Mycology Update 2017

MYCOLOGY
Margie Morgan, PhD D(ABMM)
Just the basics –
Updated 2017
Mycology

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
Molds are:
• Produce filaments or hyphae
• Produce conidia [spores]
• Colonies on solid agar are downy, fluffy, cottony
• Most mold colonies produce pigment which aid in
identification
hyphae
spores

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
• Sabouraud’s 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 30˚C
• Lower temperature than bacterial culture incubation [35˚C]
• 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

• Inoculate fungal media with specimen
• Seal plates with tape to prevent culture
contamination and escape of fungal spores
• Incubate at 30˚C 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.
Processing of Fungal Cultures

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
Mold Identification methods

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

• 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
Gram Stain
X100 oil immersion

•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!
KOH – potassium hydroxide prep

• 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.
Calcofluor white stain

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.
India Ink

Grocott’s 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.
Examination of fungi in fixed tissue

Positive staining hyphae will appear magenta – will
also stain structures containing a high proportion
of carbohydrate macromolecules (glycogen,
glycoprotein, proteoglycans) Stains living fungi.
Periodic Acid Schiff [PAS]

Mucicarmine stains the polysaccharide capsule
pink for Cryptococcus neoformans and C. gatti.
Also stains mucin in fixed tissue.
Mucicarmine [Mucin] stain

Great for cellularity, not a
specific stain for fungi.
Hematoxylin and Eosin Stain

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 <=30˚C
• Yeast or yeast like form
• Parasitic phase found in human tissue or in the lab >= 35˚C
Histoplasma capsulatum – mold
Incubated at 30˚C
Histoplasma capsulatum – yeast
from tissue and 35˚C 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

H & E PAS
Gram Wright’s
Histoplasma will stain with a variety of stains

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 30˚C
• Requires 2 – 8 weeks to grow
• Colony - white to brown and cottony
• Microscopic appearance – tuberculated macroconidia
that are large and round (8 – 16 µM) and small
microconidia (2 - 4µM)
• 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.

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 µM) are seen in
all tissues

Leishmania species
Note small round
kinetoplast next to nucleus
Toxoplasma
Histoplasma capsulatum
Beware of look alike organisms
in tissue specimens!!

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.
H. capsulatum var duboisii disease
is found in Central Africa – differs
from H. capsulatum with primary
infection in skin and bone
Unusual variant of H. capsulatum

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 30˚C
• 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

• 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.
Blastomyces culture at 35° C
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 30˚C
• 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 30°C 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

Culture at 30*C and 35*C produce hyphae with barrel shaped
alternating arthroconidia.
There is no yeast phase for C. immitis [posadasii]
No yeast phase with Coccidioides!

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

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
Coccidioides is not the only spherule
forming organism!

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 30°C for mycelial phase are usually
not performed - due to slow growth and nonspecific
sporulation
• Culture @ 37˚C
• Slow growing yeast
• Large yeast (10 – 30uM) with multiple daughter buds
(2 – 10 uM) in size
• Unique budding yeast cell known as the Mariner’s wheel or
Pilot’s 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
• 37˚C culture small oval yeast cells,
elongated 2 – 5 µM, 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

Daisy like spore arrangement
Sporothrix schenckii
Cigar shaped yeast cell
Splendore-hoeppli

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

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

Swollen extremity with draining sinuses that
drain sulfur granules
Sulfur Granule
Mycetoma

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

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.
Beware! Sulfur granule caused
by Actinomyces israelii looks
identical to one formed by
Nocardia.
Actinomycotic sulfur granule

Gram stain = filamentous Gram positive bacilli
can be poorly staining and appear speckled.
Positive [red] on the Modified Kinyoun acid fast stain.
Modified acid-fast stain
[modified Kinyoun stain]
Gram stain of sputum
Nocardia

Grows in 3 – 5 days on agar media
[Sabs, 5% Sheep’s 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
Nocardia species – Mycetoma, Pulmonary
and Brain infections

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
• Farmer’s 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
Wart like/Verrucous lesions
In subcutaneous tissue
Sclerotic Body/Medlar Body/
Copper Penny is the unique
structure found in 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
• Cladosporium-like
• Phialophora-like
• Acrotheca-like
Rhinocladiella Cladosporium
Phialophora Acrotheca

Exophiala species
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
Cladophialophora bantiana -
Brain infections

Wangiella dermatitidis
Phialophora verrucosa

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

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
• 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 µM in size and does NOT form pseudohyphae – this
species breaks all the Candida rules
pseudohyphae

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
• Growth on cornmeal agar at 48 hrs
• Rudimentary structures unique to
C. albicans
chlamydospore

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
GMS stain of Candida glabrataCandida species not
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

Positive India Ink
Urea medium demonstrating urease
enzyme activity of Cryptococcus
Observe
Budding cells
Variability in size
Positive
Mucoid colonies of
C. neoformans and
C. gattii

Mucicarmin stains the
capsular polysaccharide of
Cryptococcus Pneumocystis jiroveci (yeast like
fungus) could be confused with C.
neoformans – Careful! Central nuclear
staining in pneumocystis
C. neoformans/ C. gattii –
no nuclear staining
Cryptococcus vs. 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 µM 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
macroconidia
many micro-
conidia
Skin infection from
fomites
Red diffusible pigment

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 30˚C
• ??? 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 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 30˚C

Rhizopus Absidia
Distant rhizoids
Mucor
No rhizoids
Rhizoids
Order Mucorales

90˚ angle branching, aseptate, ribbon like hyphae
Invades vessels and can cause
infarcts and thrombi
Zygomycetes (Mucorales)

Mycology Update 2017

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Mycology Update 2017