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MYCOLOGY
Margie Morgan, PhD D(ABMM)
Just the basics – meant for board
review or brief study of this
fascinating area of microbiology!
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:
• Filamentous with hyphae
• Produce conidia [spores]
• Colonies on solid agar are downy, fluffy, cottony
• Most mold colonies are pigmented which aid in
identification
hyphae
spores
Appropriate Specimens and Transport
conditions for Fungal disease diagnosis
• Fungi are very hardy organisms
• No requirement for special transport media for culture
submission
• Sterile containers used to prevent bacterial contamination
• Numerous 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)
Common Fungal Media
• Mycosel/Mycobiotic agars
• Selective SABS with chloramphenicol and cycloheximide
• Used for culture of dermatophytes – fungi that cause skin, hair and nail
infections
• Brain heart infusion agar
• Primary recovery of all fungal organisms
• Can make it more selective by adding chloramphenicol and
cycloheximide
• Can add blood to agar to nurture systemic fungi
• Fungal cultures incubated for 4 weeks at 30˚C , room air
• Lower temperature than bacterial culture incubation [35˚C]
• If plates are used for fungal cultures the plates must be sealed with
air permeable tape for laboratory safety
What effect does Cycloheximide have
when added to media?
• Prevents rapidly growing molds from overgrowing
dimorphics and dermatophytes
• This is the positive 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
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 yeast species.
Newer methods [Mass spectrometry – MALDI-TOF and
16 sRNA sequencing
Processing of Fungal Cultures - stepwise
Lactophenol cotton blue [LCB] adhesive tape preparation is
method used for mold identification.
LCB mounting medium consists of phenol
lactic acid, glycerol and aniline cotton blue dye.
Clear adhesive (scotch) tape touches a mold colony, picking
up fungal hyphae and pressed into one drop of LCB
on a microscope slide.
If LCB prep is not able to
identify a mold 16sRNA
sequencing can be used
to identify problematic
molds in reference
laboratories.
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 – all specimen types can be Gram stained.
Can only reliably detect yeast by Gram stain.
• KOH preparation – Skin, Hair or Nails examined for
both yeast and/or hyphae
• Calcofluor white stain – all specimen types can be
stained and examined for yeast and/or hyphae
• India ink – Primarily used for CSF for the detection of
Cryptococcus neoformans and C. gattii
Yeast cells stain blue [Gram positive]. Examine for
budding cells to confirm that it is a yeast cell and not
an artifact. Examination on oil immersion lens.
You can also detect pseudohyphae on Gram stain.
Mold can be difficult to identify on a Gram stain.
pseudohyphae
mold
pseudohyphae
Gram Stain
Used to detect yeast and/or hyphae in skin, hair
and nail specimens using 40X light microscope.
KOH dissolves keratin found in cell material
and frees hyphae from the cell
KOH exams can be difficult to interpret!
KOH – potassium hydroxide prep
Yeast, pseudohyphae, and mycelial fungi bind with
the Calcofluor white stain.
Prep is interpreted using a fluorescence microscope.
Sensitivity and specificity is improved over the KOH
preparation.
Calcofluor white stain
One drop of black ink is
placed into one drop of CSF
and examined using a 40X
lens on light microscope
It is a “negative” stain,
staining the background not
the yeast cell
The clearing is the
polysaccharide capsule of
Cryptococcus neoformans or
C. gattii. Specificity is
improved if you look for
budding yeast cells.
India Ink
Methenamine Silver Stain [GMS] –
yeast and hyphae stain grey to black.
Examine the hyphae for presence of septations in the hyphae,
broad or more narrow width and angle of branching.
Examine the size and budding pattern of observed yeast.
We will observe on later slides how these criteria can assist in
identification.
Examination of fungi in fixed tissue
PAS-positive staining red against a green or
blue background
Periodic Acid Schiff [PAS]
Stain-Cryptococcus neoformans polysaccharide capsule
stains pink
Mucicarmine [Mucin] stain
Great for cellularity, but
GMS, or PAS better show
features of the fungi.
Hematoxylin and Eosin Stain
DIMORPHIC FUNGI
Important systemic pathogens with some
unique characteristics
What does Dimorphic mean?
• Two forms exist for one fungus species depending on
temperature and conditions of environment
• Mycelial form - Hyphae and conidia
• free living form found in nature and at laboratory temperature <=30˚C
• Yeast or yeast like form
• parasitic phase found in human tissue or in the lab >= 35˚
Histoplasma capsulatum – mold
from 30˚C culture
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
Histoplasma capsulatum
• World wide distribution / In USA in Ohio, Missouri,
and Mississippi River valleys considered endemic
• Associate with 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
The yeast of Histoplasma capsulatum
prefer to be intracellular and inhabit
Macrophages.
Small 2 – 4 um, regular in size, and oval to round.
Do not have a capsule, this is just a staining artifact.
H & E PAS
Gram Wright’s
Histoplasma will stain with a variety of stains
Histoplasmosis rapid
diagnosis
• Antigen detection in urine
• Quantitative Enzyme immunoassay
• Random urine specimen
• Most useful 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
• Fungal culture incubated at 30˚C
• Very SLOW growing taking 2 – 8 weeks to form colonies
• Colony is white to brown and cottony
• Microscopic appearance – tuberculated macroconidia that are
large and round (8 – 16 µM) plus small microconidia (2 - 4µM)
[see picture]
• Microconidia are the infectious particle growing in nature and
capable of penetrating deep into the lung
• DNA probe must be used to confirm identification so there is no
confusion with look alike fungi to the inexperience
• Sepedonium species looks somewhat like Histoplasma and is
considered a look a like fungus – it is a contaminate
Histoplasma capsulatum
culture at 30˚C is white and
cottony.
Microscopic exam:
Tuberculate [projections]
on the macroconidia.
Microconidia are the infectious
particle. Thin hyaline septate hyphae
Appearance in culture at 30 degrees C
Sepedonium
species
Appearance in culture at
35 degrees C
• Culture at 35˚C is yeast phase
• Grow as small yeast, round to oval, always consistent
in size and shape (2 -4 uM) narrow neck at the budding
juncture
Histoplasma capsulatum in tissue
• Granulomas are usually produced and can be either
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!!
H capsulatum var duboisii yeast cells are 8 –
10 uM in size, which is 2X the size of regular
Histoplasma capsulatum yeast cells.
H. capsulatum var duboisii disease
is found in Central Africa
Causes infection in skin and bone
The 30˚C culture is identical to
H capsulatum.
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 to the skin
and bone
Well demarcated skin lesion is typical
Scraping of 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 taking @ 4
weeks to form a colony
Yeast cell is 8 – 20 um in size and is unique
for it’s Broad Based Budding pattern and the
double contoured wall.
Blastomyces culture at 37 degrees
C
Blastomyces dermatitidis histopathology
Mixed pyogenic and granulomatous inflammation is
observed in tissue with
Broad based budding yeast cells
Coccidioides immitis
• Endemic in SW USA, Mexico,
South America, in areas known
as the Sonoran life zone with a warm climate
and desert sands
Infection is from inhalation of fungal particles
found in the sand
• Coccidioides posadasii is a genetically related
to C. immitis. The two species are located in
different endemic regions, but produce the
same disease process
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 is difficult to cure and has 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 only 2 – 3 days to grow, colony starts waxy
and becomes wooly in around 7 – 10 days
• Under the microscope one looks for foci of septated
hyphae with thick walled barrel shaped arthroconidia
with clear spaces in between. The clear spaces are
dead arthroconidia.
• Arthroconidia infectious particle in nature
• Very infectious to laboratory personnel
Coccidioides• Malbranchea species can look like C. immitis under the
microscope
• Because of look-a-like fungi one needs to confirm
identification of Coccidioides immitis with DNA probe or
similar method to be sure!
Coccidioides Malbranchea
Barrel shaped alternating arthroconidia are produced
in cultures grown at both 30 and 35 C.
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 for this fungus.
• Spherules are at 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 the Cocci spherules - they are usually > 80
uM in size. Also R. seeberi almost always cause 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
done 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 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 cell –
High likelihood it is Paracoccidioides
Granulomatous inflammation with Giant
Cells
Sporotrichosis
• Sporothrix schenckii
• Cutaneous inoculation of fungus from penetrating injury with a
spore or thorn (rose bush)
• Initial skin lesion w/wo ulceration
• Lymph-cutaneous spread – bone – systemic
• Pulmonary and CNS infections are rare but reported
Starts as one ulcerative lesion and then chains
Up the lymphatics – can involve lymph nodes
and bone
Sporothrix
schenckii
•MOLD PHASE
• 30*˚C growth in 3 -5 days
• Turns brown to black over time
• Septate hyphae with conidia in daisy wheel pattern
•YEAST PHASE
• At 37˚C small oval yeast cells,
elongated 2 – 5 µM, described as cigar bodies
Sporothrix schenckii
• Histology –
• Pyogenic – to – granulomatous inflammation
• Hard to find yeast in human tissue
• Asteroid body known as Splendore-Hoeppli phenomenon
can be seen, but not specific to Sporothrix, also seen in:
• Zygomycetes (Mucorales)
• Aspergillus
• Blastomycosis
• Candida
Daisy like spore arrangement
Sporothrix
schenckii
Green colony with red diffusable pigment
Uncommon dimorphic fungus
The only species of Penicillium that is dimorphic
Causes skin lesions in tropics and
Pneumonia in immune suppressed
Penicillium marneffei
Penicillium marneffei yeast like cells in tissue
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 describe Mycetoma:
1. Lesions lead to swollen extremities
2. Draining sinuses
3. Sulfur granules observed in tissue and found
in the weeping drainage
• Fungus grows on organic debris in soil
• Implanted into subcutaneous tissue from trauma
Swollen extremity and draining sinus with
sulfur granules
Sulfur Granule
Mycetoma
Mycetoma
• There are two types of Mycetoma:
1. Actinomycotic mycetoma – caused by higher bacteria
species
2. Eumycotic mycetoma – caused by the black molds
• Actinomycotic Mycetoma
• 98% of cases of Mycetoma
• Nocardia species most common cause
• Sulfur granules formed in tissue and the granules vary in
color and contain a matrix of the filamentous bacteria
Gram stain as filamentous Gram positive bacilli – can be
poorly staining and appear speckled.
Nocardia are positive [red] on the Modified Kinyoun stain.
Modified acid-fast stain
[modified Kinyoun stain]
Gram stain of sputum
containing Nocardia
Nocardia
Edge of granule has thin filamentous bacteria
for both bacteria – Nocardia is modified acid fast
[PAF] positive and is aerobic bacteria. Actinomyces
is PAF negative and grows anaerobically.
Beware! Sulfur granule
caused by Actinomyces
israelii looks identical.
Actinomycotic sulfur granule -
Nocardia
Requires 3 – 5 days to
grow on agar media [Sabs,
5% Sheep’s blood agar
Colony is dry and crumbly
Musty smell
Total of 85 species
Nocardia asteroides is the
most common species
isolated from human
infection
Identification by HPLC
or molecular methods
Nocardia species cause mycetoma, and
can also cause Pulmonary and Brain infections
Eumycotic mycetoma – subcutaneous
infection caused by the black molds
Numerous species of pigmented/black fungi found
naturally in the soil can cause this type of infection
Cause @ 2% of cases of mycetoma
Traumatic implantation injects the mold into the
subcutaneous tissue
Most common species of black mold include:
Cladophialophora (Cladosporium) carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Eumycotic sulfur granule – the granule is
full of a matrix of thick fungal hyphae
Chromomycosis/Chromoblastomycos
is
• Three characteristics describe Chromomycosis
• 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
This infection is caused by traumatic implantation of dark
fungi into subcutaneous tissue
• Variety of infections but nodules/lesions most common
with/without dissemination
• Dark hyphae only observed in tissue
Black molds/Dark molds
also known as Dematiaceous
fungi
Black colored colonies and the
reverse [back of colony] is also
black
Naturally brown hyphae and
spores
One of the major causes of mold
growth due to water damage!
Black Molds – Dematiaceous fungi
• Black colonies
• Brown hyphae and spores
• Numerous species
• Difficult to identify
• All have one of four types of sporulation
• Rhinocladiella-like
• Cladosporium-like
• Phialophora-like
• Acrotheca-like
Rhinocladiella type
sporulation
Phialophora type sporulation
Cladophialophora
[Cladosporium type sporulation]
Acrotheca type sporulation
Exophiala species
Black Molds that can cause
Mycetoma/Chromomycosis/Phaeohyphomycosis
These are difficult to identify but viewing is necessary!
Cladophialophora bantiana
Wangiella dermatitidis
Phialophora verrucosa
Alternaria
Other black molds of
importance:
Bipolaris
australiensis
Very invasive
fungal infection:
Skin, nasal
sinuses, bone
brain
Curvularia lunata
Center cell is the
largest
Opportunistic fungal pathogen
Reported in patients with Bone Marrow
Transplantation and Aplastic anemia
Can cause unusual erythematous skin lesions
Exserohilum rostrum
• Associated with compounded pharmaceutical [steroid]
products contaminated with dust/dirt
• Used for infections into lumbar spine and knee joints for
pain management
• Meningitis
• Spinal abscess
• Synovial infections
Scedosporium apiospermum/
Pseudallescheria boydii
Cat fur-like colony
IMPORTANT YEAST
CAUSING HUMAN
INFECTION
Candida species
Cryptococcus neoformans &
Cryptococcus gattii
Cutaneous and Superficial Mycoses
• Candida species (@ 10 found in humans)
• Opportunistic pathogen involving skin or mucous
membranes from excessive exposure to moisture,
antibiotics, or immune suppression
• Yeast is from endogenous source – found as normal
flora in the GI and GU tracts and skin
• Variety of infections including: Thrush, vaginitis, skin
lesions, nail, diaper rash, to more serious infections like
fungemia and endoarditis.
Candida species
• Candida albicans – most common species causing @
60% of human yeast infections
• Candida glabrata, C. krusei, and C. tropicalis are
becoming more common in infection
• These 3 species are more likelyto be resistant to
Fluconazole
• Candida parapsilosis has emerged as a pathogen of
children and IV lines
Candida species
• Grow in 24 – 48 hours
• SABS, IMA, BAP
• Bacteria-like colony – pasty white
• Budding yeast – oval @ 7-8 um in size
form pseudohyphae (Yeast do not detach and form
elongated hyhae) Can appear like sausage links
• Exception **Candida glabrata is @ 4 µM in size and does
NOT form any pseudohyphae
Candida albicans
Identification
• Germ tube formation
• Incubate small amount of yeast in serum for 3-4hr at 35 ˚C
• Do not incubate >4 hr – this can lead to a false positive
reaction with C. tropicalis
• C. dubliniensis also positive (uncommon yeast isolate)
• Chlamydospore formation
• Growth on cornmeal agar >48 hrs
• Rudimentary structures
C. albicans
chlamydospore
C. glabrata only forms yeast
No pseudohyphae
ChromAgar for the
identification of Candida
Chromogenic substrates
Turn different colors with
4 different yeast species
Yeast with pseudohyphae
Candida Histopathology
• Pyogenic to granulomatous
• Usually observe yeast cells, pseudohyphae and/or hyphae
appearing structures
• Candida glabrata = smaller yeast cells and no pseudohyphae
GMS stain of Candida glabrataCandida species not glabrata
Cryptococcus
neoformans
• In nature forms a 2um non-encapsulated yeast cell. It is
associated with bird droppings (esp. pigeon). C
neoformans is enriched by the nitrogen in the droppings.
• Yeast cells are inhaled – travels through the pulmonary
system with hematogenous spread to brain and meninges
• Has tropism to the meninges
• Infects mostly compromised hosts - AIDS
Cryptococcus neoformans
• Irregular sized (2 – 20uM) yeast cells
• Polysaccharide capsule is virulence factor and it’s presence is used in
diagnostic tests for C. neoformans
• India ink exam of CSF is a negative staining method/capsule not
stained,
• Sensitive test for AIDS patients (90% sensitive)
• Cryptococcal antigen test – capsular polysaccharide is detected
in both CSF and serum,
• Test for diagnosis and can also follow recovery with falling titer /more
sensitive than India ink
• Grows on mycologic agars but is sensitive to cycloheximide –
• Mucoid colonies due to capsule polysaccharide formation
• Urease enzyme + Inositol assimilation +
• Brown colonies produced on bird seed agar
Cryptococcus gattii – a closely related
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
• Mostly Pulmonary disease [Cryptococcoma] but can
develop meningitis like C. neoformans
• Culture and staining identical to C. neoformans except for
L Canavanine glycine bromthymol blue medium –
C
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
C. neoformans/C. gattii forms
brown colonies on Birdseed agar
Mucicarmine stain
Stains the capsular polysaccharide of capsule
Pneumocystis jiroveci could
be confused with C.
neoformans – Careful!
Central nuclear staining
C. neoformans/ C. gattii
CUTANEOUS AND
SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes
Malassezia furfur
• Pityriasis versicolor
• Most superficial of the dermatomycoses
• Found as 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
• Fungemia in neonates caused by skin flora
tunneling in the IV lipid feeding lines
Malassezia furfur
• Lipophilic yeast – oil required for growth
• Media used for culture must contain oil or have oil
overlay
• Small budding yeast 2 – 4 µM with collarette
Spaghetti and meatballs
Size range for Yeast
• Candida glabrata/Histoplasma capsulatum
• 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:
tinea capitis (head), t. pedis (foot)
• Usually a clinical diagnosis not requiring culture
• Can do a KOH prep or Calcofluor white prep to
visualize fungal hyphae
Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence –
thin fungal hyphae
Microsporum canis
Main cause of ringworm from dog and cat
White colony/ yellow on backside of colony
Tuberculate macroconidia [spiny projections]
Few if any microconidia
Microsporum gypseum infection from
exposure to contaminated soil
Trichophyton
rubrum
White colony with
red diffusable
pigment
Pencil shaped
Macroconidia
Many micro-
Conidia
Infection from
fomites
Red diffusible pigment
Trichophyton
tonsurans
Ballooning
Microconidia
Primary cause of
epidemic scalp
ringworm in
children from
infection of Hair
Epidermophyton floccosum
Beaver tail large spores without microconidia
Khaki green colony
Opportunistic Fungal
Pathogens
Infections in the immune suppressed host
or special circumstances
Hyaline molds
Black molds
Opportunistic Fungi - hyaline
• Hyaline – no color to the hyphae
• Regular septations in the hyphae
• Branching – angle can be helpful in identification
• Usually grow in 3 – 5 days at 30˚C
• ??? of species thousands– taxonomy changing
daily
Aspergillus species
• Hyaline with septations
• Numerous round conidia
• In tissue - Branching at 45 degree angle
• Primarily pulmonary infection in immune suppressed
• Invade vessels, cause thrombosis & infarctions
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 – only
Aspergillus species resistant to Amphotericin B
Aspergillus Galactomannan Enzyme immunoassay –
detects circulating Aspergillus antigen in the blood
and/or bronchial lavage fluid
Problems with low sensitivity and specificity
False positive reaction in patients on therapy with
Piperacillin/Tazobactam, infected with Histoplasma
capsulatum
Aspergillus fumigatus
Blue/Green colony
Phialids with spores are
Directed upward
Aspergillus flavus
Green colony
Orange colored spores
that surround the vesecle
Aspergillus niger
Black colony
Black spores surround the vesicle
Aspergillus terreus Sandy colored colony
Aleurioconidia
Resistance to Amphotericin B
Aspergillus
•fruiting head dichotomous
•branching septate hyphae
• branching at 45* angle
Pseudallescheria boydii
can be confused for
Aspergillus, hyphae is a bit
thinner. Culture separates
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 –
Soil and plants
Infections: Nail, skin, sinusitis, pulmonary and disseminated
Paecilomyces species
Isolated from soil and food
Opportunistic pathogen in the
immune suppressed
Penicillium species – most common mold
in the environment, bread mold, uncommon
cause of human disease
MUCORMYCOSIS/
ZYGOMYCOSIS
Hyaline
Broad non-septate hyphae
Mucormycosis/Zygomycosis
• Infections in diabetics, the elevated
glucose enriches fungal growth – classic
infection is rhinocerebral mucormycosis
• Sinus and pulmonary infection in the immune
suppressed host
• Broad, hyaline, aseptate hyphae produced
• Cultures grow in 24 hrs, coarse aerial hyphae
• Can be difficult to culture – tube like hyphae killed
during manipulation and plating
• Should not grind tissue
• Mince tissue and place on agar
Zygomycete – coarse, aerial hyphae after 24
hours growth on SABS agar at 30˚C
Rhizopus Absidia
Distant rhizoids
Mucor
No rhizoids
Rhizoids
90˚ angle branching, aseptate, ribbon like
Invades vessels and can cause
infarcts and thrombi
Zygomycetes (Mucorales)

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Mycology 2016

  • 1. MYCOLOGY Margie Morgan, PhD D(ABMM) Just the basics – meant for board review or brief study of this fascinating area of microbiology! Mycology
  • 2. 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)
  • 3. Starting point Molds are: • Filamentous with hyphae • Produce conidia [spores] • Colonies on solid agar are downy, fluffy, cottony • Most mold colonies are pigmented which aid in identification hyphae spores
  • 4. Appropriate Specimens and Transport conditions for Fungal disease diagnosis • Fungi are very hardy organisms • No requirement for special transport media for culture submission • Sterile containers used to prevent bacterial contamination • Numerous 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
  • 5. Fungal Culture Media • Sabouraud’s glucose agar (SABS)
  • 6. Common Fungal Media • Mycosel/Mycobiotic agars • Selective SABS with chloramphenicol and cycloheximide • Used for culture of dermatophytes – fungi that cause skin, hair and nail infections • Brain heart infusion agar • Primary recovery of all fungal organisms • Can make it more selective by adding chloramphenicol and cycloheximide • Can add blood to agar to nurture systemic fungi • Fungal cultures incubated for 4 weeks at 30˚C , room air • Lower temperature than bacterial culture incubation [35˚C] • If plates are used for fungal cultures the plates must be sealed with air permeable tape for laboratory safety
  • 7. What effect does Cycloheximide have when added to media? • Prevents rapidly growing molds from overgrowing dimorphics and dermatophytes • This is the positive 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
  • 8. Inoculate fungal media 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 yeast species. Newer methods [Mass spectrometry – MALDI-TOF and 16 sRNA sequencing Processing of Fungal Cultures - stepwise
  • 9. Lactophenol cotton blue [LCB] adhesive tape preparation is method used for mold identification. LCB mounting medium consists of phenol lactic acid, glycerol and aniline cotton blue dye. Clear adhesive (scotch) tape touches a mold colony, picking up fungal hyphae and pressed into one drop of LCB on a microscope slide. If LCB prep is not able to identify a mold 16sRNA sequencing can be used to identify problematic molds in reference laboratories. Mold Identification methods
  • 10. 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
  • 11. Direct Exams used to identify fungi directly from patient specimens • Gram stain – all specimen types can be Gram stained. Can only reliably detect yeast by Gram stain. • KOH preparation – Skin, Hair or Nails examined for both yeast and/or hyphae • Calcofluor white stain – all specimen types can be stained and examined for yeast and/or hyphae • India ink – Primarily used for CSF for the detection of Cryptococcus neoformans and C. gattii
  • 12. Yeast cells stain blue [Gram positive]. Examine for budding cells to confirm that it is a yeast cell and not an artifact. Examination on oil immersion lens. You can also detect pseudohyphae on Gram stain. Mold can be difficult to identify on a Gram stain. pseudohyphae mold pseudohyphae Gram Stain
  • 13. Used to detect yeast and/or hyphae in skin, hair and nail specimens using 40X light microscope. KOH dissolves keratin found in cell material and frees hyphae from the cell KOH exams can be difficult to interpret! KOH – potassium hydroxide prep
  • 14. Yeast, pseudohyphae, and mycelial fungi bind with the Calcofluor white stain. Prep is interpreted using a fluorescence microscope. Sensitivity and specificity is improved over the KOH preparation. Calcofluor white stain
  • 15. One drop of black ink is placed into one drop of CSF and examined using a 40X lens on light microscope It is a “negative” stain, staining the background not the yeast cell The clearing is the polysaccharide capsule of Cryptococcus neoformans or C. gattii. Specificity is improved if you look for budding yeast cells. India Ink
  • 16. Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Examine the hyphae for presence of septations in the hyphae, broad or more narrow width and angle of branching. Examine the size and budding pattern of observed yeast. We will observe on later slides how these criteria can assist in identification. Examination of fungi in fixed tissue
  • 17. PAS-positive staining red against a green or blue background Periodic Acid Schiff [PAS]
  • 18. Stain-Cryptococcus neoformans polysaccharide capsule stains pink Mucicarmine [Mucin] stain
  • 19. Great for cellularity, but GMS, or PAS better show features of the fungi. Hematoxylin and Eosin Stain
  • 20. DIMORPHIC FUNGI Important systemic pathogens with some unique characteristics
  • 21. What does Dimorphic mean? • Two forms exist for one fungus species depending on temperature and conditions of environment • Mycelial form - Hyphae and conidia • free living form found in nature and at laboratory temperature <=30˚C • Yeast or yeast like form • parasitic phase found in human tissue or in the lab >= 35˚ Histoplasma capsulatum – mold from 30˚C culture Histoplasma capsulatum – yeast from tissue and 35˚C culture
  • 22. Dimorphic Fungi capable of causing systemic infection – most common • Histoplasma capsulatum • Blastomyces dermatitidis • Coccidioides immitis • Paracoccidioides brasiliensis • Sporothrix schenckii • Penicillium marneffei
  • 23. Histoplasma capsulatum • World wide distribution / In USA in Ohio, Missouri, and Mississippi River valleys considered endemic • Associate with Bat guano (Spelunker = cave explorers) and bird droppings
  • 24. 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
  • 25. The yeast of Histoplasma capsulatum prefer to be intracellular and inhabit Macrophages. Small 2 – 4 um, regular in size, and oval to round. Do not have a capsule, this is just a staining artifact.
  • 26. H & E PAS Gram Wright’s Histoplasma will stain with a variety of stains
  • 27. Histoplasmosis rapid diagnosis • Antigen detection in urine • Quantitative Enzyme immunoassay • Random urine specimen • Most useful 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
  • 28. Histoplasma capsulatum • Fungal culture incubated at 30˚C • Very SLOW growing taking 2 – 8 weeks to form colonies • Colony is white to brown and cottony • Microscopic appearance – tuberculated macroconidia that are large and round (8 – 16 µM) plus small microconidia (2 - 4µM) [see picture] • Microconidia are the infectious particle growing in nature and capable of penetrating deep into the lung • DNA probe must be used to confirm identification so there is no confusion with look alike fungi to the inexperience • Sepedonium species looks somewhat like Histoplasma and is considered a look a like fungus – it is a contaminate
  • 29. Histoplasma capsulatum culture at 30˚C is white and cottony. Microscopic exam: Tuberculate [projections] on the macroconidia. Microconidia are the infectious particle. Thin hyaline septate hyphae Appearance in culture at 30 degrees C Sepedonium species
  • 30. Appearance in culture at 35 degrees C • Culture at 35˚C is yeast phase • Grow as small yeast, round to oval, always consistent in size and shape (2 -4 uM) narrow neck at the budding juncture
  • 31. Histoplasma capsulatum in tissue • Granulomas are usually produced and can be either 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
  • 32. Leishmania species Note small round kinetoplast next to nucleus Toxoplasma Histoplasma capsulatum Beware of look alike organisms in tissue specimens!!
  • 33. H capsulatum var duboisii yeast cells are 8 – 10 uM in size, which is 2X the size of regular Histoplasma capsulatum yeast cells. H. capsulatum var duboisii disease is found in Central Africa Causes infection in skin and bone The 30˚C culture is identical to H capsulatum. Unusual variant of H. capsulatum
  • 34. 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 to the skin and bone Well demarcated skin lesion is typical Scraping of skin lesions are full of yeast cells
  • 35. 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
  • 36. Slow growing yeast colony taking @ 4 weeks to form a colony Yeast cell is 8 – 20 um in size and is unique for it’s Broad Based Budding pattern and the double contoured wall. Blastomyces culture at 37 degrees C
  • 37. Blastomyces dermatitidis histopathology Mixed pyogenic and granulomatous inflammation is observed in tissue with Broad based budding yeast cells
  • 38. Coccidioides immitis • Endemic in SW USA, Mexico, South America, in areas known as the Sonoran life zone with a warm climate and desert sands Infection is from inhalation of fungal particles found in the sand • Coccidioides posadasii is a genetically related to C. immitis. The two species are located in different endemic regions, but produce the same disease process
  • 39. 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 is difficult to cure and has a high fatality rate. • Higher incidence of dissemination occurs in patients with: • defects in cell mediated immunity (HIV), • darker skinned ethnic groups, • pregnancy
  • 40. Coccidioides immitis [posadasii] • Culture at 30˚C • Requires only 2 – 3 days to grow, colony starts waxy and becomes wooly in around 7 – 10 days • Under the microscope one looks for foci of septated hyphae with thick walled barrel shaped arthroconidia with clear spaces in between. The clear spaces are dead arthroconidia. • Arthroconidia infectious particle in nature • Very infectious to laboratory personnel
  • 41. Coccidioides• Malbranchea species can look like C. immitis under the microscope • Because of look-a-like fungi one needs to confirm identification of Coccidioides immitis with DNA probe or similar method to be sure! Coccidioides Malbranchea
  • 42. Barrel shaped alternating arthroconidia are produced in cultures grown at both 30 and 35 C. There is no yeast phase for C. immitis [posadasii] No yeast phase with Coccidioides!
  • 43. 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 for this fungus. • Spherules are at all stages of development- fragmented spherules to well formed with endospores • Granulomatous inflammation with caseation is usually observed
  • 44. Development of Cocci spherules from the inhalation of Arthroconidia from nature
  • 45. Rhinosporidium seeberi (aquatic parasite) forms spherules but much larger than the Cocci spherules - they are usually > 80 uM in size. Also R. seeberi almost always cause oral or nasal mass lesions Oral or nasal mass lesions of Rhinosporidium seeberi Coccidioides spherules Coccidioides is not the only spherule forming organism!
  • 46. 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
  • 47. Paracoccidioides • Cultures at 30*C for mycelial phase are usually not done 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 yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast
  • 48. Tissue Exam of Paracoccidioides brasiliensis If more than 2 buds off mother cell – High likelihood it is Paracoccidioides Granulomatous inflammation with Giant Cells
  • 49. Sporotrichosis • Sporothrix schenckii • Cutaneous inoculation of fungus from penetrating injury with a spore or thorn (rose bush) • Initial skin lesion w/wo ulceration • Lymph-cutaneous spread – bone – systemic • Pulmonary and CNS infections are rare but reported
  • 50. Starts as one ulcerative lesion and then chains Up the lymphatics – can involve lymph nodes and bone
  • 51. Sporothrix schenckii •MOLD PHASE • 30*˚C growth in 3 -5 days • Turns brown to black over time • Septate hyphae with conidia in daisy wheel pattern •YEAST PHASE • At 37˚C small oval yeast cells, elongated 2 – 5 µM, described as cigar bodies
  • 52. Sporothrix schenckii • Histology – • Pyogenic – to – granulomatous inflammation • Hard to find yeast in human tissue • Asteroid body known as Splendore-Hoeppli phenomenon can be seen, but not specific to Sporothrix, also seen in: • Zygomycetes (Mucorales) • Aspergillus • Blastomycosis • Candida
  • 53. Daisy like spore arrangement Sporothrix schenckii
  • 54. Green colony with red diffusable pigment Uncommon dimorphic fungus The only species of Penicillium that is dimorphic Causes skin lesions in tropics and Pneumonia in immune suppressed Penicillium marneffei
  • 55. Penicillium marneffei yeast like cells in tissue
  • 57. 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
  • 58. Mycetoma • First observed in India and known as Madura Foot or Maduromycosis • Found in the hot temperate parts of the world • Three criteria describe Mycetoma: 1. Lesions lead to swollen extremities 2. Draining sinuses 3. Sulfur granules observed in tissue and found in the weeping drainage • Fungus grows on organic debris in soil • Implanted into subcutaneous tissue from trauma
  • 59. Swollen extremity and draining sinus with sulfur granules Sulfur Granule Mycetoma
  • 60. Mycetoma • There are two types of Mycetoma: 1. Actinomycotic mycetoma – caused by higher bacteria species 2. Eumycotic mycetoma – caused by the black molds • Actinomycotic Mycetoma • 98% of cases of Mycetoma • Nocardia species most common cause • Sulfur granules formed in tissue and the granules vary in color and contain a matrix of the filamentous bacteria
  • 61. Gram stain as filamentous Gram positive bacilli – can be poorly staining and appear speckled. Nocardia are positive [red] on the Modified Kinyoun stain. Modified acid-fast stain [modified Kinyoun stain] Gram stain of sputum containing Nocardia Nocardia
  • 62. Edge of granule has thin filamentous bacteria for both bacteria – Nocardia is modified acid fast [PAF] positive and is aerobic bacteria. Actinomyces is PAF negative and grows anaerobically. Beware! Sulfur granule caused by Actinomyces israelii looks identical. Actinomycotic sulfur granule - Nocardia
  • 63. Requires 3 – 5 days to grow on agar media [Sabs, 5% Sheep’s blood agar Colony is dry and crumbly Musty smell Total of 85 species Nocardia asteroides is the most common species isolated from human infection Identification by HPLC or molecular methods Nocardia species cause mycetoma, and can also cause Pulmonary and Brain infections
  • 64. Eumycotic mycetoma – subcutaneous infection caused by the black molds Numerous species of pigmented/black fungi found naturally in the soil can cause this type of infection Cause @ 2% of cases of mycetoma Traumatic implantation injects the mold into the subcutaneous tissue Most common species of black mold include: Cladophialophora (Cladosporium) carrionii Cladophialophora bantiana Phialophora verrucosa Fonsecaea pedrosoi Exophiala species Wangiella species
  • 65. Eumycotic sulfur granule – the granule is full of a matrix of thick fungal hyphae
  • 66. Chromomycosis/Chromoblastomycos is • Three characteristics describe Chromomycosis • 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
  • 67. Chromomycosis/Chromoblastomycosis Wart like/Verrucous lesions In subcutaneous tissue Sclerotic Body/Medlar Body/ Copper Penny is the unique structure found in tissue
  • 68. 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
  • 69. Phaeohyphomycosis This infection is caused by traumatic implantation of dark fungi into subcutaneous tissue • Variety of infections but nodules/lesions most common with/without dissemination • Dark hyphae only observed in tissue
  • 70. Black molds/Dark molds also known as Dematiaceous fungi Black colored colonies and the reverse [back of colony] is also black Naturally brown hyphae and spores One of the major causes of mold growth due to water damage!
  • 71. Black Molds – Dematiaceous fungi • Black colonies • Brown hyphae and spores • Numerous species • Difficult to identify • All have one of four types of sporulation • Rhinocladiella-like • Cladosporium-like • Phialophora-like • Acrotheca-like
  • 76. Exophiala species Black Molds that can cause Mycetoma/Chromomycosis/Phaeohyphomycosis These are difficult to identify but viewing is necessary! Cladophialophora bantiana
  • 80. Curvularia lunata Center cell is the largest Opportunistic fungal pathogen Reported in patients with Bone Marrow Transplantation and Aplastic anemia Can cause unusual erythematous skin lesions
  • 81. Exserohilum rostrum • Associated with compounded pharmaceutical [steroid] products contaminated with dust/dirt • Used for infections into lumbar spine and knee joints for pain management • Meningitis • Spinal abscess • Synovial infections
  • 83. IMPORTANT YEAST CAUSING HUMAN INFECTION Candida species Cryptococcus neoformans & Cryptococcus gattii
  • 84. Cutaneous and Superficial Mycoses • Candida species (@ 10 found in humans) • Opportunistic pathogen involving skin or mucous membranes from excessive exposure to moisture, antibiotics, or immune suppression • Yeast is from endogenous source – found as normal flora in the GI and GU tracts and skin • Variety of infections including: Thrush, vaginitis, skin lesions, nail, diaper rash, to more serious infections like fungemia and endoarditis.
  • 85. Candida species • Candida albicans – most common species causing @ 60% of human yeast infections • Candida glabrata, C. krusei, and C. tropicalis are becoming more common in infection • These 3 species are more likelyto be resistant to Fluconazole • Candida parapsilosis has emerged as a pathogen of children and IV lines
  • 86. Candida species • Grow in 24 – 48 hours • SABS, IMA, BAP • Bacteria-like colony – pasty white • Budding yeast – oval @ 7-8 um in size form pseudohyphae (Yeast do not detach and form elongated hyhae) Can appear like sausage links • Exception **Candida glabrata is @ 4 µM in size and does NOT form any pseudohyphae
  • 87. Candida albicans Identification • Germ tube formation • Incubate small amount of yeast in serum for 3-4hr at 35 ˚C • Do not incubate >4 hr – this can lead to a false positive reaction with C. tropicalis • C. dubliniensis also positive (uncommon yeast isolate) • Chlamydospore formation • Growth on cornmeal agar >48 hrs • Rudimentary structures C. albicans chlamydospore C. glabrata only forms yeast No pseudohyphae
  • 88. ChromAgar for the identification of Candida Chromogenic substrates Turn different colors with 4 different yeast species Yeast with pseudohyphae
  • 89. Candida Histopathology • Pyogenic to granulomatous • Usually observe yeast cells, pseudohyphae and/or hyphae appearing structures • Candida glabrata = smaller yeast cells and no pseudohyphae GMS stain of Candida glabrataCandida species not glabrata
  • 90. Cryptococcus neoformans • In nature forms a 2um non-encapsulated yeast cell. It is associated with bird droppings (esp. pigeon). C neoformans is enriched by the nitrogen in the droppings. • Yeast cells are inhaled – travels through the pulmonary system with hematogenous spread to brain and meninges • Has tropism to the meninges • Infects mostly compromised hosts - AIDS
  • 91. Cryptococcus neoformans • Irregular sized (2 – 20uM) yeast cells • Polysaccharide capsule is virulence factor and it’s presence is used in diagnostic tests for C. neoformans • India ink exam of CSF is a negative staining method/capsule not stained, • Sensitive test for AIDS patients (90% sensitive) • Cryptococcal antigen test – capsular polysaccharide is detected in both CSF and serum, • Test for diagnosis and can also follow recovery with falling titer /more sensitive than India ink • Grows on mycologic agars but is sensitive to cycloheximide – • Mucoid colonies due to capsule polysaccharide formation • Urease enzyme + Inositol assimilation + • Brown colonies produced on bird seed agar
  • 92. Cryptococcus gattii – a closely related 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 • Mostly Pulmonary disease [Cryptococcoma] but can develop meningitis like C. neoformans • Culture and staining identical to C. neoformans except for L Canavanine glycine bromthymol blue medium – C
  • 93. 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
  • 94. C. neoformans/C. gattii forms brown colonies on Birdseed agar
  • 95. Mucicarmine stain Stains the capsular polysaccharide of capsule Pneumocystis jiroveci could be confused with C. neoformans – Careful! Central nuclear staining C. neoformans/ C. gattii
  • 97. Malassezia furfur • Pityriasis versicolor • Most superficial of the dermatomycoses • Found as 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 • Fungemia in neonates caused by skin flora tunneling in the IV lipid feeding lines
  • 98. Malassezia furfur • Lipophilic yeast – oil required for growth • Media used for culture must contain oil or have oil overlay • Small budding yeast 2 – 4 µM with collarette Spaghetti and meatballs
  • 99. Size range for Yeast • Candida glabrata/Histoplasma capsulatum • 2 – 4 um • Candida species • 8 – 10 um plus pseudohyphae • Cryptococcus neoformans/gattii • 2 – 20 um • Blastomyces dermatitidis • 8-15 um
  • 100. 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: tinea capitis (head), t. pedis (foot) • Usually a clinical diagnosis not requiring culture • Can do a KOH prep or Calcofluor white prep to visualize fungal hyphae
  • 101. Positive KOH prep Showing thin septate fungal hyphae Calcofluor white stain with fluorescence – thin fungal hyphae
  • 102. Microsporum canis Main cause of ringworm from dog and cat White colony/ yellow on backside of colony Tuberculate macroconidia [spiny projections] Few if any microconidia
  • 103. Microsporum gypseum infection from exposure to contaminated soil
  • 104. Trichophyton rubrum White colony with red diffusable pigment Pencil shaped Macroconidia Many micro- Conidia Infection from fomites Red diffusible pigment
  • 105. Trichophyton tonsurans Ballooning Microconidia Primary cause of epidemic scalp ringworm in children from infection of Hair
  • 106. Epidermophyton floccosum Beaver tail large spores without microconidia Khaki green colony
  • 107. Opportunistic Fungal Pathogens Infections in the immune suppressed host or special circumstances Hyaline molds Black molds
  • 108. Opportunistic Fungi - hyaline • Hyaline – no color to the hyphae • Regular septations in the hyphae • Branching – angle can be helpful in identification • Usually grow in 3 – 5 days at 30˚C • ??? of species thousands– taxonomy changing daily
  • 109. Aspergillus species • Hyaline with septations • Numerous round conidia • In tissue - Branching at 45 degree angle • Primarily pulmonary infection in immune suppressed • Invade vessels, cause thrombosis & infarctions
  • 111. Aspergillus species Four species most common in human infections: 1. Aspergillus fumigatus 2. Aspergillus flavus 3. Aspergillus niger 4. Aspergillus terreus – unique and important – only Aspergillus species resistant to Amphotericin B Aspergillus Galactomannan Enzyme immunoassay – detects circulating Aspergillus antigen in the blood and/or bronchial lavage fluid Problems with low sensitivity and specificity False positive reaction in patients on therapy with Piperacillin/Tazobactam, infected with Histoplasma capsulatum
  • 112. Aspergillus fumigatus Blue/Green colony Phialids with spores are Directed upward
  • 113. Aspergillus flavus Green colony Orange colored spores that surround the vesecle
  • 114. Aspergillus niger Black colony Black spores surround the vesicle
  • 115. Aspergillus terreus Sandy colored colony Aleurioconidia Resistance to Amphotericin B
  • 116. Aspergillus •fruiting head dichotomous •branching septate hyphae • branching at 45* angle Pseudallescheria boydii can be confused for Aspergillus, hyphae is a bit thinner. Culture separates the two fungi.
  • 117. 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
  • 118. Scopulariopsis species – Soil and plants Infections: Nail, skin, sinusitis, pulmonary and disseminated
  • 119. Paecilomyces species Isolated from soil and food Opportunistic pathogen in the immune suppressed
  • 120. Penicillium species – most common mold in the environment, bread mold, uncommon cause of human disease
  • 122. Mucormycosis/Zygomycosis • Infections in diabetics, the elevated glucose enriches fungal growth – classic infection is rhinocerebral mucormycosis • Sinus and pulmonary infection in the immune suppressed host • Broad, hyaline, aseptate hyphae produced • Cultures grow in 24 hrs, coarse aerial hyphae • Can be difficult to culture – tube like hyphae killed during manipulation and plating • Should not grind tissue • Mince tissue and place on agar
  • 123. Zygomycete – coarse, aerial hyphae after 24 hours growth on SABS agar at 30˚C
  • 125. 90˚ angle branching, aseptate, ribbon like Invades vessels and can cause infarcts and thrombi Zygomycetes (Mucorales)