Mycology 2018 Update

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MYCOLOGY
Mycology Update 2018
Margie Morgan, PhD D(ABMM)
Yeast
Zygomycota Conidial Molds
Dematiacious Hyaline
Yeast

Starting point
Yeast are:
• Unicellular / budding to produce daughter cells
• Growth on solid media usually white to beige...

Starting point
Molds:
• Produce filaments or hyphae
• Produce conidia [spores]
• Growth on solid media are downy, fluffy, ...

Specimen collection and Transport
• Fungi are very hardy organisms
• No requirement for special transport media
• Sterile ...

Fungal Culture Media
• Sabouraud’s glucose agar (SABS)

Fungal Culture Media
• Mycosel/Mycobiotic agar
• Selective SABS agar with chloramphenicol and cycloheximide
• Used for sel...

•Inoculate specimen onto media
•Seal plates with tape to prevent culture from
environmental contamination and protect labo...

Yeast Identification
• Biochemical reactions identify most but not all yeast
species – manual and automated instrument ide...

Lactophenol cotton blue [LCB] adhesive tape preparation is
used for mold identification.
LCB mounting medium consists of p...

Safety in the Mycology Laboratory
•All work on molds must be performed in a BSL-2 biosafety
cabinet with HEPA filtration
•...

Gram stain
•Yeast cells are Gram positive
•Examine for budding cells to confirm that it is a yeast
cell and not an artifac...

•Detect yeast and hyphae from skin, hair, and nail
specimens
•KOH dissolves the keratin found in cellular material and
fre...

• Yeast, pseudo-hyphae, and mycelial fungi bind
with the Calcofluor white stain and fluoresce
• Read using a fluorescence ...

One drop of black ink is placed into one
drop of CSF onto a microscope slide -
examined using light microscope (40X)
It is...

Grocott’s Methenamine Silver Stain [GMS]
– yeast and hyphae stain grey to black. Will stain
both living and dead yeast and...

Positive staining hyphae are magenta – will also
stain structures containing
carbohydrate macromolecules (glycogen,
glycop...

Mucicarmine stains the polysaccharide capsule of
Cryptococcus neoformans and C. gatti pink. Will
also stain mucin in fixed...

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

DIMORPHIC FUNGI
Important systemic pathogens with some
unique characteristics

Definition of Dimorphic
• Depending on temperature and conditions of
environment one fungi demonstrates two forms:
• (1) M...

Most common Dimorphic Fungi
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis/posadasii
• Paracoc...

Histoplasma
capsulatum• Epidemiology: World wide distribution / In USA
endemic in Ohio, Missouri, and Mississippi River
va...

Histoplasmosis Disease
• 95% of infections are subclinical
• 5% infections: Pulmonary +/- dissemination, reactivation
dise...

Histoplasmosis rapid
diagnosis
• Antigen detection in urine
• Quantitative Enzyme immunoassay
• Performed on random urine ...

Histoplasma capsulatum
Culture
• Incubated at 30˚C
• Requires 2 – 8 weeks to grow
• Colony - white to brown and cottony
• ...

Histoplasma capsulatum
Culture
• Incubated at 37*C
• Requires 4 – 8 weeks to grow
• Colony – white to beige, creamy
• Micr...

Histoplasma capsulatum in fixed
tissue
• Infection begins by inhalation of
the microconidia into the lung
• Granulomas usu...

Histoplasma capsulatum - Yeast
Intracellular within macrophages
Small 2 – 4 um, regular in size, oval to round.
Appear to ...

Leishmania amastigote
Note: kinetoplast (mitochondrial
DNA) next to nucleus
Toxoplasma tachyzoites
oval to crescent in sha...

The mold phase is identical to H. capsulatum.
Difference is the size of the yeast cell: Note yeast cell of H. duboisii
is ...

Blastomyces dermatitidis
• Epidemiology
• Ohio and Mississippi River valleys
• No association with animal or activity
• Fo...

Blastomyces dermatitidis
• Culture at 30˚C
• Grows in 2- 3 weeks
• Fluffy white – buff colored mold, prickly
• Microscopic...

• Culture at 35*C
• Slow growing yeast colony (4 weeks)
• Large yeast cell is 8 – 20 um in size
• Unique Broad Based Buddi...

Blastomyces dermatitidis
histopathology
• Mixed pyogenic and granulomatous inflammation is
observed in tissue
• Broad base...

Coccidioides immitis
(posadasii)
• New species: Coccidioides posadasii is
genetically related to C. immitis. The two
speci...

Coccidioidomycosis
• 60% of infections are asymptomatic or
• 30% with limited pulmonary diseases
• The remaining 5-10% res...

Serologic diagnosis of
Coccidiodomycosis
• Enzyme immunoassay (EIA): A very sensitive and
commonly used method for diagnos...

Coccidioides Culture
• Culture at 30˚C
• Growth in 2-3 days, colony starts waxy and becomes
wooly in around 7–10 days
• Mi...

Look-a-likes
• Malbranchea species mimics C. immitis/posadasii under
the microscope /must confirm ID by MALDI or molecular...

Coccidioides Histopathology
• No yeast cell produced, instead: Thick walled spherules
(10 – 80 uM) with endospores are the...

• Rhinosporidium seeberi (aquatic parasite) forms
spherules but much larger than those of Cocci.
• Rhinosporidium spherule...

Paracoccidioides brasiliensis
• South American Blastomycosis – Brazil
80% of cases reported from Brazil
• Most prevalent s...

Paracoccidioides
• Cultures at 30*C usually not done, slow growth and
nonspecific sporulation
• Culture at 37˚C
• Slow gro...

Tissue Exam of
Paracoccidioides brasiliensis
• Granulomatous inflammation
with Giant Cells formed in tissue
GMS
GMS

Sporothrix schenckii
• Sporotrichosis
• Cutaneous inoculation from penetrating
injury, cut or scrape from thorns or sphagn...

Sporothrix
schenckii
• 30˚C culture grows in 3 -5 days
as beige mold that becomes brown
to black over time
• Microscopic: ...

Sporothrix schenckii Histology
• Pyogenic to granulomatous inflammation
• Hard to find yeast cells in human tissue
• If se...

•Disease: Skin lesions in tropics and SE Asia
•Pneumonia or systemic infection in HIV/AIDS
•Only dimorphic species of Peni...

SUBCUTANEOUS FUNGAL
INFECTIONS
Mycetoma
Chromoblastomycoses
Phaeohyphomycosis

Mycetoma
• Found in the hot temperate parts of the
world – fungus grows on organic debris
• Three criteria:
-Swollen extre...

Actinomycotic Mycetoma
• 98% of cases
• Nocardia species most common agent
• Sulfur granules formed in tissue. The granule...

Note: Edge of granule has thin filamentous bacteria:
How can you tell if it is Nocardia?
Nocardia is modified (partial) ac...

Gram stain = filamentous Gram positive bacilli
can be poorly staining and appear speckled.
Positive [red] on the Modified ...

Grows in 3 – 5 days on SABs, 5%
Sheep’s blood agar …
Colony is dry/crumbly - Musty smell
Total of 85 species:
Nocardia ast...

Eumycotic mycetoma
Caused by pigmented/black fungi
-Numerous species found in soil and debris
-2% of cases of mycetoma
-Tr...

Thermoactinomycetes
• Nocardia related Gram positive filamentous rods
• Thermoactinomyces and Saccaropolyspora
• Flourish ...

Chromomycosis/Chromoblastomycosi
s
• Three characteristics:
• Wart like lesions in subcutaneous tissue
• Sclerotic bodies ...

Prototheca wickerhamii – Protothecosis
• Algae without chlorophyll
• Causes skin lesions & nodules
• Most common in patien...

Phaeohyphomycosis
Traumatic implantation of dark fungi into subcutaneous
tissue
• Variety of infections but nodular lesion...

Black mold also known as
Dematiaceous mold
-Black colored colonies and the reverse
[underside of colony] is also black
-Na...

Black Molds
• Numerous species
• Difficult to identify
• All have one of four types of sporulation
• Rhinocladiella-like
•...

Exophiala species
Black Molds cause
Mycetoma/Chromomycosis/Phaeohyphomycosis
Also: sinusitis, pulmonary, brain, and dissem...

Wangiella dermatitidis
Phialophora verrucosa

Alternaria species– Opportunistic fungus -
sinusitis in normal host, disseminated infection in
immune suppressed.
Other mo...

Bipolaris
australiensis
Disseminated fungal
infection in both normal
and immune suppressed
hosts
Also, Skin, nasal
sinus, ...

Curvularia lunata
Center cell is the largest
Opportunistic
Bone Marrow Transplant and Aplastic anemia
patients
Can cause u...

Exserohilum rostrum
• Outbreak associated with compounded pharmaceutical
[steroid] products contaminated with dust/dirt du...

Scedosporium apiospermum/Pseudallescheria
(Scedosporium) boydii
Cat fur-like gray colony / growth in 3 – 5 days
Lollipop l...

IMPORTANT YEAST
CAUSING HUMAN
INFECTION
Candida species
Cryptococcus neoformans
Cryptococcus gattii
Trichosporon species

Candida species
• Candida species (@ 10 infect humans)
• Yeast are normal flora in GI, GU and skin
• Opportunistic pathoge...

Important Candida species
• Candida albicans – most common species causing @
60% of Candida yeast infections
• Candida gla...

Candida species
• Grow in 24 – 48 hours at 30-35*C
• SABS, IMA, Blood agar… others
• Bacteria-like colony – pasty white
• ...

Candida albicans
Identification
• Germ tube formation
• Incubate yeast in serum for 3-4hrs at 35 ˚C
• Extension from yeast...

ChromAgar for the
identification of Candida spp
using chromogenic
substrates.
The unique color formation
can identify 4 ye...

Candida Histopathology
• Pyogenic to granulomatous inflammation
• Usually observe yeast cells (8 um) and
pseudohyphae
• Ca...

Cryptococcus
neoformans
• In nature forms a 2um non-encapsulated yeast cell.
• Associated with bird droppings (esp. pigeon...

Cryptococcus gattii – a close relative of C.
neoformans
• Isolated first from forested areas of the Pacific Northwest
(Bri...

Cryptococcus neoformans
and C. gattii
• Irregularly sized yeast cells (2 – 20 um)
• Virulence factor: Polysaccharide capsu...

Grows on mycologic agars
Mucoid colonies due to
capsular polysaccharide
Both C. Neoformans & C. gattii
brown colonies on B...

Mucicarmin stains the
capsular polysaccharide of
Cryptococcus Pneumocystis jiroveci (yeast like
fungus) could be confused ...

Pneumocystis jiroveci
• Yeast like fungus
• Used to be Pneumocystis carini and considered a
protozoan
• Pneumonia in the i...

Trichosporon
• T. asahii most common – 9 species
• Commonly inhabit the soil. Colonize the skin/
gastrointestinal tract of...

CUTANEOUS AND
SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes
Microsporum
Trichophyton
Epidermophyton

Malassezia furfur
• Most superficial of the dermatomycoses
• Normal flora on human skin
• More common on oily skin or high...

Malassezia furfur
• Lipophilic yeast – oil required for growth
• Media for isolation must contain oil or use an oil overla...

Dermatophytes – Ringworm
infections
• Hair, skin and nail infections
• 3 genera of fungi
• Microsporum species (many)
• Ep...

Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence –
thin fungal hyphae

Microsporum canis
Ringworm acquired from dog or cat
White colony/ yellow on reverse of colony
Tuberculate thick walled mac...

Microsporum gypseum – skin infection from
exposure to contaminated soil, sandy colored
colony, large macroconidia are prod...

Trichophyton
rubrum
White colony with
red diffusable
pigment
Pencil shaped
macroconidia/
many micro-
conidia
Infection fro...

Trichophyton
tonsurans
White colony
No macroconidia
Ballooning
microconidia
Primary cause of
epidemic scalp
ringworm in
ch...

Epidermophyton floccosum
Khaki green colored colony
Beaver tail shaped large macroconidia – no microconidia
– most often c...

Opportunistic Fungal
Pathogens
Infections in the immune suppressed host or
special circumstances
Hyaline molds

Opportunistic Fungi
• Hyaline – no natural color to the hyphae
• Regular septations in the hyphae
• Usually grow on a vari...

Aspergillus species
• Ubiquitous in nature @ 200 species
• Hyaline with regular septation
• Numerous round conidia produce...

Aspergillus Structure

Supportive not Conclusive for Diagnosis
Aspergillus Galactomannan Enzyme immunoassay
• Detects circulating Aspergillus ant...

Aspergillus fumigatus
•Ubiquitous airborne fungus,
inhale conidia
•One of most common in immune
suppressed
•Blue/Green col...

Aspergillus flavus
•Cereals, grains, legumes
•Same disease potential as A.
fumigatus
•Green/yellow colony
•Green/yellow hu...

Aspergillus niger
•Black colony – visible black
fruiting heads
•Contaminate in fruits and
vegetables/ soil
•Invasive disea...

Aspergillus terreus •Infection in the immune
compromised host
•Sandy colored colony
•Isolated from soil
•Colorless conidia...

Aspergillus – fruiting head seldom seen in fixed
tissue usually dichotomous (continuous) branching
septate hyphae which br...

Fusarium species
•Common in nature/plants
•Fuschia colored colony
•Disease related to immune status of
host/ neutropenia
•...

• Scopulariopsis species
• Found in soil and plants
• Infections: Nail, skin, sinusitis,
pulmonary and disseminated in the...

Penicillium species – one of the most common
molds in the environment , cause of bread mold,
uncommon cause of human disea...

MUCORMYCOSIS/
ZYGOMYCOSIs
Hyaline
Broad hyphae without septation
Sporangium with spores

Mucormycosis/Zygomycosis
• Soil, rotten fruit and vegetables
• Rhinocerebral mucormycosis - classic
• Infections in diabet...

Rhizopus Absidia
Mucor
No rhizoids
Rhizoids
Order Mucorales
Distant rhizoid

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

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