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Mycology Update 2024
Margie Morgan, PhD, D(ABMM)
Mucormycetes Conidial Molds
Dematiacious Hyaline
Yeast
1
Starting points
Yeast are:
• Unicellular and bud to produce daughter cells
• Growth on solid media produce white to beige colonies in the first 24-48
hours (30*-35*C) and appear much like bacterial colonies
• A few genera produce pigment (ex. Rhodotorula = orange/red)
• Some genera produce mucoid colonies (ex. Cryptococcus)
• Taxonomic updates / new name then (old in parentheses)
Candida albicans
(5% Sheep’s blood agar)
Growth at 24hr / 35*C
2
C. neoformans
mucoid colonies
(Sabouraud agar)
Starting points
Molds:
• Produce hyphae and conidia [spores]
• Growth on solid media produce downy, fluffy, or cottony colonies
• Growth time is variable (3 – 21 days, (30-35*C) depending on genera of mold
• Most mold colonies produce pigment, which can assist with identification
• Taxonomic updates with new name then (old name in parentheses).
hyphae
spores
3
Specimen Collection and Transport
• Fungi are hardy
• Transport media usually not required
• Sterile containers for collection to prevent bacterial
contamination / room temperature transport is adequate
• Numerous anatomic sites are appropriate for culture
• Respiratory specimens – sputum, bronchial lavage, brushings, nasal
sinuses
• Tissue biopsies
• Cutaneous - skin scrapings, material from lesions
• Ocular
• Sterile body fluids, including CSF
• Blood
• Bone Marrow
4
Fungal Culture Media / most common
• Sabouraud dextrose agar (SABS)
All purpose fungal medium – antibiotic free
Will also grow bacteria from contaminated sites
Contains 2% glucose, pH @7.
Best use is for the subculture of fungi
for identification testing and susceptibility workup
• Inhibitory mold agar (IMA)
Selective and enriched medium - contains antibiotics
chloramphenicol and gentamicin to inhibit bacterial growth
Medium used for the primary recovery of pathogenic fungi
5
Fungal Culture Media
• Mycosel (Mycobiotic) agar
• Highly selective SABs-like medium containing chloramphenicol and cycloheximide
• Used primarily to culture dermatophytes – molds that cause skin, hair and nail infections
• Should not be used as a primary plating medium for cycloheximide prevents growth of some
genera of pathogenic fungi:
• Cycloheximide can suppress: Trichosporon species, Candida tropicalis and Cryptococcus
neoformans/gattii
• Brain heart infusion (BHI) medium /with or without blood
• Used for the primary recovery of fungi
• Inhibition of bacterial growth with addition of chloramphenicol and gentamicin
• Sheep’s blood can be added to the medium to nurture for fastidious systemic fungi such as
Histoplasma capsulatum
6
Mycology Laboratory Safety Precautions
• BSL-2 level safety precautions
• BSL-2 biosafety cabinet with HEPA filtration required for
working with mold cultures
• Yeast procedures can be performed on bench-top
• PPE (personal protective equipment) includes lab coat and
gloves
• Biohazardous waste must be autoclaved
• Agar plates must be sealed after inoculation to protect
those working in the laboratory from mold spores
created during culture incubation.
7
• Using Biosafety Level 2 precautions, inoculate specimen onto medium and
streak for isolation
• Seal plates with oxygen permeable tape prior to incubation
• Tubes or vials can also be used for culture, but it is more difficult to
visualize fungal growth and perform testing
• Incubate media at 30˚C for 4 weeks (extended time necessary for the
detection of slower growing systemic fungi), screen periodically
• If growth occurs - perform appropriate identification tests
Processing of Fungal
Cultures
8
Yeast Identification Methods
• Biochemical reactions have been used for decades to identify yeast.
Recent taxonomic updates and new emerging yeast pathogens have
challenged the accuracy of biochemical identification methods.
• Newer methods with improved accuracy of yeast identification include:
(1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight)
Colonies are smeared onto a target plate, superheated by laser and
converted into charged protein particles, the particles based on mass
and charge travel up a vacuum chamber to a detection plate to form
identification peaks that identify yeast to genus and species.
Most popular current method, precise, rapid, and inexpensive
(2) rRNA sequencing – particularly helpful for rare and difficult to identify
yeast species/ based on the detection of sequence differences
(polymorphisms) in the16S rRNA gene which is present in all yeast and
can be used to identify yeast to the genus/species level
9
• Lactophenol Cotton Blue [LCB] adhesive tape preparation used for
decades to presumptively identify molds using microscopic observation
• LCB mounting medium consists of phenol, lactic acid, glycerol and
aniline cotton blue dye.
• Clear adhesive tape is used to touch the colony, picking up fungal
hyphae/conidia, then pressed into one drop of LCB on a microscope
slide. Observe under light microscope using 40X.
• Can be used for identification to genus for numeorous fungi.
Newer/ more definitive
identification methods include:
(1) MALDI-TOF
(2) rRNA sequencing techniques
Mold Identification Methods
10
Gram stain
• Most yeast species stain well with Gram stain and stain positive (blue)
• Examination for budding cells can assist with confirmation that it is a yeast and not a
staining artifact
• Pseudo-hyphae Gram stain well, they are formed by yeast cells elongating with
incomplete budding, yeast cells remain attached to pseudo-hyphae
• Mycelia (produced by cell wall synthesis) may be difficult to visualize on Gram stain prep
pseudohyphae
Mold mycelia
Yeast
Pseudo-hyphae
Direct Visual Examination of Specimens
X100 oil immersion
11
•KOH mount may be used to detect yeast, pseudo-hyphae, and hyphae
most usually in skin, hair, and nail specimens
•KOH dissolves the keratin in cellular material and frees hyphae and yeast
cells so they can be better visualized on the slide preparation
•Poor sensitivity and specificity particularly for mold, the colorless hyphae
can be difficult to visualize and be confused with cell borders
•Less used as better techniques evolve
Potassium Hydroxide Prep (KOH)
X40
Light microscopy
12
• Yeast, pseudo-hyphae, and
mycelial fungi bind with the
Calcofluor white stain reagent and
fluoresce a bluish color
• Stain is read using a fluorescence
microscope (40X)
• More sensitive and specific method
than a KOH preparation for
visualization of yeast, pseudo-
hyphae and hyphae
Calcofluor White Stain
13
One drop of black ink is placed into one drop of CSF
on a microscope slide, cover with a coverslip and
examine using light microscopy (40X)
It is a “Negative” staining technique – stains the
background of prep not the yeast cell or capsule. The
clearing around the yeast cell is the polysaccharide
capsule produced by both Cryptococcus neoformans
and C. gattii.
India ink is less sensitive than the Cryptococcal
antigen test and Cryptococcal antigen is considered
the superior test for diagnosis of Cryptococcal
meningitis.
India Ink – Cryptococcus detection in CSF
14
Molecular and Sequencing Direct Detection
• Direct detection of microbial DNA from patient specimens:
• DNA is isolated from specimen and amplified by conventional PCR using a battery of broad-
range primers for yeast and mold organisms
• Amplified products are sequenced, and the organism identified using sequence data.
• Detection and identification of specific pathogens:
• DNA is isolated from a cultured organism or direct patient specimen and amplified by PCR
using primers developed specifically to detect a particular yeast or mold.
• Liquid biopsy to detect a large menu of infectious agents from plasma.
• This technique detects microbial cell–free DNA (mcfDNA) circulating in the bloodstream
from more than 1000 pathogens that cause infections. This can detect a good number of
yeast and mold species that could cause infection and circulate in the bloodstream.
Analysis of complex genomic data arrives at identification of the potential pathogen.
• These tests are most usually reference laboratory offerings.
15
Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae
stain grey to black and stains both living and dead fungi.
• Molds / Observe the width of the hyphae, the presence or absence of regular
septations, and the angle of branching
• Yeast / Observe the size and budding pattern of yeast and presence of pseudo-
hyphae, look for rounded cells to separate from debris
• Later slides will discuss specific observations that assist in identification
Examination of Fungi in Fixed tissue
16
PAS stain yeast and hyphae magenta. PAS is not specific to fungi, will
also stain structures containing carbohydrate macromolecules
(glycogen, glycoprotein, proteoglycans). PAS stains only living fungi.
Periodic Acid Schiff [PAS] Stain
17
Stains the polysaccharide capsule of Cryptococcus neoformans and
C. gatti pink. Also stains mucin in fixed tissue.
Mucicarmine [Mucin] Stain
Fontana-Masson Silver Stain detects melanin, used in the rare situation
when capsule deficient cryptococcus organisms are encountered.
Non-encapsulated Cryptococcus stain brown.
18
Helpful for the description of cellularity in tissue
and morphologic assessment of size, septations
and branching pattern of molds and the size and
budding pattern of yeast. Can also visualize
structures such as sulfur granules.
Hematoxylin and Eosin Stain
19
Sulfur Granule
Aseptate fungal hyphae
Granuloma
Intracellular yeast
Morphology of Molds in H & E Stains
Narrow, uniform Broad, irregular hyphae Pigmented hyphae/spores
hyaline septate hyphae folded appearance
Aspergillus Mucorales such as Dematiaceous fungus
Fusarium Mucor and Rhizopus Culture for definitive
Scedosporium Culture for definitive identification
Culture required for identification
definitive identification
20
Antifungal Drug Classification and Action
• Loss of cell membrane integrity:
• Polyenes: Amphotericin B and nystatin
• Action: Bind to ergosterol, a steroid-alcohol unique to Fungi. The polyene-ergosterol complex creates pores
in the fungal cell membrane, ultimately leading to electrolyte leakage, cell lysis, and cell death
• Azoles: Ketoconazole, miconazole, clotrimazole, itraconazole, isavuconazole, fluconazole,
voriconazole, and Posaconazole
• Action: Non-competitive inhibitors of the fungal enzyme lanosterol 14-alpha-demethylase, which is a rate-
limiting enzyme in the fungal biosynthetic pathway of ergosterol. This action destabilizes the fungal cell
membrane, causing cell content leakage, lysis, and eventual death.
• Loss of cell wall integrity:
• Echinocandins: Anidulafungin, caspofungin, and micafungin
• Action: Inhibit the fungal beta-(1,3)-D-glucan synthase, which is the enzyme responsible for synthesizing
beta-(1,3)-D-glucan, a key component of fungal cell walls. Losing this cell wall component leads to osmotic
instability and cell death.
21
DIMORPHIC FUNGI
Systemic pathogens with unique characteristics
22
What is a Dimorphic Fungus?
• Depending on temperature and conditions of environment, one fungi
demonstrates two forms:
• (1) Mycelial form - Hyphae with conidia (spores)
• Free living form in nature and growth in laboratory at temperatures <=30˚C
• (2) Yeast or yeast like form
• Parasitic phase found in human tissue and growth in the laboratory at temperatures >= 35˚C
• Exposure to increased body temperature transforms the mycelial form acquired in nature to
the yeast form that infects tissue
Histoplasma capsulatum –
Mycelial form in nature and growth at 30˚C
Histoplasma capsulatum – Pathogenic
Yeast form in tissue exam and growth at 35˚C
23
Dimorphic Fungi
• Histoplasma capsulatum and H. duboisii
• Blastomyces dermatitidis
• Coccidioides immitis and C. posadasii
• Paracoccidioides brasiliensis
• Sporothrix schenckii complex
• Talaromyces (Penicillium) marneffei
24
Histoplasma capsulatum
• Epidemiology: Worldwide distribution,
in the USA primary endemic region Ohio, Missouri,
and Mississippi River valleys, but global warming
has led to an increased endemic region.
• Environmental sources: Aerosolized bat guano is rich in H. capsulatum
conidia, so infections occur in spelunkers (cave explorers). Conidia found in
many species of bird droppings, including chickens, making farmers at risk.
25
Histoplasmosis
• Up to 90% of infections are asymptomatic or subclinical
• @ 10% acute pulmonary, chronic pulmonary or disseminated to
bone marrow, heart, CNS, gastrointestinal, skin & genitourinary
• Mucocutaneous lesions a unique dissemination site
• Who is most likely to have progressive Histoplasmosis?
• HIV/AIDS / both primary and reactivation disease
• Organ transplant / both primary and reactivation disease
• Taking medications such as corticosteroids or TNF-inhibitors
• Infants
• Adults aged 55 and older / both primary and reactivation disease
• Treatment for serious infections include : Itraconazole,
Fluconazole +/- Amphotericin B
26
Histoplasmosis Diagnosis
• Antigen detection in urine and/or serum
• Quantitative enzyme immunoassay
• Screening test for Histoplasmosis infection
• Performed on random collected urine or serum specimen
• Most sensitive (>=85%) for the diagnosis of disseminated and chronic
pulmonary Histoplasmosis cases
• Useful test in immune suppressed patients that do not produce a
detectable antibody response
• H. capsulatum antibody tests are available (immunodiffusion and
complement fixation.) Antibody usually appears in the second month of
infection so not as useful for acute diagnosis as antigen detection.
27
H. capsulatum Culture
• Growth of Mold at 30*C
• Mold encountered in nature microconidia are
inhaled / penetrate the lung causing infection
• Requires 2-8 weeks to grow in culture
• Colony is white to brown and cottony
• Microscopic appearance on LCB tape mount
• Slender septate hyphae
• Tuberculate macroconidia, large & round (8 – 16 µM)
• Microconidia (2 - 4µM)
• MALDI-TOF, DNA hybridization probe, or 16S rRNA
sequencing necessary to confirm identification
• Confirmation is required due to look alike fungi, such as
Chrysosporium and Sepedonium species, which are usually
considered saprophytic fungi
28
Macroconidia have tubercles
(spike protrusions)
H. capsulatum Culture
• Growth of Yeast at 37*C
• Requires 4-8 weeks to grow in culture
• Colony is white to beige with a creamy consistency
• Microscopic appearance
• 2-4 uM in size, oval to round, narrow-based budding
• Yeast is the form found in human tissue
• Increase in temperature in human induces the inhaled conidia/mycelia from nature to
undergo transformation to the yeast phase in human tissue
29
Histopathology H. capsulatum
• Granulomas produced in lung and other tissues can be either non-
caseating or caseating
• Primary infection usually in lung but can disseminate to organs of
the Reticuloendothelial System (RES) – high % of cases with
dissemination to bone marrow
30
Histoplasma capsulatum – Yeast in fixed tissue
• Small yeast 2-4 uM, minimal size variation, narrow neck bud, oval to round.
• Yeast are often intracellular within macrophages
• In H& E stain, appears to be encapsulated due to staining artifact – caused
by shrinkage away from the cell wall
• Stains well with a variety of stains
• Possible confusion due to size with N. glabrata, C. auris, pneumocystis, or
Cryptococcus spp.
H & E
PAS
Gram Wright’s
31
Appears to be
encapsulated
GMS
Leishmania donovani
amastigote
Note: kinetoplast (mitochondrial
DNA) next to nucleus, L. donovani
does not stain with GMS
Toxoplasma gondii
tachyzoites - Oval to
crescent in shape, no capsule-
like clearing around tachyzoite
Beware of these Look-a-likes and rare occurrences!
32
Histoplasma capsulatum
in aortic valve tissue pictured.
Unlike in other organs, Histoplasma
can produce variable size yeast
cells and hyphal forms on valve
tissue in cases of endocarditis.
This could be confused with other
molds.
Case History: Histoplasmosis
38 yr old male, Newly diagnosed with HIV/AIDS
HIV Viral Load =220,000 copies
CD4 count = 3
Anemia
Thrombocytopenia
Bilateral pulmonary infiltrates
Bone marrow aspiration procedure: Giemsa stain
and GMS stain of aspirate show small yeast
Culture performed, examined with LPCB prep
33
Therapy: mild cases Itraconazole, serious or immune
suppressed add Amphotericin B.
Giemsa
stain
GMS stain
Lacto-phenol
cotton blue prep
The mold phase is identical in culture growth
and appearnce on adhesive tape preparation to
H. capsulatum.
Difference is the size of the yeast cell:
Note yeast cell of H. duboisii is 6-12 um,
which is 2-3X the size of H. capsulatum yeast.
Endemic area: Central, east and west Africa
Primary infection: cutaneous with occasional
involvement of the bone
Unusual variant of Histoplasma –
Histoplasma duboisii
34
Blastomyces dermatitidis
• Epidemiology
• In the US, Ohio and Mississippi River valley and
Great Lakes region
• No association with any animal or specific activity
• Mold found in forested areas and riverbanks
• Infection from inhalation of airborne conidia
• Primarily a pulmonary pathogen
• Small % of patients disseminate to skin and bone
• Well demarcated (raised border) skin lesions /
direct examination positive for yeast
• Dissemination most often in immune suppressed
patients
35
Blastomyces dermatitidis
• Mold grows in culture at 30˚C
• Growth 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 and possibly others (?)
• Requirement to confirm identification: MALDI-TOF, DNA hybridization
probe or 16s RNA sequencing
Blastomyces Chrysosporium
36
• Yeast grows at 35*C in culture
• Slow growing taking up to 4 weeks
• Large yeast cell with size variation (8-15 um), round to slightly oval
• Unique “Broad Based Budding” pattern and thick/double contoured wall.
Blastomyces dermatitidis
Broad based bud
Double contoured wall
37
Histopathology Blastomyces dermatitidis
• Mixed neutrophilic and granulomatous inflammation is observed in tissue
• Stains well with H&E, PAS and GMS. May stain weakly positive on
mucicarmine and cause confusion with C. neoformans and C.gattii
• Broad based budding yeast cell with thick wall is the classic structure
38
PAS stain
GMS Stain
Coccidioides immitis and C. posadasii
• These two species share close genetic relatedness
• Found in different geographic regions, C. immitis mostly in
California region and both species found outside CA
• Both species are thought to have the same infectious process, react
the same in serologic tests, and are morphologically identical
• Coccidioides endemic in SW USA (San Joaquin Valley), Mexico, and
South America, in areas known as the Sonoran life zone with warm
and desert sands, common in rodent burrows in the desert areas.
Global warming has led to an increasing endemic area.
• Infection is from inhalation of fungal particles (arthroconidia) found in
the sandy soil of the Sonoran life zone areas
39
Coccidioidomycosis
• 65% of patients have asymptomatic infection to flu-like symptoms
• 30% of patients have progressive pulmonary disease, may include
erythema nodosum, may produce a coin lesion in the lung
• Usually <5% will experience disseminated infections
• Infection of skin, bones, joints, lymph nodes, adrenal glands, and CNS
• Tropism to the Central Nervous System (CNS) with high fatality rate
• Risk factors for severe or disseminated coccidioidomycosis include:
• Darker skinned individuals and those of Filipino descent
• HIV/AIDS
• Use of immunosuppressive medications
• Organ transplant
• Diabetes mellitus
• Pregnancy
40
Serologic diagnosis of Coccidioidomycosis
• Enzyme immunoassay (EIA): 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,
• Not as specific as EIA detection of antibodies and can have “false” positive
results, must confirm infection with additional testing
• Complement Fixation (CF): detects IgG antibodies and allows for
assessment of disease severity.
41
Coccidioides Culture
• Mold grows in Culture at 30ºC
• Growth in 2-3 days as a waxy colony, becoming wooly in around 7–10 days
• Microscopically (40X) one observes areas with septate hyphae and thick-
walled alternating barrel shaped arthroconidia
• Barreled arthroconidia breakoff from hyphae and become the infectious
particle in nature and is inhaled
• Beware! Laboratory workers must cautiously handle in BSL-2 biosafety
cabinet, reported source of acquired infection
40X
2-3 days 7-10 days
42
Coccidioides Look-a-likes
• Malbranchea species similar to C. immitis and C. posadasii under
the microscope using adhesive tape prep method
• Must confirm any fungi suspected to be Coccidioides spp. using
MALDI-TOF, molecular DNA hybridization probe or 16S rRNA
sequencing
Coccidioides Malbranchea
43
Histopathology Coccidioides
• No yeast cell produced in human tissue: Instead, a thick-walled spherule
variable in size (10 – 80 uM) with endospores is the structure observed.
• Spherules can be in all stages of development: fragmented spherules to
well formed mature cysts with endospores (2-5 uM)
• Hyphae may be observed in CNS infections or bronchus, but uncommon
• Granulomatous inflammation with caseation is usually observed
44
Position of two Coccidioides
spherules pushed together can
mimic the broad-based bud of
Blastomyces
Case History:
Coccidiodoimycosis
75 year old male
Right pleural effusion
Lung nodule
Past medical history: Gout,
hypertension, anemia, obesity
Fungal serology testing:
Histoplasma urine Ag: negative
Cryptococcal Ag: negative
Coccidioides IgM: Indeterminate
Coccidioides IgG: Weak Positive
Titer 1:4
Epidemiology: Lives on ranch in Simi
Valley, California
Biopsy of lung
nodule stain/ GMS
Culture of lung
nodule incubated
at 30°C
45
• Rhinosporidium seeberi (an aquatic parasite) forms spherules that
are larger than those of Coccidioides
• Rhinosporidium spherules can be > 80 uM in size compared to those of
Coccidioides <=30 uM
• R. seeberi cause oral or nasal mass lesions
Oral /nasal mass lesions
Coccidioides is not the only spherule forming organism!
46
Prototheca wickerhamii
produce structures known
as morulas that mimic
spherules. Endospores
(2-20) are in a rosette
structure.
Paracoccidioides brasiliensis
• South American Blastomycosis – 80% of cases reported
from Brazil
• Most prevalent systemic fungal infection in Latin America
• Infection acquired from inhaling infectious particles from soil
and decaying vegetation or possibly trauma, leading to skin
infections.
• Disease presentation:
• Pneumonia and/or cutaneous involvement
• Disseminated infection can occur
• Extrapulmonary lesions on the face and oral mucosa are common site
of dissemination
47
Paracoccidioides
• Mold grows at 30*C – cultures usually not performed due to
slow growth and nonspecific sporulation
• Yeast grows at 35-37˚C
• Slow growing yeast – 3 weeks
• Large (10 – 30um), thick walled, with 2 or more tear drop shaped daughter
buds (2–10 um)
• Unique multiply budding yeast cell known as the Mariner’s wheel or
Pilot’s wheel yeast Careful! Do not confuse with tuberculate
macroconidia of mold phase Histoplasma capsulatum.
48
Histoplasma capsulatum –
tuberculate macroconidia
Histopathology
Paracoccidioides brasiliensis
• Granulomatous non-necrotizing and
necrotizing or pyrogranulomatous response
• Mariner’s wheel yeast forms
GMS
GMS
49
Sporothrix schenckii complex
• Sporotrichosis
• Cutaneous inoculation from penetrating
injury, scrape with contaminated plant thorns
• Known as “Rose gardener’s disease”
• Usually begins as skin lesion with or without
ulceration and leads to a progressive
subcutaneous disease
• Lesion can progress with lymphocutaneous
spread and possible dissemination to bone and
other organs
• Pulmonary and CNS infections occur, but rarely
50
Sporothrix schenckii complex
• Mold grows at 30˚C in 3 -5 days, starts
beige eventually turns brown/black
• Microscopic: thin septate hyphae with
conidia in daisy wheel or rosette pattern
• Yeast grows at 37˚C in 7 days as oval and
elongated yeast cells, 2 x 5 µm, described as cigar bodies
51
Histopathology S. schenckii
• Pyogenic to granulomatous inflammation
• Yeast may not be observed in human tissue
• If seen, elongated, pleomorphic described as cigar shaped
• More common structure seen in human tissue is an Asteroid body
known as Splendore-Hoeppli phenomenon (SHP)
• SHP are not unique to Sporotrichosis, also seen in infections with:
• Mucormycetes (Mucor, Rhizopus)
• Aspergillus
• Blastomyces
• Candida spp
52
Asteroid body Pleomorphic yeast cell
• Disease: Cutaneous bumpy lesions
(Talaromycosis) most common but
pneumonia or systemic infection can occur
in immune deficient or HIV/AIDS
• Endemic in the soil in SE Asia, southern
China and eastern India
• Mold grows in 2-3 days (<=30*C) / green
mold with red diffusible pigment
• Microscopically mold looks like a typical
Penicillium spp with branched
conidiophore and sporulation
• Small yeast like cells seen in fixed tissue /
divide by transverse fission
Talaromyces (Penicillium) marneffei
53
54
Mold In Tissue
SUBCUTANEOUS FUNGAL
INFECTIONS
Mycetoma
Chromoblastomycoses
Phaeohyphomycosis
Protothecosis
55
Mycetoma
• Chronic, granulomatous disease of the skin and subcutaneous
tissue, which sometimes involves muscle, bone, and
neighboring organs
• Occurs in hot temperate parts of the world
• Trauma in nature, implants the fungi into the subcutaneous
tissue, farm workers are most at risk
• 3 criteria help to explain mycetoma:
• Swollen extremity from lesion progression
• Development of abscess and fistulas (sinus tracts)
• Sulfur granules drain from sinus tracts, observed in tissue histology slides
to help establish diagnosis
Two types of mycetoma:
1. Actinomycotic – infection with higher bacteria species
2. Eumycotic – infection with black molds, occasionally hyaline molds
56
Actinomycotic Mycetoma
• 98% of all mycetoma infections are actinomycotic
• Nocardia species most common infectious agents
• Sulfur granules are formed in tissue.
• The granules are composed of a matrix of the filamentous
bacteria. Observe the edge of stained granule to observe
the size and morphology of the bacteria.
57
Edge of sulfur granule show thin filamentous
bacteria: How can you tell if infection is due to
Nocardia or Actinomyces?
Perform a PAF stain and culture:
• Nocardia species are modified (partial) acid
fast [PAF] stain positive and grow aerobically
• Actinomyces species are PAF stain negative
and grow best anaerobically
Careful! Sulfur granules created by infection
with Actinomyces species (an anaerobic
Gram positive bacilli) can look very similar to
ones formed by Nocardia species. This is a
distinct infection known as Actinomycosis.
Sulfur granules in Tissue
Actinomyces
58
Nocardia
Filamentous branching Gram positive bacilli which can stain poorly and
appear speckled.
Modified Kinyoun Acid-Fast Stain (PAF) = Stain red /Partial Acid-Fast
Modified (Partial)
Kinyoun acid-fast stain
Gram stains
Nocardia species
59
• Besides Mycetoma, Nocardia spp can cause primary
Pulmonary and Brain infection in immune suppressed
• Grows at 30 – 35*C in 3-5 days on many agars
including SABs and 5% Sheep’s blood agar
• Colony is dry/crumbly with a musty odor and can vary
in color, usually white to coral
• Total of 85 species of Nocardia with the members of
Nocardia asteroides complex most common
• Identification: MALDI-TOF & 16s rRNA gene
sequencing
• Therapy is species specific and best guided by
performing susceptibility testing
Nocardia species
60
Thermoactinomycetes
• Gram positive filamentous rods related to Nocardia species
• Thermoactinomyces and Saccaropolyspora species
• Flourish in areas of high humidity and high temperature (40*-60*C)
• Grow best in moist and hot agricultural environments, like stacks of hay
• Agents of Farmer’s lung – hypersensitivity pneumonitis or extrinsic allergic
alveolitis, occupational disease
• Immunologically mediated inflammatory disease of the lung
• Inhalation exposure to the thermophilic actinomycetes
• Pathology: Loose, non-necrotizing granulomas
• Diagnosis
• Detailed environmental history
• Serology testing
61
Eumycotic Mycetoma
Infection with pigmented/black molds (dematiaceous molds)
-Numerous species of black molds found in soil and soil debris
-Skin infection by the implantation of the mold into the subcutaneous
tissue, usually a traumatic event occurring in environment
- Cause only @ 2% of mycetoma like infections
-Most common agents: Acremonium spp. and Scedosporium
apiospermum complex
Notice the thick
hyphae on the
edge of the granule
62
Dark grey black colony
Chromoblastomycosis
• Three characteristics help in the diagnosis:
• Wart like lesions into subcutaneous tissue (scarred and nodular)
• Muriform bodies observed in fixed tissue slides
• Infection caused by black pigmented fungi (dematiaceous)
• Skin trauma causes implantation of fungi into tissue
Structure in tissue: Naturally brown in
color, septate structures known as
sclerotic bodies, or copper pennies.
These structure can break up into
pieces extend into the surrounding
subcutaneous tissue
Most common agents: Fonsecaea,
Cladophialophora, and Phialophora spp
63
Prototheca wickerhamii (Protothecosis)
• Algae without chlorophyll (not a fungal organism)
• Usually related to outdoor penetrating injury
• Causes nodular skin lesions and olecranon bursa infection
• Most common in patients with suppressed immune system
• Compare morula of Protothecosis to copper penny of Chromoblastomycoses
• Morula of Protothecosis is NOT naturally brown in color and no regular septations
Prototheca
morula
Copper penny
64
Chromoblastomycoses
Phaeohyphomycosis
Traumatic implantation of black pigmented fungi into subcutaneous tissue
• Infections usually nodular lesions or cysts on the skin, controlled by an
intact immune response but may disseminate in the immune suppressed
• Disseminate in lung, nasal sinus with extension to brain
• In fixed tissue, dark brown colored swollen hyphae and yeast like cells
• Most common agents: Alternaria, Curvularia, Exophiala and Phialophora
65
Black molds /known as Dematiaceous
molds
• Black colored colonies; due to containing melanin or
melanin like pigments in the cell wall of the mold
• Commonly found in areas damaged by flooding
• Commonly found in the environment
• Difficult to identify based on morphology due to
different fungal genera sharing sporulation
characteristics, usually require MALDI-TOF or
Sequencing for identification
66
Exophiala species
A few common Black Molds
Cladophialophora
bantiana -
Associated with
dissemination infections
that can extend to brain
67
Wangiella
dermatitidis
Phialophora
verrucosa
Alternaria species
• Commonly found in nature in soil and plants
• Causative agent of phaeohyphomycosis (skin and
subcutaneous tissue), sinus, and nail in the normal host
• Emerging pathogen for invasive disease in immune suppressed
• Grows in 3 – 5 days at 30*culture on a variety of fungal media
• Black colony producing multicellular chaining conidia
68
Scotch tape exam
Curvularia lunata
Center cell is the largest
• Found on soil and plants in tropical and subtropical areas
• Phaeohyphomycoses, erythematous skin lesions, sinusitis, and
invasive infections including endocarditis.
• Emerging pathogen in the normal and immune suppressed
• Grows in 3-5 days at 30*C in culture on variety of fungal media
69
Scotch Tape Exam
Exserohilum rostrum
• Causative fungus in an outbreak from a compound pharmaceutical [steroid]
product contaminated during manufacturing with environmental dust
containing Exserohilum
• Contaminated commercial product injected into lumbar spine and knee joints
for pain management and led to serious and fatal infections:
• Meningitis
• Spinal abscess
• Synovial infections
70
Scedosporium apiospermum (Pseudallescheria boydii complex)
• Found in soil and environment
• Subcutaneous infections in normal host from trauma imbedding fungus into tissue
• Pulmonary pathogen from spore inhalation with possible dissemination in immune suppressed
Can invade vessels, particularly in the lung, and lead to infarcts
• Cat fur-like gray colony with growth in 3 – 5 days at 30 *C on variety of fungal media
• Microscopic appearance: Lollipop-like spore production branching from septate hyphae
• Difficult to distinguish from Aspergillus in fixed tissue stains – both can branch at 45* angle
• Unique: Intrinsically resistant to Amphotericin B
71
PAS stain of fungal hyphae
LCB preparation
IMPORTANT YEAST CAUSING
HUMAN INFECTION
Candida species
Cryptococcus neoformans
Cryptococcus gattii
Trichosporon species
72
Candida and related species
• Greater than 200 species but @ 10 species infect humans
• Found as normal flora in GI, GU and skin
• Opportunistic pathogens involving skin, mucous membranes and invasive
sites from excessive exposure to moisture, excessive antibiotics, or
immune suppression
• C. albicans is the most commonly-isolated yeast in human disease (@60%)
• Implicated in both superficial and systemic disease.
• Nail, ear, endocarditis, and bloodstream most often
• Risk factors include age of >=65 years, immunosuppression prior to steroid use,
leukocytosis, intensive care unit stays, or presence of intravascular or urinary catheters.
73
Thrush
Candida species
• Growth in 24 – 48 hours at 30-35*C
• Growth on many agars SABS, IMA, Sheep’s Blood agar
• Bacteria-like colony – pasty white
• Oval shaped @ 7-8 um in size**
• Most species form pseudohyphae in human tissue.
Not true hyphae for the yeast do not detach, the yeast cells elongate to
form hyphae. Appearance has been described as “links of sausage”
• C. auris and N. glabrata are two common related species that do not
produce pseudohyphae in tissue,** Also small, 2-4µm in size
• Identify using biochemicals, MALDI-TOF, or 16 sRNA sequencing
pseudohyphae
74
Candida albicans
Identification
• Germ tube formation
• Incubate yeast in serum for 3-4 hrs at 35 ˚C
• Look for an extension from yeast cell (germ tube)
• Note: C albicans and C. dubliniensis can form germ
tubes and if extend incubation >4 hrs – C. tropicalis
produces germ tubes
• Chlamydospore formation
• Growth on cornmeal agar at 48 hrs
• Form rudimentary spore (chlamydospore) structures
unique to C. albicans
chlamydospore
75
Starry appearing
Colony on BAP
Candida auris
• Nosocomial colonization and infection in critically ill patients with lines and tubes
in place who recently spent time in skilled nursing or long-term care facilities
• Additional risk factors are similar to risk factors for other types
of Candida infections: recent surgery, diabetes, broad-spectrum antibiotic and
antifungal use.
• Infections in patients of all ages, preterm infants to elderly with mortality 30 – 60%
• Very transmissible in healthcare settings through contact with contaminated
environmental surfaces or equipment, and from person to person
• Patients and healthcare workers can be colonized on skin, particularly axilla and groin, and
can persist for months
• Patient surveillance screening by PCR (most sensitive) and culture on
ChromAgar
• Intrinsic resistance to Fluconazole and Amphotericin B
77
Candida species Susceptibility to Azoles
(Fluconazole)
Unique features
C. albicans Susceptible Most common yeast / @ 60%
C. parapsilosis Susceptible
Nakaseomyces (Candida) glabrata 50% resistant Small yeast (2-4um) no
pseudohyphae produced
C. tropicalis 50-75% resistant
Pitchia
kudriavzevi (Candida krusei)
100% resistant
C. auris 100% resistant Emerging nosocomial pathogen,
resistant to Amphotericin B,
susceptible to the echinocandins.
Small yeast (2-4um) no
pseudohyphae produced
Most common Candida spp in human infection and unique features
ChromAgar for the preliminary identification of
Candida species:
When grown on this agar, a unique color is formed for
specific Candida species due to action on chromogenic
substrates contained in the medium.
Susceptibility testing of yeast is much like
bacteria, using broth dilution and Etest
methods.
78
ChromAgar
C. auris
Candida Histopathology
• Pyogenic to granulomatous inflammation produced in tissue
• Most Candida species produce oval yeast cells (@ 8 um) and
pseudohyphae
• N. glabrata and C. auris form smaller yeast cells (4 um) and no
pseudohyphae is formed. Could be confused with H. capsulatum.
Candida glabrata or C. auris GMS stain
Candida species not glabrata or auris
79
Cryptococcus
neoformans
• In nature, a 2um non-encapsulated yeast cell
• Found in pigeon droppings and soil contaminated with droppings
• C neoformans is enriched by the nitrogen, humidity and warmth in the heaped
droppings
• Small, non-encapsulated yeast cells are inhaled – travel through the
pulmonary system with hematogenous spread to brain and meninges
(neurotropic)
• Infects compromised hosts but as of late increasing reports in the more
normal host – one of the major AIDS defining infections, and less
common in corticosteroid use and chemotherapy.
80
Cryptococcus gattii – close relative of C. neoformans
• First Isolated from forested areas of the Pacific Northwest
(British Columbia, Washington, Oregon, and California)
• Associated with soil debris, decaying tree bark, and eucalyptus trees
• Infection of both normal and immune suppressed hosts
• Primarily a pulmonary disease (lung cryptococcoma) but may disseminate to the central
nervous system
• Culture, biochemical & staining characteristics identical to C. neoformans
• One defining biochemical reaction for C. gattii
L Canavanine glycine bromthymol blue medium
C. gatti = tuns blue C. neoformans = colorless
MALDI-TOF and 16sRNA sequencing will correctly ID both species
81
Cryptococcus neoformans
and C. gattii
• Variable sized yeast cells (2 – 20 um)
• Virulence factor: Polysaccharide capsule
• Polysaccharide capsule provides basis of diagnostic testing:
• India ink exam- CSF placed in one drop of black ink. It is a
negative staining method , staining the background but not the
polysaccharide capsule or yeast cell. Sensitivity >=50%
• Cryptococcal antigen test – Titer of capsular polysaccharide
leached off yeast cell and detected in CSF and serum
• Best test for diagnosis with sensitivity >=99%, can also follow
patient recovery with decreasing titer of polysaccharide present
in the sample
82
Positive India Ink
Grows on SABS and IMA in
1-3 days at 30 – 35*C culture
Mucoid colonies due to production of
polysaccharide capsule
C. neoformans & C. gattii are the only yeast
that formbrown colonies on Birdseed agar
Due to phenol oxidase activity
Gram stain /
stains as blue blobs due
to the presence of the
polysaccharide capsule
Cryptococcus
species have a
Positive urease
enzyme reaction
Cryptococcus neoformans and C. gattii
Negative
83
Positive
Cryptococcus in tissue
84
Cryptococcus neoformans
with visible clearing around
yeast cells due to capsule
Mucicarmine stains the
capsular polysaccharide of
Cryptococcus neoformans
and C. gattii.
GMS stain –Notice the yeast are
variable in size, narrow neck
budding
Fontana-Mason
stain/ stains the cell
wall of Cryptococcus, used do identify
Cryptococcus without a capsule
Yeast and Yeast-like Organisms in Tissue
Small Variable size Large
(2 - 7 um) (2 – 20 um) (8 – 15 um)
Oval Round Budding Budding
Yes No Coccidiodes No Yes
Histoplasma
N. glabrata C. neoformans/gatti
Candida spp. (narrow neck) Broad based bud
Malassezia thick double wall
Sporothrix schenckii
Talaromyces (Penicillium) marneffei Blastomyces dermatitidis
Pneumocystis jorovecii
85
Pneumocystis jiroveci
• Yeast like fungus / cannot be cultured
• Used to be named Pneumocystis carinii and
considered a protozoan parasite
• Causes pneumonia in the immunocompromised host
(PCP) particularly HIV/AIDS / alveoli become filled
with the organisms
• Diagnosis:
• Staining of Bronchial lavage, lung biopsy tissue,
induced sputum using direct fluorescent antibody
(DFA) or GMS stain.
• Positive reaction in (1.3)-ß-d-glucan assay.
DFA
86
GMS
Pneumocystis jiroveci (yeast like
fungus) could be confused with C.
neoformans – Careful! Central nuclear
staining in pneumocystis, collapsed
helmet forms.
C. neoformans/ C. gattii – no
nuclear staining
Cryptococcus vs.
Pneumocystis
Pneumocystis froth on H & E
87
Trichosporon spp
• T. asahii most common species
• Commonly inhabit soil, water, & vegetables
• Colonize skin, mouth and nails.
• Grows within 24-48 hrs in culture, becomes wrinkled over time
• Hyphae, pseudohyphae, arthroconidia, & rectangular yeast cells (3 x 8 uM)
• Urease enzyme positive
• Cause of superficial infection of the hair shaft named White Piedra, nail
(onychomycosis), and cutaneous infections
• Uncommon cause of fungemia and disseminated disease
• Most usually recovered from patients with corticosteroid use, solid tumors, HIV/AIDS, and
intravascular devices, including catheters and prosthetic heart valves
• Difficult to treat, elevated MICs to most anti-fungal drugs including echinocandins.
• Mortality 50 – 80%
88
CUTANEOUS AND SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes that cause Dermatophytoses
Microsporum species
Trichophyton species
Epidermophyton floccosum
89
Malassezia furfur
• Most superficial of the dermatomycoses
• M. furfur can be normal flora on human skin in adults and able to
colonize neonates at a very young age
• More common on oily skin or patients with high use of skin oils
• Most common in warm and humid climates
• Diseases:
• Skin infection is most common: Pityriasis versicolor / Tinea versicolor
• Macules, papules, patches, plaques on chest back and shoulders with either hypo
or hyper pigmentation – does not invade into deeper tissues
• Fungemia: caused by tunneling into bloodstream with IV catheter lipid
feeding lines (parenteral nutrition/hyperalimentation) in infants and adults
90
Malassezia furfur
• Lipophilic yeast – require long chain fatty acids for growth
• This can be provided on media with a film of olive oil
• Small budding yeast like cells 2 – 4 µm with collarette (appears like
necklace at junction of mother and daughter cell)
• Skin and tissue preparations described as “Spaghetti and Meatballs”
due to yeast and short hyphal fragments.
91
Spaghetti and meatballs with
basket weave morphology in the
keratin layer
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), tinea pedis (foot)
• Usually, a clinical diagnosis not requiring culture
• KOH or Calcofluor white prep can be used to
visualize fungal hyphae from skin scrapings
92
Calcofluor white prep
Round “ringworm” lesions
Microsporum canis
• Skin or scalp ringworm infection acquired from an infected dog or cat
• Colony grows in 5-7 days at 30* C on Mycosel or other fungal media as
a white mold with yellow pigment on reverse side of colony
• Thick-walled macroconidia, tuberculate [tiny spiny projections]
• Rare microconidia (small spores)
93
Nannizia gypseum (Microsporum gypseum )
• Mostly scalp or skin infection from exposure to contaminated soil
• Sandy colored colony that grows in 5-7 days, 30*C on Mycosel or other
fungal media
• Large macroconidia are produced, no microconidia produced
94
Trichophyton rubrum
• Mostly infects skin and nails after
exposure to fomites, such as wet
towels
• Grows in 5 – 7 days at 30*C. on
Mycosel and other fungal media.
• White mold, the back side of the
colony produces an intense red
colored pigment that diffuses into the
medium
• Pencil shaped macroconidia
produced but tear shaped micro-
conidia are most common and can
appear like “birds on a wire”
morphology along hyphae. Spiral
hyphae also present.
Red diffusible pigment
95
Trichophyton tonsurans
• White colony, grows in 5-7 days at
30* C on Mycosel and other fungal
media
• Light yellow reverse
• No macroconidia
• Only microconidia produced with
rare ballooning microconidia which
is the identification structure
• Primary cause of epidemic scalp
ringworm in children, nails &skin
96
Epidermophyton floccosum
• Infections in skin and
nails
• Yellow-Khaki green
colored colony that
grows in 5-7 days at
30*C on Mycosel and
other fungal media
• Beaver tail shaped large
macroconidia with no
microconidia produced
97
Opportunistic Hyaline Molds
Two groups:
(1) Narrow uniform septate hyphae
(2) Broad non-septate ribbon like-
hyphae
Infections usually in the immune
suppressed host or special
circumstances
98
Hyaline molds with narrow septate hyphae
• Hyaline – no color to the hyphae
• Regular septations along hyphae
• Grow on a variety of agar media
in @ 3-5 days at 30˚C or 35°C
• Preliminary identification based on growth rate,
color and texture of colony, and
microscopic structures observed on LCB prep
• Definitive identification: Using MALDI-TOF or 16sRNA sequencing
99
Serology Tests Supportive not Conclusive for Diagnosis
Aspergillus Galactomannan Enzyme Immunoassay
• Detects circulating Aspergillus antigen in serum and/or bronchial lavage,
• Most diagnostic in advanced disseminated Aspergillus infections
• Moderate sensitivity and specificity (PPV 68%/ NPV 96%)
• False positive reactions (10%) with patients on Piperacillin/Tazobactam therapy, infection with
H. capsulatum, and rice and pasta ingestion
(1,3)–Beta-D-glucan assay
• Detects1,3 Beta-D glucan in serum or BAL with the following pathogens: Candida spp.,
Acremonium, Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum,
Trichosporon spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis
jiroveci.
• Does not detect Cryptococcus or Zygomycetes
• High values more meaningful, false positive reaction can occur from colonization
100
Aspergillus species
• Ubiquitous in nature with @ 200 species
• Hyaline hyphae produced with regular septations,
occasionally irregular bulbous forms along hyphae
• Respiratory tract infection most common
• Can cause a fungus ball in the nasal sinus
• Can disseminate to other organs in immune suppressed
• Neutropenia can predispose to infection
Septations
101
Fungus ball
Aspergillus Structure
102
Aspergillus fumigatus complex
• Ubiquitous airborne fungus in nature, infection starts
by inhaling conidia
• Most common Aspergillus species causing
pulmonary infection in immune suppressed:
Aspergilloma, pulmonary nodules with cavitary formation
• Allergic bronchopulmonary syndrome in cystic
fibrosis and asthma, accompanied by elevation in
both eosinophil count and IgE
• Blue/Green colored mold, grayish with age
• Grows in 3 – 5 days at 30*C on multiple fungal media
• Phialides produce colorless conidia on upper third of
the swollen vesicle
103
Aspergillus flavus
• Normally isolated from cereals, grains,
legumes
• Same disease potential as A. fumigatus
but far less common, pulmonary
infection with possible dissemination,
sinusitis and nail infections
• Brownish green –yellow mold growing in
3-5 days at 30*C in culture
• Sometimes can visualize the
Green/yellow hue on conidia produced
on phialides that surround the swollen
vesicle
104
Aspergillus niger complex
• Contaminant in fruits and vegetables, also
found in soil and decaying vegetation
• Chronic otitis media, occasional
aspergilloma and dissemination in
immunosuppressed
• Black colony with white border – visible
black fruiting heads grows in 2-5 days at
30*C on many types of fungal media
• Dark grey to black conidia produced,
supported by phialides that surround the
vesicle
105
Aspergillus terreus complex
• Isolated from soil and decaying vegetation
• Infection primarily in the immune
compromised host
• Sandy colored mold grows in 3-5 days at
30*C on a variety of fungal media
• Colorless conidia supported by phialides,
headed upward
• Aleurioconidia produced (asexual spore
produced on the hyphae) are unique to this
species
• Note: Intrinsic resistance to Amphotericin B
106
Histopathology Aspergillus
Fruiting head seldom seen in fixed tissue
usually dichotomous (continuous) branching septate
hyphae with some pieces branching at a 45* angle,
occasional bulbous hyphal pieces. Can invade
vessels and unique for calcium oxylate crystals.
Look a like fungus in fixed tissue
slides: Scedosporium species
Growth in culture will
differentiate the two fungi.
107
Invasion of vessels
Calcium oxylate
crystals
Neocosmospora solani (Fusarium solani)
• Common in nature, grows on plants
• White to fuchsia colored mold grows in 3-5 days 30*C on fungal
media. It is inhibited by cycloheximide (Mycosel media).
• Produces septate hyaline hyphae, banana boat shaped conidia
• Disease normal host: keratitis, nail and skin infections
• Disease immune compromised: sinusitis, pneumonia, deep
cutaneous lesions, and fungemia (unlike most fungi can be isolated
from blood culture)
• Random septate hyphae in fixed tissue
• Confirm identification with culture and identification
Banana boat shaped spore
108
Scopulariopsis species
• Found in soil and grows on plant
• Infections: Nail infections in normal host and skin, sinusitis,
pulmonary, brain abscess in immune suppressed host
• White to sandy colored mold growing in 3-5 days at 30*C on a
variety of fungal media
• Round, spiny conidia that chain
• Very resistant to antifungal agents including amphotericin B
109
Penicillium species – > 300 species
• Common in air, frequent laboratory contaminate
• Cause of bread mold
• Uncommon cause of human disease
• Can appear as a culture contaminate
• Blue/green colony grows in 3-5 days 30*C on a
variety of fungal media
• Hyphae with branched phialides that produce
chains of conidia / appears like brush or bony hand
110
The Mucorales
Mucor, Rhizopus and related
molds
Fungi in the Order Mucorales
• Broad, Hyaline hyphae without
septation
• Spores produced in a sack-like
structure known as sporangium
111
Mucorales
• Found in soil, rotten fruit and vegetables
• Unique infection: Acute rhinocerebral mucormycosis
• Occurs in diabetics due to the elevated glucose enriching growth of
fungus, usually caused by Mucor or Rhizopus species
• Infection starts in single sided nasal sinusitis then spreads to the
same side orbit of eye then invades the brain / high fatality rate
• Pulmonary infection in immune suppressed: most
commonly diabetics, corticosteroids and burn patients
• Molds grow within 24-48 hr producing coarse aerial hyphae
• Microscopically, broad, hyaline hyphae without septation
seen with spores produced within a sporangium (sack)
• Mince, do not grind tissue submitted for culture, the
grinding will kill the tubular non-septate hyphal structures
112
Rhizopus species Lichtheimia complex (Absidia)
Mucor species
No
rhizoids
Rhizoids
Species within the Mucorales
Distant rhizoid
113
Cunninghamella species
Mold grows within 24-48 hrs,
filling the plate with coarse
aerial hyphae
• Unique finding of 90˚ angle branching, with rare to no septations, ribbon
like hyphae, can invade vessels and cause infarcts and thrombi
• Faintly staining particularly on GMS stain
• Distinguishing hyphae characteristic of Mucorales, from other hyphae in
quite important, for Mucorales are more resistant than many fungi to the
azoles and echinocandin antifungal agents Invades vessels and
can cause infarcts
and thrombi
Mucorales in Tissue
114
90* angle branching
115

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Mycology Update February 2024 Microbes with Morgan

  • 1. Mycology Update 2024 Margie Morgan, PhD, D(ABMM) Mucormycetes Conidial Molds Dematiacious Hyaline Yeast 1
  • 2. Starting points Yeast are: • Unicellular and bud to produce daughter cells • Growth on solid media produce white to beige colonies in the first 24-48 hours (30*-35*C) and appear much like bacterial colonies • A few genera produce pigment (ex. Rhodotorula = orange/red) • Some genera produce mucoid colonies (ex. Cryptococcus) • Taxonomic updates / new name then (old in parentheses) Candida albicans (5% Sheep’s blood agar) Growth at 24hr / 35*C 2 C. neoformans mucoid colonies (Sabouraud agar)
  • 3. Starting points Molds: • Produce hyphae and conidia [spores] • Growth on solid media produce downy, fluffy, or cottony colonies • Growth time is variable (3 – 21 days, (30-35*C) depending on genera of mold • Most mold colonies produce pigment, which can assist with identification • Taxonomic updates with new name then (old name in parentheses). hyphae spores 3
  • 4. Specimen Collection and Transport • Fungi are hardy • Transport media usually not required • Sterile containers for collection to prevent bacterial contamination / room temperature transport is adequate • Numerous anatomic sites are appropriate for culture • Respiratory specimens – sputum, bronchial lavage, brushings, nasal sinuses • Tissue biopsies • Cutaneous - skin scrapings, material from lesions • Ocular • Sterile body fluids, including CSF • Blood • Bone Marrow 4
  • 5. Fungal Culture Media / most common • Sabouraud dextrose agar (SABS) All purpose fungal medium – antibiotic free Will also grow bacteria from contaminated sites Contains 2% glucose, pH @7. Best use is for the subculture of fungi for identification testing and susceptibility workup • Inhibitory mold agar (IMA) Selective and enriched medium - contains antibiotics chloramphenicol and gentamicin to inhibit bacterial growth Medium used for the primary recovery of pathogenic fungi 5
  • 6. Fungal Culture Media • Mycosel (Mycobiotic) agar • Highly selective SABs-like medium containing chloramphenicol and cycloheximide • Used primarily to culture dermatophytes – molds that cause skin, hair and nail infections • Should not be used as a primary plating medium for cycloheximide prevents growth of some genera of pathogenic fungi: • Cycloheximide can suppress: Trichosporon species, Candida tropicalis and Cryptococcus neoformans/gattii • Brain heart infusion (BHI) medium /with or without blood • Used for the primary recovery of fungi • Inhibition of bacterial growth with addition of chloramphenicol and gentamicin • Sheep’s blood can be added to the medium to nurture for fastidious systemic fungi such as Histoplasma capsulatum 6
  • 7. Mycology Laboratory Safety Precautions • BSL-2 level safety precautions • BSL-2 biosafety cabinet with HEPA filtration required for working with mold cultures • Yeast procedures can be performed on bench-top • PPE (personal protective equipment) includes lab coat and gloves • Biohazardous waste must be autoclaved • Agar plates must be sealed after inoculation to protect those working in the laboratory from mold spores created during culture incubation. 7
  • 8. • Using Biosafety Level 2 precautions, inoculate specimen onto medium and streak for isolation • Seal plates with oxygen permeable tape prior to incubation • Tubes or vials can also be used for culture, but it is more difficult to visualize fungal growth and perform testing • Incubate media at 30˚C for 4 weeks (extended time necessary for the detection of slower growing systemic fungi), screen periodically • If growth occurs - perform appropriate identification tests Processing of Fungal Cultures 8
  • 9. Yeast Identification Methods • Biochemical reactions have been used for decades to identify yeast. Recent taxonomic updates and new emerging yeast pathogens have challenged the accuracy of biochemical identification methods. • Newer methods with improved accuracy of yeast identification include: (1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight) Colonies are smeared onto a target plate, superheated by laser and converted into charged protein particles, the particles based on mass and charge travel up a vacuum chamber to a detection plate to form identification peaks that identify yeast to genus and species. Most popular current method, precise, rapid, and inexpensive (2) rRNA sequencing – particularly helpful for rare and difficult to identify yeast species/ based on the detection of sequence differences (polymorphisms) in the16S rRNA gene which is present in all yeast and can be used to identify yeast to the genus/species level 9
  • 10. • Lactophenol Cotton Blue [LCB] adhesive tape preparation used for decades to presumptively identify molds using microscopic observation • LCB mounting medium consists of phenol, lactic acid, glycerol and aniline cotton blue dye. • Clear adhesive tape is used to touch the colony, picking up fungal hyphae/conidia, then pressed into one drop of LCB on a microscope slide. Observe under light microscope using 40X. • Can be used for identification to genus for numeorous fungi. Newer/ more definitive identification methods include: (1) MALDI-TOF (2) rRNA sequencing techniques Mold Identification Methods 10
  • 11. Gram stain • Most yeast species stain well with Gram stain and stain positive (blue) • Examination for budding cells can assist with confirmation that it is a yeast and not a staining artifact • Pseudo-hyphae Gram stain well, they are formed by yeast cells elongating with incomplete budding, yeast cells remain attached to pseudo-hyphae • Mycelia (produced by cell wall synthesis) may be difficult to visualize on Gram stain prep pseudohyphae Mold mycelia Yeast Pseudo-hyphae Direct Visual Examination of Specimens X100 oil immersion 11
  • 12. •KOH mount may be used to detect yeast, pseudo-hyphae, and hyphae most usually in skin, hair, and nail specimens •KOH dissolves the keratin in cellular material and frees hyphae and yeast cells so they can be better visualized on the slide preparation •Poor sensitivity and specificity particularly for mold, the colorless hyphae can be difficult to visualize and be confused with cell borders •Less used as better techniques evolve Potassium Hydroxide Prep (KOH) X40 Light microscopy 12
  • 13. • Yeast, pseudo-hyphae, and mycelial fungi bind with the Calcofluor white stain reagent and fluoresce a bluish color • Stain is read using a fluorescence microscope (40X) • More sensitive and specific method than a KOH preparation for visualization of yeast, pseudo- hyphae and hyphae Calcofluor White Stain 13
  • 14. One drop of black ink is placed into one drop of CSF on a microscope slide, cover with a coverslip and examine using light microscopy (40X) It is a “Negative” staining technique – stains the background of prep not the yeast cell or capsule. The clearing around the yeast cell is the polysaccharide capsule produced by both Cryptococcus neoformans and C. gattii. India ink is less sensitive than the Cryptococcal antigen test and Cryptococcal antigen is considered the superior test for diagnosis of Cryptococcal meningitis. India Ink – Cryptococcus detection in CSF 14
  • 15. Molecular and Sequencing Direct Detection • Direct detection of microbial DNA from patient specimens: • DNA is isolated from specimen and amplified by conventional PCR using a battery of broad- range primers for yeast and mold organisms • Amplified products are sequenced, and the organism identified using sequence data. • Detection and identification of specific pathogens: • DNA is isolated from a cultured organism or direct patient specimen and amplified by PCR using primers developed specifically to detect a particular yeast or mold. • Liquid biopsy to detect a large menu of infectious agents from plasma. • This technique detects microbial cell–free DNA (mcfDNA) circulating in the bloodstream from more than 1000 pathogens that cause infections. This can detect a good number of yeast and mold species that could cause infection and circulate in the bloodstream. Analysis of complex genomic data arrives at identification of the potential pathogen. • These tests are most usually reference laboratory offerings. 15
  • 16. Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black and stains both living and dead fungi. • Molds / Observe the width of the hyphae, the presence or absence of regular septations, and the angle of branching • Yeast / Observe the size and budding pattern of yeast and presence of pseudo- hyphae, look for rounded cells to separate from debris • Later slides will discuss specific observations that assist in identification Examination of Fungi in Fixed tissue 16
  • 17. PAS stain yeast and hyphae magenta. PAS is not specific to fungi, will also stain structures containing carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans). PAS stains only living fungi. Periodic Acid Schiff [PAS] Stain 17
  • 18. Stains the polysaccharide capsule of Cryptococcus neoformans and C. gatti pink. Also stains mucin in fixed tissue. Mucicarmine [Mucin] Stain Fontana-Masson Silver Stain detects melanin, used in the rare situation when capsule deficient cryptococcus organisms are encountered. Non-encapsulated Cryptococcus stain brown. 18
  • 19. Helpful for the description of cellularity in tissue and morphologic assessment of size, septations and branching pattern of molds and the size and budding pattern of yeast. Can also visualize structures such as sulfur granules. Hematoxylin and Eosin Stain 19 Sulfur Granule Aseptate fungal hyphae Granuloma Intracellular yeast
  • 20. Morphology of Molds in H & E Stains Narrow, uniform Broad, irregular hyphae Pigmented hyphae/spores hyaline septate hyphae folded appearance Aspergillus Mucorales such as Dematiaceous fungus Fusarium Mucor and Rhizopus Culture for definitive Scedosporium Culture for definitive identification Culture required for identification definitive identification 20
  • 21. Antifungal Drug Classification and Action • Loss of cell membrane integrity: • Polyenes: Amphotericin B and nystatin • Action: Bind to ergosterol, a steroid-alcohol unique to Fungi. The polyene-ergosterol complex creates pores in the fungal cell membrane, ultimately leading to electrolyte leakage, cell lysis, and cell death • Azoles: Ketoconazole, miconazole, clotrimazole, itraconazole, isavuconazole, fluconazole, voriconazole, and Posaconazole • Action: Non-competitive inhibitors of the fungal enzyme lanosterol 14-alpha-demethylase, which is a rate- limiting enzyme in the fungal biosynthetic pathway of ergosterol. This action destabilizes the fungal cell membrane, causing cell content leakage, lysis, and eventual death. • Loss of cell wall integrity: • Echinocandins: Anidulafungin, caspofungin, and micafungin • Action: Inhibit the fungal beta-(1,3)-D-glucan synthase, which is the enzyme responsible for synthesizing beta-(1,3)-D-glucan, a key component of fungal cell walls. Losing this cell wall component leads to osmotic instability and cell death. 21
  • 22. DIMORPHIC FUNGI Systemic pathogens with unique characteristics 22
  • 23. What is a Dimorphic Fungus? • Depending on temperature and conditions of environment, one fungi demonstrates two forms: • (1) Mycelial form - Hyphae with conidia (spores) • Free living form in nature and growth in laboratory at temperatures <=30˚C • (2) Yeast or yeast like form • Parasitic phase found in human tissue and growth in the laboratory at temperatures >= 35˚C • Exposure to increased body temperature transforms the mycelial form acquired in nature to the yeast form that infects tissue Histoplasma capsulatum – Mycelial form in nature and growth at 30˚C Histoplasma capsulatum – Pathogenic Yeast form in tissue exam and growth at 35˚C 23
  • 24. Dimorphic Fungi • Histoplasma capsulatum and H. duboisii • Blastomyces dermatitidis • Coccidioides immitis and C. posadasii • Paracoccidioides brasiliensis • Sporothrix schenckii complex • Talaromyces (Penicillium) marneffei 24
  • 25. Histoplasma capsulatum • Epidemiology: Worldwide distribution, in the USA primary endemic region Ohio, Missouri, and Mississippi River valleys, but global warming has led to an increased endemic region. • Environmental sources: Aerosolized bat guano is rich in H. capsulatum conidia, so infections occur in spelunkers (cave explorers). Conidia found in many species of bird droppings, including chickens, making farmers at risk. 25
  • 26. Histoplasmosis • Up to 90% of infections are asymptomatic or subclinical • @ 10% acute pulmonary, chronic pulmonary or disseminated to bone marrow, heart, CNS, gastrointestinal, skin & genitourinary • Mucocutaneous lesions a unique dissemination site • Who is most likely to have progressive Histoplasmosis? • HIV/AIDS / both primary and reactivation disease • Organ transplant / both primary and reactivation disease • Taking medications such as corticosteroids or TNF-inhibitors • Infants • Adults aged 55 and older / both primary and reactivation disease • Treatment for serious infections include : Itraconazole, Fluconazole +/- Amphotericin B 26
  • 27. Histoplasmosis Diagnosis • Antigen detection in urine and/or serum • Quantitative enzyme immunoassay • Screening test for Histoplasmosis infection • Performed on random collected urine or serum specimen • Most sensitive (>=85%) for the diagnosis of disseminated and chronic pulmonary Histoplasmosis cases • Useful test in immune suppressed patients that do not produce a detectable antibody response • H. capsulatum antibody tests are available (immunodiffusion and complement fixation.) Antibody usually appears in the second month of infection so not as useful for acute diagnosis as antigen detection. 27
  • 28. H. capsulatum Culture • Growth of Mold at 30*C • Mold encountered in nature microconidia are inhaled / penetrate the lung causing infection • Requires 2-8 weeks to grow in culture • Colony is white to brown and cottony • Microscopic appearance on LCB tape mount • Slender septate hyphae • Tuberculate macroconidia, large & round (8 – 16 µM) • Microconidia (2 - 4µM) • MALDI-TOF, DNA hybridization probe, or 16S rRNA sequencing necessary to confirm identification • Confirmation is required due to look alike fungi, such as Chrysosporium and Sepedonium species, which are usually considered saprophytic fungi 28 Macroconidia have tubercles (spike protrusions)
  • 29. H. capsulatum Culture • Growth of Yeast at 37*C • Requires 4-8 weeks to grow in culture • Colony is white to beige with a creamy consistency • Microscopic appearance • 2-4 uM in size, oval to round, narrow-based budding • Yeast is the form found in human tissue • Increase in temperature in human induces the inhaled conidia/mycelia from nature to undergo transformation to the yeast phase in human tissue 29
  • 30. Histopathology H. capsulatum • Granulomas produced in lung and other tissues can be either non- caseating or caseating • Primary infection usually in lung but can disseminate to organs of the Reticuloendothelial System (RES) – high % of cases with dissemination to bone marrow 30
  • 31. Histoplasma capsulatum – Yeast in fixed tissue • Small yeast 2-4 uM, minimal size variation, narrow neck bud, oval to round. • Yeast are often intracellular within macrophages • In H& E stain, appears to be encapsulated due to staining artifact – caused by shrinkage away from the cell wall • Stains well with a variety of stains • Possible confusion due to size with N. glabrata, C. auris, pneumocystis, or Cryptococcus spp. H & E PAS Gram Wright’s 31 Appears to be encapsulated GMS
  • 32. Leishmania donovani amastigote Note: kinetoplast (mitochondrial DNA) next to nucleus, L. donovani does not stain with GMS Toxoplasma gondii tachyzoites - Oval to crescent in shape, no capsule- like clearing around tachyzoite Beware of these Look-a-likes and rare occurrences! 32 Histoplasma capsulatum in aortic valve tissue pictured. Unlike in other organs, Histoplasma can produce variable size yeast cells and hyphal forms on valve tissue in cases of endocarditis. This could be confused with other molds.
  • 33. Case History: Histoplasmosis 38 yr old male, Newly diagnosed with HIV/AIDS HIV Viral Load =220,000 copies CD4 count = 3 Anemia Thrombocytopenia Bilateral pulmonary infiltrates Bone marrow aspiration procedure: Giemsa stain and GMS stain of aspirate show small yeast Culture performed, examined with LPCB prep 33 Therapy: mild cases Itraconazole, serious or immune suppressed add Amphotericin B. Giemsa stain GMS stain Lacto-phenol cotton blue prep
  • 34. The mold phase is identical in culture growth and appearnce on adhesive tape preparation to H. capsulatum. Difference is the size of the yeast cell: Note yeast cell of H. duboisii is 6-12 um, which is 2-3X the size of H. capsulatum yeast. Endemic area: Central, east and west Africa Primary infection: cutaneous with occasional involvement of the bone Unusual variant of Histoplasma – Histoplasma duboisii 34
  • 35. Blastomyces dermatitidis • Epidemiology • In the US, Ohio and Mississippi River valley and Great Lakes region • No association with any animal or specific activity • Mold found in forested areas and riverbanks • Infection from inhalation of airborne conidia • Primarily a pulmonary pathogen • Small % of patients disseminate to skin and bone • Well demarcated (raised border) skin lesions / direct examination positive for yeast • Dissemination most often in immune suppressed patients 35
  • 36. Blastomyces dermatitidis • Mold grows in culture at 30˚C • Growth 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 and possibly others (?) • Requirement to confirm identification: MALDI-TOF, DNA hybridization probe or 16s RNA sequencing Blastomyces Chrysosporium 36
  • 37. • Yeast grows at 35*C in culture • Slow growing taking up to 4 weeks • Large yeast cell with size variation (8-15 um), round to slightly oval • Unique “Broad Based Budding” pattern and thick/double contoured wall. Blastomyces dermatitidis Broad based bud Double contoured wall 37
  • 38. Histopathology Blastomyces dermatitidis • Mixed neutrophilic and granulomatous inflammation is observed in tissue • Stains well with H&E, PAS and GMS. May stain weakly positive on mucicarmine and cause confusion with C. neoformans and C.gattii • Broad based budding yeast cell with thick wall is the classic structure 38 PAS stain GMS Stain
  • 39. Coccidioides immitis and C. posadasii • These two species share close genetic relatedness • Found in different geographic regions, C. immitis mostly in California region and both species found outside CA • Both species are thought to have the same infectious process, react the same in serologic tests, and are morphologically identical • Coccidioides endemic in SW USA (San Joaquin Valley), Mexico, and South America, in areas known as the Sonoran life zone with warm and desert sands, common in rodent burrows in the desert areas. Global warming has led to an increasing endemic area. • Infection is from inhalation of fungal particles (arthroconidia) found in the sandy soil of the Sonoran life zone areas 39
  • 40. Coccidioidomycosis • 65% of patients have asymptomatic infection to flu-like symptoms • 30% of patients have progressive pulmonary disease, may include erythema nodosum, may produce a coin lesion in the lung • Usually <5% will experience disseminated infections • Infection of skin, bones, joints, lymph nodes, adrenal glands, and CNS • Tropism to the Central Nervous System (CNS) with high fatality rate • Risk factors for severe or disseminated coccidioidomycosis include: • Darker skinned individuals and those of Filipino descent • HIV/AIDS • Use of immunosuppressive medications • Organ transplant • Diabetes mellitus • Pregnancy 40
  • 41. Serologic diagnosis of Coccidioidomycosis • Enzyme immunoassay (EIA): 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, • Not as specific as EIA detection of antibodies and can have “false” positive results, must confirm infection with additional testing • Complement Fixation (CF): detects IgG antibodies and allows for assessment of disease severity. 41
  • 42. Coccidioides Culture • Mold grows in Culture at 30ºC • Growth in 2-3 days as a waxy colony, becoming wooly in around 7–10 days • Microscopically (40X) one observes areas with septate hyphae and thick- walled alternating barrel shaped arthroconidia • Barreled arthroconidia breakoff from hyphae and become the infectious particle in nature and is inhaled • Beware! Laboratory workers must cautiously handle in BSL-2 biosafety cabinet, reported source of acquired infection 40X 2-3 days 7-10 days 42
  • 43. Coccidioides Look-a-likes • Malbranchea species similar to C. immitis and C. posadasii under the microscope using adhesive tape prep method • Must confirm any fungi suspected to be Coccidioides spp. using MALDI-TOF, molecular DNA hybridization probe or 16S rRNA sequencing Coccidioides Malbranchea 43
  • 44. Histopathology Coccidioides • No yeast cell produced in human tissue: Instead, a thick-walled spherule variable in size (10 – 80 uM) with endospores is the structure observed. • Spherules can be in all stages of development: fragmented spherules to well formed mature cysts with endospores (2-5 uM) • Hyphae may be observed in CNS infections or bronchus, but uncommon • Granulomatous inflammation with caseation is usually observed 44 Position of two Coccidioides spherules pushed together can mimic the broad-based bud of Blastomyces
  • 45. Case History: Coccidiodoimycosis 75 year old male Right pleural effusion Lung nodule Past medical history: Gout, hypertension, anemia, obesity Fungal serology testing: Histoplasma urine Ag: negative Cryptococcal Ag: negative Coccidioides IgM: Indeterminate Coccidioides IgG: Weak Positive Titer 1:4 Epidemiology: Lives on ranch in Simi Valley, California Biopsy of lung nodule stain/ GMS Culture of lung nodule incubated at 30°C 45
  • 46. • Rhinosporidium seeberi (an aquatic parasite) forms spherules that are larger than those of Coccidioides • Rhinosporidium spherules can be > 80 uM in size compared to those of Coccidioides <=30 uM • R. seeberi cause oral or nasal mass lesions Oral /nasal mass lesions Coccidioides is not the only spherule forming organism! 46 Prototheca wickerhamii produce structures known as morulas that mimic spherules. Endospores (2-20) are in a rosette structure.
  • 47. Paracoccidioides brasiliensis • South American Blastomycosis – 80% of cases reported from Brazil • Most prevalent systemic fungal infection in Latin America • Infection acquired from inhaling infectious particles from soil and decaying vegetation or possibly trauma, leading to skin infections. • Disease presentation: • Pneumonia and/or cutaneous involvement • Disseminated infection can occur • Extrapulmonary lesions on the face and oral mucosa are common site of dissemination 47
  • 48. Paracoccidioides • Mold grows at 30*C – cultures usually not performed due to slow growth and nonspecific sporulation • Yeast grows at 35-37˚C • Slow growing yeast – 3 weeks • Large (10 – 30um), thick walled, with 2 or more tear drop shaped daughter buds (2–10 um) • Unique multiply budding yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast Careful! Do not confuse with tuberculate macroconidia of mold phase Histoplasma capsulatum. 48 Histoplasma capsulatum – tuberculate macroconidia
  • 49. Histopathology Paracoccidioides brasiliensis • Granulomatous non-necrotizing and necrotizing or pyrogranulomatous response • Mariner’s wheel yeast forms GMS GMS 49
  • 50. Sporothrix schenckii complex • Sporotrichosis • Cutaneous inoculation from penetrating injury, scrape with contaminated plant thorns • Known as “Rose gardener’s disease” • Usually begins as skin lesion with or without ulceration and leads to a progressive subcutaneous disease • Lesion can progress with lymphocutaneous spread and possible dissemination to bone and other organs • Pulmonary and CNS infections occur, but rarely 50
  • 51. Sporothrix schenckii complex • Mold grows at 30˚C in 3 -5 days, starts beige eventually turns brown/black • Microscopic: thin septate hyphae with conidia in daisy wheel or rosette pattern • Yeast grows at 37˚C in 7 days as oval and elongated yeast cells, 2 x 5 µm, described as cigar bodies 51
  • 52. Histopathology S. schenckii • Pyogenic to granulomatous inflammation • Yeast may not be observed in human tissue • If seen, elongated, pleomorphic described as cigar shaped • More common structure seen in human tissue is an Asteroid body known as Splendore-Hoeppli phenomenon (SHP) • SHP are not unique to Sporotrichosis, also seen in infections with: • Mucormycetes (Mucor, Rhizopus) • Aspergillus • Blastomyces • Candida spp 52 Asteroid body Pleomorphic yeast cell
  • 53. • Disease: Cutaneous bumpy lesions (Talaromycosis) most common but pneumonia or systemic infection can occur in immune deficient or HIV/AIDS • Endemic in the soil in SE Asia, southern China and eastern India • Mold grows in 2-3 days (<=30*C) / green mold with red diffusible pigment • Microscopically mold looks like a typical Penicillium spp with branched conidiophore and sporulation • Small yeast like cells seen in fixed tissue / divide by transverse fission Talaromyces (Penicillium) marneffei 53
  • 56. Mycetoma • Chronic, granulomatous disease of the skin and subcutaneous tissue, which sometimes involves muscle, bone, and neighboring organs • Occurs in hot temperate parts of the world • Trauma in nature, implants the fungi into the subcutaneous tissue, farm workers are most at risk • 3 criteria help to explain mycetoma: • Swollen extremity from lesion progression • Development of abscess and fistulas (sinus tracts) • Sulfur granules drain from sinus tracts, observed in tissue histology slides to help establish diagnosis Two types of mycetoma: 1. Actinomycotic – infection with higher bacteria species 2. Eumycotic – infection with black molds, occasionally hyaline molds 56
  • 57. Actinomycotic Mycetoma • 98% of all mycetoma infections are actinomycotic • Nocardia species most common infectious agents • Sulfur granules are formed in tissue. • The granules are composed of a matrix of the filamentous bacteria. Observe the edge of stained granule to observe the size and morphology of the bacteria. 57
  • 58. Edge of sulfur granule show thin filamentous bacteria: How can you tell if infection is due to Nocardia or Actinomyces? Perform a PAF stain and culture: • Nocardia species are modified (partial) acid fast [PAF] stain positive and grow aerobically • Actinomyces species are PAF stain negative and grow best anaerobically Careful! Sulfur granules created by infection with Actinomyces species (an anaerobic Gram positive bacilli) can look very similar to ones formed by Nocardia species. This is a distinct infection known as Actinomycosis. Sulfur granules in Tissue Actinomyces 58 Nocardia
  • 59. Filamentous branching Gram positive bacilli which can stain poorly and appear speckled. Modified Kinyoun Acid-Fast Stain (PAF) = Stain red /Partial Acid-Fast Modified (Partial) Kinyoun acid-fast stain Gram stains Nocardia species 59
  • 60. • Besides Mycetoma, Nocardia spp can cause primary Pulmonary and Brain infection in immune suppressed • Grows at 30 – 35*C in 3-5 days on many agars including SABs and 5% Sheep’s blood agar • Colony is dry/crumbly with a musty odor and can vary in color, usually white to coral • Total of 85 species of Nocardia with the members of Nocardia asteroides complex most common • Identification: MALDI-TOF & 16s rRNA gene sequencing • Therapy is species specific and best guided by performing susceptibility testing Nocardia species 60
  • 61. Thermoactinomycetes • Gram positive filamentous rods related to Nocardia species • Thermoactinomyces and Saccaropolyspora species • Flourish in areas of high humidity and high temperature (40*-60*C) • Grow best in moist and hot agricultural environments, like stacks of hay • Agents of Farmer’s lung – hypersensitivity pneumonitis or extrinsic allergic alveolitis, occupational disease • Immunologically mediated inflammatory disease of the lung • Inhalation exposure to the thermophilic actinomycetes • Pathology: Loose, non-necrotizing granulomas • Diagnosis • Detailed environmental history • Serology testing 61
  • 62. Eumycotic Mycetoma Infection with pigmented/black molds (dematiaceous molds) -Numerous species of black molds found in soil and soil debris -Skin infection by the implantation of the mold into the subcutaneous tissue, usually a traumatic event occurring in environment - Cause only @ 2% of mycetoma like infections -Most common agents: Acremonium spp. and Scedosporium apiospermum complex Notice the thick hyphae on the edge of the granule 62 Dark grey black colony
  • 63. Chromoblastomycosis • Three characteristics help in the diagnosis: • Wart like lesions into subcutaneous tissue (scarred and nodular) • Muriform bodies observed in fixed tissue slides • Infection caused by black pigmented fungi (dematiaceous) • Skin trauma causes implantation of fungi into tissue Structure in tissue: Naturally brown in color, septate structures known as sclerotic bodies, or copper pennies. These structure can break up into pieces extend into the surrounding subcutaneous tissue Most common agents: Fonsecaea, Cladophialophora, and Phialophora spp 63
  • 64. Prototheca wickerhamii (Protothecosis) • Algae without chlorophyll (not a fungal organism) • Usually related to outdoor penetrating injury • Causes nodular skin lesions and olecranon bursa infection • Most common in patients with suppressed immune system • Compare morula of Protothecosis to copper penny of Chromoblastomycoses • Morula of Protothecosis is NOT naturally brown in color and no regular septations Prototheca morula Copper penny 64 Chromoblastomycoses
  • 65. Phaeohyphomycosis Traumatic implantation of black pigmented fungi into subcutaneous tissue • Infections usually nodular lesions or cysts on the skin, controlled by an intact immune response but may disseminate in the immune suppressed • Disseminate in lung, nasal sinus with extension to brain • In fixed tissue, dark brown colored swollen hyphae and yeast like cells • Most common agents: Alternaria, Curvularia, Exophiala and Phialophora 65
  • 66. Black molds /known as Dematiaceous molds • Black colored colonies; due to containing melanin or melanin like pigments in the cell wall of the mold • Commonly found in areas damaged by flooding • Commonly found in the environment • Difficult to identify based on morphology due to different fungal genera sharing sporulation characteristics, usually require MALDI-TOF or Sequencing for identification 66
  • 67. Exophiala species A few common Black Molds Cladophialophora bantiana - Associated with dissemination infections that can extend to brain 67 Wangiella dermatitidis Phialophora verrucosa
  • 68. Alternaria species • Commonly found in nature in soil and plants • Causative agent of phaeohyphomycosis (skin and subcutaneous tissue), sinus, and nail in the normal host • Emerging pathogen for invasive disease in immune suppressed • Grows in 3 – 5 days at 30*culture on a variety of fungal media • Black colony producing multicellular chaining conidia 68 Scotch tape exam
  • 69. Curvularia lunata Center cell is the largest • Found on soil and plants in tropical and subtropical areas • Phaeohyphomycoses, erythematous skin lesions, sinusitis, and invasive infections including endocarditis. • Emerging pathogen in the normal and immune suppressed • Grows in 3-5 days at 30*C in culture on variety of fungal media 69 Scotch Tape Exam
  • 70. Exserohilum rostrum • Causative fungus in an outbreak from a compound pharmaceutical [steroid] product contaminated during manufacturing with environmental dust containing Exserohilum • Contaminated commercial product injected into lumbar spine and knee joints for pain management and led to serious and fatal infections: • Meningitis • Spinal abscess • Synovial infections 70
  • 71. Scedosporium apiospermum (Pseudallescheria boydii complex) • Found in soil and environment • Subcutaneous infections in normal host from trauma imbedding fungus into tissue • Pulmonary pathogen from spore inhalation with possible dissemination in immune suppressed Can invade vessels, particularly in the lung, and lead to infarcts • Cat fur-like gray colony with growth in 3 – 5 days at 30 *C on variety of fungal media • Microscopic appearance: Lollipop-like spore production branching from septate hyphae • Difficult to distinguish from Aspergillus in fixed tissue stains – both can branch at 45* angle • Unique: Intrinsically resistant to Amphotericin B 71 PAS stain of fungal hyphae LCB preparation
  • 72. IMPORTANT YEAST CAUSING HUMAN INFECTION Candida species Cryptococcus neoformans Cryptococcus gattii Trichosporon species 72
  • 73. Candida and related species • Greater than 200 species but @ 10 species infect humans • Found as normal flora in GI, GU and skin • Opportunistic pathogens involving skin, mucous membranes and invasive sites from excessive exposure to moisture, excessive antibiotics, or immune suppression • C. albicans is the most commonly-isolated yeast in human disease (@60%) • Implicated in both superficial and systemic disease. • Nail, ear, endocarditis, and bloodstream most often • Risk factors include age of >=65 years, immunosuppression prior to steroid use, leukocytosis, intensive care unit stays, or presence of intravascular or urinary catheters. 73 Thrush
  • 74. Candida species • Growth in 24 – 48 hours at 30-35*C • Growth on many agars SABS, IMA, Sheep’s Blood agar • Bacteria-like colony – pasty white • Oval shaped @ 7-8 um in size** • Most species form pseudohyphae in human tissue. Not true hyphae for the yeast do not detach, the yeast cells elongate to form hyphae. Appearance has been described as “links of sausage” • C. auris and N. glabrata are two common related species that do not produce pseudohyphae in tissue,** Also small, 2-4µm in size • Identify using biochemicals, MALDI-TOF, or 16 sRNA sequencing pseudohyphae 74
  • 75. Candida albicans Identification • Germ tube formation • Incubate yeast in serum for 3-4 hrs at 35 ˚C • Look for an extension from yeast cell (germ tube) • Note: C albicans and C. dubliniensis can form germ tubes and if extend incubation >4 hrs – C. tropicalis produces germ tubes • Chlamydospore formation • Growth on cornmeal agar at 48 hrs • Form rudimentary spore (chlamydospore) structures unique to C. albicans chlamydospore 75 Starry appearing Colony on BAP
  • 76. Candida auris • Nosocomial colonization and infection in critically ill patients with lines and tubes in place who recently spent time in skilled nursing or long-term care facilities • Additional risk factors are similar to risk factors for other types of Candida infections: recent surgery, diabetes, broad-spectrum antibiotic and antifungal use. • Infections in patients of all ages, preterm infants to elderly with mortality 30 – 60% • Very transmissible in healthcare settings through contact with contaminated environmental surfaces or equipment, and from person to person • Patients and healthcare workers can be colonized on skin, particularly axilla and groin, and can persist for months • Patient surveillance screening by PCR (most sensitive) and culture on ChromAgar • Intrinsic resistance to Fluconazole and Amphotericin B
  • 77. 77 Candida species Susceptibility to Azoles (Fluconazole) Unique features C. albicans Susceptible Most common yeast / @ 60% C. parapsilosis Susceptible Nakaseomyces (Candida) glabrata 50% resistant Small yeast (2-4um) no pseudohyphae produced C. tropicalis 50-75% resistant Pitchia kudriavzevi (Candida krusei) 100% resistant C. auris 100% resistant Emerging nosocomial pathogen, resistant to Amphotericin B, susceptible to the echinocandins. Small yeast (2-4um) no pseudohyphae produced Most common Candida spp in human infection and unique features
  • 78. ChromAgar for the preliminary identification of Candida species: When grown on this agar, a unique color is formed for specific Candida species due to action on chromogenic substrates contained in the medium. Susceptibility testing of yeast is much like bacteria, using broth dilution and Etest methods. 78 ChromAgar C. auris
  • 79. Candida Histopathology • Pyogenic to granulomatous inflammation produced in tissue • Most Candida species produce oval yeast cells (@ 8 um) and pseudohyphae • N. glabrata and C. auris form smaller yeast cells (4 um) and no pseudohyphae is formed. Could be confused with H. capsulatum. Candida glabrata or C. auris GMS stain Candida species not glabrata or auris 79
  • 80. Cryptococcus neoformans • In nature, a 2um non-encapsulated yeast cell • Found in pigeon droppings and soil contaminated with droppings • C neoformans is enriched by the nitrogen, humidity and warmth in the heaped droppings • Small, non-encapsulated yeast cells are inhaled – travel through the pulmonary system with hematogenous spread to brain and meninges (neurotropic) • Infects compromised hosts but as of late increasing reports in the more normal host – one of the major AIDS defining infections, and less common in corticosteroid use and chemotherapy. 80
  • 81. Cryptococcus gattii – close relative of C. neoformans • First Isolated from forested areas of the Pacific Northwest (British Columbia, Washington, Oregon, and California) • Associated with soil debris, decaying tree bark, and eucalyptus trees • Infection of both normal and immune suppressed hosts • Primarily a pulmonary disease (lung cryptococcoma) but may disseminate to the central nervous system • Culture, biochemical & staining characteristics identical to C. neoformans • One defining biochemical reaction for C. gattii L Canavanine glycine bromthymol blue medium C. gatti = tuns blue C. neoformans = colorless MALDI-TOF and 16sRNA sequencing will correctly ID both species 81
  • 82. Cryptococcus neoformans and C. gattii • Variable sized yeast cells (2 – 20 um) • Virulence factor: Polysaccharide capsule • Polysaccharide capsule provides basis of diagnostic testing: • India ink exam- CSF placed in one drop of black ink. It is a negative staining method , staining the background but not the polysaccharide capsule or yeast cell. Sensitivity >=50% • Cryptococcal antigen test – Titer of capsular polysaccharide leached off yeast cell and detected in CSF and serum • Best test for diagnosis with sensitivity >=99%, can also follow patient recovery with decreasing titer of polysaccharide present in the sample 82 Positive India Ink
  • 83. Grows on SABS and IMA in 1-3 days at 30 – 35*C culture Mucoid colonies due to production of polysaccharide capsule C. neoformans & C. gattii are the only yeast that formbrown colonies on Birdseed agar Due to phenol oxidase activity Gram stain / stains as blue blobs due to the presence of the polysaccharide capsule Cryptococcus species have a Positive urease enzyme reaction Cryptococcus neoformans and C. gattii Negative 83 Positive
  • 84. Cryptococcus in tissue 84 Cryptococcus neoformans with visible clearing around yeast cells due to capsule Mucicarmine stains the capsular polysaccharide of Cryptococcus neoformans and C. gattii. GMS stain –Notice the yeast are variable in size, narrow neck budding Fontana-Mason stain/ stains the cell wall of Cryptococcus, used do identify Cryptococcus without a capsule
  • 85. Yeast and Yeast-like Organisms in Tissue Small Variable size Large (2 - 7 um) (2 – 20 um) (8 – 15 um) Oval Round Budding Budding Yes No Coccidiodes No Yes Histoplasma N. glabrata C. neoformans/gatti Candida spp. (narrow neck) Broad based bud Malassezia thick double wall Sporothrix schenckii Talaromyces (Penicillium) marneffei Blastomyces dermatitidis Pneumocystis jorovecii 85
  • 86. Pneumocystis jiroveci • Yeast like fungus / cannot be cultured • Used to be named Pneumocystis carinii and considered a protozoan parasite • Causes pneumonia in the immunocompromised host (PCP) particularly HIV/AIDS / alveoli become filled with the organisms • Diagnosis: • Staining of Bronchial lavage, lung biopsy tissue, induced sputum using direct fluorescent antibody (DFA) or GMS stain. • Positive reaction in (1.3)-ß-d-glucan assay. DFA 86 GMS
  • 87. Pneumocystis jiroveci (yeast like fungus) could be confused with C. neoformans – Careful! Central nuclear staining in pneumocystis, collapsed helmet forms. C. neoformans/ C. gattii – no nuclear staining Cryptococcus vs. Pneumocystis Pneumocystis froth on H & E 87
  • 88. Trichosporon spp • T. asahii most common species • Commonly inhabit soil, water, & vegetables • Colonize skin, mouth and nails. • Grows within 24-48 hrs in culture, becomes wrinkled over time • Hyphae, pseudohyphae, arthroconidia, & rectangular yeast cells (3 x 8 uM) • Urease enzyme positive • Cause of superficial infection of the hair shaft named White Piedra, nail (onychomycosis), and cutaneous infections • Uncommon cause of fungemia and disseminated disease • Most usually recovered from patients with corticosteroid use, solid tumors, HIV/AIDS, and intravascular devices, including catheters and prosthetic heart valves • Difficult to treat, elevated MICs to most anti-fungal drugs including echinocandins. • Mortality 50 – 80% 88
  • 89. CUTANEOUS AND SUPERFICIAL MYCOSES Malassezia furfur Dermatophytes that cause Dermatophytoses Microsporum species Trichophyton species Epidermophyton floccosum 89
  • 90. Malassezia furfur • Most superficial of the dermatomycoses • M. furfur can be normal flora on human skin in adults and able to colonize neonates at a very young age • More common on oily skin or patients with high use of skin oils • Most common in warm and humid climates • Diseases: • Skin infection is most common: Pityriasis versicolor / Tinea versicolor • Macules, papules, patches, plaques on chest back and shoulders with either hypo or hyper pigmentation – does not invade into deeper tissues • Fungemia: caused by tunneling into bloodstream with IV catheter lipid feeding lines (parenteral nutrition/hyperalimentation) in infants and adults 90
  • 91. Malassezia furfur • Lipophilic yeast – require long chain fatty acids for growth • This can be provided on media with a film of olive oil • Small budding yeast like cells 2 – 4 µm with collarette (appears like necklace at junction of mother and daughter cell) • Skin and tissue preparations described as “Spaghetti and Meatballs” due to yeast and short hyphal fragments. 91 Spaghetti and meatballs with basket weave morphology in the keratin layer Spaghetti and meatballs
  • 92. 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), tinea pedis (foot) • Usually, a clinical diagnosis not requiring culture • KOH or Calcofluor white prep can be used to visualize fungal hyphae from skin scrapings 92 Calcofluor white prep Round “ringworm” lesions
  • 93. Microsporum canis • Skin or scalp ringworm infection acquired from an infected dog or cat • Colony grows in 5-7 days at 30* C on Mycosel or other fungal media as a white mold with yellow pigment on reverse side of colony • Thick-walled macroconidia, tuberculate [tiny spiny projections] • Rare microconidia (small spores) 93
  • 94. Nannizia gypseum (Microsporum gypseum ) • Mostly scalp or skin infection from exposure to contaminated soil • Sandy colored colony that grows in 5-7 days, 30*C on Mycosel or other fungal media • Large macroconidia are produced, no microconidia produced 94
  • 95. Trichophyton rubrum • Mostly infects skin and nails after exposure to fomites, such as wet towels • Grows in 5 – 7 days at 30*C. on Mycosel and other fungal media. • White mold, the back side of the colony produces an intense red colored pigment that diffuses into the medium • Pencil shaped macroconidia produced but tear shaped micro- conidia are most common and can appear like “birds on a wire” morphology along hyphae. Spiral hyphae also present. Red diffusible pigment 95
  • 96. Trichophyton tonsurans • White colony, grows in 5-7 days at 30* C on Mycosel and other fungal media • Light yellow reverse • No macroconidia • Only microconidia produced with rare ballooning microconidia which is the identification structure • Primary cause of epidemic scalp ringworm in children, nails &skin 96
  • 97. Epidermophyton floccosum • Infections in skin and nails • Yellow-Khaki green colored colony that grows in 5-7 days at 30*C on Mycosel and other fungal media • Beaver tail shaped large macroconidia with no microconidia produced 97
  • 98. Opportunistic Hyaline Molds Two groups: (1) Narrow uniform septate hyphae (2) Broad non-septate ribbon like- hyphae Infections usually in the immune suppressed host or special circumstances 98
  • 99. Hyaline molds with narrow septate hyphae • Hyaline – no color to the hyphae • Regular septations along hyphae • Grow on a variety of agar media in @ 3-5 days at 30˚C or 35°C • Preliminary identification based on growth rate, color and texture of colony, and microscopic structures observed on LCB prep • Definitive identification: Using MALDI-TOF or 16sRNA sequencing 99
  • 100. Serology Tests Supportive not Conclusive for Diagnosis Aspergillus Galactomannan Enzyme Immunoassay • Detects circulating Aspergillus antigen in serum and/or bronchial lavage, • Most diagnostic in advanced disseminated Aspergillus infections • Moderate sensitivity and specificity (PPV 68%/ NPV 96%) • False positive reactions (10%) with patients on Piperacillin/Tazobactam therapy, infection with H. capsulatum, and rice and pasta ingestion (1,3)–Beta-D-glucan assay • Detects1,3 Beta-D glucan in serum or BAL with the following pathogens: Candida spp., Acremonium, Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum, Trichosporon spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis jiroveci. • Does not detect Cryptococcus or Zygomycetes • High values more meaningful, false positive reaction can occur from colonization 100
  • 101. Aspergillus species • Ubiquitous in nature with @ 200 species • Hyaline hyphae produced with regular septations, occasionally irregular bulbous forms along hyphae • Respiratory tract infection most common • Can cause a fungus ball in the nasal sinus • Can disseminate to other organs in immune suppressed • Neutropenia can predispose to infection Septations 101 Fungus ball
  • 103. Aspergillus fumigatus complex • Ubiquitous airborne fungus in nature, infection starts by inhaling conidia • Most common Aspergillus species causing pulmonary infection in immune suppressed: Aspergilloma, pulmonary nodules with cavitary formation • Allergic bronchopulmonary syndrome in cystic fibrosis and asthma, accompanied by elevation in both eosinophil count and IgE • Blue/Green colored mold, grayish with age • Grows in 3 – 5 days at 30*C on multiple fungal media • Phialides produce colorless conidia on upper third of the swollen vesicle 103
  • 104. Aspergillus flavus • Normally isolated from cereals, grains, legumes • Same disease potential as A. fumigatus but far less common, pulmonary infection with possible dissemination, sinusitis and nail infections • Brownish green –yellow mold growing in 3-5 days at 30*C in culture • Sometimes can visualize the Green/yellow hue on conidia produced on phialides that surround the swollen vesicle 104
  • 105. Aspergillus niger complex • Contaminant in fruits and vegetables, also found in soil and decaying vegetation • Chronic otitis media, occasional aspergilloma and dissemination in immunosuppressed • Black colony with white border – visible black fruiting heads grows in 2-5 days at 30*C on many types of fungal media • Dark grey to black conidia produced, supported by phialides that surround the vesicle 105
  • 106. Aspergillus terreus complex • Isolated from soil and decaying vegetation • Infection primarily in the immune compromised host • Sandy colored mold grows in 3-5 days at 30*C on a variety of fungal media • Colorless conidia supported by phialides, headed upward • Aleurioconidia produced (asexual spore produced on the hyphae) are unique to this species • Note: Intrinsic resistance to Amphotericin B 106
  • 107. Histopathology Aspergillus Fruiting head seldom seen in fixed tissue usually dichotomous (continuous) branching septate hyphae with some pieces branching at a 45* angle, occasional bulbous hyphal pieces. Can invade vessels and unique for calcium oxylate crystals. Look a like fungus in fixed tissue slides: Scedosporium species Growth in culture will differentiate the two fungi. 107 Invasion of vessels Calcium oxylate crystals
  • 108. Neocosmospora solani (Fusarium solani) • Common in nature, grows on plants • White to fuchsia colored mold grows in 3-5 days 30*C on fungal media. It is inhibited by cycloheximide (Mycosel media). • Produces septate hyaline hyphae, banana boat shaped conidia • Disease normal host: keratitis, nail and skin infections • Disease immune compromised: sinusitis, pneumonia, deep cutaneous lesions, and fungemia (unlike most fungi can be isolated from blood culture) • Random septate hyphae in fixed tissue • Confirm identification with culture and identification Banana boat shaped spore 108
  • 109. Scopulariopsis species • Found in soil and grows on plant • Infections: Nail infections in normal host and skin, sinusitis, pulmonary, brain abscess in immune suppressed host • White to sandy colored mold growing in 3-5 days at 30*C on a variety of fungal media • Round, spiny conidia that chain • Very resistant to antifungal agents including amphotericin B 109
  • 110. Penicillium species – > 300 species • Common in air, frequent laboratory contaminate • Cause of bread mold • Uncommon cause of human disease • Can appear as a culture contaminate • Blue/green colony grows in 3-5 days 30*C on a variety of fungal media • Hyphae with branched phialides that produce chains of conidia / appears like brush or bony hand 110
  • 111. The Mucorales Mucor, Rhizopus and related molds Fungi in the Order Mucorales • Broad, Hyaline hyphae without septation • Spores produced in a sack-like structure known as sporangium 111
  • 112. Mucorales • Found in soil, rotten fruit and vegetables • Unique infection: Acute rhinocerebral mucormycosis • Occurs in diabetics due to the elevated glucose enriching growth of fungus, usually caused by Mucor or Rhizopus species • Infection starts in single sided nasal sinusitis then spreads to the same side orbit of eye then invades the brain / high fatality rate • Pulmonary infection in immune suppressed: most commonly diabetics, corticosteroids and burn patients • Molds grow within 24-48 hr producing coarse aerial hyphae • Microscopically, broad, hyaline hyphae without septation seen with spores produced within a sporangium (sack) • Mince, do not grind tissue submitted for culture, the grinding will kill the tubular non-septate hyphal structures 112
  • 113. Rhizopus species Lichtheimia complex (Absidia) Mucor species No rhizoids Rhizoids Species within the Mucorales Distant rhizoid 113 Cunninghamella species Mold grows within 24-48 hrs, filling the plate with coarse aerial hyphae
  • 114. • Unique finding of 90˚ angle branching, with rare to no septations, ribbon like hyphae, can invade vessels and cause infarcts and thrombi • Faintly staining particularly on GMS stain • Distinguishing hyphae characteristic of Mucorales, from other hyphae in quite important, for Mucorales are more resistant than many fungi to the azoles and echinocandin antifungal agents Invades vessels and can cause infarcts and thrombi Mucorales in Tissue 114 90* angle branching
  • 115. 115