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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 and appear
much like bacterial colonies
• A few Genera produce pigment (Rhodotorula = red)
• Some genera produce mucoid colonies (Cryptococcus)
Starting point
Molds:
• Produce filaments or hyphae
• Produce conidia [spores]
• Growth on solid media are downy, fluffy, cottony
• Most mold colonies produce pigment, which aids in
identification
hyphae
spores
Specimen collection and Transport
• Fungi are very hardy organisms
• No requirement for special transport media
• Sterile containers necessary 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, CSF
• Blood, bone marrow
Fungal Culture Media
• Sabouraud’s glucose agar (SABS)
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.
• Beware - Cycloheximide can suppress some species of pathogenic fungi
from growing. Inhibited fungi include Trichosporon, Candida tropicalis and
Cryptococcus neoformans/gattii
• Brain heart infusion agar
• Primary recovery of all fungi
• Inhibits bacterial growth by adding chloramphenicol and cycloheximide
• Addition of blood to agar to nurtures for systemic fungi
•Inoculate specimen onto media
•Seal plates with tape to prevent culture from
environmental contamination and protect laboratory
workers from plates opening
•Incubate plates at 30˚C for 4 weeks
•If growth occurs - perform identification methods
Processing of Fungal Cultures
Yeast Identification
• Biochemical reactions identify most but not all yeast
species – manual and automated instrument identification
• Newer methods with improved accuracy of identification:
(1) MALDI-TOF (Matrix Assisted Laser
Desorption/Ionization Time of Flight)
The yeast is superheated and converted into
charged particles (proteins) and based on
mass and charge form identification peaks
characteristic for each yeast species
(2) 16 sRNA sequencing – particularly helpful for
very unusual of difficult to identify yeast species
Lactophenol cotton blue [LCB] adhesive tape preparation is
used for mold identification.
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/conidia and pressed into one drop of LCB
on a microscope slide.
Newer (better methods)
include:
MALDI-TOF
16sRNA sequencing
Mold Identification methods
Safety in the Mycology Laboratory
•All work on molds must be performed in a BSL-2 biosafety
cabinet with HEPA filtration
•Yeast work can be performed on the bench top
Gram stain
•Yeast cells are Gram positive
•Examine for budding cells to confirm that it is a yeast
cell and not an artifact.
•Detect yeast pseudo-hyphae
•Mold can be difficult to fully visualize
pseudohyphae
mold
pseudohyphae
Direct Exams from Specimens
X100 oil immersion
•Detect yeast and hyphae from skin, hair, and nail
specimens
•KOH dissolves the keratin found in cellular material and
frees fungal hyphae and yeast cells – hyphae can be
confused with cell borders
•KOH exams are difficult to interpret!
KOH – potassium hydroxide prep
X40
Light microscopy
• Yeast, pseudo-hyphae, and mycelial fungi 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 onto a microscope slide -
examined using light microscope (40X)
It is a “negative” stain – stains the
background of slide not the yeast cell or
capsule. The clearing around the yeast
is due to the polysaccharide capsule
formed by Cryptococcus neoformans
and C. gattii.
India ink is less sensitive (50%) than
the Cryptococcal Antigen test.
India Ink – specifically for Cryptococcus
detection
Grocott’s Methenamine Silver Stain [GMS]
– yeast and hyphae stain grey to black. Will stain
both living and dead yeast and hyphae
• Observe the width of the hyphae, presence of hyphae
septation and angle of branching
• Observe the size and budding pattern of yeast
• Will explain on later slides how these observations can
assist in identification
Examination of fungi in fixed
tissue
Positive staining hyphae are magenta – will also
stain structures containing
carbohydrate macromolecules (glycogen,
glycoprotein, proteoglycans) Only stains living fungi.
Periodic Acid Schiff [PAS]
Mucicarmine stains the polysaccharide capsule of
Cryptococcus neoformans and C. gatti pink. Will
also stain mucin in fixed tissue.
Mucicarmine [Mucin] stain
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) Mycelial form - Hyphae and conidia (spores)
• Free living form in nature and laboratory temperature <=30˚C
• (2) Yeast or yeast like form
• Parasitic phase found in human tissue or in the lab >= 35˚C
• Body temperature allows mycelial form to morph to yeast form
Histoplasma capsulatum –
Mycelial form incubated at 30˚C
Histoplasma capsulatum –
Yeast form in tissue and 35˚C
Most common Dimorphic Fungi
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis/posadasii
• Paracoccidioides brasiliensis
• Sporothrix schenckii
• Penicillium marneffei (new name:
Talaromyces marneffei)
Histoplasma
capsulatum• Epidemiology: World wide distribution / In USA
endemic in Ohio, Missouri, and Mississippi River
valleys
• Environmental source: Bat guano (Spelunker =
cave explorers) bird droppings, and farmers
Histoplasmosis Disease
• 95% of infections are subclinical
• 5% infections: Pulmonary +/- dissemination, reactivation
disease in the elderly and immune suppressed
• Who gets progressive Histoplasmosis?
• HIV/AIDS
• Organ transplants
• Taking medications such as corticosteroids or TNF-inhibitors
• Infants
• Adults aged 55 and older
• Examination of bone marrow is useful in
diagnosing disseminated infections
• Mucocutaneous lesions are a unique &
common site of dissemination
Histoplasmosis rapid
diagnosis
• Antigen detection in urine
• Quantitative Enzyme immunoassay
• Performed on random urine specimen
• Most sensitive to diagnose disseminated Histoplasma
infection and chronic pulmonary disease
• Antigen is detectable in >=85% of these infections
• Helpful in diagnosis of immune suppressed patients
that do not produce a detectable antibody response
• Antibody tests are available but have been mostly
replaced by screening for antigen in urine
Histoplasma capsulatum
Culture
• Incubated at 30˚C
• Requires 2 – 8 weeks to grow
• Colony - white to brown and cottony
• Microscopic appearance on scotch tape mount
• Tuberculate macroconidia that are large and round (8 – 16 µM)
• Plus small microconidia (2 - 4µM)
• Form growing in nature, microconidia are inhaled / capable of
penetrating deep into the lung
• MALDI-TOF or Molecular testing to confirm ID
• Look alike fungus - Sepedonium species - usually not a pathogen
Histoplasma capsulatum
Culture
• Incubated at 37*C
• Requires 4 – 8 weeks to grow
• Colony – white to beige, creamy
• Microscopic appearance
• 2-4 um in size, oval to round
• Form found in human tissue
• Increase in temperature in human/laboratory induces the
mycelial phase to undergo transformation to the yeast phase
Histoplasma capsulatum in fixed
tissue
• Infection begins by inhalation of
the microconidia into the lung
• Granulomas usually produced –
both caseating or non-caseating
• Can disseminate to organs of the
Reticuloendothelial System (RES) –
with high % of dissemination to the b
bone marrow
Histoplasma capsulatum - Yeast
Intracellular within macrophages
Small 2 – 4 um, regular in size, oval to round.
Appear to be encapsulated due to staining artifact
Stains with a variety of stains.
H & E PAS
Gram Wrights
Leishmania amastigote
Note: kinetoplast (mitochondrial
DNA) next to nucleus
Toxoplasma tachyzoites
oval to crescent in shape
Histoplasma yeast
appear to have capsule
Beware of look-a-likes
The mold phase is identical to H. capsulatum.
Difference is the size of the yeast cell: Note yeast cell of H. duboisii
is 8 – 10 um which is 2X the size of H. capsulatum yeast cell.
H. capsulatum var duboisii differs from
disease H. capsulatum
1) Endemic area Central Africa
2) Primary infection is in skin and bone
Unusual variant of Histoplasma –
variate duboisii
Blastomyces dermatitidis
• Epidemiology
• Ohio and Mississippi River valleys
• No association with animal or activity
• Found in forrests and river banks
• Primarily a pulmonary pathogen with
% of dissemination to the skin and bone occurring
most often in immune suppressed patients
• Well demarcated skin lesions can
be seen in disseminated cases of
Blastomycosis.
Blastomyces dermatitidis
• Culture at 30˚C
• Grows in 2- 3 weeks
• Fluffy white – buff colored mold, prickly
• Microscopic - Pear shaped conidia at the end of
supporting hyphae – looks like a lollipop
• Look alike fungus – Chrysosporium species
• MALDI-TOF or Molecular test to confirm identification
Blastomyces
Chrysosporium
• Culture at 35*C
• Slow growing yeast colony (4 weeks)
• Large yeast cell is 8 – 20 um in size
• Unique Broad Based Budding pattern and the thick/double
contoured wall.
Blastomyces dermatitidis
Broad based bud
Double contoured wall
Blastomyces dermatitidis
histopathology
• Mixed pyogenic and granulomatous inflammation is
observed in tissue
• Broad based budding yeast cells
Coccidioides immitis
(posadasii)
• New species: Coccidioides posadasii is
genetically related to C. immitis. The two
species are found in different regions,
C. immitis (California) and C. posadasii
(outside CA),
• Same disease process
• Endemic in SW USA (San Joaquin Valley),
Mexico, and South America, in areas known
as the Sonoran life zone / warm and desert
sands
• Infection is from inhalation of fungal
particles (arthroconidia) found in the soil
Coccidioidomycosis
• 60% of infections are asymptomatic or
• 30% with limited pulmonary diseases
• The remaining 5-10% result in chronic disease,
progressive pulmonary or disseminated infections
• Tropism to the Central Nervous System (CNS)
• Dissemination to CNS carries a high fatality rate.
• Risk factors for severe or disseminated
coccidioidomycosis include:
• African-American race or Filipino ethnicity, HIV/AIDS, use of
immunosuppressive medications, organ transplant, diabetes
mellitus, or pregnancy
Serologic diagnosis of
Coccidiodomycosis
• Enzyme immunoassay (EIA): A very sensitive and
commonly used method for diagnosing
coccidioidomycosis, detects IgG and IgM antibodies
• Immunodiffusion (ID): detects IgM antibodies; positive
early in the course of infection
• Complement Fixation (CF): detects IgG antibodies
and allows for assessment of disease severity.
Coccidioides Culture
• Culture at 30˚C
• Growth in 2-3 days, colony starts waxy and becomes
wooly in around 7–10 days
• Microscopic (40X) one observes areas with septate
hyphae and thick walled alternating barrel shaped
arthroconidia
• Barreled arthroconidia breakoff / become the infectious
particle in nature
• Mature culture is infectious to laboratory personnel
40X 2-3 days 7-10 days
Look-a-likes
• Malbranchea species mimics C. immitis/posadasii under
the microscope /must confirm ID by MALDI or molecular
Coccidioides Malbranchea
Coccidioides Histopathology
• No yeast cell produced, instead: Thick walled spherules
(10 – 80 uM) with endospores are the growth structure in
tissue.
• Spherules can be in all stages of development-
fragmented spherules to well formed with endospores
• Granulomatous inflammation with caseation is usually
observed
• Rhinosporidium seeberi (aquatic parasite) forms
spherules but much larger than those of Cocci.
• Rhinosporidium spherules are usually > 80 uM in size
• R. seeberi cause oral or nasal mass lesions
Oral or nasal mass lesions
Careful!! Coccidioides is not the
only spherule forming organism!
Paracoccidioides brasiliensis
• South American Blastomycosis – Brazil
80% of cases reported from Brazil
• Most prevalent systemic fungal infection in Latin
America
• Infection acquired from inhaling 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
3. Extrapulmonary lesions on the face and oral mucosa
Paracoccidioides
• Cultures at 30*C usually not done, slow growth and
nonspecific sporulation
• Culture at 37˚C
• Slow growing yeast – 3 weeks
• Large (10 – 30um), thick walled,
2 or more tear drop daughter buds (2–10 um)
• Unique multiply budding yeast cell known as the
Mariner’s wheel or Pilot’s wheel yeast
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 sphagnum moss
• Known as “Rose gardener’s disease”
• Usually begins as skin lesion w/wo ulceration and
leads to a subcutaneous infection
• Lesion can progress/ lymphocutaneous spread,
possible dissemination to bone and other organs
• Pulmonary and CNS infections occur but rare
Sporothrix
schenckii
• 30˚C culture grows in 3 -5 days
as beige mold that becomes brown
to black over time
• Microscopic: septate hyphae with
conidia in daisy wheel pattern
• 37˚C culture grows in 7 days, small oval yeast,
elongated 2 – 5 µm, described as cigar bodies
Sporothrix schenckii Histology
• Pyogenic to granulomatous inflammation
• Hard to find yeast cells in human tissue
• If seen, elongated yeast, cigar shape
• More commonly seen is an Asteroid body known as
Splendore-Hoeppli phenomenon,
• Not specific to Sporothrix, also seen in infections with:
• Zygomycetes (Mucorales)
• Aspergillus
• Blastomyces
• Candida spp
•Disease: Skin lesions in tropics and SE Asia
•Pneumonia or systemic infection in HIV/AIDS
•Only dimorphic species of Penicillium, hence
change in Genus to Talaromyces
•Green colony with red diffusible pigment, grows in 2 – 3
days at 30 C° In tissue – yeast like
cells
Penicillium (Talaromyces)
marneffei
SUBCUTANEOUS FUNGAL
INFECTIONS
Mycetoma
Chromoblastomycoses
Phaeohyphomycosis
Mycetoma
• Found in the hot temperate parts of the
world – fungus grows on organic debris
• Three criteria:
-Swollen extremity from lesion progression
-Draining sinuses
-Sulfur granules observed in tissue and
drainage from sinus tract
• Fungus grows on organic debris in soil
• Implanted into subcutaneous tissue from
trauma
Two types of Mycetoma:
1. Actinomycotic – caused by higher bacteria
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 depending on the Nocardia species causing
infection
• Granules contain a matrix of the filamentous bacteria
Note: Edge of granule has thin filamentous bacteria:
How can you tell if it is Nocardia?
Nocardia is modified (partial) 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 (Partial) Kinyoun
acid-fast stain
Gram stain
Nocardia
Grows in 3 – 5 days on SABs, 5%
Sheep’s blood agar …
Colony is dry/crumbly - Musty smell
Total of 85 species:
Nocardia asteroides most common
Identification by MALDI -TOF
or 16s rRNA gene sequencing
Therapy: Trimethoprim/Sulfa
Nocardia species – besides Mycetoma,
Nocardia can also lead to Pulmonary and Brain
infection
Eumycotic mycetoma
Caused by pigmented/black fungi
-Numerous species found in soil and debris
-2% of cases of mycetoma
-Traumatic implantation of the fungus into the
subcutaneous tissue
Notice the thick
hyphae on the
edge of the granule
Thermoactinomycetes
• Nocardia related Gram positive filamentous rods
• Thermoactinomyces and Saccaropolyspora
• 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
Chromomycosis/Chromoblastomycosi
s
• 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
Sclerotic Body/Medlar Body/
Copper Penny is the unique
structure found in tissue –
naturally
brown
Prototheca wickerhamii – 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 nodular 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 due to melanin production
-One of the most common molds to grow
due to water damage!
Most common black molds include:
Cladophialophora carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Black Molds
• 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
Also: sinusitis, pulmonary, brain, and disseminated infections
These fungi are difficult to identify using only
morphology – require molecular methods or MALDI-TOF
Cladophialophora bantiana -
Brain infections
Wangiella dermatitidis
Phialophora verrucosa
Alternaria species– Opportunistic fungus -
sinusitis in normal host, disseminated infection in
immune suppressed.
Other more common black molds of importance:
Bipolaris
australiensis
Disseminated fungal
infection in both normal
and immune suppressed
hosts
Also, Skin, nasal
sinus, bone and
Brain infections
Curvularia lunata
Center cell is the largest
Opportunistic
Bone Marrow Transplant and Aplastic anemia
patients
Can cause unusual erythematous skin lesions
Exserohilum rostrum
• Outbreak associated with compounded pharmaceutical
[steroid] products contaminated with dust/dirt during
manufacturing
• Injected into lumbar spine and knee joints for pain
management and led to infections:
• Meningitis
• Spinal abscess
• Synovial infections
Scedosporium apiospermum/Pseudallescheria
(Scedosporium) boydii
Cat fur-like gray colony / growth in 3 – 5 days
Lollipop like spore production
Opportunistic pulmonary pathogen
Can be difficult to distinguish from Aspergillus in fixed tissue
stains / Culture is suggested
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 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.
Important Candida species
• Candida albicans – most common species causing @
60% of Candida yeast infections
• Candida glabrata, C. krusei*, and C. tropicalis are
causing higher % of infections than in the past
• These 3 species are more likely to be resistant to Fluconazole,
one of the most commonly used anti-fungals
• Candida auris – invasive infections, intrinsically resistant
to fluconazole additional anti-fungals, susceptible to
echinocandins, difficult to identify
• Candida parapsilosis pathogen of children and common
in IV line infections (forms biofilms on plastics)
Susceptible to fluconazole.
• * Taxonomy update: C. krusei now Pichia kudriavzevi
Candida species
• Grow in 24 – 48 hours at 30-35*C
• SABS, IMA, Blood agar… others
• Bacteria-like colony – pasty white
• Oval shaped yeast @ 7-8 um in size**
• Form pseudohyphae not true hyphae (Yeast do not
detach and form elongated hyphae)
• Identify using biochemicals, 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 yeast in serum for 3-4hrs at 35 ˚C
• Extension from yeast cell “tube” positive for C. albicans
• If incubate >4 hrs – C. tropicalis false positive
• C. dubliniensis is also positive for germ tubes
• Chlamydospore formation
• Growth on cornmeal agar at 48 hrs
• Rudimentary structures unique to
C. albicans - Chlamydospore
chlamydospore
ChromAgar for the
identification of Candida spp
using chromogenic
substrates.
The unique color formation
can identify 4 yeast species.
Susceptibility testing of yeast is much like bacteria, using
broth dilution and Etest methods.
Candida Histopathology
• Pyogenic to granulomatous inflammation
• Usually observe yeast cells (8 um) and
pseudohyphae
• 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)
• Infects mostly compromised hosts – one of the major
AIDS defining infections
Cryptococcus gattii – a close relative of C.
neoformans
• Isolated first from forested areas of the Pacific Northwest
(British Columbia, Washington, Oregon and California)
found in soil debris and tree species
• Infection of both normal and immune suppressed hosts
• Primarily a pulmonary disease [Cryptococcoma] but can
develop meningitis
• Culture, biochemical & staining identical to C. neoformans
• Defining reactions –
• L Canavanine glycine bromthymol blue medium –
C. gatti = blue C. neoformans = colorless
MALDI-TOF, ssequencing will also ID both species
Cryptococcus neoformans
and C. gattii
• Irregularly sized yeast cells (2 – 20 um)
• Virulence factor: Polysaccharide capsule
• Polysaccharide capsule is target in diagnostic testing:
• India ink exam- CSF placed in one drop of black ink.
It is a negative staining method – background is
stained but not the polysaccharide capsule
• Cryptococcal antigen test – Titer of capsular
polysaccharide detected in either CSF or serum
• More sensitive than India ink
• Test for diagnosis or can follow recovery with
decreasing titer
Grows on mycologic agars
Mucoid colonies due to
capsular polysaccharide
Both C. Neoformans & C. gattii
brown colonies on Birdseed agar
Gram stain
Blue blobs
Positive urea rxn
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
Pneumocystis jiroveci
• Yeast like fungus
• Used to be Pneumocystis carini and considered a
protozoan
• Pneumonia in the immunocompromised host (PCP)
particularly HIV/AIDS
• Diagnosis: Examination of Bronchial lavage, lung biopsy,
induced sputum by Direct Fluorescent antibody (DFA) and
PCR.
DFA
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
Microsporum
Trichophyton
Epidermophyton
Malassezia furfur
• Most superficial of the dermatomycoses
• Normal flora on human 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 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 use 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
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 or cat
White colony/ yellow on reverse 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
Infection from
fomites
Red diffusible pigment
Trichophyton
tonsurans
White colony
No macroconidia
Ballooning
microconidia
Primary cause of
epidemic scalp
ringworm in
children
Epidermophyton floccosum
Khaki green colored colony
Beaver tail shaped large macroconidia – no microconidia
– most often cause infectionsin skin and nails.
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 variety of
agar media in 3-5 days at 35 or 30˚C
• ID = growth rate, color/texture of colony and
microscopic appearance
• Branching of hyphae in stains of fixed
tissue – angle of branching can be
helpful in identification
• ??? of species in the thousands
Aspergillus species
• Ubiquitous in nature @ 200 species
• Hyaline with regular septation
• Numerous round conidia produced
• Pulmonary infection / disseminate in immune suppressed
• Neutropenia
• Branches at 45 degree angle in fixed tissue
• Invade vessels, cause thrombosis & infarctions
Septation
Aspergillus Structure
Supportive not Conclusive for Diagnosis
Aspergillus Galactomannan Enzyme immunoassay
• Detects circulating Aspergillus antigen in the blood and/or
bronchial lavage, most helpful in disseminated infections
• +/- sensitivity and specificity (PPV 68%/ NPV 96%)
• False positive reactions (10%) with Piperacillin/Tazobactam
therapy, infection with H. capsulatum, and rice and pasta
ingestion
(1,3)–Beta-D-glucan assay
• Detected in serum or BAL from the following pathogens: Candida
spp., Acremonium, Aspergillus spp., Coccidioides spp, Fusarium
spp., Histoplasma capsulatum, Trichosporon spp, Sporothrix
schenckii, Saccharomyces cerevisiae, and Pneumocystis jiroveci.
• No reaction for Cryptococcus and Zygomycetes
• High values more meaningful, False positive reaction from the
environment
Aspergillus fumigatus
•Ubiquitous airborne fungus,
inhale conidia
•One of most common in immune
suppressed
•Blue/Green colony
•Phialid with colorless spores are
directed upward
Aspergillus flavus
•Cereals, grains, legumes
•Same disease potential as A.
fumigatus
•Green/yellow colony
•Green/yellow hue to spores on
phialids that surround the
vesicle
Aspergillus niger
•Black colony – visible black
fruiting heads
•Contaminate in fruits and
vegetables/ soil
•Invasive disease uncommon
•Black spores supported by
phialids that surround the vesicle
Aspergillus terreus •Infection in the immune
compromised host
•Sandy colored colony
•Isolated from soil
•Colorless conidia supported by
phialids headed upward
•Aleurioconidia produced
•Intrinsic resistance to
Amphotericin B
Aspergillus – fruiting head seldom seen in fixed
tissue usually 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
•Fuschia colored colony
•Disease related to immune status of
host/ neutropenia
•Infections reported:
•Disseminated in bone marrow
transplants
•Corneal infections in contact lens
wearers
•Random hyphae in fixed tissue/
difficult to identify
Banana boat shaped spores
• Scopulariopsis species
• Found in soil and plants
• Infections: Nail, skin, sinusitis,
pulmonary and disseminated in the
immune suppressed host
• Very resistant to antifungal agents
Penicillium species – one of the most common
molds in the environment , cause of bread mold,
uncommon cause of human disease , can appear as a
culture contaminate
MUCORMYCOSIS/
ZYGOMYCOSIs
Hyaline
Broad hyphae without septation
Sporangium with spores
Mucormycosis/Zygomycosis
• Soil, rotten fruit and vegetables
• Rhinocerebral mucormycosis - classic
• Infections in diabetics, the elevated glucose
enriches fungal growth
• Starts in nasal sinus / spreads to orbit of eye –
then brain
• Sinus and pulmonary infection in the immune
suppressed host
• Broad, hyaline, aseptate hyphae produced
• Culture grows within 24 hrs, producing coarse aerial
hyphae
• Mince infected tissue and place on agar
• Do not grind infected tissue – destroys hyphae
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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Mycology 2018 Update

  • 1. MYCOLOGY Mycology Update 2018 Margie Morgan, PhD D(ABMM) Yeast Zygomycota Conidial Molds Dematiacious Hyaline Yeast
  • 2. Starting point Yeast are: • Unicellular / budding to produce daughter cells • Growth on solid media usually white to beige and appear much like bacterial colonies • A few Genera produce pigment (Rhodotorula = red) • Some genera produce mucoid colonies (Cryptococcus)
  • 3. Starting point Molds: • Produce filaments or hyphae • Produce conidia [spores] • Growth on solid media are downy, fluffy, cottony • Most mold colonies produce pigment, which aids in identification hyphae spores
  • 4. Specimen collection and Transport • Fungi are very hardy organisms • No requirement for special transport media • Sterile containers necessary 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, CSF • Blood, bone marrow
  • 5. Fungal Culture Media • Sabouraud’s glucose agar (SABS)
  • 6. 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. • Beware - Cycloheximide can suppress some species of pathogenic fungi from growing. Inhibited fungi include Trichosporon, Candida tropicalis and Cryptococcus neoformans/gattii • Brain heart infusion agar • Primary recovery of all fungi • Inhibits bacterial growth by adding chloramphenicol and cycloheximide • Addition of blood to agar to nurtures for systemic fungi
  • 7. •Inoculate specimen onto media •Seal plates with tape to prevent culture from environmental contamination and protect laboratory workers from plates opening •Incubate plates at 30˚C for 4 weeks •If growth occurs - perform identification methods Processing of Fungal Cultures
  • 8. Yeast Identification • Biochemical reactions identify most but not all yeast species – manual and automated instrument identification • Newer methods with improved accuracy of identification: (1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight) The yeast is superheated and converted into charged particles (proteins) and based on mass and charge form identification peaks characteristic for each yeast species (2) 16 sRNA sequencing – particularly helpful for very unusual of difficult to identify yeast species
  • 9. Lactophenol cotton blue [LCB] adhesive tape preparation is used for mold identification. 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/conidia and pressed into one drop of LCB on a microscope slide. Newer (better methods) include: MALDI-TOF 16sRNA sequencing Mold Identification methods
  • 10. Safety in the Mycology Laboratory •All work on molds must be performed in a BSL-2 biosafety cabinet with HEPA filtration •Yeast work can be performed on the bench top
  • 11. Gram stain •Yeast cells are Gram positive •Examine for budding cells to confirm that it is a yeast cell and not an artifact. •Detect yeast pseudo-hyphae •Mold can be difficult to fully visualize pseudohyphae mold pseudohyphae Direct Exams from Specimens X100 oil immersion
  • 12. •Detect yeast and hyphae from skin, hair, and nail specimens •KOH dissolves the keratin found in cellular material and frees fungal hyphae and yeast cells – hyphae can be confused with cell borders •KOH exams are difficult to interpret! KOH – potassium hydroxide prep X40 Light microscopy
  • 13. • Yeast, pseudo-hyphae, and mycelial fungi bind with the Calcofluor white stain and fluoresce • Read using a fluorescence microscope (40X) • More sensitive and specific than KOH preparation. Calcofluor white stain
  • 14. One drop of black ink is placed into one drop of CSF onto a microscope slide - examined using light microscope (40X) It is a “negative” stain – stains the background of slide not the yeast cell or capsule. The clearing around the yeast is due to the polysaccharide capsule formed by Cryptococcus neoformans and C. gattii. India ink is less sensitive (50%) than the Cryptococcal Antigen test. India Ink – specifically for Cryptococcus detection
  • 15. Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Will stain both living and dead yeast and hyphae • Observe the width of the hyphae, presence of hyphae septation and angle of branching • Observe the size and budding pattern of yeast • Will explain on later slides how these observations can assist in identification Examination of fungi in fixed tissue
  • 16. Positive staining hyphae are magenta – will also stain structures containing carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) Only stains living fungi. Periodic Acid Schiff [PAS]
  • 17. Mucicarmine stains the polysaccharide capsule of Cryptococcus neoformans and C. gatti pink. Will also stain mucin in fixed tissue. Mucicarmine [Mucin] stain
  • 18. Great for description of cellularity, not a specific stain for fungi. Hematoxylin and Eosin Stain
  • 19. DIMORPHIC FUNGI Important systemic pathogens with some unique characteristics
  • 20. Definition of Dimorphic • Depending on temperature and conditions of environment one fungi demonstrates two forms: • (1) Mycelial form - Hyphae and conidia (spores) • Free living form in nature and laboratory temperature <=30˚C • (2) Yeast or yeast like form • Parasitic phase found in human tissue or in the lab >= 35˚C • Body temperature allows mycelial form to morph to yeast form Histoplasma capsulatum – Mycelial form incubated at 30˚C Histoplasma capsulatum – Yeast form in tissue and 35˚C
  • 21. Most common Dimorphic Fungi • Histoplasma capsulatum • Blastomyces dermatitidis • Coccidioides immitis/posadasii • Paracoccidioides brasiliensis • Sporothrix schenckii • Penicillium marneffei (new name: Talaromyces marneffei)
  • 22. Histoplasma capsulatum• Epidemiology: World wide distribution / In USA endemic in Ohio, Missouri, and Mississippi River valleys • Environmental source: Bat guano (Spelunker = cave explorers) bird droppings, and farmers
  • 23. Histoplasmosis Disease • 95% of infections are subclinical • 5% infections: Pulmonary +/- dissemination, reactivation disease in the elderly and immune suppressed • Who gets progressive Histoplasmosis? • HIV/AIDS • Organ transplants • Taking medications such as corticosteroids or TNF-inhibitors • Infants • Adults aged 55 and older • Examination of bone marrow is useful in diagnosing disseminated infections • Mucocutaneous lesions are a unique & common site of dissemination
  • 24. Histoplasmosis rapid diagnosis • Antigen detection in urine • Quantitative Enzyme immunoassay • Performed on random urine specimen • Most sensitive to diagnose disseminated Histoplasma infection and chronic pulmonary disease • Antigen is detectable in >=85% of these infections • Helpful in diagnosis of immune suppressed patients that do not produce a detectable antibody response • Antibody tests are available but have been mostly replaced by screening for antigen in urine
  • 25. Histoplasma capsulatum Culture • Incubated at 30˚C • Requires 2 – 8 weeks to grow • Colony - white to brown and cottony • Microscopic appearance on scotch tape mount • Tuberculate macroconidia that are large and round (8 – 16 µM) • Plus small microconidia (2 - 4µM) • Form growing in nature, microconidia are inhaled / capable of penetrating deep into the lung • MALDI-TOF or Molecular testing to confirm ID • Look alike fungus - Sepedonium species - usually not a pathogen
  • 26. Histoplasma capsulatum Culture • Incubated at 37*C • Requires 4 – 8 weeks to grow • Colony – white to beige, creamy • Microscopic appearance • 2-4 um in size, oval to round • Form found in human tissue • Increase in temperature in human/laboratory induces the mycelial phase to undergo transformation to the yeast phase
  • 27. Histoplasma capsulatum in fixed tissue • Infection begins by inhalation of the microconidia into the lung • Granulomas usually produced – both caseating or non-caseating • Can disseminate to organs of the Reticuloendothelial System (RES) – with high % of dissemination to the b bone marrow
  • 28. Histoplasma capsulatum - Yeast Intracellular within macrophages Small 2 – 4 um, regular in size, oval to round. Appear to be encapsulated due to staining artifact Stains with a variety of stains. H & E PAS Gram Wrights
  • 29. Leishmania amastigote Note: kinetoplast (mitochondrial DNA) next to nucleus Toxoplasma tachyzoites oval to crescent in shape Histoplasma yeast appear to have capsule Beware of look-a-likes
  • 30. The mold phase is identical to H. capsulatum. Difference is the size of the yeast cell: Note yeast cell of H. duboisii is 8 – 10 um which is 2X the size of H. capsulatum yeast cell. H. capsulatum var duboisii differs from disease H. capsulatum 1) Endemic area Central Africa 2) Primary infection is in skin and bone Unusual variant of Histoplasma – variate duboisii
  • 31. Blastomyces dermatitidis • Epidemiology • Ohio and Mississippi River valleys • No association with animal or activity • Found in forrests and river banks • Primarily a pulmonary pathogen with % of dissemination to the skin and bone occurring most often in immune suppressed patients • Well demarcated skin lesions can be seen in disseminated cases of Blastomycosis.
  • 32. Blastomyces dermatitidis • Culture at 30˚C • Grows in 2- 3 weeks • Fluffy white – buff colored mold, prickly • Microscopic - Pear shaped conidia at the end of supporting hyphae – looks like a lollipop • Look alike fungus – Chrysosporium species • MALDI-TOF or Molecular test to confirm identification Blastomyces Chrysosporium
  • 33. • Culture at 35*C • Slow growing yeast colony (4 weeks) • Large yeast cell is 8 – 20 um in size • Unique Broad Based Budding pattern and the thick/double contoured wall. Blastomyces dermatitidis Broad based bud Double contoured wall
  • 34. Blastomyces dermatitidis histopathology • Mixed pyogenic and granulomatous inflammation is observed in tissue • Broad based budding yeast cells
  • 35. Coccidioides immitis (posadasii) • New species: Coccidioides posadasii is genetically related to C. immitis. The two species are found in different regions, C. immitis (California) and C. posadasii (outside CA), • Same disease process • Endemic in SW USA (San Joaquin Valley), Mexico, and South America, in areas known as the Sonoran life zone / warm and desert sands • Infection is from inhalation of fungal particles (arthroconidia) found in the soil
  • 36. Coccidioidomycosis • 60% of infections are asymptomatic or • 30% with limited pulmonary diseases • The remaining 5-10% result in chronic disease, progressive pulmonary or disseminated infections • Tropism to the Central Nervous System (CNS) • Dissemination to CNS carries a high fatality rate. • Risk factors for severe or disseminated coccidioidomycosis include: • African-American race or Filipino ethnicity, HIV/AIDS, use of immunosuppressive medications, organ transplant, diabetes mellitus, or pregnancy
  • 37. Serologic diagnosis of Coccidiodomycosis • Enzyme immunoassay (EIA): A very sensitive and commonly used method for diagnosing coccidioidomycosis, detects IgG and IgM antibodies • Immunodiffusion (ID): detects IgM antibodies; positive early in the course of infection • Complement Fixation (CF): detects IgG antibodies and allows for assessment of disease severity.
  • 38. Coccidioides Culture • Culture at 30˚C • Growth in 2-3 days, colony starts waxy and becomes wooly in around 7–10 days • Microscopic (40X) one observes areas with septate hyphae and thick walled alternating barrel shaped arthroconidia • Barreled arthroconidia breakoff / become the infectious particle in nature • Mature culture is infectious to laboratory personnel 40X 2-3 days 7-10 days
  • 39. Look-a-likes • Malbranchea species mimics C. immitis/posadasii under the microscope /must confirm ID by MALDI or molecular Coccidioides Malbranchea
  • 40. Coccidioides Histopathology • No yeast cell produced, instead: Thick walled spherules (10 – 80 uM) with endospores are the growth structure in tissue. • Spherules can be in all stages of development- fragmented spherules to well formed with endospores • Granulomatous inflammation with caseation is usually observed
  • 41. • Rhinosporidium seeberi (aquatic parasite) forms spherules but much larger than those of Cocci. • Rhinosporidium spherules are usually > 80 uM in size • R. seeberi cause oral or nasal mass lesions Oral or nasal mass lesions Careful!! Coccidioides is not the only spherule forming organism!
  • 42. Paracoccidioides brasiliensis • South American Blastomycosis – Brazil 80% of cases reported from Brazil • Most prevalent systemic fungal infection in Latin America • Infection acquired from inhaling 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 3. Extrapulmonary lesions on the face and oral mucosa
  • 43. Paracoccidioides • Cultures at 30*C usually not done, slow growth and nonspecific sporulation • Culture at 37˚C • Slow growing yeast – 3 weeks • Large (10 – 30um), thick walled, 2 or more tear drop daughter buds (2–10 um) • Unique multiply budding yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast
  • 44. Tissue Exam of Paracoccidioides brasiliensis • Granulomatous inflammation with Giant Cells formed in tissue GMS GMS
  • 45. Sporothrix schenckii • Sporotrichosis • Cutaneous inoculation from penetrating injury, cut or scrape from thorns or sphagnum moss • Known as “Rose gardener’s disease” • Usually begins as skin lesion w/wo ulceration and leads to a subcutaneous infection • Lesion can progress/ lymphocutaneous spread, possible dissemination to bone and other organs • Pulmonary and CNS infections occur but rare
  • 46. Sporothrix schenckii • 30˚C culture grows in 3 -5 days as beige mold that becomes brown to black over time • Microscopic: septate hyphae with conidia in daisy wheel pattern • 37˚C culture grows in 7 days, small oval yeast, elongated 2 – 5 µm, described as cigar bodies
  • 47. Sporothrix schenckii Histology • Pyogenic to granulomatous inflammation • Hard to find yeast cells in human tissue • If seen, elongated yeast, cigar shape • More commonly seen is an Asteroid body known as Splendore-Hoeppli phenomenon, • Not specific to Sporothrix, also seen in infections with: • Zygomycetes (Mucorales) • Aspergillus • Blastomyces • Candida spp
  • 48. •Disease: Skin lesions in tropics and SE Asia •Pneumonia or systemic infection in HIV/AIDS •Only dimorphic species of Penicillium, hence change in Genus to Talaromyces •Green colony with red diffusible pigment, grows in 2 – 3 days at 30 C° In tissue – yeast like cells Penicillium (Talaromyces) marneffei
  • 50. Mycetoma • Found in the hot temperate parts of the world – fungus grows on organic debris • Three criteria: -Swollen extremity from lesion progression -Draining sinuses -Sulfur granules observed in tissue and drainage from sinus tract • Fungus grows on organic debris in soil • Implanted into subcutaneous tissue from trauma Two types of Mycetoma: 1. Actinomycotic – caused by higher bacteria 2. Eumycotic – caused by black molds
  • 51. Actinomycotic Mycetoma • 98% of cases • Nocardia species most common agent • Sulfur granules formed in tissue. The granules vary in color depending on the Nocardia species causing infection • Granules contain a matrix of the filamentous bacteria
  • 52. Note: Edge of granule has thin filamentous bacteria: How can you tell if it is Nocardia? Nocardia is modified (partial) 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
  • 53. Gram stain = filamentous Gram positive bacilli can be poorly staining and appear speckled. Positive [red] on the Modified Kinyoun acid fast stain. Modified (Partial) Kinyoun acid-fast stain Gram stain Nocardia
  • 54. Grows in 3 – 5 days on SABs, 5% Sheep’s blood agar … Colony is dry/crumbly - Musty smell Total of 85 species: Nocardia asteroides most common Identification by MALDI -TOF or 16s rRNA gene sequencing Therapy: Trimethoprim/Sulfa Nocardia species – besides Mycetoma, Nocardia can also lead to Pulmonary and Brain infection
  • 55. Eumycotic mycetoma Caused by pigmented/black fungi -Numerous species found in soil and debris -2% of cases of mycetoma -Traumatic implantation of the fungus into the subcutaneous tissue Notice the thick hyphae on the edge of the granule
  • 56. Thermoactinomycetes • Nocardia related Gram positive filamentous rods • Thermoactinomyces and Saccaropolyspora • 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
  • 57. Chromomycosis/Chromoblastomycosi s • 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 Sclerotic Body/Medlar Body/ Copper Penny is the unique structure found in tissue – naturally brown
  • 58. Prototheca wickerhamii – 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
  • 59. Phaeohyphomycosis Traumatic implantation of dark fungi into subcutaneous tissue • Variety of infections but nodular lesions most common with/without dissemination • Dark hyphae observed in tissue
  • 60. 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 due to melanin production -One of the most common molds to grow due to water damage! Most common black molds include: Cladophialophora carrionii Cladophialophora bantiana Phialophora verrucosa Fonsecaea pedrosoi Exophiala species Wangiella species
  • 61. Black Molds • 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
  • 62. Exophiala species Black Molds cause Mycetoma/Chromomycosis/Phaeohyphomycosis Also: sinusitis, pulmonary, brain, and disseminated infections These fungi are difficult to identify using only morphology – require molecular methods or MALDI-TOF Cladophialophora bantiana - Brain infections
  • 64. Alternaria species– Opportunistic fungus - sinusitis in normal host, disseminated infection in immune suppressed. Other more common black molds of importance:
  • 65. Bipolaris australiensis Disseminated fungal infection in both normal and immune suppressed hosts Also, Skin, nasal sinus, bone and Brain infections
  • 66. Curvularia lunata Center cell is the largest Opportunistic Bone Marrow Transplant and Aplastic anemia patients Can cause unusual erythematous skin lesions
  • 67. Exserohilum rostrum • Outbreak associated with compounded pharmaceutical [steroid] products contaminated with dust/dirt during manufacturing • Injected into lumbar spine and knee joints for pain management and led to infections: • Meningitis • Spinal abscess • Synovial infections
  • 68. Scedosporium apiospermum/Pseudallescheria (Scedosporium) boydii Cat fur-like gray colony / growth in 3 – 5 days Lollipop like spore production Opportunistic pulmonary pathogen Can be difficult to distinguish from Aspergillus in fixed tissue stains / Culture is suggested
  • 69. IMPORTANT YEAST CAUSING HUMAN INFECTION Candida species Cryptococcus neoformans Cryptococcus gattii Trichosporon species
  • 70. 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.
  • 71. Important Candida species • Candida albicans – most common species causing @ 60% of Candida yeast infections • Candida glabrata, C. krusei*, and C. tropicalis are causing higher % of infections than in the past • These 3 species are more likely to be resistant to Fluconazole, one of the most commonly used anti-fungals • Candida auris – invasive infections, intrinsically resistant to fluconazole additional anti-fungals, susceptible to echinocandins, difficult to identify • Candida parapsilosis pathogen of children and common in IV line infections (forms biofilms on plastics) Susceptible to fluconazole. • * Taxonomy update: C. krusei now Pichia kudriavzevi
  • 72. Candida species • Grow in 24 – 48 hours at 30-35*C • SABS, IMA, Blood agar… others • Bacteria-like colony – pasty white • Oval shaped yeast @ 7-8 um in size** • Form pseudohyphae not true hyphae (Yeast do not detach and form elongated hyphae) • Identify using biochemicals, 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
  • 73. Candida albicans Identification • Germ tube formation • Incubate yeast in serum for 3-4hrs at 35 ˚C • Extension from yeast cell “tube” positive for C. albicans • If incubate >4 hrs – C. tropicalis false positive • C. dubliniensis is also positive for germ tubes • Chlamydospore formation • Growth on cornmeal agar at 48 hrs • Rudimentary structures unique to C. albicans - Chlamydospore chlamydospore
  • 74. ChromAgar for the identification of Candida spp using chromogenic substrates. The unique color formation can identify 4 yeast species. Susceptibility testing of yeast is much like bacteria, using broth dilution and Etest methods.
  • 75. Candida Histopathology • Pyogenic to granulomatous inflammation • Usually observe yeast cells (8 um) and pseudohyphae • Candida glabrata = smaller yeast cells (4 um) and absence of pseudohyphae GMS stain of Candida glabrataCandida species not glabrata
  • 76. 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) • Infects mostly compromised hosts – one of the major AIDS defining infections
  • 77. Cryptococcus gattii – a close relative of C. neoformans • Isolated first from forested areas of the Pacific Northwest (British Columbia, Washington, Oregon and California) found in soil debris and tree species • Infection of both normal and immune suppressed hosts • Primarily a pulmonary disease [Cryptococcoma] but can develop meningitis • Culture, biochemical & staining identical to C. neoformans • Defining reactions – • L Canavanine glycine bromthymol blue medium – C. gatti = blue C. neoformans = colorless MALDI-TOF, ssequencing will also ID both species
  • 78. Cryptococcus neoformans and C. gattii • Irregularly sized yeast cells (2 – 20 um) • Virulence factor: Polysaccharide capsule • Polysaccharide capsule is target in diagnostic testing: • India ink exam- CSF placed in one drop of black ink. It is a negative staining method – background is stained but not the polysaccharide capsule • Cryptococcal antigen test – Titer of capsular polysaccharide detected in either CSF or serum • More sensitive than India ink • Test for diagnosis or can follow recovery with decreasing titer
  • 79. Grows on mycologic agars Mucoid colonies due to capsular polysaccharide Both C. Neoformans & C. gattii brown colonies on Birdseed agar Gram stain Blue blobs Positive urea rxn
  • 80. 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
  • 81. Pneumocystis jiroveci • Yeast like fungus • Used to be Pneumocystis carini and considered a protozoan • Pneumonia in the immunocompromised host (PCP) particularly HIV/AIDS • Diagnosis: Examination of Bronchial lavage, lung biopsy, induced sputum by Direct Fluorescent antibody (DFA) and PCR. DFA
  • 82. 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%
  • 84. Malassezia furfur • Most superficial of the dermatomycoses • Normal flora on human 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 tunneling in with IV lipid feeding lines (parenteral nutrition) – in neonates or rarely adults
  • 85. Malassezia furfur • Lipophilic yeast – oil required for growth • Media for isolation must contain oil or use 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
  • 86. 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
  • 87. Positive KOH prep Showing thin septate fungal hyphae Calcofluor white stain with fluorescence – thin fungal hyphae
  • 88. Microsporum canis Ringworm acquired from dog or cat White colony/ yellow on reverse of colony Tuberculate thick walled macroconidia [spiny projections] Few if any microconidia
  • 89. Microsporum gypseum – skin infection from exposure to contaminated soil, sandy colored colony, large macroconidia are produced.
  • 90. Trichophyton rubrum White colony with red diffusable pigment Pencil shaped macroconidia/ many micro- conidia Infection from fomites Red diffusible pigment
  • 92. Epidermophyton floccosum Khaki green colored colony Beaver tail shaped large macroconidia – no microconidia – most often cause infectionsin skin and nails.
  • 93. Opportunistic Fungal Pathogens Infections in the immune suppressed host or special circumstances Hyaline molds
  • 94. Opportunistic Fungi • Hyaline – no natural color to the hyphae • Regular septations in the hyphae • Usually grow on a variety of agar media in 3-5 days at 35 or 30˚C • ID = growth rate, color/texture of colony and microscopic appearance • Branching of hyphae in stains of fixed tissue – angle of branching can be helpful in identification • ??? of species in the thousands
  • 95. Aspergillus species • Ubiquitous in nature @ 200 species • Hyaline with regular septation • Numerous round conidia produced • Pulmonary infection / disseminate in immune suppressed • Neutropenia • Branches at 45 degree angle in fixed tissue • Invade vessels, cause thrombosis & infarctions Septation
  • 97. Supportive not Conclusive for Diagnosis Aspergillus Galactomannan Enzyme immunoassay • Detects circulating Aspergillus antigen in the blood and/or bronchial lavage, most helpful in disseminated infections • +/- sensitivity and specificity (PPV 68%/ NPV 96%) • False positive reactions (10%) with Piperacillin/Tazobactam therapy, infection with H. capsulatum, and rice and pasta ingestion (1,3)–Beta-D-glucan assay • Detected in serum or BAL from the following pathogens: Candida spp., Acremonium, Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum, Trichosporon spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis jiroveci. • No reaction for Cryptococcus and Zygomycetes • High values more meaningful, False positive reaction from the environment
  • 98. Aspergillus fumigatus •Ubiquitous airborne fungus, inhale conidia •One of most common in immune suppressed •Blue/Green colony •Phialid with colorless spores are directed upward
  • 99. Aspergillus flavus •Cereals, grains, legumes •Same disease potential as A. fumigatus •Green/yellow colony •Green/yellow hue to spores on phialids that surround the vesicle
  • 100. Aspergillus niger •Black colony – visible black fruiting heads •Contaminate in fruits and vegetables/ soil •Invasive disease uncommon •Black spores supported by phialids that surround the vesicle
  • 101. Aspergillus terreus •Infection in the immune compromised host •Sandy colored colony •Isolated from soil •Colorless conidia supported by phialids headed upward •Aleurioconidia produced •Intrinsic resistance to Amphotericin B
  • 102. Aspergillus – fruiting head seldom seen in fixed tissue usually 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.
  • 103. Fusarium species •Common in nature/plants •Fuschia colored colony •Disease related to immune status of host/ neutropenia •Infections reported: •Disseminated in bone marrow transplants •Corneal infections in contact lens wearers •Random hyphae in fixed tissue/ difficult to identify Banana boat shaped spores
  • 104. • Scopulariopsis species • Found in soil and plants • Infections: Nail, skin, sinusitis, pulmonary and disseminated in the immune suppressed host • Very resistant to antifungal agents
  • 105. Penicillium species – one of the most common molds in the environment , cause of bread mold, uncommon cause of human disease , can appear as a culture contaminate
  • 106. MUCORMYCOSIS/ ZYGOMYCOSIs Hyaline Broad hyphae without septation Sporangium with spores
  • 107. Mucormycosis/Zygomycosis • Soil, rotten fruit and vegetables • Rhinocerebral mucormycosis - classic • Infections in diabetics, the elevated glucose enriches fungal growth • Starts in nasal sinus / spreads to orbit of eye – then brain • Sinus and pulmonary infection in the immune suppressed host • Broad, hyaline, aseptate hyphae produced • Culture grows within 24 hrs, producing coarse aerial hyphae • Mince infected tissue and place on agar • Do not grind infected tissue – destroys hyphae
  • 109. 90˚ angle branching, aseptate, ribbon like hyphae Invades vessels and can cause infarcts and thrombi Zygomycetes