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MYCOLOGY
Mycology Update 2019
Margie Morgan, PhD, D(ABMM)
Yeast
Mycormycetes Conidial Molds
Dematiacious Hyaline
Yeast
1
Starting point
Yeast are:
• Unicellular / budding to produce daughter cells
• Growth on solid media usually white to beige and appear much like
bacterial colonies
• A few genera produce pigment (Rhodotorula = red)
• Some genera produce mucoid colonies (Cryptococcus)
Yeast colonies
on blood agar
2
Starting point
Molds:
• Produce hyphae and conidia [spores]
• Growth on solid media are downy, fluffy, cottony
• Most mold colonies produce pigment, which aid in identification
hyphae
spores
3
Specimen collection and Transport
• Fungi are very hardy organisms
• No requirement for special transport media
• Sterile containers necessary to prevent bacterial
contamination
• Numerous anatomic sites are appropriate for culture
• Respiratory specimens – sputum, bronchial lavage, brushings,
nasal sinuses
• Tissue biopsies
• Cutaneous - Skin scrapings, material from lesions
• Ocular
• Sterile body fluids, CSF
• Blood, bone marrow
4
Fungal Culture Media
• Sabouraud’s glucose agar (SABS)
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5
Fungal Culture Media
• Mycosel/Mycobiotic agar
• Selective SABS agar containing chloramphenicol and cycloheximide
• Used for selective culture of dermatophytes – fungi that cause skin,
hair and nail infections.
• Beware - Cycloheximide can suppress some species of pathogenic fungi
from growing: these include Trichosporon, Candida tropicalis and
Cryptococcus neoformans/gattii
• Brain heart infusion agar / with or without blood
• Primary recovery of all fungi
• Inhibits bacterial growth by adding chloramphenicol and gentamicin
• Addition of blood to agar to nurture for systemic fungi
6
•Inoculate specimen onto media
•Seal plates with tape to prevent culture from
environmental contamination and protect laboratory
workers from plates opening
•Incubate plates at 30˚C for 4 weeks
•If growth occurs - perform identification test methods
Processing of Fungal Cultures
7
Yeast Identification
• Biochemical reactions identify most but not all yeast species – there are both
manual and automated systems used for biochemical identification
• Newer methods with improved accuracy of identification include:
(1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight)
The yeast is superheated and converted into charged particles (proteins)
based on mass and charge form identification peaks
characteristic for each yeast species
(2) 16S 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 bacteria
and yeast
8
For many years, lactophenol cotton blue [LCB] adhesive tape preparation
used for mold identification.
LCB mounting medium consists of phenol, lactic acid, glycerol and
aniline cotton blue dye.
Clear adhesive tape touches mold colony, picking up fungal
hyphae/conidia. Prep then gently pressed into one drop of LCB
on a microscope slide.
Newer/ more complete identification
methods include:
MALDI-TOF and
16S rRNA sequencing
Mold Identification methods
9
Safety in the Mycology Laboratory
•All identification work on molds must be performed in a BSL-2 biosafety cabinet
with HEPA filtration
•Yeast identification testing can be performed on the bench top
•All fungal media plates should be sealed to prevent contamination and from
spores being released from plates.
10
Gram stain
•Yeast cells stain positive (blue) using Gram stain procedure
•Examine stain for budding cells to confirm that it is a yeast and not a
staining artifact.
•Yeast pseudo-hyphae can often be seen on Gram stain
•Mold (true hyphae) can be difficult to visualize on Gram stain
pseudohyphae
mold
pseudohyphae
Direct Exam of Specimens
X100 oil immersion
11
•KOH is used to detect yeast, pseudohyphae, and hyphae from skin, hair,
and nail specimens
•KOH dissolves the keratin in cellular material and frees fungal hyphae and
yeast cells – Careful! hyphae can be confused with cell borders
•KOH exams are difficult to interpret!
KOH – potassium hydroxide prep
X40
Light microscopy
12
• Yeast, pseudo-hyphae, and mycelial fungi bind with the
Calcofluor white stain and fluoresce
• Read using a fluorescence microscope (40X)
• More sensitive and specific than KOH preparation.
Calcofluor white stain
13
One drop of black ink is placed into one drop of
CSF on a microscope slide – examine using light
microscope (40X)
It is a “negative” stain – ie. stains the
background of prep not the yeast cell or capsule.
The clearing around the yeast is due to the
polysaccharide capsule produced by
Cryptococcus neoformans and C. gattii.
India ink is less sensitive than the Cryptococcal
antigen test for meningitis diagnosis
India Ink – specifically for Cryptococcus detection
14
Grocott’s Methenamine Silver Stain [GMS] – yeast and
hyphae stain grey to black. Will stain both living and dead yeast
and hyphae
• Observe the width of the hyphae, presence of hyphae septations and angle of
branching
• Observe the size and budding pattern of yeast, is pseudohyphae present ?
• Will explain on later slides how these observations can assist in identification
Examination of fungi in fixed
tissue
15
Positive staining hyphae are magenta – will also stain structures
containing carbohydrate macromolecules (glycogen,
glycoprotein, proteoglycans) Only stains living fungi.
Periodic Acid Schiff [PAS]
16
Mucicarmine stains the polysaccharide capsule of Cryptococcus
neoformans and C. gatti pink. Will also stain mucin in fixed tissue.
Mucicarmine [Mucin] stain
Fontana-Masson silver stain that detects melanin, can be used in the rare
situation when cell wall deficient cryptococcus organisms are encountered.
17
Best for description of
cellularity, not a specific
stain for fungi.
Hematoxylin and Eosin Stain
18
DIMORPHIC FUNGI
Important systemic pathogens with some unique
characteristics
19
What is a Dimorphic Fungus?
• Depending on temperature and conditions of environment, one fungi
demonstrates two forms:
• (1) Mycelial form - Hyphae and conidia (spores)
• Free living form in nature and growth in laboratory temperature <=30˚C
• (2) Yeast or yeast like form
• Parasitic phase found in human tissue or grown in the lab at temperatures >= 35˚C
• Body temperature allows mycelial form to morph to yeast form
Histoplasma capsulatum –
Mycelial form incubated at 30˚C
Histoplasma capsulatum –
Yeast form in tissue and 35˚C
20
Most Common Dimorphic Fungi
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis and C. posadasii
• Paracoccidioides brasiliensis complex
• Sporothrix schenckii
• Penicillium marneffei
(new name: Talaromyces marneffei)
21
Histoplasma
capsulatum• Epidemiology: World wide distribution / In USA endemic in Ohio, Missouri,
and Mississippi River valleys, over time has spread over a larger
geographic area
• Environmental source: Aerosolized bat guano, infection in Spelunkers
(cave explorers), bird droppings, and farmers
22
Histoplasmosis Disease
• 95% of infections are subclinical
• 5% infections: Pulmonary +/- dissemination, reactivation disease in the
elderly and immune suppressed
• Who gets progressive Histoplasmosis?
• HIV/AIDS
• Organ transplants
• Taking medications such as corticosteroids or TNF-inhibitors
• Infants
• Adults aged 55 and older
• Examination of bone marrow is useful in
diagnosing disseminated infections
• Mucocutaneous lesions are a unique &
common site of dissemination
23
Histoplasmosis rapid
diagnosis
• Antigen detection in urine
• Quantitative enzyme immunoassay
• Performed on random urine specimen
• Most sensitive (>=85%) for the diagnosis of disseminated and chronic
pulmonary Histoplasmosis
• Helpful in Histoplasma diagnosis in immune suppressed patients that
do not produce a detectable antibody response
• H. capsulatum antibody tests are available, but have mostly been
replaced by screening for antigen in urine
24
Histoplasma capsulatum Culture
• Incubated at 30˚C - Mold
• Requires 2-8 weeks to grow
• Colony - white to brown and cottony
• Microscopic appearance on scotch tape mount
• Tuberculate macroconidia that are large and round (8 – 16 µM)
• Small microconidia (2 - 4µM)
• Mold grows in nature, microconidia are inhaled / capable of penetrating deep into
the lung and beginning infection
• MALDI-TOF, DNA probe, or 16S rRNA sequencing to confirm
identification
• Confirmation test is needed because of look alike fungi, for example Sepedonium
species – which is usually considered not to be pathogenic
25
Histoplasma capsulatum Culture
• Incubated at 37*C - Yeast
• Requires 4-8 weeks to grow
• Colony – white to beige, creamy
• Microscopic appearance
• 2-4 um in size, oval to round
• Form found in human tissue
• Increase in temperature in human/laboratory induces the mycelial phase to undergo
transformation to the yeast phase
26
Histoplasma capsulatum in fixed tissue
• Infection begins by inhalation of
the microconidia
• Granulomas usually produced –
both caseating or non-caseating
• Can disseminate to organs of the
Reticuloendothelial System (RES) –
with high % of dissemination to bone marrow
27
Histoplasma capsulatum - Yeast
Usually intracellular within macrophages
Small yeast 2-4 um, regular in size, oval to round.
Appear to be encapsulated due to staining artifact – shrinkage
of the yeast away from the cell wall
Stains well with a variety of stains.
H & E PAS
Gram Wrights
28
Leishmania amastigote
Note: kinetoplast (mitochondrial
DNA) next to nucleus
Toxoplasma tachyzoites
oval to crescent in shape, no
capsule like clearing around cell
Histoplasma yeast
appear to have capsule
Beware of look-a-likes
29
Case History:
38 year old male
Newly diagnosed HIV disease/AIDS
CD4=3, HIV VL=220,000
Anemia
Thrombocytopenia
Bilateral pulmonary infiltrate
Bone marrow aspiration
Stain and culture
30
The mold phase is identical to H. capsulatum.
Difference is the size of the yeast cell:
Note yeast cell of H. duboisii is 8-10 um,
which is 2X the size of H. capsulatum yeast cell.
Endemic area in Central Africa
Primary infection is in skin and bone
Unusual variant of Histoplasma –
variate duboisii
31
Blastomyces dermatitidis
• Epidemiology
• Ohio and Mississippi River valleys
• No association with animal or activity
• Found in forested areas and river banks
• Primarily a pulmonary pathogen with small
% of patients with dissemination to skin and bone occurring
most often in immune suppressed patients
• Well demarcated skin lesions can
be seen in disseminated cases of
Blastomycosis.
32
Blastomyces dermatitidis
• Culture at 30˚C - mold
• Grows in 2-3 weeks
• Fluffy white – buff colored mold, prickly
• Microscopic - pear shaped conidia at the end of supporting hyphae –
looks like a lollipop
• Look-alike fungus – Chrysosporium species and possibly others (?)
• Require MALDI-TOF, DNA probe of sequencing to confirm
identification
Blastomyces
Chrysosporium
33
• Culture at 35*C - yeast
• Slow growing yeast colony (4 weeks)
• Large yeast cell is 8-20 um in size
• Unique “Broad Based Budding” pattern and thick/double contoured wall.
Blastomyces dermatitidis
Broad based bud
Double contoured wall
34
Blastomyces dermatitidis
Histopathology
• Mixed pyogenic and granulomatous inflammation is
observed in tissue
• Broad based budding yeast cells
35
Coccidioides immitis and C.
posadasii
• New species: Coccidioides posadasii is genetically
related to C. immitis.
• The two species are found in different geographic regions,
C.immitis (California) and C. posadasii (outside CA)
• Identical disease process
• Endemic in SW USA (San Joaquin Valley), Mexico, and
South America, in areas known as the Sonoran life
zone / warm and desert sands
• Infection is from inhalation of fungal particles
(arthroconidia) found in the sandy soil
36
Coccidioidomycosis
• 60% of infections are asymptomatic
• 30% with limited pulmonary diseases
• The remaining 5-10% result in chronic disease, progressive
pulmonary or disseminated infections
• Tropism to the Central Nervous System (CNS)
• Dissemination to CNS carries a high fatality rate.
• Risk factors for severe or disseminated coccidioidomycosis include:
• African-American race or Filipino ethnicity, HIV/AIDS, use of
immunosuppressive medications, organ transplant, diabetes
mellitus, or pregnancy
37
Serologic diagnosis of Coccidiodomycosis
• Enzyme immunoassay (EIA): 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
• Complement Fixation (CF): detects IgG antibodies and allows for
assessment of disease severity.
38
Coccidioides Culture
• Culture at 30˚C - Mold
• Growth in 2-3 days, colony starts waxy and becomes wooly in around 7–
10 days
• Microscopic (40X) one observes areas with septate hyphae and thick
walled alternating barrel shaped arthroconidia
• Barreled arthroconidia breakoff and become the infectious particle in
nature
• Beware! Mature culture is hazardous to laboratory personnel
40X 2-3 days 7-10 days
39
Look-a-likes
• Malbranchea species can look somewhat like C. immitis and
C. posadasii under the microscope /
• Must confirm identification using MALDI-TOF, molecular probe or
sequencing Coccidioides Malbranchea
40
Coccidioides Histopathology
• No yeast cell produced, instead: Thick walled spherules (10 – 80 uM)
with endospores are the growth structure in tissue.
• Spherules can be in all stages of development- fragmented spherules
to well formed with endospores
• Granulomatous inflammation with caseation is usually observed
41
Case History:
75 year old male
Right Pleural Effusion
Lung nodule
PMH: Gout, hypertension, anemia,
obesity
Fungal serology testing:
Histoplasma urine Ag: negative
Cryptococcal Ag: negative
Coccidioides IgM: Indeterminate
Coccidioides IgG: Weak Positive
Titer 1:4
Epi: Lives on ranch in Moorpark
(Simi Valley)
Biopsy of lung
nodule stain/ GMS
Culture of lung
nodule
42
• 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 or nasal mass lesions
Coccidioides is not the only spherule forming
organism!
43
Paracoccidioides brasiliensis complex
• South American Blastomycosis – 80% of cases reported
from Brazil
• Most prevalent systemic fungal infection in Latin America
• Infection acquired from inhaling infectious particle from soil
• >95% of infections in males, possibly due to estrogen
inhibition of the fungus mycelial to yeast transformation
• Disease presentation:
1. Pneumonia
2. Disseminated infection
3. Extrapulmonary lesions on the face and oral mucosa
44
Paracoccidioides
• Cultures at 30*C – mold, cultures usually not done, slow growth and
nonspecific sporulation
• Culture at 37˚C - yeast
• Slow growing yeast – 3 weeks
• Large (10 – 30um), thick walled, with
2 or more tear drop daughter buds (2–10 um)
• Unique multiply budding yeast cell known as the Mariner’s wheel or
Pilot’s wheel yeast
45
Tissue Exam of
Paracoccidioides brasiliensis complex
• Granulomatous inflammation
with giant cells formed in tissue
GMS
GMS
46
Sporothrix schenckii
• Sporotrichosis
• Cutaneous inoculation from penetrating
injury, cut or scrape with thorns or sphagnum moss
• Known as “Rose gardener’s disease”
• Usually begins as skin lesion w/wo ulceration and leads to a
subcutaneous infection
• Lesion can progress/ lymphocutaneous spread, possible
dissemination to bone and other organs
• Pulmonary and CNS infections occur but rare
47
Sporothrix schenckii
• 30˚C culture - mold grows in 3 -5 days as
beige mold that eventually turns brown/black
• Microscopic: septate hyphae with
conidia in daisy wheel pattern or rosette
• 37˚C culture – yeast grows in 7 days as small
• oval and elongated yeast cells,2 – 5 µm, described as cigar bodies
48
Sporothrix schenckii Histology
• Pyogenic to granulomatous inflammation
• Hard to find yeast cells in human tissue
• If seen, elongated yeast, cigar shape
• More commonly seen is an Asteroid body known as Splendore-
Hoeppli phenomenon (SHP)
• SHP are not only found in Sporothrix infections, also seen with:
• Mucormycetes (Mucor, Rhizopus)
• Aspergillus
• Blastomyces
• Candida spp
49
•Disease:
• Skin lesions endemic in tropics and SE Asia
• Pneumonia or systemic infection in HIV/AIDS
•Mold is a green colony with red diffusible pigment, growing in 2-3 days
•Yeast like cells in culture at 30* C and in fixed tissue slides
Penicillium (Talaromyces)
marneffei
50
SUBCUTANEOUS FUNGAL
INFECTIONS
Mycetoma
Chromoblastomycoses
Phaeohyphomycosis
51
Mycetoma
• Found in hot temperate parts of the world – the fungi
grow in organic debris in soil
• Infection begins with trauma implanting the fungi into
the subcutaneous tissue
• Three criteria define mycetoma:
-Swollen extremity from lesion progression
-Draining sinuses
-Sulfur granules observed in tissue and drainage from
sinus tract
Two types of mycetoma:
1. Actinomycotic – caused by higher bacteria
2. Eumycotic – caused by black molds
52
Actinomycotic Mycetoma
• 98% of cases
• Nocardia species most common agent of infection
• Sulfur granules are formed in tissue. The granules vary in
color depending on the Nocardia species causing
infection
• The granules contain a matrix of the filamentous bacteria
53
Edge of sulfur granule shows thin filamentous bacteria: How can you
tell if it is Nocardia? Culture and staining will differentiate.
•Nocardia is modified (partial) acid fast [PAF] stain positive and
grows aerobically
•Actinomyces is PAF stain negative and grows best anaerobically
Careful! Sulfur granule caused by
infection with Actinomyces species looks
identical to ones formed by Nocardia.
Actinomycotic sulfur granule
Actinomyces
54
Gram stain = filamentous Gram positive bacilli which can be poorly
staining and appear speckled.
Positive [red] on the Modified Kinyoun acid fast stain.
Modified (Partial)
Kinyoun acid-fast stain
Gram stain
Nocardia
55
Grows in 3-5 days on many agars including
SABs and 5% Sheep’s blood agar
Colony is dry/crumbly with a musty odor
Total of 85 species of Nocardia with the
members of Nocardia asteroids complex most
common
Identification: MALDI -TOF
16s rRNA gene sequencing
Nocardia species – besides Mycetoma, Nocardia can
also cause Pulmonary and Brain infection
56
Eumycotic Mycetoma
Infection with pigmented/black fungi (dematiaceous)
-Numerous black fungi species found in soil and debris
-Cause @ 2% of mycetoma cases
-Traumatic implantation of the fungus into the subcutaneous
tissue
Notice the thick
hyphae on the
edge of the granule
57
Thermoactinomycetes
• Gram positive filamentous rods related to Nocardia species
• Thermoactinomyces and Saccaropolyspora
• Flourish in areas of high humidity and high temperature (40*-60*C)
• Grow in agricultural environments – 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
58
Chromoblastomycosis
• Three characteristics:
• Wart like lesions (scarred and nodular) in subcutaneous tissue
• Muriform bodies observed in fixed tissue slides
• Infection caused by black pigmented fungi (dematiaceous)
• Skin abrasion and implantation of fungi into tissue
Naturally brown in color, septate
structures known as muriform
bodies or copper pennies is the
unique structure found in tissue –
they break up and pieces extend
into the subcutaneous tissue
59
Prototheca wickerhamii
• Algae without chlorophyll
• Causes skin lesions & nodules
• Most common in patients with suppressed immune system
• Compare morula of Protothecosis to muriform body of Chromoblastomycoses
• Morula of Protothecosis is Not naturally brown in color
Prototheca
morula
Copper penny
60
Phaeohyphomycosis
Traumatic implantation of dark fungi into subcutaneous tissue
• Infections usually nodular lesions/cysts most common
with/without dissemination
• Infections usually confined to skin but can disseminate,
particularly to brain
• In fixed tissue, dark swollen hyphae and yeast like cells
61
Black mold / Dematiaceous mold
•Black colored colonies; both topside and the
reverse [underside of colony] is black
•Brown colored hyphae and spores due to melanin
production
•Most common molds to grow in areas with water
damage
Most common black molds include:
Cladophialophora carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Rhinocladiella species
62
Black Molds /Dematiacious molds
• Numerous species
• Difficult to identify requiring MALDI-TOF or 16S rRNA sequencing
• All have one of four types of sporulation
• Rhinocladiella-like
• Cladosporium-like
• Phialophora-like
• Acrotheca-like
Rhinocladiella
Cladosporium
Phialophora Acrotheca
63
Exophiala species
Most common Black Molds
Cladophialophora bantiana -
Associated with dissemination to
brain
64
Wangiella dermatitidis
Phialophora verrucosa
65
Alternaria species– Opportunistic - sinusitis in normal
host, possible dissemination in immune suppressed.
Found in nature
66
Curvularia lunata
Center cell is the largest
Opportunistic species/ disseminated infections rare in patient
with aplastic anemia of a bone marrow transplant patient/
erythematous skin lesions
67
Exserohilum rostrum
• Associated with an outbreak of compound pharmaceutical [steroid] product
contaminated with dust containing Exserohilum during manufacturing
• Injected into lumbar spine and knee joints for pain management and led to
serious and fatal infections:
• Meningitis
• Spinal abscess
• Synovial infections
68
Scedosporium complex/ previously Pseudallescheria boydii complex
Cat fur-like gray colony / growth in 3 – 5 days
Lollipop like spore production
Opportunistic pulmonary pathogen
Can invade vessels and lead to infarcts
Difficult to distinguish from Aspergillus in fixed tissue stains
Intrinsically resistant to Amphotericin B
69
IMPORTANT YEAST CAUSING
HUMAN INFECTION
Candida species
Cryptococcus neoformans
Cryptococcus gattii
Trichosporon species
70
Candida species
• Candida species (@ 10 species infect humans)
• Yeast are usually found as normal flora in GI, GU and skin
sites
• Candida can be opportunistic pathogens involving skin or
mucous membranes from excessive exposure to moisture,
antibiotics, or immune suppression
• Thrush, vaginitis, skin lesions, nail, diaper rash
• Also can cause more serious infections such as fungemia,
endocarditis, and systemic tissue infection.
71
Important Candida species
• Candida albicans – most common species causing @ 60% of Candida
infections
• Candida glabrata, C. krusei*(Pichia kudriavzevi) and C. tropicalis are
causing higher % of infections than in past
• C. glabrata and C. tropicalis are likely to be resistant to fluconazole
• C. krusei (P. kudriavzevi) is intrinsically resistant to fluconazole
• Candida auris – newly recognized as problematic yeast in the environment
and nosocomial infections
• Intrinsically resistant to fluconazole and additional anti-fungals, susceptible to
echinocandins,
• Can be difficult to identify, must use MALDI-TOF or sequencing
• Candida parapsilosis is a pathogen of children and common in IV line
infections (forms biofilms on plastics)
• Susceptible to fluconazole
72
Candida species
• Grow in 24 – 48 hours at 30-35*C
• Growth on many agars SABS, IMA, Blood agar… others
• Bacteria-like colony – pasty white
• Oval shaped yeast @ 7-8 um in size**
• Form pseudohyphae not true hyphae (Yeast do not detach and form
elongated hyphae)
• Identify using biochemicals, MALDI-TOF, or Sequencing
• Exception: **Candida glabrata is only Candida species that is small @ 4
µM in size and does NOT form pseudohyphae – this species breaks all the
Candida rules
pseudohyphae
73
Candida albicans
Identification
• Germ tube formation
• Incubate yeast in serum for 3-4 hrs at 35 ˚C
• Extension from yeast cell “tube” positive for C. albicans
• If incubate >4 hrs – C. tropicalis can have false positive reaction
• C. dubliniensis can also form germ tubes
• Chlamydospore formation
• Growth on cornmeal agar at 48 hrs
• Rudimentary structures unique to
C. albicans known as chlamydospore
chlamydospore
74
ChromAgar for the identification of
Candida species: different Candida
spp turn unique colors on this agar
based on reactions on chromogenic
substrates.
Susceptibility testing of yeast is much like bacteria, using
broth dilution and Etest methods.
75
Candida Histopathology
• Pyogenic to granulomatous inflammation
• Most Candida species have yeast cells (8 um) and pseudohyphae
• Candida glabrata - smaller yeast cells (4 um) and absence of
pseudohyphae
GMS stain of Candida glabrataCandida species not glabrata
76
Cryptococcus
neoformans
• In nature forms a 2um non-encapsulated yeast cell.
• Associated with bird droppings (esp. pigeon),
C neoformans is enriched by the nitrogen in the heaped droppings.
• Non-encapsulated yeast cells are inhaled – travel through the
pulmonary system with hematogenous spread to brain and meninges
(tropism)
• Infects mostly compromised hosts – one of the major AIDS defining
infections
77
Cryptococcus gattii – a close relative of C. neoformans
• Isolated first from forested areas of the Pacific Northwest
(British Columbia, Washington, Oregon and California)
found in soil debris and tree species
• Infection of both normal and immune suppressed hosts
• Primarily a pulmonary disease [Cryptococcoma] but can disseminate and
develop meningitis
• Culture, biochemical & staining identical to C. neoformans
• Defining reactions –
• L Canavanine glycine bromthymol blue medium –
C. gatti = blue C. neoformans = colorless
MALDI-TOF and 16sRNA sequencing will also ID both species
78
Cryptococcus neoformans
and C. gattii
• Irregularly sized yeast cells (2 – 20 um)
• Virulence factor: Polysaccharide capsule
• Polysaccharide capsule is used in diagnostic testing:
• India ink exam- CSF placed in one drop of black ink. It is a
negative staining method – background is stained but not the
polysaccharide capsule
• Cryptococcal antigen test – Titer of capsular polysaccharide,
leached off of the yeast cell and detected in CSF or serum
• More sensitive test than India ink exam
• Test for diagnosis or can follow recovery with decreasing titer of
polysaccharide
79
Grows on mycologic agars
as mucoid colonies due to
capsular polysaccharide
Both C. neoformans & C. gattii form
brown colonies on Birdseed agar
Gram stain /
stains as blue blobs due
to presence of capsule
Positive urease
enzyme reaction
Cryptococcus neoformans and gattii
Negative
80
Mucicarmin stains the
capsular polysaccharide of
Cryptococcus Pneumocystis jiroveci (yeast like
fungus) could be confused with
C. neoformans – Careful! Central
nuclear staining in pneumocystis
C. neoformans/ C. gattii – no
nuclear staining
Cryptococcus vs. Pneumocystis
Pneumocystis froth on H & E
81
Pneumocystis jiroveci
• Yeast like fungus
• Used to be named Pneumocystis carinii and considered a
protozoan (parasite)
• Causes pneumonia in the immunocompromised host (PCP)
particularly HIV/AIDS
• Diagnosis: staining of Bronchial lavage, lung biopsy, induced
sputum using direct fluorescent antibody (DFA) and GMS.
DFA
82
Trichosporon
• T. asahii most common species
• Commonly inhabit the soil
• Colonize the skin/ gastrointestinal tract of humans.
• Rectangular shaped yeast cells
• Cause of superficial infection - white piedra, a distal infection of the hair shaft,
• Commonly reported cause of disseminated yeast infections in humans
• Rare, even among patients with impaired host defenses:
• Most usually recovered from patients with corticosteroid use, solid tumors, HIV/AIDS, and
intravascular devices, including catheters and prosthetic heart valves
• Mortality 50 – 80%
83
CUTANEOUS AND
SUPERFICIAL MYCOSES
Malassezia furfur
Dermatophytes
Microsporum species
Trichophyton species
Epidermophyton floccosum
84
Malassezia furfur
• Most superficial of the dermatomycoses
• M. furfur can be normal flora on human skin
• More common on oily skin or patients with high use of skin oils
• Diseases:
• Skin: macules, papules, patches, plaques on chest back and shoulders
with either hypo or hyper pigmentation – does not invade into deeper
tissues – known as Pityriasis versicolor
• Fungemia: caused by tunneling in with IV lipid feeding lines (parenteral
nutrition) – in neonates or rarely adults
85
Malassezia furfur
• Lipophilic yeast – oil required for growth
• Media for isolation must contain oil or use an oil overlay
• Small budding yeast 2 – 4 µm with collarette (appears like necklace at
junction of mother and daughter yeast cell)
• In tissue described as “Spaghetti and Meatballs”
Spaghetti and meatballs
86
Dermatophytes – Ringworm infections
• Hair, skin and nail infections
• 3 genera of fungi
• Microsporum species (many)
• Epidermophyton floccosum
• Trichophyton species (many)
• Disease described by area of the body infected: For example:
tinea capitis (head), t. pedis (foot)
• Usually a clinical diagnosis not requiring culture
• KOH prep or Calcofluor white prep can be used to visualize
fungal hyphae from skin scrapings
87
Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence –
thin fungal hyphae
88
Microsporum canis
Ringworm infection acquired infected dog or cat
White colony/ yellow on reverse (back) of colony
Tuberculate thick walled macroconidia [spiny projections]
Few if any microconidia (small spores)
89
Microsporum gypseum –
Mostly skin infection from exposure to contaminated soil
Sandy colored colony,
Large macroconidia are produced, no microconidia producerd
90
Trichophyton rubrum
Mostly infects skin and hair
after exposure to fomites, such
as wet towels or hair brushes
White colony. The back side of
the colony has an intense red
pigment diffuses into the media
Pencil shaped macroconidia/
many micro-conidia
(different than Microsporidium)
Red diffusible pigment
91
Trichophyton
tonsurans
White colony
Light yellow reverse
No macroconidia
only microconidia
produced with rare
ballooning microconidia
Primary cause of
epidemic scalp
ringworm in children
92
Epidermophyton floccosum
Most often cause
infections in skin
and nails
Khaki green colored
colony
Beaver tail shaped
large macroconidia
– no microconidia
93
Opportunistic Hyaline
Molds
Infections in the immune suppressed host
or special circumstances
94
Opportunistic Hyaline Fungi
• Hyaline – no color to the hyphae
• Regular septations in hyphae
• Grow on a variety of agar media
in 3-5 days at 35 or 30˚C
• Identification based on growth rate,
color/texture of colony and
microscopic structures
• Definitive speciation: ID / MALDI-TOF or 16sRNA sequencing
95
Aspergillus species
• Ubiquitous in nature @ 200 species
• Hyaline with regular septation
• Numerous round conidia produced
• Most often pulmonary infection / disseminate in immune suppressed
• Neutropenia can predispose to infection
• Branches at 45 degree angle in fixed tissue
• Invade vessels, cause thrombosis & infarctions
Septation
96
Aspergillus Structure
97
Serology Tests Supportive not Conclusive for
Diagnosis
Aspergillus Galactomannan Enzyme Immunoassay
• Detects circulating Aspergillus antigen in the blood and/or bronchial lavage, most helpful in
disseminated infections
• +/- sensitivity and specificity (PPV 68%/ NPV 96%)
• False positive reactions (10%) with Piperacillin/Tazobactam therapy, infection with H.
capsulatum, and rice and pasta ingestion
(1,3)–Beta-D-glucan assay
• Detected in serum or BAL from the following pathogens: Candida spp., Acremonium,
Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum, Trichosporon
spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis jiroveci.
• No reaction for Cryptococcus or Zygomycetes
• High values more meaningful, false positive reaction from the environment contaminated
with many of these fungi
98
Aspergillus fumigatus
•Ubiquitous airborne fungus, inhale conidia in nature
•One of most common species causing pulmonary
infection in immune suppressed, can disseminate from
lung to other organs
•Blue/Green colony
•Phialides produce colorless spores, directed upward
99
Aspergillus flavus
•Normally found in cereals, grains, legumes
•Same disease potential as A. fumigatus/
pulmonary infection with possible
dissemination
•Green/yellow colony
•Green/yellow hue to conidia produced on
phialides that surround the swollen vesicle
100
Aspergillus niger
•Black colony – visible black fruiting
heads
•Contaminate fruits and vegetables/
found in soil
•Invasive disease uncommon, commonly
isolated in ear infections
•Black conidia supported by phialides
that surround the vesicle
101
Aspergillus terreus •Infection primarily in the immune
compromised host
•Sandy colored colony
•Isolated from soil
•Colorless conidia supported by phialids,
headed upward
•Aleurioconidia structure produced
•Intrinsic resistance to Amphotericin B
102
Aspergillus – fruiting head seldom seen in fixed tissue
usually dichotomous (continuous) branching septate hyphae which
branch at a 45* angle
Can appear much like that of
Scedosporium species,
Growth in culture can
differentiate the two fungi.
103
Fusarium species
•Common in nature/plants
•Fuchsia colored colony
•Disease related to immune status of host/
neutropenia
•Infections reported:
•Disseminated disease in bone marrow
transplants and corneal infections in contact lens
wearers
•Random hyphae in fixed tissue
Banana boat shaped spore
104
Scopulariopsis species
•Found in soil and grows on plant
•Infections: Nail, skin, sinusitis, pulmonary and may
disseminate in an immune suppressed host
•Very resistant to antifungal agents
105
Penicillium species –
•One of the most common molds in the environment
•Cause of bread mold
•Uncommon cause of human disease
•Can appear as culture contaminate
•Branching hyphae with conidia production
•Appears like a bony hand
106
MUCORMYCOSIS/
ZYGOMYCOSIS
Fungi in the Order Mucorales
Hyaline
Broad hyphae without septation
Sporangium (sack) with spores
107
Mucormycosis/Zygomycosis
• Found in soil, rotten fruit and vegetables
• Rhinocerebral mucormycosis - classic infection
• Diabetics, the elevated glucose enriches fungal
growth
• Infection begins in nasal sinus - spreads to orbit of eye
– then brain, high fatality rate
• Broad, hyaline, hyphae without septation produced
• Culture grows within 24 -48 hrs, producing coarse aerial
hyphae
• Mince do not grind infected tissue for culture, the
grinding will kill the aseptate hyphae
108
Rhizopus species Absidia (Lichtheimia complex)
Mucor species
No rhizoids
Rhizoids
Order Mucorales
Distant rhizoid
109
90˚ angle branching and also random branching,
no septation, ribbon like hyphae
Invades vessels and can cause
infarcts and thrombi
Zygomycete/ Order Mucorales
110

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Mycology Update 2019

  • 1. MYCOLOGY Mycology Update 2019 Margie Morgan, PhD, D(ABMM) Yeast Mycormycetes Conidial Molds Dematiacious Hyaline Yeast 1
  • 2. Starting point Yeast are: • Unicellular / budding to produce daughter cells • Growth on solid media usually white to beige and appear much like bacterial colonies • A few genera produce pigment (Rhodotorula = red) • Some genera produce mucoid colonies (Cryptococcus) Yeast colonies on blood agar 2
  • 3. Starting point Molds: • Produce hyphae and conidia [spores] • Growth on solid media are downy, fluffy, cottony • Most mold colonies produce pigment, which aid in identification hyphae spores 3
  • 4. Specimen collection and Transport • Fungi are very hardy organisms • No requirement for special transport media • Sterile containers necessary to prevent bacterial contamination • Numerous anatomic sites are appropriate for culture • Respiratory specimens – sputum, bronchial lavage, brushings, nasal sinuses • Tissue biopsies • Cutaneous - Skin scrapings, material from lesions • Ocular • Sterile body fluids, CSF • Blood, bone marrow 4
  • 5. Fungal Culture Media • Sabouraud’s glucose agar (SABS) l l p u r p o s e F u n g a l m e 5
  • 6. Fungal Culture Media • Mycosel/Mycobiotic agar • Selective SABS agar containing chloramphenicol and cycloheximide • Used for selective culture of dermatophytes – fungi that cause skin, hair and nail infections. • Beware - Cycloheximide can suppress some species of pathogenic fungi from growing: these include Trichosporon, Candida tropicalis and Cryptococcus neoformans/gattii • Brain heart infusion agar / with or without blood • Primary recovery of all fungi • Inhibits bacterial growth by adding chloramphenicol and gentamicin • Addition of blood to agar to nurture for systemic fungi 6
  • 7. •Inoculate specimen onto media •Seal plates with tape to prevent culture from environmental contamination and protect laboratory workers from plates opening •Incubate plates at 30˚C for 4 weeks •If growth occurs - perform identification test methods Processing of Fungal Cultures 7
  • 8. Yeast Identification • Biochemical reactions identify most but not all yeast species – there are both manual and automated systems used for biochemical identification • Newer methods with improved accuracy of identification include: (1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight) The yeast is superheated and converted into charged particles (proteins) based on mass and charge form identification peaks characteristic for each yeast species (2) 16S 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 bacteria and yeast 8
  • 9. For many years, lactophenol cotton blue [LCB] adhesive tape preparation used for mold identification. LCB mounting medium consists of phenol, lactic acid, glycerol and aniline cotton blue dye. Clear adhesive tape touches mold colony, picking up fungal hyphae/conidia. Prep then gently pressed into one drop of LCB on a microscope slide. Newer/ more complete identification methods include: MALDI-TOF and 16S rRNA sequencing Mold Identification methods 9
  • 10. Safety in the Mycology Laboratory •All identification work on molds must be performed in a BSL-2 biosafety cabinet with HEPA filtration •Yeast identification testing can be performed on the bench top •All fungal media plates should be sealed to prevent contamination and from spores being released from plates. 10
  • 11. Gram stain •Yeast cells stain positive (blue) using Gram stain procedure •Examine stain for budding cells to confirm that it is a yeast and not a staining artifact. •Yeast pseudo-hyphae can often be seen on Gram stain •Mold (true hyphae) can be difficult to visualize on Gram stain pseudohyphae mold pseudohyphae Direct Exam of Specimens X100 oil immersion 11
  • 12. •KOH is used to detect yeast, pseudohyphae, and hyphae from skin, hair, and nail specimens •KOH dissolves the keratin in cellular material and frees fungal hyphae and yeast cells – Careful! hyphae can be confused with cell borders •KOH exams are difficult to interpret! KOH – potassium hydroxide prep X40 Light microscopy 12
  • 13. • Yeast, pseudo-hyphae, and mycelial fungi bind with the Calcofluor white stain and fluoresce • Read using a fluorescence microscope (40X) • More sensitive and specific than KOH preparation. Calcofluor white stain 13
  • 14. One drop of black ink is placed into one drop of CSF on a microscope slide – examine using light microscope (40X) It is a “negative” stain – ie. stains the background of prep not the yeast cell or capsule. The clearing around the yeast is due to the polysaccharide capsule produced by Cryptococcus neoformans and C. gattii. India ink is less sensitive than the Cryptococcal antigen test for meningitis diagnosis India Ink – specifically for Cryptococcus detection 14
  • 15. Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Will stain both living and dead yeast and hyphae • Observe the width of the hyphae, presence of hyphae septations and angle of branching • Observe the size and budding pattern of yeast, is pseudohyphae present ? • Will explain on later slides how these observations can assist in identification Examination of fungi in fixed tissue 15
  • 16. Positive staining hyphae are magenta – will also stain structures containing carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) Only stains living fungi. Periodic Acid Schiff [PAS] 16
  • 17. Mucicarmine stains the polysaccharide capsule of Cryptococcus neoformans and C. gatti pink. Will also stain mucin in fixed tissue. Mucicarmine [Mucin] stain Fontana-Masson silver stain that detects melanin, can be used in the rare situation when cell wall deficient cryptococcus organisms are encountered. 17
  • 18. Best for description of cellularity, not a specific stain for fungi. Hematoxylin and Eosin Stain 18
  • 19. DIMORPHIC FUNGI Important systemic pathogens with some unique characteristics 19
  • 20. What is a Dimorphic Fungus? • Depending on temperature and conditions of environment, one fungi demonstrates two forms: • (1) Mycelial form - Hyphae and conidia (spores) • Free living form in nature and growth in laboratory temperature <=30˚C • (2) Yeast or yeast like form • Parasitic phase found in human tissue or grown in the lab at temperatures >= 35˚C • Body temperature allows mycelial form to morph to yeast form Histoplasma capsulatum – Mycelial form incubated at 30˚C Histoplasma capsulatum – Yeast form in tissue and 35˚C 20
  • 21. Most Common Dimorphic Fungi • Histoplasma capsulatum • Blastomyces dermatitidis • Coccidioides immitis and C. posadasii • Paracoccidioides brasiliensis complex • Sporothrix schenckii • Penicillium marneffei (new name: Talaromyces marneffei) 21
  • 22. Histoplasma capsulatum• Epidemiology: World wide distribution / In USA endemic in Ohio, Missouri, and Mississippi River valleys, over time has spread over a larger geographic area • Environmental source: Aerosolized bat guano, infection in Spelunkers (cave explorers), bird droppings, and farmers 22
  • 23. Histoplasmosis Disease • 95% of infections are subclinical • 5% infections: Pulmonary +/- dissemination, reactivation disease in the elderly and immune suppressed • Who gets progressive Histoplasmosis? • HIV/AIDS • Organ transplants • Taking medications such as corticosteroids or TNF-inhibitors • Infants • Adults aged 55 and older • Examination of bone marrow is useful in diagnosing disseminated infections • Mucocutaneous lesions are a unique & common site of dissemination 23
  • 24. Histoplasmosis rapid diagnosis • Antigen detection in urine • Quantitative enzyme immunoassay • Performed on random urine specimen • Most sensitive (>=85%) for the diagnosis of disseminated and chronic pulmonary Histoplasmosis • Helpful in Histoplasma diagnosis in immune suppressed patients that do not produce a detectable antibody response • H. capsulatum antibody tests are available, but have mostly been replaced by screening for antigen in urine 24
  • 25. Histoplasma capsulatum Culture • Incubated at 30˚C - Mold • Requires 2-8 weeks to grow • Colony - white to brown and cottony • Microscopic appearance on scotch tape mount • Tuberculate macroconidia that are large and round (8 – 16 µM) • Small microconidia (2 - 4µM) • Mold grows in nature, microconidia are inhaled / capable of penetrating deep into the lung and beginning infection • MALDI-TOF, DNA probe, or 16S rRNA sequencing to confirm identification • Confirmation test is needed because of look alike fungi, for example Sepedonium species – which is usually considered not to be pathogenic 25
  • 26. Histoplasma capsulatum Culture • Incubated at 37*C - Yeast • Requires 4-8 weeks to grow • Colony – white to beige, creamy • Microscopic appearance • 2-4 um in size, oval to round • Form found in human tissue • Increase in temperature in human/laboratory induces the mycelial phase to undergo transformation to the yeast phase 26
  • 27. Histoplasma capsulatum in fixed tissue • Infection begins by inhalation of the microconidia • Granulomas usually produced – both caseating or non-caseating • Can disseminate to organs of the Reticuloendothelial System (RES) – with high % of dissemination to bone marrow 27
  • 28. Histoplasma capsulatum - Yeast Usually intracellular within macrophages Small yeast 2-4 um, regular in size, oval to round. Appear to be encapsulated due to staining artifact – shrinkage of the yeast away from the cell wall Stains well with a variety of stains. H & E PAS Gram Wrights 28
  • 29. Leishmania amastigote Note: kinetoplast (mitochondrial DNA) next to nucleus Toxoplasma tachyzoites oval to crescent in shape, no capsule like clearing around cell Histoplasma yeast appear to have capsule Beware of look-a-likes 29
  • 30. Case History: 38 year old male Newly diagnosed HIV disease/AIDS CD4=3, HIV VL=220,000 Anemia Thrombocytopenia Bilateral pulmonary infiltrate Bone marrow aspiration Stain and culture 30
  • 31. The mold phase is identical to H. capsulatum. Difference is the size of the yeast cell: Note yeast cell of H. duboisii is 8-10 um, which is 2X the size of H. capsulatum yeast cell. Endemic area in Central Africa Primary infection is in skin and bone Unusual variant of Histoplasma – variate duboisii 31
  • 32. Blastomyces dermatitidis • Epidemiology • Ohio and Mississippi River valleys • No association with animal or activity • Found in forested areas and river banks • Primarily a pulmonary pathogen with small % of patients with dissemination to skin and bone occurring most often in immune suppressed patients • Well demarcated skin lesions can be seen in disseminated cases of Blastomycosis. 32
  • 33. Blastomyces dermatitidis • Culture at 30˚C - mold • Grows in 2-3 weeks • Fluffy white – buff colored mold, prickly • Microscopic - pear shaped conidia at the end of supporting hyphae – looks like a lollipop • Look-alike fungus – Chrysosporium species and possibly others (?) • Require MALDI-TOF, DNA probe of sequencing to confirm identification Blastomyces Chrysosporium 33
  • 34. • Culture at 35*C - yeast • Slow growing yeast colony (4 weeks) • Large yeast cell is 8-20 um in size • Unique “Broad Based Budding” pattern and thick/double contoured wall. Blastomyces dermatitidis Broad based bud Double contoured wall 34
  • 35. Blastomyces dermatitidis Histopathology • Mixed pyogenic and granulomatous inflammation is observed in tissue • Broad based budding yeast cells 35
  • 36. Coccidioides immitis and C. posadasii • New species: Coccidioides posadasii is genetically related to C. immitis. • The two species are found in different geographic regions, C.immitis (California) and C. posadasii (outside CA) • Identical disease process • Endemic in SW USA (San Joaquin Valley), Mexico, and South America, in areas known as the Sonoran life zone / warm and desert sands • Infection is from inhalation of fungal particles (arthroconidia) found in the sandy soil 36
  • 37. Coccidioidomycosis • 60% of infections are asymptomatic • 30% with limited pulmonary diseases • The remaining 5-10% result in chronic disease, progressive pulmonary or disseminated infections • Tropism to the Central Nervous System (CNS) • Dissemination to CNS carries a high fatality rate. • Risk factors for severe or disseminated coccidioidomycosis include: • African-American race or Filipino ethnicity, HIV/AIDS, use of immunosuppressive medications, organ transplant, diabetes mellitus, or pregnancy 37
  • 38. Serologic diagnosis of Coccidiodomycosis • 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 • Complement Fixation (CF): detects IgG antibodies and allows for assessment of disease severity. 38
  • 39. Coccidioides Culture • Culture at 30˚C - Mold • Growth in 2-3 days, colony starts waxy and becomes wooly in around 7– 10 days • Microscopic (40X) one observes areas with septate hyphae and thick walled alternating barrel shaped arthroconidia • Barreled arthroconidia breakoff and become the infectious particle in nature • Beware! Mature culture is hazardous to laboratory personnel 40X 2-3 days 7-10 days 39
  • 40. Look-a-likes • Malbranchea species can look somewhat like C. immitis and C. posadasii under the microscope / • Must confirm identification using MALDI-TOF, molecular probe or sequencing Coccidioides Malbranchea 40
  • 41. Coccidioides Histopathology • No yeast cell produced, instead: Thick walled spherules (10 – 80 uM) with endospores are the growth structure in tissue. • Spherules can be in all stages of development- fragmented spherules to well formed with endospores • Granulomatous inflammation with caseation is usually observed 41
  • 42. Case History: 75 year old male Right Pleural Effusion Lung nodule PMH: Gout, hypertension, anemia, obesity Fungal serology testing: Histoplasma urine Ag: negative Cryptococcal Ag: negative Coccidioides IgM: Indeterminate Coccidioides IgG: Weak Positive Titer 1:4 Epi: Lives on ranch in Moorpark (Simi Valley) Biopsy of lung nodule stain/ GMS Culture of lung nodule 42
  • 43. • 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 or nasal mass lesions Coccidioides is not the only spherule forming organism! 43
  • 44. Paracoccidioides brasiliensis complex • South American Blastomycosis – 80% of cases reported from Brazil • Most prevalent systemic fungal infection in Latin America • Infection acquired from inhaling infectious particle from soil • >95% of infections in males, possibly due to estrogen inhibition of the fungus mycelial to yeast transformation • Disease presentation: 1. Pneumonia 2. Disseminated infection 3. Extrapulmonary lesions on the face and oral mucosa 44
  • 45. Paracoccidioides • Cultures at 30*C – mold, cultures usually not done, slow growth and nonspecific sporulation • Culture at 37˚C - yeast • Slow growing yeast – 3 weeks • Large (10 – 30um), thick walled, with 2 or more tear drop daughter buds (2–10 um) • Unique multiply budding yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast 45
  • 46. Tissue Exam of Paracoccidioides brasiliensis complex • Granulomatous inflammation with giant cells formed in tissue GMS GMS 46
  • 47. Sporothrix schenckii • Sporotrichosis • Cutaneous inoculation from penetrating injury, cut or scrape with thorns or sphagnum moss • Known as “Rose gardener’s disease” • Usually begins as skin lesion w/wo ulceration and leads to a subcutaneous infection • Lesion can progress/ lymphocutaneous spread, possible dissemination to bone and other organs • Pulmonary and CNS infections occur but rare 47
  • 48. Sporothrix schenckii • 30˚C culture - mold grows in 3 -5 days as beige mold that eventually turns brown/black • Microscopic: septate hyphae with conidia in daisy wheel pattern or rosette • 37˚C culture – yeast grows in 7 days as small • oval and elongated yeast cells,2 – 5 µm, described as cigar bodies 48
  • 49. Sporothrix schenckii Histology • Pyogenic to granulomatous inflammation • Hard to find yeast cells in human tissue • If seen, elongated yeast, cigar shape • More commonly seen is an Asteroid body known as Splendore- Hoeppli phenomenon (SHP) • SHP are not only found in Sporothrix infections, also seen with: • Mucormycetes (Mucor, Rhizopus) • Aspergillus • Blastomyces • Candida spp 49
  • 50. •Disease: • Skin lesions endemic in tropics and SE Asia • Pneumonia or systemic infection in HIV/AIDS •Mold is a green colony with red diffusible pigment, growing in 2-3 days •Yeast like cells in culture at 30* C and in fixed tissue slides Penicillium (Talaromyces) marneffei 50
  • 52. Mycetoma • Found in hot temperate parts of the world – the fungi grow in organic debris in soil • Infection begins with trauma implanting the fungi into the subcutaneous tissue • Three criteria define mycetoma: -Swollen extremity from lesion progression -Draining sinuses -Sulfur granules observed in tissue and drainage from sinus tract Two types of mycetoma: 1. Actinomycotic – caused by higher bacteria 2. Eumycotic – caused by black molds 52
  • 53. Actinomycotic Mycetoma • 98% of cases • Nocardia species most common agent of infection • Sulfur granules are formed in tissue. The granules vary in color depending on the Nocardia species causing infection • The granules contain a matrix of the filamentous bacteria 53
  • 54. Edge of sulfur granule shows thin filamentous bacteria: How can you tell if it is Nocardia? Culture and staining will differentiate. •Nocardia is modified (partial) acid fast [PAF] stain positive and grows aerobically •Actinomyces is PAF stain negative and grows best anaerobically Careful! Sulfur granule caused by infection with Actinomyces species looks identical to ones formed by Nocardia. Actinomycotic sulfur granule Actinomyces 54
  • 55. Gram stain = filamentous Gram positive bacilli which can be poorly staining and appear speckled. Positive [red] on the Modified Kinyoun acid fast stain. Modified (Partial) Kinyoun acid-fast stain Gram stain Nocardia 55
  • 56. Grows in 3-5 days on many agars including SABs and 5% Sheep’s blood agar Colony is dry/crumbly with a musty odor Total of 85 species of Nocardia with the members of Nocardia asteroids complex most common Identification: MALDI -TOF 16s rRNA gene sequencing Nocardia species – besides Mycetoma, Nocardia can also cause Pulmonary and Brain infection 56
  • 57. Eumycotic Mycetoma Infection with pigmented/black fungi (dematiaceous) -Numerous black fungi species found in soil and debris -Cause @ 2% of mycetoma cases -Traumatic implantation of the fungus into the subcutaneous tissue Notice the thick hyphae on the edge of the granule 57
  • 58. Thermoactinomycetes • Gram positive filamentous rods related to Nocardia species • Thermoactinomyces and Saccaropolyspora • Flourish in areas of high humidity and high temperature (40*-60*C) • Grow in agricultural environments – 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 58
  • 59. Chromoblastomycosis • Three characteristics: • Wart like lesions (scarred and nodular) in subcutaneous tissue • Muriform bodies observed in fixed tissue slides • Infection caused by black pigmented fungi (dematiaceous) • Skin abrasion and implantation of fungi into tissue Naturally brown in color, septate structures known as muriform bodies or copper pennies is the unique structure found in tissue – they break up and pieces extend into the subcutaneous tissue 59
  • 60. Prototheca wickerhamii • Algae without chlorophyll • Causes skin lesions & nodules • Most common in patients with suppressed immune system • Compare morula of Protothecosis to muriform body of Chromoblastomycoses • Morula of Protothecosis is Not naturally brown in color Prototheca morula Copper penny 60
  • 61. Phaeohyphomycosis Traumatic implantation of dark fungi into subcutaneous tissue • Infections usually nodular lesions/cysts most common with/without dissemination • Infections usually confined to skin but can disseminate, particularly to brain • In fixed tissue, dark swollen hyphae and yeast like cells 61
  • 62. Black mold / Dematiaceous mold •Black colored colonies; both topside and the reverse [underside of colony] is black •Brown colored hyphae and spores due to melanin production •Most common molds to grow in areas with water damage Most common black molds include: Cladophialophora carrionii Cladophialophora bantiana Phialophora verrucosa Fonsecaea pedrosoi Exophiala species Wangiella species Rhinocladiella species 62
  • 63. Black Molds /Dematiacious molds • Numerous species • Difficult to identify requiring MALDI-TOF or 16S rRNA sequencing • All have one of four types of sporulation • Rhinocladiella-like • Cladosporium-like • Phialophora-like • Acrotheca-like Rhinocladiella Cladosporium Phialophora Acrotheca 63
  • 64. Exophiala species Most common Black Molds Cladophialophora bantiana - Associated with dissemination to brain 64
  • 66. Alternaria species– Opportunistic - sinusitis in normal host, possible dissemination in immune suppressed. Found in nature 66
  • 67. Curvularia lunata Center cell is the largest Opportunistic species/ disseminated infections rare in patient with aplastic anemia of a bone marrow transplant patient/ erythematous skin lesions 67
  • 68. Exserohilum rostrum • Associated with an outbreak of compound pharmaceutical [steroid] product contaminated with dust containing Exserohilum during manufacturing • Injected into lumbar spine and knee joints for pain management and led to serious and fatal infections: • Meningitis • Spinal abscess • Synovial infections 68
  • 69. Scedosporium complex/ previously Pseudallescheria boydii complex Cat fur-like gray colony / growth in 3 – 5 days Lollipop like spore production Opportunistic pulmonary pathogen Can invade vessels and lead to infarcts Difficult to distinguish from Aspergillus in fixed tissue stains Intrinsically resistant to Amphotericin B 69
  • 70. IMPORTANT YEAST CAUSING HUMAN INFECTION Candida species Cryptococcus neoformans Cryptococcus gattii Trichosporon species 70
  • 71. Candida species • Candida species (@ 10 species infect humans) • Yeast are usually found as normal flora in GI, GU and skin sites • Candida can be opportunistic pathogens involving skin or mucous membranes from excessive exposure to moisture, antibiotics, or immune suppression • Thrush, vaginitis, skin lesions, nail, diaper rash • Also can cause more serious infections such as fungemia, endocarditis, and systemic tissue infection. 71
  • 72. Important Candida species • Candida albicans – most common species causing @ 60% of Candida infections • Candida glabrata, C. krusei*(Pichia kudriavzevi) and C. tropicalis are causing higher % of infections than in past • C. glabrata and C. tropicalis are likely to be resistant to fluconazole • C. krusei (P. kudriavzevi) is intrinsically resistant to fluconazole • Candida auris – newly recognized as problematic yeast in the environment and nosocomial infections • Intrinsically resistant to fluconazole and additional anti-fungals, susceptible to echinocandins, • Can be difficult to identify, must use MALDI-TOF or sequencing • Candida parapsilosis is a pathogen of children and common in IV line infections (forms biofilms on plastics) • Susceptible to fluconazole 72
  • 73. Candida species • Grow in 24 – 48 hours at 30-35*C • Growth on many agars SABS, IMA, Blood agar… others • Bacteria-like colony – pasty white • Oval shaped yeast @ 7-8 um in size** • Form pseudohyphae not true hyphae (Yeast do not detach and form elongated hyphae) • Identify using biochemicals, MALDI-TOF, or Sequencing • Exception: **Candida glabrata is only Candida species that is small @ 4 µM in size and does NOT form pseudohyphae – this species breaks all the Candida rules pseudohyphae 73
  • 74. Candida albicans Identification • Germ tube formation • Incubate yeast in serum for 3-4 hrs at 35 ˚C • Extension from yeast cell “tube” positive for C. albicans • If incubate >4 hrs – C. tropicalis can have false positive reaction • C. dubliniensis can also form germ tubes • Chlamydospore formation • Growth on cornmeal agar at 48 hrs • Rudimentary structures unique to C. albicans known as chlamydospore chlamydospore 74
  • 75. ChromAgar for the identification of Candida species: different Candida spp turn unique colors on this agar based on reactions on chromogenic substrates. Susceptibility testing of yeast is much like bacteria, using broth dilution and Etest methods. 75
  • 76. Candida Histopathology • Pyogenic to granulomatous inflammation • Most Candida species have yeast cells (8 um) and pseudohyphae • Candida glabrata - smaller yeast cells (4 um) and absence of pseudohyphae GMS stain of Candida glabrataCandida species not glabrata 76
  • 77. Cryptococcus neoformans • In nature forms a 2um non-encapsulated yeast cell. • Associated with bird droppings (esp. pigeon), C neoformans is enriched by the nitrogen in the heaped droppings. • Non-encapsulated yeast cells are inhaled – travel through the pulmonary system with hematogenous spread to brain and meninges (tropism) • Infects mostly compromised hosts – one of the major AIDS defining infections 77
  • 78. Cryptococcus gattii – a close relative of C. neoformans • Isolated first from forested areas of the Pacific Northwest (British Columbia, Washington, Oregon and California) found in soil debris and tree species • Infection of both normal and immune suppressed hosts • Primarily a pulmonary disease [Cryptococcoma] but can disseminate and develop meningitis • Culture, biochemical & staining identical to C. neoformans • Defining reactions – • L Canavanine glycine bromthymol blue medium – C. gatti = blue C. neoformans = colorless MALDI-TOF and 16sRNA sequencing will also ID both species 78
  • 79. Cryptococcus neoformans and C. gattii • Irregularly sized yeast cells (2 – 20 um) • Virulence factor: Polysaccharide capsule • Polysaccharide capsule is used in diagnostic testing: • India ink exam- CSF placed in one drop of black ink. It is a negative staining method – background is stained but not the polysaccharide capsule • Cryptococcal antigen test – Titer of capsular polysaccharide, leached off of the yeast cell and detected in CSF or serum • More sensitive test than India ink exam • Test for diagnosis or can follow recovery with decreasing titer of polysaccharide 79
  • 80. Grows on mycologic agars as mucoid colonies due to capsular polysaccharide Both C. neoformans & C. gattii form brown colonies on Birdseed agar Gram stain / stains as blue blobs due to presence of capsule Positive urease enzyme reaction Cryptococcus neoformans and gattii Negative 80
  • 81. Mucicarmin stains the capsular polysaccharide of Cryptococcus Pneumocystis jiroveci (yeast like fungus) could be confused with C. neoformans – Careful! Central nuclear staining in pneumocystis C. neoformans/ C. gattii – no nuclear staining Cryptococcus vs. Pneumocystis Pneumocystis froth on H & E 81
  • 82. Pneumocystis jiroveci • Yeast like fungus • Used to be named Pneumocystis carinii and considered a protozoan (parasite) • Causes pneumonia in the immunocompromised host (PCP) particularly HIV/AIDS • Diagnosis: staining of Bronchial lavage, lung biopsy, induced sputum using direct fluorescent antibody (DFA) and GMS. DFA 82
  • 83. Trichosporon • T. asahii most common species • Commonly inhabit the soil • Colonize the skin/ gastrointestinal tract of humans. • Rectangular shaped yeast cells • Cause of superficial infection - white piedra, a distal infection of the hair shaft, • Commonly reported cause of disseminated yeast infections in humans • Rare, even among patients with impaired host defenses: • Most usually recovered from patients with corticosteroid use, solid tumors, HIV/AIDS, and intravascular devices, including catheters and prosthetic heart valves • Mortality 50 – 80% 83
  • 84. CUTANEOUS AND SUPERFICIAL MYCOSES Malassezia furfur Dermatophytes Microsporum species Trichophyton species Epidermophyton floccosum 84
  • 85. Malassezia furfur • Most superficial of the dermatomycoses • M. furfur can be normal flora on human skin • More common on oily skin or patients with high use of skin oils • Diseases: • Skin: macules, papules, patches, plaques on chest back and shoulders with either hypo or hyper pigmentation – does not invade into deeper tissues – known as Pityriasis versicolor • Fungemia: caused by tunneling in with IV lipid feeding lines (parenteral nutrition) – in neonates or rarely adults 85
  • 86. Malassezia furfur • Lipophilic yeast – oil required for growth • Media for isolation must contain oil or use an oil overlay • Small budding yeast 2 – 4 µm with collarette (appears like necklace at junction of mother and daughter yeast cell) • In tissue described as “Spaghetti and Meatballs” Spaghetti and meatballs 86
  • 87. Dermatophytes – Ringworm infections • Hair, skin and nail infections • 3 genera of fungi • Microsporum species (many) • Epidermophyton floccosum • Trichophyton species (many) • Disease described by area of the body infected: For example: tinea capitis (head), t. pedis (foot) • Usually a clinical diagnosis not requiring culture • KOH prep or Calcofluor white prep can be used to visualize fungal hyphae from skin scrapings 87
  • 88. Positive KOH prep Showing thin septate fungal hyphae Calcofluor white stain with fluorescence – thin fungal hyphae 88
  • 89. Microsporum canis Ringworm infection acquired infected dog or cat White colony/ yellow on reverse (back) of colony Tuberculate thick walled macroconidia [spiny projections] Few if any microconidia (small spores) 89
  • 90. Microsporum gypseum – Mostly skin infection from exposure to contaminated soil Sandy colored colony, Large macroconidia are produced, no microconidia producerd 90
  • 91. Trichophyton rubrum Mostly infects skin and hair after exposure to fomites, such as wet towels or hair brushes White colony. The back side of the colony has an intense red pigment diffuses into the media Pencil shaped macroconidia/ many micro-conidia (different than Microsporidium) Red diffusible pigment 91
  • 92. Trichophyton tonsurans White colony Light yellow reverse No macroconidia only microconidia produced with rare ballooning microconidia Primary cause of epidemic scalp ringworm in children 92
  • 93. Epidermophyton floccosum Most often cause infections in skin and nails Khaki green colored colony Beaver tail shaped large macroconidia – no microconidia 93
  • 94. Opportunistic Hyaline Molds Infections in the immune suppressed host or special circumstances 94
  • 95. Opportunistic Hyaline Fungi • Hyaline – no color to the hyphae • Regular septations in hyphae • Grow on a variety of agar media in 3-5 days at 35 or 30˚C • Identification based on growth rate, color/texture of colony and microscopic structures • Definitive speciation: ID / MALDI-TOF or 16sRNA sequencing 95
  • 96. Aspergillus species • Ubiquitous in nature @ 200 species • Hyaline with regular septation • Numerous round conidia produced • Most often pulmonary infection / disseminate in immune suppressed • Neutropenia can predispose to infection • Branches at 45 degree angle in fixed tissue • Invade vessels, cause thrombosis & infarctions Septation 96
  • 98. Serology Tests Supportive not Conclusive for Diagnosis Aspergillus Galactomannan Enzyme Immunoassay • Detects circulating Aspergillus antigen in the blood and/or bronchial lavage, most helpful in disseminated infections • +/- sensitivity and specificity (PPV 68%/ NPV 96%) • False positive reactions (10%) with Piperacillin/Tazobactam therapy, infection with H. capsulatum, and rice and pasta ingestion (1,3)–Beta-D-glucan assay • Detected in serum or BAL from the following pathogens: Candida spp., Acremonium, Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum, Trichosporon spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis jiroveci. • No reaction for Cryptococcus or Zygomycetes • High values more meaningful, false positive reaction from the environment contaminated with many of these fungi 98
  • 99. Aspergillus fumigatus •Ubiquitous airborne fungus, inhale conidia in nature •One of most common species causing pulmonary infection in immune suppressed, can disseminate from lung to other organs •Blue/Green colony •Phialides produce colorless spores, directed upward 99
  • 100. Aspergillus flavus •Normally found in cereals, grains, legumes •Same disease potential as A. fumigatus/ pulmonary infection with possible dissemination •Green/yellow colony •Green/yellow hue to conidia produced on phialides that surround the swollen vesicle 100
  • 101. Aspergillus niger •Black colony – visible black fruiting heads •Contaminate fruits and vegetables/ found in soil •Invasive disease uncommon, commonly isolated in ear infections •Black conidia supported by phialides that surround the vesicle 101
  • 102. Aspergillus terreus •Infection primarily in the immune compromised host •Sandy colored colony •Isolated from soil •Colorless conidia supported by phialids, headed upward •Aleurioconidia structure produced •Intrinsic resistance to Amphotericin B 102
  • 103. Aspergillus – fruiting head seldom seen in fixed tissue usually dichotomous (continuous) branching septate hyphae which branch at a 45* angle Can appear much like that of Scedosporium species, Growth in culture can differentiate the two fungi. 103
  • 104. Fusarium species •Common in nature/plants •Fuchsia colored colony •Disease related to immune status of host/ neutropenia •Infections reported: •Disseminated disease in bone marrow transplants and corneal infections in contact lens wearers •Random hyphae in fixed tissue Banana boat shaped spore 104
  • 105. Scopulariopsis species •Found in soil and grows on plant •Infections: Nail, skin, sinusitis, pulmonary and may disseminate in an immune suppressed host •Very resistant to antifungal agents 105
  • 106. Penicillium species – •One of the most common molds in the environment •Cause of bread mold •Uncommon cause of human disease •Can appear as culture contaminate •Branching hyphae with conidia production •Appears like a bony hand 106
  • 107. MUCORMYCOSIS/ ZYGOMYCOSIS Fungi in the Order Mucorales Hyaline Broad hyphae without septation Sporangium (sack) with spores 107
  • 108. Mucormycosis/Zygomycosis • Found in soil, rotten fruit and vegetables • Rhinocerebral mucormycosis - classic infection • Diabetics, the elevated glucose enriches fungal growth • Infection begins in nasal sinus - spreads to orbit of eye – then brain, high fatality rate • Broad, hyaline, hyphae without septation produced • Culture grows within 24 -48 hrs, producing coarse aerial hyphae • Mince do not grind infected tissue for culture, the grinding will kill the aseptate hyphae 108
  • 109. Rhizopus species Absidia (Lichtheimia complex) Mucor species No rhizoids Rhizoids Order Mucorales Distant rhizoid 109
  • 110. 90˚ angle branching and also random branching, no septation, ribbon like hyphae Invades vessels and can cause infarcts and thrombi Zygomycete/ Order Mucorales 110