested PCR, and Polysaccharide Detection Using Sera from Patients with
Deep-Seated Trichosporonosis
Patient No. Sex/Age (Year)a Underlying Diseaseb Blood Culturec PCR Productsc Titer of PS Antigen
1 M/64 AML + + 1:4096
2 M/47 ML + + 1:32
3 F/15 AML − − 0
4 F/55 AML − − 0
5 M/29 MI ND + 0
6 M/63 MM ND + 0
a M, male; F, female.
b AML, acute myelogenic leukemia; MI, multiple injuries, ML, malignant lymphoma; MM, multiple myeloma.
c +, positive; −, negative; ND, not done.

Trichosporon 685
TABLE 77.4
Laboratory Features of Trichosporonosis in the Patient
Day after transplant 59 61 62 68 72 75 76 78 81 83 86 90 104
Blood culture − + − −
Urine culture + + + −
Beta-d-glucan (pg/dL) ND 95 60 22 ND
Serum glucuronoxylomannan − + + + −
PCR (number of plasmid copy) ND 63 480 642 190 52 ND
Note: ND, not detected.
day 61, Trichosporon species was cultured from a urine sam- 7. Matsue K et al. Breakthrough trichosporonosis in patients
ple, but not from a blood sample. The patient was treated with with hematologic malignancies receiving micafungin. Clin.
micafungin. On day 76, beta-d-glucan in serum was posi- Infect. Dis. 42, 753–757, 2006.
8. Asada N et al. Successful treatment of breakthrough
tive and on day 78, Trichosporon was cultured from a blood
Trichosporon asahii fungemia with voriconazole in a patient
sample. On day 80, the antifungal therapy was switched with acute myeloid leukemia. Clin. Infect. Dis. 43, e39–e41,
from micafungin to voriconazole. From day 59 to day 104, 2006.
the serum T. asahii DNA concentration was assessed serially 9. Bayramoglu G et al. Breakthrough Trichosporon asahii
using real-time PCR. Fungal DNA was detected in serum fungemia in neutropenic patient with acute leukemia while
16 and 14 days before the respective days the positive blood receiving caspofungin. Infection 36, 68–70, 2008.
culture and positive blood beta-d-glucan were obtained. The 10. Mekha N et al. Genotyping and antifungal drug susceptibil-
concentration of T. asahii DNA correlated well with the ity of the pathogenic yeast Trichosporon asahii isolated from
Thai patients. Mycopathologia 169, 67–70, 2010.
patient’s clinical course. 11. Kalkanci A et al. Molecular identification, genotyping, and
drug susceptibility of the basidiomycetous yeast pathogen
Trichosporon isolated from Turkish patients. Med. Mycol. 48,
77.3 Conclusion 141–146, 2010.
For pathogenic Trichosporon species, it is clinically impor- 12. Sugita T et al. Sequence analysis of the ribosomal DNA
tant to be able to detect and identify the causative agent of intergenic spacer 1 regions of Trichosporon species. J. Clin.
Microbiol. 40, 1826–1830, 2002.
deep-seated trichosporonosis. After the introduction of can-
13. Kustimur S et al. Nosocomial fungemia due to Trichosporon
din antifungal agents, the number of patients with break- asteroides: Firstly described bloodstream infection. Diagn.
through trichosporonosis infections has increased. Early Microbiol. Infect. Dis. 43, 167–170, 2002.
detection of the causative agent is needed for a diagnosis. 14. Sugita T et al. Two new yeasts, Trichosporon debeurmannia-
Several molecular-based detection methods have been devel- num sp. nov. and Trichosporon dermatis sp. nov., transferred
oped, including a quantitative assay. A retrospective study from the Cryptococcus humicola complex. Int. J. Syst. Evol.
of these methods has been conducted using clinical speci- Microbiol. 51, 1221–1228, 2001.
mens. A prospective study using molecular-based methods 15. Koyanagi T et al. Autopsy case of disseminated Trichosporon
inkin infection identified with molecular biological and bio-
is expected. chemical methods. Pathol. Int. 56, 738–743, 2006.
16. David C et al. Disseminated Trichosporon inkin and
Histoplasma capsulatum in a patient with newly diagnosed
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78 Ustilago and Pseudozyma
Dongyou Liu
Contents
78.1 Introduction...................................................................................................................................................................... 687
78.2 Methods............................................................................................................................................................................ 688
78.2.2.1 PCR Identification of Ustilago maydis ................................................................................................. 689
78.2.2.2 Pan-Fungal Detection............................................................................................................................ 689
78.3 Conclusion........................................................................................................................................................................ 690
References.................................................................................................................................................................................. 690
78.1 Introduction Spitzenkörper consists of a heterogeneous population of vesi-

cles surrounding a core that contains polysomes, microtubules,
78.1.1 Classification, Morphology, and Biology and actin, and serves as a supply center for the distribution of
The genera Ustilago and Pseudozyma are basidiomy- vesicles containing materials necessary for tip extension.
cetous yeasts belonging to the family Ustilaginaceae, U. maydis is capable of switching from one form to the
order Ustilaginales, class Ustilaginomycetes, phylum other (the dimorphic switch). The dimorphic switch in the
Basidiomycota. The family Ustilaginaceae covers 26 gen- U. maydis life cycle is controlled by two mating type loci,
era: Anomalomyces, Cintractia, Dermatosorus, Farysia, a and b. The a locus has two alleles (a1 and a2), codes for
Franzpetrakia, Gymnocintractia, Heterotolyposporium, components of a signal transduction pathway (pheromone
Leucocintractia, Macalpinomyces, Melanopsichium, mito- precursor and receptor genes), and governs cell fusion of hap-
sporic Ustilaginaceae, Moesziomyces, Moreaua, Mycosyrinx, loid cells and filamentous growth of the dikaryon in vitro but
Parvulago, Pericladium, Portalia, Restiosporium, not in planta. The b locus has 25 naturally occurring alleles
Schizonella, Sporisorium, Stegocintractia, Tolyposporium, (b1…b25), codes for a combinatorial homeodomain protein,
Tranzscheliella, Trichocintractia, Ustilago, and Websdanea and is the major determinant of filamentous growth, in vitro
as well as some unclassified Ustilaginaceae [1–4]. and in planta, and of pathogenicity. During interaction with
The genus Ustilago consists of 42 recognized species as its plant host, U. maydis shows an increased repertoire of
well as 11 unassigned species, which are known as smut fungi morphologies, suggesting that plant signals play an important
with sophisticated life cycles including sexual and asexual role in generation of additional morphologies [6,7].
(yeast) morphologies and pathogenic to corn [1,5]. The best On fungal culture media, Ustilago grows slowly and
known member of this genus is Ustilago maydis (obsolete matures in 20 days. Colonies are moist, compact, and yeast-
synonym: U. zeae), which is smut fungus causing diseases like, later becoming wrinkled and membrane-like with pro-
in maize, and is occasionally involved in human infections. fuse budding in the medium. The color is cream to yellow
Ustilago maydis, a Basidiomycete fungus infecting maize initially and turns to tan to dark brown with age. Reverse
and teosinte, demonstrates two basic morphologies: a yeast- is pale. Blastoconidia are spindle shaped, elongated, and
like form and a filamentous form. The yeast-like form is irregular, resembling arthroconidia. Pseudohyphae consist of
haploid, unicellular, and elongated with tapered ends (cigar spindle-shaped cells. Short hyphae with clamp connections
shaped), divides by budding (once per cell cycle at one of may appear when old.
the cell poles), with the bud growing by tip extension, and is Ustilago maydis harbors a genome of approximately
nonpathogenic. The filamentous form is dikaryotic, divides 20 Mb, which contains the machinery for recognition and
at the apical cell, grows by tip extension, and is pathogenic. interpretation of the budding yeast axial and bipolar land-
A fungal-specific phase-contrast opaque body, the marks as well as for cell polarity establishment, exocytosis,
Spitzenkörper (apical body), located at or just below the tip actin and microtubule organization, microtubule plus end–
of the apical cell, is thought to drive hyphal growth. The associated proteins, kinesins, and myosins [8].
687

The genus Pseudozyma, the only member of the mito- in culture as yeast-like cells. Haploid strains of opposite
sporic Ustilaginaceae group, comprises 15 recognized mating type fuse and form a filamentous, dikaryotic cell
species: Candida tsukubaensis, Pseudozyma antarctica, type that invades plant tissue to reinitiate infection. Several
Pseudozyma aphidis, Pseudozyma flocculosa, Pseudozyma invasion-associated genes have been identified in U. maydis.
fusiformata, Pseudozyma graminicola, Pseudozyma hubei- An ortholog of YAP1 (for Yeast AP-1-like) functions as a
ensis, Pseudozyma jujuensis, Pseudozyma parantarctica, redox sensor and regulates the oxidative stress response in
Pseudozyma prolifica, Pseudozyma pruni, Pseudozyma U. maydis. The fungus utilizes its Yap1-controlled detoxifi-
rugulosa, Pseudozyma shanxiensis, Pseudozyma siamensis, cation system for coping with early plant defense responses
and Pseudozyma thailandica, in addition to 33 unassigned [16,17]. In addition, extracellular effector proteins Hum3 and
species [1]. Pseudozyma species are heterobasidiomycetous Rsp1 are pathogenicity proteins that share an essential func-
yeasts related to the smut fungi in the genus Ustilago [9]. tion in early stages of the infection [18].
While many Pseudozyma spp. are usually isolated from Ustilago and Pseudozyma are susceptible to amphotericin
corns [6], several (P. Antarctica, P. parantarctica, and P. B, ketoconazole, itraconazole, simeconazole, which may be
thailandica) have been identified from the blood of human used for effective treatment of these fungi [10,13].
patients [10], and P. aphidis is recently implicated in a pedi-
atric case of central venous catheter (CVC) infection [11].
After 4 days of incubation on Sabouraud dextrose agar 78.1.3 Laboratory Diagnosis
at 37°C in ambient air, Pseudozyma colonies are tan-yellow Traditionally, laboratory identification of Ustilago and
and wrinkled; blastoconidia are fusiform, spindle shaped, Pseudozyma yeasts are based on assessment of their macro-
and elongated. scopic and microscopic features. Several stains (e.g., KOH,
lactophenol, calcofluor white, periodic acid-Schiff [PAS],
78.1.2 Clinical Features and Pathogenesis Gomori methenamine silver [GMS]) may be employed.
Standard fungal media are useful for isolation of these organ-
Ustilago and Pseudozyma are widely distributed in soil and isms for further characterization. More recently, molecular
are pathogens of seeds and flowers of cereals, wheat, corn, techniques are increasingly relied upon for identification,
and grasses [26]. Occasionally, these yeasts are associ- detection, and phylogenetic analysis of these fungi [19].
ated with human diseases such as skin and CVC infections Through examination of the cytochrome b gene sequences,
[10–13]. Since Ustilago and Pseudozyma are associated with it was shown that basidiomycetous yeasts can be sepa-
corn, these organisms may enter the host through diet. rated into two main clusters: cluster 1 includes Tremellales,
In a Ustilago-related skin infection, the patient showed Filobasidiales, and their anamorphs (which possess xylose in
a chronic skin rash with scaly erythematous plaques over the cell wall and have dolipore septa) and cluster 2 includes
the nasal alae and philtrum [13]. Airborne Ustilago spores Ustilaginales, Sporidiales, and their anamorphs (which pos-
have also been implicated in hypersensitivity pneumonitis, sess no xylose in the cell wall and have a simple pore septum).
asthma, and allergic rhinitis [14,15]. Thus, use of cytochrome b sequences enables both species
The first documented case of CVC infection due to identification and the study of phylogenetic relationships
Pseudozyma species involved a 7-year-old child, who was [20]. Similarly, DNA sequencing analysis of the internal
born with gastroschisis and ileocolonic atresia resulting in transcribed spacer (ITS) region of the nuclear rRNA genes
short bowel syndrome. As a result, the patient relied on a revealed the monophyly of a bipartite genus Sporisorium and
long-term indwelling central line for parenteral nutrition. On the monophyly of a core Ustilago clade and indicated a puta-
admission, the patient presented with intermittent fevers (up to tive connection between Ustilago maydis and Sporisorium
39.7°C) accompanied by chills, malaise, and fatigue. Culture [21]. Furthermore, combined analyses of ITS and LSU rRNA
of the blood specimen on chocolate and Sabouraud dextrose sequences resolved three major groups of almost identical
agars yielded mature, moist yeast-like, tan-yellow, and wrin- composition: Sporisorium, Ustilago, and a basal assemblage
kled colonies after 4 days of incubation at 37°C in ambient air. of both Ustilago and Sporisorium species [21].
Microscopic examination of the isolate on wet mount revealed By using oligonucleotides corresponding to a specific
fusiform spindle-shaped blastoconidia that were elongate and region downstream of the homeodomain of the bE genes, a
slightly irregular, suggestive of a yeast belonging to the order 500 bp product was amplified from U. maydis DNA only.
Ustilaginales. Sequencing analysis using fungal-specific Use of this method permitted rapid and precise detection
primers ITS5 and ITS4 targeting the intervening transcribed and identification of U. maydis organism from infected
spacer region of the rRNA confirmed its P. aphidis identity. tissues [22].
Prompt removal of the CVC in conjunction with oral itracon-
azole therapy resulted in a successful outcome [11].
U. maydis interacts with its plant host through a reciprocal 78.2 Methods
process of signal exchange, leading to alterations in cell mor-
phology of both host and fungus. The fungus induces tumors
on host plants and forms masses of diploid teliospores, which Clinical specimens are examined under microscope for
germinate to form haploid meiotic strains that can propagate mycotic elements. Portions of the samples are inoculated on

Ustilago and Pseudozyma 689
inhibitory mold agar, modified Sabouraud agars, or potato (5′-CTCGAGGTTCATCAGCTCA-3′) and 1370 (5′-GCTG
dextrose agar and incubated at various temperatures for a few AGTTCTGGAGTCG-3′). These primers target a conserved
days. region downstream of the homeodomain of the bE genes
After growth for 1–7 days on potato dextrose agar slants, from U. Maydis, with primer 1369 being located at nt 645
lysates are prepared from approximately 1 cm2 of mycelia of bE1 and bE2, and nt 489 of bE5; with primer 1370 being
with IDI lysis kits (GeneOhm Sciences, San Diego, CA). located at nt 1147 of bE1 and bE2, and nt 992 of bE5.
Briefly, in a biological safety cabinet, mycelia are collected Interestingly, the life cycle of U. maydis is regulated by
by scraping the slant with a sterile stick in 1 mL of ster- the mating-type loci a and b. The a locus (consisting of two
ile, molecular-grade H2O. The material is transferred to a idiomorphs, a1 and a2 ) is required for cell-to-cell recogni-
2 mL screw-cap tube. The tubes are centrifuged for 1 min tion during the mating process and for the maintenance of
at 6000 × g. If the mycelia do not pellet, the material is con- filamentous growth. Each idiomorph encodes a pheromone
tained with a pediatric blood serum filter (Porex Corp.). and a receptor for the pheromone synthesized by the com-
After removal of supernatant, the material is resuspended patible partner strain. The b locus (consisting of at least
in 200 μL of IDI sample buffer and transferred to the lysis 25 alleles at each of two genes, bE and bW) regulates the
tube, which contains glass beads. Lysis tubes are vortexed on steps in sexual development that occur after fusion of hap-
the highest setting for 5 min. The tubes are placed in a boil- loid cells. Different a and b alleles in mating partners are
ing water bath for 15 min. Tubes are centrifuged for 5 min at necessary to trigger mating, filamentous growth, and tumor
16,000 × g. The supernatant is stored at −20°C until ampli- induction [22].
fication [23].
Procedure
Alternatively, 1 cm 2 of fungal material is transferred
to a 2 mL Eppendorf tube containing a 2:1 (wt/wt) mix-
ture of silica gel and Celite (silica gel H, Merck 7736/ 1. PCR mixture (50 μL) is made up of 100 μM dNTPs,
Kieselguhr Celite 545; Machery) and 300 μL of TES 2 mM MgCl2, 1× PCR buffer (Gibco), 2.5 U of DNA
buffer (2 g Tris [hydroxymethyl]-aminomethane, 0.38 g Taq polymerase (Gibco), extracted fungal DNA, and
Na-ethylenediaminetetraacetic acid (EDTA), and 2 g primers 1369 and 1370.
sodium dodecyl sulfate in 80 mL of ultrapure water [pH 2. Amplification is conducted with 30 cycles of dena-
8]). The fungal material is ground with a micropestle for turation at 95°C for 1 min, annealing at 55°C for
1–2 min. The volume is adjusted by adding 200 μL of TES 1 min, and extension at 72°C for 1 min, followed by
buffer. After vigorous shaking and the addition of 10 μL of a final extension for 10 min at 72°C.
a 10 mg/mL concentration of proteinase K to the tube, the 3. PCR products (5 μL) are separated by electropho-
mixture is incubated at 65°C for 10 min. With the addition resis on a 1.5% agarose gel, stained with ethidium
of 140 μL of 5 M NaCl solution, the mixture is combined bromide, and observed under ultraviolet light.
with 1/10 volume (∼65 μL) of cetyltrimethylammonium
bromide (CTAB) buffer 10%, followed by incubation for Note. The PCR assay using primers 1369 and 1370 generates
another 30 min at 65°C. One volume (∼700 μL) of chloro- a 502-bp product from DNA of U. maydis, but not from DNA
form-isoamyl alcohol (vol/vol = 24/1) is added and mixed of other fungi unrelated to U. maydis.
by inversion. After incubation for 30 min at 0°C (on ice
water) and centrifugation at 14,000 rpm at 4°C for 10 min, 78.2.2.2 Pan-Fungal Detection
the top layer is transferred to a clean Eppendorf tube. The
sample is added with 225 μL of 5 M NH4 -acetate and incu-
bated for at least 30 min (on ice water) and centrifuged.
The supernatant is transferred to a clean sterile Eppendorf
tube and mixed with a 0.55 volume (∼510 μL) of ice-cold
reverse (5′-TCCTCCGCTTATTGATATGC-3′), which cov-
isopropanol. After centrifugation for 7 min at 14,000 rpm
ers ITS1-5.8S-ITS2 rRNA gene cluster. The identity of the
and 4°C (or room temperature), the supernatant is decanted.
fungi is verified by subsequent sequencing analysis.
The pellet is washed with ice-cold 70% ethanol twice and
dried by using a vacuum dryer. The powder is resuspended Procedure
in 48.5 μL of Tris-EDTA buffer with 1.5 μL of 10 mg of
RNase/mL, incubated at 37°C for 15–30 min, and stored at 1. PCR mixture is composed of 1× Lightcycler
−20°C until use [24]. FastStart DNA Master Hybridization Probes mix-
ture (Roche Applied Science) (containing deoxy-
78.2.2 Detection Procedures nucleoside triphosphates, FastStart Taq DNA
polymerase, and 1 mM MgCl2, additional MgCl2 is
78.2.2.1 PCR Identification of Ustilago maydis added to a final concentration of 4.6 mM), 0.4 μM
Martínez-Espinoza et al. [22] designed a poly- each of ITS1 forward and ITS4 reverse primers, 1×
merase chain reaction (PCR) assay for specific iden- SYBR green (Molecular Probes), and 3 μL tem-
tification of Ustilago maydis using primers 1369 plate DNA.

2. Thermal cycling parameters include 95°C for References

10 min; 50 cycles at 95°C for 5 s, 60°C for 20 s, and
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3. The quality of the amplicon is determined using the 2. Begerow, D. and Bauer, R. (2000). Phylogenetic placements
derivative of the melt analysis curve (55°C–99°C, of ustilaginomycetous anamorphs as deduced from nuclear
45 s hold at 55°C, 5 s/°C) using the RotorGene 3000 LSU rDNA sequences. Mycol. Res. 104, 53–60.
(Corbett Robotics, Inc.). 3. Begerow, D., Stoll, M., and Bauer, R. (2006). A phyloge-
4. The amplified product is purified for bidirec- netic hypothesis of Ustilaginomycotina based on multiple
tional sequencing using ExoSAP-IT (USB Corp.). gene analyses and morphological data. Mycologia 98(6),
906–916.
Five microliters of Big Dye Terminator Ready 4. Fell, J.W. et al. (2000). Biodiversity and systematics of basid-
Reaction Mix v. 1.1 (Applied Biosystems) is added iomycetous yeasts as determined by large-subunit rDNA D1/
to 4 μL of each primer (0.8 pmol/μL) and 3 μL of D2 domain sequence analysis. Int. J. Syst. Evol. Microbiol.
purified PCR product. Cycle sequencing is per- 50, 1351.
formed with a 9700 thermal cycler (ABI), using 5. Stoll, M., Begerow, D., and Oberwinkler, F. (2005). Molecular
25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C phylogeny of Ustilago, Sporisorium, and related taxa based
for 4 min. Sequencing reaction products are passed on combined analyses of rDNA sequences. Mycol. Res. 109,
342–356.
through a Sephadex G-50 fine column to remove
6. Martinez-Espinoza, A.D., Garcia-Pedrajas, M.D., and Gold,
unincorporated dye terminators. Purified sequenc- S.E. (2002). The Ustilaginales as plant pests and model sys-
ing reaction products are run on an ABI Prism 3100 tems. Fungal Genet. Biol. 35, 1–20.
Genetic Analyzer with a 50 cm capillary array. 7. Perez-Martin, J. and Castillo-Lluva, S. (2006). Pathocycles:
5. Sequences are analyzed with the SmartGene Ustilago maydis as a model to study the relationship between
Integrated Database Network software version cell cycle and virulence in pathogenic fungi. Mol. Genet.
3.2.3 vr. SmartGene is a web-based software and Genom. 276, 211–220.
database system with reference sequences derived 8. Kamper, J. et al. (2006). Insights from the genome of the bio-
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from the National Center for Biological Information 97–101.
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a genus for yeast-like anamorphs of ustilaginales. J. Gen.
Note. If real time PCR instrument is unavailable, stan- Appl. Microbiol. 41, 359–366.
dard PCR may be performed with primers ITS1 and ITS4, 10. Sugita, T. et al. (2003). The first isolation of ustilaginomyce-
and the resulting amplicon is sequenced with the same tous anamorphic yeasts, Pseudozyma species, from patients’
primers. Sequence-based identifications are defined by blood and a description of two new species: P. parantarctica
and P. thailandica. Microbiol. Immunol. 47, 183–190.
percent identity: species, ≥99%; genus, 93%–99%; and
11. Lin, S.S. et al. (2008). Central venous catheter infection asso-
inconclusive, ≤93%. For strains producing discrepant iden- ciated with Pseudozyma aphidis in a child with short gut syn-
tification between the methods based on phenotypic char- drome. J. M ed. Microbiol. 57, 516–518.
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the large-subunit RNA gene is amplified with primers NL1 Ustilago species. Clin. Infect. Dis. 21, 1043–1044.
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 13. Teo, L.H. and Tay, Y.K. (2006). Ustilago species infection in
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14. Santilli, J., Rockwell, W.J., and Collins, R.P. (1985). The sig-
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78.3 Conclusion
15. Yoshida, K. et al. (1996). Hypersensitivity pneumonitis
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belonging to the family Ustilaginaceae. Besides causing dis- 650–651.
16. Molina, L. and Kahmann, R. (2007). An Ustilago maydis
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and corns, they may gain entry into human host through 17. Howlett, B.J., Idnurm, A., and Heitman, J. (2007). Fungal
food intake [26]. Patients with underlying conditions such pathogenesis: Gene clusters unveiled as secrets within the
as short gut syndrome seem vulnerable to the infection by Ustilago maydis code. Curr. Biol. 17(3), R87–R90.
Ustilago and Pseudozyma spp. Assessment of the macro- 18. Müller, O., Schreier, P.H., and Uhrig, J.F. (2008).
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their determination; however, it suffers from the drawbacks esis-related proteins in Ustilago maydis. Mol. Genet. Genom.
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19. Menzies, J.G. et al. (2003). Use of inter-simple sequence
techniques facilitates rapid, sensitive and specific identifi- repeats and amplified fragment length polymorphisms to ana-
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Pseudozyma spp. cies of Ustilago. Phytopathology 93, 167.

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20. Biswas, S.K. et al. (2001). Molecular phylogenetics of the 23. Pounder, J.I. et al. (2007). Discovering potential pathogens
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79 Wallemia
Dongyou Liu
Contents
79.1 Introduction...................................................................................................................................................................... 693
79.1.3 Diagnosis.............................................................................................................................................................. 694
79.2 Methods............................................................................................................................................................................ 694
79.2.2.1 PCR for Wallemia sebi.......................................................................................................................... 695
79.3 Conclusion........................................................................................................................................................................ 696
References.................................................................................................................................................................................. 696
79.1 Introduction fertile hypha arises and fragments to form conidia. Conidia
are catenate, cubical, 1.5–2.5 μm in diameter, becoming
79.1.1 Classification, Morphology, and Biology spherical or subspherical, finely verruculose, subhyaline, and
The genus Wallemia is the only member of the family brown in mass. W. sebi conidiogenesis is characterized by
Wallemiales incertae sedis, order Wallemiales, class the basauxic development of fertile hyphae, basipetal segre-
Wallemiomycetes, phylum Basidiomycota, kingdom Fungi. gation of conidial units, and disarticulation of conidial units
As a mold lacking a known sexual state, the genus Wallemia into mostly four arthrospore-like conidia [4].
is often considered as a member of the Fungi Imperfecti. Analyses of the nuclear small subunit ribosomal RNA
Currently, the genus Wallemia (obsolete synonym: (SSU rRNA) place Wallemia into a clade together with
Hemispora) consists of three xerophilic species Wallemia Ustilaginomycetes and Hymenomycetes (Basidiomycota).
ichthyophaga, Wallemia muriae (obsolete synonym: Torula Within this clade, Wallemia occupies an isolated position
epizoa var. muriae), and Wallemia sebi (obsolete synonyms: distantly related to the Filobasidiales (reflecting their simi-
Hemispora stellata, Sporendonema sebi, and Wallemia lar parenthesome ultrastructures). Wallemia demonstrates a
ichthyophaga) and two unassigned species (Wallemia sp. rare xerotolerance among the Basidiomycota. Of the three
F53 and Wallemia sp. HLS205) [1]. These unassigned species recognized Wallemia species (W. ichthyophaga, W. sebi, and
are phenotypically distinguished by the size range of conidia W. muriae), W. sebi has the smallest conidia; W. ichthyophaga
differs from W. sebi and W. muriae in numerous nucleo-
and by the degree of their xerophily as well as genetically by
tides of the SSU and ITS rRNA. This high variation within
sequence analysis of the rRNA internal transcribed spacer
Wallemia suggests the existence of at least two cryptic genera
(ITS) regions [2]. Within the genus, Wallemia sebi is the only
not distinguishable by morphological characters [2].
species that has been implicated in cutaneous or subcutane-
Wallemia sebi is a mold with a ubiquitous distribution and
ous infections as well as allergies in humans. Collectively, the
has been isolated from agricultural and indoor environments
infections due to Wallemia sebi is referred to as “hemisporio-
(e.g., soil, hay, hypersaline water of man-made salterns and
sis” after its now obsolete synonym Hemispora stellata [3].
textiles) and foods (e.g., jams, dates, fruits, bread, cakes,
Wallemia sebi grows slowly on malt extract agar and other
salted beans, maize flour, crystalline sugar, fish, bacon, dairy
common fungal culture media and is often obscured by the
products, and salt). Wallemia is known to cause food spoilage
fast-growing fungi. Use of selective media for xerophilic
[5]. Given that W. sebi conidia are rough-surfaced spheres of
fungi enhances the growth and recovery of W. sebi from
1.5–2.5 μm in diameter, they can reach the respiratory bron-
environmental and clinical specimens. Wallemia sebi colo-
chioles when inhaled [6].
nies reach a diameter of 2–4 mm after 2 weeks at 25°C and
are elevated, irregular in shape, and orange–brown to black-
ish brown in color. Smooth and subhyaline conidiophores
are slender, cylindrical, and usually unbranched, and show Being a mold with common presence in all environments,
a swollen upper part from which a cylindrical, verrucose Wallemia sebi has been shown as a causative agent for
693

human cutaneous or subcutaneous infections. In addition, exacerbated by the fact that W. sebi tends to display a slower
airborne W. sebi spores have been implicated in human aller- growth than other molds and may be discarded as contami-
gies, particularly bronchial asthma [6,7]. Some asthmatic nants without detailed investigation.
individuals often show immediate type hypersensitivity to Molecular techniques are independent of the morphologi-
W. sebi [8,9]. The organism has been identified as playing a cal and biochemical characteristics of W. sebi, and offer a
role in farmer’s lung disease (FLD) (together with Eurotium promising alternative to the conventional detection methods,
amstelodami and Absidia corymbifera), with symptoms of with an added benefit of detecting the presence of microor-
dyspnea, cough, tiredness, headaches, occasional fever/night ganisms in a sample regardless of their culturability. PCR
sweats, and general feeling of sickness [4,10–13]. FLD tends amplification and sequencing analysis of the 18S and 28S
to occur in cattle-rearing areas, during the cold and rainy rRNA genes and their internal transcribed spacer (ITS)
during the indoor feeding season (winter) [13]. regions enable rapid and precise determination of W. sebi
In a case of Wallemia sebi-related subcutaneous phaeo- and other fungi [16–18]. The recent development of W. sebi-
hyphomycosis, a 43-year-old woman in northern India pre- specific PCR targeting 18S rRNA gene sequence allows sen-
sented with a nonhealing ulcer, which had a ragged margin sitive amplification of W. sebi from composite environmental
and was covered with slough, on the dorsum of left foot. samples [19]. Application of this PCR revealed a high con-
Having started 8 months earlier as an itchy papule that grad- centration of W. sebi spores in aerosol and surface samples
ually progressed to its present size (8 × 6 cm), the lesion was from farms handling hay and grain and from cow barns,
erythematous at its base, mildly warm (to touch), and mini- highlighting the potential role of this fungus in FLD [19].
mally tender. Histopathological examination of lesion biopsy
using Grocott stain revealed septate hyphae. A portion of
the biopsy sample (minced into tiny pieces) was cultured 79.2 Methods
on Sabouraud dextrose agar (SDA; Difco) containing chlor-
amphenicol (0.05 mg/mL) and SDA with chloramphenicol
and cycloheximide (0.5 mg/mL). This led to the isolation of Smears of scrapings from the lesion are stained with fungal
Wallemia sebi [14]. stains and examined under microscope for mycotic elements.
Wallemia sebi produces a toxic metabolite, walleminol A, Air samples are collected by 25 mm diameter polycarbonate
which is a tricyclic dihydroxy compound, with structural fea- filters with a pore size of 0.4 μm (Isopore; Millipore), which
tures characteristic of a sesquiterpene possessing an isolated is mounted in a 25 mm cassette IOM sampler (SKC Inc.). Air
double bond. The minimum inhibitory dose of walleminol A is drawn through the filter with an Aircheck Sampler model
in the bioassays is approximately 50 mg/mL, which is com- 224-PCXR7 (SKC Inc.). After sampling, 1.5 mL of suspen-
parable with a number of mycotoxins such as citrinin and sion buffer (50 mM Tris–HCl pH 7.5, 50 mM ethylenediami-
penicillic acid [15]. netetraacetic acid [EDTA], 2% sodium dodecyl sulfate, 1%
Triton-100) is added to each sampling cassette. The cassettes
are shaken on a shaker for 10 min to suspend the particles.
79.1.3 Diagnosis
From each suspension, a 500 μL aliquot is serially diluted in
Wallemia sebi is a dematiaceous anamorphic fungus that is 0.05% Tween 80. Colony counting is performed by spread-
a causative agent of subcutaneous phaeohyphomycosis and ing 100 μL of each dilution onto DG18 agar plates in dupli-
FLD. Phaeohyphomycosis is an infectious disease of skin, cate and incubating at room temperature (22°C) for 14 days.
subcutaneous tissues, and internal organs due to dematia- Another 750 μL of the particle suspension is used for DNA
ceous (melanized) fungi that produce pigmented hyphae and/ extraction with a Qiagen kit; and 1:100 dilution of the eluate
or yeast-like cells in culture and frequently in the infected is used for PCR testing [16,19].
tissue. Besides Wallemia sebi, other dematiaceous fungi Alternatively, 1 cm2 of fungal material is transferred to
(e.g., Alternaria, Bipolaris, Curvularia, Cladophialophora, a 2 mL Eppendorf tube containing a 2:1 (wt/wt) mixture of
Cladosporium, Exophiala, Exserohilum, Phaeoacremonium, silica gel and Celite (silica gel H, Merck 7736/Kieselguhr
or Phialophora) are also capable of causing phaeohyphomy- Celite 545; Machery) and 300 μL of TES buffer (2 g Tris
cosis. Thus, it is important to differentiate Wallemia sebi [hydroxymethyl]-aminomethane, 0.38 g Na-EDTA, and
from other dematiaceous fungal species in order to imple- 2 g sodium dodecyl sulfate in 80 mL of ultrapure water
ment appropriate treatment strategies. [pH 8]). The fungal material is ground with a micropestle for
Conventional methods for the identification of W. sebi 1–2 min. The volume is adjusted by adding 200 μL of TES
are dependent on observation of mycotic elements in clini- buffer. After vigorous shaking and the addition of 10 μL of
cal samples and cultured isolates with the help of a variety a 10 mg/mL concentration of proteinase K to the tube, the
of stains such as KOH, lactophenol, calcofluor white, peri- mixture is incubated at 65°C for 10 min. With the addition
odic acid-Schiff (PAS), Gomori methenamine silver (GMS), of 140 μL of 5 M NaCl solution, the mixture is combined
etc. Since the macroscopic and microscopic features of with 1/10 volume (∼65 μL) of cetyltrimethylammonium bro-
W. sebi demonstrate close resemblances to those of dema- mide (CTAB) buffer 10%, followed by incubation for another
tiaceous fungi, it is technically challenging to make correct 30 min at 65°C. One volume (∼700 μL) of chloroform–
calls on the basis of phenotypic criteria alone. This is further isoamyl alcohol (vol/vol = 24/L) is added and mixed by

Wallemia 695
inversion. After incubation for 30 min at 0°C (on ice water) in temperature to 95°C at a rate of 0.5°C/10 s with continu-
and centrifugation at 14,000 rpm at 4°C for 10 min, the top ous acquisition of fluorescence decline. Melt-curve analysis
layer is transferred to a clean Eppendorf tube. The sample is is used to observe melting characteristics of the amplicon and
added with 225 μL of 5 M NH4-acetate and incubated for at to determine the presence of the specific product.
least 30 min (on ice water) and centrifuged. The supernatant Note. Without other background DNA, 4.2 × 10 −4 ng of
is transferred to a clean sterile Eppendorf tube and mixed W. sebi genomic DNA could be detected with either of the
with a 0.55 volume (∼510 μL) of ice-cold isopropanol. After primer sets Wall-SYB4/6 or Wall-SYB7/8 in conventional
centrifugation for 7 min at 14,000 rpm and 4°C (or room tem- PCR. When W. sebi DNA was mixed with DNA from 10
perature), the supernatant is decanted. The pellet is washed other fungi, 2.1 × 10 −4 ng of W. sebi DNA could be detected
with ice-cold 70% ethanol twice and dried by using a vacuum against a background of 11.2 ng of unrelated fungal DNA.
dryer. The powder is resuspended in 48.5 μL of Tris-EDTA Assuming one fungal genome is ca. 4.0 × 10 −5 ng of DNA,
buffer with 1.5 μL of 10 mg of RNase/mL, incubated at 37°C the conventional PCR could potentially detect 5–10 fungal
for 15–30 min, and stored at −20°C until use [20]. spores in a reaction. In the real-time PCR, all isolates of
W. sebi gave strong positive fluorescent signals after 12–20
79.2.2 Detection Procedures cycles, while the other 36 fungal DNAs produced very faint
signals only after 36 cycles that were identified by melt-curve
79.2.2.1 PCR for Wallemia sebi
analysis as primer dimers [19].
Zeng et al. [19] designed two pairs of primers from the nuclear
18S rRNA sequence (GenBank accession number AF548107) 79.2.2.2 P an-Fungal Real-Time PCR and
for identification of Wallemia sebi. These primers displayed Sequencing Analysis
a mismatch of >25% to any other fungal sequences in the
GenBank database. While primer pair Wall-SYB4/6 ampli-
fies a 371 bp fragment, primer pair Wall-SYB7/8 generates a
328 bp fragment from W. sebi DNA (Table 79.1).
Procedure reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity
For conventional PCR detection, PCR mixture (25 μL) is
composed of 10 pmol of each primer (Wall-SYB4/6 or Wall- Procedure
SYB7/8), 0.75 U of Taq DNA polymerase (Invitrogen Life
Technologies), 200 μM of each deoxynucleoside triphosphate, 1. PCR mixture is composed of 1× Lightcycler FastStart
1.5 mM MgCl2, and 1–5 ng of template DNA. Amplification DNA Master Hybridization Probes mixture (Roche
is conducted with an initial 94°C for 3 min; 30 cycles at 94°C Applied Science) (containing deoxynucleoside tri-
for 30 s, 54°C for 30 s, and 72°C for 30 s; and a final 72°C for phosphates, FastStart Taq DNA polymerase, and
3 min. PCR products (3 μL) are analyzed by electrophoresis 1 mM MgCl2, additional MgCl2 is added to a final
on 1.4% agarose gels in 1× Tris-acetate-EDTA buffer. A 1 kb concentration of 4.6 mM), 0.4 μM each of ITS1 for-
Plus ladder (Invitrogen Life Technologies) is used as a DNA ward and ITS4 reverse primers, 1× SYBR green
size standard. The gels are stained with ethidium bromide (Molecular Probes), and 3 μL template DNA.
and visualized under UV light. 2. Thermal cycling parameters include 95°C for
For real-time PCR detection, PCR mixture (25 μL) is made 10 min; 50 cycles at 95°C for 5 s, 60°C for 20 s, and
up of 10 pmol of each primer (Wall-SYB4/6 or Wall-SYB7/8), 76°C for 30 s; and a final extension at 72°C for 2 min.
12.5 μL of iQ SYBR Green Supermix with an iCycler iQ real- 3. The quality of the amplicon is determined using the
time PCR detection system (Bio-Rad), and 1–5 ng of template derivative of the melt analysis curve (55°C–99°C,
DNA. Amplification is performed with an initial 95°C for 45 s hold at 55°C, 5 s/°C) using the RotorGene 3000
3 min followed by 40 cycles at 95°C for 10 s and 60°C for (Corbett Robotics, Inc.).
60 s (for Wall-SYB4/6) or 30 s (for Wall-SYB7/8). A melt- 4. The amplified product is purified for bidirectional
curve analysis is conducted immediately after amplification sequencing using ExoSAP-IT (USB Corp.). Five
at 95°C for 60 s, cooling to 60°C for 60 s, and a slow rise microliters of Big Dye Terminator Ready Reaction
TABLE 79.1
W. sebi-Specific Primers
Primer Sequence (5′–3′) Position Amplicon (bp)
Wall-SYB4 GTAGTGAACTATATTGAAGAA 621–991 371
Wall-SYB6 ATGAGTCAATAATATAACGTC
Wall-SYB7 GATTGGATGACGTTATATTAT 963–1290 328
Wall-SYB8 ACAACAAAATGTCGTACCG

Mix v. 1.1 (Applied Biosystems) is added to 4 μL of 5. Vindelov, J. and N. Arneberg. (2001). Interactions between
each primer (0.8 pmol/μL) and 3 μL of purified PCR Zygosaccharomyces mellis and Wallemia sebi in diluted
product. Cycle sequencing is performed with a 9700 molasses. Int. J. Food Microbiol. 63:73–79.
6. Hanhela, R., K. Louhelainen, and A.-L. Pasanen. (1995).
thermal cycler (ABI), using 25 cycles at 96°C for
Prevalence of microfungi in Finnish cow barns and some
10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing aspects of the occurrence of Wallemia sebi and Fusaria.
reaction products are passed through a Sephadex Scand. J. Work Environ. Health 21:223–228.
G-50 fine column to remove unincorporated dye ter- 7. Lappalainen, S. et al. (1998). Serum IgG antibodies against
minators. Purified sequencing reaction products are Wallemia sebi and Fusarium species in Finnish farmers. Ann.
run on an ABI Prism 3100 Genetic Analyzer with a Allergy Asthma Immunol. 81:585–592.
50 cm capillary array. 8. Sakamoto, T. et al. (1989a). Allergenic and antigenic activities
5. Sequences are analyzed with the SmartGene of the osmophilic fungus Wallemia sebi asthmatic patients.
Arerugi 38:352–359.
Integrated Database Network software version 9. Sakamoto, T. et al. (1989b). Studies on the osmophilic fun-
3.2.3 vr. SmartGene is a web-based software and gus Wallemia sebi as an allergen evaluated by skin prick test
database system with reference sequences derived and radioallergosorbent test. Int. Arch. Allergy Appl. Immunol.
from the National Center for Biological Information 90:368–372.
(NCBI) GenBank repository. 10. Roussel, S. et al. (2004). Microbiological evolution of hay and
relapse in patients with farmer’s lung. Occup. Environ. Med.
Note. If real time PCR instrument is available, standard PCR 61(1):e3.
11. Roussel, S. et al. (2005a). Evaluation of salting as a hay pre-
may be performed with primers ITS1 and ITS4, and the result-
servative against farmer’s lung disease agents. Ann. Agric.
ing amplicon is sequenced with the same primers. Sequence- Environ. Med. 12:217–221.
based identifications are defined by percent identity: species, 12. Roussel, S. et al. (2005b). Farmer’s lung disease and
≥99%; genus, 93%–99%; and inconclusive, ≤93%. microbiological composition of hay: A case-control study.
Mycopathologia 160(4):273–279.
13. Gbaguidi-Haore, H. et al. (2009). Multilevel analysis of the
79.3 Conclusion impact of environmental factors and agricultural practices
Wallemia sebi is a dematiaceous anamorphic fungus that on the concentration in hay of microorganisms respon-
occasionally causes subcutaneous phaeohyphomycosis and sible for farmer’s lung disease. Ann. Agric. Environ. Med.
16:219–225.
FLD in humans. Due to its presence in the environment and
14. Guarro, J. et al. (2008). Subcutaneous phaeohyphomyco-
its relatively slow growth in standard fungal culture media in sis caused by Wallemia sebi in an immunocompetent host.
comparison with other molds, Wallemia sebi may be deemed J. Clin. Microbiol. 46(3):1129–1131.
as contaminants and discarded without further analysis, lead- 15. Wood, G.M. et al. (1990). Studies on a toxic metabolite from
ing to a possible underestimation of this fungus in human the mould Wallemia. Food Addit. Contam. 7:69–77.
disease process. With the development and application of 16. Wu, Z. et al. (2003). 18S rRNA gene variation among com-
rapid, sensitive, and specific PCR assays, it is envisaged that mon airborne fungi, and development of specific oligonucle-
otide probes for the detection of fungal isolates. Appl. Environ.
a true picture of W. sebi’s involvement in human infections
Microbiol. 69:5389–5397.
will emerge in future, contributing to the improved control 17. Pounder, J.I. et al. (2007). Discovering potential pathogens
and prevention of human diseases due to this fungus. among fungi identified as nonsporulating molds. J. Clin.
Microbiol. 45(2):568–571.
18. Bagyalakshmi, R. et al. (2008). Newer emerging pathogens
References
of ocular non-sporulating molds (NSM) identified by poly-
1. The UniProt Consortium. Available at http://www.uniprot. merase chain reaction (PCR)-based DNA sequencing tech-
org/, accessed on August 1, 2010. nique targeting internal transcribed spacer (ITS) region. Curr.
2. Zalar, P. et al. (2005). Taxonomy and phylogeny of the osmo- Eye Res. 33(2):139–147.
philic genus Wallemia (Wallemiomycetes and Wallemiales, cl. 19. Zeng, Q.-Y. et al. (2004). Detection and quantification
et ord. nov.). Anton Leeuwen 87:311. of Wallemia sebi in aerosols by real-time PCR, conven-
3. Moore, R.T. (1986). A note on Wallemia sebi. Anton Leeuwen tional PCR, and cultivation. Appl. Environ. Microbiol.
52:183–187. 70:7295–7302.
4. Reboux, G. et al. (2001). Role of molds in farmer’s lung 20. Zeng, J.S. et al. (2007). Spectrum of clinically relevant
disease in Eastern France. Am. J. Respir. Crit. Care Med. Exophiala species in the United States. J. Clin. Microbiol.
163:1534–1539. 45(11):3713–3720.

Part III
Entomohpthoromycotina and Mucoromyotina

80 Apophysomyces
Arunaloke Chakrabarti, Shiv Sekhar Chatterjee, and Varghese K. George
Contents
80.1 Introduction...................................................................................................................................................................... 699
80.1.2 Ecology and Epidemiology................................................................................................................................... 700
80.1.4 Diagnosis.............................................................................................................................................................. 705
80.1.5 Treatment.............................................................................................................................................................. 705
80.2 Methods............................................................................................................................................................................ 706
80.2.2.1 PCR Identification.................................................................................................................................. 706
80.2.2.2 Restriction Fragment Length Polymorphism......................................................................................... 706
80.2.2.3 Microsatellite Fingerprinting................................................................................................................. 706
80.3 Conclusion........................................................................................................................................................................ 707
References.................................................................................................................................................................................. 707
80.1 Introduction The colonies of A. elegans on Sabouraud dextrose agar

(SDA) are fast growing, covering the agar plate over 7 days
Apophysomyces elegans, an emerging human pathogen, is at 25°C.5–12 The colonies are floccose with erect mycelial
an elegant zygomycotic fungus with an impressive bell- or growth, white when young, turning cream white to yellow
funnel-shaped distinct apophyses. on aging. However, the growth is non-pigmented on reverse.
Misra et al., in 1979, isolated this fungus from soil speci- Colonies often fail to sporulate on routine SDA medium or
mens from a mango orchard in northern India.1 The first on deficient media like corn meal agar and potato dextrose
isolation from human was reported by Ellis and Ajello in agar (PDA). The fungus grows abundantly without sporula-
1982. They isolated this fungus from the bronchial washings tion at 25°C, 37°C, and 42°C (identification point), though
of a patient from the United States.2 Two more cases were occasionally9 it may sporulate at the higher temperatures. It
reported around the same time when A. elegans was isolated can grow up to 45°C.
from traumatic lesions of limbs.3,4 However, the first compre- The sporulation is best induced by growth on 1% water
hensive report of such infection is attributed to the study by agar2 or by the modified method of Padhye and Ajello, 1988.7
Wieden et al.5 They reported A. elegans cutaneous infection According to the Ellis and Ajello recommendation, fungus
on the lower limb of a diabetic patient. The patient survived is grown on corn meal-glucose-sucrose-yeast extract agar at
after a below-knee amputation followed by amphotericin B 30°C for 7 days, and then 3 mm3 agar blocks permeated with
therapy.5 hyphae are cut aseptically and placed on the surface of steril-
ized 1% water agar in a petri dish. This plate is incubated at
30°C for 5–7 days to initiate sporulation. According to the
modified method of Padhye and Ajello, agar blocks perme-
Taxonomical position6 ated with hyphal growth of the isolate on SDA are cut asep-
tically and transferred on to a Petri plate containing sterile
Domain Eukaryota distilled water. Three drops of 10% yeast extract solution are
Kingdom Fungi added to the water and incubated at 37°C for 15 days. Most
Phylum Zygomycota isolates sporulate within 10–12 days.
Subphylum Zygomycotina On lactophenol cotton blue mount, coenocytic, broad,
Class Zygomycetes sparsely septate hyphae typical of Zygomycetes is found.
Order Mucorales Rhizoids are thin walled, subhyaline, and predominantly
Family Mucoraceae unbranched. The sporangiophores arise at right angles to the
699

Few infections are reported as a result of traumatic inocula-

tion of cactus spines, other plant materials, and insect parts,
which have presumably been contaminated with soil.15–17
Interestingly, this agent has also caused fatal infections in
marine animals.18
Disease due to A. elegans has been considered rare, but
not so rare in tropical and subtropical climates.5,19,20 Cases
have been documented from southern United States, India,
North and Western Australia, Mexico, Caribbean islands,
Columbia, and Venezuela. Of nearly 100 cases published
in the literature, a major proportion (∼60%) of cases was
reported from India (Table 80.1).9,12,19–32 Next in frequency
was the United States (∼20% cases). Like other zygomycosis,
A. elegans infection has been documented predominantly in
males, though the reason is not known.
Zygomycosis due to A. elegans is often a disease of the
Figure 80.1 Thick-walled, darkly pigmented sporangiophore immunocompetent host.15,19,33,34 Trauma is the single most
with champagne glass-shaped apophysis and sporangiospores ×200 common (72%) factor associated with cutaneous A. elegans
(insert X400). infections5,19 and can range from trivial ones to severe road
traffic accidents with multiple fractures. However, contact
aerial hyphae and often have a septate basal segment resem- with soil or plant material is observed in many cases of trau-
bling the “foot cell” seen in Aspergillus spp. The sporangio- matic A. elegans infections. Of note, two tsunami victims
phores are unbranched, straight, or curved, slightly tapering with extensive polytrauma have been reported with severe and
toward the apex, nearly 200 μm long (100–200 μm in young multifocal A. elegans cutaneous infections.35,36 Cutaneous
cultures, and 200–300 μm in mature cultures), 3–5 μm in zygomycosis due to A. elegans acquired in the hospital is
width near the apophyses, hyaline when young and turning often associated with burns, intramuscular injections,19,23 and
into sepia or brown in older cultures. A conspicuous pig- surgical wounds.21 Diabetes mellitus,9,19,33,37 corticosteroid
mented subapical thickening (10–16 μm below the apophy- therapy, hematological malignancy, myelofibrosis,31 renal
ses), which constricts the lumen of the sporangiophore, is transplantation,38 and chronic alcoholism19 are other predis-
a distinctive feature. The sporangia are multi-spored, small posing factors noted for the development of A. elegans infec-
(20–50 μm in diameter), typically pyriform in shape, hyaline tion. Extensive burn wounds (more than 25% of body surface
at first, sepia colored when mature, columellate, and strongly area) are especially associated with zygomycotic infection.
apophysate. The columella are hemispherical, and the apoph- Contact with soil during the act of extinguishing the flames is
yses distinctly funnel or bell shaped giving the sporangia a possibly the reason for acquiring A. elegans infection in few
characteristic “cocktail glass” look. The sporangiospores are patients. In one such case A. elegans has been isolated from
smooth-walled, mostly oblong, occasionally subglobose (3–4 the implicated soil.8 Rhino-orbito-cerebral (ROC) zygomy-
× 5–6 μm), and subhyaline to sepia en masse. A. elegans can cosis due to A. elegans has been recorded in farm laborers,
be easily distinguished from other members of the family and in persons with a history of intimate contact with soil.39,40
Mucoraceae, especially the morphologically similar Absidia Pregnancy has been associated on two occasions.21,41 Due to
corymbifera, by growth at 42°C, bell-shaped apophyses, the rarity of the A. elegans infection and the lack of case con-
hemispherical columella, a distinctive foot cell and the pig- trol studies, the risk factors are only empirically understood.
mented subapical thickening in the sporangiophore (Figure
80.1).
80.1.3 Pathogenesis and Clinical Features
Like other fungi, Apophysomyces elegans can be pre-
served for long periods in soil, on oil-covered slants, at In the more common scenario of traumatic A. elegans zygo-
−20°C to −70°C, in liquid nitrogen and by lyophillization.13 mycosis, route of entry is through the skin.5,8,42,43 The trau-
This fungus can even be preserved most conveniently on matic episode can range from an innocuous insect and spider
PDA slants at −70°C for a prolonged period of time. bite/sting,15,33 skin patch test,17 intramuscular injections,19
burns, blunt trauma,19 surgical wounds,12 and road traffic
accidents.20 Contact with soil and vegetation containing the
80.1.2 Ecology and Epidemiology
fungus greatly enhances the chances of A. elegans infec-
A. elegans is found abundantly in tropics and subtropical tion.8,16,21,33,43 Even inhalation of soil particles contaminated
areas. Following the first isolation in India from soil samples with A. elegans has been proposed to be the mode of entry
obtained from a mango orchard,1 it was isolated from soil and of the organism in a case of ROC A. elegans infection.39
dust of air filters in north Australia.8,14 A. elegans may pro- After gaining entry into the body, A. elegans may invade
duce infection by traumatic inoculation of the fungus in soil- blood vessels and disseminates to other organs like other
contaminated wounds, particularly after accidents and burns. zygomycotic infection.33 Contiguous spread of A. elegans

Apophysomyces
Table 80.1
Apophysomyces elegans Infections Reported in Human
Number
Reference of Patients Clinical Type Location Risk Factors Treatment Outcome
Ellis and Ajello 2 1 Pulmonary (bronchial washings) USA Information not available Information not available Information not
available
Winn et al.3 2 Cutaneous Texas, USA Trauma Amp-B, extensive debridement Survived—1, Died—1
Winn et al.4 1 Cutaneous Texas, USA Trauma Amp-B, extensive debridement Survived
Wieden et al.5 1 Cutaneous Arizona, USA Diabetes mellitus, vascular Amp-B, amputation Survived
disease, trauma
Lawrence et al.46 1 Renal Texas, USA None Amp-B, nephrectomy Survived
Newton et al.59 1 Cutaneous Mississippi Gulf Coast, Trauma Amp-B, hyperbaric oxygen, Recovered
USA extensive debridement
Cooter et al.8 1 Cutaneous Northern Territory, Australia Burn, soil contact Amp-B, amputation Survived
Huffnagle et al.61 1 Cutaneous Texas, USA Burn, soil and vegetation Amp-B, multiple amputations Survived
contact
McGinnis et al.62 1 Cutaneous (necrotizing fasciitis) — Trauma Surgical debridement Died
later spread to lungs, kidney, aortic
wall, cecum, appendix, vertebrae
Weinberg et al.33 1 Cutaneous Florida, USA Bite/sting Amp-B, resection Survived
Lakshmi et al.12 1 Cutaneous Andhra Pradesh, India Surgery (trauma) Amp-B, extensive debridement Died
Okhuysen et al.42 1 Cutaneous with contiguous spread Texas, USA Trauma Amp-B, multiple debridement, Survived
to kidney nephrostomy
Eaton et al.11 1 Cutaneous Florida, USA Trauma due to cut end of a tree Amp-B, extensive debridement Survived
branch
Meis et al.10 1 Cutaneous Aruba, Netherland Antilles, None Amp-B, amputation Survived
Caribbean Islands
Radner et al.34 1 ROC Guadalajara, Mexico Trauma (fall into a ditch) Amp-B, orbital exenteration, Survived
(treated in California, maxillectomy, multiple debridement
USA)
Naguib et al.38 1 Cutaneous Oklahoma, USA Trauma, renal transplant, Amp-B lipid complex, debridement Survived
immunosuppressive agents
Chugh et al.32 1 Renal Chandigarh, India None Amp-B, bilateral nephrectomy Death
Chakrabarti et al.23 1 Cutaneous Chandigarh, India Trauma (intra-muscular Surgical debridement Recovered
injection)
Chakrabarti et al.31 1 Craniofacial (ROC) Chandigarh, India Myelofibrosis, hyperglycemia, Amp-B, orbital exenteration, Died
thrombocytopenia extensive debridement
Mathews et al.21 1 Cutaneous Vellore, South India Pregnancy, surgery (trauma) Amp-B, extensive debridement Survived
Sedralis et al.14 1 ROC Australia None Amp-B, orbital exenteration, multiple Survived
(7) debridement, hyperbaric oxygen
Cáceres et al.43 1 Cutaneous Caracas, Venezuela Trauma Amp-B, itraconazole, debridement Survived
(continued)
701
702
Apophysomyces elegans Infections Reported in Human
Number
Reference of Patients Clinical Type Location Risk Factors Treatment Outcome
Burrell et al. 16 1 Cutaneous with contiguous spread Arizona, USA Trauma (cactus spine) Lip Amp-B, multiple (7) debridement Survived
to kidneys, spleen, diaphragm,
colon, and retroperitoneal tissues
Brown et al.40 1 ROC Arizona, USA Contact with soil (chiseling of Amp-B, bilateral debridement of Survived
soil), steroids maxillary sinuses
Kimura et al.60 2 Patient 1—cutaneous with Osaka-Sayama, Japan Trauma Amp-B, itraconazole, extensive Died
widespread involvement of viscera debridement
(kidney, spleen, tail of pancreas,
left colon, jejunum)
Patient 2—cutaneous (leg wound) Alcoholic cirrhosis, trauma, Amp-B changed to lip Amp-B, Survived
contact with soil multiple debridement, hyperbaric
oxygen
Fairley et al.63 1 ROC Brisbane, Australia Water-jet injury while cleaning Lip Amp-B, local Amp-B, orbital Survived
air-conditioner exenteration, multiple debridement (2)
Garcia-Covarrubias 1 ROC Columbia, USA Polytrauma with facial Lip Amp-B, orbital exenteration, Survived
et al.58 fractures multiple debridement, hyperbaric
oxygen, G-CSF
Chakrabarti et al.22 3 ROC Chandigarh, India Information not available Information not available Information not
available
4 Cutaneous
1 Renal
Page et al.64 2 Cutaneous Western Australia Case-1—diabetes, obesity Amp-B, wound debridement, below Death

knee-amputation
Case-2—none Lip Amp-B, followed by Amp-B, Survived
multiple debridement
Blair et al.66 1 Cutaneous Arizona, USA Trauma with plant material Lip Amp-B, debridement Survived
(patch test)
Wang et al.65 1 Cutaneous (with disseminated Texas, USA Alcoholic cirrhosis, trauma, Amp-B, extensive debridement Died
spread) renal failure
Lesueur et al.17 1 Cutaneous Arizona, USA Contact with snapdragon plant Lip Amp-B, multiple debridement Survived
(Antirrhinum majus) (patch
test), diabetes mellitus
Chakrabarti et al.19 8 ROC-1 Chandigarh, India None Amp-B, orbital exenteration, Survived
extensive debridement
Cutaneous—3 Intramuscular infections-2, Local debridement, amp-B-1, Survived-2, Left against
unknown-1 Itraconazole-1, No antifungal-1 medical advice-1
Renal—3 Alcoholism—1 Amp-B (2 patients), Surgical Survived-2, Died-1
drainage (2 patients), local
debridement (1 patient)
Apophysomyces
Cutaneous extending to kidney and Trauma Amp-B, local debridement Died
spleen—1
Carter and 1 Cutaneous Alabama, USA Trauma, pregnancy Amp-B, multiple debridement, Died
Ulusarac41 amputation
Kordy et al.45 1 Cutaneous Riyadh, Saudi Arabia Trauma Lip Amp-B, multiple debridement Survived
Ruiz et al.67 1 Cutaneous (necrotizing fasciitis) Medellin, Colombia Trauma Information not available Died
Andresen et al.36 1 Multifocal cutaneous Sri Lanka (reported from Trauma (tsunami), soil contact Lip Amp-B, debridement, hyperbaric Survived
Australia) (found in a paddy field) oxygen
Pitisuttithum 1 Brain abscess (right parietal, mixed Information not available Severely immunocompromised Lip Amp-B changed to posaconazole Died
et al.47 infection with a basidiomycetes) (not stated precisely)
Sridhara et al.30 3 ROC Chandigarh, India None Amp-B, orbital exenteration, All survived
extensive debridement
Schütz et al.39 1 ROC Kuwait Farm laborer (probable contact Amp-B, local Amp-B, orbital Died
with soil), steroid exenteration, extensive debridement
(delayed)
Liang et al.37 1 ROC Minnesota, USA Diabetes mellitus, Lip Amp-B, orbital exenteration, Survived
corticosteroid therapy maxillectomy, sphenoidectomy,
multiple debridement
Jain et al.29 5 Cutaneous (necrotising fascitis) Chandigarh, India Trauma-2 cases, burn-1 case, Information not available Information not
none-2 cases available
Suryanarayan Rao 5 ROC, 1 patient also had a Chandigarh, India None Amp-B, orbital exenteration, Survived-4, Died-1
et al.27 para-pharyngeal abscess debridement
Chakrabarti et al.28 1 ROC Chandigarh, India Information not available Information not available Information not
available
12 Cutaneous
3 Renal
1 Disseminated
Kindo et al.20 1 Cutaneous Tamil Nadu, India Trauma Amp-B, extensive debridement Died
Goyal et al.26 1 Cutaneous (cervico-facial) with Lucknow, India Diabetes mellitus, trauma Lip Amp-B, debridement Died
salivary gland involvement
Snell and 1 Cutaneous Thailand Trauma (tsunami survivor) Lip Amp-B, hyperbaric O2, Survived
Tavakoli35 debridement, split-skin graft
Ferguson et al.57 1 ROC Georgia, USA Diabetes mellitus, trauma Lip Amp-B changed to posaconazole, Survived
multiple debridement (2)
Reddy et al.9 1 Cutaneous Andhra Pradesh, India Diabetes mellitus, trauma Amp-B, itraconazole, extensive Survived
debridement
Thomas et al.24 1 Renal Tamil Nadu, India None (except hypertension) Amp-B, nephrectomy Survived
Devi et al.25 1 Cutaneous Pondicherry, India Trauma (tractor accident) Amp-B, multiple debridement (2) Survived
Saravia-Flores 1 Cutaneous Guatemala Trauma (spider bite) Amp-B, multiple debridement, Died
et al.15 amputation leg
Note: ROC, Rhino-orbito cerebral; Lip Amp-B, Liposomal Amphotericin B; Amp-B, Amphotericin B.
703
through tissue layers has also been documented on multiple infection45 and was treated empirically with parenteral antibi-
occasions.33 Rapid spread of lesions is a reason provided for otics. Fungal etiology was suspected after failure in response
repeated debridement and amputations in cases of A. elegans to antibiotic therapy and repeated debridement. Direct micros-
necrotizing fasciitis.33 copy and culture of the debrided tissue often help in diagnosis.
The overwhelming association of cutaneous zygomyco- Cutaneous zygomycosis due to A. elegans may lead to fatality
sis and A. elegans has been hypothesized on the basis of the because of delay in diagnosis and management.39
thermophillic nature of the fungus. Rapid growth at higher ROC zygomycosis: ROC zygomycosis was involved in
temperature has been suggested as a reason behind successful nearly 25% patients with A. elegans infection (Table 80.1).
proliferation of A. elegans from cutaneous to deep tissues in ROC zygomycosis due to A. elegans, unlike that due to other
a febrile host. However, proof to such hypothesis is lacking. agents like Rhizopus spp., is a disease occurring mainly in
Metabolic acidosis and local ischemia contribute to the immunocompetent individuals.27,34,37 However, myelofibrosis,
deterioration of infectious process. Along with A. elegans, systemic corticosteroid therapy, and diabetes mellitus have
Staphylococcus spp., Escherichia coli, Pseudomonas aeru- been documented in few patients.19,37 Majority of reported
ginosa, Enterobacter spp., and Enterococcus spp. are some- cases are adult males. ROC zygomycosis due to A. elegans
times isolated from the culture of the necrosed tissue.25,33,42 may manifest in two situations: (i) patients have traumatic
Around one-third of patients with A. elegans infection suc- inoculation of A. elegans in the orbit and face, which leads to
cumb to illness, though the patient may be immunocompe- orbital cellulitis and further spread into the contiguous areas
tent.39 Poor general condition of those patients due to burns, of the brain and sinuses39; and (ii) A. elegans enters through
trauma, surgical procedure, and general anesthesia possibly nasal or sinus mucosa and spreads into the orbit and brain.
contributes to such high mortality.39 No specific virulence fac- In a farm laborer, inhalation of soil particles contaminated
tor of this fungus has been identified till date. Experimental with A. elegans was suspected as the route of inoculation.39
animal studies indicate that even a 250–2300-fold lower Patients typically present with facial pain, and swelling, nasal
size inoculum of A. elegans can match the mortality due to congestion, rhinitis, frontal headache, redness and swelling
R. microsporus and Absidia corymbifera infections.44 of eyes, periorbital pain, chemosis, diplopia, impaired ocular
Cutaneous zygomycosis: A. elegans commonly involves motility, decreased vision, ptosis, facial drooping, numbness,
cutaneous and subcutaneous tissue. Of all reported cases with and fever.37,39 Tenderness, erythema, necrosis, periorbital
A. elegans infection, nearly 60% had cutaneous involvement echymoses, periorbital edema, and proptosis have been noted
(Table 80.1). Cutaneous zygomycosis may be divided into pri- during physical examination.37 Presenting signs and symp-
mary cutaneous zygomycosis and those secondarily involved toms of the disease are thus similar to the classic features of
in disseminated zygomycosis. In primary cutaneous zygo- ROC fungal infection due to other Zygomycetes.
mycosis, the route of infection is trans-cutaneous (infections Renal zygomycosis: A. elegans renal infections may pres-
at sites of injury—intravenous access, traumatic inoculation, ent as isolated renal zygomycosis and have been documented
adhesive tapes, occlusive dressing, burn wound infections, and in 12% cases (Table 80.1). Lawrence et al. described the first
postoperative surgical wound infections); while in second- case of isolated renal A. elegans infection in 198646. The
ary cutaneous zygomycosis, infection spreads to skin either infection may spread contiguously from skin and subcutane-
hematogenously (in cases of disseminated zygomycosis), or ous tissue.16,19,42 In a patient with cactus spine injury extensive
by contiguous spread from underlying diseases. The second- lesions developed involving the retroperitoneum, spleen, left
ary cutaneous zygomycosis due to A. elegans is not clearly diaphragm, colon and kidney.16 Another case of A. elegans
established. Primary cutaneous zygomycosis is by far the renal infection (of the contralateral kidney) has been described
most common manifestation of A. elegans disease. Traumatic following extensive traumatic cutaneous zygomycosis of the
implantation of the agent is often the initiator of the disease.19,43 right flank and retroperitoneal tissue.19 Most patients with iso-
Iatrogenic introduction of the fungus has been documented lated renal disease due to A. elegans have been documented
following surgery12. An erythematous lesion with raised, dark- from different centers in India.19,24,32 The typical patient is
ened, tender eschar at the center usually appears within a few a young adult male, previously healthy, with no known risk
days of inoculation.33,43 Extensive damage of soft tissue, local factors.19,24 However, chronic alcoholism and systemic corti-
ischemia, and metabolic acidosis characterize such infec- costeroid therapy as predisposing factors were found in few
tions.39,43 Sloughing, purulent discharge, and indurated mar- patients.19 Clinical features of isolated renal zygomycosis
gins are observed around wounds, which are grossly infected due to A. elegans include flank pain (unilateral or bilateral),
with multiple microorganisms. Later white cheesy material weight loss, abdominal distension, pedal edema, uremia, dys-
may come out from pockets of such lesions following antibi- uria, pyuria, passage of whitish flakes per urine, and fever.19,24
otic therapy.25 Obvious mold-like growth over the lesion is an Pulmonary zygomycosis: No case of pulmonary zygomy-
interesting finding.43 Leucocytosis with neutrophilia is com- cosis due to A. elegans had been reported except for the isola-
monly detected on peripheral blood smear examination.9,20,45 tion of the fungus from bronchial washings of a patient with
Cutaneous lesions often extend to produce extensive necro- respiratory tract infection.2
tizing fascitis5 and osteomyelitis.10,11 Contiguous spread may Brain abscess: Mixed infection due to A. elegans and a
also involve kidneys, spleen, diaphragm, and other visceral Basidiomycetes spp. has been implicated to cause fatal brain
organs. Initial diagnosis in most of those cases was bacterial abscess in a severely immunocompromised adult male patient.47

Apophysomyces 705
Other infection sites: Goyal et al. reported a patient who attempted using exo-antigens of A. elegans.50 Three specific
developed otogenic cervico-facial zygomycosis with salivary A. elegans precipitins were produced when A. elegans antisera
gland abscess due to A. elegans.26 was absorbed with S. vasiformis. A. elegans antigen had little
cross-reaction with those of S. vasiformis and A. corymbifera.
80.1.4 Diagnosis
80.1.4.1 Conventional Techniques Identification. Molecular techniques have also been employed
Early clinical suspicion of zygomycotic infection is most for the identification of A. elegans using database of 18S and
important for management. Otherwise, the disease may 28S ribosomal DNA sequences of 42 isolates of zygomyce-
disseminate to many organs resulting in mortality. Tissue tes including A. elegans.51 By using the aligned 28S rDNA
specimens are the best samples and superficial swabs are not sequences, 13 taxon-specific PCR primer pairs were designed,
recommended. Direct examination of potassium hydroxide which correctly identified those 42 isolates. However, these
wet mount of the tissue, and histopathological examination primers should be evaluated on a larger number of zygomy-
help in early diagnosis. A black necrotic eschar on nasophar- cetes before its recommendation for routine use. Chakrabarti
ynx/palate/skin may be the clue to diagnosis. Material from et al.19 analyzed the internal transcribed spacer (ITS) region
the eschar and underlying tissue should be obtained. Biopsy of eight A. elegans isolates. PCR-RFLP of the ITS regions of
or curettage samples are transported quickly to the laboratory. A. elegans ribosomal DNA using restriction enzymes MboI,
For renal and pulmonary lesions, ultrasound and computed MspI, or HinfI distinguished this fungus from other common
tomography-guided aspiration from the site of lesion may be zygomycetes like Rhizopus oryzae, Saksenaea vasiformis,
useful. Both histopathology and isolation of fungi should be Rhizomucor pusillus, Mucor cercinelloides, and Basidiobolus
attempted from the sample. Calcofluor white stain may help ranarum.
in detection of broad, sparsely septate hyphae on fluorescent Real time PCR assay was attempted for the detection
microscope. Various special fungal stains like periodic acid of zygomycetes both in culture and tissue specimens.52
schiff stain (PAS) and gomori’s methanamine silver stain In that assay a 167 bp conserved region (found in Absidia,
(GMS) may be performed on histopathology sections. These Apophysomyces, Cunninghamella, Mucor, Rhizopus, and
allow better identification of the wide (3–25 μm wide), right Saksenaea) of the multicopy cytochrome b gene was targeted.
angled branching, sparsely septate, ribbon like hyphae in tis- The identification at the Genus level could be achieved in
sue. Frequently these hyphae have focal bulbous dilatation approximately 4 h. The assay did not show any cross reaction
and non-dichotomous, irregular branching. Generally, sur- with other fungi like Candida, Aspergillus, Fusarium or even
rounding reaction is often acute inflammatory or necrotic. bacteria. Future studies in this area may help in development
The fungi are stained black with GMS and red with PAS of molecular diagnosis of A. elegans infection.
stain. Zygomycetes may not take up special fungal stain Typing. Chakrabarti et al. attempted to type 12 A. elegans
(PAS, GMS) as deeply as other filamentous fungi, but may strains using microsatellite primers. The method was not dis-
be detected in tissue sections stained with hematoxylin and criminatory, as 10 strains isolated at different times, had the
eosin stain (H&E). Hyphae invading tissue and blood vessels same pattern, though 2 strains from the United States had a
with or without thrombosis are often observed. Differential different pattern.19
diagnosis of A. elegans on histopathological examination
include other members of the class Zygomycetes, namely
80.1.5 Treatment
Rhizopus, Absidia, Mucor, Cuninghamella, Conidiobolus and
Basidiobolus spp. Hence fungal culture is recommended in The best management of A. elegans infections is presumed
all cases. The fungus grows within 4–6 days when adequate to be aggressive surgical debridement combined with anti-
samples are inoculated on SDA. Importantly, tissue samples fungal therapy and control of the predisposing factor where
should be minced and not crushed before inoculation. The possible.53–55 Surgery is considered necessary due to massive
necrotic material may be gently teased apart with scissors in amount of tissue necrosis that may prevent entry of antifungal
a sterile Petri plate. Upon primary isolation, the fungus may agents to the site of infection. Additionally surgery is supposed
not sporulate; hence special techniques like water agar may to minimize the fungal load in the tissue.53,55,56 Extensive and
be required to induce sporulation. multiple debridements may be necessary in invasive cutane-
Serologic methods had been attempted for diagnosis ous A. elegans lesions.41 Orbital exenteration and partial facial
of zygomycosis including immunodiffusion and indirect resection are often necessary in A. elegans ROC disease.37,57
ELISA.48,49 Most of these serological studies had been done Amphotericin B (conventional or liposomal) is chosen as the
using different antigen preparations of the more common first line of therapy.44,53,55 In recent years posaconazole as a
zygomycetes namely Rhizopus arrhizus, Absidia corymbifera, substitute for amphotericin B is gaining popularity especially
and Rhizomucor pusillus. No such test has been documented as salvage therapy.47,57 However, in-vitro susceptibility testing
in A. elegans infection. against multiple A. elegans isolates has not been performed.
As the fungus fails to sporulate on routine media, exoan- Control of predisposing factor or boosting the immunity with
tigen testing may provide a simpler and more rapid method the use of cytokines may help to restore immune function.53
of identification. A simple immunodiffusion test has been Hyperbaric oxygen therapy has been used successfully in a

few cases of cutaneous A. elegans infections; though, true Each PCR mixture (50 μL) is prepared by adding the fol-
value of this approach remains uncertain.21,35,36,58–60 lowing components:
5× PCR buffer with MgCl2 10 μL

80.2 Methods Primers forward and reverse 10–30 pmol of each primer
dNTPs 10 mM
DNA polymerase 1.5 U (0.5 μL of 3 U/μL)
Whole cell DNA from each isolate is extracted as follows: Template DNA ∼500 ng
the isolate is grown on SDA plates for 5–7 days. Once luxuri- DDW up to 50 μL
ant growth is obtained, subcultures are made onto Sabouraud
dextrose broth and incubated at 37°C in a rotating shaker- PCR cycling. A common PCR program starts with an
incubator at 120 rpm for 3–5 days. The mycelial mat is recov- initial denaturation step at 95°C for 2–5 min followed by 30
ered by filtration and washed with normal saline. About cycles of denaturation at 94°C for 1 min, primer annealing at
0.2–0.3 g of the mycelial mat is placed in a clean mortar. 45°C (decided by primer Tm) for 1 min and primer extension
Liquid nitrogen is added to the mat and ground quickly with at 72°C for 1 min followed by a final extension step at 72°C
a pestle till a fine powder is obtained. The resultant powder for 5–10 min.
is then transferred to a clean sterile 1.5 mL microcentrifuge Verifying PCR amplification. About 2–5 μL of PCR prod-
tube to which 600 μL of lysis buffer (100 mM Tris HCl pH uct is run on a 1%–2% agarose gel to check for the presence of
8.0, 50 mM EDTA, 3% SDS) is added and vortexed briefly. fungal specific DNA in the sample. Molecular weight markers,
Twenty microliters proteinase K at a concentration of 20 mg/ positive and negative control samples are also run together.
mL is then added and incubated at 56°C for 1 h. Finally, The gel is visualized by staining with ethidium bromide. A
DNA is extracted using 600 μL phenol:chloroform (25:24). successful PCR amplification should display a specific band
After centrifugation (10,000 rpm for 5 min), equal volume with the expected size without nonspecific bands and smear.
of chloroform:isoamylalcohol (24:1) is added to the aqueous Some important facts. (i) The quality and concentration of
phase and mixed thoroughly. The mixture is centrifuged at DNA templates can directly affect outcome of PCR; (ii) Proper
10,000 rpm for 5 min and the aqueous phase transferred to a concentration (usually 0.1–1 μM) and appropriate primers are
microcentrifuge tube and DNA is precipitated with 1 mL iso- very critical for successful PCR. The optimal primer size is
propanol and 10 μL 3 M sodium acetate. The pellet is then usually between 18–20 bases. Both forward and reverse prim-
washed with 70% alcohol, treated with 100 μL of Tris-EDTA- ers should have melting temperature within limits of 2°C–5°C
RNase (10 mM Tris HCl pH 7.5, 1 mM EDTA, 50 μg of RNase of each other. Avoid complementary sequences between prim-
per mL) at 37°C for 2 h, re-precipitated with absolute alcohol, ers to reduce primer dimer formation. Primers with Tm higher
and finally dissolved in 100 μL of TE (10 mM Tris HCl pH 7.5, than 50°C will generally give more specific results; and (iii)
1 mM EDTA) and stored at −20°C for further use. Magnesium concentration is critical to the success rate of PCR
amplification. It may affect DNA polymerase activity. Excess
magnesium results in accumulation of nonspecific products.
80.2.2 Detection Procedures Desirable magnesium concentration is at 0.5–2 mM.
80.2.2.1 PCR Identification
80.2.2.2 Restriction Fragment Length Polymorphism
After confirming the purity of the DNA obtained by gel elec-
trophoresis, the desired genes are amplified by PCR. The technique may be used to detect macroevolutionary
Primers. At first the ITS region is amplified in order to changes in genomes. After PCR, the ITS amplified products
carry out any further analysis of the DNA. The ITS region has are subjected to digestion using different restriction enzymes.
been used as an important tool for phylogenetic and epidemio- For A. elegans, restriction enzymes like MboI, MspI, HinfI,
logical studies because of its moderate rate of evolution. The BamHI, and BglII have been used in our laboratory. The
ITS primers are designed from the conserved regions of the restriction mixture (30 μL), which contains 3 μL of 10× buf-
18S and 28S rRNA genes, respectively. The ITS regions are fer, 1 μL (10–12 U) of restriction enzymes, 10 μL of amplified
amplified with two primers namely the ITS1 (forward) and the product and 16 μL of DDW is incubated at 37°C for 3 h. After
ITS4 (reverse): pITS1F (5′-TCCGTAGGTGAACCTGCGC-3′) digestion, 15 μL of each digested product is electrophoresed
and pITS4R (5′-TCCTCCGCTTATTGATATGC-3′). in a 1.5%–2.5% agarose gel for 30 min to 2 h depending on
PCR setup. The total volume per reaction mixture is usu- product size. The bands are visualized by ethidium bromide
ally 10, 25 or 50 μL. All the reaction components can be staining for 1 h in the dark and destaining in 1× TAE (Tris-
mixed together in a 0.5 mL PCR tube in any sequence except acetate-EDTA) buffer for 1 h.
for DNA polymerase, which should be added at the end. It
is recommended to mix all the components just before PCR 80.2.2.3 Microsatellite Fingerprinting
cycling. Positive control (DNA from known A. elegans isolate) High discriminatory value and reproducibility of microsatel-
and negative control (sterile double-distilled water [DDW]) lite fingerprinting technique makes it a much better typing
must always be tested in each run. method than RFLP. For A. elegans, a single primer, pMS1

Apophysomyces 707
([GTG]5 or pMS2 [GAC]5) may be used. All components of 7. Padhye, A.A. and Ajello, L., Simple method of inducing sporu-
PCR mixture are the same as mentioned previously for ITS lation by Apophysomyces elegans and Saksenaea vasiformis, J.
amplification. The cycling parameters used for microsatellite Clin. Microbiol., 26, 1861, 1988.
8. Cooter, R.D. et al., Burn wound zygomycosis caused by
fingerprinting are; initial denaturation for 5 min at 94°C, fol-
Apophysomyces elegans, J. Clin. Microbiol., 28, 2151, 1990.
lowed by 40 cycles of 1 min at 94°C, 2 min at 45°C (for pMS1) 9. Reddy, I.S. et al., Primary cutaneous mucormycosis (zygomy-
or 55°C (for pMS2), and 3 min at 72°C, final extension for cosis) caused by Apophysomyces elegans, Indian J. Dermatol.
10 min at 72°C and cooling at 4°C. The amplified product is Venereol. Leprol., 74, 367, 2008.
then separated by gel electrophoresis and analyzed. 10. Meis, J.F. et al., Severe osteomyelitis due to the zygomycete
11. Eaton, M.E. et al., Osteomyelitis of the sternum caused
80.3 Conclusion by Apophysomyces elegans. J. Clin. Microbiol., 32, 2827,
Apophysomyces elegans, an emerging pathogen belonging 1994.
12. Lakshmi, V. et al., Zygomycotic necrotizing fasciitis caused by
to class Zygomycetes and the order Mucorales, is a relatively
newer agent in this order, being isolated from soil, dust, and 13. Pasarell, L. and McGinnis, M.R., Viability of fungal cul-
vegetation matter in tropical and subtropical countries. By far, tures maintained at −70°C, J. Clin. Microbiol., 30, 1000,
the agent most commonly causes cutaneous zygomycosis in 1992.
apparently healthy hosts and traumatic implantation into skin 14. Sedralis, T., Krishnan, S., and Holland J., Martini glass mucor-
following contact with soil and vegetation containing the fun- mycosis: Apophysomyces elegans infection in an immunocom-
gus is considered the route of entry. However, in recent years petent host, Aust. J. Otolaryngol., 2, 600, 1997.
rhino-cerebral and renal zygomycosis due to A. elegans are 15. Saravia-Flores, M., Guaran, D.M., and Argueta, V., Invasive
cutaneous infection caused by Apophysomyces elegans associ-
increasingly reported in immunocompetent or immunosup-
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pressed individuals. The mode of entry in those infections is 16. Burrell, S.R. et al., Apophysomyces elegans infection associ-
not clearly understood. Majority of the cases with A. elegans ated with cactus spine injury in an immunocompetent pediatric
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excellent mycelial growth is seen on standard culture media, dolphins (Tursiops truncatus), a killer whale (Orcinus orca),
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48. Jones, K.W. and Kaufman, L., Development and evaluation of 67. Ruiz, C.E. et al., Necrotizing fasciitis in an immunocompe-
an immunodiffusion test for diagnosis of systemic zygomyco- tent patient caused by Apophysomyces elegans, Biomedica, 24,
sis: Preliminary report, J. Clin. Microbiol., 7, 97, 1978. 239, 2004.

81 Cokeromyces
Dongyou Liu
Contents
81.1 Introduction...................................................................................................................................................................... 709
81.1.3 Laboratory Diagnosis............................................................................................................................................710
81.2 Methods.............................................................................................................................................................................711
81.2.2.1 PCR for Cokeromyces recurvatus..........................................................................................................711
81.2.2.2 Sequence Analysis of ITS Regions.........................................................................................................711
81.3 Conclusion........................................................................................................................................................................ 712
References.................................................................................................................................................................................. 712
81.1 Introduction and an unassigned species Cokeromyces sp. SW078 within

the genus Cokeromyces. A former species Cokeromyces
According to the most recent classification system, the poitrasii is now known as Benjaminiella poitrasii.
fungi kingdom is divided into seven phyla: Ascomycota Cokeromyces recurvatus grows slowly at 30°C and more
(Dikarya), Basidiomycota (Dikarya), Blastocladiomycota, rapidly at 37°C. Colonies measure 15–21 mm in diameter at
Chytridiomycota, Glomeromycota (containing part of 5 days on potato flakes and Sabouraud dextrose agars at 25°C
the former phylum Zygomycota), Microsporidia, and and are yeast-like, pasty in texture, containing large, spheri-
Neocallimastigomycota as well as Fungi incertae sedis (con- cal, thick-walled cells (of 15–30 μm in size) bearing one to
taining the remainder of the former phylum Zygomycota) [1]. multiple buds. On potato dextrose agar (PDA) slide cultures
Fungi incertae sedis, covering remaining molds of at 30°C for 10 days, the fungus is mold-like, producing hya-
the displaced phylum Zygomycota (commonly known line, smooth-walled, unbranched, broad sporangiophores
as “sugar” and “pin” molds), is separated into four sub- (usually 300–500 μm long), each terminating in a spherical
phyla: Entomophthoromycotina, Kickxellomycotina, vesicle (13–31 μm in diameter). Elongate, recurved stalks
Mucoromycotina, and Zoopagomycotina, with most or pedicels (each bearing globose sporangiola of 10–12 μm
opportunistic human and animal pathogens belonging to in diameter) arise from a vesicle at the apex of the sporan-
Entomophthoromycotina and Mucoromycotina [1–3]. giophore. Each sporangiolum contains 12–20 oval, smooth-
While the subphylum Entomophthoromycotina con- walled sporangiospores. Being homothallic, C. recurvatus
sists of a single-order Entomophthorales, which is sepa- requires only one mating type for sexual reproduction and
rated into four families: Ancylistaceae, Basidiobolaceae, zygospores are generated within a single isolate. Spherical,
Entomophthoraceae, and Neozygitaceae; the subphylum rough-walled, dark brown–black zygospores (33.5–54.5 μm
Mucoromycotina contains three orders: Endogonales, in diameter) borne on opposed suspensors from the hyphae
Mortierellales, and Mucorales, in addition to some unclassi- are visible at higher temperatures (up to 37°C) under anaero-
fied Mucoromycotina [1]. bic candle jar incubation [5–9].
Being a dimorphic fungus, C. recurvatus grows as a felt-
like (mold-like) mat on animal excreta in nature and appears
as large yeast-like, budding cells in vaginal, urine, and stool
Previously assigned to the family Choanephoraceae, the genus specimens, resembling morphologically to the yeast phase
Cokeromyces is classified now in the family Thamnidiaceae, of Paracoccidioides brasiliensis as well as a spherule of
order Mucorales, subphylum Mucoromycotina. The family Coccidioides immitis [10].
Thamnidiaceae is made up of 12 genera: Backusella, As a sporangiola-forming species of zygomycete,
Cokeromyces, Dicranophora, Ellisomyces, Fennellomyces, C. recurvatus is commonly present in soil contaminated with
Helicostylum, Kirkomyces, Phascolomyces, Pirella, rodent, lagomorph, or lizard dung in parts of Mexico and
Thamnidium, Thamnostylum, and Zychaea [4]. Currently, such U.S. states as California, Arizona, Illinois, Michigan,
there are one recognized species Cokeromyces recurvatus Florida, and Texas. The fungus has been identified from the
709

colon and genitourinary tract of healthy individuals as well after 2 days of incubation. Examination of biopsy specimen
as patients in the United States only. It is possible that human using Gomori’s methenamine silver and periodic acid-Schiff
clinical cases of C. recurvatus infection may arise from pre- (PAS) stains showed no evidence of tissue infection. The
vious colonization of the involved sites (stomach, genitouri- patient went into cardiopulmonary arrest and died on day 32
nary tract, or colon). of hospitalization [14].
Significantly, in most of the clinical cases reported, no
obvious tissue invasions by C. recurvatus have been noted.
It is therefore possible that disease caused by this fungus is
Cokeromyces recurvatus, a sporangiola-forming dimorphic mediated by one or more of its extracellular mycotoxins.
fungus, is a rare cause of urogenital and gastrointestinal Underlying conditions (e.g., pregnancy, diabetes mellitus,
infections, cystitis, peritonitis, and myositis in humans, both alcoholism, and immunosuppression) often increase suscep-
immunocompetent and immunosuppressed (with underly- tibility to C. recurvatus infection.
ing ulcer, diabetes mellitus, myeloma, and cancer as well as C. recurvatus infection may be treated with a 10 day
chronic alcoholism) [6,11]. course of nystatin or a 2-week course of terconazole [13,15].
C. recurvatus was implicated in a case of hemorrhagic Its response to amphotericin B is variable.
cystitis involving a 72-year-old male. Large yeast-like cells
with one to multiple buds, similar in appearance to P. brasil-
iensis, were detected in the urine specimens. However, the
fungus was absent in the bladder wall. The patient responded Because Cokeromyces recurvatus is commonly present in
to amphotericin B therapy [12]. C. recurvatus was also the environment, and clinical manifestations of C. recurvatus
responsible for causing severe watery diarrhea in a patient infection and other zygomycosis are indistinguishable, a
under post-transplantation immunosuppression therapy. definitive diagnosis of infection due to C. recurvatus requires
Cokeromyces was detected in the stool and intestinal biopsy a combination of mycological, histopathological, and clini-
specimens. The patient recovered from the infection fully cal evidence. Although culture isolation of the fungus cul-
with high-dose oral nystatin [13]. Similarly, a 66-year-old ture is useful for confirmation of C. recurvatus infection in
man presented with rash, nausea, and vomiting after under- patients with diabetes or neutropenia, direct observation of
going bone marrow transplantation for chronic myelogenous the organism in clinical specimens gives additional weight
leukemia. The patient succumbed to the disease shortly after to a positive culture result. Repeated positive fungal cultures
hospital admission. Culture of sputum collected on the day and a response to specific antifungal therapy in the absence
of death showed heavy growth of C. recurvatus 6 days later. of concurrent conditions also provide support that the organ-
Microscopic examination of the lungs uncovered numerous ism is the causative agent of the disease.
thick-walled, non-budding spherules of 40–80 μm in size [10]. C. recurvatus is a dimorphic zygomycete. In clinical
In a separate report, a 64-year-old man with a 10-year specimens stained with calcofluor white or lactophenol cot-
history of peptic ulcer disease and alcohol abuse presented ton blue, C. recurvatus appears as a large (30–90 μm), thick-
with severe abdominal pain, pulmonary edema, and bilat- walled yeast often surrounded by a crown of smaller yeast
eral pleural effusions. Culture of peritoneal and pleural buds (so called “mariner’s wheel”), which is morphologically
fluid specimens on chocolate and blood agars at 35°C grew similar to the yeast phase of Paracoccidioides brasiliensis
molds. A tease mount of the fungus with one drop of phenol and a spherule of Coccidioides immitis. However, P. brasil-
revealed sporangia with nonseptate hyphae (typical of zygo- iensis and C. immitis often show early evidence of internal
mycetes). A scotch tape preparation with lactophenol ani- cleavage or contain endospores [15]. While hematoxylin and
line blue showed no rhizoids. Colonies on potato flakes and eosin (H&E) can be used to observe C. recurvatus hyphae
Sabouraud dextrose agar subcultures were tan, thin, and radi- in tissue sections, the Grocott technique gives high contrast
ally wrinkled, with gray central areas due to the production with minimal background.
of sporangiophores, and turning brown later with the produc- In standard fungal culture media, C. recurvatus may be
tion of zygospores. Microscopically, the isolate showed dis- both yeast-like and mold-like. The yeast-like colonies (of
tinct sporangiophores forming terminal, enlarged vesicles, 3–5 mm in size) grow on brain heart infusion agar with 5%
from which sporangiolar stalks (pedicels) arose, elongated sheep blood at 37°C in 2 days and are pasty, gray, and slightly
and recurved toward the vesicle, and subsequently produced wrinkled, with large, spherical, thick-walled cells bearing
smooth-walled, ovoid to elliptical sporangiospores. Sexual multiple buds. The mold-like colonies (of 15–30 mm in size)
zygospores, which developed between opposing suspensory appear in 3–5 days at room temperature, containing branch-
cells on the same hyphae, were spherical, rough edged, and ing, broad, generally nonseptate hyphae (5–15 μm in diam-
dark brownish black in color. Large, spherical yeast-like cells eter) and thick-walled, dark brown–black zygospores borne
(up to 20 mm in diameter) with sparse, but distinct, budding on suspensors. After 10 days of incubation at 30°C, hyaline,
developed at 35°C on brain heart infusion agar containing smooth-walled, broad sporangiophores with terminating vesi-
5% sheep blood, and colonies appeared pasty in texture, gray, cles, elongated, recurved stalks or pedicels, globose sporangi-
and slightly wrinkled and measured 3–5 mm in diameter ola and sporangiospores as well as zygospores are visible [6].

Cokeromyces 711
Use of molecular techniques such as PCR and sequenc- Procedure

ing allows early and accurate diagnosis of C. recurvatus
infection in comparison with conventional methods. The 1. PCR mixture (50 μL) is made up of 0.225 mM of
most common gene targets for fungal identification com- each deoxynucleotide, 25 pmol of each primer,
prise 18S and 28S ribosomal RNA (rRNA) internal tran- 50 mM KCl, 10 mM Tris–Cl (pH 8.4), 2.5 mM
scribed spacer (ITS) as well as other conserved gene MgCl2, 0.1 mg of gelatin/mL, and 1.25 U of
regions [16–19]. By using the aligned 28S rRNA sequences, AmpliTaq polymerase (Perkin-Elmer), and approxi-
C. recurvatus-specific PCR primers were designed, facili- mately 10–20 ng of genomic DNA.
tating its rapid and precise determination [20]. 2. Amplification is performed with an initial 94°C for
2 min; 40 cycles at 94°C for 30 s, 60°C for 30 s, and
72°C for 90 s; a final 72°C for 10 min.
81.2 Methods
3. PCR product (434 bp) is fractionated in agarose
81.2.1 Sample Preparation gel, stained with ethidium bromide, and visualized
under UV light.
For direct microscopic examination, portions of clinical
specimens are stained with KOH, calcofluor white, or lac-
81.2.2.2 Sequence Analysis of ITS Regions
tophenol blue; and tissue sections are stained with PAS and
other fungal stains. Standard fungal media containing anti- Fungus-specific universal primers ITS1 (5′-TCCGTAGGTG
biotics (e.g., malt agar, Sabouraud dextrose agar, Sabouraud AACCTGCGG-3′) and ITS4 (5′-GCATATCAATAAGCGG
glucose agar, and PDA) are inoculated with portions of the AGGA-3′) [21] can be used to amplify the ITS1 and ITS2
samples and incubated at 25°C and 30°C for a few days. Both regions. Subsequent sequencing analysis of the resulting
yeast-like and mold-like colonies are examined microscopi- amplicon facilitates identification of a range of fungal patho-
cally by wet mount with lactophenol aniline blue stain for gens including Cokeromyces recurvatus [19].
characteristic features of C. recurvatus [6]. Procedure
Mycelium for DNA isolation is grown in YM broth (0.3%
yeast extract, 0.3% malt extract, 0.5% peptone, 2% dextrose; 1. PCR mixture (50 μL) is made up of 10 mM Tris–
Difco) at room temperature for 2–5 days. DNA is isolated HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM
from lyophilized mycelium according to the CTAB (hexacet- of each deoxynucleoside triphosphate, 1.2 U of Taq
yltrimethylammonium bromide; Sigma) miniprep protocol. DNA polymerase, 0.4 μM of each primer (ITS1/
Namely, approximately 50 mg of pulverized mycelium is ITS4), 2 μL (1–5 ng) of DNA template. A negative
resuspended in 700 μL of CTAB extraction buffer (100 mM control is included in each run by replacing the tem-
Tris–Cl pH 8.4, 1.4 M NaCl, 25 mM EDTA, 2% CTAB) and plate DNA with sterile water in the PCR mixture.
vortexed for 10 s. Following extraction, an equal volume of 2. Amplification is conducted with an initial 94°C for
chloroform is added to each tube, vortexed for 5 s, and then 3 min; 30 cycles at 94°C, 60°C, and 72°C for 1 min
spun for 10 min at 12,300 × g in a microcentrifuge. A 500 μL each; and a final 72°C for 3 min.
portion of the upper phase is removed to a new 1.5 mL tube, 3. After checking the PCR products on 1.5% aga-
and DNA is precipitated by the addition of an equal volume rose gel, the amplicons are purified using a com-
of −20°C isopropanol. After centrifugation at 12,300 × g in a mercial PCR cleanup kit. The DNA fragments are
microcentrifuge for 1 min, the supernatant is discarded and sequenced using an ABI Prism 377 automated DNA
the pellet is washed with 70% ethanol and resuspended in sequencer (Applied Biosystems) with a BigDye
200 μL of TE buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA Terminator cycle sequencing kit (version 3.1;
pH 8.0). For PCR amplifications, 8 μL of the genomic DNA Applied Biosystems). All amplicons are sequenced
stock is diluted in 1 mL of deionized water and stored at on both strands using primers ITS1 and ITS4.
−20°C until use. For PCR experiments, 25 μL of the diluted 4. The resulting sequence is compared with those avail-
genomic DNA is added to an equal volume of a 2 × PCR able at the GenBank databases using the BLAST
master mix [20]. sequence analysis tool (http://www.ncbi.nlm.nih.
gov//BLAST//) (blastn) with default settings except
81.2.2 Detection Procedures that sequences are not filtered for low complexity.
Species identification is determined from the lowest
81.2.2.1 PCR for Cokeromyces recurvatus expected value of the BLAST output.
Voigt et al. [20] designed a pair of Cokeromyces recurvatus
primers from its 28S rRNA gene for rapid identification Note. For strains producing discrepant identification
of this fungus. The primers Cr1 (5′-GTGAGAATCCC between the methods based on phenotypic characteris-
GTGAATTCAC-3′) and Cr2 (5′-CAAAGCACTCAACTAT tics and ITS sequence analysis, the D1–D2 region of the
TTCGC-3′) amplify a specific fragment of 434 bp from large-subunit RNA gene is amplified with primers NL1
C. recurvatus DNA only. (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4

(5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for 8. de Hoog GS et al., Atlas of Clinical Fungi. Baarn, the
species clarification. Netherlands: Centraalbureau voor Schimmelcultures; 2000.
9. Nielsen C et al., Isolation of Cokeromyces recurvatus, ini-
tially misidentified as Coccidioides immitis, from peritoneal
81.3 Conclusion fluid in a cat with jejunal perforation. J Vet Diagn Invest.
2005;17:372–378.
Cokeromyces recurvatus is a ubiquitous fungus that is occa- 10. Ryan LJ et al., Fatal Cokeromyces recurvatus Pneumonia:
sionally associated with human diseases, ranging from uro- Report of a case highlighting the potential for histopathologic
genital and gastrointestinal infections, cystitis to peritonitis, misdiagnosis as Coccidioides. Int J Surg Pathol. 2009.
especially in patients with underlying illnesses such as ulcer, 11. Ramani R et al., Cokeromyces recurvatus as a human patho-
diabetes mellitus, myeloma, and cancer. As the fungus does genic fungus: Case report and critical review of the published
literature. Pediatr Infect Dis J. 2000;19:155–158.
not normally invade host tissues, its pathogenicity may be
12. Axelrod P et al., Chronic cystitis due to Cokeromyces recur-
due to the effects of extracellular mycotoxins it produces. vatus: A case report. J Infect Dis. 1987;155(5):1062–1064.
As a dimorphic zygomycete, C. recurvatus often appears 13. Alvarez OA et al., Severe diarrhea due to Cokeromyces
in clinical samples as large yeast-like, thick-walled cells with recurvatus in a bone marrow transplant recipient. Am J
multiple buds (“mariner’s wheel”), which may be confused Gastroenterol. 1995;90(8):1350–1351.
with the yeast phase of Paracoccidioides brasiliensis and a 14. Munipalli B, Rinaldi MG, Greenberg SB. Cokeromyces recur-
spherule of Coccidioides immitis morphologically. In cul- vatus isolated from pleural and peritoneal fluid: Case report.
ture, C. recurvatus may be both yeast-like and mold-like. J Clin Microbiol. 1996;34(10):2601–2603.
15. Tsai TW et al., Cokeromyces recurvatus infection in a bone
Microscopic identification of this fungus is aided by its for- marrow transplant recipient. Bone Marrow Transplant.
mation of hyaline, smooth-walled, broad sporangiophores 1997;19(3):301–302.
with terminating vesicles, elongated, recurved stalks or 16. Makimura K, Murayama SY, Yamaguchi H. Detection of a
pedicels, globose sporangiola and sporangiospores as well as wide range of medically important fungi by the polymerase
zygospores. Recent application of PCR and sequencing ana chain reaction. J Med Microbiol. 1994;40:358–364.
lysis permits improved diagnosis of C. recurvatus infection. 17. Haynes KA, et al., Rapid detection and identification of
pathogenic fungi by polymerase chain reaction amplifica-
tion of large subunit ribosomal DNA. J Med Vet Mycol.
References 1995;33:319–325.
18. Sandhu GS et al., Molecular probes for diagnosis of fungal
1. Hibbett DS et al., A higher-level phylogenetic classification of infections. J Clin Microbiol. 1995;33:2913–2919.
the fungi. Mycol Res. 2007;111:509–547. 19. Leaw SN et al., Identification of medically important yeast
2. Elgart ML. Zygomycosis. Dermatol Clin. 1996;14:141–146. species by sequence analysis of the internal transcribed spacer
3. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in regions. J Clin Microbiol. 2006;44:693–699.
human disease. Clin Microbiol Rev. 2000;13:236–301. 20. Voigt K, Cigelnik E, O’donnell K. Phylogeny and PCR
4. The UniProt Consortium. Available at http://www.uniprot. identification of clinically important Zygomycetes based on
org/, accessed on August 1, 2010. nuclear ribosomal-DNA sequence data. J Clin Microbiol.
5. Kwon-Chung KJ, Bennett JE. Medical Mycology. 1999;37(12):3957–3964.
Philadelphia, PA: Lea & Febiger; 1992. 21. White TJ et al., Amplification and direct sequencing of
6. Kemna ME et al., Cokeromyces recurvatus, a mucoraceous fungal ribosomal RNA genes for phylogenetics. In: Innis
zygomycete rarely isolated in clinical laboratories. J Clin MA et al., Eds., PCR Protocols: A Guide to Methods and
Microbiol. 1994;32(3):843–845. Applications. San Diego, CA: Academic Press, Inc.; 1990,
7. Weitzman I et al., Zygospores: The last word in identification pp. 315–322.
of rare or atypical zygomycetes isolated from clinical speci-
mens. J Clin Microbiol. 1995;33:781–783.

82 Cunninghamella
Nancy L. Wengenack and D. Jane Hata
Contents
82.1 Introduction...................................................................................................................................................................... 713
82.1.1.1 Classification.......................................................................................................................................... 713
82.1.1.2 Morphology............................................................................................................................................ 713
82.1.2 Epidemiology.........................................................................................................................................................714
82.1.3 Pathogenesis.......................................................................................................................................................... 715
82.1.5 Virulence Factors...................................................................................................................................................716
82.2 Methods.............................................................................................................................................................................717
82.2.1 Conventional Techniques for Diagnosis of Cunninghamella Infection................................................................717
82.2.2 Molecular Diagnosis of Cunninghamella Infection..............................................................................................717
82.2.3 Sequence-Based Identification of Cunninghamella spp........................................................................................717
82.2.4 PCR Methods for Identification of Cunninghamella spp......................................................................................718
References.................................................................................................................................................................................. 720
82.1 Introduction Members of the Mucorales associated with human dis-

eases include Rhizopus, Mucor, Absidia, Syncephalastrum,
82.1.1 Classification and Morphology Rhizomucor, Apophysomyces, Saksenaea, Cokeromyces, and
82.1.1.1 Classification Cunninghamella. For additional details on taxonomy, the
reader is referred to the excellent review by Ribes et al.3
Cunninghamella species are ubiquitous environmental fungi
found in soil and plant materials worldwide with seven spe-
cies described.1 C. bertholletiae has been the primary spe- 82.1.1.2 Morphology
cies reported as a human pathogen, although C. echinulata Characteristic of the zygomycetes are zygospores or sex-
has also been reported in association with diseases. Only ual reproductive structures that form as a result of contact
rarely has C. elegans been reported as an agent in human between two rapidly growing hyphal branches known as
infection.2 In 1958, the first description of a disseminated zygophores. Upon contact, and subsequent formation of a
Cunninghamella infection was described in 1958.3 structure known as a gametangium, nuclear fusion and mix-
The phylum Zygomycota consists of two classes: the ing of cytoplasm occur, with formation of the zygosporan-
Trichomycetes and the Zygomycetes. Members of the gium. These sexual structures are rarely seen in the human
Trichomycetes are obligate symbiotes of arthropods. host and are generally not used as a basis of laboratory
Zygomycetes are divided into two orders: Mucorales and identification.
Entomophthorales (Ancylistaceae, Basidiobolaceae), which As a result of asexual reproduction, sporangiospores are
contain the human pathogens Basidiobolus and Conidiobolus. produced in a sack-like structure known as a sporangium.
Production of actively expelled asexual sporangioles is a Sporangiospores have been implicated as agents of transmis-
unique characteristic and distinguishes Entomophthorales sion due to their small size and ability to remain airborne for
from the Mucorales. long periods of time. Unfortunately, this characteristic may
Cunninghamella was first described by Blakeslee in 1904. also contribute to contamination within the laboratory and
As a member of the phylum Zygomycota, class Zygomycetes potential confusion in clinical diagnosis.3
in the order Mucorales, these eukaryotic molds are charac- Although most zygomycetes may typically pro-
terized by the formation of aseptate or pauciseptate, wide duce hundreds of sporangiospores within a sporangium,
hyaline hyphae (coenocytic hyphae) with branching from Cunninghamella is unique, as only a single spore is con-
45° to 90°. Wide, ribbon-like hyphae are a key feature that tained within a vesicle-like sporangiola. Single sporan-
allows microscopic distinction of the Zygomycetes from giola may appear hyaline or brownish when observed in a
other filamentous pathogenic fungi, such as Aspergillus spp. mass. The sporangiola are supported by short stalks known
713

Sporangiola
Sporangiospore
Vesicle Sterigmata
Sporangiophore
FIGURE 82.1 Microscopic morphology of Cunninghamella spp.
as sterigmata, which are mounted on swollen vesicles (15– methenamine silver (GMS), or hematoxylin–eosin (H&E)
59 μm), and may be subglobose, pyriform, or ellipsoidal. may stain zygomycetes inconsistently or weakly. This may
Vesicles are supported by a structure known as a sporangio- be due in part to the thinner hyphal walls characteristic of
phore. In Cunninghamella, sporangiophores are up to 20 μm this genus, as compared to other fungi.6 Chlamydospores
wide with single or verticillate branches supporting the sub- may be H&E or PAS positive, but stain poorly with GMS.
globose or pyriform vesicles. Lateral branches of the sporan- However, sporulation in tissue is rarely reported.4
giophores may also produce vesicles. These vesicles may also Rapid invasion of hyphal elements into vascular tissues
be ellipsoidal (3–30 μm). Unlike Rhizopus or Rhizomucor, (angioinvasion) is a characteristic of zygomycetes. Involved
rhizoids are primitive and rarely noted extending from the soft tissues are swollen and erythematous, becoming black
sporangiophores (Figure 82.1). with disease progression. Extensive thrombosis and necrosis
Macroscopic colony morphology of Cunninghamella sp. often occur, resulting in tissue infarction. Vascular stenosis
is similar to other zygomycetes. Colonies grow optimally may occur due to thromboembolic fungal dissemination.7
at 25°C–30°C; the maximal temperature tolerated is 50°C. Perineural invasion is commonly encountered, resulting
Colonies are white and cottony, becoming dark gray with time. in rhinocerebral zygomycosis. It is theorized that secondary
Colony reverse may be buff or colorless. Cunninghamella to vascular invasion, fungi may extend into the optical orbit,
grows extremely rapidly and may completely fill a culture meninges, and brain. Invasion via neural tissue may also
plate within 2–3 days. Due to the propensity of these fungi occur. The mechanisms by which vascular or neural tropism
to lift the lid off of culture dishes, sealing of culture plates is occur are not currently known, although iron uptake or physi-
recommended.4 Although some species of zygomycetes have cal characteristics of tissues may play a role.6
been reported to exhibit thermal dimorphism, this has not Pulmonary manifestations can range from solitary nodu-
been reported among Cunninghamella sp.3 Growth is most lar lesions on x-ray to lobar involvement, with progression
robust on mycological culture media such as Sabouraud dex- to wedge infarcts. Radiographic findings in zygomycosis are
trose agar (SDA) or inhibitory mold agar (IMA). Growth of nondescript and may result in initial misdiagnoses of bacte-
Cunninghamella sp. and other zygomycetes are inhibited on rial pneumonia. Darrisaw et al. reported improved survival
media containing cycloheximide (i.e., Mycosel). in Cunninghamella pulmonary infections associated with
Recovery of zygomycetes from tissues can be problematic. wedge resection or lobectomy in conjunction with ampho-
In some studies, recovery of a zygomycete by culture was only tericin B therapy.8
achieved in 50% of cases.5 The fragile reproductive structures
of the mold are easily disrupted by grinding of tissues or other
82.1.2 Epidemiology
aggressive processing procedures prior to inoculation of cul-
ture media. Use of more gentle methods, such as mincing of As with other members of zygomycetes, Cunninghamella
tissue samples will result in improved fungal recovery.3 spp. are commonly isolated from a variety of environmen-
As with other zygomycetes, Cunninghamella spp. exhibit tal sources. These origins may vary from soils, to dead
hyaline, broad ribbon-like pauciseptate hyphae. In histologi- wood, to edible fruits and vegetables. C. bertholletiae is fre-
cal preparations, hyphae are prone to twisting due to their quently isolated from Brazil nuts. Environmental isolation
thinner cell walls. The appearance of folding may be misin- of Cunninghamella sp. is noted predominantly in temperate
terpreted as hyphal septations. Hyphal branching of 45°–90° climates.
is a helpful characteristic in differentiation of zygomycetes Interestingly, C. elegans has been reported to have a
from other acutely branching fungi such as Aspergillus sp. number of biotechnological applications, ranging from chi-
Visualization in tissue is highly dependent upon appropri- tin production9 to neutralization of organic pesticides10 and
ate staining techniques. Although traditionally used, histo- aromatic hydrocarbons11 and as a microbial model of cyto-
logical stains such as periodic acid-Schiff (PAS), Gomori chrome P-450 hepatic metabolism.12

Cunninghamella 715
Disease transmission by zygomycetes has been reported the period 1995–1999 invasive infections with zygomycete
to occur via inhalation of spores, through percutaneous species (including Cunninghamella) nearly doubled as com-
exposure (needlesticks, catheter insertion), or via inges- pared to the previous 5-year period.17
tion. Acquisition of infection with Cunninghamella sp. may In a more recent study also at a large cancer center,
occur through ingestion of sporangioles carried on food. Kontoyiannis et al. also noted increased overall incidence
Transmission may also occur via percutaneous implanta- of zygomycosis among BMT recipients during a 2002–2004
tion of sporangioles secondary to trauma. Motohashi et al. study period. This was higher overall (13/834 patients;
reported a localized cutaneous infection with C. bertholle- 1.55%) as compared to 1984–1998 (10/4020 patients; 0.25%).
tiae in a patient with chronic myelogenous leukemia. In this The incidence of invasive aspergillosis (IA) had decreased
case, the lesion was associated with application of an elasti- during the 2002–2004 study period. A multivariate analy-
cized tape dressing after thoracentesis for a pleural effusion.13 sis comparing patients with zygomycosis to patients with IA
Most cases of infection due to Cunninghamella sp. are determined voriconazole prophylaxis to be a significant risk
believed to be acquired via inhalation, resulting in sinus or factor in the development of zygomycosis vs. IA. It is of note
pulmonary disease. Several case studies report the predomi- that zygomycetes are inherently resistant to most expanded-
nance of pulmonary infections due to Cunninghamella sp.8,14 spectrum azole drugs, including voriconazole. These authors
The small size of sporangiospores (approximately 6 μm) raised the concern that prophylaxis with agents that lack anti-
allow for ease of environmental dissemination and persis- fungal activities against non-Aspergillus molds (i.e., zygo-
tence in currents of air. mycetes), could lead to increased incidence of breakthrough
In one of the largest reviews of epidemiology and out- infections in highly susceptible transplant populations.15
comes in zygomycosis, Roden et al. report infection specifi-
cally with Cunninghamella sp. as a significant risk factor
82.1.3 Pathogenesis
for mortality among patients with a confirmed diagnosis of
zygomycosis.5 In this study of 465 patients, infection with Zygomycosis refers to invasive infection caused by any of
C. bertholletiae was associated with the greatest mortal- the zygomycetes known to be associated with human dis-
ity rate (76%), as compared to other zygomycete genera. eases: Rhizopus, Mucor, Absidia, Rhizomucor, Saksenaea,
Mortality due to C. bertholletiae was second only to Rhizopus Apophysomyces, Cokeromyces, and Cunninghamella. As
stolonifer among patients studied. Cunninghamella sp. was previously discussed, the immune status of the host plays a
also considered an independent risk factor for development crucial role in pathogenesis and progression of disease due to
of pulmonary zygomycosis (odds ratio = 7.75).5 Cunninghamella sp.
Proven infections with Cunninghamella sp., and indeed Inflammatory responses in tissue are variable but may
with any of the zygomycetes, are highly dependent upon the commonly consist entirely of neutrophils. In a study by Frater
immunological status of the host. Hematological malignancy et al., mixtures of granulomas and neutrophils were noted.
is most commonly reported as an underlying disease associ- Primary granulomatous reactions have been described, or,
ated with Cunninghamella sp. infections.8 Other associated in some cases, no tissue inflammatory reactions at all have
conditions may include bone marrow transplant (BMT), dia- been reported.6 Patients who develop functional deficiencies
betes mellitus, graft vs. host disease (GVHD), and malnu- or numerical deficiencies in monocytes, macrophages, or
trition.15 Generalized disseminated zygomycosis was noted neutrophils are at increased risk of development of zygomy-
most frequently in patients undergoing deferoxamine ther- cosis. Recovery of neutrophil numbers may play a signifi-
apy.5 Prabhu and Patel reported only a rare association of cant role in reduction of or recovery from infections due to
zygomycete infection in patients with solid tumors.16 Cunninghamella sp.7,16 Neutrophils are primarily protective
A review by Roden et al. reported a clear association of against fungal hyphae, whereas macrophages may engulf
zygomycosis due to Cunninghamella sp. with the male sex. and destroy sporangiospores through release of oxidative
However, it was unclear whether this association was due to by-products and proteases.18 Phagocytosis by macrophages
physiological factors such as male hormones or environmen- results in oxidative destruction of spores, thus preventing
tal factors (i.e., trauma). Estrogen has been shown to have initiation of hyphal growth. When macrophage function is
protective effects in paracoccidioidomycosis; however, the impaired, clearance of spores is diminished.19 It is believed
role of estrogen in zygomycosis has not been investigated.5 that dysfunction in T cells does not appear to play a major
Several studies have suggested increasing overall inci- role in immune defense against zygomycete infection as
dence of zygomycosis in specific transplant populations. demonstrated by the lack of reported disease in individuals
Marr et al. reported that among allogenic hematopoietic stem infected with HIV.18
cell transplant (HSCT) patients, severe GVHD and underly- Systemic physiological states, such as increased iron con-
ing myelodysplastic syndrome were significant factors in the centrations secondary to chelation therapy, administration of
development of zygomycete infection. In addition, infections deferoxamine, diabetes, or metabolic acidosis are risk fac-
tended to manifest themselves >90 days after transplantation. tors for development of zygomycosis.18 The chemotactic and
In 1993, the practice of routine antifungal prophylaxis with phagocytic characteristics of neutrophils are inhibited by
fluconazole for 75 days after HSCT was initiated. In a study decreased serum pH.19 Steroid therapy or other immunosup-
of non-Aspergillus mold infections, it was noted that during pressive therapies play a contributory role in the development

of disease either by directly suppressing production of inflam- of amphotericin B plus surgical debridement when possible.
matory mediators, inhibition of inflammatory cell migration, Early diagnosis of infection is essential to improve patient
or induction of a diabetic state in the host.3 outcome.
82.1.4 Clinical Features 82.1.5 Virulence Factors

In addition to pulmonary diseases, Cunninghamella spp. Iron appears to play a special role contributing to increased
have been isolated from infections in sites such as the gastro- risk of zygomycosis. In a recent review by Ibrahim et al., it
intestinal tract, lymph nodes, and brain. Cutaneous infections was noted that high serum iron levels predispose individuals
may be localized secondary to inoculation or may result in to zygomycete infection but not to infections with Candida or
disseminated disease. Cutaneous disease is the most common Aspergillus. Iron is a requirement for enhanced growth and
presentation of zygomycosis in individuals with no underly- virulence by many fungal pathogens.21 In acid pH conditions
ing medical conditions.5 Abscess formation, production of (i.e., metabolic acidosis), the transferrin system functions at
pus, and tissue swelling are noted in local infections. Lesions a lower efficiency, thereby allowing additional unbound iron
may become necrotic and result in large ulcers that are foci to circulate in the serum.19 Physiologically, iron must be in
of secondary bacterial infection. Subcutaneous tissues, fat, the ferrous (Fe2+) ionization state in order to be accumulated
fascial layers, and muscle may be invaded by fungal hyphae. intercellularly. However, excess intercellular iron must be
Necrotizing fasciitis with resultant high mortality rates may stored in order to prevent iron-catalyzed production of free
occur. Surgical excision of infected sites may be curative but radicals, which cause oxidative damage to cellular substrates.
may result in substantial disfigurement.3 Cutaneous disease Zygomycetes are unique in their ability to store iron as part
may be underestimated as a source of hematogenous dissem- of protein complexes known as ferritins. Mycoferritin, bacte-
ination, leading to a systemic syndrome. In their case series, rioferritin, and zygoferritins have all been identified in zygo-
Roden et al. reported that 20% of 176 patients developed dis- mycetes; zygoferritin is unique to this genera.
semination of zygomycete infection from a cutaneous site to Three mechanisms of enhanced iron uptake have been
another organ. This resulted in a mortality rate of 94% within described in zygomycetes. Siderophores chelate ferric (Fe3+)
this subgroup.5 iron from the environment, reduce it to the ferrous (Fe2+)
Development of disseminated disease may manifest itself form, and transfer it into the fungal cell via a siderophore
as multiple, rapidly developing skin lesions. Disseminated permease. Zygomycetes secrete rhizoferrin, a siderophore
disease is most commonly associated with hematogenous that supplies iron to the cell via an energy-dependent recep-
spread from the lung. In a case report from Darrisaw et al., a tor-mediated process. Rhizopus has been shown to also
46-year-old female with acute GVHD secondary to unrelated utilize siderophores secreted by other organisms (xenosidero-
allogenic BMT for acute myelogenous leukemia developed phores) to enhance fungal iron uptake. The exogenous chelat-
numerous erythematous skin lesions with necrotic centers ing agent, deferoxamine, can be utilized as a siderophore by
3 days after development of bilateral pulmonary infiltrates. Rhizopus and other zygomycetes, thus supplying supplemen-
Skin biopsies indicated fungal hyphae with dichotomous tal iron to spur fungal growth. In patients with chronic renal
branching and occasional septation. Autopsy findings failure, deferoxamine treatment has proven to be a significant
included fungal pneumonia with hyphal masses in addition factor in development of zygomycete infections. However,
to disseminated fungal dermatitis with Cunninghamella sp.8 deferasirox, an orally bioavailable iron chelator, has been
Spread of infections typically occurs via the hematogenous shown to chelate iron from R. oryzae and is cidal against
route. A review by Darrisaw and colleagues of 26 cases of zygomycetes in vitro. Use of this agent may have substantial
invasive Cunninghamella sp. infections reported pulmonary contribution to survival in patients who must undergo iron
infection as a primary site in 19/26 (73%) cases. Mortality in chelation therapy.
this case group was 77%. Dissemination to other sites such Reductive iron uptake involves an iron reductase, which
as the brain, sinuses, cervical lymph nodes, and joint spaces reduces Fe3+ to Fe2+, combined with an iron permease, which
were reported. Both localized skin lesions and disseminated brings Fe2+ into the fungal cell. A high-affinity iron permease
cutaneous infections were described in this case series.8 An in R. oryzae has been cloned and found to have significant
additional case series from Garey et al. reported a mortality DNA sequence homology to high-affinity Fe2+ permeases
rate of 81%, with lung as the most common site of involve- noted in C. albicans and S. cerevisiae. In addition, disrup-
ment. A troubling observation was noted in 10 cases with an tion of Fe2+ permease expression reduced R. oryzae virulence
increase in reported amphotericin B MIC values. In seven in a mouse model of ketoacidiosis.
of these cases, Cunninghamella MICs ranged from 12.5 to The third mechanism for iron uptake involves hemin as
100 μg/mL. Mortality in this group was 100%.20 a source of iron. In addition to receptors for hemin binding,
Human infection with Cunninghamella spp., and indeed an oxygenase is required for iron assimilation into fungal
any zygomycete, constitutes a medical emergency and should cells. Sequencing of the Rhizopus genome has described two
not be discounted. Some studies have indicated increased homologues of a putative C. albicans heme oxygenase gene.
pathogenicity of Cunninghamella as compared to other These homologues may provide a mechanistic example by
members of the zygomycetes. Treatment generally consists which Rhizopus and other zygomycetes may obtain iron from

Cunninghamella 717
host hemoglobin. It may also provide some insight into the demonstrates broad, pauciseptate hyphae containing long,
angioinvasive tendencies of zygomycetes.21 branched sporangiophores that terminate in swollen vesicles,
Mechanisms of antifungal resistance may also be con- which are 30–65 μm in diameter. The vesicles are covered
sidered factors contributing to increased virulence of with sterigmata that each terminates in a single round to oval-
zygomycetes. Antifungals of the azole class are generally shaped sporangiola containing a single spore.25 The sporan-
ineffective against zygomycetes. Aside from posaconazole, giola often is covered with small hair-like crystals. There are
azole MICs (itraconazole, voriconazole) can range from 0.03 certain instances in which identification using conventional
to 64 μg/mL.22 Azoles inhibit lanosterol 14-α-demethylase, techniques such as microscopic morphology is not possible or
resulting in reduced synthesis of ergosterol required for is not optimum for patient care. For example, it is not infre-
development and maintenance of fungal cell membranes. quent that surgical samples are obtained for pathologic stain
Although genus-specific data are limited, zygomycete resis- and examination but a microbiologic culture is not consid-
tance to azoles are most likely due to decreased membrane ered. Although, histopathology may indicate the presence of
permeability to drugs, upregulation of membrane-bound a zygomycete, often identification to the genus and/or species
efflux transporter pumps, interference of azole activity in level is not possible from these specimens. In addition, there
inhibition of lanosterol 14-α-demethylase, or overexpression are instances when direct identification from patient speci-
of this particular enzyme target.23 mens without the need to wait for cultures to grow is desired
Echinocandin agents have no effect against zygomyce- because, in spite of their rapid growth characteristics, zygo-
tes. This class of antifungals inhibit β-1,3-d-glucan synthe- mycete culture still often requires several days. Therefore,
tase, responsible for synthesis of β-1,3 glucan, an essential molecular methods of identification, such as those discussed
component of the fungal cell wall. Mutation in Fks1, a gene in the next section, may speed the turnaround time for identi-
that encodes the major subunit of glucan synthetase, is fication of these organisms and have the potential to positively
believed to be the principal resistance mechanism to echi- impact patient care.
nocandins, as demonstrated in Aspergillus and Candida. As
glucan synthetase activity in Cryptococcus and other molds 82.2.2 Molecular Diagnosis of
is strongly inhibited by echinocandins, it is postulated that
Cunninghamella Infection
an alternative glucan synthesis mechanism, independent
of Fks1 and unaffected by echinocandin agents, may be Molecular methods for the detection of Cunninghamella spp.
involved in the resistance of zygomycetes to this antifungal have not yet been widely reported in the literature but, in gen-
class. In addition, reduced cell permeability for echinocan- eral, two molecular approaches have been used to date. The
dins may also play a role in resistance. Unfortunately, spe- first approach uses DNA sequence analysis to rapidly iden-
cifics of these mechanisms have not been elucidated for the tify Cunninghamella after it is grown in pure culture from
zygomycetes.24 patient specimens. The second approach uses PCR to directly
Aside from these general discussions of iron uptake and identify Cunninghamella from patient specimens. The PCR
antifungal resistance as applied to zygomycetes as a whole, approaches utilize traditional PCR, nested PCR, or real-time
little information is available concerning specific virulence PCR methods, each of which differ in their turnaround time
factors in Cunninghamella sp. When compared to other and potential for false-positive results due to environmental
zygomycetes, Cunninghamella does exhibit thermotolerant contaminants. Both DNA sequencing from culture and PCR
growth properties, which may contribute to invasive cerebral from specimen are described in Sections 82.2.3 and 82.2.4
and pulmonary infection.3 An early study from Weitzman in detail.
and Crist demonstrated robust growth of C. bertholletiae
at temperatures up to 45°C, whereas C. elegans exhibited 82.2.3 Sequence-Based Identification
growth only at 25°C–37°C.1
of Cunninghamella spp.
Alastruey-Izquierdo et al. used sequencing of the internal

82.2 Methods transcribed spacer (ITS) region to identify 77 clinical strains
82.2.1 Conventional Techniques for Diagnosis of Mucorales species.26 The ITS region of the rRNA gene
has been demonstrated to be useful for the differentiation of
of Cunninghamella Infection
Mucorales genera including Cunninghamella spp. from other
Cunninghamella spp. are traditionally identified from human fungal genera and from other members of the Mucorales.
specimens using a combination of macroscopic and micro- PCR amplification of the ITS target was done using primers
scopic techniques. As mentioned earlier, colonies grow opti- ITS1 and ITS4 (Table 82.1) in a GeneAmp 9700 PCR system
mally at 25°C–30°C and are initially fluffy and white, turning (Applied Biosystems). For the sequencing reaction, 2 μL sam-
gray over time. The reverse side of the colony is typically ple of ReadyReaction mixture from the BigDye terminator
white to buff colored. Cunninghamella grows extremely rap- cycle sequencing kit (Applied Biosystems) was used along with
idly and may completely fill a culture plate within 2–4 days. 1 μM of primers (ITS1 and ITS4) and 3 μL of the PCR reaction
Growth is inhibited by cycloheximide. The microscopic product. The resulting nucleotide sequence was assembled and
morphology is usually sufficient to allow identification and edited using the SeqManII and EditSeq software packages

TABLE 82.1
Primer and Probe Sequences for Amplification and Detection of
Cunninghamella
Primer/Probe Sequence Reference
U1 5′-GTG AAA TTG TTG AAA GGG AA-3′ Kobayashi33
U2 5′-GAC TCC TTG GTC CGT GTT-3′
Cu1 5′-GGA TTG TAA ACT AAA GTT TTC-3′
Cu2 5′-AAA TTC TCT AAT TAT TCC CTC-3′
ITS1 5′-TCC GTA GGT GAA CCT GCG G-3′ Alastruey-Izquierdo26
ITS4 5′-TCC TCC GCT TAT TGA TAT GC-3′
ZM1 5′-ATT ACC ATG AGC AAA TCA GA-3′ Bialek34
ZM2 5′-TCC GTC AAT TCC TTT AAG TTT C-3′
ZM3 5′-CAA TCC AAG AAT TTC ACC TCT AG-3′
ITS4 5′-TCC TCC GCT TAT TGA TAT GC-3′ Alvarez28
ITS5 5′-GGA AGT AAA AGT CGT AAC AAG G-3′
CU(+) sense 5′-TAG TCA GCC AGG TAA ATA AGT-3′ Kasai37
CU(−) sense 5′-TCG TCA ATA TTT AGC TTT AGG-3′
CU FITC 5′-GCT TGG AAA CGA AGA GTC AGG TTG-3′
CU RD 640 5′-TGG GAA TGC AGC CTA AAA TGG GAG TGA-3′
Cu1 5′-GGA TTG TAA ACT AAA GTT TTC-3′ Voigt32
Cu2 5′-AAA TTC TCT AAT TAT TCC CTC-3′
(Lasergene, DNAstar, Inc., Madison, WI).27 Sequencing of the Myocladus corymbifera (5.3%), Rhizopus pusillus (3.7%),
ITS region of 77 clinical strains of Mucorales species allowed Cunninghamella bertholletiae (3.2%), Mucor indicus (2.6%),
the authors to identify all 77 strains including 2 isolates of Cunninghamella echinulata (1%), and Apophysomyces
Cunninghamella spp. elegans (0.5%). The authors also noted a high genetic vari-
Alvarez et al. examined the spectrum of zygomycete ability in the 5.8S rRNA gene of Cunninghamella spp. and
species identified at a mycology reference laboratory from Apophysomyces spp., leading them to conclude that the iden-
clinically significant specimens in the United States using tification of a Cunninghamella ITS region amplicon distinct
sequencing of the ITS region of the fungal ribosomal DNA.28 from either C. bertholletiae or C. echinulata using phyloge-
The fungal DNA was extracted and purified using the netic analysis supports the idea that there may be other spe-
FastDNA kit (Bio101), which was slightly modified by per- cies of Cunninghamella that have yet to be recognized.
forming a homogenization step three times using a FastPrep Voigt et al. sequenced a portion of the 18S small ribo-
F120 instrument (ThermoSavant, Holbrook, NY). The ITS somal DNA subunit and the D1/D2 domains of the 28S large
region of the rDNA was amplified using primers ITS4 and ribosomal DNA subunit in order to construct a sequence
ITS5 (Table 82.1)29,30 following a protocol described by database for the clinically significant zygomycetes including
Gilgado et al.31 The length of the amplicons were in the Cunninghamella.32 The 28S sequences were aligned and 13
range of 500–800 bp with Cunninghamella bertholletiae at separate primer pairs were constructed from the consensus
602–631 bp and Cunninghamella echinulata at 770–810 bp. sequence to allow for PCR amplification in order to rapidly
The PCR mixture included 10 mM Tris–HCl pH 8.3, 50 mM identify causative agents of mucormycosis without cross-
KCl, 2.5 mM MgCl2, 100 μM of each dNTP, 1 μM of each reacting with related genera. The Cunninghamella-specific
primer, and 1.5 U of AmpliTaq DNA polymerase. The ampli- primer pair sequences are listed in Table 82.1.
fication program consisted of 35 cycles with denaturation
at 95°C for 30 s, annealing for 1 min at 52°C, and exten- 82.2.4 PCR Methods for Identification
sion for 1 min at 72°C. The PCR products were sequenced
of Cunninghamella spp.
using the dideoxy chain termination method on an ABI
Prism 310 instrument. Sequences were compared to those Kobayashi et al. used a pan-fungal PCR reaction followed
deposited in the GenBank/EMBL database with an identity by direct DNA sequencing of the PCR reaction product to
level of ≥98% required for identification to the species level. identify C. bertholletiae as the cause of a fatal case of pul-
Identity levels below 98% were reported only to the genus monary mucormycosis in a host with acute lymphocytic
level. Of 190 isolates morphologically identified as zygomy- leukemia.33 Serum samples (200 μL) were extracted using
cetes, the molecular analysis indicated that Rhizopus oryzae a High-Pure viral nucleic acid kit (Roche Diagnostics).
represented almost half of the isolates (44.7%) followed by Sputum was extracted using traditional phenol-chloroform
Rhizopus microsporus (22.1%), Mucor circinelloides (9.5%), technique and formalin-fixed, paraffin-embedded lung

Cunninghamella 719
tissue was extracted using DEXPAT (Takara Shuzo, Tokyo, were sequenced and sequences were compared to BLAST
Japan). PCR amplification was done using pan-fungal (U1 search results from the GenBank databases. For the zygomy-
and U2) or Cunninghamella-specific primers (Cu1 and Cu2) cete targets, a 100% homology to the database sequence was
(Table 82.1). required to be reported.
The PCR reaction mixture consisted of 5μL of 10× PCR Of 23 cases of zygomycosis diagnosed by histopathology,
buffer, 2.5 mmol MgCl2, 0.25 mmol of the four dNTPs, PCR resulted in genus- or species-specific identification in 14
100 pmol of each primer, and 2.5 U of Taq polymerase in cases. The authors concluded that the availability of a definitive
a total volume of 50 μL. Thermal cycling conditions were diagnosis of zygomycosis to the genus or species level can help
5 min at 95°C followed by 50 cycles of 30 s at 95°C, 1 min to optimize antifungal therapy. Organisms identified included
at 50°C, and 2 min at 72°C. A 5 min 72°C hold cycle fol- Rhizopus spp., Rhizomucor spp., Mucor sp., and Myocladus
lowed. PCR products were separated on an agarose gel to (formerly Absidia) corymbifera but Cunninghamella-specific
obtain either a 371 bp product using the U1/U2 primer set DNA was not detected in the study specimens.
or a 571 bp product using the CU1/CU2 primer set. Direct Rickerts et al. used the assay described above34 and com-
sequencing of the PCR product was done using the BigDye pared the ability of histopathologic analysis, culture, and
terminator cycle sequencing kit (Applied Biosystems) using PCR to detect invasive mold infections in tissue biopsy spec-
the Cu1 primer. A BLAST search of the sequence obtained imens.35 Mold hyphae, including zygomycetes (n = 6), were
using the Cunninghamella-specific primer product matched detected in 27/56 histological specimens (48%) while culture
the C. bertholletiae 28S rRNA sequence exactly. Sequence detected 17/27 specimens (63%), and a seminested PCR strat-
analysis using the pan-fungal primer product demonstrated a egy detected mold in 26/27 (96%) specimens. Identification
single mismatch in the U1 primer region. to the genus and/or species level by PCR correlated with cul-
Bialek et al. described a seminested PCR assay that tar- ture identification in 16/18 culture-positive cases, leading the
geted the 18S rDNA of zygomycetes including C. bertholletiae authors to conclude that PCR is superior to culture in patients
to the exclusion of Aspergillus species.34 Paraffin-embedded with invasive mold infections.
tissue from sinus, lung, CNS, liver, and skin biopsies were We recently developed a real-time PCR method for the
screened. The paraffin wax was removed by adding 1 mL of detection of zygomycetes including Cunninghamella spe-
xylene to an Eppendorf tube containing two 5 μm sections cies (C. bertholletiae, C. blakesleeana, C. echinulata,
of the embedded tissue. Following incubation, the extract C. elegans).36 The assay utilized primers and fluoresence
was washed with xylene and ethanol and DNA was extracted resonance energy transfer (FRET) probes targeting a 167 bp
using a QIAamp tissue kit (Qiagen), which was modified by region of the multicopy cytochrome b gene. The clinical sen-
the authors through the addition of three cycles of freezing in sitivity and specificity of the assay from culture isolates was
liquid nitrogen for 1 min followed by boiling for 5 min to dis- determined to be 100% and 92%, respectively. The assay
rupt the fungal cell wall. Two outer primers (ZM1 and ZM2, detects the genera Absidia, Apophysomyces, Cunninghamella,
Table 82.1) were used for the first PCR reaction and produced Mucor, Rhizopus, and Saksenaea through the use of melt
a product corresponding to nucleotide positions 711–1117 of curve analysis, although the melt curves are not sufficiently
Rhizopus arrhizus (AF113440). A second nested PCR reac- resolved to allow identification to the genus level. The assay
tion utilized primers ZM1 and ZM3, which produced a prod- is also able to detect zygomycetes from fresh tissue as well
uct of about 176 bp depending on the species amplified. as from formalin-fixed, paraffin-embedded tissue. The closed
The PCR reaction mixture contained 10 μL of extracted format of real-time PCR significantly reduces the potential for
DNA in a total volume of 50 μL with 10 mM Tris/HCl pH contamination from environmental zygomycetes.
8.3, 50 mM KCl, 2.5 mM MgCl2, 1 μM of each outer primer, Kasai et al. described a quantitative real-time PCR assay
1.5 U of AmpliTaq polymerase (Roche), and 100 μM of each for the detection of Cunninghamella species.37 The assay
dNTP (Promega). For the nested reaction, the same mixture targeted the 28S rRNA gene and used consensus sequence
was used with 1 μL of the first PCR reaction replacing the alignment of Cunninghamella species from GenBank.
extracted DNA, 50 μM of each dNTP, and 1 μM of each The mastermix for PCR contained 0.5 μM primers, 1×
inner primer. PCR conditions were 94°C for 5 min, 94°C for PCR buffer, 4 mM MgCl2, 0.025% bovine serum albumin,
30 s over 35 cycles, 50°C for 30 s, 72°C for 1 min followed 0.025 U/mL Platinum Taq DNA polymerase, 0.2 mM dNTPs,
by 72°C for 5 min. A human β globin gene (nucleotides and 0.2 μM FRET probes labeled with fluorescein isothiocy-
70400–70667) was used as a control of amplification inhi- canate (FITC) and RD640 (Table 82.1). Uracil-N-glycosylase
bition. Globin primers were G1 (5′-GAA GAG CCA AGG (UNG) was also incorporated to prevent amplicon carryover.
ACA GGT AC-3′) and G2 (5′-CAA CTT CAT CCA CGT The amplification conditions consisted of denaturation at
TCA CC-3′), and conditions were identical to those used 95°C for 0 s (slope, 20°C/s), annealing at 57°C for 5 s (slope,
above with the exception of 5 μL of extracted DNA. Three 10°C/s), and extension at 72°C for 15 s for 50 cycles. A melt
millimolar MgCl2 a measured quantity was used and the curve was generated at the end of the run to assure product
extension time was lowered to 45 s. Primary PCR products specificity; the conditions were 90°C for 0 s (slope, 20°C/s),
were analyzed using electrophoresis on a 1.8% agarose gel 40°C for 30 s (slope, 2°C/s) and 75°C for 0 s (slope, 0.2°C/s).
and ethidium bromide staining for detection. Cloned plasmid Furthermore, the assay was able to detect 10–100 copies of
DNA was used as a positive control. Nested PCR products target and had high specificity. Further, the assay was able to

distinguish between C. bertholletiae and C. echinulata using 4. Richardson, M.D. and Koukila-Kahkola, P., Rhizopus,
melt curve analysis. Although not tested directly, C. elegans Rhizomucor, Absidia, and other agents of systemic and sub-
may also be detected by virtue of its sequence homology to cutaneous Zygomyceses, in Manual of Clinical Microbiology,
p. 1839, Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry,
the primer and probe sequences used for this assay. The clini-
M.L., and Pfaller, M.A. (Eds.), ASM Press, Washington, DC,
cal sensitivity of the assay from an experimental model of 2007.
pulmonary zygomycosis in both BAL fluid and lung tissue 5. Roden, M.M. et al., Epidemiology and outcome of zygomy-
samples was excellent at 100% for both specimen sources. cosis: A review of 929 reported cases, Clin. Infect. Dis., 41,
634, 2005.
6. Frater, J.L., Hall, G.S., and Procop, G.W., Histologic features
82.3 C
onclusions and
of zygomycosis: Emphasis on perineural invasion and fungal
Future Perspectives morphology, Arch. Pathol. Lab. Med., 125, 375, 2001.
7. Ortin, X. et al., Cunninghamella bertholletiae infection
Zygomycosis appears to be increasing in frequency particu-
(mucormycosis) in a patient with acute T-cell lymphoblastic
larly in the immunocompromised host possibly due to prophy- leukemia, Leuk. Lymphoma, 45, 617, 2004.
laxis regimens using antifungal agents that have gaps in their 8. Darrisaw, L. et al., Cunninghamella infection post bone mar-
spectrum of coverage for these organisms. The recent study by row transplant: Case report and review of the literature, Bone
Alvarez et al. indicates that Cunninghamella species account Marrow Transplant., 25, 1213, 2000.
for 3%–4% of all zygomycetes identified from clinically signif- 9. Andrade, V.S. et al., A factorial design analysis of chitin pro-
icant sources at one reference laboratory in the United States. duction by Cunninghamella elegans, Can. J. Microbiol., 46,
Given that Cunninghamella spp. appear to be more pathogenic 1042, 2000.
10. Pothuluri, J.V. et al., Metabolism of metolachlor by the fungus
than other zygomycete genera, the need for rapid, accurate Cunninghamella elegans, Arch. Environ. Contam. Toxicol.,
diagnostics of Cunninghamella is important in order to pro- 32, 117, 1997.
vide the best possible chance for therapeutic success in these 11. Lisowska, K. and Dlugonski, J., Concurrent corticosteroid
extremely challenging clinical situations. Traditional methods and phenanthrene transformation by filamentous fungus
of identification such as morphology can be challenging, slow Cunninghamella elegans, J. Steroid Biochem. Mol. Biol., 85,
and requires trained, experienced mycologists who are in short 63, 2003.
supply in many laboratories; therefore, molecular diagnostics 12. Parshikov, I.A. et al., Transformation of artemisinin by
Cunninghamella elegans, Appl. Microbiol. Biotechnol., 64,
provide an important opportunity to accurately and rapidly dif-
782, 2004.
ferentiate Cunninghamella spp. from other zygomycetes. To 13. Motohashi, K. et al., Cutaneous zygomycosis caused by
date, there has not been a significant emphasis in the clinical Cunninghamella bertholletiae in a patient with chronic
diagnostic laboratory on the development of molecular meth- myelogenous leukemia in blast crisis, Am. J. Hematol., 84,
ods for zygomycetes including Cunninghamella. No doubt this 447, 2009.
is secondary to the relative rarity of these infections. Nucleic 14. Rickerts, V. et al., Cluster of pulmonary infections caused
acid sequencing has proven to be a useful tool once the organ- by Cunninghamella bertholletiae in immunocompromised
ism has been grown in culture but for optimal patient care, patients, Clin. Infect. Dis., 31, 910, 2000.
15. Kontoyiannis, D.P. et al., Zygomycosis in a tertiary-care
the ideal scenario is to directly identify Cunninghamella spp. cancer center in the era of Aspergillus-active antifungal ther-
from patient specimens without waiting for culture growth. A apy: A case-control observational study of 27 recent cases,
few PCR methods described in this chapter have been useful J. Infect. Dis., 191, 1350, 2005.
for direct detection of Cunninghamella spp. although many 16. Prabhu, R.M. and Patel, R., Mucormycosis and entomoph-
were designed to detect the more commonly encountered gen- thoramycosis: A review of the clinical manifestations,
era of Zygomycetes such as Rhizopus or Mucor rather than diagnosis and treatment, Clin. Microbiol. Infect., 10, S31,
focusing specifically on Cunninghamella. The laboratory is 2004.
17. Marr, K.A. et al., Epidemiology and outcome of mould infec-
an essential partner in patient care and management, and as
tions in hematopoietic stem cell transplant recipients, Clin.
such the development of molecular methods able to rapidly and Infect. Dis., 34, 909, 2002.
accurately detect these organisms will be critical to continue 18. Walsh, T.J. et al., Infections due to emerging and uncom-
success in the battle against the high mortality associated with mon medically important fungal pathogens, Clin. Microbiol.
Cunninghamella infection. Infect., 10, S48, 2004.
19. Gonzalez, C.E., Rinaldi, M.G., and Sugar, A.M., Zygomycosis,
Infect. Dis. Clin. North Am., 16, 895, 2002.
References 20. Garey, K.W. et al., Cunninghamella bertholletiae infection
1. Weitzman, I. and Crist, M.Y., Studies with clinical isolates in a bone marrow transplant patient: Amphotericin lung pen-
of Cunninghamella. I. Mating behavior, Mycologia, 71, 1024, etration, MIC determinations, and review of the literature,
1979. Pharmacotherapy, 21, 855, 2001.
2. Jayasuriya, N.S. et al., An unusual presentation of rhinofacial 21. Ibrahim, A.S., Spellberg, B., and Edwards, J. Jr., Iron acquisi-
zygomycosis due to Cunninghamella sp. in an immunocom- tion: A novel perspective on mucormycosis pathogenesis and
petent patient: A case report and literature review, Oral Dis., treatment, Curr. Opin. Infect. Dis., 21, 620, 2008.
12, 67, 2006. 22. Kanafani, Z.A. and Perfect, J.R., Antimicrobial resistance:
3. Ribes, J.A., Vanover-Sams, C.L., and Baker, D.J., Zygomy Resistance to antifungal agents: Mechanisms and clinical
cetes in human disease, Clin. Microbiol. Rev., 13, 236, 2000. impact, Clin. Infect. Dis., 46, 120, 2008.

Cunninghamella 721
23. Canuto, M.M. and Rodero, F.G., Antifungal drug resistance to 31. Gilgado, F. et al., Molecular phylogeny of the Pseudallescheria
azoles and polyenes, Lancet Infect. Dis., 2, 550, 2002. boydii species complex: Proposal of two new species, J. Clin.
24. Perlin, D.S., Resistance to echinocandin-class antifungal Microbiol., 43, 4930, 2005.
drugs, Drug Resist. Updat., 10, 121, 2007. 32. Voigt, K., Cigelnik, E., and O’Donnell, K., Phylogeny and
25. Larone, D.H., Medically Important Fungi: A Guide to PCR identification of clinically important Zygomycetes based
Identification, ASM Press, Washington, DC, 2002. on nuclear ribosomal-DNA sequence data, J. Clin. Microbiol.,
26. Alastruey-Izquierdo, A. et al., Activity of posaconazole and 37, 3957, 1999.
other antifungal agents against Mucorales strains identified 33. Kobayashi, M. et al., Molecular polymerase chain reac-
by sequencing of internal transcribed spacers, Antimicrob. tion diagnosis of pulmonary mucormycosis caused by
Agents Chemother., 53, 1686, 2009. Cunninghamella bertholletiae, Respirology, 9, 397, 2004.
27. Alastruey-Izquierdo, A. et al., Prevalence and susceptibility 34. Bialek, R. et al., PCR based identification and discrimination
testing of new species of Pseudallescheria and Scedosporium of agents of mucormycosis and aspergillosis in paraffin wax
in a collection of clinical mold isolates, Antimicrob. Agents embedded tissue, J. Clin. Pathol., 58, 1180, 2005.
Chemother., 51, 748, 2007. 35. Rickerts, V. et al., Comparison of histopathological analy-
28. Alvarez, E. et al., Spectrum of zygomycete species identi- sis, culture, and polymerase chain reaction assays to detect
fied in clinically significant specimens in the United States, invasive mold infections from biopsy specimens, Clin. Infect.
J. Clin. Microbiol., 47, 1650, 2009. Dis., 44, 1078, 2007.
29. Mitchell, T.G. et al., Unique oligonucleotide primers in 36. Hata, D.J. et al., Real-time PCR method for detection of zygo-
PCR for identification of Cryptococcus neoformans, J. Clin. mycetes, J. Clin. Microbiol., 46, 2353, 2008.
Microbiol., 32, 253, 1994. 37. Kasai, M. et al., Detection of a molecular biomarker for zygo-
30. Viebahn, M. et al., Assessment of differences in ascomycete mycetes by quantitative PCR assays of plasma, bronchoalveo-
communities in the rhizosphere of field-grown wheat and lar lavage, and lung tissue in a rabbit model of experimental
potato, FEMS Microbiol. Ecol., 53, 245, 2005. pulmonary zygomycosis, J. Clin. Microbiol., 46, 3690, 2008.

83 Entomophthorales
Johannes E. Rothhardt, Volker U. Schwartze, and Kerstin Voigt
Contents
83.1 Introduction...................................................................................................................................................................... 723
83.1.1 Classification......................................................................................................................................................... 723
83.1.2 Morphology.......................................................................................................................................................... 724
83.1.2.1 Conidiobolus coronatus........................................................................................................................ 724
83.1.2.2 Basidiobolus ranarum........................................................................................................................... 724
83.1.4.1 Characteristic Features of Entomophthoromycoses.............................................................................. 726
83.1.4.2 Unusual Cases of Entomophthoromycoses............................................................................................ 727
83.1.5 Diagnosis.............................................................................................................................................................. 728
83.2 Methods............................................................................................................................................................................ 729
83.2.1.1 Sample Recovery................................................................................................................................... 729
83.2.1.2 Sample Preparation for Light Microscopy and Fluorescent Microscopy.............................................. 729
83.2.1.3 Preparation of Genomic DNA for Molecular Detection........................................................................ 730
83.2.2.1 Morphological Detection in Pure Cultures............................................................................................ 730
83.2.2.2 Morphological Detection in Human Tissue........................................................................................... 730
83.2.2.3 Immunological Detection...................................................................................................................... 730
83.2.2.4 Molecular Detection Based on Polymerase Chain Reaction................................................................. 731
References.................................................................................................................................................................................. 733
83.1 Introduction these analyses, the phylogenetic position of Basidiobolus is

pivotal in resolving a natural classification. Multigene gene-
83.1.1 Classification alogies place Basidiobolus either with the Chytridiomycota3
The intraordinal and interordinal phylogenetic relation- or the Entomophthorales.1 In contrast to the ambiguous phy-
ships of the order Entomophthorales are still unclear. logenetic position of Basidiobolus, Conidiobolus, and other
The Entomophthorales includes, primarily, insect-killing Entomophthoralean genera consistently group within the
fungi, although a few exhibit a certain pathogenic poten- Entomophthorales.
tial to infect animals and humans. The Entomophthoralean The classification of the Entomophthorales using morpho-
group traditionally belongs to the phylum Zygomycota, but logical and ecological characteristics has changed several
recent phylogenetic reevaluations show that the Zygomycota times after the first description by Winter4 and is still under
are polyphyletic.1 The phylogenetic position of the controversial discussion. The molecular phylogenetic analy-
Entomophthorales and the degree of phylogenetic relatedness sis using, for instance, exonic nucleotide sequences from the
to its putative connatural fungal groups are still unresolved. gene encoding the nuclear RNA polymerase subunit 1 (RPB1)
Recently, the Zygomycota have been divided into several separated the Basidiobolaceae from the Entomophthorales.5
subphyla and the Entomophthorales are included as a sepa- A recently published comprehensive phylogenetic analysis
rate subphylum, the Entomopthoromycotina.2 In addition, confirms the uncertain position of Basidiobolus within the
the traditional family structure within the Entomophthorales fungi.2
has changed several times based on a multitude of phyloge- Using 18S rRNA sequences for phylogenetic analysis
netic analyses and algorithms applied to different loci. In produces results placing Basidiobolus inside a cluster of
723

Chytridiomycota.3 However, a comprehensive study1 using primary spores form hair-like structures or multiple small
six genes of taxa representing almost all fungal or fungus-like spherical structures resembling a corona.10,11 Another type of
group places Basidiobolus with the chytrid Olpidium bras- spore, a chlamydospore, serves as a resting spore. This is an
sicae as a sistergroup to Entomophthora and Conidiobolus asexual spore and is formed by modification of the vegetative
constituting the Entomophthorales. Summarizing using hyphae, which become thick walled.
molecular, morphological, and ecological data, the phylog- Observations have shown that the fungal mycelium grows
eny of Entomophthorales excluding Basidiobolus seems to fast and flat on most standard media (e.g., Sabouraud [SABG]
be clear. Basidiobolus may probably represent a phylogenetic agar) at 28°C, different strains of C. coronatus growing at
link between the chytrids and Entomophthorales, but this different rates in vitro. The time taken to overgrow a Petri
position is still unclear and requires further research based dish differs from 2 to 4 days depending on the strains tested.
on more phylogenetic marker genes. The ambiguity between The cause of the rapid growth is the early and multitudinous
phylogenetic position and morphology-based taxonomy of spore production that results in the ejection of ballistospores
Basidiobolus among the alliance of Entomophthoralean fungi extending the area of the growing mycelium with satellite
requires further discussion in the light of the genealogical colonies. Macroscopically, the mass of these large spores
concordance phylogenetic species recognition (GCPSR) con- gives the colony a white-cream, rough, and powdery appear-
cept, which is based on the multigene genealogy-dependent ance. In addition to these morphological features, the myce-
definition of phylogenetic species.6 lium desiccates the agar to produce a radially folded colony.
In accordance to the classification adopted in the 10th edi- This is a unique and reliable macromorphological diagnostic
tion of the Dictionary of the Fungi, the Entomophthorales character.
consist of five families: Ancylistaceae (5 genera, e.g.,
Conidiobolus), Completoriaceae (1 genus Completoria), 83.1.2.2 Basidiobolus ranarum
Entomophthoraceae (17 genera, e.g., Entomophthora and Basidiobolus ranarum shows morphological and ecologi-
Entomophaga), Meristacraceae (6 genera), Neozygitaceae cal affinities to Conidiobolus spp., and it also exhibits some
(3 genera), and Incertae sedis (1 genus).7 The Basidiobolaceae, characteristics resembling features of C. coronatus. The
which are part of the Incertae sedis group among the phylum discharged ballistospores of B. ranarum germinate imme-
Zygomycota, comprises solely two genera, Basidiobolus and diately under suitable conditions like those of C. coronatus.
Amphoromorpha.7 Under inappropriate growth conditions, the spores can form
a new sporophore directly with the new spore reaching matu-
rity within 2–3 h.8 The germinating spores form hyphae of
83.1.2 Morphology
8–20 μm width (similar to the hyphae of C. coronatus) with
Although the Entomophthorales do not probably belong somewhat randomly produced septa providing the mycelial
to the abolished polyphyletic phylum Zygomycota, they segmentation typical for the Entomophthorales (Figure 83.1G
still share many morphological and ecological characteris- and H).
tics with the members traditionally assigned to this taxon. During the process of spore formation, which is less pro-
The medically important species Basidiobolus ranarum, lific and not as rapid as in C. coronatus, the hyphae become
Conidiobolus coronatus, and perhaps Conidiobolus incon- increasingly septate. The short sporophores grow with posi-
gruus possess mainly nonseptate mycelium and they pro- tive phototropism and enlarge to form single-celled sporan-
duce large and very hydrophobic single-celled spores gia. The sporangium and the upper part of the sporophores
(10–40 μm), which are forcibly discharged. Zygospores, so are forcibly discharged for up to 1–2 cm. It is a characteristic
far produced, develop after fusion of a pair of two gametan- of B. ranarum that the apexes of these sporophores break
gia (a process called gametangiogamy) forming the suspen- after the spores were actively released. Subsequently, the
sors after the initialization of genesis and maturation of the sporangia (ballistospores) form new satellite colonies on the
zygospore. Asymmetric zygospore formation on a single agar. Due to this multiplication of growth initiation points,
suspensor occurs frequently and is the characteristic for the the expansion of the colony increases faster than the normal
Entomophthorales.8 growth of the fungal hyphae.11
Zygospores are formed after conjugative hyphal fusion
83.1.2.1 Conidiobolus coronatus by homothallic gametangiogamy. They are produced in
The unbranched sporophores of C. coronatus are positively high quantity and possess a thick wall with one beak-like
phototropic and discharge spores that are able to germinate tip (Figure 83.1J and K). This tip is a characteristic remnant
immediately (Figure 83.1C–F). The typical papilla, the for- of the gametangiogamy process. It is conceivable that the
mer connection between sporophore and spore, is a 5–10 μm zygospores serve as dormant spores ensuring survival dur-
projection on the spore and it is not involved in spore ger- ing conditions unsuitable for growth. However, the germi-
mination. The emerging germ tube is able to develop into a nation of zygospores is not unambiguously and reproducibly
mycelium comprising hyphae up to 6–25 μm in diameter and documented.
nonparallel, pleomorphic walls (Figure 83.1A and B).9 On the The colony morphology of B. ranarum cannot be eas-
other hand, the germ tube is also capable to form a secondary ily distinguished from that of C. coronatus. The hyphal
dumbbell-shaped spore. It is described in few studies that the system of B. ranarum might be more compact than that of

Entomophthorales 725
(A) (B) (C)
(D) (E) (F)
(G) (H)
(I) (J) (K)
FIGURE 83.1 Micromorphological observations on Conidiobolus coronatus (A–F) and Basidiobolus ranarum (G–K). A: hyphae, B: chla-
mydospore-like resting spores, C–F: unispored sporangiole (mitospore), C and D: mitospores with typical tips, F: bipolar spore germination,
G and H: hyphae with swollen and pleomorphic hyphal tips typical for Basidiobolus, I: germinating mitospore bearing a beak-like tip, J and
K: zygospores. The scale bars indicate 10 μm each.
C. coronatus. The color is whitish cream and the surface 83.1.3 Biology and Epidemiology
is rough with the typical powdery appearance. The agar
medium also becomes folded and desiccated as in the case The Entomophthorales are commonly distributed and ubiq-
of Conidiobolus. uitous. All genera are more or less able to infect and kill
In comparison, both Entomophthoralean fungi are able arthropods including a very broad spectrum of taxa such as
to sporulate rapidly after ballistospore germination, while Tardigrada,12 Coleoptera,13 and Diptera.14 Species of the gen-
the initial mycelium is just starting to grow. In addition, the era Basidiobolus and Conidiobolus can also infect humans
medium becomes desiccated, a process which is associated and other mammals such as horses15 and sheep.16,17
with a folded colony morphology. This feature is apparently Species of Conidiobolus, with C. coronatus as the most
more pronounced in Basidiobolus ranarum. common one, can be isolated from soil, decaying vegetation,
The beak-like tip of the zygospores and sporophores the feces of amphibians, from reptiles, insectivorous bats,
with a swollen area below the ballistospore is typical for and other animals and also from dead insects overgrown by
B. ranarum. various other fungi.18,19 In addition to humans, horses20 and
Even though zygospores are unknown in C. coronatus, its sheep21 infections with Conidiobolus spp. in a dolphin22 and a
asexual chlamydospores may serve as analogues. In addition, chimpanzee23 are also reported. The fungus can live as a sap-
C. coronatus develops spores with typical papillae giving the robe, commensal, or parasite in very different environmental
spores a zygospore-like appearance.11 conditions and has a worldwide distribution. However, high

humidity (95%–100%) and high temperature (25°C–37°C) for a successful infection and establishment of entomophtho-
appear to be important for its satisfactory propagation and romcoses. Furthermore, a number of gastrointestinal basid-
the germination of the spores.18,24 iobolomycoses in Arizona have recently emerged even if
Almost all described cases of conidiobolomycosis caused the number of patients (at seven cases) remains still small.32
by Conidiobolus coronatus are located in tropical and sub- Possible reasons might be (i) an increased exposure to
tropical area of Africa, Asia, Southeast Asia, the Middle amphibians and reptiles,37 (ii) camping near a water body,
East, Central, and South America.25 Only a few cases are or (iii) accompanying diseases like diabetes mellitus, peptic
reported from Europe and North America and for some of ulcer disease, or pica.32 Treatment with ranitidine might be
these scattered instances, the reports relate to immigrants a risk factor as its major effect is a reduced stomach acid-
from the main regions of prevalence.26 Only few cases are ity. This allows the fungi to pass the stomach more easily
infections caused by C. incongruus known,27 but these were and results in more frequent infections of the gastrointestinal
comparatively invasive.18 tract.32
Basidiobolus ranarum is as prevalent as species of A more or less intensive thermotolerance (28°C–37°C)
Conidiobolus. It is common worldwide, but usually causes and the ability to use a wide spectrum of carbon sources
infections in subtropical and tropical regions. The fungus was (these features can differ strongly among strains) are possi-
first isolated in 1886 from a frog28 and can be isolated from ble factors contributing to an essential pathogenicity of these
the same habitats as species of Conidiobolus: soil, decay- fungi (own unpublished data).
ing plants, insects, and the gastrointestinal tract or the feces
of amphibians and reptiles. Moreover, B. ranarum can also
be detected in substances of mammalian origin with case
reports related to bats,29 horses,15 dogs,30 and also humans. The infections caused by Entomophthoralean fungi are
Both pathogens induce the rare entomophthoromycosis in referred to by a number of different terms in the literature.
mostly immunocompetent human hosts. In addition, conid- Usually these cases are called zygomycosis. The detailed
iobolomycosis and basidiobolomycosis are usually subcuta- names to characterize the etiologic agent of the mycosis are
neous infections and every genus has its own predilection entomophthoromycosis (to differentiate them from mucor-
site. mycosis), basidiobolomycosis (caused by Basidiobolus
Conidiobolus coronatus mainly afflicts adults and causes ranarum), and conidiobolomycosis (caused by Conidiobolus
facial infections with a chronic sinusitis.10,31 Infections by coronatus and C. incongruus).
C. incongruus are very rarely described, but the few reported The etiologic agents of these fungal infections are
cases have almost always resulted in a fatal outcome, inde- B. ranarum, C. coronatus, and, in very rare cases, C. incon-
pendent of the immune status of the infected host. The infec- gruus. The two main Entomophthoralean pathogens differ
tions caused are usually more invasive and have a higher in pathogenesis and clinical features, but still have many
dissemination rate than infections caused by C. coronatus.27 similarities. Usually both fungi cause chronically subcuta-
Basidiobolus ranarum causes chronic subcutaneous neous infection of the human tissue of immunocompetent
and peripheral infections of the trunk, limbs, buttocks, and patients, whereas the predilection site differs. B. ranarum
arms.31 Only a few cases (15–20) of fatal gastrointestinal normally afflicts the skin between feet and shoulder: trunk,
basidiobolomycosis are reported.32,33 Basidiobolomycosis of limbs, buttocks, and arms.31 In addition, few (10–15) gastro-
male and female patients of every age are known, although intestinal cases of basidiobolomycoses are known and well
male children seem to be more frequently afflicted.9,18 documented.32,33,36,38 In contrast, C. coronatus causes mainly
Conidiobolus coronatus and Basidiobolus ranarum are subcutaneous infections of the sinusitis and facial skin.10,26
able to disseminate in the human host and cause extremely Furthermore, both fungi can rarely cause disseminated infec-
dangerous infections. Conidiobolomycoses seem to be less tions, which may prove to be fatal.
abundant than basidiobolomycoses. All cases are very infre-
quently reported. The dependency between infection and 83.1.4.1 C haracteristic Features of
the immune status of the host is still unclear. Disseminated Entomophthoromycoses
entomophthoromycoses occur in patients with and without Patients infected with C. coronatus display in most cases
immunodeficiency but without a tendency of prevalence for a a swollen nose or a swelling of the upper lip or the entire
special immune status.18,34,35 face.9,10,26,39 The infection is almost always present in the
It is likely that there are specific preconditions for the rare paranasal sinus and extends to nearly all contiguous tissues.18
but occasionally occurring infections caused by Conidiobolus The swollen area is firm and feels rubbery. As different tis-
coronatus, C. incongruus, and Basidiobolus ranarum. Even sue can be infected, the symptoms vary from case to case.
though the fungi are ubiquitous, only 167 cases of conidiobo- The infected skin may ache or be pain-free and subcutaneous
lomycosis10 are reported worldwide and a similar number nodules may be present or absent.10,26
can be assumed for basidiobolomycoses.32,36,37 A possible The clinical picture of subcutaneous infections caused
explanation might be the climatic influence of tropical and by B. ranarum is usually as follows. The infection becomes
subtropical regions on the etiology and manifestation of the apparent as a solid, hardened subcutaneous painless nodule
disease. Warm and humid climate appears to be important on arms, the trunk, buttocks, and limbs. It enlarges gradually

toward the periphery as a clearly defined flat mass attached disseminating C. incongruus. The patient, who was granulo-
to the overlying skin. The cutaneous area is intact and might cytopenic due to lymphocytic lymphoma, showed a rapidly
be bluish or purple at the region with active growth. A func- progressive pneumonia and fatal pericardial tamponade. The
tional impairment is not present as long as the joints are not fungus, which was resistant to amphotericin B and flucyto-
blocked by the swollen tissue, which may reach enormous sine in vitro, was determined using the morphology of the
proportions.36 culture.
Normally, cross sections of samples show broad, thin- Cases of disseminated basidiobolomycosis are rarely
walled hyphae with surrounding eosinophilic granulocytes, reported, whereas gastrointestinal infections are more fre-
called Splendore-Hoeppli phenomenon. However, the pres- quently described. A case of dissemination of a basidiobolo-
ence or absence of this feature is a sufficient criterium for the mycosis is reported by Bigliazzi et al.37 A healthy 40-year-old
medical outcome of an entomophthoromycosis. Hence, three immunocompetent woman died after 4 months of indetermi-
techniques combined—histopathology, molecular detection, nate medical conditions. The patient showed low-grade fever,
and culture of the fungus—are the most reliable approach for increasing eosinophilia, and increasing immunoglobulin E.
the verification of an entomophthoromycosis. Furthermore, an airflow obstruction was diagnosed, an oxy-
gen saturation of 98% while on room air was noted, and chest
83.1.4.2 Unusual Cases of Entomophthoromycoses radiographs revealed a right upper lobe infiltrate. Biopsies
One of the first well-documented cases of disseminating from bronchus and middle right lobe showed only eosinophil
conidiobolomycosis was reported by Eckert et al.40 The infiltration and necrosis without organisms. However, the his-
authors describe a deep infection with Conidiobolus in a tology of biopsied subcutaneous nodules showed eosinophilic
15-month-old boy, who showed a brassy and hoarse cough, pannicultis with hyphal elements. The next day the patient
weight loss, temperature, and irritability. Investigations led to died in the emergency room due to cardiorespiratory arrest
a thoracotomy, which revealed a hard, posterior mediastinal after sudden acute dyspnea and hypotension. The autopsy
mass invading the left main stem bronchus and pericardium. report identified fungal hyphae with Splendore-Hoeppli phe-
Histology showed a Splendore-Hoeppli phenomenon, which nomenon in the brain, kidneys, lungs, pancreas, spleen, and
is characteristic for entomophthoromycosis. The culture of a stomach. Macroscopic examination indicated necrotic white-
biopsy sample led to the identification of a fungus of the genus yellow opacities (3–4 mm diameter) in the pericardium, pari-
Conidiobolus. A determination to the species level was not etal pleura of the right hemithorax, visceral surface of the
carried out. Kamalam and Thambiah report a case of lymph right lung, liver, spleen, kidneys, adrenal glands, pancreas,
node invasion by C. coronatus in a 40-year-old woman from uterus, and peritoneum.37
India with a subcutaneous infection of facial tissue with this Another case of fatal basidiobolomycosis is reported from
kind of etiologic agent.41 Another case is described by Walker the Netherlands by Van den Berk et al.34 A 61-year-old man
et al.35 A renal transplant patient infected with cytomegalo- suffering from abdominal pain and constipation for several
virus and the fungus Histoplasma capsulatum, and addition- months died of a septic shock. First diagnosis by colonos-
ally with C. coronatus. This caused a disseminated infection copy revealed a large obstructing tumor in the descend-
and blood vessel invasion, which suggests that C. coronatus ing colon and histology showed an inflammatory reaction.
can cause disseminated infections in immunocompetent and However, organisms were not implicated at this stage. A few
immunocompromised humans as well. weeks later, abdominal discomfort in the right upper colon
Case reports for C. incongruus infections are very rare, developed. Six weeks after colonoscopy a CT scan showed
but almost all cases are invasive and fatal, independent of a large mass of 6 cm diameter in the central position in the
the immune status of the host. Busapakum et al. describe right liver lobe. Treatment against an amebic liver abscess
a case of a 20-year-old immunocompetent woman from with metronidazole was unsuccessful. After developing an
Thailand with a disseminated infection of C. incongruus.27 eosinophilia, a liver biopsy was performed. Furthermore, a
She suffered from a subcutaneous mass in her left breast, second CT scan showed that the size of the hepatic mass had
low-grade fever, weight loss, and cough with hemopty- increased to 9 cm diameter. A review of the samples showed
sis. Biopsy of the skin and subcutaneous tissue showed the fungal hyphae and the Splendore-Hoeppli phenomenon. The
Splendore-Hoeppli phenomenon around fungal hyphae. Six patient developed a septic shock, while Escherichia coli and
weeks after admission, the patient died despite antifungal Clostridium perfringens were isolated from blood. A therapy
therapy. The autopsy examination showed fungal filaments with antibiotics and amphotericin B was unsuccessful and
with Splendore-Hoeppli phenomena in lymph nodes, medi- the patient died of multiple-organ failure a few days later.
astinum, esophagus, lung, liver, and jejunum. To identify the After autopsy, B. ranarum was cultured from liver, gallblad-
fungal pathogen, the biopsy samples of skin and subcutane- der, and sigmoid colon, each of which was severely afflicted.
ous tissue were cultured and the characteristic morphology These cases show clearly that rapid molecular detection of
of the culture resulted in a diagnosis of C. incongruus as the the etiologic agent is extremely important, especially when
etiologic agent. the organism cannot be identified by pathohistology and
Another fatal case of disseminated infection is reported culture.
by Walsh et al.42 In this case, an immunocompromised Cases of gastrointestinal basidiobolomycosis are more
32-year-old woman from the United States was infected with frequently reported than disseminated infection with

B. ranarum, but they are still rare and usually nonfatal. In are a few described cases of gastrointestinal infections. Both
2001, Lyon et al. summarized seven cases of gastrointestinal pathogens have to be isolated from the host by biopsy of the
basidiobolomycosis, which occurred in Arizona from April infected soft tissue rather than from the surface of the skin.
1994 to May 1999.32 The patients’ most common symptom Axenic cultivation of Basidiobolus and Conidiobolus on
was abdominal pain, but discharged mucus was also pre- commercially available cultivation media (e.g., SABG or
sented and also Leukocytosis and eosinophilia in all cases. cornmeal) is easy and neither time- nor cost-intensive. In con-
After excision of the inflammatory mass, these features dis- trast to most of the other Entomophthoralean genera, which
appeared. The characteristic Splendore-Hoeppli phenomenon include obligate parasites which rarely grow in pure cultures,
was not uniformly noted, although eosinophilic inflamma- species of Basidiobolus and Conidiobolus are saprobes.
tion was prominent in all cases. The identification of the Macroscopic (colony morphology) and microscopic (spore
etiologic agent was always done by histology and in some morphology) observations are easy to perform and applica-
cases an additional culture was performed. Remarkably, four ble to the unequivocal identification of both these organisms.
of the seven cases of basidiobolomycosis were preoperatively The formation of asexual, aplanetic, mitospores (also known
diagnosed as cancer, two as inflammatory bowel disease, and as capilliconidia, conidia, sporangia, or sporangiola) can be
one as diverticulitis.32 Another case of gastrointestinal basid- observed on SABG agar.10 In addition to the asexual apla-
iobolomycosis was described by Khan et al. from Kuwait nospores, zygospores can be found for B. ranarum growing
in 2001.33 A 41-year-old male patient, who was otherwise on SABG.38 Since the mitospores of C. coronatus are large
healthy, was taken to a hospital after 20 days of abdominal (around 25 μm diameter) and actively discharged, they can
pain. He was febrile and had a tender and distended abdo- be found on the lid of the Petri dish as a fine white powdery
men with a palpable, nodular mass. Ultrasound confirmed deposit.
the presence of a thick mass in the right iliac fossa. His pros- The observation of fungal hyphae in tissue samples is
tate was enlarged and his colon showed fecal impaction. The more difficult, although many staining methods are available
presumptive diagnosis of intestinal tuberculosis turned out to to visualize them. In addition to staining techniques used
be negative. for light microscopy, which are mostly time-consuming and
After the patient had developed difficulty in passing urine, labor-intensive, fluorescent dyes for fluorescence microscopy
a culture of his urine sample was made. Some days later, provide fast and reliable alternatives to conventional staining
B. ranarum was identified by colony morphology and micromethods. Staining with optical brighteners like Calcofluor
scopic examination.33 (Bayer, Leverkusen, Germany) or Blancophor (Sigma,
These cases demonstrate clearly that nonspecific and Taufenkirchen, Germany) is easy, rapid, and inexpensive.46
diverse symptoms of gastrointestinal basidiobolomycosis Optical brighteners form strong, noncovalent bonds to gly-
may lead to wrong diagnoses followed by medication det- cosides in the fungal cell wall.46,47 Since such glycosides
rimental to the well-being of the patients and fatal in worst- are ubiquitous in fungal cell walls (e.g. chitin) and absent in
case scenarios. human tissue, this method results a high contrast between
The treatment of entomophthoromycosis is highly vari- the fungal tissue and the host tissue, including immune cells.
ably implemented. Amphotericin B, terbinafin, potassium A common feature of infections by Entomophthoralean
iodide, fluconazole, itraconazol, and other antimycotic fungi is the Splendore-Hoeppli phenomenon.10,37,48,49 It is
agents are administered in various doses, combinations, characterized by intensively eosinophilic material surround-
and chronologies.9,10,18,26,39 Although the in vitro susceptibil- ing the pathogen.50,51 To visualize this, different staining
ity testing can show resistant isolates, the cure with these techniques can be used. Staining with methenamine-silver
antifungals may still be achievable.26 An additional surgical nitrate or periodic acid-Schiff (PAS) distinguishes hyphae of
resection of the infected tissue is described in several case fungi well. Staining with hematoxylin and eosin for histology
reports,33,43 especially in cases of gastrointestinal basidiobo- is also possible.
lomycosis.32,34,44,45 The need for surgery has to be carefully Since biopsy material does not show typical structures
assessed in each case taking into account the potential for in all instances, isolation from infected tissue is not always
spreading the infection. successful; alternative methods using samples of serum have
In summary, a fatal outcome of entomophthoromycosis is been developed.
rare and the cure is highly dependent on a positive detection An immunodiffusion test for serodiagnosing has been
of the fungus in biopsy material, using molecular detection developed by Kaufman et al.52 Antigens of the pathogen are
and culture methods. employed to observe interactions with serum of the patient.52
This method is inexpensive and takes just 24 h.
83.1.5 Diagnosis
83.1.5.1 Conventional Techniques Molecular techniques are fast and potentially exact tech-
Infections with C. coronatus are mostly subcutaneous and niques for the identification of fungal pathogens. For the two
restricted to the facial region10,31 whereas those of B. rana- best-known Entomophthoralean pathogens, Conidiobolus
rum cause chronic subcutaneous and peripheral infections coronatus and Basidiobolus ranarum, specific primers are
of the trunk, limbs, buttocks, and arms.31 In addition, there available.53 Both primers are based on specific regions of

the 28S ribosomal DNA and in a polymerase chain reac- containing 0.5% zinc acetate, 0.5% zinc chloride, and 0.05%
tion (PCR); using these primers, there is only amplification calcium acetate in Tris base buffer (pH 7–7.4) at room tem-
of fragments if DNA of the target organism is present. By perature. After fixation, samples can be embedded in par-
employing this method it is possible to use samples from an affin wax. For cultivation analysis, samples should not be
infected region to amplify DNA even though there might be pretreated with formalin or formaldehyde to retain maximum
DNA from the patient or other microorganisms. The result viability of the fungal material. If necessary, samples can be
can be easily visualized by gel electrophoresis in which a stored in 20% glycerol at −20°C. Cultivation of species from
band of expected size can be found in the case of infection both genera should be performed on SABG agar or cornmeal
with the pathogen. Thus, diagnosis without the need for cul- agar (CMA). SABG contains 10 g/L peptone, 100 mM glu-
tivation of the pathogen is possible. The main disadvantage cose, 4 mM MgSO4 × 7 H2O, and 7 mM KH2PO4. CMA con-
of the use of taxon-specific primers is the limitation on the tains 20 g/L cornmeal, 20 g/L peptone, and 20 g/L glucose.
range of detectable pathogens by the availability of specific For solid medium, 15 g/L agar should be added. Optimal
primers. In addition, it may be the case that not all patho- temperature for cultivation is 28°C–34°C. For microscopy of
genic species of the Entomophthorales are described. For the cultured mycelium, hyphae are scraped off and transferred to
genus Basidiobolus, there is another primer available that a slide. To maintain the intactness of the micromorphologi-
is capable of detecting every described species based on a cal structures, a few drops of 10%–35% potassium hydroxide
specific sequence of the internal transcribed spacer (ITS) (KOH) can be added to the slide. Further, optical brighteners
region of the genome (own unpublished data). It has not like Calcofluor white (disodium salt of 4,4′-bis-[Canilino-bis-
been possible, thus far, to design an equivalent primer for the diethylamino-S-triazin-2-ylaminol-2,2′-stilbene-disulfonic
genus Conidiobolus. This is mainly due to the lack of ITS acid; Bayer, Leverkusen, Germany) or Blancophor (Sigma,
sequences in public databases. To increase the concentration Taufenkirchen, Germany) can be added to enhance the con-
of DNA in samples taken from biopsy material, nested PCR trast. A similar rapid method represents the fluorescence
can be performed. Since the primers for both C. coronatus microscopy using Calcofluor white and Blancophor.46 Thin
and B. ranarum are for the 28S ribosomal DNA, the first sections of the material are transferred to a slide and are cov-
amplification step is performed with universal primers for ered with a 0.1% (w/v) Calcofluor white solution. For stain-
this region. The amplicon can then be amplified with the ing with Blancophor, paraffin has to be previously removed
specific primer in a second PCR. from the sample. The paraffin-free sample is incubated for
If cultures of the pathogen are available, sequence analy- 3–30 min in 0.08 mg/mL in Tris buffer followed by two
ses are possible using universal primers to amplify the DNA washes for 1 min each with Tris buffer and embedding in
for specific regions such as the 18S or 28S ribosomal DNA mounting medium.46 Samples should be kept in the dark
and the ITS followed by sequencing. Many commercial since Calcofluor and Blancophor are light-sensitive and not
organizations offer these services. stable at daylight.
A more complex staining procedure for the detection
of fungi in host tissue is a combined staining with methe-
83.2 Methods
namine-silver nitrate and hematoxylin and eosin, accord-
83.2.1 Sample Preparation ing to Huppert et al.55 This method applies a combination
of techniques, which are also parts of other protocols, such
83.2.1.1 Sample Recovery as Grocott’s staining or Gomori methenamine silver (GMS)
Material for the detection of C. coronatus can be taken from stain. For the staining, several solutions have to be prepared.
the nose cavity or other infected area under a local anes- The sample has to be deparaffinized and hydrated in water.
thetic.10 Biopsy material infected with B. ranarum should After incubation for 1 h in 4% chromic acid and a subsequent
be recovered directly from the infected region under general washing step for 1 min with water, the slice is incubated for
anesthesia. 1 min in 1% sodium bisulfite and then rinsed for 5–10 min
Kaufman et al. designed an immunodiffusion assay for the in water. After this, the samples are washed four times with
diagnosis of infections with C. coronatus and B. ranarum.52 distilled water. The section is then incubated at 58°C–60°C
For this assay, serum from the whole blood of the patient has in methenamine-silver nitrate containing 5% silver nitrate in
to be generated in order to raise antibodies against the fungal 20 volumes hexamythelentetramine diluted in equal volume
pathogens. of distilled water and 8% (v/v) 5% sodium borate. The stain-
ing reaction is stopped by rinsing six times in distilled water
83.2.1.2 Sample Preparation for Light Microscopy until the microscopical sections look golden brown (mini-
and Fluorescent Microscopy mum 1 h). The sample is incubated for 4 min in 0.1% gold
Samples for microscopy can be fixed by neutral buffered for- chloride followed by rinsing with distilled water and incu-
malin or 10% formaldehyde. Alternatively, fixation according bation for 4 min in 2% sodium thiosulfate. The sections are
to Beckstead can be performed in order to obtain better pres- washed under running water for 4–5 min and then incubated
ervation of the antigenic surface proteins and morphologi- for 30 min in hematoxylin solution and 2% glacial acetic acid
cal integrity of the fungi.54 In this method, fresh samples of followed by washing for 30 s in running water. For differen-
approximately 3 mm3 are incubated for 8–12 h in zinc fixative tiation, it is now quickly dipped in acid alcohol containing

70% ethanol and 1% hydrochloric acid followed by rinsing other methods, for example, the hexadecyl-trimethylammo-
in water. The sample is finally treated with ammonia water nium bromide (CTAB) method61 or kits for DNA purification
containing 0.1% ammonium hydroxide until it is blue fol- commercially available (e.g., Quiagen plant tissue kit, ana-
lowed by washing under running tap water for 5–10 min. If lytic Jena innuPrep Plant DNA Kit, Invitek InviMag Plant
the differentiation was successful, the fungi can be observed DNA Mini Kit, Macherey & Nagel NucleoSpin Plant L) can
in black and nuclei in blue to blue-black in comparison with a be used.
colorless background. If the differentiation was not success-
ful, the steps starting from the incubation with acid alcohol 83.2.2 Detection Procedures
have to be repeated. For counterstaining, the section is incu-
bated for 30–60 s in eosin–phloxin solution containing 11% 83.2.2.1 Morphological Detection in Pure Cultures
(v/v) eosin stock (1% eosin Y in 95% ethanol), 1% phloxin Cultivation of C. coronatus on SABG agar produces flat fun-
stock (1% phloxin), and 0.5% glacial acetic acid in 95% etha- gal colonies where almost no aerial hyphae can be observed.
nol. Finally, the slice is dehydrated, cleared, and mounted. Large spores of about 10–40 μm diameter are formed and
Alternatively, the detection can be performed via hema- forcibly discharged, resulting in multiple satellite colonies.
toxylin and eosin staining in combination with PAS stain- Macroscopically, the formation of these spores in large quan-
ing; the PAS procedure was described by McManus56,57 and tities results in a white-cream colony with a rough and pow-
Kligman and Mescon.58 For this method, the fixed samples dery texture. In addition, spores can be found adhering to
are first dehydrated and embedded in paraffin and sectioned. the lid of the Petri dish. The broad pleomorphic, essentially
Sections are deparaffinized and dehydrated with absolute nonseptate, hyphae with refractive inclusions and large and
alcohol, washed in distilled water, and then incubated for typically papillate spores formed on the tip of unbranched
5 min in 1% periodic acid following washing under run- sporophores can be easily observed under a simple light
ning tap water for 15 min. The sections are then stained with microscope using 80–400× magnification (Figure 83.1A–F).
Schiff reagent for 10–15 min. Schiff regent is prepared as Cultivation of B. ranarum on SABG agar leads also to
follows: 0.5 g of basic fuchsine is dissolved in 100 mL boil- more or less identical colonies, which are thus not easy to
ing distilled water. After cooling to 50°C, the solution is fil- distinguish from C. coronatus. In contrast to C. coronatus,
tered and 10 mL of 1 N hydrochloric acid and 0.5 g anhydrous cultures of B. ranarum usually dry out and fold the agar sur-
potassium metabisulfite are added to the filtrate. The sec- face. Light microscopy at 80–400× magnification can be
tions are incubated twice in 0.5% potassium metabisulfite in used to observe the broad, essentially nonseptate, although
0.05 N hydrochloric acid and twice in 5% thionyl chloride for rarely septate, hyphae which form short sporophores, which
5 min each, and then washed for 10 min under running water. bulge to form solitary spores (Figure 83.1G–K).
Counterstaining is performed with light green. Finally, the
sections are dehydrated, cleared, and mounted. 83.2.2.2 Morphological Detection in Human Tissue
For detection of hyphae in human tissues, the staining
83.2.1.3 P reparation of Genomic DNA procedures described in Section 83.2.1.2 should be used.
for Molecular Detection Fluorescence microscopy with optical brighteners, performed
Genomic DNA can be purified by the method according at 365–395 nm, enables the hyphal cell wall to be detectable
to Cenis.59 Fungal cultures for DNA preparation should be as a blue region, although spores, which are not formed dur-
cultivated in liquid SABG medium. Filtered mycelium is ing growth in host tissue, cannot be detected. Thus, it is not
ground in a pestle and mortar under constant supply of liquid possible to reliably distinguish between C. coronatus and
nitrogen to fine powder, from which 50–100 mg are trans- B. ranarum, but it helps to discriminate entomophthoromyco-
ferred to a reaction tube with 300 μL lysis buffer containing sis from other zygomycosis. Staining by the method accord-
200 mM Tris–HCl, 250 mM sodium chloride (SDS), 25 mM ing to Huppert et al. visualizes fungal hyphae and cells of
ethylenediaminetetraacetic acid (EDTA), and 0.5% SDS in the host immune system. Fungal hyphae are stained black
accordance to Raeder and Broda.60 The sample is vigorously while nuclei appear dark blue.55 With this method, it is also
stirred for 3 min and then incubated for 10 min at 65°C for possible to detect the asteroid Splendore-Hoeppli reaction,
an increased cell lysis. After an additional 3 min stirring, which is a typical feature of entomophthoromycoses.10,37,48,49
150 μL 3 M sodium acetate at pH 5.2 is added, and the mix The fungal hyphae are surrounded by intensely eosinophilic
is incubated for 10 min at −20°C. After centrifugation at material. This phenomenon is also detectable by PAS and
13,000 rpm for 15 min, the supernatant is transferred into hematoxylin–eosin staining.37
a new reaction tube and an equal volume of isopropanol is
added. The mix is incubated at room temperature for 15 min 83.2.2.3 Immunological Detection
and then centrifuged at 13,000 rpm for 15 min. The pellet is To use the immunodiffusion test according to Kaufman
washed twice with 70% isopropanol, dried, and resuspended et al.,52 specific antigens of C. coronatus and B. ranarum
in 10 mM Tris–HCl (pH 8) or TE buffer, containing 10 mM have to be generated. For that, C. coronatus and B. ranarum
Tris–HCl (pH 8) and 100 mM EDTA (pH 8). The sample can are cultivated in brain heart infusion broth at 37°C under con-
be stored at −20°C until required for PCR-based analysis. stant shaking with 150 rpm. The cultures are inactivated with
This is a rapid and inexpensive procedure. However, also 0.02% merthiolate and filtered through Whatman no. 1 paper

(Whatman, Inc., Clifton, NJ). Afterwards, an additional fil- EDTA) and electrophoretically separated in a electric field of
tering step through membrane with a pore size of 0.45 μm is 5–10 V/cm field strength. The fragments are visualized and
performed and the filtrate is concentrated via ultracentrifuga- photodocumented on a UV transilluminator (wavelength of
tion. Petri dishes with 0.25% phenolized–1% purified agar 321 nm) after staining of the agarose gel in 50 μg/mL ethid-
are used for the immunodiffusion assay. Holes, 4 mm diam- iumbromide solution for 10 min. PCR fragments appear as
eter, are punched in pairs with 4 mm distances from each orange fluorescent bands by intercalating the ethidium bro-
other on the agar, so that one pair of holes corresponds to a mide molecules in the DNA double helix.
single antigen–antibody reaction and is more distantly sepa-
rated from the other pairs. Sera and antigens are applied to 83.2.2.4.1 Taxon-Specific Amplification of
one of each of a pair of wells followed by incubation for 24 h Conidiobolus and Basidiobolus
in a humid chamber at room temperature. Positive antigen– The molecular detection can reliably be performed using
antibody reactions can be observed as white bands in the in vitro amplification of species-specific DNA fragments
agar between each pair of wells. The white bands result from by PCR using oligonucleotide primers inhabiting different
coagulation of the antigen with its respective antibody in the taxonomic specificities (Table 83.1). For the PCR-mediated
serum following a compatible reaction. detection of C. coronatus, an annealing temperature of
55°C and the taxon-specific primers Cc1 and Cc2 targeting
83.2.2.4 Molecular Detection Based on the D1/D2 domain of the nuclear large subunit (LSU, 28S)
Polymerase Chain Reaction ribosomal DNA are recommended. In assays positive for the
All PCR amplification mixtures should contain 1 μL genomic pathogen, a fragment of 419 bp in length can be detected in
DNA (around 50–100 ng), 0.2 mM of each deoxynucleotide the lane respective to the sample. For the species-specific
(from a 2 mM stock solution mixture), 10 pmol of each primer PCR detection of Basidiobolus ranarum, the taxon-spe-
(from 10 pmol/μL stock solutions), 1.5–3.0 mM magnesium cific primers Ba1 and Ba2 are used. The primer pair Ba1/2
chloride, 1–2 units of Taq polymerase (e.g., Dream Taq from detects B. ranarum and B. haptosporus. The PCR tempera-
MBI Fermentas or InnuTaq from Analytik Jena), and a buffer ture profile is much the same like for Cc1/2-primed PCR
system according to the manufacturer’s recommendations. amplifications with the exception of the application of an
The temperature profile of the PCR includes an initial dena- increased annealing temperature (60°C) to prevent false
turation step of 5 min at 95°C, 30 cycles of 0.5 min at 95°C positive fragments in consequence of unspecific primer
to denature the template DNA followed by 30 cycles of 1 min annealing. After separation of the fragments on 1% agarose
at 52°C–60°C for primer annealing, 1 min at 72°C for primer gel, a fragment of 651 bp can be detected in PCR assays
extension, 0.5 min at 95°C for denaturation of the novel syn- positives for both species of Basidiobolus. Considering a
thesized DNA double strands, and a final primer extension putative pathogenic potential of Basidiobolus species other
step of 10 min at 72°C to ensure the completion of double- than B. ranarum and B. haptosporus, an additional taxon-
stranded amplicons. To visualize the PCR fragments after specific primer pair, Bs1 and Bs2, was developed that is
finalization of the PCR temperature profile, the PCR mix specific for the ITS1 and ITS2 region including 5.8S of the
is loaded with one-fourth volume of loading buffer (0.2 g/L nuclear rDNA cluster from the species of the entire genus
bromphenol blue, 0.2 g/L xylene cyanol, 10 mM EDTA, 30% Basidiobolus. For the application of that primer pair, the
glycerol) on a 1% agarose gel (Seakem LE, Serva) in 1× TAE annealing temperature as outlined for the PCR with Cc1 and
buffer (40 mM Tris [pH 7.8], 20 mM sodium acetate, 1 mM Cc2 should be used.
TABLE 83.1
Oligonucleotide Primers for PCR Detection of Entomophthorales
Primer Specificity Target Primer Sequence (5′–3′) Amplicon (bp) Reference
Bs1 Basidiobolus spec. ITS ACTGTTRAMGTATGCTTTGGTAG 465 Authors’s own unpublished data
Bs2 Basidiobolus spec. ITS CTTGCGACGCCTCCAACTAG Authors’s own unpublished data
Cc1 Conidiobolus coronatus 28S rDNA TCTCTTAACTTGCTTCTATGCC 419 Voigt et al. [53]
Cc2 Conidiobolus coronatus 28S rDNA CTTTAATTAAGCTAATCAACATG Voigt et al. [53]
Ba1 Basidiobolus ranarum 28S rDNA AAAATCTGTAAGGTTCAACCTTG 651 Voigt et al. [53]
Ba2 Basidiobolus ranarum 28S rDNA TGCAGGAGAAGTACATCCGC Voigt et al. [53]
NS1 Pan-eukaryotic 18S rDNA GTAGTCATATGCTTGTCTC ∼1140 White et al. [63]
NS41 Pan-eukaryotic 18S rDNA CCCGTGTTGAGTCAAATTA O’Donnell et al. [64]
NL1 Pan-eukaryotic 28S rDNA GCATATCAATAAGCGGAGGAAAAG ∼680 O’Donnell et al. [70]
NL4 Pan-eukaryotic 28S rDNA GGTCCGTGTTTCAAGACGG O’Donnell et al. [70]
ITS1 Pan-eukaryotic TCCGTAGGTGAACCTGCGG White et al. [63]
ITS4 Pan-eukaryotic TCCTCCGCTTATTGATATGC White et al. [63]

83.2.2.4.2 Universal Amplification of Conidiobolus with universal oligonucleotide primers (Cc1/NL4, NL1/Cc2,

and Basidiobolus from Axenic Cultures Ba1/NL4, or NL1/Ba2) in the second step. The detection of
As taxon-specific PCR approaches often do not detect all positive PCR signals is the same as described in more detail
potential pathogenic species, it is advisable to use universal in Section 83.2.2.4.1.
primers for the PCR-driven molecular identification (Table
83.1). A prerequisite for that procedure are pure cultures of
the isolated fungi with pathogenic potential in axenic cultures
83.3 C
onclusions and
in order to eliminate cross-reactions with the host or other Future Perspectives
fungal contaminants. The primer pairs ITS1/4, NS1/41, and Entomopthoralean fungi are distributed worldwide and can
NL1/4 according to the ITS, the nuclear small (SSU, 18S), be found in soil, decaying vegetation, in or on different ani-
and large subunit (LSU, 28S) ribosomal DNA, respectively, mals including insects and mammals, and also in vertebrate
universally hybridize to the ribosomal DNA cluster of all feces. The systematic relationships on an inter- and intraordi-
eukaryonts. Twenty sequences of the ITS from reference spe- nal level are still unclear. Infections with Entomophthoralean
cies for Basidiobolus and 3 ITS sequences of Conidiobolus fungi in human are uncommon in Europe and North America
are available from the International Nucleotide Sequence but widespread in Africa, Asia, Southeast Asia, the Middle
Database Collaboration, whereas just 6 (4/2) entries exist for East, Central, and South America.25 The infections are sub-
the 18S/28S rDNA of Basidiobolus but 16 (11/5) for the SSU/ cutaneous, prolonged but usually painless. Almost no funda-
LSU Conidiobolus, respectively (as of September 28, 2009). mental research has been done. Most articles are based on case
Those nucleotides sequences can easily be used as refer- reports. Since the immune status of the host seems to be less
ence sequences in BLAST searches, targeting regions of the important for the infection, the causes, circumstances, and
nuclear ribosomal DNA as molecular barcodes as outlined preconditions for an infection remain unclear. This situation
by Hoffmann et al.62 The oligonucleotides ITS1, ITS4, and is aggravated by the fact that the pathogenic potential of many
NS1 originate from White et al.63 To overcome the neces- species is not known. Since most Entomophthoralean fungi are
sity of primer walking using internal sequencing primers pathogenic to insects, the correlation between pathothenicity to
for sequencing completion, the reverse primer NS41 should insects and human is yet to be determined. This necessitates an
be used.64 Amplification with primer pair NS1/41 results exhaustive screening with imaging techniques appropriate for
an amplicon of approximately 1.3 kb in size, which can be the detection of infected areas of the bowels for local and deep
directly sequenced through using NS 1 and NS41 as sequenc- Entomophthoralean infections in both humans and insects.
ing primers. The temperature profile for the amplification of Moreover, there are plans for the use of Entomoph
the SSU and the LSU is very the same as used for the taxon- thoralean fungi as biocontrol agent including species of
specific primers besides that the annealing temperature Conidiobolus.14,65 The knowledge relating to the pathogenic
should be 52°C in order to reach a universal amplification of potential of these fungi should help to avoid the use of poten-
the pure fungal DNA. tially harmful fungi in such applications. Diagnostic tools for
the detection of entomophthoromycosis are not well devel-
83.2.2.4.3 Taxon-Specific, Cultivation-Independent oped. This is mostly caused by the rare occurrence of such
Amplification of Species of Conidiobolus infections. The sensitivity of the immunodiffusion test for
and Basidiobolus in Clinical Samples zygomycosis designed by Kaufman et al.52 is around 66%.66
Crude extracts from biopsy material can be used for total However, the sensitivity could be increased by the use of
DNA extraction. In order to gain an increased specificity indirect enzyme-linked immunosorbent assay (ELISA) by
and a decreased cross reaction between the fungal and the which a sensitivity of 81% was achieved.66 This assay has
human background DNA, it becomes advantageous to con- not been demonstrated for Entomophthoralean fungi but only
duct nested PCR approaches. The nested PCR procedure for the mucoralean Rhizopus arrhizus and Rhizomucor pusil-
applies a two-step PCR strategy using the universal amplifi- lus. It is likely that the investigation of this technique would
cation of eukaryotic or fungal DNA in a first and the taxon- improve the detection of entomophthoromycosis. Although
specific amplification of DNA from species of Basidiobolus PCR-based methods are used to the advantage for the detec-
and Conidiobolus in a second step. The first step aims at the tion of fungal infections, there are some major disadvan-
increased accumulation of target DNA, which will be used as tages. Real-time PCR is a standard technique in diagnostics
template in the second PCR. Using the ITS region as molecu- and it has been reported that the sensitivity and specificity is
lar barcode marker, the primer pairs ITS1/4 can be used for increased compared to classical PCR.67 This technique facili-
the enrichment of the ITS region in the first step following tates the rapid and highly sensitive detection of fungal patho-
subsequent amplification with primer pairs Bs1/2, Bs1/ITS4, gens without the need of cultivation, but no real-time PCR
or ITS1/Bs2 in the second step. Using the D1/D2 domain of assay has currently been established for the specific demands
the 28S rDNA (SSU) as molecular barcode marker, the primer of Entomophthoralean fungi. The detection of an infection
pairs NL1/4 may be used for the enrichment of the 5′ region with C. coronatus via real-time fluorescence PCR using a
of the SSU rDNA in a first step following subsequent ampli- broad-range panfungal primer was shown to be sufficient.67
fication with primer pairs Cc1/Cc2, Ba1/Ba2 or combinations In combination with the specific primers for C. coronatus

and B. ranarum described above, this technique could be a 9. Pfaller, M.A. and Diekema, D.J., Unusual fungal and pseu-
big advance toward the reliable and rapid PCR-based and dofungal infections of humans, J. Clin. Microbiol, 43, 1495,
cultivation-independent detection of entomophthoromycoses. 2005.
10. Yang, X. et al., Rhinofacial conidiobolomycosis caused by
For the future, a molecular detection using DNA chips with
Conidiobolus coronatus in a Chinese rice farmer, Mycoses:
specific nucleotide probes specific for the pathogen would be Letter to the Editor, April 2009.
preferable. Comparable procedures and kits for other patho- 11. Larone, D.H., Medically Important Fungi: A Guide to
gens are available.68 Since zygomycoses have become more Identification, Vol. 4, ASM Press, Washington, DC, 2002.
common since the mid-1990s, the reliable diagnostics of such 12. Drechsler, C., An Entomophthoraceous tardigrade parasite
infections become more and more important.69 Moreover, producing small conidia on propulsive cells in spicate heads,
climate changes might affect the distribution of entomoph- B. Torrey. Bot. Club, 78, 183, 1951.
thoromycoses. Infections with Entomophthoralean fungi 13. Wheeler, A.G., “Violent deaths” of soldier beetles
(Coleoptera: Cantharidae) revisited: New records of the
seem to be restricted to tropical or subtropical regions and fungal pathogen Eryniopsis lampyridarum (Zygomycetes:
global warming could thus lead to an increasing number of Entomophthoraceae), Coleopterists Bull., 43, 233, 1988.
infections in, particularly, northern temperate regions, con- 14. Scholte, E.J. et al., Entomopathogenic fungi for mosquito
currently not significantly affected by cases of entomophoro- control: A review, J. Insect Sci., 4, 19, 2004.
mycoses. Since the mode of infection is still unclear, it is not 15. Miller, R. and Pott, B., Phycomycosis of the horses caused by
possible to rule out the influence of a putative insect vector, Basidiobolus haptosporus, Aust. Vet. J., 56, 224, 1980.
which could spread to northern temperate regions as a result 16. Sutton, D.A., Fothergill, A.W., and Rinaldi, M.G., Guide to
Clinically Significant Fungi, The Williams and Wilkins Co.,
of major climatic changes.
Baltimore, MD, 1998.
17. Silva, S.M.M.S. et al., Conidiobolomycosis in sheep in Brazil,
Acknowledgments Vet. Pathol., 44, 314, 2007.
18. Ribes, J.A., Vanover-Sams, C.L., and Baker, D.J.,
We wish to express their gratitude to Paul M. Kirk (CAB Zygomycetes in human disease, Clin. Microbiol. Rev., 13,
International Egham, Surrey, U.K.) for his expert advice and 236, 2000.
critical revision of the manuscript. We would like to thank 19. Beneke, E.S. and Rogers, A.L., Medical Mycology and
Human Mycoses, Star Publishing Co., Belmont, CA, 1996,
Rolf Beutel (Institute of Systematic Zoology and Evolutionary
pp. 182–206.
Biology, University of Jena, Germany) for sharing his zoolog- 20. Mendoza, L. and Alfaro, A.A., Equine subcutaneous zygomy-
ical and cladistic expertise. We are grateful to our colleagues cosis in Costa Rica, Mykosen, 28, 545, 1985.
at the Centraalbureau voor Schimmelcultures in Utrecht (The 21. Morris, R.J., Müller, C.B., and Godfray, H.C.J., Field experi-
Netherlands) for sharing strains of Conidiobolus. We would ments testing for apparent competition between primary para-
also like to thank Claudia Kesselboth for her excellent assis- sitoids mediated by secondary parasitoids, J. Anim. Ecol., 70,
tance in light microscopical observations and for assembling 301, 2001.
Figure 83.1. JR and VS equally contributed to this chapter. 22. Rippon, J.W., Medical Mycology: The Pathogenic Fungi and
Actinomycetes, The W.B. Saunders Co., Philadelphia, PA,
1987, pp. 690–699.
23. Roy, A.D. and Cameroon, H.M., Rhinophycomycosis
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84 Lichtheimia (Absidia-Like Fungi)
Kerstin Hoffmann and Kerstin Voigt
Contents
84.1 Introduction...................................................................................................................................................................... 735
84.1.1 History of the Classification of Absidia-Like Fungi and Lichtheimia................................................................. 735
84.1.2 Chronological Summary of Absidia sensu lato and Its Generic Synonyms........................................................ 739
84.1.4 Diagnosis.............................................................................................................................................................. 740
84.2 Methods............................................................................................................................................................................ 742
References.................................................................................................................................................................................. 745
84.1 Introduction able to parasitize on several other mucoralean fungi (e.g., spe-
cies of the genera Thamnidium, Zygorhynchus, Mucor, and
84.1.1 History of the Classification of Blakeslea), the mycoparasitic potential on compatible host
Absidia-Like Fungi and Lichtheimia species for Le. zychae is not known.6,7 Both species exhibit
features typical for mycoparasites such as low growth rates
Most fungi in the order Mucorales (Mucoromycotina1) are
and thin mycelia with sucker-like branches in the substrate
saprobic and live as ubiquitous inhabitants of soil. Few of
mycelium (see Figure 2A in Hoffmann and Voigt 20096).
them are able to facultatively parasitize on other fungi or
Both species are homothallic, forming their sexual repro-
plants. Furthermore, there are few genera and species that
ductive structures, the zygospores, within a single thallus or
occur as opportunistic pathogens and cause mucormycoses
colony (in contrary to heterothallic species, which cross only
in humans and animals.2–4 The trademark of all mucora-
between two mycelia of compatible mating types designated
lean fungi is the columella, a bulbous and sterile vesicle at
as “plus” and “minus” in the literature). The globose zygo-
the sporangiophore apex, which rejuvenates in a funnel-like
spores appear dark brown with warty exospore and opposed
manner into the apophysis giving the appearance of pear-
suspensors lacking appendages. A recently proposed genus
shaped (pyriform) sporangia typical for the genus Absidia
for these two species, Siepmannia Kwaśna & Nirenberg
(Figure 84.1A, B, M–R).
2008, comprises in addition to the synonyms S. parricida
The traditional morphology-defined genus Absidia
and S. zychae also S. pineti and S. lariceti. However, this
Tiegh. (Etym.: absis, arcus) is one of few genera within
study is based mainly on a single analysis of internal tran-
the order Mucorales comprising all habitats and lifestyles
scribed spacer (ITS) sequences (ITS1 and 2, including 5.8S
mentioned above. But recently, molecular-based phylog-
ribosomal DNA [rDNA]) and morphological comparisons.8,9
enies suggest a trichotomous separation in three distinct
Critical reevaluation of the stated morphological features
genera. This differentiation is supported by morphological
could not assign the two species of Lentamyces with absolute
and physiological traits.5,6 Psychrophilic and mesophilic spe-
certainty to Siepmannia as claimed by Kwaśna & Nirenberg.8
cies with optimal temperatures for growth below 30°C and
Since living cultures of the type strain from Siepmannia
37°C currently belong to the genera Lentamyces K. Hoffm.
are not available, the relationship between Siepmannia and
& K. Voigt 2008 (Etym.: lentus, slow) and Absidia Tiegh.
Lentamyces remains to be investigated.6,8–10
sensu stricto, respectively. Species with an increased opti-
Currently, the genus Absidia sensu stricto comprises sap-
mum temperature around and above 37°C are combined in
robic species displaying the typical Absidia-specific char-
the genus Lichtheimia (Cohn) Vuill. 1903 (Etym.: after
acters (Figure 84.1): sporangia arising from stolons (tillers);
Prof. Lichtheim, Bern, Switzerland).
sporangiophores not opposite the rhizoids; sporangia pyri-
Lentamyces is a genus with saprobic, mycoparasitic, and
form, with apophysis and deliquescent wall; and zygospores
potentially mycoparasitic species and constitutes Le. zychae
surrounded with appendages originating from one or both
and its type taxon Le. Parricida.6 Whereas Le. parricida is
735

(D)
(A) (B) (C) (E)
(F) (H) (J)
(G) (I) (K)
(L) (M) (N) (O) (P) (Q) (R)
(T) (W) (Y)
(U) (V) (X) (Z)
Figure 84.1 Micromorphological characteristics of Absidia sensu lato. (A–E) Absidia sensu stricto. (A) Pyriform sporangia with
apophysis; (B) columellae with apical projection; (C) typical whorl of sporangia; (D) young zygospores with appendages originating from
a single suspensor (left sided); (E) mature zygospores with appendages; (F–K) Lentamyces; (F–H) globose sporangia, not in whorls; (I)
young zygospore; (J and K) mature zygospores, warty exospore, and suspensors without appendages; (L–Z) Lichtheimia; (L) apophysate
sporangium; (M–R) differently shaped columella, predominantly dome-shaped; (T) gametangiogamy, initialization of zygospore forma-
tion; (U) young zygospore; (V) mature zygospore with lipid droplets, suspensors without appendages; (W–Z) variously shaped giant cells.
Scale bar: (A–V) 20 μm; (W–Z) 50 μm.
suspensors.11 The sporangia are commonly multispored and A. cylindrospora, A. fusca, A. glauca, A. heterospora,
possess a columella with an apical projection (Figure 84.1B). A. macrospora, A. pseudocylindrospora, A. psychrophilia,
Homothallic and heterothallic species exists within the and A. repens. Absidia repens may probably represent a
genus. Homothallic species are A. anomala and A. spinosa. complex of different cryptic species as accomplished by
Heterothallic species with plus and minus mating typespe- molecular barcode analyses on different loci.12 The mono-
cific mycelia are A. caerulea, A. californica, A. cuneospora, phyletic genus Absidia sensu stricto is trichotomous divided

Lichtheimia (Absidia-Like Fungi) 737
on its own.5 The subclades are well supported by sporan- a probable misapplication consisting of at least two differ-
giospore morphology and molecular phylogenetic analyses. ent species making M. verticillatus an invalid type taxon for
Globose sporangiospores occur in A. caerulea, A. califor- a family.19,25 The oldest available synonyms described for
nica, A. glauca, and A. macrospora, whereas oval to cylin- Absidia species with naked zygospores were Pseudoabsidia
drical spores are formed by A. anomala, A. cylindrospora, (as “Pseudo-Absidia”26) and Lichtheimia.27 The type taxon
A. pseudocylindrospora, A. psychrophilia, A. repens, and of Pseudoabsidia, P. vulgaris, was originally described as
A. spinosa. Conical shaped spores are present in the interme- Absidia dubia.28 The correction of the misapplied epithet
dia species A. cuneospora, which represents an intermedi- was done by Sydow,29 who recombined P. vulgaris and
ate between the globose and the cylindrical spored groups of Absidia dubia to P. dubia, which was later on transferred
Absidia sensu stricto.5–7,13 The latest species described so far to Lichtheimia as L. dubia.27,30 The type of Lichtheimia is
for Absidia is A. idahoensis,14 probably a misidentification L. corymbifera, described as Mucor corymbifer, a patho-
since A. idahoensis shows greater similarities in morphology genic fungus in rabbits.27,30,31 Lichtheimia is now the type
and molecular analyses to Circinella umbellata than to any genus of the family Lichtheimiaceae K. Hoffm., G. Walther
other species of Absidia (K. Hoffmann, unpublished data). & K. Voigt.25 This family includes the following species:
Together with the sibling genera Chlamydoabsidia Hesselt. L. corymbifera, L. hyalospora, L. ornata, L. ramosa, and
& J.J. Ellis 1966, Cunninghamella Matr. 1903, Gongronella L. sphaerocystis.32 Due to the difficulty of the synonymous
Ribaldi 1952, Halteromyces Shipton & Schipper 1975, and treatment of L. corymbifera, L. ramosa, and L. ornata in the
Hesseltinella H.P. Upadhyay 1970, Absidia is summarized in past and their reerection as distinct species,17,25,32,33 an easy
the family Absidiaceae Arx 1982.5,10,15 separation of species isolated and described during that time
Furthermore, in literature, several species were described is rather difficult. Therefore, isolates assigned to L. corym-
as members belonging to Absidia, which either lack a proper bifera between the years 1974–200932–34 should be handled
description or are now considered as lost ones, because such with caution regarding their current species affiliation.
species were once encountered and described. Consequently, Reliable reference strains (if not type strains) are given under
with respect to genus and species affiliations, the follow- Section 84.1.2 (Table 84.1).
ing species were not considered here: A. scabra Cocc. 1899; In recent years, one new species of Lichtheimia was
A. septata Tiegh. 1876; A. reflexa Tiegh. 1876; A. aegyptia- described—the thermotolerant variety of A. idahoensis,
cum Satory, Meyer, & Tawfik 1939; A. capillata Tiegh. 1876; originating from soil in the Chinese province Yunnan.35
A. clavata B.S. Mehrotra & Nand 1967; A. fassatiae Vánová A. idahoensis var. thermophila displayed many features typi-
1971; A. griseola H. Nagan. & Hirahara 1970; A. inflata J.H. cal for Lichtheimia and its membership in this genus was
Mirza, S.M. Khan, S. Begum, & Shagufta 1979; A. nara- confirmed by molecular analyses, reducing the species to a
yanai Subrahamanyam 1990; A. robusta Raciborski 1899; synonym of L. ramosa.32
A. tuneta Renner & Muskat 1958; A. ushtrina S.C. Ararwal Distinguishing species of Lichtheimia requires profound
1974 (but see Ellis & Hesseltine 1965,16 196617). knowledge and experience in recognition and evaluation of
Species within the genus Lichtheimia display features observed characteristics. Unfortunately, zygomycetes pos-
quite different from Absidia sensu stricto. Although all spe- sess only few characteristics of phylogenetic importance.36
cies currently grouped here were originally placed within Moreover, exclusively through a combination of unique mor-
Absidia, molecular, morphological, and physiological fea- phological features, physiological and biochemical parame-
tures justify their separation in their own genus. One main ters, and molecular or genetic barcodes (nucleotide signature
criterion is zygospores with nonappendaged suspensors. The sequences), a precise identification and taxonomic delimita-
first species described for Absidia with such zygospores was tion is possible. Within the zygomycetous order Mucorales,
A. verticillata18 originally described as Mycocladus verticil- 9 families with 51 genera and 205 species are recognized.37
latus Beauverie 1900. The original culture of Beauverie was Common features of the Mucorales encompass (i) the com-
lost, but Zycha described a similar fungus, which in return monly nonseptated vegetative mycelia (irregularly septae
he named A. verticillata.18,19 Within a reexamination, this predominantly occur near reproductive structures, injured
name was not accepted and changed in A. zychae because of hyphae, or in older cultures), (ii) the occurrence of rhizoids,
morphological differences.20,21 Nevertheless, the aim to dis- and (iii) the reproduction by the formation of sexually pro-
tinguish between morphological and physiological different duced zygospores or various asexual spores produced in
Absidia species resulted in the application of “Mycocladus” differently shaped sporangia or as chlamydospores or arthro-
to species with nonappendaged zygospores and optimum and spores. All these structures could vary in color, size, shape,
maximum temperatures for growth higher than 37°C (pro- or surface texture.20,38,39
posed as subgenus in Absidia by Hesseltine & Ellis 1964,22 But in the course of identification of specimens, one of the
accepted by Schipper 1990,7 and described as separate genus most controversially discussed questions is still the defini-
by Mirza et al. 197923 and Vánová 199124). Based on these tion of the term “species.” Although a “species” is the basal
publications, the thermotolerant species of Absidia were unit in systematics, “species recognition” could rely on dif-
designated as Mycocladus in the family Mycocladaceae (as ferent criteria, for example, biology, morphology, physiology,
“Mycocladiaceae”5). But unfortunately, the morphological ecology, and phylogeny. For fungal systematics, the phylo-
appearance of M. verticillatus gave considerable reason to genetic approach with analyses of several molecular marker

Table 84.1
Chronological Summary of Absidia sensu lato and Its Generic Synonyms, Including Information about Type Strains,
Original Publication and Some Important Taxonomical Synonyms, as well as the Corresponding MycoBank
Number
Species, Current Name Type Strain MycoBank No. Reference Important Taxonomical Synonyms
Absidia sensu stricto

A. repens CBS115583 (IT) MB223578 Van Tieghem11 A. japonica, Mycocladus hyalinus,
Tieghemella repens, T. japonica
A. caerulea NRRL1315 (NT) MB351936 Bainier102 A. orchidis, Mucor saccardoi,
T. caerulea, T. orchidis
A. spinosa var. spinosa NRRL2797 (T) MB224063 Lendner103 T. spinosa
A. cylindrospora var. NRRL1317 (T) MB427391 Hagem104 T. cylindrospora, Mycocladus altaini
cylindrospora
A. glauca NRRL1328 (T) MB221208 Hagem104 A. sphaerosporangioides, A. septata,
T. glauca
A. heterospora NRRL2800 (T) MB252572 Ling-Young105 None
A. fusca NRRL2793 (T) MB252285 Linnemann106 None
A. spinosa var. azygospora NRRL2841 (T) MB346488 Boedijn107 None
A. cuneospora NRRL2632 (T) MB292052 Orr and Plunkett108 None
A. cylindrospora var. NRRL2771 (T) MB348992 Hesseltine and Ellis63 None
rhizomorpha
A. pseudocylindrospora NRRL2770 (T) MB325715 Hesseltine and Ellis63 A. brasiliensis
A. anomala NRRL1807 (T) MB325709 Hesseltine and Ellis22 None
A. psychrophilia NRRL3044 (T) MB325716 Hesseltine and Ellis22 None
A. cylindrospora var. nigra NRRL3060 (T) MB353238 Hesseltine and Ellis22 None
A. spinosa var. NRRL3033 (T) MB348993 Rall and Solheim109 None
biappendiculata
A. californica NRRL2968 (T) MB325710 Ellis and Hesseltine16 None
A. macrospora CBS697.68 (T) MB325712 Vánová110 None
Lichtheimia
L. corymbiferaa NRRL2981 (NT) MB416447 Vuillemin27 (originally as Mucor A. corymbifera, A. ginsan, A. gracilis,
corymbifer by Cohn 1884) A. hesseltinei, A. regnieri, L. regnieri,
Mucor lichtheimii, M. regnieri,
Mycocladus corymbifer
L. ramosaa NRRL1309 (NT) MB416448 Vuillemin27 (originally as Rhizopus A. italiana, A. ramosa, L. ramosa,
ramosus by Zopf 1890) L. italiana, L. italica Mucor ramosus,
Mycocladus ramosus, M. lutetiensis,
Tieghemella italiana
L. hyalospora NRRL2916 (NT) MB512830 Hoffmann et al.25 (originally as A. cristata, A. hyalospora, A.
Tieghemella hyalospora by Saito 1906) merdaria, Mycocladus hyalospora
(for A. blakesleeana, L. blakesleeana,
Mycocladus blakesleeanus, see
Alastruey-Izquierdo et al. 2010)
L. ornataa NRRL10293 (IT) NN Alastruey-Izquierdo et al.32 (originally as
Absidia ornata by Sarbhoy 1965)
L. sphaerocystis NN NN Alastruey-Izquierdo et al.32 None
Lentamyces
L. parricida NRRL2409 (T) MB511980 Hoffmann and Voigt (originally as Absidia Absidia parasitica, Siepmannia
parricida by Renner & Muskat ex parricida
Hesselt. & J.J. Ellis 1964)
L. zychae NRRL2806 (T) MB511981 Hoffmann and Voigt (originally as Absidia Siepmannia zychae
zychae by Hesselt. and Ellis 1966)
Abbreviations: NT, neotype; IT, isotype; T, type; NRRL, Northern Regional Research Laboratories, strain collection of the National Center of Agricultural
Utilization Research Peoria, Illinois; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands.
a …since L. corymbifera, L. ramosa, and L. ornata were synonymous in recent years, a separation of taxonomical synonyms is rather difficult.

regions (multigene genealogies) proved to be a useful method Absidia was described by Van Tieghem11 [MycoBank no.
in species recognition named phylogenetic species recogni- MB20001] and placed in the family Absidiaceae [MB81973]
tion (PSR).40 In this vein, defined species descriptions may be by Von Arx.15 Although this family was treated as a syn-
verified and refined by a combination of PSR with biological onym of the Mucoraceae37,39 [MB81030], its independent
and morphological aspects. status is more than justified (as substantiated by Hoffmann
The classical genus Absidia was defined by its morphol- et al.5; Voigt et al.36; Hoffmann 201010). Tieghemella Berl.
ogy (morphospecies), but by PSR, a trichotomous genus & de Toni (1888) [MB20576] and Proabsidia Vuill. (1903)
separation was recognized, which could be supported by a [MB20441] are now considered synonyms of Absidia sensu
redefinition of characters, which delimit more precisely the lato (discussed in Hesseltine & Ellis 196422) (Table 84.1).
morphological, physiological, and biological features and Lichtheimia was described by Vuillemin27 [MB20308],
which are independent on physiological variances.5–7 With placed within Absidia by Lendner,43 and is now raised to its
the exception of molecular data provided by the PSR con- own family, the Lichtheimiaceae25 [MB508680]. Generic
ception, the species delimitation within Lichtheimia depends synonyms for this genus are Mycocladus Beauverie (1900)
now solely on slight differences in spore morphology and [MB20354], Pseudoabsidia Bainier (1903) [MB20445],
optimum growth temperature treated as a complex trait in and Protoabsidia Naumov (1935) [MB20443].22 Although
the right relation.5,32 L. corymbifera, L. ramosa, and L. ornata were originally
Although the upper temperature limit is not a fixed value described as different species, they were treated as synony-
for all isolates of one species, the observed average is never- mous species,7,34 making it rather difficult separating them
theless of diagnostic importance. Whereas L. corymbifera, again32,33 (Table 84.1).
L. ramosa, and L. ornata show optimal growth (in the mean Since the genus Lentamyces [MB511979] is not closely
of growth velocity) at temperatures above 37°C, L. hyalos- related to any other mucoralean genus, a separation in a
pora and L. sphaerocystis possess an optimum for growth distinct genus is reasonable.6 Whether the proposed genus
below that temperature.5,32 Some isolates of L. ramosa are Siepmannia is a synonymous one, it requires further investi-
capable of growing still at 53°C, whereas the maximum for gation6,8–10 (Table 84.1).
L. corymbifera is reached at 50°C and at 46°C for L. ornata.
L. hyalospora should not grow above 40°C and L. sphaero-
cystis not above 37 (40)°C.32 But these values are mean val-
ues since few isolates of, for example, L. hyalospora (as Although opportunistic infections associated with members
L. blakesleeana) were reported to grow up to 50°C.5 The of the Mucorales are rare if compared to other fungal infec-
reason for the capability of the species L. corymbifera, tions like aspergilloses and candidoses, the constant increase
L. ramosa, and L. ornata and the putative incapability of in reported cases of mucormycoses indicates their impor-
L. hyalospora and L. sphaerocystis to colonize endothermic tance in clinical settings.3,44
organisms is not yet understood. Since opportunistic infections of man and animals require
the capability to grow at least at body temperature, thermotol-
84.1.2 Chronological Summary of Absidia erant fungal species benefit from their physiological possibil-
ities over mesophilic species not capable to grow at elevated
sensu lato and Its Generic Synonyms
temperatures. Whereas all species of Absidia sensu stricto
Since the first description of species of Absidia, several grow only below 40°C, all species of Lichtheimia could
nomenclatural changes and interchanges of synonymies have also grow above this limit, which is suppressive for all non-
occurred That makes it rather difficult to keep track of cur- pathogenic Absidia spp. Although all species of Lichtheimia
rently used and accepted species names. In the following, an are thermotolerant growing at temperatures permissive for
overview is given for the genus Asidia sensu lato including the human pathogenic Absidia-like spp., only few turned out to
current state of family affiliations, species names with a ref- be causative agents of mucormycoses. The first etiological
erence of their original publication, as well as important syn- agent of human mucormycoses was described already by
onyms. For each generic name, an entry in MycoBank (http:// Platauf,45 which is most likely the same pathogenic agent
www.mycobank.org) exists. Mycobank is an online database identified in rabbits as L. corymbifera (formerly Absidia
for the management of the mycological nomenclature includ- corymbifera and Mycocladus corymbifer) 1 year earlier in
ing species descriptions and occasionally illustrations.41,42 1884 by Cohn, who described it as Mucor corymbifer.
MycoBank is associated to the fungal strain collection of the The mode of transmission of Lichtheimia to an efficient
CBS (Centraalbureau voor Schimmelcultures, Utrecht, the causative agent of lichtheimiamycosis does not really dif-
Netherlands, http://www.cbs.knaw.nl) and to IndexFungorum fer from that of any other mucoralean human pathogen.
(http://www.indexfungorum.org). The asexually produced sporangiospores of Mucorales are
To distinguish species with morphological and physiologi- highly airborne and could be easily inhaled leading to pul-
cal differences, several genera were proposed over the years, monary and rhinocerebral infections46 (demonstrated in ani-
which represent now synonyms of the currently accepted mal models by Singh et al.47 and Sondhi et al.48). Also the
genera of Absidia sensu lato, namely, Absidia sensu stricto, spores could dwell on injured skin resulting in (sub)cutane-
Lichtheimia, and Lentamyces (Table 84.1). ous mycosis. But ingestion of spores via contaminated food

is also possible followed up by gastrointestinal infections. In general, the identification of fungal agents down to spe-
Furthermore, a nosocomial transmission via transplanted cies level in pure (axenic) culture is always easier than that
organs, bone marrow, or clinical instruments and person- from infected tissue or liquid cultures. However, the cultiva-
nel is also probable.49–51 Moreover, lichtheimiamycosis tion of the fungal agent on artificial media is not always pos-
caused by L. corymbifera was observed as secondary infec- sible and successful, because it depends on the type and the
tion following immune suppression in a leukemic patient.52 age of tissue conservation, but should not be neglected. As a
Dissemination from the primary spot of infection (metasta- rule of thumb, biopsy material recovered from the infected
ses), invasion of blood vessels (vasculotropism), as well as tissue should be as fresh and native as possible in order to
the development of allergic symptoms or even an asymptom- retain the viability and gain a successful axenic cultivation
atic colonization of the human tissue cannot be precluded. of the fungal agent. Freezing of the biopsy material without
Whereas mycoses after traumata with fungal inoculation are cryoprotectant additives (e.g., glycerol) and fixation in form-
rare, most infections occur in diabetic patients (enforced by aldehyde should be prevented, because these actions dra-
ketoacidosis), in immunocompromised patients or in patients matically decrease the viability of the fungus. Although a
with neutropenia.3,53,54 A probable correlation with voricon- wide range of methods for genomic DNA preparation exist
azole prophylaxis and zygomycoses is also assumed.55,56 The to purify total genomic DNA from pure culture or from
clinical manifestations of lichtheimiamycoses were reviewed infected tissue, the purification of fungal DNA in sufficient
in detail by Ribes et al.2 and Chayakulkeeree et al.57 quality and quantity for a DNA-based identification is still
Since species of Lichtheimia are opportunistic fungi, problematic.
their dominant mode of nutrition is saprobic in their natu-
ral habitat soil and decaying plants displaying a worldwide 84.1.4.1 Conventional Techniques
distribution. Lichtheimia ranks as the second most caus- The traditional identification of all fungal species predomi-
ative agent of mucormycoses,57 causing approximately 5% nantly based on morphological criteria with support by
of the total incidents of human mycoses.44 However, the physiological (mostly growth temperature) or biochemical
mainly morphology-based diagnostics was not supported by (e.g., iodine staining) traits. Since zygomycetes usually do
a DNA-based detection method in most cases, this number is not display many morphological features, the relevance of
likely to increase.55,58 traditional morphological parameters in species recognition
Like all infectious Mucorales, Lichtheimia forms broad is limited. Although most features are of nonmonophyletic
ribbon-like hyphae with wide-angle branchings in the origin and are therefore less relevant in evolutionary phylog-
infected tissue. Although the prevailing understanding of eny, the recognition of monophyletic species is possible.36
zygomycetes is the nonseptated coenocytic mycelium, the Differences are eminent in (i) the mycelium, (ii) its mode
occurrence of septae in hyphae should never exclude the of septation (unseptated, unregularly septated, and septated
diagnosis for a mucormycosis. near reproductive structures or injured hyphae), (iii) the mor-
phology of the sexually formed zygospores (with distinguish-
ing characters as displayed in the texture of the epispor as
84.1.4 Diagnosis
well as the morphology of the appendages and suspensors),
Because of the existence of wide-ranged diagnostic tools for and (iv) the morphology of the asexually formed mitospores
specific causative agents of mycoses other than those main- (sporangiospores, chlamydospores, and arthrospores). The
tained under the term zygomycoses and mucormycoses (e.g., appearance of the asexual reproductive structures is quite
Aspergillus, Fusarium), the awareness to unravel the agents diverse, ranging form variously shaped sporangia and spo-
behind zygomycoses and mucormycoses is accelerating.59 rangiola to merosporangia. Few species produce smaller and
Nevertheless, with the exception of aspects on cultivation, few-spored secondary sporangia in older cultures. The spo-
morphological, and physiological differences, the diagno- rangiophores may differ in their mode of branching, benting,
sis of lichtheimiamycoses still depends on methods used or in their ability for positive phototropism. Furthermore,
also for the detection of mucormycotic agents other than size and color of all structures could vary as well as the over-
Lichtheimia. all colony appearance.61
The diagnosis starts first with the anamnesis elucidating Apart from all disadvantages of morphological over
the history of the patients symptomatology, which is easily molecular identification, a big and undeniable advantage
supplemented by the application of imaging techniques like of morphology is its universal applicability on all scientific
radiography. The interpretation of the radiograms in combi- fields of interest. Independently of physiological, ecological,
nation with the anamnesis requires expertise and experiences evolutionary, or simply diagnostic aspects, morphological
in the differentiation of mycotic proliferation, because symp- features are always easy to access, but require an experi-
toms developed through fungal infections are well known enced eye in terms of the comparability of the observed
to be quite similar over a broad range of fungal agents. characters. All morphological features are prone to envi-
Therefore, additional steps should be considered for a more ronmental changes, for example, decreasing/increasing/
convenient identification, because the therapeutic application arresting mycelial growth, occurrence, size, and appear-
of antifungal drugs harbor dangerous side effects and display ance of reproductive structures by variations in nutrition,
different susceptibilities against fungal species.60 temperature, pH, day–night rhythm, humidity, or salinity.

Nevertheless, all clinical important species of Lichtheimia 4a Densely branched giant cells, 380–760 (−900) × 320–660
grow well in axenic cultures on artificial media. For an (−770) μm, present in 2-week-old YEA (yeast extract agar)
unambiguous detection and identification based on morphol- cultures L. ornata
ogy combined with growth measurements, we recommend
4b Giant-cells absent from 2-week-old YEA cultures
the media used in the respective description keys to avoid
L. corymbifera
ambivalent results.
Besides the constantly updated “Dictionary of the [1]L. hyalospora is now treated as a synonym of L. blakeslee-
Fungi,”37 keys and descriptions specially adapted to zygo- ana.7,32 L. hyalospora was originally separated from
mycetous orders are provided by Zycha et al.,20 Benjamin,38 L. blakesleeana by the formation of larger and unusual hya-
Benny,62 and Benny et al.39 Additionally, the homepage of line mitospores. A prospective new separation of both spe-
Gerald. L. Benny (Department of Plant Pathology, University cies in varieties or formae cannot be excluded.
of Florida, Gainesville, FL; http://www.zygomycetes.org)
offers a highly valuable survey about the taxonomy of zygo- In addition to the possibility of a morphological dis-
mycetes and the literature. The system is constantly updated crimination between L. corymbifera and L. ramosa, the
following the actual developments in zygomycete systemat- utilization of the oligosaccharides melecitose and palati-
ics and research. nose (isomaltulose) as carbon sources by L. ramosa, but not
The genus Absidia sensu lato was described in detail by by L. corymbifera is possible.33 Unfortunately, nothing is
Hesseltine & Ellis/Ellis & Hesseltine16,17,22,63 and Schipper,7 reported about the assimilation of these sugars by other spe-
who also provided several significant keys. cies of Lichtheimia.
Alastruey-Izquierdo et al.32 published a description key to Well-defined and well-described reference strains are
single, accepted species within Lichtheimia. The overall col- stored and maintained in several online-accessible culture col-
ony morphology is represented by rapid and uniform growth lections like ATCC (American Type Culture Collection; http://
at temperatures above 34°C with initially white mycelium, www.lgcpromochem-atcc.com), ARS-NRRL (Agricultural
turning gray in age. Research Service Culture Collection; http://ncaur.usda.gov),
CBS (Centraalbureau voor Schimmelcultures; http://cbs.
Key to the genus Lichtheimia (Vuill. 1903; Lichtheimiaceae knaw.nl), DSMZ (German Collection of Microorganisms
K. Hoffm., G. Walther, & K. Voigt 2009): this key is origi- and Cell Cultures; http://www.dsmz.de), or the JMRC (Jena
nally published in Alastruey-Izquierdo et al.32; pictures are Microbial Reference Centre; http://www.prz.uni-jena.de).
presented there. The morphology was assessed on malt Many of the reliable reference strains are given in the
extract agar (30 g/L MEA). Section 84.1.2. Type strain material has the important advan-
tage of an unambiguous and functional attribution of a spe-
1a Sporangia dark brown or dark gray to black; colony diam-
cies description and to an individual microorganism—a fact
eter after 72 h at 43°C <2 mm; mature sporangiospores rough
which should always be taken into account in species identi-
and/or >6.5 μm in their longest extension 2
fication. Information about type strains can be retrieved from
1b Sporangia light brownish gray; colony diameter after MycoBank (http://www.mycobank.org).
72 h at 43°C >14 mm; mature sporangiospores smooth and If the cultivation of infectious material is impossible or
<6.5 μm in their longest extension 3 too time-consuming, the existence of zygomycete contami-
nants in infected tissue can also be proven in liquid samples,
2a Giant-cells consistently globose, 60–150 μm in diameter
scrape or swab tests, as well as biopsy tissue. In combina-
L. sphaerocystis
tion with appropriate staining techniques, hyphae typical for
2b Giant-cells (if present) more hypha-like, irregularly swol- Lichtheimia becomes detectable directly in infected tissue.
len, simple to strongly branched, never consistently globose However, these tests depend on the locality of the sample
L. hyalospora[1] drawing. If the biopsy needle did not hit directly the infected
tissue, the detection might be not always positive. False nega-
2ba Mature sporangiospores small (<5.5 μm), rough, and
tive detection may also be achieved in cases with observed
brownish small-spored variants of L. hyalospora[1]
septae matching the hyphal morphology of ascomycetes.
2bb Mature sporangiospores larger (on the majority >5.5 μm),
smooth or rough, hyaline or brownish large-spored variants 84.1.4.2 Molecular Techniques
of L. hyalospora[1] One and probably the largest disadvantage of the single applica-
tion of molecular data is its inability to unequivocally identify
3a Colony diameter at 43°C after 72 h >40 mm (average
a fungal organism if used as a single criterion without the com-
growth rate of 1.3 mm/h, growth rate range 0.5–3.2 mm/h);
bination of morphological observations. The more additional
spores ellipsoidal to cylindrical or subglobose to broadly
information about the morphology, physiology, or biochemis-
ellipsoidal L. ramosa
try or even whole-genome data exist, the more proper and reli-
3b Colony diameter at 43°C after 72 h <27 mm (average able is the identity and species designation of a specimen.
growth rate of 0.4 mm/h, growth rate range 0.1–1.0 mm/h); But, with a deliberate choice of the molecular markers
spores never consistently ellipsoidal to cylindrical 4 used for species identification and a well-established reliable

database, which consists of well-defined reference speci- mucormycosis-inducing agents in multiplex PCR75 and dif-
mens, even a single molecular marker can result in a high ferences in gene expression as revealed by real-time PCR.76
approximation of the identity of the fungal agent in question. However, none of those methods are explicitly adapted for
However, such databases do not enable the identification of the molecular detection of lichtheimiamycosis. Until now, no
new or undescribed species. Therefore, a close collaboration reliable serological detection method is available for routine
is recommended between clinical laboratories, research insti- use in zygomycete detection.77
tutions, and reference laboratories, which not only enables Several online-accessible databases of the International
early detection of novel fungi with pathogenic potential but Nucleotide Sequence Database Collaboration store and main-
also facilitates the establishment of a platform for new or tain sequence information from fungal strains. Open access
improved methods. allows free up- and download of generated sequence mate-
One basic and in most diagnostic laboratories well- rial, but requires very high responsibilities on underlying
established method is the polymerase chain reaction (PCR)64,65 background information about the fungal organisms used for
aiming at the amplification of DNA fragments, which local- data generation. Some databases are DBBJ (DNA Database
ize between two short oligonucleotide primers. One univer- of Japan; http://www.ddbj.nig.ac.jp), EMBL (European
sally applicable molecular marker useful for the identification Molecular Biology Laboratory; http://www.ebi.ac.uk/embl),
of most fungi including zygomycetes is the region between GenBank at NCBI (http://www.ncbi.nlm.nih.gov), PDB
the nuclear small subunit (SSU) rDNA (18S rDNA) and the (Protein Data Bank; http://www.rcsb.org/pdb), and UniProt
nuclear large subunit rDNA (28S rDNA). The variable regions (Universal Protein Resource; http://www.expasy.uniprot.org).
of the ITS1 and ITS2 include also the 5.8S rDNA, which is Approximately 20% of the sequences currently deposited at
highly conserved in fungi. Without evolutionary pressure, the GenBank may be not annotated correctly.78 Thus, BLAST
ITS region is highly heterogenous at the above-species level results should be handled with care and verified by the align-
and in zygomycetes at the below-species level.12,66 Therefore, ment to reference sequences from database platforms spe-
the ITS is a suitable barcode marker for species discrimina- cialized on clinically relevant fungi (see also Section 84.3).
tion and recognition for all Mucorales including Lichtheimia.
However, the application of ITS in species recognition is
tedious. The ITS is homogenous at the above-species level and 84.2 Methods
not suitable for the species discrimination in Penicillium.67
Although the ITS region possesses few disadvantages aris-
ing from its repetitive nature and discontinuities in sequence Direct microscopy of samples like scrapings, smears, blood,
homogeneity among individual elements at the infraindi- or sputum obtained from patients or biopsy material from
vidual level, it has been widely used in the characterization infected tissue could be used to visualize the characteristi-
of type and reference strains. Consequently, a wide range of cally unseptated hyphae typical for Lichtheimia and all other
ITS reference sequences are accessible in public databases. Mucorales, though just in material sufficiently charged with
If compared with ITS sequences of unknown species desig- fungal material. But fungal hyphae observed here do not
nation, reliable reference sequences play an essential rule in allow a differentiation down to species level. Fluorescence
the validity of BLAST searches. The majority of reference staining combined with optical brighteners like Calcofluor
sequences target the ITS. Thus, it is not remarkable that ITS or Blancophor79 facilitates the universal identification of
markers are discussed as the golden standard for molecular Mucorales.
barcodes suitable for the reliable species identification of Cultures could be grown from scrapings or smears
zygomycetes including Lichtheimia 68 (recommended by the obtained from patients or from liquids containing fungal
Clinical and Laboratory Standards Institute, CLSI 200769). material. However, cultures from blood, urine, sputum, or
In this chapter, the term molecular barcoding is treated as bronchoalveolar lavage are less successful.80,81 The utiliza-
synonym for the terms molecular identification or molecu- tion of biopsy samples for the inoculation of media should be
lar detection of fungal taxa, because all procedures have in handled with caution, because a forceful crushing of tissue
common the use of specific nucleotide sequences as molecu- could easily destroy fungal hyphae causing a false negative
lar markers or barcodes in order to gain genetic signatures, result in axenic cultures. Because hyphae of zygomycetes
which are specific for a certain taxon, for example, genus-, lack regular septation, a reliable and successful inoculation of
species, or pathotype-specific. culture media on Petri dishes is not guarantied.3,81 Species of
Alternative genetic markers are the adjacent loci of the Lichtheimia as well as other mucoralean fungi grow well on
nuclear SSU and LSU rDNA (18S, 28S) or exonic sequences MEA (30 g/L), PDA (potato dextrose agar; DSMZ media no.
of single- till few-copy protein-coding genes encoding actin 129; http://www.dsmz.de), or Sabouraud glucose agar (20 g/L
(act), translation elongation factor 1 alpha (tef),5,70–72 or the glucose; e.g., Carl Roth, Karlsruhe). These media could eas-
high affinity iron permease (FTR).73 Apart from nucleotide ily be supplemented with a cocktail of single or multiple
sequence-based detection, additional methods, which gen- antibiotics (e.g., ampicilline, streptomycine, tetracycline,
erate DNA polymorphisms, were also proposed including neomycine, or chloramphenicol in final concentrations of 1.9,
restriction length polymorphism procedures74 or variations 1.2, 0.6, 1.4, and 1.4 mg/mL, respectively) in order to effi-
of PCR targeting the multiple species-specific detection of ciently suppress bacterial growth. Each individual medium

will influence the overall appearance of the growing fungal oil conserves the morphological features and viability of
colony and is to be evaluated during the course of identifica- fungi.89,90
tion. Fungal colonies should be cultivated at different tem-
peratures to ensure their optimum (and maximum) growth.
The standard procedure for observations on the phenotype of
fungal cultures should be applied, where possible.82 Purification of genomic DNA could either be performed
Microscopic observations on fungal colonies grown in from pure cultures or directly from tissue material. If using
axenic cultures should be done using a binocular dissecting tissue-purified fungal DNA, the genomic target has to be fun-
microscope for the assessment of the colony morphology and gal specific because the preparation does not discriminate
for micromorphological observations on the reproductive between human and fungal DNA.
structures (sporangia, chlamydospores, etc.). Under optimum DNA extraction can be manually performed using dif-
conditions, the overall appearance of colonies of Lichtheimia ferent kits, which are commercially available, for example,
is white at the beginning, turning gray in age. The colony the DNeasy Plant Kit series (Qiagen, Venlo, the Netherlands)
is rapidly and uniformly growing with an uncolored reverse or the innuPREP Kit series (Analytik Jena, Jena, Germany).
side. The latter one is also available for automatic purification
Micromorphological observations were carried out and may also be used for tissue embedded in paraffin. The
from pure fungal cultures grown on solid agar media. The innuSPEED Kit series (Analytik Jena, Jena, Germany) are
preparation of the microscopic slides should be done with also suitable for DNA extraction in combination with a tis-
juvenile and proliferating cultures in order to obtain young, sue homogenizator or speed mill distributed by the same
spore-bearing sporangia and to observe the periphery and company. In order to increase yield and quality of the puri-
growth front of the colony. Either a “tape prep” or a “tease fied fungal DNA from human samples, improved lysis of
prep” preparative procedure facilitates the display of mor- the fungal hyphae is recommended during grounding the
phological features. The “tape prep” method applies the mycelium in liquid nitrogen or bead-mediated maceration
adhesive side of a tape to fix parts of a fungal colony. The of the mycelium in a speed mill prior proceeding with the
tape is then placed sticky sidedown on a slide for micros- purification steps. If the DNA purification has to be done
copy. The “tease prep” includes careful preparation of parts from formalin-fixed, paraffin-embedded tissue samples, the
from the fungal colony in a drop of lactophenol cotton blue, potential degradation of DNA during the fixation process has
which is covered with a coverslip and used for micros- to be considered.
copy. The latter method is more recommended because it A rapid protocol for fungal DNA based on a modified
ensures a higher biological safety. Another method is the method was described by Cenis.91 This method is optimized
traditional slide culture. Here a sterile coverslip is placed for pure fungal cultures. The mycelium is frozen and ground
on agar medium prior to fungal inoculation. After inocula- to a fine powder in liquid nitrogen. Approximately 100 mg of
tion the fungus will partially grow over the coverslip. The fungal mycelium is suspended in extraction buffer (200 mM
coverslip can be removed and subjected to microscopy. All Tris–HCL [pH 8.5]; 250 mM NaCl; 25 mM EDTA [pH 8.0];
microscopic preparations can be stained with lactophenol 0.5% sodium dodecyl sulfate [SDS])92 and vigorously mixed
cotton blue dye solution to gain a higher contrast during for 3 min. After incubation at 65°C for 10 min and repeated
lightmicroscopical observation.2 All observations have to agitation, an amount of 0.15 mL 3 M sodium acetate (pH 5.2)
be compared to the descriptions and keys presented here or is added and tubes are placed for 10 min at −20°C. Separation
elsewhere.7,17,20,21,32 of the DNA is achieved at 13,000 rpm for 15 min and followed
For direct histopathological microscopy of biopsy mate- by precipitation of the supernatant with an equal volume of
rial, the probes should be fixed in neutral buffered or 10% isopropanol. After incubation for 15 min at room tempera-
buffered formol saline83 and embedded in paraffin for the ture, and centrifugation for 15 min at 13,000 rpm, the DNA
preparation of microscopic sections in a microtome. A fixa- pellet is washed twice in 70% isopropanol. The air-dried
tion method with less toxicity and improved survival of anti- DNA pellet is resuspended in up to 50 μL TE buffer (10 mM
gens for immunohistochemistry is described by Beckstead84 Tris–HCl [pH 7.6]; 1 mM EDTA). Storage of the DNA is pos-
using zinc as primary fixative. sible at −25°C.91 The amount of DNA for successful ampli-
Tissue sections could then be stained with hematoxylin fication in a PCR depends on the complexity and number
and eosin. For verification of fungal infections in the tissue, of genomic copies of the desired region in the DNA sample
staining with Grocott’s methenamine-silver nitrate, periodic used as template for PCR. Average concentrations from 1 pg
acid-Schiff or similar fluorescence staining methods with for plasmid DNA till 1 μg for mammalian DNA are required
optical brighteners are useful.85–87 Tissue samples, which per PCR reaction.93 Alternatively, the DNA extraction may
were not embedded in paraffin, could also be viewed for fun- be carried out using CTAB (hexacetyltrimethylammonium
gal hyphae after homogenization in a tissue grinder in 0.9% bromide; Sigma) as described by Voigt et al.70 and modified
NaCl.2,79 Even simple phase-contrast microscopy is sufficient by Schwarz et al.68
for the detection of zygomycetous hyphae.88 Amplification of the ITS as target region (ITS1–5.8S
Samples for cultures could not be treated with formalde- rDNA–ITS2) is conducted using the universal primers
hyde. But preservation in 10% glycerine or sterile mineral ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4

(5′-TCCTCCGCTTATTGATATGC-3′).94 For fungus-spe- barcode-based designation to a species or even the molec-

cific amplification of the ITS, the fungal universal primers ular barcode-based identification of a new species rather
V9D (5′-TTAAGTCCCTGCCCTTTGTA-3′) and LS266 tedious. Such thresholds are under discussion12,97 but will
(5′-GCATTCCCAAACAACTCGACTC-3′) are appropri- never replace taxonomical expertise, experimental experi-
ate as well.95 One PCR assay typically contains 2–6 μg ence, and exploration of additional data.
genomic DNA of the fungal pathogen, 10 pmol of each Schwarz et al.68 proposed a protocol for rapid analysis of
primer, 0–4 mM MgCl2; 5 μL of 2 mM dNTPs (Fermentas), zygomycetes directly from infected tissue. Amplification and
10× PCR DreamTaq™ Buffer (Fermentas), and 1.25 U of sequencing of ITS turned out to be a reliable method for spe-
DreamTaq™ Polymerase (Fermentas) in a total volume of cies identification in animal models. If this method is also
50 μL. Polymerases with 3′–5′ exonucleolytic proofreading reliable in human, tissue samples need to be tested.
activity, for example, Pfu polymerase (Fermentas), are rec- Molecular methods for the generation of sequences
ommended in order to obtain amplicons with low error rate. of fixed and already embedded tissue samples are appli-
PCR fragments are amplified using a FlexCycler (Analytik cable with varying success. Prior to PCR, the fixed tissues
Jena, Jena, Germany) and the following temperature pro- need to be washed with xylene and 95% alcohol in order
file: an initial denaturation step of 5 min at 95°C following to remove the wax. The samples are then equilibrated in
30 cycles of denaturation for 30 s at 95°C, annealing for 60 s buffer (Tris–EDTA, SDS, proteinase K) and incubated at
at 55°C, and primer elongation for 60 s at 72°C, with a final 55°C for 24 h under constant shaking.76 With a subsequent
primer extension reaction for 4 min at 72°C. PCR amplicons LightCycler platform-based real-time PCR assay, the detec-
may be purified by adsorption on glass particles (GeneClean tion of Lichtheimia was principally possible but resulted in a
II, BIO 101, Vista, CA) based on a protocol of Vogelstein & decreased sensitivity for fixed tissue samples.76
Gillespie96 if an intermediate cloning step is required prior
sequencing of the fragment. The PCR assay is spiked with 84.3 C
onclusions and
a threefold volume of 6 M NaI (Roth, Karlsruhe, Germany)
Future Perspectives
and 5 μL glass milk particles (MP Biomedicals Heidelberg,
Germany). After vigorous agitation, the tubes are incubated Mucormycoses seem to be highly abundant during systemic
at 55°C for 5 min under occasional gentle agitation. The glass and rhinocerebral infections. The clinical manifestation
particles are spun down for 10 s and washed twice, each wash- of mucormycoses is advanced by airborne transmission in
ing step with 200 μL New Wash (50% Ethanol, 20 mM Tris– patients with an underlying disease.44 Recently, an increase
HCl [pH 7.6], 1 mM EDTA, 0.1 M NaCl). The DNA is eluted of incidents for cutaneous lichtheimiamycoses98 as well as an
twice from the air-dried glass particles each with 10–20 μL increasing number of nosocomial infections in hospitals were
bidistilled water at 65°C for 10 min. The glass particle-free reported.99 The growing awareness of life-threatening zygo-
eluate is stored at −25°C until performance of the PCR. mycoses and their causative agents makes a taxonomic dis-
Cycle sequencing is performed using the Big Dye™ fluo- crimination of the infectious agents down to the species level
rescent-labeled terminator DyeDeoxy protocol with AmpliTaq indispensable. Mycologists and taxonomists keep constantly
polymerase (Applied Biosystems) and single oligonucleotide going on renaming and recombining fungal species in order
PCR primers as sequencings primers. Reaction products are to gain an improved systematics, which is concordant with
analyzed on an ABI Prism™ 310 automated sequencer apply- phylogenetic relationships. That mannerism makes it very
ing a one-capillary system (Applied Biosystems). For high- difficult for the personnel of medical laboratories to keep
throughput sequence analyses, the application of alternative track of the changes. Nevertheless, its necessity becomes
equipment is recommended, for example, a combination of more and more accepted also by nontaxonomists especially
the SpeedCycler (Analytik Jena AG, Jena, Germany) and clinicians, because the phylogenetic relationships will help
the ABI 3730xl DNA analyzer applying a 96-capillary block to understand the similarities and differences observed not
system (Applied Biosystems) together with adapted protocols only at the morphological and physiological level but also
are adequate methods to reduce pure analytic time to less at the level of the susceptibility to antifungal agents with
than 1 h. Sequences are edited and manually manipulated consequences to treatment and therapy. Therefore, the
using Chromas Lite version 2.01 (Technelysium, Helensvale, separation of Absidia sensu lato in three distinct genera
Queensland, Australia). not only allows the establishment of a monophyly-based
Sequence analysis for species identification is done by natural system but also facilitates the distinction of harm-
BLAST searches against available reference databases of less and noncausative Absidia-like genera and their species
the network from the International Nucleotide Sequence (Lentamyces, Absidia sensu stricto) from harmful Absidia-
Database Collaboration, for example, GenBank. Sequence like genus (Lichtheimia) with species possessing pathogenic
similarities of 100% correlate with a highly supported potential. Nomenclatural changes did yet not ease the course
approximation in species identification. Values below of identification of the specimens. Although new and fast
100% should be treated with care, because the designation methods exist to identify fungal organisms, the tradition-
to a distinct species is less confirmed. The lack of thresh- ally morphology-based methods should not be neglected.
old definitions for sequence similarity makes a molecular Just a combined application of both strategies will withdraw

disadvantages of the individual methods if applied in single For example, the comparison of isolates from Absidia repens
analyses. The concatenation of colony- and micromorpho- and other Absidia-like fungi revealed enormous differences
logical criteria with growth physiological and molecular in the ITS sequences of isolates from different continents of
markers (genetic signature sequences, carbon source utili- the northern hemisphere. That variability highly correlates
zation, and other chemotaxonomic profiles) will inevitably with the geographic distribution.12 In order to avoid such
result in an increased reliability of the identification and the variances, a wide taxon sampling is required over a broad
discrimination between nonpathogenic species from patho- range of individual isolates recovered from different patients
genic species belonging to Lichtheimia. and hospitals in different geographic localities. The more
Although the classical phenotypic (morphological) all-embracing that survey is, the more reliable and power-
approach of species identification by morphological crite- ful the molecular diagnostics will be. The more criteria other
ria is still essential for definite new species assignments, it than molecular markers will be used in combination with the
has been proven to be error-prone for daily routine identi- methods of molecular identification, the higher the resolution
fication because its outcome is subjective and it is costly in and dependability in the species discrimination.
terms of time and labor.55 In most cases, the fungal agents After successful evaluations of the intraspecific variabil-
need to be identified directly in the infected tissue and ity, the long-term storage of the involved fungal specimens
the proof by an axenic culture is often missing. But most and type material in culture collections and the reexamina-
fungi, also Lichtheimia, develop their typical morphol- tion of the maintained specimens are essential prerequisites
ogy just in pure cultures, and several modes of molecular for research on the distribution and the epidemiology as well
detection are less time-consuming and are therefore pref- as the evolution of fungal speciation.
erably used over classical morphological identification. Although the number of available methods for molecular
However, molecular detection methods are cost-intensive detection is relatively low and not fully taxon-discriminative
and rely on consistent reference material. The lack of pur- within zygomycetes, their diagnostic value imply a powerful
suant platforms providing congruent data about clinical supplement of classical methods traditionally used in species
symptomatology and fungal type material hamper the dis- identification of Absidia-like fungi including Lichtheimia.
covery of new species. Consequently, molecular data can One major breakthrough toward the achievement of an
solely account for an approximation on the fungal identity, increased diagnostic power of molecular markers would
whereas morphological traits can support and verify the probably be the establishment of a pan-fungal DNA chip-
diagnosis. But in cases of a successful cultivation of the based assay for all clinical important fungi.68,100,101
fungus, the incidence of possible air-borne contaminations During the clinical daily routine analysis of Lichtheimia
during cultivation and false-positive signals in molecular and its discrimination from harmless Absidia-like fungi or
detection due to fungal spores inhaled or ingested prior other harmful human pathogens, the decision between a
sample recovery has to be eliminated. Both air-borne con- detection-first or a therapy-first action is influenced by the
taminations and incorporated fungal spores lack clinically celerity and accuracy of the method of choice. Thus, risks
relevant manifestations. and feasibility of a probable rapid proliferation have to be
Another problem arises from the fact that for many patho- meticulously calculated if suspicion for a lichtheimiamy-
genic fungi the number of available isolates is limited. The cosis is entertained. As long as the spectrum for applicable
deprivation of morphological and molecular information antifungal chemotherapy is limited and not fungus-specific,
from a broad range of different isolates leads to a deprived a profound determination of the exact fungal species of
and inadequate assessment of the intra- and interspecies vari- Lichtheimia will just play an ancillary role in order to serve
ability. Morphological and growth physiological criteria tend the patient’s benefits. Consequently, with the development of
to develop intraspecific variations. However, especially sugar novel and more species-effective antifungal drugs accom-
assimilation profiles provide easy and cheap methods for panied with advancement of rapidity and precision of the
the discrimination of L. corymbifera, which does not utilize molecular diagnostic methods, the prognosis of lichtheimia-
melecitose and palatinose (isomaltulose) as carbon sources, mycosis will markedly be improved.
and L. ramosa, which does so.33 Apart from the elucidation of
the utilization of these sugars by two species of Lichtheimia,
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85 Mortierella
Tamás Papp, Kerstin Hoffmann, Ildikó Nyilasi, Tamás Petkovits,
Lysett Wagner, Csaba Vágvölgyi, and Kerstin Voigt
Contents
85.1 Introduction...................................................................................................................................................................... 749
85.1.2 Clinical Features, Pathogenesis, and Epidemiology............................................................................................. 750
85.1.3 Diagnosis...............................................................................................................................................................751
85.2 Methods............................................................................................................................................................................ 752
85.2.1.1 Samples for Direct Microscopic Investigation........................................................................................753
85.2.1.2 Samples for Morphological Investigation...............................................................................................753
References.................................................................................................................................................................................. 755
85.1 Introduction the Mortierella isabellina group described by Zycha et al.11

Antigenic properties of their extracellular polysaccharides12
Members of the genus Mortierella are saprobic zygomy- and their sterol and fatty acid composition13,14 supported this
cetous fungi belonging to the order Mortierellales. Among separation. Phylogenetic analysis of 18S and 28S rDNA, act,
them, the thermophilic Mortierella wolfii is recognized as a and tef sequences demonstrated that Umbelopsis–Micromucor
pathogen, being an important casual agent of bovine mycotic represents a basal divergent group of Mucorales.4,5,8 Later,
abortion, pneumonia, and systemic mycosis (mortierellosis based on internal transcribed spacer (ITS) rDNA sequences,
or mortierellomycosis). a new monophyletic family, Umbelopsidaceae, belonging to
the Mucorales, was proposed for this group.15
The large genus Mortierella has been divided into “sec-
The genus Mortierella (Mortierellaceae, Mortierellales) is the tions” based on the grouping of presumed related spe-
largest genus of zygomycetous fungi with about 100 species cies.10,11,16 However, sequence comparison of the ITS regions
recognized. It was placed under the family Mortierellaceae of more than 80 independently obtained isolates suggested
and originally treated as a family of the traditionally circum- a variety of natural groups with monophyletic origin and
scribed Mucorales1,2 but was later excluded and placed in revealed that the traditional morphology-based classification
the newly proposed Mortierellales.3 The status of this clas- of Mortierellaceae is highly artificial and further molecular
sification was supported by results from several molecular studies are needed to resolve the phylogeny of this taxon.17
phylogenetic studies based on ribosomal RNA gene (rDNA) The genus contains the animal pathogen M. wolfii, which
sequences4–7 and sequences of the protein-coding genes for differs from the other species of Mortierella in its thermophilic
actin (act)8 and the translation elongation factor 1α (tef).5,8 nature. This species was placed in the section Mutabilis11
These studies detected a deep dichotomy between the two together with M. mutabilis and M. sterilis. In a recent phylog-
orders and thus confirmed the new classification. eny inferred from sequences of the ITS region, the small and
At present Mortierellales contains one family, the large subunit rDNA genes and a fragment of the tef gene of
Mortierellaceae, comprising six genera—Aquamortierella, several strains of Mortierella, Gamsiella, and Dissophora and
Dissophora, Gamsiella, Lobosporangium, Modicella, and strains of M. wolfii formed a distinct, well-supported clade,
Mortierella.9 Within the genus Mortierella, Gams10 distin- which could be clearly distinguished from the other species of
guished two subgenera, Mortierella subgen. Mortierella and Mortierella using the determined molecular markers.18
Mortierella subgen. Micromucor (syn. Umbelopsis), based on Species of Mortierella generally produce unusually fine
morphological characters. The latter subgenus also contained or cottony, coenocytic or irregularly septate mycelium in
749

which anastomosing hyphae may occur. They produce small 85.1.2 Clinical Features, Pathogenesis,
sporangiospores in one- to many-spored, globose sporan- and Epidemiology
gia. Columellae are often absent or rudimentary and never
protrude into the sporangium. The lack of pronounced Although some early publications reported sporadic cases of
columellae together with non-apophysate sporangia are primarily cutaneous infection of humans caused by M. wolfii,
diagnostic characters of members of the Mortierellales and these reports are suspected to be due to possible misidentifi-
distinguish them from Mucorales.19 They may form smooth- cations and this species of Mortierella is actually not consid-
or rough-walled chlamydospores and zygospores, if known, ered as a human pathogen.22 However, M. wolfii is regarded
are smooth or dimpled with generally apposed and unequal as a true animal pathogen22 and has been reported as an
suspensors.9,20 agent of bovine mycotic abortion, pneumonia, and systemic
M. wolfii form white to grayish white colonies, which, mycosis in New Zealand,23,24 Australia,25,26 Japan,27 Great
depending on the culturing conditions, often have a broadly Britain,24 and the United States28,29 (Table 85.1).
zonate (rosette-like) surface appearance (Figure 85.1). The col- Unlike other types of mycotic abortions, 20% of cows
onies may have a garlic-like odor, characteristic of other spe- that abort as a consequence of M. wolfii infection will sub-
cies of Mortierella. The fungus is thermophilic; its maximum sequently develop fatal mycotic pneumonia and occasionally
temperature of growth is 48°C. It has 90–250 μm long spo- other systemic mycoses.29 In addition to the lungs, systemic
rangiophores, which can be simple or branched immediately mycosis may involve damage to the uterus, the liver, and
below the apex. Sporangia are small (15–50 μm in diameter), the brain; histology often reveals lesions, necrosis, destruc-
multispored, and without a columella, and after deliques- tion of the blood vessels, and hemorrhage in the affected
cence, a conspicuous collar can frequently be observed at the organs.27,29,30 Concerning the pathogenesis of M. wolfii, caus-
apex of the sporangiophore (Figure 85.1). Sporangiospores ing bovine abortion and pneumonia, it has been suggested that
are short, cylindrical to reniform, and 6–10 × 3–5 μm in size. the fungus first infects the lungs and subsequently reaches
Chlamydospores of varying shape but often carrying short the uterus via the blood stream, where it causes endometritis
appendages (often referred to as stylospores) may be present; and abortion. After abortion, depending on the degree of the
zygospores have never been observed.20,21 damage to the uterine blood vessels, the fungus may reenter
(a) (k)
(b) (i) (j)
(c) (d) (l) (m)
(e)
(f) (g) (h) (n) (o) (p)
FIGURE 85.1 Lobed growth of Mortierella sp. (i) Micromorphology of Mortierella wolfii (a–h). Sporangiophores can be simple or
branched subterminally, tapering from the base to the tip (b–e, g–h). Sporangia are without a columellae, leaving a conspicuous collar
after deliquescence at the apex of the sporangiophore (c–d). Sporangiospores are short and cylindrical to reniform (a). Chlamydospore
with short appendages (f). Micromorphology of Mortierella polycephala (j–p). The sporangiophores possess a broadened base and taper
to the apex; branches occur mostly in the upper part of the sporangiophores; the sporangia are spherical, leaving a collar at the apex
of the sporangiophore after dehiscence (j, n–p). Sporangiospores are oval to irregular in shape (k). Stylospores are produced on stalks,
verrucose to echinulate (l–m). The lengths of scale bars indicate 10 μm for the panels c, d, f, k, and l and 25 μm for the panels a, b, e, g,
h, j, and m–p.

Mortierella 751
TABLE 85.1
Strains of M. polycephala and M. wolfii Available from Public Culture Collections
Including Information about MycoBank Number, Taxonomically Important Synonyms,
and Substrate of Isolation
Strain, Current Name, MycoBank Numbers Taxonomic Synonyms
Culture collection numbers Substrate of isolation
M. wolfii Mehrotra & Baijal 1963, [MB334519] Actinomortierella wolfii
CBS209.69 Coal spoil tip soil
CBS611.70, IMI149020, IP1706.87 Cow, lung, dying from mycotic pneumonia
CBS612.70 Decayed hay
CBS614.70 Decayed hay
CBS552.93 Unknown
CBS651.93 Compost for mushrooms
ATCC24296 Unknown
ATCC36820 Stomach contents of an aborted bovine fetus
ATCC64116 Cattle, Australia
M. polycephala Coem. 1863, [MB145769] M. raphani, M. vantieghemi var. raphani, M. polycephala
var. raphani, M. vantieghemi, M. polycephala var.
canina, M. canina, M. crystallina, M. lemonnieri
CBS293.34 Lupinus
CBS227.35, FSU696 Unknown
CBS456.66, DSMZ1212, FSU759 Dung of wood mouse
CBS649.68, ATHUM2571, IHEM3807, NRRL A-12030 Unknown
CBS327.72, FSU866 Salt-marsh soil under Spartina townsendii
CBS328.72, FSU867 Soil
the circulation and cause embolic pneumonia and infections in Japan.27 Apart from Aspergillus spp., M. wolfii was found
in other organs.29 to be the second major fungal pathogen responsible for
Strains of M. wolfii may produce a heat labile (at 60°C) 15%–20% of this condition in Australia.25,34 In New Zealand,
and trypsin-sensitive nephrotoxin, which is active over a M. wolfii is considered as the major and most common cause
wide pH range31–33 and is able to cause death in experimental of bovine mycotic abortion.29,35 In Europe, Mortierella was
models. It has been suggested that this toxin is responsible frequently reported in Great Britain since 196736 and even
for most of the symptoms caused by the fungus.22 However, more recently.24,37,38
when only the toxin was administered to rabbits and mice, A mild/warm and wet climate and frequent use of grass
it damaged primarily the kidneys and its effects proved to silage for feeding are thought to be the main factors resulting
be distinct from those associated with M. wolfii infections. in the high incidences in some regions.22,25,29 It is generally
Thus, the possible role of the toxin in natural infections suggested that transmission of the fungus occurs primarily
remained unclear.33 by inhalation of spores originating from moldy silage, but
Species of Mortierella are ubiquitous and are among transmission via infected semen or ulcerations in the alimen-
the most common fungi in the soil.20 M. wolfii has a world- tary tract were also suggested by certain authors.22,39
wide distribution and is common in the soil in tropical and In earlier studies, single cases of animal mycoses were
subtropical regions of the world. In the temperate zone, it reported as caused by some other species of Mortierella,
may be found in thermophilic environments, such as over- e.g., M. polycephala, M. alpina, M. hyalina, and M. zychae.
heated, spoiled silage and hay. Infections caused by M. wolfii However, the maximum growth temperature of these species
have been reported from almost all regions of the world but is below 36°C, and, thus, it is likely that the identifications of
with very different frequencies. Previous reports recorded the causal agent associated with these cases were incorrect
that more than 60% of cases of bovine mycotic abortion and were most probably misidentifications of M. wolfii.20,40
in the United States were caused by Aspergillus fumiga-
tus, but Zygomycetes accounted for about 20% of the total 85.1.3 Diagnosis
cases followed by a wide range of opportunistic fungi and
yeasts involved in the remaining 20% cases.28 Concurrently, 85.1.3.1 Conventional Techniques
M. wolfii has been identified in 0.5% of all bovine mycotic A serological assay was described for detection of M. wolfii in
abortions in whole North America29 and only occasionally cows41 but no other Zygomycetes were included in this study

although similar antigens in other species and genera were Nevertheless, use of such databases could not identify new,
reported earlier.42 Nevertheless, M. wolfii appears to possess undescribed species, enforcing further methods to be applied
fewer antigenic similarities when compared to Mucorales,43 in collaboration work with scientific research/reference
but no serological tests are applied in routine labs. laboratories.
The advantages, disadvantages, difficulties, and require-
85.1.3.2 Molecular Techniques ments of molecular applications are discussed in Chapter 34
In addition to conventional techniques, molecular-based and by Balajee et al.44
methods present very promising approaches to identify the The molecular method of choice is currently the com-
fungi involved in human and animal diseases. Although parative sequence-based identification of fungi.45 Based on
molecular approaches are often regarded as sole identifica- amplification and sequencing of short DNA regions followed
tion methods, relying on them in isolation should be avoided. by comparison with available reference sequences. Although
Accepting the advantage of their easy performance because the first polymerase chain reaction (PCR) application on fun-
they can be carried out following standardized protocols, gal infections in animals was to identify M. wolfii,29 no stan-
these methods are not able to identify all species with dardized recommended method and no reference database
unquestionable certainty. Nevertheless, reference databases exists so far for Mortierella. In addition, GenBank currently
of molecular markers could provide a resource through which has only a few relevant sequences available (Table 85.2).
a very close approximation to the identity of the investigated
organism could be reached. However, such databases would
be required to fulfill several prerequisites: 85.2 Methods
1. The reference specimens should encompass a vari-
ety of isolates of the same species from different Since infections with M. wolfii are only confirmed for ani-
locations so as to include possible population and mals, the methods described here were not commonly applied
geographical variations. in diagnosing animal infections and could therefore only be
2. The molecular marker of choice should have a assumed to be applicable in detection of human infections
high-level of interspecies variation to differenti- with this fungus. Infections of animals are mainly discov-
ate between species and a low-level of intraspecific ered after death, an option not acceptable in human infec-
variation. tions. However, as there is no prior experience of detection
3. The amplification of the marker must be easy by the of M. wolfii in human, the following methods are only an
application of universal primers to avoid debarment adaptation from commonly known and applied methods for
of key player specimens. detection of zygomycosis.
4. The database must encompass more than the target In combination with any method applied for detection of
species to ensure the correct species is identified. fungal infection, a test for infections caused by bacteria and/
TABLE 85.2
Nucleotide Sequences of M. polycephala and M. wolfii Available from GenBank at http://www.ncbi.nlm.nih.gov
Species GenBank Acc. Numbers Gene Region Strain Collection Numbers Reference
M. polycephala AB476414 ITS NBRC 6335 H. Sekiguchi, A. Masunaka, Y. Hashimoto, and
S. Takenaka (unpublished)
AJ287169 Actin, partial NRRL22890 Voigt and Wöstemeyer8
X89436 18S rRNA, partial Unknown K. O’Donnell (unpublished)
AF157261 Tef, partial NRRL22890 Voigt and Wöstemeyer8
AF113464 28S rRNA, partial NRRL22890 Voigt et al.4
M. wolfii AJ287171 Actin, partial CBS611.70, NRRL28640 Voigt and Wöstemeyer8
AB154774 // 28S rRNA, partial IFM47051, CBS651.93 // H. Miyano, S. Endo, N. Obara, M. Haritani, N.
AB154775 // IFM52979, CBS611.70, Tanimura, K. Kimura, A. Sano, K. Yokoyama, K.
AB154776 IMI149020 // Kamei, M. Miyaji, and K. Nishimura (unpublished)
IFM52980, CBS612.70
Note: Accession numbers of the sequenced region, strain numbers of the strain collection, and references are also indicated.
Abbreviations: NBRC, NITE Biological Resource Center (NITE, National Institute of Technology and Evaluation), Japan; NRRL, Northern Regional Research
Laboratories, Strain Collection of the National Center of Agricultural Utilization Research Peoria, IL; CBS, Centraalbureau voor Schimmelcultures,
Utrecht, the Netherlands; IFM, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan.

Mortierella 753
or viruses is also recommended so as to assist in determining required to maintain the viability of hyphal fragments.54,55
if the fungal infection is the primary or secondary causative Species of Mortierella grow well on a wide variety of artifi-
agent of the observed disease. cial media, e.g., MEA (30 g/L malt extract agar), PDA (potato
dextrose agar; DSMZ media no. 129; http://www.dsmz.de),
85.2.1.1 Samples for Direct Microscopic Investigation OA (oatmeal agar; DSMZ media no. 425), Sabouraud glucose
If a tissue displays mycotic lesions, samples should be taken agar (20 g/L glucose; e.g., Carl Roth, Karlsruhe, Germany),
under sterile conditions, avoiding secondary inoculation or BHIA (brain heart infusion agar; DSMZ media no. 215,
with environmental microorganisms. Most infections with supplemented with 5% sheep blood). Each medium, however,
Mortierella were described in animals rather than humans will have an influence on the overall appearance of the fun-
and were observed after fetal abortion. Therefore, biopsy gal colony, and, thus, for confident identification or during
samples from the fetus are the main material for exami- examination by inexperienced investigators, media used in
nation. The biopsy material should be subjected to direct the published descriptions and keys are recommended for
microscopy or observed after appropriate staining. Using culturing.11–56 In addition these media can be supplemented
simple phase-contrast microscopy on smears from the tis- with antibiotics to suppress bacterial growth, applied single
sue or direct tissue samples ground in a homogenizer should or in combinations (e.g., ampicillin, chloramphenicol, neo-
reveal the presence of fungal hyphae.22,46,47 Careful prepara- mycin, tetracycline, or streptomycin in final concentrations of
tion of small tissue samples on a microscopic slide and stain- 1.9, 1.4, 1.4, 0.6, and 1.2 mg/mL, respectively). Furthermore,
ing with Parker ink in potassium hydroxide (KOH) can also incubation should be at various temperatures to evaluate the
be used to display fungal structures. cultures under optimal and maximal growth conditions. The
Samples should also be embedded in paraffin after fixa- cultures should also be observed and maintained for 4 weeks
tion in 10% or neutral buffered formalin saline.48 The use of to account for slow growing fungi. Where possible, the stan-
zinc as primary fixative improves the survival of antigens dard procedure for phenotypic observations of fungal cul-
and therefore allows for immunohistochemistry.49 Sections tures should be applied.21
prepared with a microtome are then stained with hematoxylin The macromorphological appearance of M. wolfii and M.
and eosin (H&E) for general histopathological examination polycephala are illustrated in Figure 85.1, where the typi-
and with periodic acid Schiff reaction (PAS) and Grocott’s cal undulate or wavy growth as found in various species of
methenamine silver stain (GMS) for the investigation of fun- Mortierella can be seen. This typical lobed growth is not
gal hyphae. Other fluorescent stains are also possible like present in every isolate of a species and may not form on all
the optical brighteners Calcofluor and Blankophor.47,50,51 types of media. Colonies often produce a garlic-like odor.11
Although these staining techniques are not specific for the The members of the order Mortierellales are described as
genus Mortierella or even for other species of Zygomycetes, forming coenocytic, anastomosing, and often arachnoid veg-
specific characteristics of this group should be observable and etative hyphae and sporangiophores, which are commonly
provide a means of excluding, e.g., ascomycetous fungi like awl-shaped and characterized by a reduced columella, which
Aspergillus. Sufficiently infected tissue will show the char- is either septum-like or slightly convex. The sporangial wall
acteristically nonseptate hyphae of Zygomycetes. Although is fugacious and the zygosporangia are hyaline with apposed
some septa may be observed typically associated with spore- suspensors.57
bearing structures or in old vacuolated hyphae, they are not The sporangiophores of the type species of Mortierella,
as regularly distributed as in the hyphae of Ascomycetes. M. polycephala, arise single or in tufts. They possess a
Distinguishing features like asexual or sexual reproductive broadened base and taper to the apex; rhizoid-like structures
structures are not very likely to be produced in infected tis- are sometimes formed at the base. Branches are arranged
sue. Therefore, in addition to the direct, first investigation of racemosely and occur mostly in the upper part of the spo-
suspected tissue, the underlying species should be identified rangiophores; secondary or tertiary branching is possible.
by further methods based on morphological and/or molecu- The sporangia are spherical, 37–75 μm, leaving a collar at
lar data. the apex of the sporangiophore after dehiscence. The spo-
rangiospores are smooth, oval to irregular in shape, and
85.2.1.2 Samples for Morphological Investigation 5.5–13 × 5.5–11 μm in size. Stylospores (sporangiola) are pro-
Samples for axenic cultures should not be treated with form- duced on stalks and are single spored and verrucose to echi-
aldehyde or other harsh fixatives as this action will reduce nulate. Chlamydospores occur intercalary or terminally and
viability. Like any other terrestrial fungi, Mortierella can are typically smaller than sporangiospores. Zygospores are
also be stored via cryopreservation using spores and hyphae surrounded by a dense hyphal network and the suspensors
in 10%–20% glycerol or in active cultures overlaid with are slightly unequal. At temperatures above 13°C only stylo-
mineral oil like it is done by strain collections for short- and spores may be encountered, whereas sporangia are produced
long-term conservation of viability as well as preservation of abundantly at low temperatures11,21,56 (Figure 85.1).
morphological features.52,53 Axenic cultures should be grown The sporangiophores of M. wolfii often arise from rhi-
from any infected material containing spores or hyphae but zoids formed in the aerial mycelium. The sporangiophores
the tissue samples should not be forcefully crushed because are broad at the base and taper to the apex, mostly branched
the hyphae of Zygomycetes posses less septa, which are subterminally. The branches arise cymosely, mostly in

whorls, sometimes displaying a helicoid appearance just gov), such a reference database is far from being available
below the tip. Secondary branches are possible. The sporan- and molecular identification is thus only possible in a rudi-
gia are spherical, 11–68 μm, leaving a prominent reflexed mentary way. Based on the available sequences of the small
collar at the apex of the sporangiophore after dehiscence; ribosomal subunit (SSU), a restriction map for molecular
secondary sporangia are smaller. The sporangiospores are diagnosis is proposed in the Atlas of Clinical Fungi21 and
smooth, ellipsoidal to reniform, and 2–5.5 × 3.5–13 μm in augmented here (Figure 85.2). Generating new sequences
size. Chlamydospores are infrequently described and are of including amplification, sequencing, and sequence analysis
various shapes with lobed appendages. No stylospores are from isolated specimens is also described in Chapter 34.
produced. Zygospores are unknown. Colonies of M. wolfii
are cottony, white, fine, and often with a garlic-like odor 85.3 C
on grass compost media. Good growth at 37°C, maximum
Perspectives
at 48°C. Isolates originate from materials characterized by
elevated temperatures, e.g., rotten silage, hay, silage casing With a growing awareness to life-threatening zygomycoses,
soil, and coal spoil tips11,21,56 (Figure 85.1). past and future case reports will gain importance. The infec-
Micromorphological observations should be made on fun- tious agents belong to the Mucorales, Entomophthorales,
gal colonies bearing slightly immature, spore-containing spo- and Mortierellales with most agents in the first order, lead-
rangia. Older sporangia are very likely to loose their spores ing to the term “mucormycosis.” This was further supported
during slide preparation. With an adhesive tape, fungal struc- because in early years most infections were originally identi-
tures can be directly fixed from the colony. The tape is then fied as species of the genus Mucor. However, later research
placed sticky side down on a microscopic slide (“tape prep”). revealed that the most important causal agents belong to
For observation of fungal structures at higher resolution, a the genus Rhizopus (reviewed by Ribes et al.22 and Roden
few parts of the colony are carefully removed and placed in a et al.60). Therefore, zygomycosis is currently applied with a
drop of lactophenol cotton blue and covered with a coverslip somewhat overlapping synonymous relationship to mucor-
(“tease prep”). The high-contrast staining with cotton blue mycosis. But referring to zygomycosis caused by a variety
makes observations somewhat easier. Using the traditional of different genera in one sentence with mycosis caused by
slide culture is another option. Here, a sterile coverslip or single genera from the Ascomycetes termed, e.g., candido-
sterile cellophane is placed on top of the medium prior to sis or aspergillosis will not help solving the problem of cor-
inoculation. The fungus eventually grows on the coverslip rectly referring to the diversity of disease-causing species.
or cellophane, which can then be removed and subjected to Naming the mycosis after the causal genus will probably help
direct microscopy.22,58 The observed morphological charac- to broaden the insight into their epidemiology. This problem
teristics have to be compared with the original description of is already addressed like rhizomucormycosis due to infec-
the presumed fungus.11,56,59 tions with Rhizomucor61 or lichtheimiamycosis caused by
Lichtheimia corymbifera (former Mycocladus [ex Absidia]
corymbifer[a], Chapter 34).
Determination of the correct species causing the mycosis
The preparation of genomic DNA for any molecular analysis will also reveal specimens with pathogenicity to humans or
should be performed directly from infected tissue or from animals only.
pure cultures. The methods currently available for DNA Although M. wolfii and M. polycephala are reported
extraction are extensively described in Chapters 23 and 34. as pathogenic to animals, some reasonable doubts may be
Purifying DNA from tissue samples may not be as effective entertained about the clinical relevance of M. polycephala as
and reliable in terms of diagnostic signal as from pure fungal causative agent of mortierellomycoses. Exclusively, a single
cultures since host DNA will also be retrieved. Furthermore, case was reported due to the infection with M. polycephala,62
the DNA of the host will be in much higher concentration which appears to be undoubtedly a misidentification. In that
compared to that of the fungal DNA, thus requiring a method report, few morphological traits suggest M. wolfii, e.g., the
with high fungal sensitivity for DNA extraction and subse- production of chlamydospores and the double-walled spo-
quent identification of the pathogen. This can be achieved rangiospores. However, the fungus was reported to grow
by fungal-specific oligonucleotide primers targeting genomic well at 37°C, clearly above the accepted maximal growth
regions that are unique for fungi. The primers of choice for temperature for M. polycephala, which is below 30°C, and
identification of Mortierella are not easy to define. First of thus below body temperature.20 On the contrary, M. wolfii is
all a reliable reference database is needed, which is acces- able to grow well at body temperature, although confirmed
sible by the investigator and shared with the scientific com- infections in humans have not been documented.63 Currently,
munity as a whole in order to make the results transparent. M. wolfii is thought of as a sole pathogen to animals, but the
Such a database should contain information about as many awareness of its potential to infect humans also should be
specimens as possible regarding molecular data and infor- continued.
mation related to, e.g., isolation, distribution, morphology, Although molecular methods for identification of fungi
physiology, and ecology. Since there are only few sequences gain importance, traditional methods should not be neglected.
available in GenBank (Table 85.2; http://www.ncbi.nlm.nih. Although there are still fungal infections reported that are

Mortierella 755
Sau3AI
HaeIII
HaeIII
HinfI
HinfI
RsaI
M. wolfii, 1727 bp
Sau3AI
Sau3AI
Sau3AI
HaeIII
HaeIII
Sau3AI
HaeIII
HaeIII
HinfI
HinfI
HinfI
HinfI
HinfI
RsaI
RsaI
RsaI
RsaI
M. polycephala, 1719 bp
RsaI
HaeIII
RsaI
(A)
HaeIII
HaeIII
RsaI
HinfI
RsaI
M. wolfii, 715 bp
HaeIII
HinfI
HinfI
HinfI
HinfI
RsaI
RsaI
Sau3AI
HaeIII
HaeIII
(B)
Sau3AI
Sau3AI
HaeIII
HaeIII
HaeIII
HinfI
HinfI
RsaI
RsaI
M. wolfii, 807 bp
Sau3AI
Sau3AI
Sau3AI
HaeIII
HaeIII
HinfI
RsaI
RsaI
RsaI
Sau3AI
Sau3AI
Sau3AI
RsaI
(C)
FIGURE 85.2 Restriction maps for molecular diagnosis of M. wolfii and M. polycephala. Restriction enzymes used for the endonucleolytic
digestions are: HaeIII, HinfI, RsaI, and Sau3AI. Restriction sites present in both strains are indicated between both schematic sequences,
whereas unique restriction sites are displayed aside the schematic nucleotide sequences. (A) Restriction map based on the sequences NCBI
AF113425 (M. wolfii) and X89436 (M. polycephala) coding for 18S rRNA. (B) Restriction map based on the sequences NCBI AF113465,
AB154774, AB154775, AB154776 (M. wolfii), and AF113464 (M. polycephala) coding for 28S rRNA. (C) Restriction map based on the
sequences NCBI AJ287171 (M. wolfii) and AJ287169 (M. polycephala) coding for actin (cDNA).
identified solely through histopathology, making the same critical revision of the manuscript. We would like to thank
mistake using solely molecular identification methods should our colleagues at the Centraalbureau voor Schimmelcultures
be avoided. The diagnosed specimen should always be sup- in Utrecht (the Netherlands) for sharing strains of Mortierella.
ported by morphological identification since histopathology This project was supported by a bilateral grant between
or molecular markers are not able to determine the cause of Hungarian Scientific Research Fund (OTKA-NN75255) and
an infection to species level. Molecular-based identification German Research Foundation (DFG; project no. Vo 772/9-1).
will only be an approximation as long as whole-genome com- TP and KH contributed equally to this review.
parisons are not possible. Nevertheless, morphology alone is
also not error free, and thus in combination with all other
available methods previously unrecognized species could References
be revealed. Until now the molecular detection of M. wolfii
1. Hesseltine, C.W. and Ellis, J.J., Mucorales, in: The Fungi, an
and its related species is accomplished in few designated Advanced Treatise, vol. IVb. A Taxonomic Review with Keys:
laboratories worldwide. A publicly available database may Basidiomycetes and Lower Fungi, Ainsworth, G.C., Sparrow,
serve as a platform, which unifies different methods, strate- F.K., and Sussman, A.S., Eds., Academic Press, New York,
gies, and techniques of molecular detection in combination pp. 187–217, 1973.
with traditional identification in order to make the expertise 2. Benjamin, R.K., Zygomycetes and their spores, in: The Whole
more transparent and shape the reliability of the diagnostics Fungus, The Sexual-Asexual Synthesis, vol. 2, Kendrick, B.,
toward an increased screening for mortierellomycoses in Ed., National Museums of Canada, Ottawa, Canada, pp. 573–
616, 1979.
clinical environments. 3. Cavalier-Smith, T., A revised six-kingdom system of life,
Biol. Rev., 73, 203, 1998.
Acknowledgments 4. Voigt, K., Cigelnik, E., and O’Donnell, K., Phylogeny and
PCR identification of clinically important Zygomycetes based
We wish to express their gratitude to Paul M. Kirk (CAB on nuclear ribosomal-DNA sequence data, J. Clin. Microbiol.,
International Egham, Surrey, U.K.) for his expert advice and 37, 3957, 1999.

5. O’Donnell, K. et al., Evolutionary relationships among muco- 25. Connole, M.D., Review of animal mycoses in Australia,
ralean fungi (Zygomycota): Evidence for family polyphyly on Mycopathology, 111, 133, 1990.
a large scale, Mycologia, 93, 286, 2001. 26. Gabor, L.J., Mycotic pneumonia in a dairy cow caused by
6. Kwaśna, H., Ward, E., and Bateman, G.L., Phylogenetic rela- Mortierella wolfii, Aust. Vet. J., 81, 409, 2003.
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rDNA sequences, Mycol. Res., 110, 501, 2006. wolfii, Mycopathology, 101, 89, 1988.
7. White, M.M. et al., Phylogeny of the Zygomycota based on 28. Knudtson, W.U. and Kirkbride, C.A., Fungi associated with
nuclear ribosomal sequence data, Mycologia, 98, 872, 2006. bovine abortion in the northern plains states (USA), J. Vet.
8. Voigt, K. and Wöstemeyer, J., Phylogeny and origin of Diagn. Invest., 4, 181, 1992.
82 Zygomycetes from all 54 genera of the Mucorales and 29. Munday, J.S. et al., Meningoencephalitis in an adult cow due
Mortierellales based on combined analysis of actin and trans- to Mortierella wolfii, J. Vet. Diagn. Invest., 18, 619–622, 2006.
lation elongation factor EF-1a genes, Gene, 270, 113, 2001. 30. Uzal, F.A. et al., Mortierella wolfii isolated from the liver of a
9. Benny, G.L., Zygomycetes, published on the Internet at http:// cow in Australia, Vet. Rec., 145, 260, 1999.
www.zygomycetes.org, 2005, accessed on November 11, 31. Davey, G., Smith, J.M., and Kalmakoff, J., Purification and
2009. properties of a toxin isolated from Mortierella wolfii, Infect.
10. Gams, W., A key to the species of Mortierella, Persoonia, 9, Immun., 8, 882, 1973.
381, 1977. 32. Davey, G. and Kalmakoff, J., Evidence that the nephro-
11. Zycha, H., Siepmann, R., and Linnemann, G., Mucorales. Eine toxin from the fungus Mortierella wolfii is a protein, Can. J.
Beschreibung aller Gattungen und Arten dieser Pilzgruppe, Microbiol., 20, 1513, 1974.
J Cramer, Lehre, 1969. 33. Corbel, M.J. and Edades, S.M., Observations on the experi-
12. de Ruiter, G.A. et al., Approaches to the classification of the mental pathogenicity and toxigenicity of Mortierella wolfii
Mortierella isabellina group: Antigenic extracellular polysac- strains of bovine origin, Br. Vet. J., 147, 504, 1991.
charides, Mycol. Res., 97, 690, 1993. 34. McCausland, I.P., Slee, K.J., and Hirst, F.S., Mycotic abortion
13. Amano, N. et al., Chemotaxonomic significance of fatty in cattle. Aust. Vet. J., 64, 129, 1987.
acid composition in the genus Mortierella (Zygomycetes, 35. di Menna, M.E., Carter, M.E., and Cordes, D.O., The identi-
Mortierellaceae), Mycotaxon, 44, 257, 1992. fication of Mortierella wolfii isolated from cases of abortion
14. Weete, J.D. and Gandhi, S.R., Sterols and fatty acids of the and pneumonia in cattle and a search for its infection source,
Mortierellaceae: Taxonomic implications, Mycologia, 91, Res. Vet. Sci., 13, 439, 1972.
642, 1999. 36. Hugh-Jones, M.E. and Austwick, P.C.K., Epidemiological
15. Meyer, W. and Gams, W., Delimitation of Umbelopsis studies in bovine mycotic abortion. I. The effect of climate on
(Mucorales, Umbelopsidaceae fam. nov.) based on ITS incidence, Vet. Rec., 85, 273, 1967.
sequence and RFLP data, Mycol. Res., 107, 339, 2003. 37. MacDonald, S.M. and Corbel, M.J., Mortierella wolfii infec-
16. Domsch, K.H., Gams, W., and Anderson, T.H., Compendium tions in cattle in Britain. Vet. Rec., 109, 419, 1981.
of Soil Fungi, vols. 1, 2, Academic Press, New York, 1980. 38. Johnson, C.T., Lupson, G.R., and Lawrence, K.E., The bovine
17. Nyilasi, I. et al., Phylogenetic and physiological characteriza- placentome in bacterial and mycotic abortions, Vet. Rec., 12,
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Acta Microbiol. Immunol. Hung., 56, 219, 2009. 39. Smith, J.M., An interesting bovine mycotic complex in New
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2009. production of food ingredients, such as arachidonic acid,
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structural and phylogenetic characterization of zygomyce- 41. Skilbeck, N.W., Serological diagnosis of bovine abortion due
tes from different ecological habitats and climatic regions: to Mortierella wolfii. Aust. Vet. J., 61, 238, 1984.
Limitations and utility of nuclear ribosomal DNA barcode 42. Kaufman, L., Turner, L.F., and McLaughlin, D.W., Indirect
markers, in: Current Advances in Molecular Mycology, enzyme linked immunosorbent assay for zygomycosis,
Gherbawy, Y., Mach, R.L., and Rai, M., Eds., Nova Science J. Clin. Microbiol., 27, 1979, 1989.
Publishers, Inc., New York, pp. 263–312, 2009. 43. Hessian, P. and Smith, J.M.B., Antigenic characteriza-
20. Schipper, M.A.A. and Stalpers, J.A., Zygomycetes: The order tion of some potentially pathogenic mucoraceous fungi,
Mucorales. in: Pathogenic Fungi in Humans and Animals, 2nd Sabouraudia, 20, 209, 1982.
edn., Mycology Series, vol. 16, Howard, D.H., Ed., Marcel 44. Balajee, S.A. et al., Sequence-based identification of
Dekker, New York, 2002, pp. 67–125. Aspergillus, Fusarium and Mucorales species in the clinical
21. de Hoog, D.S. et al., Atlas of Clinical Fungi, 2nd edn., mycology laboratory: Where are we and where should we go
Centraalbureau voor Schimmelcultures, the Netherlands, from here?, J. Clin. Microbiol., 47, 877, 2009.
2000. 45. Summerbell, R.C. et al., Microcoding: The second step in
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Zygomycetes in human disease, Clin. Microbiol. Rev., 13, 360,1897, 2005.
236, 2000. 46. Roberts, G.D., Detection of fungi in clinical specimens by
23. Carter, M.E. et al., Fungi isolated from bovine mycotic abor- phase contrast microscopy, J. Clin. Microbiol., 2, 261, 1975.
tion and pneumonia with special reference to Mortierella 47. Rüchel, R. and Schaffrinski, M., Versatile fluorescent staining
wolfii, Res. Vet. Sci., 14, 201, 1973. of fungi in clinical specimens by using the optical brightener
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cattle, Med. Mycol., 25, 115, 1987. Golgi element, Quart. J. Micr. Sci., 85, 1, 1944.

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49. Beckstead, J.H., A simple technique for preservation of 56. Mehrotra, B.S. and Baijal, U., Species of Mortierella from
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50. Luna, L.G., Manual of Histological Staining Methods of the in Botany No. 2, Department of Botany, University of Georgia,
Armed Forces Institute of Pathology, 3rd edn., McGraw Hill Athens, GA, 1979, p. 257.
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Mukherji, K.G. et al., Eds., Aditya Books, New Delhi, India, Mortierella polycephala et II. Martensella pectinata, Bull.
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86 Mucor
Peter C. Iwen
Contents
86.1 Introduction...................................................................................................................................................................... 759
86.1.1 Classification......................................................................................................................................................... 759
86.1.2 Pathogenicity and Epidemiology...........................................................................................................................761
86.1.4 Diagnosis.............................................................................................................................................................. 764
86.1.4.1 General Growth Characteristics in Culture........................................................................................... 764
86.1.4.2 Conventional Diagnostic Methods......................................................................................................... 764
86.2 Methods............................................................................................................................................................................ 767
86.2.2.1 Identification of Mucor Species by Conventional PCR and Sequence Analysis................................... 767
86.2.2.2 Identification of Mucor Species by Conventional PCR with RFLP Analysis....................................... 768
86.3 Conclusion........................................................................................................................................................................ 768
References.................................................................................................................................................................................. 769
86.1 Introduction phylum, which has subsequently been divided into two
classes, the Trichomycetes and the Zygomycetes, with
Zygomycosis (also referred to as mucormycosis) traditionally the latter class containing the human pathogens.11 Recent
refers to an infection caused by fungi in the class Zygomycota reports now suggest that the polyphyletic nature of this
and the order Mucorales. Numerous fungi within this order group supports a new classification.13–16 Hibbett et al. sug-
(collectively referred to as the zygomycetes) have been asso- gested that the traditional phylum Zygomycota consists
ciated with human disease—with Rhizopus species the most of four subphyla described as incertae sedis to include the
common known cause and Mucor species or Absidia species Mucormycotina, Kickxellomycotina, Zoopagomycotina, and
listed as a distant second, dependent on the patient group Entomophthoromycotina.17 Others have also shown that the
being affected.1–5 Although zygomycosis has historically classification of this group is highly artificial with more work
been considered a rare disease, an emergence of this infec- needed to define the taxonomic details.14,16 The most cur-
tion in immunocompromised individuals has been noted, as rent taxonomy includes the genus Mucor in the subphylum
technological advances occur in medicine.6,7 The number of Mucormycotina within the order Mucorales in the family
cases of disease is probably much higher than that reported Mucoraceae.17 Currently, the GenBank database (National
due to limitations in diagnostic techniques for zygomycosis Center for Biotechnology Information, Washington, DC)
and the high mortality associated with this disease, where lists the phylum for Mucor as “fungi incertae sedis” and
infection may go unrecognized. The problems in diagnosis gives a taxonomic description for this genus as “basal fungal
makes separation of the species difficult, which is evident in lineages” within the subphylum Mucormyocotina.
the numerous articles available in the literature that review Over 250 validly named species of Mucor with multi-
these cases.1,5–12 This chapter will provide information on ple varieties, forma, and subspecies are listed at the Index
what is known about the zygomycetes classified within the Fungorum website (http:// www.indexfungorum.org/Names/
genus Mucor, with an emphasis on the recent area of fungal Names.asp). The number of species within this genus, how-
molecular diagnostics. ever, continues to undergo changes as new taxon are added
and previously named species are reclassified following
molecular sequence studies. Currently, five Mucor species
are reported to cause human disease.6,11,18 These include the
The classification of species within the genus Mucor has thermo tolerant species of M. circinelloides, M. indicus, and
remained unresolved due to questions surrounding the pre- M. ramosissimus, as well as M. hiemalis, which only grows
viously named phylum Zygomycota. Original taxonomic poorly at 37°C. Mucor racemosus has also been suggested
schemes had the zygomycetes placed into this distinctive as a cause of human disease, but this association has been
759

questioned due to the organisms inability to grow at the ele- evaluated using a BLAST search of the GenBank database,
vated temperatures.18 the M. circinelloides sequence was shown to be highly simi-
Although sequencing using genomic targets within the lar to those of Rhizomucor variabilis, Amylomyces rouxii,
rRNA complex has been shown to be reliable for the spe- and Mucor racemosus (Table 86.1). This close relationship
cies identification of the zygomycetes, this process has also between M. circinelloides and R. variabilis was also shown
shown that a number of the currently named species within by Alvarez et al. in their evaluation of 18 strains of M. cir-
the genus Mucor and other closely related genera are highly cinelloides.2 They showed these strains to have a sequence
similar.2,19–22 This similarity suggests that synonymy among similarity of >99% with the type strain of R. variabilis var
some of these species is present.23 When the internal tran- regularior (CBS 103.93T). These authors suggested that it
scribed spacer (ITS)1-5.8S-ITS2 sequences of the type strains seemed logical to consider these two species to be synonyms
of the Mucor species associated with human infection were of each other. Additionally, Schwarz et al. also showed that
TABLE 86.1
Sequence Analysis Using the Internal Transcribe Spacer Regions of the rDNA Complex to Determine Relatedness
within the Various Mucor Species Associated with Human Infectiona
Significant Alingments with Source Speciesc Significant Alingments with Related Speciesc
Alignment Sequenceb GenBank GenBank
GenBank Isolate No. Accession No. Sequences f Accession No. Species
Mucor Speciesd Accession No. Sourcee (% Identify) (% Identity)
M. circinelloides DQ118991 CBS 195.68T (99.9) DQ118988 >20 (98.7) EF583641 R. variabilis
(99.8)g AY243943 (98.7) EF583640
(99.5) DQ118987 (98.7) DQ119007T
(99.5) AM933549 (98.7) EF151444
(99.5) AM933548 (98.7) GQ150159 A. rouxiih
(98.8) EF203695
(98.7) AJ878933 M. racemosus
(98.8) AJ243940
(98.6) AY243941
M. hiemalis DQ118992 CBS 201.65T (99.5) AJ876490 14 None
(99.5) AJ876489
(100) EU484263
(100) EU484253
(100) EU484277
M. racemosus AY213659 CBS 260.68T (100)g DQ118996 11 (99.0) EU484245 M. plumbeus
(99.8) AB369913 (99.0) EU484261
(99.8) FJ345353 (98.9) EU484262
(99.4) AY213661 (98.9) EU484241
(100) AY625074 (98.9) EU484235
M. indicus DQ118994 CBS 226.29T (100) DQ118993 5 None
(100)g AB113026
(100) DQ113027
(100) AB113027
(100) EU798706
M. ramosissimus DQ118997 CBS 135.65T (100)g AY213663 1 None
Abbreviations: R., Rhizomucor; A., Amylomyces; T, type strain.

a Only includes GenBank sequences for analysis that had a complete ITS1-5.8S-ITS2 sequence where the sequence was referenced with a species name.
b The alignment sequences were obtained from the type species.
c Included only up to five of the sequences with the top bit values (E-scores) following a BLAST search where the percentage identity was >98.5%. The
sequences listed are ordered from highest to lowest bit scores.

d No attempt was made to correlate results among the various forma or varieties within the Mucor species.
e Accession code from the culture collection at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands.
f Total number of sequences with a 99% identity to the alignment sequence.
g Duplicate sequence obtained from the type strain.
h Current validated species name reported in the Index Fungorum database (http://www.indexfungorum.org/) is Rhizopus arrhizus var. arrhizus.

Mucor 761
these two species had a high level of similarity when evaluat- species within the genus Mucor will likely lead to future
ing the spacer sequences.22 Voigt et al. when utilizing the 18S changes in the classification of this group. Multigene-based
gene and 28S gene tree topology, also supported the transfer phylogeny projects such as the Assembling the Fungal Tree
of R. variabilis to the genus Mucor.24 of Life project will most certainly have an impact on future
The close genomic sequence similarity between changes in the classification of fungal species including those
Amylomyces rouxii (formerly called Mucor rouxii) and M. in the genus Mucor (http://aftol.org/).
circinelloides has also been reported.22,25 Although A. rouxii,
has not been described as a cause of human disease, sug-
86.1.2 Pathogenicity and Epidemiology
gestions are that the taxonomic positioning of this species
should be questioned.25 Previous classifications had this Mucor species are ubiquitous saprophytes and found in the
species included in the genus Mucor, while current classi- soi1 and dust worldwide.11 Infections by these opportunistic
fication within the Index Fungorum database has the valid pathogens are usually by inhalation of spores into the respi-
name for this species as Rhizopus arrhizus var. arrhizus. ratory tract (pulmonary disease) or by direct inoculation of
Morphological similarities between these two species have spores into a wound (cutaneous or subcutaneous disease).4,11,28
also been described.25 Instances of gastrointestinal (GI) infection are also known
The high sequence similarity between the type strain of to occur by the ingestion of food contaminated with Mucor
M. circinelloides and three GenBank sequences of Mucor species29 as well as disseminated disease resulting from any
racemosus was reported to be possibly due to a misidenti- primary site of infection because of the organisms’ ability to
fication of M. racemosus strains.25 In this report, the type rapidly invade blood vessels.4
strain of M. racemosus (CBS 260.68T) only had a low Once the pathogen enters into the human body, studies
sequence similarity (<96%) to the type strain of M. circinel- have shown that the killing and removal of the Mucor as well
loides, which was also subsequently shown by Alvarez et al. as other Mucorales is mediated by both the neutrophils and
when evaluating multiple sequences of M. circinelloides.2 the macrophages.30 Conditions that inhibit the formation of
An alignment of the type strain M. racemosus sequence neutrophils such as the administration of cytotoxic drugs and
showed good similarity to 11 other sequences of M. racemo- those that impair phagocytic function such as diabetes mel-
sus in the GenBank database (Table 86.1). Similarly, these litus or steroid therapy are known risk factors that allows for
species also differ in thermotolerance, where M. circinel- the spores to germinate and form hyphae.11 Virulence factors
loides grows well at 37°C, M. racemosus is not known to for Mucor species are not well understood, but growth at an
grow at this elevated temperature.18,26 The alignment of the elevated temperature appears to also be a major factor in the
type strain of M. racemosus also showed this species to be organisms’ ability to infect human tissue.11 Although thermo-
closely related to M. plumbeus. Kwasna et al. also described tolerance is a characteristic of some Mucor species, many of
this close relationship in their analysis of the ITS1/2 region the species will not grow at an elevated temperature, which
in their studies, showing phylogenetic relationships within suggests why infections, for instance those caused by M. hie-
the Zygomycota.27 malis, only appear on cooler areas of the body such as on
The ITS sequences of the three other species associated the skin. Additional studies have also shown that an increase
with human disease did not overlap with other species sug- in the availability of iron, as occurs with the administration
gesting that these were divergent enough to validate the of iron chelating deferoxamine can serve as a sideophore to
position of separate species although a limited number of provide iron to enhance fungal growth.4,10 Also, the ability of
sequences were available for evaluation (Table 86.1). A report the pathogen to invade arteries once established in the host
shows M. hiemalis and R. variabilis var. variabilis were phy- allows the disease to rapidly spread, leading to thrombosis
logenetically closely related; however, the ITS sequences of and tissue destruction.
the two type strains (DQ118992, M. hiemalis and DZ119006,
R. variabilis) appeared to only have a similarity of 88.4%.2
This difference was also reported by Kwasna et al. in an eval-
uation of the ITS1/2 regions.27 Numerous review articles have been written to describe the
Finally, although many forma and varieties exist among clinical features of zygomycosis.4,9–12 These articles describe
the Mucor species, the ability to separate these by sequence the features of disease collectively caused by the zygomyce-
analysis is still not possible due to the lack of a taxonomi- tes following recognition of the pathogen in tissue showing
cally robust number of isolates with sequence information characteristic histological features. These features include
for comparison. Reports have shown that there is some vari- hyphae with a variable width (5–20 μm) and a haphazard
ability within the ITS regions of M. circinelloides, which branching pattern with rare septations, as well as the ability
may support subspecies grouping.22,25 Schipper described of the fungus to be angioinvasive.10
four forma of M. circinelloides, based on differences in the Roden et al., in a review of 929 patients with zygomycosis,
shape of the spores and columellae as well as in the ther- showed that sinus involvement was the most common infec-
motolerance (f. circinelloides, f. janssenii, f. griseocyanus, tion site (39% of cases) followed by pulmonary (24%), cuta-
and f. lusitanicus).26 Comparative analysis of multiple gene neous (19%), cerebral (9%), and GI (7%).4 Of the 465 culture
sequences from a broad representation of well-characterized positive cases in this review, only 85 (18%) were positive with

a Mucor species, with Rhizopus species the most common Additionally, most of the infections involved the skin and
organism group identified (47%). Other reports from various subcutaneous tissues (10 cases), with a survival rate of these
patient groups also showed that Mucor species were uncom- cases at 78.9%, which was not unexpected because of the
mon causes of disease.1,5,6,10,31,32 Additionally, even though more localized superficial nature of the infection, although in
involvement of the sinus was the most common reported clin- general the survival rate of zygomycosis in the compromised
ical feature of zygomycosis, Mucor species have only been patient has historically been considered poor.8,9,11
rarely detected as causing this infection.1,3,33 Michael et al. in Mucor circinelloides was identified as the most common
a review of fungal sinusitis cases from patients at a tertiary Mucor species reported as a cause of human disease in the
care hospital, reported on 72 cases of invasive disease, where literature (8 cases).25,34–40 In five cases, trauma was consid-
culture was obtained.3 Of these, 33 were culture positive for a ered the predisposition to infection with the primary disease
zygomycete, 30 of these were positive for Rhizopus arrhizus, including cutaneous and subcutaneous areas (Cases 1–4 and
two were positive for nonsporulating zygomycetes and one 6). Two of the cases resulted in fungemia (Cases 5 and 8).
was positive for a Rhizomucor species and a nonsporulating Diabetes mellitus was the most common underlying disease
zygomycete. No Mucor species were detected in this study. In reported in four of these cases with two of them described as
a review of 106 solid organ transplant patients with histology a patient in a “metabolic acidotic” condition. The etiological
proven zygomycosis from a variety of sites, Almyroudis et agents in three of the most recent cases were identified fol-
al. reported that 67 cases were culture positive with 49 (73%) lowing molecular sequencing of the ITS regions (Cases 6–8).
identified as a Rhizopus species followed by 9 (13%) identi- Mucor indicus was the second most common Mucor spe-
fied as a Mucor species and the rest identified as other zygo- cies identified in the literature associated with invasive infec-
mycete species.1 In this study, sinus involvement was also the tion in seven cases.29,41–46 The most interesting fact about this
most common site of infection (31%) followed by pulmonary species was the association with GI disease (five cases). Deja
(24%), cutaneous (15%), and GI (11.2%). Finally, Neofytos et et al. had suggested that this increased predilection to the
al., in a review of hematopoietic stem cell transplant recipi- GI tract was probably through ingestion, since M. indicus is
ents with invasive fungal disease, showed that zygomycetes used as a starter for food fermentation of rice and manioc.42
were associated with 18 of the 250 cases of infection with However, in only one of the cases of GI involvement was the
Mucor/Rhizomucor described as rare causes of disease in source of infection described (Case 12). This case was sug-
three cases (1.2% of the total).33 Older literature describing gested due to ingestion of a naturopathic medicine identified
cases of zygomycosis prior to molecular sequencing usually as containing M. indicus.45 In two cases, invasion outside of
did not list the pathogen because of the difficulty in cultur- the GI tract to the liver was noted with both patients having
ing and the lack of expertise to identify the organism spe- an underlying cancer and being treated with immunosup-
cies using micromorphological methods once cultured. Also, pressive therapies (Cases 12 and 13). The etiological agent
reclassification of Rhizomucor pusillus from Mucor, which in two of the most recent cases was also identified using ITS
was proposed by Schipper and subsequently confirmed by sequencing.
rRNA gene sequencing, has dramatically decreased the num- Three cases each of zygomycosis caused by M. hiema-
ber of Mucor species associated with infection.14,26 lis47–49 and M. ramosissimus50–52 were also described. Two
This inability to culture a zygomycete from clinical mate- cases of M. hiemalis infection were noted to occur in indi-
rial and the limited capacity to recognize individual spe- viduals who did not have an underlying disease (Cases 9
cies when a culture is available has restricted the ability to and 11) and both reports described disease progression over
describe diseases association with the various Mucor spe- an extended time period (5 years for Case 9 and 10 months
cies. Alvarez et al., in an evaluation of 190 clinically signifi- for Case 11). Although trauma was indicated for two of the
cant zygomycete isolates submitted for testing to a reference cases, the third case did not indicate trauma; although it
laboratory, showed that Mucor species were the second most would have been difficult for the patient to recall this after a
common genus recognized (14.2%) following Rhizopus spe- 5 year course of the disease. The predilection for a primary
cies (66.8%).2 Of the 29 Mucor species morphologically iden- cutaneous infection in all these cases may have been due to
tified in this study, 18 were confirmed by molecular testing the organisms’ known inability to grow at the high core body
(using the ITS region targets) as M. circinelloides, 5 were temperature.18
confirmed as M. indicus, and 6 were identified as Mucor spe- Mucor ramosissimus was reported in two cases as causes
cies only. The latter 6 Mucor species, although identified by of mucocutaneous and rhinocerebral infections (Cases 19 and
morphologic methods as M. racemosus (three isolates) and 20). More recently, this organism was described as a cause
M. ramosissimus (three isolates), could not be validated to of cutaneous zygomycosis in a patient with aplastic anemia
species by sequence analysis. on immunosuppressive therapy.52 None of the etiological
In the literature, only 21 cases of histology proven inva- agents in these cases were identified by molecular methods.
sive infection with positive culture of a confirmed Mucor spe- This lack of molecular analysis for species identification and
cies were described (Table 86.2). A majority of the reported the fact that the species does not grow well at elevated tem-
cases involve patients with some underlying condition such peratures make the association of this species to human dis-
as immunosuppression or diabetes mellitus, although eight ease questionable.18 Additionally, Alvarez et al. showed that
cases did not have any reported underlying condition. M. ramosissimus, previously identified by morphological

Mucor 763
TABLE 86.2
Mucor Species Reported in the Literature as Causes of Zygomycosisa
(Species)
Underlying Primary Molecular
Case No. Predisposition Disease Disease Type Targetb Outcome Reference
(Mucor circinelloides)
1. 34, F Farmer, trauma? None Cutaneous Not donec Resolved [40]
2. 23, F Trauma, neutropenia AML Cutaneous Not donec Resolved [38]
3. 63, M Trauma, steroids, DMID, cirrhosis Cutaneous, Not donec Death [36]
metabolic acidosis subcutaneous
4. 62, F Trauma MDS Cutaneous Not donec Resolved [34]
5. 48, M Abdominal surgery TPNd Short gut syndrome Fungemia Not donec Resolved [35]
6. 90, M Trauma, pancytopenia MDS, DMID Cutaneous ITS1, ITS2e Resolved [25]
metabolic acidosis
7. 40, M Tooth extraction DMNID Rhinosinusitis ITS1, ITS2e 28S Resolved [39]
rRNA gene
8. 83, F Intensive care DMNID Disseminatedf ITS1, ITS2e Death [37]
(Mucor hiemalis)
9. 14, F None None Cutaneous Not reportedg Resolved [48]
10. 78, M Trauma DMID Cutaneous, Not donec Resolved [47]
subcutaneous
11. 10, F Trauma None Cutaneous Not reportedg Resolved [49]
(Mucor indicus)
12. 39, M GVHD, steroids MDS Gastrointestinalh Not donec Resolved [45]
13. 27, F HDC Leukemia Gastrointestinalh Not donec Death [46]
14. 66, M None None Gastrointestinal Not donec Resolved [43]
15. 82, F Burni None Subcutaneous Not donec Resolved [44]
16. 34, M None None Gastrointestinalj Not describedk Resolved [29]
17. 48, M Acute head injury None Gastrointestinal ITS1, ITS2e Resolved [42]
18. 6mo, F LVAD implant Cardiomyopathy Endovascularl ITS1, ITS2e Death [41]
(Mucor ramosissimus)m
19. 39, F None None Mucocutaneous Not listedn Resolved [51]
20. 39, F Metabolic acidosis DMo Rhinocerebral Not listedn Resolved [50]
21. 31, F Immunotherapy Aplastic anemia Cutaneous Not donec Resolved [52]
Abbreviations: F, female; M, male; DM, diabetes mellitus; DMID, diabetes mellitus, insulin dependent; DMNID, diabetes mellitus, non-insulin
dependent; AML, acute myelogenous leukemia; MDS, myelodysplastic syndrome; TPN, total parenteral nutrition; LVAD, left ventricular
assist device; HDC, high dose chemotherapy; GVHD, graft-vs.-host disease.
a Only included those cases reported in the literature where the diagnosis was made by culture from a sterile body site with characteristic histo-
pathology in tissue.
b When done, a molecular sequence of the listed target site was used to confirm the identity of the Mucor species.
c Identification of species made on the basis of macro- and micromorphologic features with or without temperature studies.
d TPN-induced hyperglycemia.
e Ribosomal DNA including the complete internal transcribed spacer (ITS)1-5.8S-ITS2 region.
f Multiple positive blood cultures with subsequent dissemination to the skin.
g Isolate submitted to a reference laboratory for confirmation. The method of confirmation was not reported.
h Dissemination to the liver.
i Suggested that hot compresses is association with a cosmetic clay composed of mashed onions suggested as the source.
j Fungemia occurred following involvement of the gastrointestinal tract.
k Species described as identified by both phenotypic and genotypic methods with no target listed.
l Involvement of the ascending aorta.
m A case of disseminated septic arthritis was described in abstract form which was unavailable for review81.
n Species identification method was not described.
o Newly diagnosed diabetes mellitus with hyperglycemic diabetic coma.

methods, could not be validated to species using a molecu- to rapidly and reliable identify species from tissue, however,
lar method.2 In Case 21, the report describes M. ramosissi- is still not routinely available in the clinical laboratory. The
mus as being identified using micromorphological methods, reality of diagnosis for zygomycosis is still dependent on his-
temperature growth studies, and biochemical tests. Although tological evidence of characteristic hyphal forms in tissue.
deposited in the culture collection of the Centralbureau voor Subsequently, when species identification is made from cul-
Schmimmelcultures (Baarn, the Netherlands), under the ture, this result is frequently not available until days to weeks
name Mucor aff. ramosissimus (CBS 144.93), this species after the specimen had been collected and therefore the result
was not included in their commercial lists of cultures because is only useful for epidemiological purposes and not for pri-
of some structures that were described as “atypical.” Also, mary management of the patient.
as problematic as including this species as a cause of human
disease is that none of the molecular sequences currently 86.1.4.1 General Growth Characteristics in Culture
deposited in the GenBank database appear to represent any Mucor species grow well on most standard fungal culture
species of M. ramosissimus that have been associated with medium such as Sabouraud dextrose agar. For most of the
human infection. species associated with human disease, the growth is gener-
Overall, of the 21 cases of zygomycosis reported in the ally rapid with mycelial elements covering the entire plate
literature, where the Mucor species was indicated, only 5 had within 2–3 days after incubation at 30°C. Unfortunately, the
the etiological agent identified using molecular methods, with recovery of a zygomycete from tissue has been described as
all using the ITS1/ITS2 region target for sequence analysis. difficult where negative results are reported although histo-
Due to the difficulty in micromorphological identification of logical evidence of a zygomycete was present.4 One reason
Mucor species, it is reasonable to assume that some of the for this inability to recover the fungus appears to be partly
species for the other 16 cases could be questioned. Although related to the aggressive processing of the specimen, which
morphological methods for the identification of Mucor spe- may damage the organism.11 Roden et al. in an evaluation of
cies has been shown to be fairly reliable, misidentifications 929 reported cases of zygomycosis showed that only 465 of
were possible, especially when identifying Mucor species these cases had microbiological findings listed.4 They also
other than M. circinelloides.2 Unfortunately, most of the iso- showed that there was a clear increase in culture positivity
lates associated with the reported cases appear unavailable over time with 71% of all cases, since 2000 diagnosed on the
in a reference collection for future verification testing, using basis of culture result. This improvement was suggested due
a molecular assay. to better training, a greater understanding of specimen pro-
cessing, improved culture techniques, and increased access
to sophisticated reference laboratories.
86.1.4 Diagnosis
The diagnose of zygomycosis has relied mostly on the char- 86.1.4.2 Conventional Diagnostic Methods
acteristic finding from a histopathological observation of The vegetative mycelium of all species in the Mucorales is
tissue.10 Although typical hyphal elements in tissue are used composed of wide, predominately aseptate hyaline hyphae.
in the diagnosis of a zygomycetous infection, atypical struc- The general characteristic for the identification within the
tures may also be present making the diagnosis more dif- Mucor genus is a rapidly growing colony and the produc-
ficult. Some Mucor species associated with human disease, tion of sporangiophores containing a multispored sporangia
to include M. circinelloides and M. racemosus, have been without basal rhizoids.18 Detailed microscopic characteristics
known to produce structures referred to as “spherule-like such as the presence of sporangiophores, which are branched
bodies,” both in culture and in tissue.11,53 These structures or unbranched, the size and shape of the sporangia and spo-
have been describe as chlamydospores that resemble those rangiospores, the features of the columellae, and the pres-
observed in culture25 and as a dimorphic growth character- ence or absence of chlamydospores, are subsequently used to
istic associated with the fungus.54 The genes involved in the identify Mucor species.18 Additional physiological methods
control of dimorphism in M. circinelloides have been further to help in the identification is to define the maximum tem-
evaluated by Wolff et al.54 perature growth and the organisms’ ability to assimilate eth-
Culture of the etiological agent is needed to identify the anol.18 Unfortunately, the identification of Mucor species on
species involved in infection. However, culture has not been morphological features alone is difficult and time consuming
reliable from clinical material, and in cases where a posi- and requires an assessment by individuals with experience in
tive culture has been successfully obtained, the identification fungal identification. Alvarez et al., retrospectively analyzed
of the species is not easily performed. Thus, the diagnosis 190 clinical isolates of zygomycetes that had been identi-
of zygomycosis caused by a validated Mucor species has fied to species by morphological methods only.2 They used
only rarely been reported. Additionally, most of the species the ITS1-5.8S-ITS2 region as a molecular target and showed
identification using morphological methods was performed an overall correlation of 92.6% between morphological and
at reference laboratories by individuals who have expertise molecular identification of all the zygomycetes evaluated.2 In
in fungal identification. Recently however, a shift to using this study, all 18 of the M. circinelloides and 5 of 6 M. indicus
molecular methods on culture material has been shown to be correlated between morphological and molecular identifica-
reliable to identify the zygomycete species.2,19,22,55 The ability tion. However, none of the three morphologically identified

Mucor 765
M. racemosus or the three M. ramosissimus was confirmed been reported to be a reliable sequence for the identification
by molecular testing. All of these six isolates, however, were of most Mucorales, including the Mucor species.2,20,22,27,66
identified to the correct genus by sequence comparison anal- This region has also been proposed as the prime fungal bar-
ysis. Although they concluded that ITS region sequencing code for fungal species identification by the International
was a useful tool to identify most of the clinically significant Subcommission on Fungal Barcoding (http://www.allfungi.
zygomycetes, they also showed that neither phenotypic meth- com/its-barcoe.php).
ods nor sequencing methods alone were able to identify all Methodologies. Numerous molecular methods using both
of the species present. They also concluded that R. variabilis nonsequence- and sequence-based approaches for analy-
var regularior should be considered a synonym of M. circi- sis have been used for the identification of the zygomycetes
nelloides, an observation that has been suggested by other (Table 86.3). Nonsequence-based approaches for analysis
research groups.22,25,56 have included conventional PCR with RFLP analysis,27,64,68
The accurate identification of heterothallic zygomy- multiplex PCR using a specific DNA probes in a microar-
cetes, such as Mucor species, has also been shown to be ray-based assay,66 real-time PCR using melting curve anal-
enhanced by observation of zygospores in mating studies.57 ysis,62,67 and seminested PCR using Luminex micro bead
This method was successfully employed by Weitzman et al. hybridization technology.20
to identify an isolate of M. circinelloides.57 However, others Restriction fragment length polymorphism (RFLP)
have shown that mating studies do not often yield a positive analysis of a PCR product has been shown to be reliable
result.25,26 Schipper in a study of fertility among M. circi- to differentiate Mucor species using a variety of restriction
nelloides and its formae revealed that isolates from differ- enzymes. Nyilasi et al. used conventional PCR on 26 zygo-
ent formae could mate with each other, which thus confused mycete strains to include four different species of Mucor
the broad-species concept for M. circinelloides but that some using a Backusella/Mucor-specific primer set and the FTR1
isolates within a formae failed to mate.26 Other researchers gene target.68 Following amplification and digestion with
showed that mating was not successful even though high sim- the AluI enzyme, all of the zygomycete species provided a
ilarities in ITS sequences among identical species were pres- unique fragment pattern allowing for separation of the vari-
ent.25 Nevertheless, the limited number of studies evaluating ous species tested. Machouart et al. also used conventional
mating studies does not allow for an accurate assessment PCR, where 36 Mucorales species were evaluated to include
of this process, and additional studies are needed to further 2 species of Mucor using a zygomycete-specific primer
research this process. Conversely, the difficulty in perform- set that targeted a variable region within the 18S rDNA
ing mating studies because of the need to maintain a library gene.64 The amplicons in this case were treated with mul-
of tester strains does make this methodology unrealistic for tiple restriction enzymes. The AflII enzyme was shown to
most clinical laboratories. be Mucor genus-specific while the XmnI enzyme was shown
to be species-specific for M. circinelloides, M. racemosus,
86.1.4.3 Molecular Techniques M. ramosissimus, and M. plumbeus, but did not cut ampli-
Recent advances in molecular testing for the identification cons obtained from M. hiemalis and M. indicus. Their stud-
of fungal pathogens have had a significant impact in the ies showed the utility of using PCR-RFLP-based assay for a
diagnostic mycology laboratory.55,58,59 A general consensus rapid and precise identification of mucorlean strains. They
addressing these changes suggest that approaches to the spe- suggested that additional studies were needed to explore this
cies identification of many fungi today should include both technology as a diagnostic tool on biological samples from
a combination of morphologic and molecular testing meth- patients with zygomycosis. Finally, Kwasna et al. showed
ods.55 This combination approach is especially relevant for that PCR-RFLP analysis of the ITS1/2 rDNA target could
the identification of molds and more specifically for spe- be used to discriminate between genera and species of
cies identification of the zygomycetes. Numerous papers are the Mucorales.27 They evaluated 18 species of Mucorales
available to describe the overall review of molecular testing including 6 Mucor strains representing 4 different species.
for medically important fungi.23,55,58–60 The nine different restriction enzymes evaluated showed
Targets. A variety of genomic targets have been used for that the best discrimination among species of Mucorales
the identification of Mucor species. The ITS regions, the could be achieved with the AluI, Hinf I, and Sau3AI restric-
D1/D2 domains of the 28S rDNA gene, and the V9 region tion enzymes.
of the 18S rDNA gene, all within the rDNA complex, are As a means to decrease the time for detection, numerous
the most common sequenced fungal loci for the identifica- studies have applied rapid cycle PCR methods as the test-
tion of Mucor species.2,20,22,24,27,61–66 Other molecular targets ing platform. Real-time PCR using the 28S rDNA target62,63
that have been used with limited success include the cyto- and the cytochrome b gene target67 have most recently been
chrome b gene67 and the high-affinity iron permease (FTR1) done. Kasai et al. used the 28S rDNA gene with zygomy-
gene.68 No studies to evaluate specifically the Mucor spe- cete-specific primers using a quantitative method of PCR
cies have been done to compare multiple genomic targets followed by melt curve analysis.62 They evaluated nine dif-
for identification. The nuclear ribosomal ITS region (ITS1, ferent species of zygomycetes to include three species of
5.8S rRNA gene, ITS2) located between the nuclear small- Mucor. In this evaluation, the results showed that melt curve
(18S rRNA gene) and large-(26–28S rRNA gene) genes has analysis was able to distinguish among the Rhizopus, Mucor,

TABLE 86.3
Molecular Assays and Genomic Targets Used for the Identification of Mucor Speciesa
PCR Technique/Analysis
Method Source Genomic Targetb Specificity Mucor Species(Number Evaluated) Reference
Conventional/sequencing Culture 28S rDNA 18S rDNA Zygomycetes M. amphiborum (1), M. circinelloides (1), [24]
M. hiemalis (1), M. indicus (1), M. mucedo
(1), M. racemosus (1), M. ramosissimus (1)
Conventional/sequencingc Culture, clinical 28S rDNA Universal M. circinelloides (2), M. hiemalis(1), [61]
M. racemosus (1)
Conventional/sequencing Culture, clinical ITS1-5.8S-ITS2 Zygomycetes M. circinelloides (5), M. hiemalis (1), [22]
M. indicus (2), M. racemosus (1),
M. ramosissimus (1)
Conventional/sequencing Culture ITS1-5.8S-ITS2 Universal M. circinelloides (18), M. indicus (5) Mucor [2]
spp.d (7)
Conventional/RFLPe Culture 18S rDNA Zygomycetes M. circinelloides (1), M. hiemalis (1) [64]
Conventional RFLPf, Culture ITS1-5.8S-ITS2 Universal M. circinelloides (1), M. hiemalis (2), [27]
sequencing M. plumbeus (1), M. racemosus (2)
Conventional/RFLPg, Culture FTR1 Zygomycetes M. circinelloides (1), M. rouxii (1), [68]
sequencing M. plumbeus (1), M. racemosus (1)
Multiplex/DNA probes Culture, clinical ITS1 Mucor M. racemosus (1) [66]
Real-time/sequencing Culture, clinical 28S rDNA Universal M. hiemalis (1), M. flavus (1) [63]
Real-time/melting curve Culture, clinical 28S rDNA Zygomycetes M. circinelloides (1), M. ramosissimus (1), [62]
M. indicus (1)
Real-time/melting curve Culture, clinical Cytochrome b Zygomycetes M. circinelloides (1), M. flavus (1), [67]
M. hiemalis (1) M. mucedo (1),
M. racemosus (1), Mucor spp.d (1)
Seminested Luminex Culture ITS2 Universal M. racemosus (1), M. mucedo (1) [20]
probesh
Abbreviations: M, Mucor; ITS, internal transcribed spacer; RFLP, restriction fragment length polymorphism; FTR1, high-affinity iron permease gene.
a Only included studies were multiple species or strains of zygomycetes were evaluated.
b The 18S, 5.8S, and 28S genes as well as the spacer regions are all included in the rDNA complex of the genome.
c Utilized the MicroSeq D2 large-subunit rDNA fungal sequencing kit (Applied Biosystems, Foster City, CA).
d Species not specified.
e Discriminatory enzymes used were AluI, HinfI, Sau3AI, and HaeIII.
f Used the XmnI enzyme (species-specific) and the AflII enzyme (Mucor-specific).
g Restriction enzyme used was AluI.
h Utilized the Luminex microbead hybridization technology (Luminex Corporation, Austin, TX).
and Rhizomucor genera, but not within the various species. More recent molecular technologies to evaluate Mucor
They suggested that the advantage of this assay was that the species included the application of PCR to microarray
three genera within the class Zygomycete could be detected analysis and to other hybridization-based probe technolo-
in a single result by melt curve analysis without the need gies. Spiess et al. established a sensitive and specific DNA
for further procedures such as sequencing, which ultimately microarray combining multiplex PCR and consecutive DNA
results in a faster diagnosis time. They also successfully chip hybridization to detect fungal genomic DNA in clini-
applied this method to tissues from a rabbit model of experi- cal samples.66 This assay involved primer sets and probes
mental pulmonary zygomycosis caused by M. circinelloides. to amplify and detect 14 clinically relevantly fungal species
Hata et al. applied real-time PCR for the detection of that included both M. racemosus and Rhizopus microsporus.
zygomycetes from both culture and tissue specimens (both This proof-of-concept evaluation showed that fungal patho-
fresh and fixed).67 This assay targeted a region of the mul- gens such as the zygomycetes could be detected and identi-
ticopied cytochrome b gene, where 44 culture isolates of fied from clinical samples, which ultimately would improve
zygomycetes that included 5 different Mucor species were the diagnosis of invasive fungal infections.
evaluated. Although their study showed this assay to be a Landlinger et al. utilized the Luminex xMAP hybridiza-
rapid and accurate method to detect zygomycetes, they also tion technology for species-specific identification of a wide
noted that overlapping melting curves were obtained with range of clinically relevant fungal pathogens to include
Absidia, Apophycomyces, and Mucor species, thus prevent- the zygomycetes.20 Their preliminary studies showed that
ing the identification to the genus level. this technology was able to successfully identify numerous

Mucor 767
fungal pathogens from clinical specimens to include two spe- with vortexing of the samples. Other pretreatment processes
cies of Mucor. Further development of this high-throughput may include heating at 95°C–100°C and freezing at −70°C or
assay for a broad range of pathogenic fungi will be relevant in liquid nitrogen.
in the context for the diagnosis of invasive fungal infections. Concurrent with or following the mechanical disruption
Most of the sequence-based analysis methods have been process, a variety of extraction methods have been used.
done using conventional PCR with universal primers to More labor intensive kit-based methods (such as the Qiagen
target sequences within the 28S rDNA gene61 or the ITS1- DNeasy plant mini kit or the QIAmp DNA blood kit [Qiagen,
5.8S-ITS2 target area.2,27 Additionally, Vollmer et al. used a Hilden, Germany]) or the automated DNA extraction sys-
real-time PCR approach also with sequencing as the analysis tems such as the NucliSens easyMAG (bioMerieux) and the
method.63 They evaluated 39 fungal species to include two MagNA Pure LC system (using either the NA isolate kit I or
Mucor species and showed that this method was useful as a the DNA Isolation kit III) have all been successfully used for
means to rapidly screen for fungal infections in various clini- the extraction of fungal DNA. Varieties of enzymes have also
cal specimens. Additionally, all of the case reports, where been employed in the process and include proteinase K, lyti-
Mucor species were identified, used sequencing and align- case, and lysozyme. Recently, a new novel method for fungal
ment analysis of the ITS regions (Table 86.2).25,37,39,41,42 DNA extraction was described by Borman et al. and involved
The availability of a nucleotide-based sequence database the utilization of chemically impregnated cellulose devices
that has the phylogenetic breadth and taxonomic accuracy (FTA Cards, Whatman, Inc., Florham Park, NJ) for quickly
needed to identify the zygomycetes is desirable. The largest preparing DNA for PCR-based applications.71 This technique
database currently used for sequence-based identification is the has been shown to be reliable for extraction of genomic DNA
publicly available GenBank database. Unfortunately, due to the from a variety of pure organisms in culture to include Mucor
fact that this is an open source database, reports suggest that up racemosus, as well as other zygomycetous molds.
to 20% of the sequence entries listed are associated with erro- An additional approach is the extraction of DNA from
neously identified species.55 Currently, over 500 Mucor nucleo- Formalin-fixed, paraffin-embedded tissue. This frequently
tide base sequences representing 18 species (accessed October leads to a DNA product that is not useful for PCR. The major
2009) are available in the GenBank database. A majority of reason suggested for this is due to the extensive cross-link-
these sequences involve three species with M. circinelloides ing of tissue proteins after fixation in formaldehyde, which
having the highest number of sequences available followed by results in nucleic acid fragmentation and thus inhibition of
M. hiemalis and M. racemosus. The partial 18S rDNA, com- the PCR process.67 Utilization of a shorter amplicon as the
plete ITS1-5.8S-ITS2, and partial 28S rDNA sequence target target may somewhat mitigate this effect.
are the most common areas represented followed by regions of Procedure for DNA extraction from Formalin-fixed tissue.
the 18S rDNA gene and 28S rDNA gene targets. Only a small
number of sequences are available for other targets such as the 1. Cut one 50 μm section from the paraffin block using
act-1 gene for actin, the cytochrome b gene, the beta-tubulin a microtome and place into a 1.5 mL screw-top tube.
gene, and the translocation elongation factor 1-alpha gene. The 2. Prepare a tissue lysate by washing the tissue section
need for sequences from a variety of genomic targets in a data- with xylene to remove wax followed by washing
base that is phylogenetically represented with accurate species with 95% alcohol.
identification is clearly needed for the zygomycetes. Although 3. After air-drying, place the tissue in a mixture of
multiple locus sequencing and alignment comparisons has been 500 μL of Tris-EDTA buffer, 50 μL of 10% sodium
done using a variety of mucoralean species, additional studies dodecyl sulfate, and 100 μL of proteinase K and
using this approach are also needed to determine the optimal incubate for 24 h at 55°C while mixing at 500 rpm.
target or targets for Mucor species identification. Studies simi-
lar to what has been done using multiple targets to evaluate for Note: Lyticase may be added in combination with proteinase
the identification of the aspergilli69 and the fusaria70 should be K to maximize the efficiency of the fungal DNA extraction
considered for the zygomycetes. process.72

86.2 Methods
86.2.2.1 Identification of Mucor Species by
86.2.1 Sample Preparation Conventional PCR and Sequence Analysis
The preparation of specimens for DNA extraction of Mucor The molecular identification of Mucor species has been
species is similar to the preparation for any other fungal performed using multiple targets with conventional PCR
agent. Briefly, the extraction process, whether processing followed by sequence analysis. These methods have had
from culture material or from clinical samples such as blood, utilization in not only identification of species, but also in
bronchial alveolar lavage specimens, and tissue, requires showing the phylogenetic relationship of the zygomyce-
some type of mechanical disruption prior to DNA extraction. tes. Described below are methods utilizing the universal
Generally, the original culture or tissue material is mechani- 28S rDNA gene target24 and the universal ITS1-5.8S-ITS2
cally disrupted using some type of homogenization along genomic region target.25

Procedure for conventional PCR using 28S rDNA target 86.2.2.2 Identification of Mucor Species by
Conventional PCR with RFLP Analysis
1. Prepare a PCR mixture (50 μL) containing 10–20 ng
Multiple applications using restriction enzymes to iden-
of genomic DNA, 0.255 mM each deoxynucleotide,
tify Mucor species have been attempted using both the ITS
25 pmol of NL-1 primer (5′-GCA TAT CAA TAA
regions and the variable region of the 18S rDNA gene.27,64 The
GCG GAG GAA AAG-3′), 25 pmol of NL-2 primer
variety of enzymes used has shown this process to have some
(5′-GGT CCG TGT TTC AAG ACG G-3′), 50 mM
utility, although some specificity for Mucor species is noted.
KCL, 10 mM Tris–Cl (pH 8.4), 2.5 mM MgCl2,
0.1 mg of gelatin/mL, and 1.25 U of Ampli Tag Procedure
polymerase (Perkin-Elmer, Foster City, CA).
2. PCR amplification is performed in a Thermocycler 1. Prepare a PCR mix (50 μL) containing 5 ng of
using a cycler program consisting of a denaturation extracted DNA, 2 mM MgCl2, 200 μM of each
step at 94°C for 2 min, 40 cycles of 30 s at 94°C for deoxynucloside triphosphate, 20 pmol of MucL1
DNA denaturation, 30 s at 60°C for primer anneal- primer (5′-TGA TCT ACG TGA CAT ATT CT-3′),
ing, and 90 s at 72°C for primer extension; a final 20 pmol of MR1 primer (5′-AGT AGT TTG TCT
extension of 10 min at 72°C and a 4°C soak. TCG GKC AA-3′), and 2 U of Fast start Tag poly-
3. Amplicons are visualized electrophoretically in merase (Roche, Meylan, France).
1.5% agarose gel, following staining with ethidium 2. PCR conditions consists of denaturation for 3 min at
bromide. 94°C, followed by 30 amplification cycles at 94°C
4. The PCR products are directly sequenced using for 1 min, 60°C for 1 min, and 72°C for 1 min and
both the NL-1 and the NL-2 primers with a resulting one final extension cycle at 72°C for 5 min.
sequence analyzed to produce a consensus sequence 3. For RFLP analysis, the amplicon is digested for 1 h
that is evaluated, following a BLAST search of the at 37°C in a total volume of 20 μL using 10 μL of
GenBank databases. the PCR product and 5 U of the selected restric-
tion enzyme. The restriction enzyme AflII (restric-
Procedure for conventional PCR using the ITS1-5.8S-ITS2 tion site 5′-CTTAAG-3′) has expected fragment
rDNA target sizes of 750 and 87 base pairs for Mucor spe-
cies while the enzyme XmnI (restriction sites,
1. Prepare a PCR mixture (50 μL) containing 5 μL of 5′-GAATAGCTTC-3′ or 5′-AGCTTCGGT-3′) has
DNA (prepared following extraction on the MagNA expected fragment sizes of 613 and 224 base pairs.
Pure LC DNA isolate kit II for bacteria and fungi)
(Roche, Mannheim, Germany). The PCR master 86.3 Conclusion
mix consisted of 31.2 μL of UltraPURE distilled
water, 5 μL of 10× high-fidelity buffer, 2.5 μL of The zygomycetes, including Mucor species, will continue
stock 2 mM deoxynucleoside triphosphate mix, to emerge as causes of invasive disease as immunosuppres-
3 μL of 50 mM MgSO4, 0.75 μL of 20 μM ITS1-M sive therapies expand with the use of more advanced medi-
primer (5′-GGA AGT AAA AGT CGT AAC AAG cal treatment options.7 Current management of a patient with
G-3′), 0.75 μL of 20 μM ITS4 primer (5′-TTC TCC zygomycosis starts with recognition of characteristic symp-
GCT TAT TGA TAT GC-3′), 1.5 μL of stock 50% toms in a high-risk patient.11 These symptoms might include
dimethyl sulfoxide, and 0.3 μL of 5 U/μL high- headache, visual disturbance, facial or orbital swelling, or
fidelity Platinum Taq DNA polymerase (Invitrogen other features such as a pulmonary lesion, necrotic lesion
Corporation, Carlsbad, CA). on the skin, or rarely a positive culture from a sterile site.
2. A total of 35 cycles of amplification are performed Aggressive management at this point involves radiological
after initial denaturation of DNA at 95°C for imaging along with a biopsy with or without total surgical
4.5 min. Each cycle consisted of a denaturation step debridement, followed by empiric antifungal therapy when
at 95°C for 30 s, an annealing step at 50°C for 30 s, medically indicated. Subsequently, microscopic evidence
and an extension step at 72°C for 1 min with a final of a zygomycete in clinical material from a deep tissue
extension at 72°C for 3 min following the last cycle. biopsy is evidence enough for the diagnosis of zygomycosis.
3. The PCR product is directly sequenced using both Unfortunately, due to the aggressive nature of the zygomyce-
the ITS1-M and ITS4 primers. tes, positive cultures will not be available to help in the initial
4. The consensus sequence is submitted to GenBank management of the patient. Thus, even with the successful
for a BLAST search of the database for sequence isolation and identification of Mucor species from clinical
alignment. material, knowing this result will generally not at the present
time change the course of therapy.
Note: Application of this process has successfully been used Recent studies now show that the in vitro susceptibility
in phylogenetic studies with the help of the software program for Mucor species to antifungal agents has some variation.74
MEGA 4.0.2,73 Although amphotericin B has historically been the agent

Mucor 769
of choice to treat zygomycosis with a reported 94%–100% 5. Zaoutis, T.E. et al. Zygomycosis in children: A systematic
susceptibility to Mucor in vitro, toxicity associated with this review and analysis of reported cases. Pediatr Infect Dis J 26,
agent with subsequent intolerance make the consideration 723–727 (2007).
6. Chayakulkeeree, M., Ghannoum, M.A., and Perfect, J.R.
of other agents for therapy a necessity. Studies now suggest
Zygomycosis: The re-emerging fungal infection. Eur J Clin
that posaconazole be considered the first choice as alternative Microbiol Infect Dis 25, 215–229 (2006).
therapy to treat zygomycosis.75–78 However, in vitro studies 7. Greenberg, R.N., Scott, L.J., Vaughn, H.H., and Ribes, J.A.
show that M. circinelloides, the most common Mucor spe- Zygomycosis (mucormycosis): Emerging clinical impor-
cies recognized as causing zygomycosis, is frequently resistance and new treatments. Curr Opin Infect Dis 17, 517–525
tant to this agent.74 In a study, where Mucor species were not (2004).
differentiated into species, only 70% of the isolates tested 8. Brown, J. Zygomycosis: An emerging fungal infection. Am J
were susceptible to posaconazole.74 Also, most of the other Health Syst Pharm 62, 2593–2596 (2005).
9. Eucker, J., Sezer, O., Graf, B., and Possinger, K.
antifungal agents evaluated showed a high level of in vitro Mucormycoses. Mycoses 44, 253–260 (2001).
resistance to strains of Mucor, including high levels of resis- 10. Freifeld, A.G. and Iwen, P.C. Zygomycosis. Sem Resp
tant to voriconazole (minimum inhibitory concentration CritCare Med 25, 221–231 (2004).
>2).74 This resistance to voriconazole is especially trouble- 11. Ribes, J.A., Vanover-Sams, C., and Baker, D.J. Zygomycetes
some since reports now imply that a significant increase in in human disease. Clin Microbiol Rev 13, 236–301 (2000).
zygomycosis may be due to prior exposure to voriconazole, 12. Takakura, S. Zygomycosis. Nippon Rinsho 66, 2356–2361
whether used as prophylaxis or in treatment of other fungal (2008).
13. James, T.Y. et al. Reconstructing the early evolution of fungi
conditions.22,30,79,80
using a six-gene phylogeny. Nature 443, 818–822 (2006).
Currently, the best therapy to treat zygomycosis caused 14. O’Donnell, K., Lutzoni, F.M., Ward, T.J., and Benny,
by Mucor species is not known. For optimal management G.L. Evolutionary relationships among mucoralean fungi
of a patient with a Mucor-associated infection, the current (Zygomycota): Evidence for family polyphyly on a large
need is for the development of a reliable and rapid molecu- scale. Mycologia 93, 286–296 (2001).
lar diagnostic assay to identify the etiological agent directly 15. Tanabe, Y., Watanabe, M.M., and Sugiyama, J. Evolutionary
from clinical material. Limitations to the development of this relationships among basal fungi (Chytridiomycota and
Zygomycota): Insights from molecular phylogentics. J Gen
type of assay, however, are that the disease is still considered
Appl Microbiol 51, 267–276 (2005).
rare, which makes clinical studies difficult; the organism is 16. White, M.M. et al. Phylogeny of the Zygomycota based on
a common environmental contaminate and thus correlation nuclear ribosomal sequence data. Mycologia 98, 872–884
of organism with causing a disease is more complicated; and (2006).
the need for an invasive procedure in a severely compromised 17. Hibbett, D.S. et al. A higher-level phylogenetic classification
patient makes evaluation of this type of testing under clinical of the fungi. Mycol Res 111, 509–547 (2007).
conditions more of a challenge. 18. de Hoog, G.S., Guarro, J., Gene, J., and Figueras, M.J. In
In conclusion, the importance of zygomycosis as an emerg- Atlas of Clinical Fungi, pp. 84–93, Centraalbureau voor
Schimmelcultures, Utrecht, the Netherlands, 2000.
ing infectious disease in compromised patients enhances the
19. Iwen, P.C., Hinrichs, S.H., and Rupp, M.E. Utilization of the
need for better methods to diagnose and treat this condition. internal transcribed spacer regions as molecular targets to
Preliminary studies have shown great promise in the devel- detect and identify human fungal pathogens. Med Mycol 40,
opment of molecular assays for the diagnosis of invasive fun- 87–109 (2002).
gal infections. As these assays undergo further improvement, 20. Landlinger, C. et al. Species-specific identification of a
the accessibility to reliable and rapid tests to diagnose this wide range of clinically relevant fungal pathogens by use of
devastating infection show some promise. On the other hand, Luminex xMAP technology. J Clin Microbiol 47, 1063–1073
(2009).
more clinical studies are needed to make this technology in
21. Rakeman, J.L. et al. Multilocus DNA sequence comparisons
the near future a reality. rapidly identify pathogenic molds. J Clin Microbiol 43, 3324–
3333 (2005).
22. Schwarz, P. et al. Molecular identification of zygomyce-
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87 Rhizopus
Dongyou Liu and Frank W. Austin
Contents
87.1 Introduction...................................................................................................................................................................... 773
87.1.2.2 Pathogenesis........................................................................................................................................... 776
87.1.2.3 Treatment............................................................................................................................................... 776
87.1.3.2 Genotypic Identification........................................................................................................................ 776
87.2 Methods............................................................................................................................................................................ 777
87.2.1.1 Fungal Strains and Cultivation.............................................................................................................. 777
87.2.1.2 Microscopic Examination...................................................................................................................... 777
87.2.1.3 DNA Isolation........................................................................................................................................ 777
87.2.2.1 PCR Identification of Individual Rhizopus Species............................................................................... 777
87.2.2.2 PCR-RFLP Identification of Rhizopus spp............................................................................................ 777
87.2.2.3 Nested PCR for Mucorales.................................................................................................................... 778
87.2.2.4 Real Time PCR for Genera Rhizopus, Mucor, Absidia, Cunninghamella, Apophysomyces,
and Saksenaea....................................................................................................................................... 778
87.2.2.5 Real Time PCR for Genera Rhizopus, Mucor, and Rhizomucor........................................................... 778
87.2.2.6 Sequence Analysis of 18S and 28S rRNA Genes.................................................................................. 779
87.3 Conclusion........................................................................................................................................................................ 779
References.................................................................................................................................................................................. 779
87.1 Introduction R. delemar, R. formosaensis, R. japonicus, R. javanicus, R.

kasanensis, R. maydis, R. nodosus, R. oryzae, R. thermosus,
87.1.1 Classification, Morphology, and Biology R. tonkinensis, R. tritici, R. trubinii and R. usamii), Rhizopus
The genus Rhizopus is a zygomycetous mold classified azygosporus, Rhizopus caespitosus, Rhizopus circinans,
under the family Mucoraceae, order Mucorales, sub- Rhizopus homothallicus, Rhizopus microsporus (consisting
phylum Mucoromycotina, kingdom Fungi. The fam- of varieties microsporus, oligosporus, and rhizopodiformis;
ily Mucoraceae currently consists of 24 genera: Absidia, obsolete synonyms: R. oligosporus, R. rhizopodiformis,
Actinomucor, Ambomucor, Amylomyces, Apophysomyces, Mucor rhizopodiformis, and R. cohnii), Rhizopus niveus,
Chlamydoabsidia, Circinella, Circinomucor, Gongronella, Rhizopus schipperae, Rhizopus sexualis, and Rhizopus sto-
Halteromyces, Hyphomucor, Lentamyces, Mucor, lonifer (synonym R. nigricans) plus 17 unassigned species [1].
Parasitella, Phycomyces, Protomycocladus, Rhizomucor, Due to their close molecular and antigenic relationships,
Rhizopus, Siepmannia, Spinellus, Sporodiniella, Syzygites, R. arrhizus and R. oryzae are now considered synonymous.
Thermomucor and Zygorhynchus in addition to some unclas- R. schipperae resembles R. microsporus var. microspo-
sified Mucoraceae [1]. Within the family Mucoraceae, mem- rus, with similar sporangiophore, sporangium, sporangio-
bers of the genera Rhizopus, Mucor, Absidia, Rhizomucor, spore, and columellar morphology and similar temperature
and Apophysomyces have been implicated in human diseases, growth dependence. However, R. schipperae differs from
with Rhizopus being the most common cause of human zygo- R. microsporus var. microsporus by having fastidious
mycosis [2,3]. sporulation requirements and distinctive organization of
The genus Rhizopus is divided into 10 recognized species, sporangiophores in large clusters of up to 10 sporangio-
namely, Rhizopus arrhizus (obsolete synonyms: R. batatas, phores. Additionally, R. schipperae also shows some limited
773

morphologic similarities to R. caespitosus, which produces Transmission of Rhizopus spp. is often by the respiratory,
large clusters of sporangiophores. Rhizopus azygosporus also percutaneous, and gastrointestinal or oral routes, with most
appears to be related to the R. microsporus group, since mat- Rhizopus infections involving the rhinocerebral and pulmo-
ing of R. microsporus var. microsporus and R. microsporus nary sites, occurring secondary to inhalation of spores into
var. rhizopodiformis results in the production of azygosporus the lungs or sinuses. The use of spore-contaminated adhesive
strains, which have very similar morphology to R. azygospo- bandages to hold dressings in place over surgical wounds has
rus. However, the species designation of R. azygosporus is contributed to percutaneous transmission of Rhizopus (ory-
upheld due to its obligate azygosporic nature. zae) arrhizus or R. microsporus var. rhizopodiformis. Insect
While Rhizopus spp. are cosmopolitan filamentous fungi bite, intramuscular injection, catheter insertion, intravenous
that are present in soil, decaying fruit and vegetables, animal drug use, and oropharyngeal examination (using spore-con-
feces, and old bread, some are pathogenic to plants, humans, and taminated wooden tongue depressors) may be also responsi-
animals, with seven distinct species and varieties of Rhizopus ble for sporadic cases of Rhizopus zygomycosis. In addition,
being implicated in human disease. The term mucormycosis or ingestion of fermented milk with dried bread products, fer-
the preferred term zygomycosis is used to describe the angio- mented porridges, and alcoholic drinks derived from corn
invasive disease due to the mucorales including Rhizopus spp. may promote gastric zygomycosis.
Indeed, Rhizopus arrhizus (=Rhizopus oryzae) is the most Sequencing analysis of Rhizopus (oryzae) arrhizus strain
common cause of human zygomycosis, accounting for 60% of 99–880, isolated from a fatal case of zygomycosis, reveals a
the reported culture positive cases, and nearly 90% of the rhi- highly repetitive 45.3 Mb genome, 20% of which are trans-
nocerebral forms of infection. Often used in the production of posable elements (TEs) containing paralogous gene pairs. A
fermented foods and alcoholic beverages in Indonesia, China, total of 13,895 protein-coding genes are predicted. There is
and Japan, R. oryzae also generates the ergot alkaloid agro- evidence that an ancestral whole-genome duplication (WGD)
clavine with toxicity for humans and animals. Other notable event may have resulted in the duplication of nearly all sub-
human-infecting Rhizopus species/varieties include Rhizopus units of the protein complexes associated with respiratory
microsporus var. rhizopodiformis, R. microsporus var. oli- electron transport chains, the V-ATPase, and the ubiquitin-
gosporus, R. microsporus var. microsporus, R. azygosporus, proteasome systems. Along with recent gene duplications,
R. schipperae, and R. stolonifer [4–6]. the WGD has contributed to the expansion of multiple gene
The morphological characteristics of Rhizopus spp. families related to cell growth, signal transduction, secreted
include the presence of stolons and pigmented rhizoids, the aspartic protease, and subtilase protein families, which are
formation of globose to ovoid, one-celled, hyaline to brown known fungal virulence factors. It is notable that the dupli-
and striate sporangiophores, which appear singly or in clus- cation of the ergosterol biosynthetic pathway, especially the
ters from nodes directly above the rhizoids, and apophysate, major azole target, lanosterol 14-α-demethylase (ERG11),
columellate, multi-spored, generally globose sporangia. may have played a role in altering R. oryzae response to dif-
Following the release of spores, the apophyses and columella ferent azole drugs (e.g., voriconazole and posaconazole) [8].
often collapse to form an umbrella-like structure [7].
On agar plates, Rhizopus colonies grow very rapidly, and 87.1.2 Clinical Features and Pathogenesis
fill the Petri dish surface with a dense cotton-candy growth
that matures (produces sporulation) in 4 days. From the 87.1.2.1 Clinical Features
front, the colony is white in color and turning gray to yel- Rhizopus spp., especially R. (oryzae) arrhizus and R.
lowish brown, while the reverse is white to pale. Broad thin- microsporus var. rhizopodiformis, are responsible for about
walled, hyaline hyphae (6–15 μm in diameter) are coenocytic 90% of human zygomycosis, which may occur as sinusitis/
(aseptate) or sparsely septate. Solitary or clustering sporan- rhinocerebral, pulmonary, cutaneous/subcutaneous, gastro-
giophores are brown in color and unbranched. Rhizoids (root- intestinal, genitourinary, and disseminated diseases, as well
like hyphae) are situated at the point where the stolons and as cystitis, external otitis, and allergic disease [9–33].
sporangiophores meet. Round sporangia (40–350 μm in diam- The hallmarks of human zygomycosis caused by muco-
eter) with flattened bases are at the tip of the sporangiophores. rales such as Rhizopus spp. are angioinvasion, thrombosis,
Columellae are hemispherical. Unicellular sporangiospores infarction, and necrosis of involved tissues. An acute inflam-
(4–11 μm in diameter) are round to ovoid in shape, hyaline to matory response is noticeable in non-neutropenic patients,
brown in color, and smooth or striated in texture (Table 87.1). with thick, necrotic fluids aspirating from areas of abscess
The length of rhizoids and sporangiophores, the diameter formation.
of sporangia, the shape of columellae, and the size, shape and The most common sites of zygomycosis are sinuses, rhino-
surface texture of sporangiospores are useful morphologi- cerebral structures, and the lungs (with or without dissemina-
cal criteria for the differentiation of Rhizopus species from tion). Rhinocerebral disease accounts for nearly one-third to
each other. The maximum growth temperature may be also one-half of all cases of zygomycosis, as a result of inspiration
employed for discrimination of Mucorales genera. For exam- of fungal spores in the sinuses, especially in the setting of
ple, the best growth temperatures for Mucor, Mortierella, diabetic ketoacidosis [34]. From initial sinus pain, drainage,
Apophysomyces, Absidia/Rhizopus, and Rhizomucor are and soft tissue swelling, the disease may progress rapidly
<37°C, 40°C, ≥42°C, 45°C, and 54°C, respectively. and extend into neighboring tissues, with signs of periorbital

Rhizopus
TABLE 87.1
Morphological and Physiological Characteristics of the Clinically Important Rhizopus Species
Species Colony Sporangium Columella Apophysis Sporangiospore Rhizoids Sporangiophore Zygospores Growth Temperature
R. arrhizus Floccose Globose; Globose to oval, Inconspicuous, Oval to Abundant; Medium (750– Heterothallic; Growth at 36°C, but not
colonies, powdery 40–75 μm by often ellipsoidal, brown, with 2000 μm, usually reddish brown, at >46°C
initially white, and gray to 60–130 μm collapsing at 6–8 μm by four to eight 1500 μm); singly or in round to flattened
turning yellow, black; sporulation 4.5–6 μm; branches groups from stolon with conical
pale brown, dark medium striated projections;
brown, or gray, (100– usually 80–140 μm
and collapsing 200 μm in
with age diameter)
R. azygosporus Floccose white Globose; Oval hyaline Discrete Oval, 4–6 μm by Simple, pale Short (usually Abundant round Growth at 28°C–45°C;
mycelim, turns small collumella 6–7 μm; faintly brown 350–500 μm by azygospores, poor growth at up to
gray to black (35–100 μm striated 6–14 μm) 30–70 μm in 50°C; sporangium and
with age in diameter) diameter azygospore forming at
25°C–37°C
R. microsporus Floccose Globose; Pyriform, Discrete Variable size and Simple, no Short (usually Heterothallic; small Variable
mycelium; small ellipsoidal, shape branching 800–1000 μm) (80–100 μm)
variable color (about cylindrical, red-brown
100 μm) conical,
globose, or
subglobose
R. schipperae Thin white Globose; Subglobose to Discrete Oval, 4–7 μm; Simple, Short (100–410 μm by Unknown; Growth sporulation at
nonsporulating small conical faintly striated brown, 5–15 μm) in clusters chlamydospores 30°C–35°C; tolerating
mycelium (<80 μm in columella rarely of 10 (20 μm in up to 45°C, but not
diameter) (5.8–6.8 μm by branched diameter) abundant >48°C
4.8–5.0 μm)
R. stolonifer floccose white Globose; Oval to Distinct Round to oval, Abundant; Long (up to 2000 μm), Heterothallic; black, Growth and sporulation
mycelium with black; large subglobose, 13 μm; black; 300–350 μm pigmented, and round zygospores at 15°C–30°C, but not
black dots (up to 70–120 μm in prominently long; unbranched; in groups with irregular >33°C
275 μm) diameter; pale striated intensely of 1–3 surface
brown ramified decorations;
150–200 μm wide;
equal suspensors
Source: Adapted from Ribes, J.A. et al., Clin. Microbiol. Rev., 13, 236, 2000.
775
edema, proptosis, and tearing; pain, blurring, or loss of vision In immunocompetent hosts, zygomycosis with Rhizopus
in the infected eye; painful, black, necrotic ulcerations into often follows a disruption of the mucocutaneous barrier (e.g.,
the hard palate in the mouth; and a bloody nasal discharge underlying trauma or surgical wound) or antibiotic or ste-
due to invasion of the terbinates and the brain. Prolonged roid use. In diabetic hosts, the monocytes/macrophages are
exposure to Rhizopus spores causes lung damage, with thick- dysfunctional and incapable of suppressing the germination
ened alveolar walls, isolated solitary nodular lesions, cavi- of Rhizopus spores, and the impaired neutrophil activation
tary lesions, granulomas, and interstitial infiltrates [35]. This further hampers the killing of proliferating hyphal elements.
is a significant health problem in the sawmill (“woodcutter’s
disease”) and malt industries. 87.1.2.3 Treatment
Cutaneous and/or subcutaneous sites are also involved in Amphotericin B, either alone or together with other interven-
Rhizopus infections. This is often associated with the use of tions (e.g., surgery) provides a clinically useful treatment for
nonsterile adhesive bandages, which introduce spores either zygomycosis caused by the Mucorales [38,39]. Posaconazole
directly into the surgical wound or indirectly into the trau- is also useful for treating Rhizopus infections [40–42].
matized skin at the time of bandage removal. Growth of Without early detection and aggressive treatment with intra-
the fungus in a preexisting lesion often generates an acute venous amphotericin B and surgical debridement, zygomy-
inflammatory response with pus, abscess formation, tissue cosis is frequently fatal, with mortality rates approaching
swelling, and necrosis. Sloughing-off in necrotic tissue may 80% in infected transplant recipients [43].
result in large ulcers. Primary cutaneous disease is highly
invasive locally, involving the cutaneous and subcutaneous 87.1.3 Laboratory Diagnosis
tissues as well as the fat, muscle, and fascial layers beneath.
Necrotizing fasciitis may occur secondary to cutaneous or 87.1.3.1 Phenotypic Identification
subcutaneous zygomycosis [36]. Initial identification of Mucorales including Rhizopus spp.
Disseminated Rhizopus disease can affect any organs in often relies on microscopic observation of hyphae and
the body, including the skin, central nervous system, liver, other fungal elements in clinical specimens, with the help
spleen, kidney, and gastrointestinal tracts. In fatal cases of of 10% KOH and other fungal specific stains such as calco-
disseminated R. azygosporus, infections involving a prema- fluor white stain, Gomori methenamine silver stain (GMS)
ture infant, angioinvasion, tissue infarction, and granuloma- and periodic acid-Schiff (PAS). Typically, Zygomycetes in
tous inflammation with the production of giant cells were cytologic specimens show wide, ribbon-like, hyaline, pre-
noted, and the cause of death was attributable to peritoneal, dominantly aseptate hyphae, which may branch at 45°–90°
gastrointestinal, kidney, or liver involvement by the fungus. angle. In tissue sections stained with calcofluor white stain
In gastrointestinal zygomycosis, necrotic gastric or intestinal or GMS, zygomycosis induces abscess formation with central
ulcers may occur whose rupture may result in peritonitis [37]. tissue necrosis, acute inflammatory exudate, and invasion of
Other unusual symptoms of zygomycosis due to Rhizopus peripheral blood vessels (angio-invasion) by hyphal elements.
spp. include inguinal abscess, bone, renal, and genitourinary The hyphae may appear crinkled or gnarled in the tissue sec-
diseases. tions (so-called crinkled cellophane).
In vitro culture and subsequent examination are neces-
87.1.2.2 Pathogenesis sary for determination of zygomycetes to the species level.
Rhizopus spp. are opportunistic pathogens that may take Colonies of the genus Rhizopus grow rapidly on standard
advantage of breakdown in host’s immune system defenses mycological agars without cyclohexamide, with coarse and
to establish infection and cause disease. Due to their angio- floccose aerial mycelia, stolons, pigmented rhizoids, brown
invasive nature and their ability to grow at or above core pigmented sporangiophores singly or in groups, multi-spored
body temperature, Rhizopus spp. spread readily from the globose terminal sporangia that are both apophysate and
sites of entry to other organs. This infection process is aided columellate, and one-celled, hyaline to brown striated or
by the production of a variety of glycosidic, proteolytic, and grooved sporangiospores (Table 87.1).
lipolytic enzymes, proteins, and by-products with pathogenic Other phenotypic techniques such as indirect immuno-
potential. Several ergot alkaloids (e.g., agroclavin, ergosine, fluorescence, crossed immunoelectrophoresis and counter-
and ergosamine) and mycotoxins (e.g., rhizonin A) produced immunoelectrophoresis, and ELISA have been utilized for
by Rhizopus spp. are toxic to mammalian hosts. the diagnosis of disease caused by Rhizopus with variable
The most frequent predisposing factors for zygomycosis success.
include diabetic ketoacidosis, immunosuppression due to
organ transplantation and cancers (e.g., leukemia, lymphoma, 87.1.3.2 Genotypic Identification
other hematologic malignancies), deferoxamine/desferriox- Nucleic acid amplification techniques targeting small subunit
amine treatment for iron or aluminum overload, hyperglyce- 18S and large subunit 28S rRNA genes, internal transcribed
mia, renal failure, extensive burns, trauma, intravenous drug spacer regions (ITS), cytochrome b, actin, high-affinity iron
use, heatstroke, contaminated adhesive tapes and wooden permease 1 (FTR1), and translation elongation factor (EF-1α)
tongue depressors, protein calorie malnutrition, diarrhea, genes have proven valuable for rapid and accurate detection
typhoid fever, gastric or intestinal ulcers, and amebic colitis. and identification of the etiological agents of zygomycosis

Rhizopus 777
[44–55]. Several species- and group-specific PCR assays is transferred to a new 2-mL microcentrifuge tube, and DNA
have been reported [56–60]. Recent development of real- is precipitated by the addition of an equal volume of 2-propa-
time PCR further permits rapid, convenient detection, and nol. DNA is pelleted at 14,000 × g for 1 min. After discard-
quantification of zygomycosis-causing agents directly in ing supernatant, the pellet is washed with 100% ethanol and
clinical specimens [50,61,62]. PCR followed by sequencing resuspended in 200 μL of TE buffer (10 mM Tris–HCl pH
analysis provides a reliable way to precisely identify a muco- 8.0, 1 mM EDTA). Genomic DNA is stored at −20°C [46].
ralean species [57]. The Microseq D2 sequencing kit target- Alternatively, DNA from cultures or from the clinical
ing the D2 domain of the large rRNA gene subunit enables samples is extracted using the High Pure PCR template
the convenient sequencing of clinically important filamen- preparation kit (Roche). Briefly, an aliquot of mycelium is
tous fungi. suspended in 200 μL of tissue lysis buffer and incubated
for 30 min at 37°C in the presence of lysozyme (10 U/μL,
final concentration). DNA is then extracted by following the
87.2 Methods instructions of the manufacturer. Finally, the concentrations
87.2.1 Sample Preparation are determined with an Ultrospec 2100 spectrophotometer
(Amersham). Samples are kept at −20°C until used [45].
87.2.1.1 Fungal Strains and Cultivation
Zygomycetes strains are stored by lyophilization or in liquid
nitrogen vapor (−175°C). Strains are subcultured from frozen 87.2.2 Detection Procedures
stocks on Sabouraud-chloramphenicol agar slants for 7 days 87.2.2.1 P CR Identification of Individual
at 20°C, 28°C, or 35°C, depending on the optimum growth Rhizopus Species
temperature for each species (Table 87.1). From the cultures, Voigt et al. [57] designed species-specific primers from the
spore suspensions are prepared in 0.9% NaCl. Twenty mil- 28S rRNA sequences for identification of Rhizopus azygospo-
liliters of RPMI 1640 (Sigma–Aldrich) with l-glutamine but rus/Rhizopus microsporus, Rhizopus oryzae, and Rhizomucor
without sodium bicarbonate buffered to pH 7 with 0.165 M miehei/Rhizomucor pusillus (Table 87.2). Use of these prim-
morpholinepropanesulfonic acid (Sigma) is inoculated with ers at an annealing temperature of 60°C results in the ampli-
the spore suspension, and the mycelium is grown for 48–72 h fication of PCR products of 469, 413, and 305 bp, respectively.
at 30°C with agitation. The mycelium is washed once with
20 mL 0.9% NaCl before use [46]. Alternatively, mycelium 87.2.2.2 PCR-RFLP Identification of Rhizopus spp.
for DNA isolation is grown in YM broth (0.3% yeast extract, Machouart et al. [45] utilized a pair of primers (RpL1,
0.3% malt extract, 0.5% peptone, 2% dextrose; Difco) at 5′-TGATCTACGTGACAAATTCT-3′; MR1, 5′-AGTAGTTT
room temperature for 2–5 days. Strains grown on YM agar GTCTTCGGKCAA-3′) for amplification of a 830 bp frag-
(2% Difco agar) are examined morphologically to confirm ment from the 18S rRNA gene of Rhizopus spp. Subsequent
their identity [57]. digestion with restriction enzymes BmgBI and AseI permits
confirmation of the identity of Rhizopus spp. and Rhizopus
87.2.1.2 Microscopic Examination
microsporus or Rhizopus azygosporus.
Mycelium is mounted on slides, or stained with KOH, lac-
tophenol cotton-blue, or other fungal stains. The presence Procedure
of wide, ribbon-like, nonseptate, hyaline hyphae suggests
zygomycetes. Rhizopus spp. are identified on the basis of 1. PCR mixture (50 μL) is composed of 2 mM MgCl2,
microscopy showing the presence of stolons, brown pig- 200 μM of each deoxynucleoside triphosphate,
mented rhizoids, sporangiophores, and globose sporangia, 20 pmol of each primer, 2 U of Fast Start Taq poly-
both apophysate and columellate (Table 87.1). merase (Roche), and 5 ng of the extracted DNA.
2. Amplification is conducted with an initial 94°C for
87.2.1.3 DNA Isolation 3 min, 30 cycles of 94°C for 1 min, 60°C for 1 min,
Genomic DNA from mycelium may be isolated according to and 72°C for 1 min; a final 72°C for 5 min.
the CTAB (hexacetyltrimethylammonium bromide; Sigma) 3. Following amplification, 5 μL of amplicons is elec-
miniprep protocol. Briefly, approximately 50 mg mycelium is trophoresed in 2% agarose gels in the presence of
homogenized in a 50-mL tube containing 1 ml CTAB extrac- ethidium bromide and visualized under UV light (to
tion buffer (100 mM Tris–HCl pH 8.4, 1.4 M NaCl, 25 mM confirm the 830 bp product).
EDTA, 2% CTAB), three glass beads (0.5 cm) (Sigma), and 4. Then, 10 μL of the specific PCR product is digested
500 mg of 425- to 600-μm-diameter glass beads (Sigma). with 5 U of restriction enzyme BmgBI or AseI in a
The suspension is vigorously vortexed and put into liquid volume 20 μL for 1 h at 37°C; the digested samples
nitrogen for 1 min followed by immersion at 37°C for 1 min. are analyzed on 1.5% BET-agarose electrophoresis
After vortexing again for 1 min, 700 μL of the suspension is gels (QBiogen).
transferred into a 2-mL microcentrifuge tube. An equal vol-
ume of chloroform is added to the mixture, vortexed for 5 s, Note: Digestion of the 830 bp amplicon with enzyme BmgBI
and spun for 10 min at 14,000 × g. The upper phase (500 μL) results in two fragments of 600 and 230 bp, indicating the

TABLE 87.2
The 28S rRNA Gene-Based Primers for Specific Identification of
Rhizopus Species
Species Primer Sequence (5′–3′) Product (bp)
Rhizopus azygosporus Rh1 TTTTCCAGGCAAGCCGGACCG 469
Rhizopus microsporus Rh2 TATTCCCAGCCAACTCGCCAAAT
Rhizopus oryzae Ro1 AGCATTTGCCTTTTGTGATACGC 413
Ro2 ACCGTAGTACCTCAGAAAACC
Rhizomucor miehei Rm1 TCTATTGCGATGCATGCTCC 305
Rhizomucor pusillus Rm2 GGTCTCTTTAGACTCCAAAGC
presence of Rhizopus spp. On the other hand, digestion probes 5′-ACAATTTTCTTATTCTTCTTAGTATTAG-3′

with enzyme AseI yields a 750 bp fragment (and undetected (anchor probe) (3′ fluorescein labeled) and 5′-TTTATTCTTA
80 bp fragment for Rhizopus microsporus or Rhizopus TTCTATGCTCCAAATA-3′ (donor probe) (5′ LCRED-
azygosporus. 640 labeled) facilitate detection of the amplicon on the
LightCycler RT instrument (Roche).
87.2.2.3 Nested PCR for Mucorales
Procedure
Hofman et al. [54] described a modified nested PCR for detec-
tion of Mucorales genera, with primers derived from the 28S
1. Real time PCR mixture (15 μL) is composed of 1×
rRNA sequences. The first round PCR primers are Mucor1
LightCycler FastStart DNA master hybridization
(5′-WTTACC RTG AGC AAA TCA GA-3′) and Mucor2
probe buffer, 3 mM MgCl2, 0.5 μM forward and
(5′-CAA TCY AAG AAT TTC ACC TCTAG-3′), and the
reverse primers, 0.2 μM fluorescein-labeled probe,
second round PCR primers are Mucor3 (5′-AGC ATG GAA
0.4 μM RED 640-labeled probe, and 5 μL of the
TAA TRA AAY A-3′) and Mucor4 (5′-AGC ATG GGA TAA
extracted DNA from clinical specimens or control
CGG AAT A-3′), yielding a final amplicon of 124 bp.
material.
Procedure 2. Amplification is carried out with 1 cycle of 95°C for
1. PCR mixture (50 μL) is made up of 2.5 μL each of 72°C for 15 s. A melting curve is generated using
primers Mucor1 and Mucor2 (final concentration the following profile: 95°C for 0 s, 59°C for 20 s, and
3 mM) for the first round [or 2.5 μL each of primers 34°C for 20 s, with a 0.3°C/s transition, and 85°C for
Mucor1 and Mucor2 for the second round], 25 μL 0 s, with a 0.3°C/s transition, using a LightCycler v.
Master Mix Gold (Applied Biosystems), and 100– 1.2 system with LightCycler v. 3.5 software.
500 ng of DNA.
2. Amplification is performed with an initial 94°C for Note: The six genera (Rhizopus, Mucor, Absidia,
10 min; 30 cycles of 94°C, 55°C, and 72°C for 30 s Cunninghamella, Apophysomyces, and Saksenaea) exhibit a
each for both primer sets. range of melt temperatures from 41°C to 55°C, with three
3. Ten microliters of each amplification product is ana- genera (Absidia, Aphophysomyces, and Mucor) having melt
lyzed by electrophoresis in an ethidium bromide- temperatures within approximately 1°C of each other. As
containing 2% agarose gel. expected, Rhizomucor, Condiobolus, and Syncephalastrum
spp. are not detected by the assay.
Note: The nested PCR recognizes several zygomycosis-caus-
ing genera in the family Mucorales. The species identity of 87.2.2.5 Real Time PCR for Genera Rhizopus,
the PCR amplicon can be verified by sequencing. Mucor, and Rhizomucor
Francesconi et al. [61] described a quantitative real time PCR
87.2.2.4 Real Time PCR for Genera Rhizopus, for detection of Mucorales genera Rhizopus, Mucor, and
Mucor, Absidia, Cunninghamella, Rhizomucor. The primers Zygo-F1 (5′-TTCAAAGAGTCA
Apophysomyces, and Saksenaea GGTTGTTTGG-3′), and Zygo-R1 (5′-CAGTCTGGCTCC
Hata et al. [50] developed a real time PCR for detec- AAACGGTTC-3′) generate a 180 bp amplicon from the 28S
tion of Mucorales genera Rhizopus, Mucor, Absidia, rRNA gene sequence, which is detected with hybridization
Cunninghamella, Apophysomyces, and Saksenaea, targeting fluorescence resonance energy transfer probes ZYGO FITC
a highly conserved, multicopy gene, the cytochrome b gene. (5′-GGCGAGAAACCGATAGCGAAC-fluorescein isothio-
The forward (5′-TAGGAATTACAGCAAAT-3′) and reverse cyanate-3′) and ZYGO RD 640 (5′-RD640-GTACCGTGAG-
(5′-CCAATGCAAACTCC-3′) primers amplify a specific 167- GGAAAGATGAAAAGAACTTTGAAA-3′) using a
bp region of the cytochrome b gene, and FRET hybridization LightCycler 2.0 instrument (Roche).

Rhizopus 779
Procedure columns (SuperFine; Pharmacia), the sequenced

products are run on an Applied Biosystems model
1. PCR master mix (15 μL) is composed of 0.025% 377 automated DNA sequencer.
bovine serum albumin, 3 mM MgCl2, 0.025 U 4. Following initial alignment with CLUSTAL W (ver-
Platinum Taq DNA polymerase, 0.2× PCR buf- sion 1.60), sequence alignments are manipulated
fer, 0.2 mM PCR Nucleotide Mix Plus, 0.002 U/μL visually with TSE, a DOS text software program
HK-UNG, 0.25 μM each of Zygo-F1 and Zygo-R1 (SemWare; Marietta, GA). Unweighted phyloge-
primers, and 0.1 μM each of ZYGO FITC and netic analyses are performed on the individual and
ZYGO RD 640 probes. To 15 μL of master mix, combined data sets by using the heuristic search
5 μL of extracted specimen is added. option in PAUP*4.0b1, with 1000 stepwise random
2. Uracil is released by incubating at 37°C for 15 min, addition sequences. The partition-homogeneity test
and then enzyme is inactivated at 95°C for 3 min. (PHT) implemented with PAUP is used to evaluate
Touchdown PCR cycling includes 95°C for 0 s the concordance of the 18S and 28S rRNA data sets,
(20°C/s), followed by annealing in 1°C steps by using 1000 replicates with MAXTREES set to
between 68°C and 54°C for 5 s (10°C/s), each with 5000. Uninformative characters are excluded from
a 72°C extension of 15 s (3°C/s) for each cycle. the PHT. A second incongruence test, the Wilcoxon
Touchdown cycling is followed by 35 cycles of 95°C signed-ranks Templeton test, is implemented with
for 0 s (20°C/s), 54°C for 5 s (10°C/s), and 72°C for PAUP, by using the most parsimonious tree (MPT)
15 s (3°C/s). and a 70% majority rule bootstrap tree as constraints
3. A post-amplification melt analysis is performed by in a separate analysis. Clade stability is estimated
cooling from 96°C to 40°C for 30 s (20°C/s), fol- from 1000 bootstrap replications with PAUP and by
lowed by a gradual increase in temperature (2°C/s) decay indices calculated with TreeRot.
to 75°C for 0 s (0.2°C/s).
87.3 Conclusion
Note: Quantitation standards (10-fold serial dilutions of R.
oryzae genomic DNA ranging from 1 × 103 fg to 1 × 101 fg) Among the human pathogenic genera (Rhizopus, Mucor,
are run in conjunction with each set of samples to assess Absidia, Rhizomucor, and Apophysomyces) in the family
assay sensitivity and linearity and qPCR results. A crossover Mucoraceae, Rhizopus is noted for its ability to cause zygo-
value of ≤22 cycles is considered positive [62]. mycosis in humans, with symptoms ranging from sinusitis/
rhinocerebral, pulmonary, cutaneous/subcutaneous, gastroin-
87.2.2.6 Sequence Analysis of 18S testinal, genitourinary, and disseminated diseases, as well as
and 28S rRNA Genes cystitis, external otitis, and allergic disease. Of the 10 recog-
Voigt et al. [57] utilized primer pairs PNS1–NS41 and NS51– nized species in the genus Rhizopus, 5 (R. arrhizus, R. azygos-
NS8Z or NS5–NS8Z to amplify the 18S rRNA as two over- porus, R. microsporus [consisting of varieties microsporus,
lapping fragments, and sequenced the amplicons with NS2, oligosporus and rhizopodiformis], R. schipperae and R. sto-
NS3, NS5, NS7, NS8; PNS1, NS6Z, NS8Z; and NS41 and lonifer) are implicated as opportunistic human pathogens.
NS51. In addition, they also utilized primer pair NL1–NL4 Due to their close morphological and biochemical similarity,
to amplify the 5′ end of 28S rRNA spanning domains D1 and precise identification of Rhizopus spp. through macroscopic
D2, and sequenced the amplicon with primers NL1, NL4, and microscopic examinations is difficult and time-con-
ZNL2A, and ZNL3A. suming. By focusing the 18S and 28S rRNA and other gene
sequences, nucleic acid amplification procedures such as
Procedure PCR and sequencing offer a rapid, sensitive, and specific
approach for detection and differentiation of Rhizopus spp.
1. PCR mixture (50 μL) is made up of 0.225 mM each Future characterization of additional species-specific gene
dNTP, 25 pmol of each primer, 50 mM KCl, 10 mM regions will enhance the utility of molecular techniques for
Tris–Cl pH 8.4, 2.5 mM MgCl2, 0.1 mg of gelatin/ diagnosis of zygomycosis, contributing to the effective treat-
mL, 1.25 U of AmpliTaq polymerase (Perkin- ment and control of this potentially fatal disease.
Elmer), and 10–20 ng of genomic DNA.
2. PCR amplification is performed with an initial 94°C
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gal infections in 1,963 thoracic organ transplant recipi- mon but life-threatening infection. Am J Perinatol. 2009
ents—A multicenter retrospective study. Transplantation. Sep;26(8):565–573.
2000;70:112–116. 33. Wilkins RM, Hahn DB, and Blum R. Bread mold osteomyeli-
13. Nosari A et al. Mucormycosis in hematologic malig- tis in the femur. Orthopedics. 2009;32(5):362.
nancies: An emerging fungal infection. Haematologica. 34. Garg J et al. Nosocomial cutaneous zygomycosis in
2000;85:1068–1071. a patient with diabetic ketoacidosis. Int J Infect Dis.
14. Kantarcioğlu AS et al. A Rhizopus oryzae strain isolated 2009;13(6):e508–e510.
from resected bone and soft tissue specimens from a sinona- 35. Lekakis LJ et al. Fatal rhizopus pneumonia in allogeneic stem
sal and palatal mucormycosis case. Report of a case and in cell transplant patients despite posaconazole prophylaxis:
vitro experiments of yeastlike cell development. Med Mycol. Two cases and review of the literature. Biol Blood Marrow
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2007;56:1243–1245. 38. Rodríguez MM et al. Posaconazole combined with amphoteri-
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patient with alcoholic encephalopathy. J Hand Surg Am. 2007 Rhizopus oryzae in a renal transplant recipient successfully
March;32(3):384–388. treated with liposomal amphotericin B. Chang Gung Med J.
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Rhizopus oryzae in a 71-year-old man with normal immune 40. Kok J et al. Early use of posaconazole in the successful treat-
status. Mycoses. 2007 January;50(1):82–84. ment of rhino-orbital mucormycosis caused by Rhizopus ory-
20. Uçkay I et al. Invasive zygomycosis in transplant recipients. zae. J Infect. 2007 September;55(3):e33–e36.
Clin Transplant. 2007;21:577–582. 41. Peel T et al. Posaconazole as first line treatment for dissemi-
21. Ustun C et al. Early fatal Rhizopus infection on voriconazole nated zygomycosis. Mycoses. 2008;51(6):542–545.
prophylaxis following allogeneic stem cell transplantation. 42. Schlemmer F et al. Breakthrough Rhizopus infec-
Bone Marrow Transplant. 2007 June;39(12):807–808. tion on posaconazole prophylaxis following alloge-
22. Zaoutis TE et al. Zygomycosis in children: A systematic neic stem cell transplantation. Bone Marrow Transplant.
review and analysis of reported cases. Pediatr Infect Dis J. 2008;42(8):551–552.
2007;26(8):723–727. 43. Vyzantiadis TA et al. Rhino-cerebral zygomycosis resis-
23. Bonifaz A et al. Palatal zygomycosis: Experience of 21 cases. tant to antimycotic treatment: A case report. Mycoses.
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an orthotopic heart transplant recipient. Transpl Infect Dis. nation of agents of mucormycosis and aspergillosis
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25. Johnson KE et al. An atypical case of fatal zygomycosis: November;58(11):1180–1184.
Simultaneous cutaneous and laryngeal infection in a patient 45. Machouart M et al. Genetic identification of the main oppor-
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46. Schwarz PS et al. Molecular identification of zygomyce- 55. Woo PC et al. Internal transcribed spacer region sequence
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xMAP technology. J Clin Microbiol. 2009;47:1063–1073. identification of clinically important Zygomycetes based on
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Pathol. 2010;5:1. 2008;46(11):3690–3702.

88 Rhizomucor
Tamás Papp, Ildikó Nyilasi, Miklós Takó, László G. Nagy, and Csaba Vágvölgyi
Contents
88.1 Introduction...................................................................................................................................................................... 783
88.1.2.1 Rhizomucor pusillus.............................................................................................................................. 785
88.1.2.2 Rhizomucor variabilis........................................................................................................................... 785
88.1.3 Diagnosis.............................................................................................................................................................. 785
88.2 Methods............................................................................................................................................................................ 786
88.2.1.1 Media and Culturing Conditions........................................................................................................... 787
88.2.1.2 Purification of Genomic DNA............................................................................................................... 787
Acknowledgment....................................................................................................................................................................... 788
References.................................................................................................................................................................................. 788
88.1 Introduction these species were originally classified in the genus Mucor,
some authors still cite R. miehei and R. pusillus as Mucor
Members of the genus Rhizomucor are Zygomycetes belong- miehei and M. pusillus, but these designations are obsolete.
ing in the order Mucorales. These saprophytic fungi are ubiq- R. tauricus had been isolated only once. On the basis of
uitous in nature and can be isolated from soil and decaying carbon source utilization studies and isoenzyme and ITS-
organic materials, worldwide. Rhizomucor spp., and espe- RFLP analyses, Vágvölgyi et al.3 questioned the validity
cially R. pusillus and R. variabilis, are also known as agents of this species and suggested that the single original isolate
of zygomycosis, e.g., frequently fatal mycotic diseases in might well be a mutant heterothallic R. pusillus strain. Later,
immunocompromised patients (infections caused by muco- two additional thermophilic species, Rhizomucor nainitalen-
ralean fungi are often referred to as mucormycosis too).1 An sis4 and Rhizomucor pakistanicus,5 were described, but they
expanding body of data is becoming available that demon- are now regarded as synonyms of R. miehei and R. pusillus,
strates the opportunistic pathogenic potential of these fungi. respectively.6
Two mesophilic species were also described as new mem-
88.1.1 Classification, Morphology, bers of the Rhizomucor genus: Rhizomucor endophyticus, an
endophyte of wheat,7 and Rhizomucor variabilis (with two
and Epidemiology
varieties, R. variabilis var. regularior and R. variabilis var.
The genus Rhizomucor (Mucoromycotina, Mucorales) com- variabilis), a causative agent of cutaneous zygomycoses in
prises Mucor-like fungi inasmuch as they have nonapoph- humans.8–10 Molecular phylogenies inferred from ribosomal
ysate sporangia and branching sporangiophores, but the RNA (rRNA) gene sequences11 and from protein coding
formation of rhizoids and stolons clearly distinguishes them sequences of the actin and the translation elongation factor
from Mucor species. Rhizomucor differs from Rhizopus by 1α (tef) genes12 demonstrated that the thermophilic R. mie-
the absence of apophysis and the presence of branched spo- hei and R. pusillus are closely related to each other, while
rangiophores, and from Absidia by the absence of apophysis the mesophilic R. variabilis proved to be phylogenetically
and the presence of globose sporangia (Figure 88.1). In con- related to Mucor species, e.g., to M. hiemalis and M. mucedo.
trast with Rhizopus, Rhizomucor develops the rhizoids inter- In a recent phylogenetic study based on gene sequences of
nodally, e.g., they arise between and not at the base of the the rRNA cluster, two strains identified previously as M. hie-
sporangiophores (Figure 88.1). malis f. hiemalis displayed 97% sequence identity with the
The genus as accepted by Schipper2 contains three ther- examined R. variabilis isolates, and R. variabilis has formed
mophilic species, R. miehei, R. pusillus, and R. tauricus. As a well-supported sister group with M. hiemalis f. hiemalis.13
783

c c
c
r c
c
r
c
r
r
c
r
(A) (B)
FIGURE 88.1 Light microscopic images of Rhizomucor pusillus WRL CN(M)231 (A) and Rhizomucor miehei NRRL 5282 (B). Strains
were cultured on MEA for 4 days at 36°C. Scale bars indicate 20 μm in each panels. C, columella; r, rhizoid.
TABLE 88.1
Differential Characteristics of Rhizomucor Speciesa
Species R. miehei R. pusillus R. variabilis
Max. growth temperature 57°C 55°C 38°C
Colony
Color Gray to deep-olive-gray Gray to hair-brown Whitish to ochraceous
Height 1 μm 1–2 μm 2 μm
Rhizoids May be present May be present Abundant
Branching of the sporangiophores Sympodial Monopodial and sympodial Simply or once branched
Size of sporangia 50–75 μm in diameter 80–100 μm in diameter Up to 120 μm in diameter
Columellae 28 × 25 μm, obovoid 45 × 38 μm, obovoid 40 μm in diameter, spherical
Sporangiospores 3–4 μm, subglobose 3–4 μm, subglobose 3–11 μm, subspherical to ellipsoidal
Chlamydospores Not produced Not produced Abundant
Zygospores Up to 50 μm in Up to 75 μm in diameter Unknown
diameter
Thiamine dependence of growth Dependent Independent Unknown
Sources: Based on the descriptions of Schipper, M.A.A., Stud. Mycol., 17, 53, 1978; de Hoog, D.S. et al., Atlas of Clinical Fungi, 2nd
edn., Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, 2000; Ribes, J.A. et al., Clin. Microbiol. Rev., 13,
236, 2000; Wang, A. et al., Chin. J. Dermatol., 27, 51, 1994 (Chinese); Lu, X. et al., Clin. Infect. Dis., 49, e39, 2009.
Carbon assimilation profiles indicated that R. variabilis and temperatures, the abundance of rhizoids and the presence
R. variabilis var. regularior are much closer to Mucor spe- or absence of chlamydospores. R. miehei and R. pusillus are
cies than to Rhizomucor species.14 A personal communica- highly similar to each other, but the colonies of R. miehei
tion from Schipper led de Hoog et al.15 to postulate whether are generally more darkly pigmented than those of R. pusil-
R. variabilis is a degenerate culture of M. hiemalis. Although lus. Moreover, the latter produces somewhat less branched
the molecular phylogeny of R. endophyticus has not been sporangiophores and smaller sporangia and zygospores than
studied in detail to date, it is considered to be a synonym of R. pusillus. R. miehei is homothallic, while R. pusillus is mainly
Mucor genevensis.16,17 All the results so far suggest that the heterothallic with rarely occurring homothallic isolates.
genus Rhizomucor consists of only two thermophilic species, R. miehei and R. pusillus are cosmopolitan fungi that
R. miehei and R. pusillus, while the “mesophilic species” are have been reported from Europe, North America, Asia, and
actually not valid members of this taxon. Africa1,18; they can readily be isolated from soil and decaying
Table 88.1 summarizes the main morphological and or composting organic matter and wastes.1 Both species are
growth characteristics of R. miehei, R. pusillus, and R. vari- known as pathogens in various animals, and they are among
abilis. R. miehei and R. pusillus can be easily distinguished the most frequently isolated causative agents of bovine mycotic
from R. variabilis on the basis of their maximum growth abortion and bovine mastitis.19 At the same time, R. pusillus is

Rhizomucor 785
a rare human pathogen; it may cause opportunistic infections immunocompetent persons, mainly agricultural workers liv-
in patients with a challenged or weakened immune system. ing in underdeveloped areas. From these facts, together with
Human infection caused by R. miehei has not been described1; the strict geographical distribution of the known cases, Lu
this fungus is rather of substantial biotechnological impor- et al.10 assumed that environmental exposure after trauma or
tance as an excellent producer of proteases and lipases. surgery was the main risk factor for these infections. In the
R. variabilis causes primary cutaneous zygomycosis few susceptibility tests performed, R. variabilis exhibited
in humans; it is able to infect immunocompetent persons. sensitivity to amphotericin B, but it proved to be resistant
Although our knowledge concerning the distribution of to other tested antifungal agents (such as azoles, terbin-
R. variabilis is limited, this fungus seems to have the dis- afine, flucitozine, and caspofungin), similarly to many other
tinct geographical feature that it has been isolated almost Zygomycetes.10,35 Accordingly, in the majority of cases,
exclusively in the central and eastern regions of China, rais- amphotericin B therapy was used to treat the disease.
ing the suggestion that R. variabilis may be an endemic
pathogen.10 88.1.3 Diagnosis
Although noteworthy advances in the diagnosis of zygomy-
88.1.2.1 Rhizomucor pusillus coses have been achieved in the past decade, many infections
Ribes et al.1 reported that 19 cases of human zygomycosis are still classified only as zygomycosis or mucormycosis,
caused by R. pusillus had been described in the literature up without species- or at least genus-level identification.36–38
to the year 2000. The majority of those cases involved pul- Histopathological examination is currently regarded as the
monary and disseminated zygomycosis, but primary cutane- definitive diagnosis.37,39,40 Apart from the routine hematoxy-
ous infection, rhinofacial disease, and mycotic endocarditis lin and eosin staining, hyphal elements can be examined by
had also been described. In the past 10 years, primary pul- staining with periodic acid-Schiff, Gomori methenamine sil-
monary infections,20,21 rhinoorbital and rhinocerebral dis- ver, or Calcoflour White stain.25,40
ease,22–24 and posttraumatic soft tissue and bone infection23 In tissues, R. pusillus presents the typical hyaline, ribbon-
have been reported. The transmission of R. pusillus generally like coenocytic hyphae characteristic of Mucorales fungi
occurs by the inhalation of sporangiospores1 and dissemi- that are often associated with necrosis and angioinvasion.1
nated cases most frequently arise from primary pulmonary R. variabilis appears in histopathological samples, as simi-
infections. Several cases have been reported as results of nos- lar broad, ribbon-like, coenocytic hyphae with right-angled
ocomial infections.25–28 Most of the human infections have branches.10,35
been associated with leukemia and profound neutropenia,1,24 Culturing of isolates from tissue samples is required for
but uncontrolled diabetes, immunosuppressive therapy, and the genus- and species-level identification of Rhizomucor.
treatment with steroids have also been implicated as predis- However, this is often difficult, because hyphal elements may
posing factors.1,23,29 For the successful treatment of R. pusil- be scarce in the specimens and they can lose their viability
lus infections, amphotericin B or its lipid formulations, often during sample preparation for histopathology. If the patho-
in combination with surgical intervention, have been used gen has been successfully isolated, it is important to maintain
most frequently. The in vitro susceptibilities of Zygomycetes it for further analysis; strain maintenance methods are well
were examined by Dannaoui et al.30 with the involvement of established for Zygomycetes.
R. pusillus and R. miehei strains; besides amphotericin B, Their characteristic morphological features, such as the
these Rhizomucor spp. proved to be sensitive to itraconazole presence of stolons, rhizoids, and repeatedly branched spo-
and posaconazole with a range of MICs of 0.03–0.25 and rangiophores clearly distinguish Rhizomucor from most
0.06–0.25 μg/mL, respectively. members of the Mucorales. Nonetheless, misidentifications
Although no cases of human disease caused by R. mie- frequently occur and morphology-based identifications dis-
hei have yet been noted, information available from animal agree with the results of DNA-based methods in some cases
mycoses and tests in animal models suggest a potentially (particularly in certain earlier studies).41 The detection of
similar pathogenic behavior as that reported for R. pusillus.1,31 zygospore formation in mating studies has been successfully
used for the identification of Rhizomucor species.42 However,
88.1.2.2 Rhizomucor variabilis this method requires a set of testing strains, and it is therefore
All cases of R. variabilis infections reported in the litera- primarily applicable by reference laboratories.24
ture have involved primary cutaneous zygomycosis.8–10,32–35 Based on their thermophily and some morphological char-
Cutaneous infections caused by other Mucorales species acteristics mentioned above (Table 88.1), true Rhizomucor
generally have an aggressively invasive feature and primar- species (i.e., R. pusillus and R. miehei) can be distinguished
ily occur in immunocompromised patients. In contrast, R. from R. variabilis by conventional methods. At the same
variabilis causes superficial or subcutaneous infections, time, discrimination between R. pusillus and R. miehei is
which generally remain localized for several years without often problematic, frequently resulting in misidentifica-
dissemination or involvement of the adjacent tissues and the tions, or the species names remain to be determined. In
blood vessels.10 All infections reported so far have affected earlier studies, determination of the number of nuclei in the

sporangiospores43 or measurement of the diameter of the them from each other. The commercially available molecu-
zygospores (<50 and >50 μm for R. miehei and R. pusillus, lar identification system, the MicroSeq fungal sequencing kit
respectively)2,42 was of some value for species delimitation, (Applied Biosystems), also uses the D2 LSU rDNA.48 In an
but the results were not clear-cut in most cases. evaluation of this method, the one R. miehei isolate involved
Carbon assimilation tests can also be carried out to differ- could be identified correctly.49 However, a satisfactory deter-
entiate these species. In an earlier study, Vastag et al.44 estab- mination of the suitability of the method for the identification
lished differences between the two species in the utilization of Rhizomucor demands the inclusion of further R. miehei
of sucrose, glycine, phenylalanine, and β-alanine as sole and R. pusillus isolates into the evaluation.
carbon sources. Recently, Schwarz et al.14 used the commer- Recent studies have demonstrated that the comparison of
cialized and standardized kits ID32C and API 50 CH (bio- ITS sequences is a reliable tool for the precise species-level
Mérieux) to evaluate carbon assimilation profiles as a tool for identification of this fungal group.48,50 Hsiao et al.51 developed
the identification of clinically important Zygomycetes. These an oligonucleotide array method that involves hybridization
tests not only clearly distinguished R. variabilis from R. mie- with species- or group-specific probes after PCR amplifica-
hei and R. pusillus; but also differentiated between the latter tion of the ITS region, using universal primers to identify 64
species. Thiamine dependence of growth can likewise be a clinically important fungal species. Unfortunately, the oligo-
differentiating character: the growth of R. miehei is stimu- nucleotide probe designed for the delimitation of R. pusillus
lated by thiamine, whereas R. pusillus does not require this also identified R. miehei. Later, Schwarz et al.52 compared the
compound for growth.28 Lukács et al.45 described a simple ITS regions of 54 strains belonging in 16 species to examine
and reliable method for the accurate differentiation of the two the value of ITS sequencing for the molecular identification of
species, based on the observation that R. pusillus is much medically important Zygomycetes: all three species (R. pusil-
more sensitive than R. miehei to lovastatin. A limitation of lus, R. miehei, and R. variabilis) were easily distinguished
all these methods is that their reliable evaluation requires the via their ITS sequences. Moreover, in the case of R. pusillus,
maintenance and application of appropriate reference strains. it was demonstrated that DNA extraction, PCR amplification,
sequencing, and identification is possible directly from the
88.1.3.2 Molecular Techniques tissues of experimentally infected mice. Indeed, in clinical
The first attempts to use molecular methods to identify and practice, the determination and analysis of the ITS region
characterize Rhizomucor species involved isoenzyme analy- was successfully utilized to identify R. pusillus causing a
sis,44 random amplified polymorphic DNA (RAPD) PCR,46,47 sinus–orbital zygomycosis in a patient with acute myelog-
and PCR-restriction fragment length polymorphism (PCR- enous leukemia,24 and R. variabilis as the agent of primary
RFLP).3 RAPD analysis is a rapid method with which to cutaneous zygomycosis in an immunocompetent host.35 In
study intra- and interspecies genetic variability without a both cases, the fungal agent was isolated and cultured prior
prior knowledge of DNA sequence data. In this technique, to the DNA extraction and molecular identification.
decamer random oligonucleotide primers are used to amplify Sequences of some protein-coding genes, primarily of the
DNA, generating characteristic amplicon patterns. The actin and the translation elongation factor-l alpha (EF-1α)
method allows the identification of specific amplicons dif- genes, have been determined from numerous Zygomycetes
ferentiating between R. pusillus and R. miehei. PCR-RFLP and used to reveal phylogenies of Mucorales.12,53 Rhizomucor
detects specific restriction fragment patterns obtained by the species were involved in these studies and the correspond-
digestion of PCR-amplified fragments and may be a useful ing sequences are available from nucleotide sequence data-
identification method for fungi, especially in laboratories that bases (e.g., EMBL and GenBank); however, their usefulness
do not have access to instrumentation for the sequencing and for species identification has not been analyzed in detail. In
analysis of the PCR products.48 PCR-RFLP of the internal a recent study, Nyilasi et al.54 have described an identifica-
transcribed spacer (ITS) region was found to be appropriately tion method based on PCR amplification and sequencing of
efficient to distinguish R. miehei and R. pusillus and could be the high-affinity iron transporter gene (FTR1) for several
used to clarify the taxonomic position of R. tauricus.3 Zygomycetes (primarily for Rhizopus species). Although
The majority of recent molecular identification methods primer pairs discriminating the thermophilic Rhizomucor
developed for clinically important Zygomycetes are based species from other Zygomycetes were established, motifs of
on the PCR amplification of nuclear ribosomal RNA gene value for the design of primers that clearly resolve R. miehei
sequences (rDNA). The first comprehensive DNA sequence and R. pusillus, were not found in the FTR1 sequences of
database for the identification of clinically important these two species.
Zygomycetes was constructed by Voigt et al.11 who deter-
mined the sequences of the small-subunit (18S) rDNA and
domains D1 and D2 of the nuclear large-subunit (LSU) (28S) 88.2 Methods
rDNA from 42 Zygomycetes. They also designed 13 taxon-
specific primer pairs on the basis of the 28S rDNA to identify
the medically most relevant Zygomycetes. The Rhizomucor- DNA extraction and molecular identification directly from
specific primer pair described identified R. pusillus and clinical specimens are poorly documented for Rhizomucor
R. miehei at a genus level, but it was unable to distinguish species. Iwen et al.24 extracted DNA for PCR amplification

Rhizomucor 787
from sinus tissue according to the procedure originally Another DNA purification method used for Zygomycetes
described by Henry et al.55 for the identification of Aspergillus by Nyilasi et al.54 is as follows. Mycelium (500 mg) is dis-
species. Schwartz et al.52 demonstrated that DNA extraction rupted with pestle and mortar in liquid nitrogen. After
and PCR can be performed directly from homogenized ani- 1.25 mL lysis buffer (50 mM Tris–HCl pH 8.0; 20 mM EDTA;
mal tissues. However, in most of the published cases, isola- 1% N-lauroylsarcosine) has been added to the mycelial pow-
tion and culturing of the fungal agent have preceded sample der, it is allowed to thaw to room temperature and mixed gen-
preparation for molecular diagnosis. tly until a homogenous slurry is obtained. This slurry is next
incubated for 15 min at 65°C and centrifuged (at 5000 × g for
88.2.1.1 Media and Culturing Conditions 10 min, at 4°C) to pellet the cell debris. The supernatant is
Similarly to other Mucorales fungi, Rhizomucor species transferred to another centrifuge tube and extracted twice
can be cultured with ease on several commonly used media, with a 25:24:1 mixture of phenol:chloroform:isoamyl alcohol
such as on malt extract agar or potato dextrose agar or in and once with chloroform:isoamyl alcohol (24:1). The DNA is
RPMI 1640, which is often chosen by clinical laboratories. then precipitated from the upper aqueous phase with 2.5 vol-
Although Sabouraud agar is among the less recommendable umes of ethanol at −20°C. After centrifugation (at 10,000 × g
media for the cultivation of Zygomycetes,31 it has been suc- for 10 min), the resulting pellet is dried under vacuum, and
cessfully applied to culture R. pusillus and R. variabilis for resuspended in 100–200 μL sterile distilled water.
molecular identification.24,35 Alternatively, commercial DNA purification kits can be
To obtain sporangiospores for inoculum preparation, malt used for rapid sample preparation. Two examples of com-
extract agar 3,45,46 (MEA; 5 g malt extract, 5 g yeast extract, mercial kits tested for Rhizomucor species: the MasterPure™
5 g glucose, 10 g KH2PO4), yeast extract-glucose medium Yeast DNA Purification Kit (Epicenter), which has been
(YEG; 5 g yeast extract, 20 g glucose) and potato dextrose tested for Zygomycetes (in this case, the procedure starts
agar 24 (PDA, PDB; 50% potato filtrate obtained by boil- with 20–50 mg mycelium and finally DNA is redissolved
ing 300 g diced potato in 500 mL water and filtering, 20 g in 30–50 mL water), and the DNA QIAamp Mini DNA
sucrose) can be used (all media are given for 1 L and contain Extraction Kit (Qiagen).35
15 g agar). Spore suspensions are prepared in 0.9% NaCl.
Genomic DNA can be extracted from mycelia grown
either on solid or in liquid media. Mycelia are generally
grown for 24–72 h; the appropriate cultivation tempera- Sequence analysis of the ITS region seems to be the best
tures are 36°C for R. pusillus and R. miehei and 24°C for choice for the species-level identification of Rhizomucor.
R. variabilis and R. variabilis var. regularior. For DNA Universal primer pairs designed for amplification of the
extraction, Schwarz et al.52 applied 50 mg mycelium obtained complete or partial ITS1-5.8S-ITS2 region work well for
from cultures in 20 mL RPMI 1640 with l-glutamine and Rhizomucor species.3,24,35,51,52 Composition of the reaction
without sodium bicarbonate (buffered to pH 7 with 0.165 M mixture and the PCR conditions are well documented 52,56,57
MOPS) incubated for 48–72 h with agitation. In several stud- and they may vary, depending on the applied primers and the
ies,3,44,46,54 R. pusillus and R. miehei were cultured in yeast recommendations of the supplier of the polymerase enzyme.
extract–glucose liquid medium with agitation for 72 h on a The primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)
rotary shaker at 200 rpm. Zhao et al.35 cultured R. variabilis and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) described
on Sabouraud glucose agar at 25°C for 7 days before extrac- by White et al.56 have been used in several experiments.3,22 To
tion was performed. amplify the ITS region, the recommended reaction mixture
contains 1 U Taq polymerase, 1 × Taq buffer (according to the
88.2.1.2 Purification of Genomic DNA description of the supplier), 2.5 mM MgCl2, 400 μM of each
Several well-established methods of genomic DNA extrac- dNTPs, 0.16 μM of each primer, and 20 ng genomic DNA as
tion are known for Zygomycetes including Rhizomucor, template. Amplification conditions involve an initial denatur-
such as the CTAB (hexacetyltrimethylammonium bromide) ing step of 5 min at 95°C, followed by 35 cycles at 95°C for
protocol described by Voigt et al.11 Briefly, approximately 30 s, 48°C for 30 s, and 72°C for 1 min; the final cycle is fol-
50 mg lyophilized mycelium is pulverized and resuspended lowed by an extension step at 72°C for 7 min.
in 700 μL CTAB buffer (100 mM Tris–Cl, pH 8.4; 1.4 M Schwarz et al.52 used the universal fungal primers
NaCl; 25 mM EDTA; 2% CTAB) and vortexed for 10 s. After V9D (5′-TTAAGTCCCTGCCCTTTGTA-3′) and LS266
that, an equal volume of chloroform is added to the samples, (5′-GCATTCCCAAACAACTCGACTC-3′).57 The reaction
they are vortexed for 5 s, and spun for 10 min at 12,300 × g in mixtures contained 5 μL genomic DNA, 2.5 μL of 20 μM
a microcentrifuge. A 500-μL portion of the upper phase is concentrations of each primer, 10 μL of each dNTPs (each at
removed to a new microcentrifuge tube, and DNA is precipi- 2.5 mM), 10 μL of 25 mM MgCl2, 6.25 U of AmpliTaq poly-
tated with an equal volume of 220°C isopropanol. After the merase (Roche), and 10 μL of 10 × PCR buffer (Roche); the final
DNA is pelleted by centrifugation and gently washed with volume was 100 μL. The cycling parameters for the PCR were
70% ethanol, it is resuspended in 200 μL TE buffer (10 mM initial denaturation for 10 min at 94°C, followed by 30 cycles
Tris–Cl, pH 8.0; 1 mM EDTA, pH 8.0) or sterile distilled of denaturation for 30 s at 94°C, annealing for 30 s at 58°C,
water. and elongation for 30 s at 72°C, with a final extension step for

10 min at 72°C. For sequencing, PCR products were purified on comparison of the determined sequence with the data depos-
P100 gel fine (Bio-Rad). Purified amplification products were ited in public databases may not bend to a correct identifi-
sequenced in both directions with the primers V9D and LS266. cation, because of the occasionally false annotation of the
The results of sequencing can be compared with nucleotide deposited sequences. Such problems can be avoided by using
sequences deposited in international databases (GenBank, sequences of safely identified reference strains.13
EMBL, and DDBJ) by using BLAST58; several ITS sequences The main criterion of a marker useful for nucleotide
for R. miehei, R. pusillus, and R. variabilis are available. sequence-based identification is that it should be diverse
Schwarz et al.52 determined the ITS sequences of the type or enough to distinguish single species. For Rhizomucor, the
the neotype strains of these three species, including R. variabi- ribosomal ITS region merits this condition, and in the few
lis var. regularior, which can be used as references during the cases described to date,24,35,52 this technique, complemented
identification (accession numbers are DQ118995, DQ119005, with histopathology and culturing of the fungus, has been
DQ119006, and DQ119007 for R. miehei, R. pusillus, R. vari- effective in identifying Rhizomucor species. However, the
abilis, and R. variabilis var. regularior, respectively).35 development of new PCR protocols involving the use of
Nyilasi et al.54 developed a method based on the ampli- taxon- or species-specific primer pairs that generate ampli-
fication of a fragment of the FTR1 gene to detect clini- cons with sizes characteristic of the different species would
cally important Zygomycetes, which is able to distinguish make easier and faster the identification process, allowing the
the thermophilic Rhizomucor species from other fungi. detection of the fungus without sequencing of the amplifica-
An advantage of this rapid procedure is that it does not tion product. The method described by Nyilasi et al.54 for the
require further sequencing and analysis; identification detection of amplification products from the FTR1 gene can
occurs only via gel electrophoresis of the PCR products be considered a first step in this direction.
through the detection of DNA bands with characteristic Besides identification of the pathogenic agent, the usage
sizes. Primers R1 (5′-GGAAACCGATGCYTTGCA-3′) and of species- and strain-specific PCR-based methods may
R2 (5′-CRTCACCRCCTTCTTCGGC-3′) were designed reveal important data as concerns the epidemiology of these
to detect a 432- and a 441-bp amplicon for R. miehei and fungi. Epidemiological investigations can be useful for con-
R. pusillus, respectively. The reaction mixture contained 1.25 firmation of the possible nosocomial spread of R. pusillus.1,25
U of Pfu polymerase (Fermentas), 2.5 μL of 10 × reaction Such studies may also be of help to prove the hypothesis that
buffer, 2.5 mM MgSO4, 400 μM each dATP, dCTP, dGTP R. variabilis is an agent of endemic mycoses and to exam-
and dTTP (Fermentas), 2 μM primers, and 20 ng of genomic ine the suggestion that environmental exposure after trauma
DNA. The amplification conditions comprised 94°C for or surgery could be the main risk factor for such disease.10
3 min, followed by 35 cycles of 94°C for 30 s, 68°C for 30 s, These questions can be answered if typing methods with suf-
and 72°C for 1 min, with a final extension at 75°C for 10 min. ficient discriminative capacity are available.
Correct identification requires the application of reference Finally, molecular studies conducted in order to develop
strains for comparison. identification methods and infer phylogenies may have taxo-
nomic implications. The genus Rhizomucor contains three
animal and/or human pathogenic species, the thermophilic
88.3 C
onclusions and R. miehei and R. pusillus and the mesophilic R. variabilis.
Future Perspectives In spite of the morphological similarities, molecular phylo-
There is an increasing need for improved diagnostic tools genetic studies have revealed that the genus is polyphyletic
available to clinical microbiology to cope with the escalating and R. variabilis is related to Mucor species. Although this
problems of emerging pathogenic fungi, which are often dif- finding is well supported by molecular, biochemical, physio-
ficult to culture and/or differentiate. For decades, most of the logical, and pathological evidence, taxonomic revision of the
Zygomycetes infections were identified only as zygomycosis genus and the species R. variabilis is called for. Broader com-
or mucormycosis, without further species or at least genus parative morphological, physiological, and molecular studies
determination. The main cause of this practice was that histo- with the involvement of new isolates and the determination
pathological examination of the clinical specimens was able of new molecular markers may help to resolve this situation.
only to prove the presence of the Zygomycete and not to iden-
tify the fungus. Morphological examination of the cultured Acknowledgment
isolates has frequently led to misidentifications, especially
This work was supported by a grant of the Hungarian
at the species level. The possibility to analyze molecular
Scientific Research Fund (OTKA CK 80188).
sequence data obtained with PCR techniques has radically
changed this situation and improved the reliability of the
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89 Saksenaea
Eric Dannaoui
Contents
89.1 Introduction...................................................................................................................................................................... 791
89.1.1.1 Classification.......................................................................................................................................... 791
89.1.1.2 Morphology............................................................................................................................................ 791
89.1.2 Biology, Ecology, and Epidemiology.................................................................................................................... 792
89.1.2.1 Biology................................................................................................................................................... 792
89.1.2.2 Ecology and Epidemiology.................................................................................................................... 792
89.1.4 Diagnosis.............................................................................................................................................................. 794
89.2 Methods............................................................................................................................................................................ 795
89.2.1.1 Culture................................................................................................................................................... 795
89.2.1.2 Tissues.................................................................................................................................................... 796
References.................................................................................................................................................................................. 797
89.1 Introduction showed a 90%–91% identity between sequences of a clinical

isolate of S. vasiformis and isolates of A. elegans [6]. These
89.1.1 Classification and Morphology molecular data showed that S. vasiformis and A. elegans are
89.1.1.1 Classification closely related although they are currently belonging to dif-
ferent families.
Saksenaea vasiformis has been originally described by
Saksena in 1953 [1]. This new genus and species have been
erected for an unknown fungus isolated in 1950 and 1952 89.1.1.2 Morphology
from forest soil near the city of Sagar, Madhya Pradesh state, Although the sporulation is obtained only after culturing the
located in the central part of India. The genus was named fungus on specific media, the morphological characteristics
after Dr. R.K. Saksena and the species name refers to the are very unique, allowing an easy identification [1,7]. A sche-
characteristic shape of the sporangium. There is currently matic diagram of S. vasiformis microscopic morphology is
only one species in the genus Saksenaea. A new family, presented in Figure 89.1. Sporangiophores (24–65 μm × 6.5–
Saksenaeaceae, was created in 1974 [2] to accommodate this 9.5 μm) are generally single and bear a terminal sporangium.
genus because its initial belonging to the Mucoraceae was Well-developed rhizoids are present at base of the sporangios-
not supported by its peculiar morphology. More recently, phore. These rhizoids are strongly dichotomously branched
phylogeny of Zygomycetes has been studied by molecular and became brown colored at maturity. The sporangium is
approaches. A recent study based on sequences of the small flask shaped with a spherical venter (22–52 μm × 16–44 μm)
subunit ribosomal DNA (SSU rDNA) and the variable regions and a long neck (54–200 μm × 6.5–11 μm). The collumella is
of the large subunit ribosomal DNA (LSU rDNA) has been hemispherical. The wall of the sporangium is smooth and
performed to uncover the phylogenetic relationships among hyaline. The distal part of the neck is closed by a gelatinous
all clinically important Zygomycetes [3]. It was shown that plugthat eventually dissolved. The sporangiospores are then
S. vasiformis clustered with Apophysomyces elegans, a mem- passively released. The sporangiospores are subcylindrical
ber of the Mucoraceae. The same close relationship between (2.8–4.2 μm × 1.5–2 μm), hyaline, and smooth.
S. vasiformis and A. elegans was also supported by other In only one study [8], morphology of different strains
phylogenetic studies based on actin and elongation factor isolated from three geographic areas (India, Ethiopia,
(EF-1alpha) sequences [4,5]. Moreover, LSU rDNA analysis and Taiwan) were compared. Very interestingly, some
791

Mucilaginous plug posaconazole, 0.015–0.03 μg/mL for itraconazole, 0.125–2 μg/

mL for amphotericin B, and higher MICs of 0.5–4 for vori-
Neck
conazole and 1–64 μg/mL for fluconazole [16]. In another
report, an isolate from French Guyana, tested with the
Sporangiospores
EUCAST microdilution reference method [17], showed high
Sporangium
MIC of >8 μg/mL for voriconazole, caspofungin, and ampho-
Venter tericin B, while itraconazole (MIC of 2 μg/mL) and posacon-
azole (MIC of 0.5 μg/mL) were more active [18]. Another
study also reported the low MIC of 0.01 μg/mL for itracon-
azole against one isolate [19]. Clearly more data are needed
to have a clear picture of the susceptibility of this fungus to
Sporangiophore
the different available antifungal drugs. In particular, it would
be very important to know if amphotericin B is less active
than posaconazole, which is the case for some Zygomycetes
such as Cunninghamella spp. and Apophysomyces elegans
Rhizoids
[20,21]. Animal models of S. vasiformis infection have not
been reported and therefore the in vivo activity of systemic
antifungal drugs has not been tested in animals.
FIGURE 89.1 Schematic diagram of S. vasiformis.
89.1.2.2 Ecology and Epidemiology
morphological differences were noted. In particular, the iso- Overall, the precise geographical distribution of this fungus
late from India had hyaline and smooth sporangiophores, is very poorly known. This could be explained, at least in
while the isolate from Ethiopia had brown and distinctly ver- part, by the difficulty to obtain the characteristic sporulation
rucose sporangiophores. These observations suggest that the necessary for identification. The geographical distribution
studied isolates may belong to different varieties or even dif- can be inferred from environment sampling studies as well
ferent species. as from human case reports (Table 89.1).
Although the isolation of S. vasiformis from the environ-
ment has been rarely reported, the fungus has been found
89.1.2 Biology, Ecology, and Epidemiology in different parts of the world, suggesting a wide distribu-
tion. Several studies reported the presence of S. vasiformis
89.1.2.1 Biology in India. After the first isolation in central India by Saksena
Concerning the biology of S. vasiformis, very few data are [1], it has been cultured from rice husk and grain in Puttur
available. At least, one detailed study has been performed Taluk, Karnataka, located in the south-west of the coun-
to evaluate the physiology of the fungus in culture [9]. It try [22]. More recently, S. vasiformis has been cultured
has been shown that S. vasiformis is able to grow at tem- at a concentration of 7 × 10 4 CFU/g in clay loam soil from
perature ranging from 15°C to 39°C, with an optimum at Annamalai Nagar, Tamil Nadu state, located in southeast of
30°C–35°C. A subsequent study showed that the maximum India [23].
growth temperature is 44°C [7]. By culturing S. vasiformis Hodges [24] isolated S. vasiformis from soil from forest
in different carbon sources, it has been shown that those car- tree nurseries in Georgia, United States, sampled between
bohydrates that supported poor growth (such as arabinose, 1956 and 1961. This was the first recovery of the fungus from
sorbose, galactose) were able in contrast to support good to the environment in the United States. It has also been iso-
excellent sporulation [9]. An interesting study shown that two lated from soil, sampled in 1952, in Barro Colorado Island in
serotypes of S. vasiformis may exist and that the two groups Panama [25]. Mo [26] found S. vasiformis in turtle nest sand
showed different morphologies, serotype 1 isolates having on the Nancite beach in Costa Rica sampled in 1987. Goos
smaller sporangia and shorter neck compared to serotype 2 [27] isolated S. vasiformis from soil of banana-producing
isolates [10]. These observations further support the hypoth- areas collected between 1958 and 1960 in Cortes province,
esis that S. vasiformis may be a species complex. Honduras. In the Middle East, S. vasiformis was isolated
Other biological characteristics of fungi, which have also in Israel from soil of groundnut fields in 1964 [28]. In Asia,
clinical implications, are their susceptibility to antifungal S. vasiformis was recovered from soil sample collected in
drugs. In vitro data on antifungal susceptibility of S. vasifor- 1986 near the seashore in Su-au County, I-lan prefecture,
mis are very scarce. In selected cases, in vitro susceptibility Taiwan [8]. S. vasiformis has also been found in Japan where
testing has been attempted but failed due to poor growth of it has been isolated from pineapple field soil in Okinawa
the fungus [11–13]. In one case, the strain has been found to [29,30]. Finally, the fungus was also found in Africa, where it
be susceptible to amphotericin B with an minimal inhibitory was isolated from intertidal driftwoods near Mitsiua located
concentration (MIC) of 0.08 μg/mL [14]. More recently, four along the Red Sea coast in Ethiopia [8].
isolates have been tested following the CLSI microdilution ref- Geographical distribution may also be inferred from clin-
erence method [15] and showed MICs of 0.015–0.25 μg/mL for ical cases. Infection by S. vasiformis has been reported in

Saksenaea 793
Some of these cases have been reported in more than one

TABLE 89.1 publication. Conversely, other cases of infection have been
Geographical Distribution of Clinical Cases of mentioned but not reported in details such as the case of post-
S. vasiformis Infections and Occurrence of traumatic wound infection after the 2004 tsunami [69]. Of
S. vasiformis in Soil note, cases have also been reported in animals [70,71].
The first human case has been published in 1976 [33] and
Number of Cases
Occurrence subsequently, cases have been reported regularly. Nevertheless,
Region and Country By Region By Country in Soil more cases (n = 10) have been reported in the recent years
Europe 6 (2008 and 2009). Human cases have been reported worldwide
France 2 with a clear predominance from tropical and subtropical areas
Spain 3 (Table 89.1). In particular, cases originated not only from
Greece 1 Australia and New Zealand (n = 13) and Asia (n = 11) mainly
North America 6 India but also Thailand and the Middle East (Iraq, Israel, and
(United States) Saudi Arabia). Other cases have been reported from North
California 1 America in the South and West part of the United States
Louisiana 1
(California, Louisiana, Mississippi, Nevada, Texas), and from
Mississippi 1
South America (Colombia, Ecuador, French Guiana). One
Nevada 1
case originated from North Africa. Interestingly, cases have
Texas 1
also been described in Europe (France, Spain, and Greece).
NS 1
Among the 41 cases, there were 27 males (66%) and 14
Georgia 0 +
South America 3
females (34%) (sex ratio 1.9). The patient age ranged from
Colombia 1
3 months to 84 years (mean age 43.9 years). Considering
Ecuador 1 underlying diseases, most of the patients (90%) were
French Guiana 1 immunocompetent. Only four patients (10%) were consid-
Panama 0 + ered immunocompromised (one had a bladder cancer, one
Costa Rica 0 + received previous chemotherapy for a metastatic gastric
Honduras 0 + cancer, one had an acute lymphoblastic leukemia, and one
Africa 1 a pre-leukemic syndrome treated with chemotherapy). Five
Tunisia 1 patients had diabetes. Among the 31 patients without any
Ethiopia 0 + known underlying diseases, most have sustained trauma
Asia 11 or skin injury. In five cases, animal (such as spider, scor-
Thailand 1 pion, or bird) bites were considered the portal of entry
India 6 + [6,36,47,63,68]. In one patient, the infection developed after
Iraq 1 a tattoo [14]. Five cases were of nosocomial origin: infection
Israel 2 + developed in two cases after surgery [38,55], in two cases
Saudi Arabia 1 at site of injection [52,56], and in one case at site of arterial
Taiwan 0 + catheter [

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MOLECULAR

© 2011 by Taylor & Francis Group, LLC

Boca Raton London New York

CRC Press is an imprint of the

© 2011 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4398-1241-9 (eBook - PDF)

© 2011 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

Printed in the United States of America on acid-free paper

International Standard Book Number: 978-1-4398-1240-2 (Hardback)

© 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC

Chapter 1 Introductory Remarks.............................................................................................................................................. 1

Chapter 4 Bipolaris and Drechslera....................................................................................................................................... 49

Chapter 6 Botryosphaeria and Lasiodiplodia........................................................................................................................ 61

Chapter 10 Fusicoccum and Scytalidium................................................................................................................................. 93

© 2011 by Taylor & Francis Group, LLC

Chapter 12 Leptosphaeria...................................................................................................................................................... 105

Chapter 13 Macrophomina..................................................................................................................................................... 109

Chapter 15 Neodeightonia...................................................................................................................................................... 129

Chapter 16 Phoma and Phomopsis......................................................................................................................................... 135

Chapter 18 Pyrenochaeta....................................................................................................................................................... 145

Chapter 19 Ramichloridium.................................................................................................................................................... 151

Chapter 20 Ulocladium........................................................................................................................................................... 157

Chapter 21 Acrophialophora.................................................................................................................................................. 163

Chapter 22 Arthrographis....................................................................................................................................................... 167

Chapter 24 Blastomyces.......................................................................................................................................................... 189

Chapter 25 Chrysosporium..................................................................................................................................................... 203

© 2011 by Taylor & Francis Group, LLC

Chapter 26 Cladophialophora................................................................................................................................................ 209

Chapter 28 Cyphellophora...................................................................................................................................................... 231

Chapter 29 Emmonsia............................................................................................................................................................. 235

Chapter 30 Epidermophyton................................................................................................................................................... 241

Chapter 31 Exophiala............................................................................................................................................................. 247

Chapter 32 Fonsecaea............................................................................................................................................................. 255

Chapter 33 Histoplasma......................................................................................................................................................... 263

Chapter 34 Lacazia................................................................................................................................................................. 275

Chapter 35 Lecythophora....................................................................................................................................................... 281

Chapter 36 Microsporum........................................................................................................................................................ 285

Chapter 37 Myriodontium....................................................................................................................................................... 299

Chapter 38 Onychocola.......................................................................................................................................................... 303

Chapter 39 Paecilomyces........................................................................................................................................................ 309

© 2011 by Taylor & Francis Group, LLC

Chapter 41 Penicillium: Mycoses and Mycotoxinoses........................................................................................................... 329

Chapter 42 Phialophora.......................................................................................................................................................... 345

Chapter 44 Trichophyton........................................................................................................................................................ 357

Chapter 45 Veronaea.............................................................................................................................................................. 377

Chapter 46 Acremonium......................................................................................................................................................... 385

Chapter 47 Beauveria............................................................................................................................................................. 391

Chapter 48 Chaetomium......................................................................................................................................................... 397

Chapter 49 Colletotrichum..................................................................................................................................................... 401

Chapter 52 Microascus, Including Scopulariopsis................................................................................................................. 435

© 2011 by Taylor & Francis Group, LLC

Chapter 53 Myceliophthora and Thielavia............................................................................................................................. 445

Chapter 54 Neocosmosporas.................................................................................................................................................. 449

Chapter 55 Ochroconis........................................................................................................................................................... 459

Chapter 56 Phaeoacremonium............................................................................................................................................... 469

Chapter 57 Phialemonium...................................................................................................................................................... 481

Chapter 58 Pseudallescheria and Scedosporium................................................................................................................... 485

Chapter 59 Sarcopodium........................................................................................................................................................ 493

Chapter 60 Sporothrix and Sporotrichosis.............................................................................................................................. 497

Chapter 63 Verticillium........................................................................................................................................................... 535