MYCOLOGY

Mycology

Margie Morgan, PhD D(ABMM)

Just the basics

Updated 2017
Starting point
Yeast are:
unicellular / produce budding daughter cells
colony on solid media are usually white to beige and
appear much like bacterial colonies
some genera produce mucoid colonies (Cryptococcus)
Starting point
spores
Molds are: hyphae
Produce filaments or hyphae
Produce conidia [spores]
Colonies on solid agar are downy, fluffy, cottony
Most mold colonies produce pigment which aid in
identification
Specimen collection and Transport
Fungi are very hardy organisms
No requirement for special transport media
Sterile containers required to prevent bacterial
contamination
Numerous anatomic sites are appropriate for culture
Respiratory specimens sputum, bronchial lavage, brushings,
nasal sinuses
Tissue biopsies
Cutaneous - Skin scrapings, material from lesions
Ocular
Sterile body fluids including CSF
Blood, bone marrow
 Fungal Culture Media
Sabourauds glucose agar (SABS)
All purpose Fungal media No antibiotics
Best used for subculture of fungi for identification workup
Contains 2% glucose, pH @7.0

Inhibitory mold agar (IMA)

Selective and enriched agar with chloramphenicol and gentamicin to
inhibit bacteria
Good for the primary recovery of pathogenic fungi
Saprophytic fungi and dermatophytes somewhat inhibited
 Fungal Culture Media
Mycosel/Mycobiotic agar
Selective SABS agar with chloramphenicol and cycloheximide
Used for selective culture of dermatophytes fungi that cause skin,
hair and nail infections

Brain heart infusion agar

Primary recovery of all fungi
Can inhibit bacterial growth by adding chloramphenicol and
cycloheximide
Can add blood to agar to nurture systemic fungi

Fungal cultures are incubated for 4 weeks at 30C

Lower temperature than bacterial culture incubation [35C]
If agar plates are used for fungal cultures the plates must be sealed
with air permeable tape to prevent spore dissemination from plates
What effect does Cycloheximide have
when added to media?
Prevents rapidly growing environmental molds
from overgrowing pathogenic fungal species
This is the good aspect of cycloheximide in media

Beware: it is not all good, it can suppress

important fungi from growing. Inhibited fungi
include:
Trichosporon beigelii
Candida tropicalis
Cryptococcus neoformans
Yeast phase of Blastomyces
Yeast phase of Histoplasma
Processing of Fungal Cultures
Inoculate fungal media with specimen

Seal plates with tape to prevent culture

contamination and escape of fungal spores

Incubate at 30C for 4 wk

If growth occurs - perform proper identification methods:

Yeast identification methods

Manual and automated biochemical reactions capable of
identifying most but not all yeast species.

Newer methods (1) Mass spectrometry MALDI-TOF and

(2) 16 sRNA sequencing are more precise methods with
the expanding taxonomy of yeast species.
Mold Identification methods
Lactophenol cotton blue [LCB] adhesive tape preparation is
standard method used for mold identification.

The LCB mounting medium consists of phenol, lactic acid,

glycerol and aniline cotton blue dye.

Clear adhesive tape touches a mold colony, picking

up fungal hyphae and pressed into one drop of LCB
on a microscope slide.
Newer (better methods) for
unusual molds:
MALDI-TOF
16sRNA sequencing
Safety in the Mycology Laboratory
If a culture is growing a mold, it cannot be opened on the
bench top
All mold work must be performed in a BSL-2 biosafety
cabinet with Hepa filtration
Yeast identification can be performed on the bench top
Direct Exams used to identify fungi directly
from patient specimens
Gram stain Most specimen types can be Gram
stained. Reliable detection of yeast

KOH preparation Skin, Hair or Nails examined for

both yeast and/or hyphae

Calcofluor white stain Most specimen types can

be stained and examined for yeast and/or hyphae

India ink Primarily used on CSF for the detection of

Cryptococcus neoformans and C. gattii
 Gram Stain
Yeast cells stain blue [Gram positive].
Examine stain for budding cells to confirm that it is a
yeast cell and not an artifact.
You can also detect pseudo-hyphae on Gram stain.
Mold can be difficult to detect on a Gram stain.

pseudohyphae
mold
pseudohyphae

X100 oil immersion

KOH potassium hydroxide prep
Detect yeast and/or pseudo-hyphae in skin, hair and
nail specimens using 40X light microscopy.
KOH dissolves keratin found in cellular material
and freeing fungal hyphae and yeast cells.
KOH exams can be difficult to interpret!
Calcofluor white stain

Yeast, pseudo-hyphae, and mycelial fungi will bind

with the Calcofluor white stain and fluoresce.
Read using a fluorescence microscope (40X)
More Sensitive and Specific than KOH preparation.
India Ink
One drop of black ink is placed
into one drop of CSF
On microscope slide - examined
using light microscope (40X)

It is a negative stain which

stains the background not the
yeast cell.
The clearing around the yeast is
due to the polysaccharide
capsule of Cryptococcus
neoformans and C. gattii.
Examination of fungi in fixed tissue
Grocotts Methenamine Silver Stain [GMS] yeast
and hyphae stain grey to black. Will stain living
and dead fungi

Observe the width of the hyphae, presence of septations

and angle of branching.
Observe the size and budding pattern of yeast.
Observe on later slides how these oservations can assist
in identification.
Periodic Acid Schiff [PAS]
Positive staining hyphae will appear magenta will
also stain structures containing a high proportion
of carbohydrate macromolecules (glycogen,
glycoprotein, proteoglycans) Stains living fungi.
Mucicarmine [Mucin] stain

Mucicarmine stains the polysaccharide capsule

pink for Cryptococcus neoformans and C. gatti.
Also stains mucin in fixed tissue.
Hematoxylin and Eosin Stain

Great for cellularity, not a

specific stain for fungi.
DIMORPHIC FUNGI
Important systemic pathogens with some
unique characteristics
What does Dimorphic mean?
Depending on temperature and conditions of
environment one fungi demonstrates two forms
Mycelial form - Hyphae and conidia (spores)
Free living form in nature and laboratory temperature <=30C
Yeast or yeast like form
Parasitic phase found in human tissue or in the lab >= 35C

Histoplasma capsulatum mold Histoplasma capsulatum yeast

Incubated at 30C from tissue and 35C culture
Dimorphic Fungi capable of causing systemic
infection most common

Histoplasma capsulatum
Blastomyces dermatitidis
Coccidioides immitis
Paracoccidioides brasiliensis
Sporothrix schenckii
Penicillium marneffei (Talaromyces marneffei)
Histoplasma capsulatum
World wide distribution / In USA considered
endemic in Ohio, Missouri, and Mississippi River
valleys

Environmental source: Bat guano (Spelunker =

cave explorers) and bird droppings
Histoplasmosis Disease
95% of infections are subclinical
5% infections:
Progressive pulmonary
Chronic systemic infection with dissemination to the RES
system including bone marrow
Acute fulminating systemic disease (fatal)
Reactivation disease can occur in elderly and
immunosuppressed (AIDS is a good example)
Bone marrow exam is useful in diagnosing disseminated
infections
Mucocutaneous lesions are a unique &
common site of dissemination
Histoplasma capsulatum - Yeast
Intracellular within macrophages
Small 2 4 um, regular in size, and oval to round.
Do not have a capsule, it is a staining artifact.

H & E stain GMS

Histoplasma will stain with a variety of stains
H&E PAS

Gram Wrights
 Histoplasma rapid diagnosis

Antigen detection in urine

Quantitative Enzyme immunoassay
Random urine specimen
Most sensitive for disseminated infection and
chronic pulmonary disease
Antigen is detectable in >=85% of these infections
Good for diagnosis of immune suppressed patients
that do not produce a detectable antibody response
Antibody tests are available but have been replaced
mostly by Antigen detection
 Histoplasma
capsulatum

Culture incubated at 30C

Requires 2 8 weeks to grow
Colony - white to brown and cottony
Microscopic appearance tuberculated macroconidia
that are large and round (8 16 M) and small
microconidia (2 - 4M)
Microconidia are the infectious particle growing in
nature and capable of penetrating deep into the lung
DNA probe necessary to confirm identification
Sepedonium species looks somewhat like Histoplasma
usually a contaminate. It is not dimorphic.
Histoplasm capsulatum
Histoplasma capsulatum in tissue

Granulomas are usually produced

caseating or non-caseating
Infection usually begins by breathing in the
microconidia and infecting the Lung
Infection disseminates to organs of the
Reticuloendothelial System (RES) with high % of
dissemination to the Bone Marrow
Intracellular budding yeast (2 4 M) are seen in
all tissues
Beware of look alike organisms
in tissue specimens!!

Toxoplasma

Leishmania species
Note small round
kinetoplast next to nucleus
Histoplasma capsulatum
Unusual variant of H. capsulatum
H. capsulatum var duboisii disease
is found in Central Africa differs
from H. capsulatum with primary
infection in skin and bone

The mold phase of H capsulatum var. duboisii is identical to

H. capsulatum.
The difference is the size of the yeast cells: Note yeast cell is
8 10 uM which is 2X the size of H. capsulatum yeast cell.
Blastomyces dermatitidis
Epidemiology
Ohio and Mississippi River valleys
No association with specific animal or activity
Forrest and river banks?
Primarily a pulmonary infection which may
disseminate primarily to the skin and bone

Well demarcated skin lesions are

often seen in disseminated
Blastomycosis.
Skin lesions are full of yeast cells.
Blastomyces dermatitidis
Culture at 30C
Grows in 2- 3 weeks
Fluffy white buff colored mold, prickly
Pear shaped conidia at the end of supporting
hyphae looks like lollipops
Look alike fungus Chrysosporium species
Do DNA probe test to confirm identification

Blastomyces
Chrysosporium
Blastomyces culture at 35 C
Slow growing yeast colony (4 weeks to
form a colony)
Yeast cell is 8 20 um in size
Unique Broad Based Budding pattern and
the double contoured wall.

Broad based bud

Blastomyces dermatitidis histopathology
Mixed pyogenic and granulomatous inflammation is
observed in tissue
Broad based budding yeast cells
Coccidioides immitis
Endemic in SW USA, Mexico,
South America, in areas known as the
Sonoran life zone/ warm and desert sands
Infection is from inhalation of fungal
particles found in the sand
New: Coccidioides posadasii is genetically
related to C. immitis. These two species are
located in different endemic regions, but
produce the same disease process
Coccidioidomycosis
 Coccidioidomycosis
95% of infections are asymptomatic or with limited
symptoms
The remaining 5% are focal pulmonary, progressive
pulmonary or disseminated infections.
Dissemination to the central nervous system carries a high
fatality rate.
Higher incidence of dissemination occurs in patients with:
defects in cell mediated immunity (HIV),
darker skinned ethnic groups,
pregnancy
Coccidioides immitis [posadasii]

Culture at 30C
Requires 2 3 days to grow, colony starts waxy and
becomes wooly in around 7 10 days
Under the microscope (40X) one observes foci with
septate hyphae with thick walled barrel shaped
arthroconidia - clear spaces in-between. The clear
spaces are dead arthroconidia.
Arthroconidia are the infectious particle in nature
Culture is very infectious to laboratory personnel
 Coccidioides 30C Culture
Malbranchea species mimics C. immitis/posadasii under
the microscope
Because of look-a-like fungi, one needs to confirm
identification of Coccidioides immitis/posadasii with DNA
probe (molecular) or similar method to be accurate

Coccidioides Malbranchea
No yeast phase with Coccidioides!

Culture at 30*C and 35*C produce hyphae with barrel shaped

alternating arthroconidia.
There is no yeast phase for C. immitis [posadasii]
 Coccidioides Histopathology
Thick walled spherules (10 80 uM) with endospores
are seen in tissue. This is the second form of Cocci.
No yeast cells are produced in tissue.
Spherules are in all stages of development-
fragmented spherules to well formed with endospores
Granulomatous inflammation with caseation is usually
observed
Development of Cocci spherules from the
inhalation of Arthroconidia from nature
Coccidioides is not the only spherule
forming organism!
Rhinosporidium seeberi (aquatic parasite) forms spherules
but much larger than those of Cocci. Rhinosporidium
spherules are usually > 80 uM in size. Also R. seeberi
usually cause only oral or nasal mass lesions

Oral or nasal mass lesions

of Rhinosporidium seeberi
Coccidioides
spherules
Paracoccidioides brasiliensis
South American Blastomycosis
endemic area Brazil, Venezuela, Columbia
Inhale infectious particle from soil
>95% of infections in males, possibly due to
estrogen inhibition of mycelial to yeast
transformation
Disease presentation:
1. Pneumonia
2. Disseminated infection to 2 or more organs
3. Extrapulmonary lesions on the face and oral mucosa
 Paracoccidioides
Cultures at 30C for mycelial phase are usually
not performed - due to slow growth and nonspecific
sporulation
Culture @ 37C
Slow growing yeast
Large yeast (10 30uM) with multiple daughter buds
(2 10 uM) in size
Unique budding yeast cell known as the Mariners wheel or
Pilots wheel yeast
Tissue Exam of
Paracoccidioides brasiliensis

If more than 2 buds off mother yeast cell

High likelihood it is Paracoccidioides
Granulomatous inflammation with Giant
Cells formed in tissue
Sporothrix schenckii
Sporotrichosis
Cutaneous inoculation of fungus from penetrating injury with a
spore or thorn (rose bush)
Begins as single skin lesion w/wo ulceration
Lesion can progress to many lesions and lymphocutaneous
spread, possibly to bone and other organs
Pulmonary and CNS infections are rare but reported
Sporothrix
schenckii
30*C culture grows in 3 -5 days as mold
Beige colony/ turns brown to black over time
Septate hyphae with conidia in daisy
wheel pattern

37C culture small oval yeast cells,

elongated 2 5 M, described as cigar bodies
Sporothrix schenckii Histology
Pyogenic to granulomatous inflammation
Hard to find yeast cells in human tissue
More commonly an Asteroid body known as Splendore-
Hoeppli phenomenon can be seen, but this is not specific
to Sporothrix, also seen in:
Zygomycetes (Mucorales)
Aspergillus
Blastomycosis
Candida
Sporothrix schenckii
Cigar shaped yeast cell

Daisy like spore arrangement

Splendore-hoeppli
Penicillium (Talaromyces) marneffei
Green colony with red diffusable pigment
grows in 2 3 days at 30 C
Uncommon dimorphic fungus yeast like cells
produced in tissue
Only dimorphic species of Penicillium
Diseases: skin lesions in tropics and
Pneumonia or systemic disease in advanced
HIV
Penicillium (Talaromyces) marneffei
SUBCUTANEOUS FUNGAL
INFECTIONS
Very unique structures in tissue!
Subcutaneous Fungal Infections
Most common will be described

Mycetoma [2 types]
Actinomycotic caused by higher bacteria
Eumycotic caused by dark pigmented molds
Chromomycosis [Chromoblastomycosis]
Phaeohyphomycosis
Sporotrichosis
Mycetoma
First observed in India and known
as Madura Foot or Maduromycosis
Found in the hot temperate parts of
the world

Three criteria define Mycetoma:

-Traumatic lesion leads to swollen extremity
-Draining sinuses evolve
-Sulfur granules observed in tissue and found in the
weeping drainage from sinus tract

Fungus grows on organic debris in soil

Implanted into subcutaneous tissue from trauma
Mycetoma
Swollen extremity with draining sinuses that
drain sulfur granules

Sulfur Granule
 Actinomycotic Mycetoma
There are two types of Mycetoma:
1. Actinomycotic caused by higher bacteria species
2. Eumycotic caused by black molds

Actinomycotic Mycetoma
98% of cases
Nocardia species most common agent
Sulfur granules formed in tissue. The granules vary in color
based on Nocardia species and contain a matrix of the
filamentous bacteria
Actinomycotic sulfur granule
Beware! Sulfur granule caused
by Actinomyces israelii looks
identical to one formed by
Nocardia.

Edge of granule has thin filamentous bacteria: How can

you tell if it is Nocardia?
Nocardia is modified acid fast [PAF] positive and is an
aerobic bacteria. Actinomyces is PAF negative and grows
anaerobically.
Nocardia
Gram stain = filamentous Gram positive bacilli
can be poorly staining and appear speckled.
Positive [red] on the Modified Kinyoun acid fast stain.

Gram stain of sputum Modified acid-fast stain

[modified Kinyoun stain]
Nocardia species Mycetoma, Pulmonary
and Brain infections
Grows in 3 5 days on agar media
[Sabs, 5% Sheeps blood agar]

Colony is dry/crumbly - Musty smell

Total of 85 species:
Nocardia asteroides most common

Therapy: Trimethoprim/Sulfa

Identification by Maldi-TOF
or 16s rRNA gene
sequencing
Thermoactinomycetes
Gram positive filamentous rods related to Nocardia
Thermoactinomyces and Saccharopolyspora
Flourish in areas of high humidity and high temperatures (40-60*C)
Grow in agricultural environments like stacks of hay
Farmers lung hypersensitivity pneumonitis or extrinsic
allergic alveolitis, occupational disease
Immunologically medicated inflammatory disease of the lung
Inhalation exposure to the thermophilic actinomycetes
Diagnosis
Detailed environmental history
Serology testing
Eumycotic mycetoma

Caused by pigmented/black fungi due to melanin

-Numerous species found in soil
-2% of cases of mycetoma
-Traumatic implantation implants the mold into
the subcutaneous tissue
-Most common black molds include:
Cladophialophora (Cladosporium) carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Eumycotic sulfur granule the granule is
composed of a matrix of thick fungal hyphae
 Chromomycosis/Chromoblastomycosis
Three characteristics:
Wart like lesions in subcutaneous tissue
Sclerotic bodies observed in tissue
Growth of dark/pigmented fungi

Black mold naturally found in the soil cause

infection through abrasion/ implantation

Black molds that can cause Chromomycosis:

Cladophialophora [Cladosporium] carrionii
C. bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Chromomycosis/Chromoblastomycosis

Sclerotic Body/Medlar Body/

Copper Penny is the unique
structure found in tissue

Wart like/Verrucous lesions

In subcutaneous tissue
Prototheca wickerhamii the cause of Protothecosis

Algae without chlorophyll

Causes skin lesions & nodules
Most common in patients with suppressed immune
system
Compare morula of Protothecosis to sclerotic body of
Chromomycoses
Phaeohyphomycosis
Traumatic implantation of dark fungi into subcutaneous
tissue
Variety of infections but nodules/lesions most common
with/without dissemination
Dark hyphae observed in tissue
Black mold also known as
Dematiaceous mold

-Black colored colonies and the

reverse [underside of colony] is also
black
-Natural-colored brown hyphae and
spores (melanin)
-One of the common molds that grow
due to water damage!
Black Molds Dematiaceous fungi
Black colonies
Brown hyphae and spores (melanin)
Numerous species
Difficult to identify
All have one of four types of sporulation
Rhinocladiella-like Rhinocladiella Cladosporium
Cladosporium-like
Phialophora-like
Acrotheca-like Phialophora Acrotheca
Black Molds cause
Mycetoma/Chromomycosis/Phaeohyphomycosis plus
sinusitis, pulmonary, brain, and disseminated infections.
These fungi are difficult to identify using only
morphology require sequencing/MALDI for speciation

Exophiala species
Cladophialophora bantiana -
Brain infections
Wangiella dermatitidis

Phialophora verrucosa
Other black molds of importance:
Alternaria species Opportunistic fungus - sinusitis
in normal host, dissemination in immune
suppressed.
Bipolaris
australiensis

Disseminated fungal
infection in both normal
and immune suppressed
hosts:
Skin, nasal
sinuses, bone
brain
Curvularia lunata

Center cell is the largest

Opportunistic fungal pathogen

Reported in Bone Marrow
Transplantation and Aplastic anemia
Can cause unusual erythematous skin lesions
Exserohilum rostrum
Outbreak associated with compounded pharmaceutical
[steroid] products contaminated with dust/dirt
Used for infections into lumbar spine and knee joints for
pain management and led to infections:
Meningitis
Spinal abscess
Synovial infections
Scedosporium apiospermum/Pseudallescheria boydii
Cat fur-like colony/growth in 3 5 days, lollipop like spores
Opportunistic pulmonary pathogen. Can be difficult to
distinguish from Aspergillus in fixed tissue stains.
IMPORTANT YEAST
CAUSING HUMAN
INFECTION
Candida species
Cryptococcus neoformans &
Cryptococcus gattii
Candida species

Candida species (@ 10 infect humans)

Yeast are normal flora in GI, GU and skin
Opportunistic pathogen involving skin or mucous
membranes from excessive exposure to moisture,
antibiotics, or immune suppression
Thrush, vaginitis, skin lesions, nail, diaper rash
Also can cause more serious infections such as
fungemia, endocarditis, and systemic tissue
infection.
Candida species

Candida albicans most common species causing @

60% of human yeast infections
Candida glabrata, C. krusei, and C. tropicalis are causing
higher % of infections than in the past
Fluconazole is a very useful oral and IV anti-fungal
These 3 species are more likely to be resistant to Fluconazole
New species Candida auris outbreaks in hospitals,
intrinsically resistant to most anti-fungals
Candida parapsilosis has emerged as a pathogen of
children and common in IV line infections (forms biofilms
on plastics)
C.parapsilosis is susceptible to fluconazole.
Candida species
Grow in 24 48 hours 30-35*C
pseudohyphae
Many agars: SABS, IMA, Blood agar
Bacteria-like colony pasty white
Budding yeast oval @ 7-8 um in size**
Form pseudohyphae not true hyphae (Yeast do not
detach and form elongated hyhae)
Identify using biochemical rxn, MALDI, or Sequencing

Exception: **Candida glabrata is only Candida species

@ 4 M in size and does NOT form pseudohyphae this
species breaks all the Candida rules
Candida albicans
Identification
Germ tube formation
Incubate small amount of yeast in serum for 3-4hrs at 35 C
If incubate >4 hrs C. tropicalis will also form germ tubes
C. dubliniensis is also positive
Specific ID from biochemical reactions, MALDI-TOF or
Sequencing
Chlamydospore formation chlamydospore
Growth on cornmeal agar at 48 hrs
Rudimentary structures unique to
C. albicans
 ChromAgar for the
identification of Candida
using chromogenic
substrates.
The unique colors can
identify 4 yeast species.

Yeast with pseudohyphae

 Candida Histopathology
Pyogenic to granulomatous
Usually observe yeast cells (8 um), pseudohyphae and/or hyphae-
like structures
Candida glabrata = smaller yeast cells (4 um) and absence of
pseudohyphae

Candida species not GMS stain of Candida glabrata

glabrata
 Cryptococcus
neoformans

In nature forms a 2um non-encapsulated yeast cell.

Associated with bird droppings (esp. pigeon).
C neoformans is enriched by the nitrogen in the
heaped droppings.
Non-encapsulated yeast cells are inhaled travel
through the pulmonary system with hematogenous
spread to brain and meninges
Tropism to the meninges
Infects mostly compromised hosts one of the major
AIDS defining infections
Cryptococcus gattii a close relative of C. neoformans
Isolated from forested area of the Pacific Northwest
(British Columbia, Washington, Oregon and California)
found in soil debris and tree species
Infection of normal and immune suppressed hosts
Primarily a pulmonary disease [Cryptococcoma] but can
develop meningitis like C. neoformans
Culture, biochemical & staining identical to C. neoformans
Defining reactions
L Canavanine glycine bromthymol blue medium
C. gatti = blue C. neoformans = colorless
MALDI-TOF will also ID both species
Cryptococcus neoformans
and C. gattii
Irregularly sized yeast cells (2 20 um)
Polysaccharide capsule is the primary virulence factor
Polysaccharide capsule is detected in diagnostic testing:
India ink exam- CSF placed in one drop of black ink. It is a
negative staining method background is stained not the
capsule
Sensitive test for AIDS patients (90% sensitive)
Cryptococcal antigen test Titer of capsular polysaccharide
detected in either CSF or serum
More sensitive than India ink for all patient groups
Test for diagnosis or can follow recovery with falling titer
Grows on mycologic agars / sensitive to cycloheximide
Mucoid colonies due to capsule polysaccharide formation
Urease enzyme + Inositol assimilation +
Brown colonies produced on bird seed agar
Both C. Neoformans & C. gattii produce
brown colonies on Birdseed agar
 Mucoid colonies of Variability in size
C. neoformans and
C. gattii

Urea medium demonstrating urease

enzyme activity of Cryptococcus

Positive
Positive India Ink
Observe
Budding cells
Mucicarmin stains the
capsular polysaccharide of
Cryptococcus
C. neoformans/ C. gattii
no nuclear staining
Cryptococcus vs. Pneumocystis
Pneumocystis jiroveci (yeast like
fungus) could be confused with C.
neoformans Careful! Central nuclear
staining in pneumocystis
Pneumocystis froth on H & E
Trichosporon
T. asahii most common 9 species
Commonly inhabit the soil. Colonize the skin/
gastrointestinal tract of humans.
Rectangular shaped yeast cells
Cause of superficial infection - white piedra,
a distal infection of the hair shaft,
Second most commonly reported cause of
disseminated yeast infections in humans
Rare, even among patients with impaired host defenses:
Corticosteroid use, solid tumors, HIV/AIDS, and intravascular
devices, including catheters and prosthetic heart valves
Mortality 50 80%
CUTANEOUS AND
SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes
 Malassezia furfur

Most superficial of the dermatomycoses

Normal flora on the skin,
More common on oily skin or high use of skin oils
Diseases:
Skin: macules, papules, patches, plaques on chest back
and shoulders with either hypo or hyper pigmentation
does not invade into deeper tissues known as
pityriasis versicolor
Fungemia: caused by skin flora tunneling in with IV lipid
feeding lines (parenteral nutrition) in neonates or rarely
adults
 Malassezia furfur
Lipophilic yeast oil required for growth
Media for isolation must contain oil or us an oil overlay
Small budding yeast 2 4 M with collarette (appears like
necklace at junction of mother and daughter yeast cell)
In tissue described as Spaghetti and Meatballs

Spaghetti and meatballs

Size range for Yeast
Candida glabrata/Histoplasma capsulatum/
M. furfur
2 4 um
Candida species
8 10 um plus pseudohyphae
Cryptococcus neoformans/gattii
2 20 um
Blastomyces dermatitidis
8-15 um
 Dermatophytes Ringworm infections
Hair, skin and nail infections
3 genera of fungi
Microsporum species (many)
Epidermophyton floccosum
Trichophyton species (many)
Disease described by area of the body infected:
For example: tinea capitis (head), t. pedis (foot)
Usually a clinical diagnosis not requiring culture
KOH prep or Calcofluor white prep can be used to
visualize fungal hyphae from skin scrapings
Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence
thin fungal hyphae
Microsporum canis
Ringworm acquired from dog and cat
White colony/ yellow on backside of colony
Tuberculate thick walled macroconidia [spiny
projections] Few if any microconidia
Microsporum gypseum skin infection from
exposure to contaminated soil, sandy colored
colony, large macroconidia are produced.
Trichophyton
rubrum

White colony with

red diffusable
pigment

Pencil shaped
Red diffusible pigment
macroconidia
many micro-
conidia

Skin infection from

fomites
Trichophyton
Tonsurans

White colony
grows in @ 5
days
No macroconidia
Ballooning
microconidia

Primary cause of
epidemic scalp
ringworm in
children
 Epidermophyton floccosum

Beaver tail large macroconidia no microconidia

Khaki green colored colony most often cause infections
in skin and nails.
Opportunistic Fungal
Pathogens
Infections in the immune suppressed host or
special circumstances
Hyaline molds
Opportunistic Fungi - hyaline
Hyaline no color to the hyphae
Regular septations (walls) in the hyphae
Branching of hyphae in stains of fixed tissue
angle of branching can be helpful in
identification
Usually grow in 3 5 days at 35 or 30C
??? of species in the thousands taxonomy
changing daily
Aspergillus species
Hyphae - Hyaline with septations
Numerous round conidia produced
In tissue - Branching at 45 degree angle
Usually pulmonary infection but can disseminate
Invade vessels, cause thrombosis & infarctions

Septation
Aspergillus Structure
Aspergillus species
Four species most common in human infections:
1. Aspergillus fumigatus
2. Aspergillus flavus
3. Aspergillus niger
4. Aspergillus terreus unique and important intrinsic
resistance to Amphotericin B

Aspergillus Galactomannan Enzyme immunoassay

detects circulating Aspergillus antigen in the blood
and/or bronchial lavage fluid
Low sensitivity and specificity
False positive reaction from therapy with Piperacillin/Tazobactam,
infection with Histoplasma capsulatum
Aspergillus fumigatus

Blue/Green colony grows

in 3 5 days

Phialids with colorless

spores are directed upward
Aspergillus flavus

Green/yellow colony

Orange colored sporeson

phialids that surround the
vessicle
Aspergillus niger

Black colony visible black heads

Black spores supported by phialids

surround the vesicle
Aspergillus terreus Sandy colored colony
Colorless conidia supported
by phialids headed upward

Aleurioconidia produced

Intrinsic resistance to
Amphotericin B
Aspergillus fruiting head seldom seen in fixed
tissue usually only dichotomous (continuous) branching
septate hyphae which branch at a 45 angle

Can appear much like that

of Pseudallescheria boydii
(Scedosporium) Growth in
culture can differentiate the
two fungi.
Fusarium species
Common in nature/plants
Disease related to immune
status of host
Infections reported:
Disseminated in bone marrow
transplants
Corneal infections in contact lens
wearers
Scopulariopsis species found in soil and plants
Infections: Nail, skin, sinusitis, pulmonary and
disseminated
Penicillium species one of the most common
mold in the environment , cause of bread mold,
uncommon cause of human disease
MUCORMYCOSIS/
ZYGOMYCOSIS
Fungi in the Order Mucorales
Hyaline
Broad hyphae without septations
Mucormycosis/Zygomycosis
Rhinocerebral mucormycosis
Infections in diabetics, the elevated glucose
enriches fungal growth
Sinus orbit of eye - brain
Sinus and pulmonary infection in the immune
suppressed host
Broad, hyaline, aseptate hyphae produced
Cultures grow in 24 hrs, producing coarse aerial
hyphae
Mince infected tissue and place on agar
Do not grind tissue grinding will kill the
aseptate hyphae
Order Mucorales coarse, aerial hyphae
after 24 hours on SABS agar at 30C
Order Mucorales
Rhizopus Absidia
Rhizoids

Distant rhizoids

Mucor

No rhizoids
 Zygomycetes (Mucorales)
90 angle branching, aseptate, ribbon like hyphae

Invades vessels and can cause

infarcts and thrombi