– and their tributaries

SYSTEMIC MYCOSES • zone extends into

Introduction – Kentucky
DIMORPHIC FUNGI – Carolinas
 Blastomycosis – Appalachian region
o Blastomyces dermatitidis – Canada
 Coccidioidomycosis – and Wisconsin
o Coccidioides immitis Reservoirs
 Histoplasmosis • The exact ecologic niche has not been determined
o Histoplasma capsulatum • but the fungus has been found in the following:
MONOMORPHIC FUNGI: – moist environments
 Paracoccidioidomycosis – wood
o Paracoccidioides brasiliensis – tree bark
 Cryptococcosis – rotting vegetation
o Cryptococcus neoformans – animal habitats
– manure
Various appearances: – wet acid soil from the banks of rivers
 Yeast cells: MOT
o Histoplasma capsulatum • airborne routes
o Cryptococcus neoformans but is only rarely infectious
 Sporangia • small threat to people with competent immune
o Coccidioides immitis systems
• although it can cause fatal infections to immuno-
 Spherules
compromised patients
o Coccidioides immitis
Human Infection
 Granules
• Blastomycosis
o Sporothrix schenkii
– Gilchrist's disease / blastomycosis
– honoring the first man to publish reports of
Lab Dx:
the infection
o KOH and Culture
– North American Blastomycosis
o Biopsy (Histopath)
– a chronic granulomatous and suppurative
o Immunoflourescence
disease that may affect the following:
o Immunohistochemistry
• Skin and mucous membrane
o Molecular study: PCR • Bones
• Lungs
Different stains used to demonstrate fungi in Histopath • GUT
1. PAS: bright red-purple • Cutaneous Blastomycosis
2. GMS: black – (skin) Blastomycosis
3. H & E: pink to purple - elevated, macerated, ill-defined, scaly
4. Mucicarmine: pink (Cryptococcus borders, central ulcer
5. Fontana-Masson: dematiaceous fungi – (mucous membrane and skin)
6. Calcoflour-white: fluorescence Blastomycosis
7. Gridley: purple to magenta – Chronic cutaneous blastomycosis (with or
without lung involvement)
BLASTOMYCES DERMATITIDIS • verrucose
Distribution • ulcerated
• endemic in • suppurative
– North America • Systemic blastomycosis:
– Mississippi – involve any organ of the body or a
– Missouri combination of organs
– Ohio Rivers – Pulmonary Blastomycosis
 • Inhalation --- lungs --- • Enzyme Immunoassay (EIA) - sensitivity of 80%,
disseminated specificity of 98%
• Round pneumonia • Complement Fixation (CF) method
• "Mass" like density with • Chemiluminescent DNA probe method
air bronchogram Special Precautions
– Osteoarticular Blastomycosis • Laboratory personnel who are working with any
• Affects the spine, ribs, long bones fluffy white colony should take special precautions
• painful debilitating arthritis or and adhere stringently to laboratory procedures.
osteomyelitis • As a minimum, cultures should always be studied
– GUT Blastomycosis within a biologic Class II safety cabinet.
• Affects the prostate and • Slide cultures should never be made
epidydimis in males Culture Media
Specimen Sources • Modified SDA or BHIA with antibiotics at
• skin scrapings temperatures from 25° to 30°C,
• crusts from skin lesions • SDA without antimicrobials at temperatures above
• aspirated pus 30°C
• Sputum • Use BHIA containing blood for culture of specimens
and other pulmonary specimens from body sites that are normally sterile
• oropharyngeal scrapings • Biphasic culture bottles containing both BHI broth
• biopsied tissue and BHI agar are recommen-ded for blood cultures
• blood • PDA or PFA may be used for subculture to encourage
• prostate secretions conidia production
• Cerebrospinal Fluid Temperature Considerations
• urine • Mould form:
Specimen Collection and Handling • Optimal temperature for growth is 25° to
• Specimens should be collected aseptically according 30°C on routine media
to the standard protocol for the type of specimen • Yeast form:
• Transported to the laboratory without delay and • 37°C is needed to induce the yeast phase
plated promptly • Primary cultures for B. dermatitidis should be held
• Direct examination: for 4 to 8 weeks before being discarded as "no
• 10% KOH added to the mounting fluid to growth."
clear the specimen before it is examined Macroscopic (Colony) Morphology
• Mucus and other thick substances should be treated  Mould-form colonies at 25°C
with N-acetyl-L-cysteine before the wet mount is  on modified SDA grow slowly
done  mature colonies typically grow in 6 to 21 days
• Tissue specimens: • white or beige to brown at first
• fixed and stained with • with a waxy or glabrous texture
• H&E • some isolates may be fluffy
• GMS stain • are prickly in the center
• Giemsa stain
Immunology
• two antigens: A and B
• A is reported to be the more useful of the
two
• Currently no skin test for blastomycosis is
commercially available because of the lack of
purified antigens
Methods
• Reverse pigment of the colonies is tan to brown
• Immunodiffusion (ID) tests - specificity of 84% to
100%, sensitivity of 57% to 62%
 •

•
• Later, colonies tend to become fluffy or woolly, and
some develop concentric rings.

• Yeast form
• At 37 oC
• Hyaline
• large (up to 20 um)
• spherical to pyriform with thick walls
• broad based budding yeast
•
• "figure eight," or hourglass, configuration of
• At 37°C, colonies of yeast appear after 10 to 15 days
the parent-daughter combination
on enriched media such as BHIA with blood.
• reproduce by forming buds that are
• They are white to light tan, with a wrinkled or
typically attached to the mother cell by
folded topography and a waxy texture.
broad (4 to 5 um) necks.
• Rarely the daughter cell buds without
separating from the parent, creating a
"threesome."
Stains
• In GMS stain the wall of the organisms stains black.
• In  PAS stain the internal structure of the organism is
red.

•
Laboratory Identification
• is a thermally dimorphic organism.
Microscopic Morphology
• It can be converted from the mould form to the
• Mold form
yeast form (using enriched media at 37°C) to confirm
• The thick, smooth walls of the conidia are
identification
doubly refractile (double image)
• Blastomyces can be identified more safely by the
• In bad preparations Blastomyces
specific
dermatitidis is difficult to identify because it
• exoantigen test
does not have unique structures. The round
• DNA probe
conidia, septate, hyaline hyphae, and
• Exo-Antigen or immunoidentification
sticklike conidiophores make it resemble a
• Simple
number of other fungi.
• cost effective method of identifying or
• Conidia:
confirming the suspected identity of the
• hyaline, pyriform or globose
organism, even in the absence of typical
• average diameter of 6 um.
morphology
• Borne directly or laterally
Helpful Features for Identification of Blastomyces
(“lollipop”)
dermatitidis:
• The thick, smooth walls of the
 Culture:
conidia are doubly refractile
• Mold form: white or beige to brown, waxy
(double image)
or glabrous texture to fluffy colonies
 • Yeast form: waxy, wrinkled, light brown
colonies
 Morphology:
o RT: lollipop-like mycelial forms
o 37C: thick-walled broad based budding
yeast (“figure of 8”or “hour glass”)
 Causes: Coccidioidomycosis
o aka: “San Joaquin fever”; “Desert fever”
 C. immitis is probably the most virulent of all agents
of human mycoses
o Very contagious
o requires biosafety level 3 cabinet

Organisms from Which Blastomyces dermatitidis Must Be Forms of Coccidioidomycosis:

Differentiated o Cutaneous Coccidioidomycosis
• Cryptococcus neoformans o Primary Pulmonary Coccidioidomycosis
– Non-encapsulated  Segmental pneumonia with LAD &
• Paracoccidioides brasiliensis pleural effusion
– Detached buds  CXR: “egg-shaped deformity”
o Chronic Pulmonary Coccidioidomycosis
 Hemoptysis due to cavitations
COCCIDIOIDES IMMITIS  fatal
Epidemiology o Disseminated Coccidioidomycosis
 first recognized in 1892 & as a fungal infection in  Fatal
1900  Meningitis & hydrocephalus:
 endemic in:  most common
o Southwestern United States complication
o Mexico  Common cause of
o Central and South America mortality
 incidence in the United States of 100,000 o Coccidioidomycosis in AIDS patients
infections/yr. - acquired by AIDS patients
 Reservoir: exposed to endemic areas
o Alkaline desert soil - pulmonary in form
o Dust storms
Life cycle of Coccidioides
o Desert rodents (vectors)
 Outbreaks are associated
o dust storms
o archeological digs
o climatic conditions
o earthquakes

Unique Risk Factors

 Competent immune system:
o Relatively small threat
 More severe in:
o Dark skinned people Specimen source
o Pregnancy  sputum
 High levels of ESTRADIOL and  pulmonary specimens
PROGESTERONE in pregnant  aspirated pus
women account for an increased  skin scrapings
risk of infection during pregnancy  biopsied tissue
 Occupational hazard:
 CSF
o Construction workers
 blood
o Farmers
 urine

Human Infection
Laboratory Diagnosis:  Surface = Partially or completely covered with a
Immunology cottony aerial mycelium that resembles COBWEB
 2 substances used as antigen
o Coccidioidin: Is a filtrate prepared from Mold colony
mould cultures
o Spherulin: Extract from a tissue culture of
the yeast form
 Skin test
o conversion from a negative to a positive
skin test is diagnostic of infection
 CF
o quite specific, but a few cross-reactions 
with other mycotic infections
 TP Test
o Highly specific with very few cross-reactions
 LA
o being reevaluated because of reports of up
to 10% false-positive results with CSF and
diluted sera.
 DNA probes
o for culture confirmation
 exoantigen test
Microscopic Morphology
o to speed identification of the fungus using
 Hyphae
indirect FA
o septate and hyaline
 FA Tests
o RACQUET hyphae may also be observed in
o rare cross-reaction
culture
o excellent screening test
 Disjunctors (disjuncture cells)
o contains the arthroconidia
Culture Media
o they fragment when mature, freeing the
 Modified SDA
arthroconidia to disperse
 SDA with antimicrobials
 Arthroconidia
 BHIA
o single celled, barrel-shaped or rectangular
 BHIB
o May round up in tissues --- spherules
o Mature: contains endospores or asteroid
 Temperature
bodies
o Optimum temp. = 25-35oC for the mould
form.
Direct Examination
o Tissue culture = 37-40oC with increased
 In host tissues
CO2.
o Spherules are round and refractive and
o Tmax = 54C
usually have thick walls.
o Endospores are freed when the mature
Macroscopic Morphology
spherule ruptures
 colonies are white and floccose at first
o Can be seen in intact or rupturing spherules
 Mature colonies
in tissue sections and other specimens
o white to gray, but strains with lavender,
buff, cinnamon, yellow or brown pigment
o Reverse: tan to dark brown or orange
o Texture: Powdery
 membranous or glabrous with hyphae that are
adherent to the agar
Coccidioides immitis
“barrel-shaped arthroconidia”
Hyphae and developing spherules (in lung tissue)
Coccidioides immitis:
arthroconidium; ruptured spherule
Helpful features for identification:
 Rapidly growing colonies with early appearance of
white cottony aerial mycelium and areas of adherent
surface hyphae
 Barrel-shaped arthroconidia alternating with
disjuncture cells at 25 to 35oC
 Spherules containing endospore in tissue or at 40C in
special media
CRYPTOCOCCUS NEOFORMANS
 Aka: Torulosis, European Blastomycosis
Introduction:
 22 strains; 5 serotypes (A-D & AD);
 3 human variants:
o neoformans (AIDS &
immunocompromised)
o gatti, grubi (non-immunocompromised)
 WORLDWIDE distribution– pigeon roosts
 Viable for 2 years or more – loses capsule
 MOT: inhalation of yeast cells
 Pathogenesis: inhaled – alveolar spaces of host’s
lung, establish colonies and produce a capsule --- BV
--- CNS
 Sources:
o Var. neoformans: weathered pigeon
droppings
o Var. gatti: Eucalyptus tree (red gum)
o Contaminated dairy products, fruits and
vegetables,
Unique Risk Factor
 Opportunistic pathogen, exposure to soil
contaminated with pigeon’s excrement.
Forms of Cryptococcosis:
 Cutaneous Cryptococcosis
o painless pustules/papules/nodules,
hemorrhagic, waxy, umbilicated & ulcerated
 Pulmonary Cryptococcosis
o Acute: immunocompromised patients
ü asymptomatic to mild flu-
like S/Sx
o Chronic: lobar pneumonia, cavitations due
to production of granulomas with
encapsulated fungi at the center
 Disseminated Cryptococcosis
o mainly CNS: Subacute/Chronic meningitis
o S/Sx: papilledema, visual loss, seizures,
hydrocephalus
o Other organs: endopthalmitis,
chorioretinitis, conjunctivitis, sinusitis,
pericarditis, gastritis, bone infection
Cutaneous cryptococcis
(face: papules)
Cutaneous cryptococcosis (arm: hemorrhagic/ulcerated
nodules)
 Laboratory Diagnosis
 Specimen Sources:
ü Tissue, CSF, blood, urine,
respiratory secretions, pus
and or biopsied tissue
from skin lesions
Cutaneous cryptococcosis (extremities)  Direct Examination:
ü Concentrate specimens by
filtration or centrifugation
ü Filtration – preferred
(centrifugation may
collapse the yeast cells,
making them inviable;
aerosol formation)
ü India ink preparation (not
sensitive but fast)
Cutaneous cryptococcosis (face: plaque)

Immunology
 Four Serotypes:
o Serotype A – most human infections
o Serotype B – US West Coast, AIDS - rare
o Serotype C – tropical areas* (Philippines)
o Serotype D – Europe
 Measuring Antibody Titers:
o Fluorescent Antibody Test
Subcutaneous cryptococcosis (arm) • tissue studies & serotyping
cultures
o Whole Yeast Cell Tube Agglutination Test
and EIA
• Cryptococcus in serum
•
*Early diagnosis of infection - improves prognosis
(appropriate treatment can be started)
 Antigen Test: (more specific)
o Rapid LA Test (simple)
CT with intravenous contrast medium injection. Nodular cystic – polysaccharide capsular antigen
lesion with enhancing capsule and central core, in the parietal in serum and CSF
region on the left side. - cross reaction with rheumatoid
factor or disseminated
Trichosporon beigelii
o infections (remedy: treat serum with
Na2EDTA or 5 mg of pronase/mL of serum)
o significant titer: >1:2 – active
cryptococcosis

Culture Media and Temperature Considerations

 Media:
o Modified SDA
o Niger Seed Agar
o Dopamine agar
 o (w/ or w/o antibiotics – sensitive to
cycloheximide) Cryptococcus: India Ink
ü Capsule production –
enhanced on CAP @ 35oC,
5%-10% CO2 or 1%
peptone

Macroscopic Morphology
 Modified SDA; 25oC-37oC, 2-4d
o Dome shaped, shiny white to tan, yellow to
light pink or light brown Cryptococcus (EM)
o mucoid colonies (+ capsule)
o Yeast form ONLY!!!
o *Dry and dull – age

Microscopic Morphology
 Yeast:
o Thin-walled globose or oval-shaped
o Singly or in pairs with narrow points of
attachment between the mother and
daughter cells
o NO pseudohyphae nor true hyphae (very Cryptococcus: LA stain
rare strains – rudimentary hyphae on CMT
agar and in tissue)
o refractile mucopolysacchardide capsule
ü Capsule is related to the
strength of the host’s
immune response
ü Capsules form to protect
the organism from the
host
o India ink preparation
 fungus in CSF, replaced by LA test
for capsular antigen (greater speed Cryptococcus: H & E
and sensitivity)
 India ink prep – good technique,
viewing encapsulated yeasts in
culture

Diagramatic Structure

Cryptococcus: Mucicarmine stain


Laboratory Identification
 special methods for examining microscopic
morphology
o Biochemical tests:
 Carbohydrate and nitrate
utilization:
 dextrose, maltose, sucrose,
galactose cellobiose, inositol,
xylose, raffinose, trehalose,
dulcitol, starch
 Urease production +
 Phenoloxidase test +
 brown colonies in Caffeic acid/bird
seed/thistle/niger seed agar
Treatment
o Non-immunocompromised:
 Amphotericin B + Flucytosine
o Immonucompromised:
 Amphotericin B + Flucytosine +
Fluconazole
o AIDS px:
 longer tx; Fluconazole as
maintenance
PARACOCCIDIOIDES BRASILIENSIS
 A.K.A. Paracoccidioidomycosis or South American
Blastomycosis
 P. brasiliensis, hyaline hyphomycete
 Endemic: Holdridge Life Zones, northwestern,
central, and southeastern South America, Central
America, and southern Mexico
 Reservoir & Unique Risk Factors
o Saprobic mould form – acid soil in humid
areas (endemic)
o Plants
o Armadillos - carrier
 MOT: Airborne (i.e., plants)
 Risk factors:
o Adult males – combined effect of hormonal
makeup and occupations
o Malnutrition and immunocompetence
Human Infection/s
 Pathogenesis:
o Primarily an oral lesion (mouth, palate,
nasal) --- BV & lymphatics --- disseminated
esp to the lungs
 Primary Pulmonary Paracoccidioidomycosis –
asymptomatic or subclinical
o self-limiting
F subacute primary pulmonary disease mild changes
in the lungs; positive skin test
F Secondary asymptomatic infections - pulmonary
disease or dissemination
 Disseminated disease in any organ (esp GIT)
 Pyogenic abscesses and ulcers - granulomatous
 Lymphadenitis - common
Specimen Sources
 Sputum (other pulmonary
specimens)
 Pus aspirated from lymph
nodes
 Skin scrapings or biopsied
tissue – edge of ulcers
 Biopsied lung tissue
 Crusts from skin lesions
Specimen Collection and Handling
 Aseptic technique
 Direct examination:
o Simple wet mounts, with added stains
o KOH – clear debris
o N-acetyl-L-cysteine (NALC) – mucus and
thick substances
o Tissue specimens (fixed and stained) –
Papanicolaou stain, H&E, or Giemsa stain
o Specific FA Test – detect yeast form in
tissue
Immunology
 Paracoccidioidin - E2 antigen extract from yeast
1. Precipitin Test – band 1
2. Skin Tests – intradermal injection
F epidemiological tool
F first serologic test to be positive
F do not differentiate between past
exposure and current condition
 F negative skin test person who was
previously positive indicates the
anergy of disseminated infection
3. CF Test
a. yeast filtrate antigens: recommended
serologic test (titers appear in late and
remain detectable for several months –
cured
b. cross reactions low titers in patients with
acute histoplasmosis and blastomycosis.
4. ID Test
o concentrated yeast antigens and reference
sera are available
o Sensitivity, 94% and highly specific
o (+) one to three precipitin continuous bands
or identical with the reference sera is
indicative of infection
o Band 1 is the first to disappear during
treatment
o number of bands is correlated with the CF
titer
o Low titers: localized infection
o high titers: acute infection or dissemination.
o When symptoms are present a combination
of ID and CF tests is 98% specific for
diagnosis of paracoccidioidomycosis.
5. Exoantigen Methods
a. – useful for speeding the identification of
cultures
6. Direct FA Tests
a. detects P. brasiliensis cells in smears of
clinical materials
Culture Media and Temperature Considerations
 Modified SDA with antimicrobials except
cycloheximide (primary isolation contaminated with
bacteria; slows fungus growth) at 25oC to 30oC
 SABHI and BHIA with blood (sterile sites)
 No antibiotics above 30C
 Yeast Extract Agar – used for primary culture to
encourage initial growth and conidiation
 PDA and PFA – for subculture to encourage
conidiation
Macroscopic Morphology
 Mould Form; 25oC, Mod. SDA
o Slow maturation – 2 cm in diameter after 2
to 3 weeks
 Young: White to cream, with short, downy aerial
mycelia and elevated centers.
 Mature: Flat, with a membranous or velvety texture
and cerebriform or folded topography.
 Pigment: Beige or brown, with a yellow-brown
reverse in mature colonies.
 Mycelial growth at 24°C on Mycosel Agar, 6 weeks of
incubation. Note multiple colonies.
Microscopic Morphology
Mould Form; 25oC to 30oC
 Modified SDA
 Hyphae: Very fine, hyaline and septate
 Conidia: Few small oval to pyriform truncate on
short conidiophores or sessile hyphae
 Chlamydoconidia: Terminal and intercalary, w/
racquet and coiled hyphae
Yeast Form:
 Yeast Extract Agar (deficient in glucose)
 Conidia: Large number of oval to pyriform with
thick-walled arthroconidia in alternating pattern
 Yeast Form; 37C
o Large spherical to pyriform cells with thick
walls
o Reproduce by multiple budding with buds
that cover the entire surface of the parent
cell... “MARINER’s WHEEL” appearance
o BUDS: Attached by thin necks and easily
dislodged.
o mariner’s wheel
Microscopic: septated hyphae and chlamydospore formation
(cotton-blue preparation) (40 X)
Yeast culture at 36°C in trypticase soy agar. Observe multiple
budding yeast cells characteristic of Paracoccidioides
brasiliensis (cotton blue preparation) (40 X).  1-3 wks incubation
 Non-specific flu-like S/Sx; (-) AFS;
 resolves w/o tx
 CXR: infiltrates to pleural effusion
 complications:
o aseptic arthritis/arthralgia
o erythema multiforme
o LAD
2. Chronic Pulmonary Histoplasmosis
 no LAD, no pleural effusion
 assoc with COPD
 Patho: pneumonia --- fibrosis --- cavitation --- lung
destruction --- pleural thickening
 CM: cough, hemoptysis, pleuritic pain
Helpful Features  CXR: interstitial infiltrates in apex of lungs
 Slow-growing colonies – white cottony aerial 3. Disseminated Histoplasmosis
mycelium and heaped-up topography with  seen in AIDS and immunocompromised patients
membranous or velvety @ 25oC,  involves the tongue, lungs, liver, GIT, adrenal glands,
 Folded colony of yeast cells @ 37oC, blood
 Large, thick-walled, multiple budding yeast cells  Treatment: Amphotericin B, Itraconazole
(mariner’s wheel”) at 37oC
Lab Diagnosis
1. Microscopic examination
2. Culture
HISTOPLASMA CAPSULATUM 3. immunology
Epidemiology
Specimen
 Phil: 1992 1st reported case
 pulmonary specimen
 Prevalent in:
o Central North America  pus / abscess
o Central South America  skin scrapings
 biopsied tissues
o Africa
 bone marrow
o Australia
 blood
o India
 csf and urine
o Malaysia
 Darling’s disease
Direct Examination
 intracellular
Reservoir
o Wright-Giemsa
 Soil with high nitrogen content
o not KOH
 Caves
o histology lab
 birds droppings (birds, chicken, bats)
 H&E
 Pap’s stain
Human Infection
 Mucous (NALC)
 Histoplasmosis
 FA tests
 MOT: inhalation
 CXR:
Culture Media
o Fibrosis
 BAP
o “coin lesions”
 mod SDA: moist
 BHIA / BHIB: from sterile sites
Forms of Histoplasmosis
 Yeast Extract PO4: inhibits candida
1. Acute Pulmonary Histoplasmosis
  PDA: encourages conidiation o H – active
 Primary cultures should be held for 10 to 12 weeks o M – early/after recovery
before discarding as “no growth”  RIA
o in reference labs
Microscopic Morphology o highly sensitive
 very fine hyphae o low specificity
 septate and hyaline o detects histoplasma polysaccharide antigen
 ropelike in BA  Exoantigen test
 microconidia on short conidiophore o detects H and M Ags
 macroconidia o faster method
o on short hyphae o identical bands
o conidiophore at right angle  test and reference
o pyriform
o echinulate Helpful features
At 25 to 30 degree celsius  slow – intermediate growth at 250C
 “sunflower” tuberculate macroconidia at RT
 waxy, wrinkled, tan yeast at 370C
 small blastoconidia with narrow neck at 370C
 intracellular yeast in RES tissue, BM and WBC
 histopath: granuloma, giant cells (diff dx: TB)
 Conversion of mycelial form to yeast form
contributes to its virulence

 “sunflower in bloom” D/dx

 resembles Candida and Blastomyces  Blastomyces dermatitidis
 blastoconidia are small and globose o nonbudding yeast
 smaller end of mother cell  Cryptococcus neoformans
 narrow neck of attachment o thinner capsule
 Paracoccidioides brasiliensis
At 37 degree celsius
o detached buds
 Coccidioides immitis
o endospores/young spherules
 Candida (Torulopsis glabrata)
o (-) Germ tube

Immunology
 histoplasmin (H and M Ag)
 skin test
 CF test
o 1:8 and 1:32
o histoplasmin/yeast extract
o 2 – 3 weeks
o 4 fold change
 ID and CIE tests
o precipitin bands