Osler fungi 10 2020

Ramon L. Sandin, MD, MS, FCAP, ABP-MM
Senior Member Emeritus,
Department of PATHOLOGY
Moffitt Cancer Center;
Founding Medical Director,
Clinical Microbiology & Virology Labs,
Consultant & Lecturer
Professor of Oncologic Sciences &
Pathology and Cell Biology, USFCOM
12902 Magnolia Drive, Tampa, Florida
33612-9497
email: ramonluis2009@me.com
This material may not be copied, posted to the Web or distributed in any form without the consent of R. L. Sandin, MD

Scheme to be followed:
I. Overview & General Considerations in identifying
fungi
II. Comprehensive list of mycoses & their fungal agents,
according to extent of involvement or body site
affected (framework)
III. The more important individual agents will be
presented in more depth: Infectious Disease
Pathology (Mycology), Microbiology, Clinical pearls

I. Overview & General Considerations
1 Fungi are eukaryotes. Morphologically-speaking, they
can be divided into two basic groups:
• yeasts
• molds
2 Yeasts are unicellular, budding fungi that can
reproduce by sexual and/or asexual means. On
culture, yeasts are often “slimy” or “mucoid”.
3 Molds are filamentous fungi that grow by forming
long chains of cells called hyphae. A mass of hyphae
is called a mycelium. On culture, molds are often
downy, fluffy, granular, cottony, or wooly.

4 Some fungi are “dimorphic” or “diphasic”, which
means that they have 2 forms or phases:
5 A mycelial (filamentous) form found as the free living
or ‘saprophytic’ form in nature, & at room
temperature (or 30°C) in the culture plate;
6 And a yeast or “yeast-like” parasitic phase found
when growing at 37°C in a culture plate or in the
histopathology slides from tissues
7 Since the form switch in most (not all) of these agents
occurs exclusively in response to changes in
temperature: thermodimorphic.

8 Examples of 7 medically-important dimorphic
fungi:
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Sporothrix schenckii
• Black molds involved in chromoblastomycosis
• Penicillium (now Talaromyces) marneffei
Penicillium marneffei in culture.
(Image courtesy of Imperial College London)

8 Examples of dimorphic fungi include:
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Sporothrix schenckii
• Black molds involved in chromoblastomycosis
• Penicillium marneffei
Penicillium marneffei in culture.
(Image courtesy of Imperial College London)

9 Fungi can best be classified according to the
body site affected or extent of involvement:
• Superficial & cutaneous mycoses
• Subcutaneous mycoses
• Deep or systemic mycoses
• Opportunistic mycoses

10 Thus, there is a multitude of types of specimens that can
be received in the Mycology laboratory for culture:
• Respiratory specimens: bronchoalveolar lavages,
bronchial washings & brushings, biopsy tissues &
sputum
• Skin: scrapings, exudates, pus, pieces of tissue,
excisional biopsies
• Biopsies: internal organs, lymph nodes, bone marrow
• Body fluids: pleural, peritoneal, CSF, urine, blood

11 Culture media for primary isolation (from a clinical
sample) vary between labs: a combination of selective
& non-selective agars, that can include:
• Sabouraud’s glucose agar (SAB), non-selective
• Selective SAB, with chloramphenicol
• Selective SAB, with chloramphenicol plus
cycloheximide (Actidione)
• Blood Brain Heart Infusion (BBHI), non-selective
• BBHI with gentamicin (G) & chloramphenicol (C)
• Selective BBHI with G, C & Actidione; & others

12. Customary procedure is to first incubate
plates at 30C (corresponds to “room
temperature”). For any growths suspicious for
a dimorphic: attempt conversion by subculture
to BBHI and incubate at 37C

13. Various available & useful histopathologic
stains include:
A GMS (Gomori/Grocott Methenamine Silver)
stain. Fungal cell walls stain black, but so can
collagen fibers, RBCs, etc. Staining pattern plus
morphology are indispensable.
B PAS (Periodic Acid Schiff) stain. Fungal cell
walls stain pink/red. Other carbohydrate material
& small lipid droplets may also take up the stain.

13. Various histopathologic stains include:
GMS stain: hyphae with septations (cross walls);
hyphal varicosities- or ballooning hyphae- can be
seen with many filamentous molds.
However, it is very frequent with Fusarium
in tissue sections. While NOT pathognomonic, it is
suggestive of Fusarium.
Courtesy of Ramon L. Sandin, MD

13. Various histopathologic stains include:

C Mucicarmine (Mayer’s or Southgate’s)
stain. Used to demonstrate the capsular
polysaccharide of C. neoformans, which
stains pink/red.
1. There are hypocapsular, or (rarely) acapsular isolates
of C. neoformans
2. The Fontana-Masson stain for melanin becomes useful
in such cases (brown cell-wall staining).

D H&E stains most fungi, although some stain
faintly. Not always dependable!
E Tissue modified Gram stains (Brown &
Brenn, or Brown & Hopps) should highlight
most fungi (as most are Gram positive) but
may stain too faintly!
1. When in doubt, request GMS or PAS;
2. preference is personal, I prefer GMS

II. Full List of mycoses and their fungal agents, according
to extent of involvement or body site affected (“laundry list”)
Type Disease Agent(s)
I. Deep or Systemic
Mycoses
1. Histoplasmosis Histoplasma capsulatum
2. Blastomycosis
Coccidioides immitis/ Coccidioides posadasii
3. Coccidioidomycosis
Pseudallescheria boydii, Madurella mycetomatis, Curvularia, Exophiala
Leptosphaeria, Neotestudina, and many others
4. Paracoccidioidomycosis
5. Lobomycosis
Blastomyces dermatitidis
Paracoccidioides brasiliensis
II. Subcutaneous
Mycoses
Fonsecaea pedrosoii, Fonsecaea compactum, Phialophora verrucosa,
Cladosporium carionii, Exophiala jeanselmei, Rhinocladiella aquasperma,
and others
3. Sporothrichosis
2. Chromoblastomycosis
1. Eumycotic Mycetoma
Sporothrix schenckii
4. Rhinosporidiosis Rhinosporidium seeberi, NOW RECLASSIFIED AS AN AQUATIC PROTISTAN
PARASITE
Loboa loboii now renamed Lacazia loboii

II. List of mycoses and their fungal agents, according to
extent of involvement or body site affected (cont.)
III. Cutaneous &
Superficial
Mycoses
1. Mucocutaneous & Candida albicans and several other species of the genus Candida
2. Tinea versicolor
3. Dermatophytoses
(Tineas)
Microsporum spp., Epidermophyton floccosum, Trichophyton spp.
Other minor superficial mycoses:
Cutaneous
Candidiasis
Malassezia furfur
Miscellaneous & rare mycoses and algoses
Hortaea (prev. Phaeoannellomyces, prev. Exophiala. prev. Cladosporium) werneckii
Tinea Nigra
Palmaris/Plantaris
Whita Piedra
Black Piedra Piedraea hortae
Trichosporon beigelii

II. List of mycoses and their fungal agents, according to
extent of involvement or body site affected (cont.)
IV. Opportunistic
Mycoses
1. Cryptococcosis Cryptococcus neoformans
2. Hyalohyphomycosis Species of Aspergillus, Penicillium, Fusarium,
Paecilomyces, Trichosporon, Geotrichum, Scopulariopsis,
Acremonium, Trichoderma, Gliocladium, Sepedonium,
Beauveria, Chrysosporium, & many others
3. Phaeohyphomycosis Alternaria spp., Anthopsis deltoidea, Aureobasidium
pullulans var. pullulans, Bipolaris spp., Botryomyces
caespitosis, Chaetomium spp., Cladosporium spp.,
Curvularia spp., Dactylaria constricta var. gallopava,
Exophiala spp., Exserohilm spp., Hormonema
dermatioides, Lecythophora spp., Nattrassia mangiferae
(Hendersonula toruloidea), Phaeoannellomyces spp.,
Phialemonium spp., Phialophora spp., Phoma spp.,
Pleurophoma spp., Pseudallescheria boydii (Scedosporium
apiospermum), Scedosporium prolificans (Scedosporium
now Lomentospora inflatum), Scytalidium dimidiatum
Xylohypha spp., & many others
4. Zygomycosis Species of Rhizopus, Mucor, Absidia, Rhizomucor,
Cunninghamella, Syncephalastrum, Circinella, Mortierella,
Conidiobolus, Basidiobolus, etc.
New emerging pathogen: Cryptococcus gattii
Wangiella dermatitidis,

III. The most important agents of each type
of mycosis will now be discussed
• Histoplasmosis
• Blastomycosis
• Coccidioidomycosis
• Paracoccidioidomycosis
Deep or Systemic Mycoses

A. Clinical pearls
• guano & debris from birds (Starlings) & bats
• 95% of cases inapparent/subclinical/completely benign, flu-like
syndrome
• 5% of cases: chronic pulmonary/mucocutaneous/systemic; acute
fulminating
1. Histoplasma capsulatum & Histoplasmosis
Beneke ES, Rippon JW, Rogers AL. 1984. Human Mycoses, a Scope Publication.
8th ed. Kalamazoo: The Upjohn Company.

B Microbiology Culture at 30°C
• Slow grower (2-8 wks)
• White to brown mycelium, cottony, NOT pathognomonic
• Perform ‘slide culture’ or scotch tape mount
• Tuberculated (echinulate, spiny) MACROconidia, large round,
8-16 µM (diagnostic); & small MICROconidia
• DDX: Sepedonium (no microconidia)

Tape Mounts
Tease mounts are not optimal in mycology
for morphologic preservation.
Gloves stick to the tape, become a hazard!
Mandatory to wash hands in adjacent sink
as soon as done working in the hood!

Histoplasma capsulatum at 30C:
fluffy mold with TUBERCULATED
MACROCONIDIA (spiny large
spores)
http://botit.botany.wisc.edu/toms_fungi/images/hcap1.jpg

C Microbiology Culture at 37°C
• moist & pasty typically; at times dry (coral-like,
cerebriform), slow to convert, sometimes does not
• small, round to oval budding yeasts, 2-4 µM, narrow
neck

Histoplasma capsulatum at 37C in tissue sections or culture:
small budding yeasts, 2-4 uM, narrow neck between mother
and daughter yeasts
http://botit.botany.wisc.edu/toms_fungi/images/hcapyeast.jpg
Size-wise (2-4 uM),
differential at 37C:
1. M. furfur
2. P. marneffei
3. A little parasite

D Histopathology
• epithelioid granulomas, tuberculoid, caseating
• can also produce non-caseating granulomas
• old histoplasmomas: CXR, coin lesion, R/I or R/O CA, lobectomy or
wedge resection, fibrocaseous or fibrocalcific lesion microscopically
with small yeasts that appear irregular (are dead or dying & rarely do
any grow out in culture)
• acute disease in RES cells: intracellular budding yeasts, usually alive, 2-
4 µM (like 37°C culture), pseudocapsule (artifact)
• mediastinal fibrosis & collagenosis with matted lymph nodes, SVC
syndrome, fistulation. Also: broncho-centric granulomatosis
Frequent
calcification!
http://www.sflorg.com/sciencenews/images/imscn042706_02_01.jpg

A non-fungus morphologic
‘look-alike’ to Histo in tissues
(intracellular, 2-5 uM, RES)
has a kinetoplast (remnants of flagella
that never developed, ‘amastigotes’) does
not grow on fungal media, stains with
Giemsa, not with GMS.
Identity?
H. capsulatum on
GMS
Histo ‘look-alike’ on Giemsa
Leishmania

2. Blastomyces dermatitides & Blastomycosis
A Clinical Pearls:
• Primary disease is pulmonary, like all systemics. Very dermatotropic
(chronic skin and bone involvement)
Eastern third of USA
Medical Mycology, by J. Rippon
One of several verrucous
lesions;
CXR negative,
no respiratory sx
Early picture of disseminated
Blasto

B. Microbiology Culture at 30°C:
• Fluffy, white-buff mold, “prickly stage”
• Pear-shaped (“pyriform”) conidia, resembles Chrysosporium,
P. boydii. “Lollipops”.
Blastomyces dermatitidis at 30C: fluffy mold with pear-shaped spores
(pyriform) conidia (‘lollipops’) (not fully pathognomonic)

C. Microbiology at 37°C tissues or culture:
• Large mother yeasts (range 8-20 µM, up to 50 µM,
with ave. 16 uM). Round to oval. Thick, double-
contoured (double-refractile) cell walls. Single
broad-based buds. Multinucleate
D. Histopathology
• Mixed pyogenic & granulomatous inflammation,
microabscesses common, calcification rare
• Yeasts as in culture at 37°C

Blastomyces dermatitidis
at 37C: large mother yeasts, with
thick double-contoured walls,
single broad-based buds
Medical Mycology Manual; ES Beneke
& AL Rogers,
1970, Burgess Publishing Company
Differential diagnosis
size- and
morphology-wise:
Ø
Pasty, coral-like,
cerebriform
colony
Beneke ES, Rippon JW, Rogers AL. 1984.
Human Mycoses, a Scope Publication.

3. Coccidioides immitis & Coccidioidomycosis
A Clinical Pearls:
• SW USA, Mexico, desert sands. Inhalation of infected sand or dust storms
• Majority of people infected are asymptomatic/flu-like sx/ “Valley fever”
• 0.5% disseminated/systemic disease; very dermatotropic/osseotropic
• MCC: Residual pulmonary cavities “coin lesions” (‘coccidioidomas’), R/O
CA

B Microbiology Culture at 30°C:
• Rapid growth; wooly-white mold
• Very infectious - CAREFUL!!! A reason why we do
NOT sniff plates in mycology, which we may do with
some bacterial colonies
• Septated hyphae with chains of thick-walled,
BARREL-shaped arthroconidia, with dead cells in
between
• DDX: Several saprophytes, esp. Malbranchea
• Confirm with exoantigen test, DNA probe, etc.

Coccidioides immitis at 30C:
fluffy white mold;
Barrel-shaped ARTHROCONIDIA
with dead-cells in between

C. Microbiology Culture at 37°C:
• Is tissue-dimorphic, more than thermodimorphic
• No transformation usually in BBHI. Requires
special broth conversion media & culture at 40°C

D. Histopathologic morphology, 37C:
• Thick-walled spherules (10-80 µM, ave. 50 µM), with
endospores; look for all stages of development in acute lesions
• In old lesions (coccidioidomas):
• fragmented spherules
• empty spherule walls
• or clusters of endospores without spherule walls
• fibrocaseous/fibrocalcific coccidioidomas with calcification
• DDX: Rhinosporidium seeberi: spherules 100-300 µM (ave.
200 µM). Endospores that are mucicarmine +. Also
myospherulosis (GMS -, PAS -)
• Granulomatous inflamm., with caseation. Pyogenic Rx at
areas where endospores are discharged

Histopathology in acute disease; all stages of spherule development can be seen

Old Coccidioidoma, a
fibrocaseous or fibrocalcific
lesion, with spherule
remnants

DDx:
Rhinosporidium seeberi:
polyps, larger spherules
with
mucicarmine-positive
endospores
Medical Mycology Manual; ES Beneke
& AL Rogers, 1970, Burgess Publishing Company
Pathologic Diagnosis of Fungal Infections; Chandler & Watts, 1987, ASCP Press

Nomenclature pearl:
Coccidioides immitis/posadasii complex
• C. immitis complex is now recognized as two species:
1. C. immitis
2. C. posadasii
• Morphologically identical!!!
• Only phenotypic difference: have different rates of growth in high salt
concentrations
1. C posadasii grows more slowly
• Distinguished by genetic analysis
• Geographic distribution:
1. C immitis: limited to California’s San Joaquin Valley region
2. C. posadasii: all USA SW desert, plus Mexico and South America
3. Both species probably co-exist in desert regions of USA

4. Paracoccidioides brasiliensis
A Clinical Pearls:
• “South American Blastomycosis”; Brazil, Venezuela, Colombia
• Pulmonary & disseminated disease occur
• In dissemination: mucocutaneous and lymphangitic disease, CNS,
adrenals, GI perforation, facial involvement
Courtesy of the teaching collections of ES Beneke & AL Rogers;
Beneke ES, Rogers AL. 1980. Medical Mycology Manual. 4th ed.
Minneapolis: Burgess Publishing Company.

B. Microbiology Culture at 30°C”:
• Slow, white-buff fluffy mold
• Pyriform conidia, like B. dermatidis or P. boydii (thus, like
Blasto at 30C: not fully pathognomonic)
30C
Beneke ES, Rippon JW, Rogers AL. 1984. Human Mycoses, a
Scope Publication.
30C 37C
37C
Beneke ES, Rogers AL. 1980. Medical Mycology Manual. 4th ed. Minneapolis:
Burgess Publishing Company.

C Microbiology at 37°C, culture & histopathology:
• Culture: slow, coral-like (cerebriform, waxy, stony)
• Large mother yeasts, ave. 10-20 µM (up to 60 µM), multiple
buds/daughter yeasts. Narrow necks
• “Mariner’s wheel”, “Pilot’s wheel”, “ship’s steering wheel”
Bulmer GS. 1978. Medical Mycology, a Scope Publication.
1st ed. Kalamazoo: The Upjohn Company.
GMS
Mariner’s Wheel of Paracoccidioidomycosis
Differential diagnosis
size- and
morphology-wise:
Ø

Subcutaneous Mycoses
• Mycetoma
• Chromoblastomycosis
• Sporotrichosis
• Rhinosporidiosis (rare, now parasite)
• Lobomycosis (rare)

1. Mycetoma
A Clinical Pearls:
• First observed in India, district of Madura, early 20th century
• “Madura Foot”, “Maduromycosis”
• Trilogy: swollen lesion usu. extremity, draining sinuses, grains/
granules
• Traumatic implantation of soil saprophytes or plant pathogens

1. Mycetoma
B Microbiology & Pathology
• Two types:
– Actinomycotic mycetoma
– Eumycotic mycetoma
• Actinomycotic mycetoma
(98% of cases; members of
the actinomycetales which
are higher bacteria) most
commonly caused by
Nocardia brasiliensis, N.
asteroides, N. caviae.
Actinomadura madurae,
Streptomyces pelletieri, S.
somaliensis and Actinomyces
israelii
• Eumycotic mycetoma (2%
of cases) predominantly (not
always) caused by black
molds, most commonly by:
Pseudallescheria boydii,
Madurella spp.,
Pyrenocheta spp.,
Leptosphaeria spp.,
Curvularia spp., Exophiala
jeanselmei, Neotestudina
rosatii, and others
• Granules in tissue, of
various colors
• Within those granules: true
hyphae vs. bacterial
filaments

Clinical Triad in a case that presented with actinomycotic mycetoma:
swollen extremity, draining sinuses, and granules

Actinomycotic mycetoma, will
have clusters or clumps
of filamentous branching bacteria.
When agent in the granules is the
genus Actinomyces, the asteroid
body or Splendore-Hoeppli
phenomenon may be observed in
the periphery of granule.
“Sulfur granules”: yellowish hue
when unstained.
All slides courtesy of Ramon L. Sandin, MD
Low-power view
High-power view
“Sulfur granules” of
Actinomyces
Molar-tooth colonies
of Actinomyces

Nocardia

Nocardia:
•positive (‘red snapper’) on modified acid-fast stain,
•in tissues, the Fite or Fite-Faraco modification is useful for
Nocardia and M. leprae, weakly acid-fast agents;
we use weakened version of the decolorizer so they retain the
carbol-fuchsin red stain
‘Pearl necklace look on Gram stain’

Pseudallescheria boydii
Front of plate Back of plate
Microscopic morphology:
pear-shaped conidia
Medical Mycology Manual; ES Beneke & AL Rogers, 1970, Burgess Publishing Company
Eumycotic mycetoma,
granules will show true
hyphae

A Clinical Pearls:
• chronic granulomatous disease of skin & subQ
• dematiaceous soil saprophytes, via abrasions
• wart-like lesion, grows into verrucous (“cauliflower-like”)
lesions, +/- ulcerations
Beneke ES, Rippon JW, Rogers AL. 1984. Human Mycoses, a Scope Publication.

B. Microbiology & Pathology
• black molds in culture: Fonsecaea pedrosoi, Fonsecaea
compactum, Phialophora verrucosa, Cladosporium
carrionii, Exophiala jeanselmei, Rhinocladiella spp.
• In culture, the genus Fonsecaea shows up to 4 types of
conidiation: “Fonsecaea-type”, “Rhinocladiella-type”,
“Phialophora-type”, “Cladosporium-type”
Medically Important Fungi. A Guide to Identification; Davise H. Larone, ASM Press
Cladosporium

• In tissue: sclerotic bodies
(copper pennies, Medlar
bodies); brown, thick-
walled large cells with
several sub-cells inside,
the product of irregular
(planate) cell divisions
• Pyogenic and
granulomatous rx (mixed
reaction); in skin,
pseudoepitheliomatous
hyperplasia
Medical Mycology Manual; ES Beneke & AL Rogers,
Medical Mycology Manual; ES Beneke & AL Rogers,
“Copper pennies” in tissues

3. Sporotrichosis & Sporothrix schenckii
A Clinical Pearls:
• Cutaneous inoculation due to penetrating injury with a
spore-contaminated thorn (“rose-gardener’s disease”)
• Lymphatic involvement (lymphocutaneous), may become
systemic, to bone
• Pulmonary sporotrichosis, rare

• Dimorphic dematiaceous mold
• At 30°C: rapid growth (3-5 d). Turns brown
or black. Conidia septate. Apex of
conidiophores bears many small conidia
(rosette or flower-like) “daisywheel pattern”,
or singly along the conidiophore

S. schenckii at 30C: black mold
with ‘florettes’
Front of plate
Reverse of plate
Microscopic morphology

• At 37°C: thin yeasts,
elongated, 2-5 µM, up
to 10 uM long, “cigar
bodies”, oval or
fusiform
• Pyogenic, later
granulomatous,
inflammation
• In tissue, hard to find
yeasts. But, just as
with Actinomyces, look
for asteroid body
(Splendore-Hoeppli
phenomenon)
Pasty ‘yeasty’colony
S. schenckii at
37C: ‘cigar-
bodies”
http://microbiology.mtsinai.on.ca/mig/images/direct/fig04-dm.jpg

4. Rhinosporidiosis
A Clinical Pearls:
• Rhinosporidium seeberi. In water & as a fish disease in India,
Sri-Lanka, Brazil
• Chronic granulomatous infs. of mucous membranes of nose,
eyes, ears, larynx
• Polypoid tumors, sessile or pedunculated
B Microbiology & Pathology:
• Spherule & endospore producer, DDx of C. immitis. Spherules
larger than cocci (100-300 µM, ave. 200 µM)
• Endospores: mucicarmine +, as well as inner aspect of spherule
wall

4. Rhinosporidiosis
C. Taxonomical Update: aquatic protistan parasite of a
novel clade, the Mesomycetozoans
A reclassification based purely on sequence analysis of 18S small-subunit
rDNA
B a clade of fish parasites branching in the evolutionary tree near the
animal-fungus divergence
C does NOT grow on synthetic media nor in human or animal cell lines

5. Lobomycosis
Synonyms
• Keloidal blastomycosis or Lobo's disease
• rare chronic cutaneous & subQ infection caused by Lacazia
loboi previously called Loboa loboi
• keloids, verrucoid to nodular lesions, crusty plaques, and
tumors
• fungus grows as globose cells connected to each other by a
narrow neck; may form branching chains
• developing lesions well defined, smooth, painless
• older lesions typically verrucoid and ulcerative with
satellite lesions resulting from autoinoculation
www.doctorfungus.org

5. Lobomycosis
Histopathology
• subepidermal histiocytic granulomas, minimal fibrosis
with large numbers of giant cells and histiocytes
• in older lesions: pyogenic infiltrates & acanthosis
• chains of globose cells 7-14 µm (average 10 µm) connected by a
narrow neck, sequential budding may produce chains of up to
6 to 10 yeasts
• yeasts may lie within giant cells and macrophages; asteroid
bodies are common
• Isolation in culture: has never been achieved, except in mice foot pads
• Natural habitat: Unknown, but the infection is also found in dolphins
www.doctorfungus.org

Cutaneous and Superficial
Mycoses
• Mucocutaneous and Cutaneous Candidiasis
• Tinea Versicolor
• Dermatophytoses (true ‘tineas’, ringworms)

A Clinical Pearls:
• Endogenous: member indigenous microbiota GI, GU tracts & skin
• Oral thrush, vaginitis, skin lesions, endocarditis, pyelonephritis,
paronychia, onychomycosis, diaper rash, moist skin areas, chronic
mucocutaneous candidiasis
1. Mucocutaneous & Cutaneous Candidiasis
Thrush
Diaper rash Onychomycosis
Chronic Mucocutaneous
Candidiasis

B Microbiology:
• Multiple species, C. albicans still most prevalent
• C. glabrata, krusei & tropicalis are manifesting R to fluconazole; rarely,
also R to Ampho B
• Rapid growth on plates, usu. pasty colonies. Traditionally, the genus
Candida is spoken of as a yeast
• Technically, however, Candida has 3 possible phases (ie., is triphasic):
budding yeasts (7 µ), pseudohyphae (sausage link) & hyphae (much less
frequent, no pinching, thinner than mold hyphae, no 45 degree
bifurcations)
• In culture media, manipulation of carbohydrate content allows for a preponderance of one or another
of these phases in that medium
• C. albicans: + chlamydoconidia on CMA
• Germ tube +: C. albicans (C. stellatoidea; rarely C. tropicalis)
C Pathology
• Pyogenic, granulomatous or inert inflammatory background, as per
host’s underlying condition
• In tissue, when Candida is invasive & non-saprophytic, 2 of the 3 forms
or all 3 forms can be seen: pseudohyphae, budding yeasts +/- hyphae.
Different species of Candida can do this & are not distinguishable from
each other in tissue.

Pasty yeast colony
Budding yeasts
Pseudohyphae
Hyphae

Candida auris
➢Originally isolated from ear (L.: aurus) infection in patient in Japan 2009
➢Some isolates R to all 3 classes of antifungal drugs (MDR-C. auris)
➢Now, a nationally notifiable disease of Public Health Concern
➢9/9/2019 Florida State Health Dept. issued Memo to all FL MD’s that it was
evaluating several cases in Miami-Dade and Broward counties
➢Additionally, has capacity adhere to surfaces & form protective biofilms to be further
shielded from antifungals; can spread from patient to patient
➢CAN BE identified now by newer updated FDA-approved databases for Maldi-TOF
systems; Biomerieux FDA-approved IVD version 3.2 or RUO libraries; Bruker FDA-
approved Biotyper CA system library version claim 4 or RUO libraries
➢NOT identified by Remel RapID Yeast system
➢Previously misidentified as:
➢C. haemulonii
➢Identified accurately by sequencing of D1-D2 region of 28s rDNA
➢Isolate is from normally-sterile body sites that type as C haemulonii & are multi-
resistant: sequencing

2. Tinea versicolor (Pityriasis versicolor)
A Clinical Pearls:
• Name of the agent: Malassezia furfur
• Macules, papules, patches, plaques; on chest, back, satellite
lesions
• Hypopigmented or hyperpigmented
• M. furfur, other than in tinea versicolor:
• also Normal skin flora in absence of T.V.
• can also cause Fungemia & death in patients on
IV lipid therapy & central catheters
• Folliculitis in BMT patients, neutropenic
Tinea versicolor:
hypo- and hyper-pigmented macules
Folliculitis
Fungemia

B Microbiologic morphology
• Technically, M. furfur is a yeast
• Lipophilic yeast, requires exogenous source medium to
long-chain fatty acids
• Small budding yeast, 2-4 µM, broad-base single bud, small
circumferential thickening at bud attachment (collarette),
urease positive, usu. lacks reactivity in miniaturized
biochemical cards or strips
• Size-wise, similar to Histo in size, but Histo has a thin-neck
to the buds

Malassezia
furfur
folliculitis
H&E
PAS
All images courtesy of Ramon L. Sandin, MD

No growth Olive Oil added
Growth after 2 days
Urease +

C Pathology
• In deeper disease (folliculitis or in systemic disease): only
yeasts are found
• However, in the one specific clinical manifestation of Tinea
Versicolor (which is superficial): small hyphal fragments
surrounding clusters of small, spherical (3-8 µM), round
spores with occasional budding (“spaghetti and meatballs”
picture) seen in skin scrapings or sections

Tinea versicolor: ‘spaguetti and meatballs’
Disseminated
disease: yeasts
only

3. Dermatophytes & Tineas
A Clinical Pearls:
• Affect keratinized tissues: hair, skin & nails
• ‘Ringworm’ fungi, 3 genera with multitude of species:
Microsporum, Epidermophyton, Trichophyton
• Geographic location in body: tinea capitis, t. corporis, t.
pedis, t. cruris, onychomycosis
• Usu. a clinical diagnoses; alternatively, KOH scrapings with
microscopy; biopsy; culture.

Named by geographic body location:
Tinea faciale
Tinea unguium
Tinea pedis
Tinea capitis by a zoophylic dermatophyte,
such as Microsporum canis

B Microbiology:
• Microscopic morphologic diagnosis following culture
is definitive in terms of species
• Microsporum species: MACRO conidia,
multicellular, spiny (tuberculated) are diagnostic
• Trichophyton species: MICRO conidia, are
diagnostic, variable arrangements
• Epidermophyton floccosum: smooth (non-spiny)
MACRO conidia, “beaver-tail-like”

B Microbiology:
http://www.asm.org/Division/c/photo/mcanis1.JPG

B Microbiology:
http://www.medmicro.wisc.edu/resources/imagelib/mycology/images/trichophyton_rubrum.html

c
o
Microsporum canis: tuberculated,
elongated (spindle-shaped),
multicellular MACROconidia
Epidermophyton floccosum:
smooth, beaver tail-like,
multicellular
MACROconidia

C. Pathology:
• In skin, either biopsy or KOH smear: septated hyphae, may be seen
breaking up into arthroconidia;
• In hair, involvement can be endothrix (hyphae with arthroconidia inside
the hair) or ectothrix (outside of the hair)
Hyphae in tissue section or KOH smear
Endothrix
hair
invasion
Ectothrix
hair invasion
Medical Mycology Manual; ES Beneke & AL Rogers, 1970, Burgess Publishing
Company
Medical Mycology; Glenn S. Bulmer, 1978, UpjohnCompany
Medical Mycology; Glenn S. Bulmer, 1978, UpjohnCompany

Opportunistic Mycoses
• Cryptococcosis
• Hyalohyphomycosis
• Phaeohyphomycosis
• Zygomycosis

1. Cryptococcus neoformans and
Cryptococcosis
A Clinical Pearls:
• Yeast, assoc. with
pigeon, chicken or
turkey droppings
• Commonest
manifestations:
CNS, skin
• Disseminated
infection
• Host
immunocompromise
is usually present
Sub-acute or chronic meningitis

B Microbiology morphology:
• Irregular, budding yeast cells (monomorphic, no filamentation, only
extremely rarely)
• 2-20 µM; great variability in size and shape!!!! with thin walls;
surrounded by polysaccharide capsules; daughter cells attached by
narrow thread, one or 2 daughter yeasts/cell
• Mucoid colonies may slide down a tilted plate or slant. Urease positive
• **Cryptococcal Antigen Latex Agglutination Test: very sensitive. CSF,
plasma, urine
C. ** Pathology:
• Mucicarmine + (red-pink) for capsular polysaccharide
• In cases of hypocapsular or acapsular strains, Fontana-Masson
(FM) stain may be useful
• FM stains the small amount of melanin present in cryptococcal cell
wall, not in the capsule, so staining here would be capsule-
independent
• Purulent, granulomatous or inert inflam. reaction
• If granulomatous, could be either caseating or “sarcoid” type
granulomas

Mucoid colony
India Ink (Nigrosine) stain; central yeast
with large polysaccharide capsule
GMS
Mucicarmine

Fontana- Masson Stain

• The majority of opportunistic agents are
filamentous molds which are either:
– hyaline (light colored) filamentous fungi -
HYALOHYPHOMYCETES
– dematiaceous or black filamentous fungi -
PHAEOHYPHOMYCETES

2. Hyalohyphomycosis
A Clinical Manifestations:
• saprophytes (‘free-living’) in soil, plants, nature
• entire spectrum, including disseminated disease
B Microbiology:
• hyaline, hyphae that are well septated (many cross-
walls), branch at approx. 45° angles (‘bifurcations’)
• Aspergillus is the prototype. Culture necessary for
speciation
• Many others: Penicillium, Fusarium, Paecilomyces,
Scopulariopsis, Trichosporon, Geotrichum, Acremonium,
Trichoderma, Gliocladium, Sepedonium, Beauveria,
Chrysosporium, etc.
Aspergillus terreus on
smear from agar plate
Culture plate
with A.
fumigatus

2. Hyalohyphomycosis
C Pathology:
• Vasoinvasive, thrombosis, infarctions
• Suppurative inflammation, coagulative necrosis
• A word of caution: NO sure WAY to differentiate
aspergillosis on histology alone from other hyalo-
& some phaeohyphomycetes, unless an air-
containing space is involved in vivo, is represented
in the sampled tissue, & fruiting bodies are seen in
tissue (not very common)

Conidiophore Vesicle Rows of conidia
Aspergillus terreus

H&E slide:
fruiting body formation in tissues,
rare phenomenon,
(usually lungs),
implies access to air-containing space,
as in emphysematous cavity,
evacuated tumor center, etc
“Flask-shaped vesicles
present, with
early rows of conidia
c/w Aspergillus spp.
Await culture
confirmation for
definitive identification.”
H&E
If NO pathognomonic fruiting
bodies are present in tissue, and
only regular-sized, septated
hyphae with bifurcations are
present, your safest call in tissue
is: ‘septated hyphae are present
c/w a filamentous mold, await
culture confirmation for
definitive identification’.

Penicillium
Fusarium
Images are courtesy of Ramon L. Sandin, MD
Medically Important Fungi. A Guide to Identification; Davise H. Larone,
ASM Press
Banana-shaped or sickle-shaped
MACROconidia in culture
Toe & toenail infections
Hyphal varicosities or
ballooning hyphae in
tissue sections

3. Phaeohyphomycoses and Black
Molds
A Clinical Manifestations
• Saprophytes in soil, plants, nature
• Can cause entire spectrum
• Could be, and frequently are, categorized by the
disease processes which they incite and with which
they are more commonly associated. These diseases
include:
* eumycotic mycetoma
* chromoblastomycosis
* phaeohyphomycosis as a generic term for infections which
do not fit the clinical features of the prior two

B Microbiology & Pathology
• Pigmented (brown to black) due to melanin-like pigment,
amount of pigment varies considerably between the hundreds of species, (as well
as speed of color development; ex. Pseudallescheria is slow to develop black color
(3-7 d) & some textbooks even classify it as a hyalohyphomycete).
• in tissue sections, produce septated hyphae which may bifurcate & be similar in
shape to the hyalohyphomycetes
• Alternaria spp., Anthopsis deltoidea, Aureobasidium
pullulans var. pullulans, Bipolaris spp., Botryomyces
caespitosis, Chaetomium spp., Cladosporium spp.,
Curvularia spp., Dactylaria constricta var. gallopava,
Exophiala spp., Exserohilum spp., Hormonema
dermatioides, Lecythophora spp., Nattrassia mangiferae
(Hendersonula toruloidea), Phaeoannellomyces spp.,
Phialemonium spp., Phialophora spp., Phoma spp.,
Pleurophoma spp., Pseudallescheria boydii (Scedosporium
apiospermum), Scedosporium prolificans (Scedosporium
inflatum) now LOMENTOSPORA prolificans, Scytalidium
dimidiatum, Wangiella dermatitidis, Xylohypha spp., etc

If you do not have culture available to help you make a call, Phaeohyphomycosis as a tissue diagnosis
may be possible on H&E, for agents that are very dematiaceous and produce at the individual hyphal level
a wine-red, brownish, or black coloration in the cell walls.
All images are courtesy of Ramon L. Sandin, MD
Inguinal
lesion
pre-
treatment
Post-
treatment
with
Itraconazole
(several years
back)
H&E

4. Zygomycosis and the order Mucorales
Clinical Manifestations:
• Saprophytes in decaying vegetable matter, etc.
• Rhinocerebral; pulmonary; GI; disseminated disease. Also
primary cutaneous
• Predisposing conditions: debilitating illnesses; diabetic
ketoacidosis, burns, malnutrition, iron overload esp.
hemodialysis patients receiving deferoxamine therapy
Quick progression is possible.
This is 8h later, gangrenous
tissue destruction

4. Zygomycosis and the Mucorales
B. Microbiology:
• Most common genera: Rhizopus, Mucor, Absidia
• Absidia spp. was later renamed Myocladus spp. and are now
named Lichtheimia spp.
• Broad, ribbon-like hyposeptate hyphae, 10-20 µM
branching at right angles, with spores within
sporangia
• Mince, do not grind, tissues for culture, to enhance
recovery
Absidia

B. Microbiology:
sporangia
recovery
Absidia

B. Microbiology:
sporangia
recovery

C. Pathology:
• Purulent inflammation, & in older infections,
granulomatous rx
• Broad hyposeptated hyphae, ribbon-like, irregular
(may fold onto themselves), may appear like “moose
antlers” or like empty tubes
• Hyphae may be difficult to see on H&E, even stain
lightly with silver stains
• Vasoinvasive fungal behavior, necrotizing vasculitis,
thrombosis, infarction

Patient from our center with
culture and biopsy proven
left cheek involvement by
Mucor
Left orbital exenteration,
prophylactic

One month
later, following
convulsions:
Broad, hyposeptated, ribbon-like hyphae, but not in optic nerve
Craniotomy
All images are courtesy of
Ramon L. Sandin, MD

Cross-section
Longitudinal sections:
broad, ribbon-like hyphae

Fruiting body:
sporangium.
sporangiophore,
sporangiospores
Very ‘wooly’ mold,
Mucor
Hyposeptated, not aseptated
(a few possible cross-striations are present) Clipping

Finally, let’s review
Fungal case
studies/questions/answers

SANDIN Osler FUNGI question #1;
10-2020
Clinical Microbiology & Virology;
33612-9497

• 30-year old Army recruit experiences fever, flu-like illness, dyspnea
• develops disseminated papular lesions skin & mucocutaneous sites
• lives at military base in Kentucky
• spends time hunting for starlings, abundant in fields next by
• explores caves in surrounding mountains
• skin lesion is biopsied; specimens sent separately to Pathology &
Microbiology
• organism grown in culture at 30C & 37C
• gross & microscopic appearance from 37C culture shown (Figures 1
& 2)
• peripheral blood smear taken during febrile stage (see Figure 3)
• (a bone marrow aspirate is also shown from a different patient, a
leukemic being worked up for relapse , microscopic morphology, see
Figure 4)

Figure 1: gross 37C culture

http://botit.botany.wisc.edu/toms_fungi/images/hcapyeast.jpg
Figure 2: microscopic smear from 37C culture; small 2-5 uM yeasts, thin necks

Figure 3: peripheral blood smear
finding

http://www.sflorg.com/sciencenews/images/imscn042706_02_01.jpg
Figure 4: a separate leukemic patient in work up for relapse, bone marrow aspirate

?
The agent that is most compatible with
these clinical, pathological
and microbiological findings, is:
1. Cryptococcus neoformans
2. Blastomyces dermatitidis
3. Coccidioides immitis
4. Histoplasma capsulatum
5. Penicillium marneffei

Answer:

Histoplasma capsulatum is a dimorphic fungus placed within the
group of the deep or systemic fungal pathogens. Guano from
starlings in the fields, as well as from bats inside caves, can lead to
pulmonary infection which is
usually benign and flu-like in symptoms. Rarely, the organism can
spread and lead to systemic involvement, most commonly of cells
within the reticuloendothelial system as well as skin or
mucocutaneous membranes.
Colonial appearance at 37C in culture consists of
dry, coral-like, cerebriform colonies; microscopic appearance on
wet preps in lactophenol cotton blue consists of small
budding yeasts, 2-4 uM, with narrow necks between mother and
daughter yeasts and with usually a single bud per yeast. In
disseminated disease, smears from aspirates of bone marrow or
-more rarely- peripheral blood may show intra- or extracellular
yeasts with morphology similar to that seen in culture at 37C.

• Cualing H, Bhargava P, Sandin R. L. 2012. Chpt
9: Clinically-relevant yeasts, pp 165-195. In: Non-
Neoplastic Hematopathology and Infections,
Wiley-Blackwell, New York, New York
• Beneke ES, Rippon JW, Rogers AL. 1984. Human
Mycoses, a Scope Publication. 8th ed. Kalamazoo:
The Upjohn Company

10-2020
33612-9497

• 40-year old farmer from southern Tennessee
• appearance of pruritic papular lesion above upper lip:
large, verrucous, crusted
• similar lesions L axilla & R thigh, with pus
• past history basal & squamous cell CA of skin
• consulted dermatologist fearful of another skin CA, biopsy
done & swab from pus taken
• Specimens sent to Pathology & Microbiology
• organism grows in culture at 30C & 37C
• gross & microscopics from 30C culture (Figures 1 & 2)
• microscopic morphology from wet mount of pus (Figure 3)
• Biopsy histopathology (Figure 4)

Figure 1: 30C culture

Figure 2: 30C culture, microscopic scotch tape mount
shows hyaline mold with these pear-shaped conidia

Figure 3: wet mount from pus shows details from the agent’s cell wall ;
thick walls with broad-based buds

Figure 4: biopsy tissue, touch prep; very
large 16 uM yeast cells

?

Answer:

Blastomyces dermatitidis is a dimorphic fungus placed within the group
of the deep or systemic fungal pathogens. While most systemic
involvement appears to be the result of dissemination following
inhalation of spores, this fungus is very dermatotropic/osseotropic and
the first manifestation may be a skin lesion, as was the case with this
patient. Colonial appearance at 30C in culture consists of
a fluffy, white to buff-colored mold which is not pathognomonic in and
of itself, manifesting pear-shaped conidia which may look like lollipops
when seen on a scotch tape mount or slide culture prepared from the
colony. The appearance, however, at 37C in culture as well as from
smears of pus or in histopathologic sections, is pathognomonic. It
consists of large mother yeasts, single or budding, with average cell
diameter of 16 uM and ranging from 8-16 uM with rare yeasts larger at
up to 20-30uM. They have a rigid and thick ‘doubly-contoured’ or
doubly refractile cell wall, with multinucleate cytoplasm and broad-
based budding between mother and daughter yeast cells. Rare
microforms have been described (2-5 uM), similar to the size of
Histoplasma.

10-2020
33612-9497

• 19-year old college student from Florida, travels to Phoenix
• returns to Tampa with flagrant pneumonitis, severe
decompensation
• admitted to Tampa General Hospital for workup &
treatment
• Asian background
• recently diagnosed as HIV +
• Pulmonary performs bronchoscopy, BAL samples to
Microbiology
• bronchoscopically-obtained lung biopsy to Surgical
Pathology
• organism grows in culture at 30C: gross & microscopic in
Figs 1-2
• Microscopics from histopathology shown in Figures 3 & 4

Figure 1: culture 30C

Figure 2: lactophenol cotton blue smear from culture at 30C

Figure 3. H & E stain,
400X magnification
Courtesy of Ramon L. Sandin, MD Courtesy of Ramon L. Sandin, MD
Figure 4. H & E stain,
1,000X magnification

Coccidioides immitis/posadasii complex is a dimorphic fungus abundant in the soil &
desert sands of the SW USA where inhalation of infected sand or dust may lead to a flu-
like illness referred to as Valley Fever. More rarely, or with underlying conditions of
immunocompromise, the agent may lead to disseminated disease affecting any organ
system but very commonly skin and bone. Colonial appearance at 30C in culture consists
of a fluffy, white mold with septated hyphae that break up into barrel-shaped
arthroconidia separated by dead cells as highlighted in a lactophenol cotton blue wet
prep.
The appearance in histopathologic sections is pathognomonic. In acute disease, it consists
of thick-walled spherules (10-80 µM, ave. 50 µM), with endospores; look for all sizes &
stages of development. Figure 4 shows intra-spherular cytoplasm compartmentalizing
into individual endospores which, once mature, are released into the surrounding
parenchyma. The wall of a spherule breaks down as it begins to release its content.
In old lesions, sections would reveal fragmented spherules, empty spherule walls, or
clusters of endospores without spherule walls. These are fibrocaseous/fibrocalcific lesions
and in lung are referred to as coccidioidomas.

• Cualing H, Bhargava P, Sandin R. L.
2012. Chpt 9: Clinically-relevant
yeasts, pp 165-195. In: Non-Neoplastic
Hematopathology and Infections,
Wiley-Blackwell, New York, New York
• Beneke ES, Rippon JW, Rogers AL.
1984. Human Mycoses, a Scope
Publication. 8th ed. Kalamazoo: The
Upjohn Company

10-2020
33612-9497

• 65 year old bone marrow transplant recipient develops
follicular rash on back & shoulders while neutropenic
• biopsies of a lesion taken, one sent to Path & one to Micro
• Figures 1, 2 & 3 show histopathology
• agar medium inoculated & incubated at 30C for 2 days;
Figure 4 shows plate that was incubated
• olive oil overlay was added to the same plate, shown in
Figure 5
• that plate was placed back in incubator; 2 days later, plate
growth is shown on Figure 6
• selective rapid urea slant was stabbed with colonies from
plate, evaluated after 20 min incubation & results are
shown in Figure 7

Figure 2. H&E,
400X mag
Figure 1. H&E,
100X mag
Figure 3. PAS stain,
1,000X mag; small
yeasts, broad base
All slides courtesy of Ramon L. Sandin, MD, MS

Figure 4: plate following 2
day incubation
Figure 7: urea medium
stabbed with organism
from the plates
Figure 6: the plate with olive
oil following 2 additional days
of incubation
Figure 5: same plate,
now with olive oil
overlay
All slides courtesy of Ramon L. Sandin, MD, MS

?
2. Candida albicans
4. Malassezia furfur

2. Candida albicans
4. Malassezia furfur

Malassezia furfur is a small, urease-positive, lipophilic yeast which requires an
exogenous source of medium to long-chain fatty acids to be able to grow on
synthetic media. It may be normal skin flora but it may also cause tinea versicolor
as well as folliculitis –as in the patient discussed- and in rare instances, fungemia
and possible death in patients on IV lipid therapy and with central catheters,
especially ‘premies’ and the very immunocompromised. Morphologically, the yeast
may be single or budding, measures 2-4 uM in diameter & has each single bud
attached to the mother yeast by a broad-base. The agent is similar in size to
Histoplasma capsulatum but the daughter cells of this dimorphic agent are
connected by thin-necks. Malassezia undergoes unipolar budding- all buds are
produced from the same site in the mother cell- and as a result, a small
circumferential thickening is formed at the budding site of the mother cells which
becomes very useful diagnostically and which is described by the term ‘collarette’.
The walls of the agent are relatively thick and multilayered. In cases of sepsis or
folliculitis, tissue sections will only show yeasts, but in the one specific clinical
scenario of tinea (pityriasis) versicolor, sections or smears may show a combination
of short, truncated-end, hyphal fragments in addition to the roundish yeasts, which
is described by the term “spaguetti and meatballs”.
It must be emphasized that with the present practice of prophylaxing all PBSCT
patients with antifungals during the neutropenic period following transplant,
finding folliculitis from Malassezia is extremely rare.

Addenda
Diagnostic features of some “newly-
inducted” members into the Fungal
world (from the parasite world) !!!
For Reference Purposes Only, WILL
NOT BE COVERED IN CLASS.

As reported on CNN and Larry King Live, patient treated at the U. of
Louisville for Severe Facial Deformities due to Rhinocerebral Mucormycosis
• 45 year old healthy, non-diabetic, hard-working
(4 jobs) husband and father
• Woke up on a Sunday morning (2/2000) with
sinus headache. By Monday, pain intensified,
incoherent speech began, taken to hospital, quick
diagnosis as mucormycosis
• Unfortunately, with stunning speed, raging
disease invaded and destroyed face & eyes.
Reasons behind this remain uncertain
• Radical surgery followed: included bilateral
orbital exenterations, removal of nose, palate,
part of upper jaw including upper teeth. Total of
11 surgeries
• Two months in a coma
• Prosthesis was created, acrylic with silicone
‘skin’, by maxillofacial prosthodontist
• Subsequently, suffered several strokes, R side of
body partially paralyzed, continues on multiple
medications

Courtesy of:
Soon Bahrami, M.D.,
Univ. of Louisville

Important for the pathologist to committ him/herself when broad, hyposeptated ribbon-like, irregular hyphae are seen.
Vfend (Voriconazole) ineffective with most of the Mucorales. Newest FDA-approved drugs are effective: Isavuconazonium
& Posaconazole, in addition to the older amphotericin B

MALDI-TOF Mass Spectrometry
a type of proteomics using ribosomal proteins for identification:
new uses for Mycobacterium, yeasts and filamentous fungi id
✓ New applications of MALDI beyond bacteria
✓ Easier, less expensive, faster, more accessible to routine
microbiology labs, will likely become method of choice for routine
id in the future; for now, use pure cultures
✓ Special issues for mycobacteria: must process to kill the
organism, disrupt clumped cells, break open cell walls; databases
for mycobacterial id are under development; can not distinguish
between members of the MTB complex and between other groups
of related species
✓ For yeasts and filamentous molds: use bead processing to break
open the organisms, followed by protein extraction

Molecular diagnostic technology now available or in
development for
YEASTS
✓ BioFire BCID (Blood Culture Identification Panel) from a drop of
positive blood culture fluid
✓ T2 Systems (uses T2 Magnetic Resonance technology) for use
directly from whole blood (non-culture based)
✓ PNA-FISH (Peptide Nucleic Acid-FISH)
✓ Others

Just FDA-approved
for 21 targets

Molecular diagnostic technology now available or in
development for
MOLDS:
✓ Broad PCR-based id of yeasts & molds-Roche LightCycler pan-
fungal PCRs
✓ AsperGenius or the MycAssay: multiplex real-time PCR assays
for rRNA targets
✓ Mucorales broad Real-time PCR
✓ Aspergillus lateral flow cartridge assays
✓ Aspergillus Breath Volatile Metabolite testing

Coccidioides immitis at 30C:
fluffy white mold;
Barrel-shaped ARTHROCONIDIA
with dead-cells in between

Histopathology in acute disease;
all stages of spherule development can be seen

• black molds in culture: Fonsecaea pedrosoi, Fonsecaea
compactum, Phialophora verrucosa, Cladosporium
carrionii, Exophiala jeanselmei, Rhinocladiella spp.
• In culture, the genus Fonsecaea shows up to 4 types of
conidiation: “Fonsecaea-type”, “Rhinocladiella-type”,
“Phialophora-type”, “Cladosporium-type”

Phialophora verrucosa
Phialides or
‘flower heads’
Medical Mycology Manual; ES Beneke & AL Rogers, 1970, Burgess Publishing Company

Botryomycosis
1. L. ‘Botrys’= bunch of grapes
2. Bacterial Pseudomycosis, often confused with actinomycotic
mycetoma
3. Chronic infection of skin, soft tissues & viscera caused by
various non-filamentous bacteria
4. Most common causative agents: Staphylococcus aureus,
Pseudomonas, and other gram + & - organisms
5. Inflammatory mass, draining sinuses; some associated with local
trauma or penetrating injury, visceral form has hematogenous
origin
6. Granules or grains with radiating Splendore-Hoeppli
eosinophilic clubs in the midst of PMNs and granulation tissue

Atlas of Fungal Pathology; K. Salfelder, 1990, Kluwer Academic Publishers

J Clin Microbiol. 43(4): 1495-1504 April 2005
For Reference Purposes Only

Addendum for Dermatologists:
DTM and routine fungal media for
primary isolation of dermatophytes
(ie., directly from clinical samples)

• Dermatophyte Test Medium (DTM) is a specialized agar used in
medical mycology. It is based on added cycloheximide (Actidione)
to inhibit saprophytic growth, antibacterial antibiotics (gentamicin
and tetracycline) to inhibit bacterial growth, and phenol red a pH
indicator. The pH indicator is useful in distinguishing a
dermatophyte fungus, which utilizes nitrogenous material for
preferred metabolism producing alkaline by-products which
impart a red color change to the medium. Typical saprophytic
fungi utilize carbohydrates in the medium that produce acidic by-
products and do not allow a red color change to occur from phenol
red.
publications.royalcanin.com/renvoie.asp?type=...

www.drugs.com/vet/fungassay-can.html
http://panacea.med.uoa.gr/extra/3914.jpg

Medically Important Fungi. A
Guide to Identification; Davise H.
Larone, ASM Press
www.findoutaboutringworm.com/.../all/p16.html

Fungemia

Cryptococcus gattii
• Emerging pathogen: previously C. neoformans var. gattii
• Serotypes B & C; grows well on eucalyptus trees
• Distinguishing features from C. neoformans:
– Generally, it used to have a more restricted (tropical) geographic distribution than
C. neoformans (Australia, Papua New Guinea, SE Asia, India), but recent outbreaks
Vancouver Island, Canada and Pacific NW USA shatter that pearl
– It also can affect NON-immunocompromised hosts, producing lung and brain
infections, unlike C. neoformans which is a true opportunist
– Has reduced susceptibility to certain antifungals
– We can use two selective & differential media to tell them apart: Niger seed agar
and CGB agar
• Hydrolysis of urea: both +
• Growth on cycloheximide medium: both –
• Current miniaturized biochemical methods (API, VITEK, Microscan etc) do NOT
differentiate between them
• Current Cryptococcal Latex Agglutination Tests do NOT differentiate between them

www.mycology.adelaide.edu.au/images/crypto1.gif
•Niger or bird seed agar, aka caffeic acid or Staib agar:
•dark brown colonies for both C. neoformans & C. gattii;
•due to melanin production by way of phenol oxidase enzyme;
•may also use caffeic acid disk tests placed on regular fungal media
C. gatti or C. neoformans
Candida
albicans

www.cher.ubc.ca/cryptococcus/new/methods.htm
www.mycology.adelaide.edu.au/images/crypto1.gif
- +
• CGB (Canavanine-Glycine-Bromothymol Blue) yellow media:
•C. gatti + ( growth & deep cobalt blue color to media)
within 2-5 d at room temperature;
•C. neoformans – ( poor or no growth, yellow color)
•CGB false +s: C laurentii, C curvatus & C luteolus;
but all fail to grow on Niger seed agar

For
Reference
Purposes
Only

Gross Pathologic findings may range from…
Nodular Infarcts

….. to cavitary, necrotizing, abscess-like lesions with total tissue drop-out;
which gross pathology ensues is an interplay between
the implicated species of the genus and the immune status of the host.
All images courtesy of Ramon L.
Sandin, MD

Parenchyma Wall of
vessel
Lumen
bifurcation

Case of sternal osteomyelitis
Hyphae in the bone

Aspergillus fumigatus
Dark Olive-green
colony
Flask-shaped
vesicle

Tissue ‘window’left open for healing

Candida
krusei,
one of the newer
mighty foes, with
resistance to
fluconazole
Grossly on the
plate, it looks almost
‘moldy’, has
little feet…

• EM - 5 Pathognomonic features of
genus Malassezia
• thick multilayered walls
• unipolar budding
• broad-based budding
• collarette
• corrugations or foldings of cell wall
& membrane
For Reference Purposes Only

Penicillium
Fusarium

Paecilomyces
Scopulariopsis

Brain infarct
Spleen infarct
Kidney infarct
Hyphae and conidia
inside glomeruli
PAS stain
Case of disseminated
Scedosporium prolificans
(inflatum)

Recent case of disseminated
Scedosporium prolificans (inflatum),
now Lomentospora prolificans
Front of plate
Young Old
Reverse of plate

PCP (Pneumocystis jirovecii (carinii)

• P. carinii (jirovecii) is being considered a
fungus for several reasons: there are glucans in
the cyst wall; the mode of spread is airborne;
greater RNA homology with fungi than with
parasites; trophozoite formation inside cysts is
similar to ascospore formation in yeasts; stains
with fungal stains like GMS and Calcofluor
White

LABORATORY DIAGNOSIS OF
Pneumocystis carinii (jirovecii)
• Relies on microscopic id of organism in BAL, induced
sputum, tissue biopsy or impression smear
• Identify cyst wall and/or trophozoites (intracystic
bodies=bradyzoites; extracystic bodies=tachyzoites)
• 3 types of stains: cyst stains, organism stains,
immunologic stains

H & E with ‘frothy intra-
alveolar exudates’

PCP Stains
1. Cyst Stains
• GMS
• Toluidine blue O
• Gridley
• Cresyl-etch violet
• Gram-Weigert
• Calcofluor White

• Cyst stains: stain cyst wall, not trophs either
inside or free; 5-6 µM, cup-shaped, collapsed,
round, to crescentic, no buds, minimal
variation in size.
• Look for the ‘black dot’ by GMS (concretion of
cell wall)

GMS
Black dot
GMS

2. Organism Stains
• Giemsa
• Wright-Giemsa
• Diff-Quick

• Organism stains: Giemsa, Wright-Giemsa,
Diff-Quik, stain free trophozoites & intracystic
organisms but not the cyst wall, seen as halo.
• Trophs 2-5 µM, roundish in tissue

Giemsa
Intracystic bodies
with the cyst wall
as a halo

3. Immunologic Stains
• Fluorescence-labelled (FITC)
monoclonals
• Colorimetric (enzyme)-labelled
monoclonals

• Immunologic stains: FITC-labelled
monoclonals, several available kits for
fluorescent microscopy.
• Some kits pick up all stages: cyst wall,
intracystic bodies, extracystic bodies, and the
matrix protein (‘frothy intra-alveolar exudate’
in formalin-fixed paraffin-embedded lung
slides)
• Enzyme-labelled monoclonals also available for
light microscopy

Lung, Pneumocysitis carinii Immunohistochemical Stain

Microsporidia
▪ Obligate intracellular protozoan (now mycological) parasites
belonging to phylum Microsporidia
▪ >1,200 species within 143 genera infect vertebrate &
invertebrate hosts
▪ At least 14 species affect humans
▪ Produce resistant spores with unique organelle: polar tubule
or polar filament coiled inside spore
▪ Spore size of species affect humans: 1-4 uM
http://www.dpd.cdc.gov/dpdx/HTML/Microsporidiosis.htm

Microsporidia
▪ Human pathogens:
▪ Brachiola algerae, B. connori, B. vesicularum
▪ Encephalitozoon cuniculi, E. hellem, E. intestinalis (Septata intestinalis)
▪ Enterocytozoon bieneusi
▪ Microsporidium ceylonensis, M. africanum
▪ Nosema ocularum
▪ Pleistophora sp.
▪ Trachipleistophora hominis, T. anthropophthera
▪ Vittaforma corneae (Nosema corneum)
▪ Some domestic & wild animals may be infected by some of the
above human species, including birds (esp. parrots)

Microsporidia Life Cycle

•Clinical Features: affects predominantly patients with AIDS
Most common: diarrhea, keratoconjunctivitis, GU and respiratory tract

Microsporidian species Clinical manifestation
Brachiola algerae Keratoconjunctivitis, skin and deep muscle infection
Enterocytozoon bieneusi* Diarrhea, acalculous cholecystitis
Encephalitozoon cuniculi and
Encephalitozoon hellem
Keratoconjunctivitis, infection of respiratory and genitourinary tract,
disseminated infection
Encephalitozoon intestinalis (syn.
Septata intestinalis)
Infection of the GI tract causing diarrhea, and dissemination to ocular,
genitourinary and respiratory tracts
Microsporidium (M. ceylonensis and
M. africanum) Infection of the cornea
Nosema sp. (N. ocularum), Brachiola
connori Ocular infection
Pleistophora sp. Muscular infection
Trachipleistophora anthropophthera Disseminated infection
Trachipleistophora hominis Muscular infection, stromal keratitis, (probably disseminated infection)
Vittaforma corneae (syn. Nosema
corneum) Ocular infection, urinary tract infection

Microsporidia: Laboratory Diagnosis
▪ Light Microscopic exam of stained smears (esp. fecal)
▪ Chromotrope 2R stain: spore and wall stain bright pink red
▪ “Quick-Hot Gram Chromotrope” technique, faster; spores stain violet
▪ Modified Trichrome stain
▪ Fluorescence microscopic exam
▪ Chemofluorescent agents like Calcofluor White with greenish color
▪ Specific Immunofluorescence assays with mono- or polyclonal
antibodies are in development
▪ Transmission Electron Microscopy for speciation; expensive,
time consuming
▪ Molecular amplification methods under development

Encephalitozoon, nasal samples. Courtesy of Yezid Gutierrez, MD
Giemsa-H&E, 320X
Giemsa-H&E, 840X
Warthin-Starry,
840X
Silver stain, 840X

Enterocytozoon bieneusi, fecal smear. Courtesy of Yezid Gutierrez, MD
Chromotrope 2R Stain, 840X

Pleistophora sp., brain tissue. Courtesy of Yezid Gutierrez, MD
840X
840X
320X

Osler fungi 10 2020

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Osler fungi 10 2020

Similar to Osler fungi 10 2020 (20)

Recently uploaded

Recently uploaded (20)

Osler fungi 10 2020