Introduction DIMORPHIC FUNGI y Blastomycosis o Blastomyces dermatitidis y Coccidioidomycosis o Coccidioides immitis y Histoplasmosis o Histoplasma capsulatum MONOMORPHIC FUNGI: y Paracoccidioidomycosis o Paracoccidioides brasiliensis y Cryptococcosis o Cryptococcus neoformans Various appearances: y Yeast cells: o Histoplasma capsulatum o Cryptococcus neoformans y Sporangia o Coccidioides immitis y Spherules o Coccidioides immitis y Granules o Sporothrix schenkii Lab Dx: o o o o o KOH and Culture Biopsy (Histopath) Immunoflourescence Immunohistochemistry Molecular study: PCR

Different stains used to demonstrate fungi in Histopath 1. PAS: bright red-purple 2. GMS: black 3. H & E: pink to purple 4. Mucicarmine: pink (Cryptococcus 5. Fontana-Masson: dematiaceous fungi 6. Calcoflour-white: fluorescence 7. Gridley: purple to magenta

Distribution ‡ endemic in ± North America ± Mississippi ± Missouri ± Ohio Rivers

± and their tributaries zone extends into ± Kentucky ± Carolinas ± Appalachian region ± Canada ± and Wisconsin Reservoirs ‡ The exact ecologic niche has not been determined ‡ but the fungus has been found in the following: ± moist environments ± wood ± tree bark ± rotting vegetation ± animal habitats ± manure ± wet acid soil from the banks of rivers MOT ‡ airborne routes but is only rarely infectious ‡ small threat to people with competent immune systems ‡ although it can cause fatal infections to immunocompromised patients Human Infection ‡ Blastomycosis ± Gilchrist's disease / blastomycosis ± honoring the first man to publish reports of the infection ± North American Blastomycosis ± a chronic granulomatous and suppurative disease that may affect the following: ‡ Skin and mucous membrane ‡ Bones ‡ Lungs ‡ GUT ‡ Cutaneous Blastomycosis ± (skin) Blastomycosis - elevated, macerated, ill-defined, scaly borders, central ulcer ± (mucous membrane and skin) Blastomycosis ± Chronic cutaneous blastomycosis (with or without lung involvement) ‡ verrucose ‡ ulcerated ‡ suppurative ‡ Systemic blastomycosis: ± involve any organ of the body or a combination of organs ± Pulmonary Blastomycosis ‡

‡ SDA without antimicrobials at temperatures above 30°C ‡ Use BHIA containing blood for culture of specimens from body sites that are normally sterile ‡ Biphasic culture bottles containing both BHI broth and BHI agar are recommen-ded for blood cultures ‡ PDA or PFA may be used for subculture to encourage conidia production Temperature Considerations ‡ Mould form: ‡ Optimal temperature for growth is 25° to 30°C on routine media ‡ Yeast form: ‡ 37°C is needed to induce the yeast phase ‡ Primary cultures for B.specificity of 84% to 100%. ribs. ‡ Slide cultures should never be made Culture Media ‡ Modified SDA or BHIA with antibiotics at temperatures from 25° to 30°C. sensitivity of 57% to 62% ‡ Enzyme Immunoassay (EIA) . dermatitidis should be held for 4 to 8 weeks before being discarded as "no growth.Inhalation --.sensitivity of 80%. long bones ‡ painful debilitating arthritis or osteomyelitis ± GUT Blastomycosis ‡ Affects the prostate and epidydimis in males Specimen Sources ‡ skin scrapings ‡ crusts from skin lesions ‡ aspirated pus ‡ Sputum and other pulmonary specimens ‡ oropharyngeal scrapings ‡ biopsied tissue ‡ blood ‡ prostate secretions ‡ Cerebrospinal Fluid ‡ urine Specimen Collection and Handling ‡ Specimens should be collected aseptically according to the standard protocol for the type of specimen ‡ Transported to the laboratory without delay and plated promptly ‡ Direct examination: ‡ 10% KOH added to the mounting fluid to clear the specimen before it is examined ‡ Mucus and other thick substances should be treated with N-acetyl-L-cysteine before the wet mount is done ‡ Tissue specimens: ‡ fixed and stained with ‡ H&E ‡ GMS stain ‡ 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 ‡ Immunodiffusion (ID) tests . cultures should always be studied within a biologic Class II safety cabinet." Macroscopic (Colony) Morphology  Mould-form colonies at 25°C  on modified SDA grow slowly  mature colonies typically grow in 6 to 21 days ‡ white or beige to brown at first ‡ with a waxy or glabrous texture ‡ some isolates may be fluffy ‡ are prickly in the center ‡ ‡ Reverse pigment of the colonies is tan to brown . ‡ As a minimum.lungs --disseminated ‡ Round pneumonia ‡ "Mass" like density with air bronchogram ± Osteoarticular Blastomycosis ‡ Affects the spine. specificity of 98% ‡ Complement Fixation (CF) method ‡ Chemiluminescent DNA probe method Special Precautions ‡ Laboratory personnel who are working with any fluffy white colony should take special precautions and adhere stringently to laboratory procedures.

wrinkled. The round conidia. They are white to light tan. In PAS stain the internal structure of the organism is red. even in the absence of typical morphology Helpful Features for Identification of Blastomyces dermatitidis:  Culture: ‡ Mold form: white or beige to brown. and some develop concentric rings. configuration of the parent-daughter combination ‡ reproduce by forming buds that are typically attached to the mother cell by broad (4 to 5 um) necks. creating a "threesome." In GMS stain the wall of the organisms stains black. pyriform or globose ‡ average diameter of 6 um. smooth walls of the conidia are doubly refractile (double image) Laboratory Identification ‡ is a thermally dimorphic organism. ‡ It can be converted from the mould form to the yeast form (using enriched media at 37°C) to confirm identification ‡ Blastomyces can be identified more safely by the specific ‡ exoantigen test ‡ DNA probe ‡ Exo-Antigen or immunoidentification ‡ Simple ‡ cost effective method of identifying or confirming the suspected identity of the organism. ‡ Borne directly or laterally ( lollipop ) ‡ The thick. smooth walls of the conidia are doubly refractile (double image) ‡ In bad preparations Blastomyces dermatitidis is difficult to identify because it does not have unique structures. and sticklike conidiophores make it resemble a number of other fungi. ‡ Conidia: ‡ hyaline. light brown colonies  Morphology: . colonies tend to become fluffy or woolly." or hourglass. septate. waxy or glabrous texture to fluffy colonies ‡ Yeast form: waxy. ‡ Yeast form o ‡ At 37 C ‡ Hyaline ‡ large (up to 20 um) ‡ spherical to pyriform with thick walls ‡ broad based budding yeast ‡ "figure eight. colonies of yeast appear after 10 to 15 days on enriched media such as BHIA with blood. hyaline hyphae.‡ ‡ Later. ‡ ‡ ‡ ‡ At 37°C. Stains ‡ ‡ ‡ Microscopic Morphology ‡ Mold form ‡ The thick. with a wrinkled or folded topography and a waxy texture. ‡ Rarely the daughter cell buds without separating from the parent.

o o RT: lollipop-like mycelial forms 37C: thick-walled broad based budding yeast ( figure of 8 or hour glass ) y C.pulmonary in form Life cycle of Coccidioides Specimen source y sputum y pulmonary specimens y aspirated pus y skin scrapings y biopsied tissue y CSF y blood y urine Laboratory Diagnosis: Immunology y 2 substances used as antigen . Desert fever Forms of Coccidioidomycosis: o Cutaneous Coccidioidomycosis o Primary Pulmonary Coccidioidomycosis  Segmental pneumonia with LAD & pleural effusion  CXR: egg-shaped deformity o Chronic Pulmonary Coccidioidomycosis  Hemoptysis due to cavitations  fatal o Disseminated Coccidioidomycosis  Fatal  Meningitis & hydrocephalus: y most common complication y Common cause of mortality o Coccidioidomycosis in AIDS patients . y Reservoir: o Alkaline desert soil o Dust storms o Desert rodents (vectors) y Outbreaks are associated o dust storms o archeological digs o climatic conditions o earthquakes Unique Risk Factors y Competent immune system: o Relatively small threat y More severe in: o Dark skinned people o Pregnancy  High levels of ESTRADIOL and PROGESTERONE in pregnant women account for an increased risk of infection during pregnancy y Occupational hazard: o Construction workers o Farmers Human Infection y Causes: Coccidioidomycosis o aka: San Joaquin fever . 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 Differentiated ‡ Cryptococcus neoformans ± Non-encapsulated ‡ Paracoccidioides brasiliensis ± Detached buds COCCIDIOIDES IMMITIS Epidemiology y first recognized in 1892 & as a fungal infection in 1900 y endemic in: o Southwestern United States o Mexico o Central and South America y incidence in the United States of 100.acquired by AIDS patients exposed to endemic areas .000 infections/yr.

cinnamon. but strains with lavender. barrel-shaped or rectangular o May round up in tissues --. = 25-35 C for the mould form. o Endospores are freed when the mature spherule ruptures o Can be seen in intact or rupturing spherules in tissue sections and other specimens Macroscopic Morphology y colonies are white and floccose at first y Mature colonies o white to gray.o o y Coccidioidin: Is a filtrate prepared from mould cultures Spherulin: Extract from a tissue culture of the yeast form Mold colony y y y y y y Skin test o conversion from a negative to a positive skin test is diagnostic of infection CF o quite specific. o o Tissue culture = 37-40 C with increased CO2. DNA probes o for culture confirmation exoantigen test o to speed identification of the fungus using indirect FA FA Tests o rare cross-reaction o excellent screening test y Culture Media y Modified SDA y SDA with antimicrobials y BHIA y BHIB y Temperature o o Optimum temp.spherules o Mature: contains endospores or asteroid bodies Direct Examination y In host tissues o Spherules are round and refractive and usually have thick walls. 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. buff. o Tmax = 54C Microscopic Morphology y Hyphae o septate and hyaline o RACQUET hyphae may also be observed in culture y Disjunctors (disjuncture cells) o contains the arthroconidia o they fragment when mature. yellow or brown pigment o Reverse: tan to dark brown or orange o Texture: Powdery y membranous or glabrous with hyphae that are adherent to the agar y Surface = Partially or completely covered with a cottony aerial mycelium that resembles COBWEB Coccidioides immitis barrel-shaped arthroconidia . freeing the arthroconidia to disperse y Arthroconidia o single celled.

umbilicated & ulcerated y Pulmonary Cryptococcosis o Acute: immunocompromised patients  asymptomatic to mild flulike S/Sx o Chronic: lobar pneumonia. gatti: Eucalyptus tree (red gum) Cutaneous cryptococcosis (arm: hemorrhagic/ulcerated nodules) Cutaneous cryptococcosis (extremities) . visual loss. grubi (non-immunocompromised) y WORLDWIDE distribution pigeon roosts y Viable for 2 years or more loses capsule y MOT: inhalation of yeast cells y Pathogenesis: inhaled alveolar spaces of host s lung. 5 serotypes (A-D & AD). establish colonies and produce a capsule --. fruits and vegetables. y 3 human variants: o neoformans (AIDS & immunocompromised) o gatti. seizures. Forms of Cryptococcosis: y Cutaneous Cryptococcosis o painless pustules/papules/nodules. sinusitis. cavitations due to production of granulomas with encapsulated fungi at the center y Disseminated Cryptococcosis o mainly CNS: Subacute/Chronic meningitis o S/Sx: papilledema. chorioretinitis.o Hyphae and developing spherules (in lung tissue) Contaminated dairy products. exposure to soil contaminated with pigeon s excrement. pericarditis. conjunctivitis.BV --. neoformans: weathered pigeon droppings o Var.CNS y Sources: o Var. gastritis. bone infection Cutaneous cryptococcis (face: papules) Coccidioides immitis: arthroconidium. waxy. Unique Risk Factor y Opportunistic pathogen. ruptured spherule Helpful features for identification: y Rapidly growing colonies with early appearance of white cottony aerial mycelium and areas of adherent surface hyphae y Barrel-shaped arthroconidia alternating with o disjuncture cells at 25 to 35 C y Spherules containing endospore in tissue or at 40C in special media CRYPTOCOCCUS NEOFORMANS y Aka: Torulosis. hydrocephalus o Other organs: endopthalmitis. hemorrhagic. European Blastomycosis Introduction: y 22 strains.

rare o Serotype C tropical areas* (Philippines) o Serotype D Europe y Measuring Antibody Titers: o Fluorescent Antibody Test ‡ tissue studies & serotyping cultures o Whole Yeast Cell Tube Agglutination Test and EIA ‡ Cryptococcus in serum ‡ *Early diagnosis of infection . shiny white to tan. Laboratory Diagnosis y Specimen Sources:  Tissue. making them inviable. respiratory secretions. aerosol formation)  India ink preparation (not sensitive but fast) Immunology y Four Serotypes: o Serotype A most human infections o Serotype B US West Coast. 5%-10% CO2 or 1% peptone Macroscopic Morphology o o y Modified SDA.collapse the yeast cells.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 y Media: o Modified SDA o Niger Seed Agar o Dopamine agar o (w/ or w/o antibiotics sensitive to cycloheximide)  Capsule production enhanced on CAP @ 35oC. blood.improves prognosis (appropriate treatment can be started) y Antigen Test: (more specific) o Rapid LA Test (simple) polysaccharide capsular antigen in serum and CSF . pus and or biopsied tissue from skin lesions y Direct Examination:  Concentrate specimens by filtration or centrifugation  Filtration preferred (centrifugation may . Nodular cystic lesion with enhancing capsule and central core. yellow to light pink or light brown o mucoid colonies (+ capsule) Cutaneous cryptococcosis (face: plaque) Subcutaneous cryptococcosis (arm) CT with intravenous contrast medium injection. in the parietal region on the left side. urine. 25 C-37 C. CSF. 2-4d o Dome shaped. AIDS .

viewing encapsulated yeasts in culture Diagramatic Structure Cryptococcus: LA stain Cryptococcus: H & E Cryptococcus: Mucicarmine stain Cryptococcus: India Ink Cryptococcus (EM) Laboratory Identification y special methods for examining microscopic morphology o Biochemical tests: . replaced by LA test for capsular antigen (greater speed and sensitivity)  India ink prep good technique.o o Yeast form ONLY!!! *Dry and dull age Microscopic Morphology y 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 rare strains rudimentary hyphae on CMT agar and in tissue) o refractile mucopolysacchardide capsule  Capsule is related to the strength of the host s immune response  Capsules form to protect the organism from the host o India ink preparation  fungus in CSF.

dulcitol.. Paracoccidioidomycosis or South American Blastomycosis P. positive skin test 4. H&E. yeast filtrate antigens: recommended serologic test (titers appear in late and remain detectable for several months cured b.common Specimen Sources y Sputum (other pulmonary specimens) y Pus aspirated from lymph nodes y Skin scrapings or biopsied tissue edge of ulcers y Biopsied lung tissue y Crusts from skin lesions Specimen Collection and Handling y Aseptic technique y Direct examination: o Simple wet mounts. and southern Mexico Reservoir & Unique Risk Factors o Saprobic mould form acid soil in humid areas (endemic) o Plants o Armadillos . Precipitin Test band 1 Skin Tests intradermal injection epidemiological tool first serologic test to be positive do not differentiate between past exposure and current condition negative skin test person who was previously positive indicates the anergy of disseminated infection CF Test a.granulomatous Lymphadenitis . or Giemsa stain o Specific FA Test detect yeast form in tissue Immunology y Paracoccidioidin . sucrose. xylose. Carbohydrate and nitrate utilization:  dextrose. Central America. trehalose. starch  Urease production +  Phenoloxidase test +  brown colonies in Caffeic acid/bird seed/thistle/niger seed agar Treatment o o Non-immunocompromised:  Amphotericin B + Flucytosine Immonucompromised:  Amphotericin B + Flucytosine + Fluconazole AIDS px:  longer tx. cross reactions low titers in patients with acute histoplasmosis and blastomycosis. brasiliensis. inositol. . Fluconazole as maintenance y y y Secondary asymptomatic infections .BV & lymphatics --. raffinose. and southeastern South America.A. palate. hyaline hyphomycete Endemic: Holdridge Life Zones. northwestern. central.carrier MOT: Airborne (i. 2.disseminated esp to the lungs y Primary Pulmonary Paracoccidioidomycosis asymptomatic or subclinical o self-limiting subacute primary pulmonary disease mild changes in the lungs. plants) Risk factors: o Adult males combined effect of hormonal makeup and occupations o Malnutrition and immunocompetence y y y 3.pulmonary disease or dissemination Disseminated disease in any organ (esp GIT) Pyogenic abscesses and ulcers . nasal) --. Human Infection/s y Pathogenesis: o Primarily an oral lesion (mouth. galactose cellobiose.K. ID Test o concentrated yeast antigens and reference sera are available o Sensitivity.e.E2 antigen extract from yeast 1. 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. maltose. 94% and highly specific o (+) one to three precipitin continuous bands or identical with the reference sera is indicative of infection o PARACOCCIDIOIDES BRASILIENSIS y y y A.

Observe multiple budding yeast cells characteristic of Paracoccidioides brasiliensis (cotton blue preparation) (40 X). with a yellow-brown reverse in mature colonies. useful for speeding the identification of cultures Direct FA Tests a. Microscopic Morphology o o Mould Form. y Mycelial growth at 24°C on Mycosel Agar. detects P. o mariner s wheel Microscopic: septated hyphae and chlamydospore formation (cotton-blue preparation) (40 X) Culture Media and Temperature Considerations y Modified SDA with antimicrobials except cycloheximide (primary isolation contaminated with bacteria. hyaline and septate y Conidia: Few small oval to pyriform truncate on short conidiophores or sessile hyphae y Chlamydoconidia: Terminal and intercalary. 6 weeks of incubation. 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. 6. slows fungus growth) at 25oC to 30oC y SABHI and BHIA with blood (sterile sites) y No antibiotics above 30C y Yeast Extract Agar used for primary culture to encourage initial growth and conidiation y PDA and PFA for subculture to encourage conidiation Macroscopic Morphology y Mould Form. SDA o Slow maturation 2 cm in diameter after 2 to 3 weeks y Young: White to cream. o When symptoms are present a combination of ID and CF tests is 98% specific for diagnosis of paracoccidioidomycosis. y Large. Mod. 25oC. y Pigment: Beige or brown. with a membranous or velvety texture and cerebriform or folded topography. o y Folded colony of yeast cells @ 37 C.. with short. downy aerial mycelia and elevated centers. multiple budding yeast cells (mariner s wheel ) at 37oC . y Mature: Flat. Note multiple colonies. 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. Helpful Features y Slow-growing colonies white cottony aerial mycelium and heaped-up topography with membranous or velvety @ 25oC.5. MARINER s WHEEL appearance o BUDS: Attached by thin necks and easily dislodged. brasiliensis cells in smears of clinical materials o Yeast Form: y Yeast Extract Agar (deficient in glucose) y Conidia: Large number of oval to pyriform with thick-walled arthroconidia in alternating pattern y Yeast Form. Exoantigen Methods a. thick-walled. 25 C to 30 C y Modified SDA y Hyphae: Very fine.. w/ racquet and coiled hyphae Yeast culture at 36°C in trypticase soy agar.

y resolves w/o tx y CXR: infiltrates to pleural effusion y complications: o aseptic arthritis/arthralgia o erythema multiforme o LAD 2. lungs. bats) Human Infection y Histoplasmosis y MOT: inhalation y CXR: o Fibrosis o coin lesions Forms of Histoplasmosis 1. no pleural effusion y assoc with COPD y Patho: pneumonia --.HISTOPLASMA CAPSULATUM Epidemiology y Phil: 1992 1st reported case y Prevalent in: o Central North America o Central South America o Africa o Australia o India o Malaysia y Darling s disease Reservoir y Soil with high nitrogen content y Caves y birds droppings (birds. immunology Specimen y pulmonary specimen y pus / abscess y skin scrapings y biopsied tissues y bone marrow y blood y csf and urine Direct Examination y intracellular o Wright-Giemsa o not KOH o histology lab  H&E  Pap s stain y Mucous (NALC) y FA tests Culture Media y BAP y mod SDA: moist y BHIA / BHIB: from sterile sites y Yeast Extract PO4: inhibits candida y PDA: encourages conidiation y Primary cultures should be held for 10 to 12 weeks before discarding as no growth Microscopic Morphology y very fine hyphae y septate and hyaline y ropelike in BA y microconidia on short conidiophore y macroconidia o on short hyphae o conidiophore at right angle o pyriform o echinulate At 25 to 30 degree celsius . Culture 3. adrenal glands. liver. (-) AFS. GIT.fibrosis --. blood y Treatment: Amphotericin B. hemoptysis. Acute Pulmonary Histoplasmosis y 1-3 wks incubation y Non-specific flu-like S/Sx. Chronic Pulmonary Histoplasmosis y no LAD. Itraconazole Lab Diagnosis 1.pleural thickening y CM: cough. chicken.lung destruction --. Microscopic examination 2. pleuritic pain y CXR: interstitial infiltrates in apex of lungs 3. Disseminated Histoplasmosis y seen in AIDS and immunocompromised patients y involves the tongue.cavitation --.

tan yeast at 370C 0  small blastoconidia with narrow neck at 37 C  intracellular yeast in RES tissue. giant cells (diff dx: TB)  Conversion of mycelial form to yeast form contributes to its virulence . wrinkled.y y y y y sunflower in bloom resembles Candida and Blastomyces blastoconidia are small and globose smaller end of mother cell narrow neck of attachment D/dx y y y At 37 degree celsius y y Blastomyces dermatitidis o nonbudding yeast Cryptococcus neoformans o thinner capsule Paracoccidioides brasiliensis o detached buds Coccidioides immitis o endospores/young spherules Candida (Torulopsis glabrata) o (-) Germ tube Immunology y histoplasmin (H and M Ag) y skin test y CF test o 1:8 and 1:32 o histoplasmin/yeast extract o 2 3 weeks o 4 fold change y ID and CIE tests o precipitin bands o H active o M early/after recovery y RIA o in reference labs o highly sensitive o low specificity o detects histoplasma polysaccharide antigen y Exoantigen test o detects H and M Ags o faster method o identical bands  test and reference Helpful features  slow intermediate growth at 250C  sunflower tuberculate macroconidia at RT  waxy. BM and WBC  histopath: granuloma.

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