Background Mycology Review Melissa B. Miller, Ph.D. April 4, 2008 Yeasts Unicellular Divide by budding or binary fission Moulds Filamentous hyphae interweave to form mycelium Saprobic phase: airborne, infectious cells produced in environment Parasitic phase: host, respiratory mucosa Background Spores Sexual: meiosis preceded by fusion of protoplasm/nuclei of 2 cells (teleomorph( teleomorph) Asexual: mitosis only (anamorph( anamorph) Sporangiospores: : spores produced within containing structure (sporangia); i.e., Zygomycetes Conidia: born naked; i.e., Aspergillius, Penicillium, dermatophytes Usually, only anamorphs seen in clinical specimens Specimen Collection RT storage if received within 2h Extended storage 30 C- CSF or other sterile sites 4 C C for specimens likely to contain bacteria (< 24h) Specimen Prep N-acetyl-cysteinecysteine or dithiothreitol- mucolytic agents 10% KOH clears keratinous material Nails and biopsies should be minced NOT homogenized Methods Microscopy Phase-contrast 1000x to see conidia Wet mount Stains Gram stain (yeast) Calcofluor white Calcofluor White Binds chitin and cellulose in cell wall Used in conjunction with 10% KOH Sensitivity and specificity 90-95% 95% 1
Methods Stains Lactophenol cotton blue/acid fuchsin Giemsa for Histo and P. carinii trophs Masson Fontana- melanin Dematiaceous fungi Cryptococcus Mucicarmine- Crypto vs. others (stains mucopolysaccharide capsule red) H&E: most fungus; can be hard to see Candida and Aspergillus GMS and PAS better Methods Culture At least one selective and one non-selective plate (BHI, IMA) Incubated at 30 C C (or RT) Blood culture system Routine for Candida and Crypto Isolator system for dimorphics and Malassezia (+olive oil) and all bone marrow aspirates Lysis centrifugation system Media Inhibitory substances Cyclohexamide: saprobic (and some pathogenic) fungi Crypto, Candida, Trichosporon, dimorphic yeasts, zygomycetes are inhibited Chloramphenicol, gentamicin and/or penicillin to inhibit bacterial contaminants Media Birdseed agar (niger( seed agar): Crypto Blood-glucose glucose-cysteine: : mould to yeast conversion BHI w/ sheep blood: all fungi including dimorphics (conversion media) Cornmeal agar: Candida differentiation based on mycelial characteristics Media Dermatophyte Test Medium Inhibitory Mould Agar: isolates cyclohexamide sensitive fungi (Crypto,( zygomycetes,, some Aspergillus spp., Fusarium) Mycobiotic (Mycosel)) Agar: cyclohexamide (dimorphic moulds, dermatophytes) Potato Dextrose Agar: Sporulation medium; facilitates ID Sabouraud Dextrose Agar: general purpose Identification Methods Tease Prep Tape Prep Slide Culture 2
Identification Methods Identification Methods DNA probes (rrna( rrna) RNA Amplicon + Unhybridized Probe Select Hybridized Probe Select Probe 60 o C 15 Min. Detect Detect No Light Light Luminometer Antigen detection Cryptococcus Histoplasma Aspergillus Antibody detection Aspergillus Blastomyces Coccidioides Histoplasma Adapted from Hill, Gene-Probe, Inc. Candida C. albicans germ tube formation Germ tube + : C. albicans,, C. dubliniensis C. dubliniensis won t t grow at 45 C C. albicans: : feet on blood containing media C.tropicalis and C. krusei have mycelial fringe C. glabrata: : rapid trehalose + Smaller cells and colonies Other Candida spp. identified using carbohydrate assimilation tests (API 20C) along with morphology on cornmeal agar CHROMagar Candida Cryptococcus neoformans Gram Stain from CSF cytospin 3
Cryptococcus neoformans Aged pigeon droppings Specimens: blood (isolator best), CSF, urine, respiratory, biopsies India Ink +: Crypto, rare Candida and Rhodotorula Phenol oxidase test +: caffeic acid substrate Rapid urease + No pseudohyphae Cell wall is fragile- crescent shapes in tissue India Ink + Urease + Cryptococcus neoformans Antigen test Detects polysaccharide antigen 100% sensitive in AIDS patients 56-70% sensitive in other immunocompromised patients T. beigelii, Capnocytophaga canimorus,, and rheumatoid factor can be false positives False negatives: prozone effect or immune complexes Serology not good: capsule prevents ab formation Cryptococcus neoformans var. gattii Tropical and subtropical areas Associated with eucalyptus trees Pneumocystis P. jiroveci (carinii) Non-filamentous fungi Persists only in mammalian lung Human-to to-human transmission; no known environmental reservoir Pneumonia in immunocompromised patients PCP leading opportunistic infection in AIDS Adhere to type I pneumocytes; ; alveoli become filled with organisms Pneumocystis H&E will not stain; silver stain will Extrapulmonary generally only seen at autopsy: LN, spleen, BM, liver Specimens HIV: induced sputum, BAL, (trach( asp) Non-HIV: BAL, NO induced sputum Pneumocystis Microscopy Cyst wall thickenings = double comma Raisin-like; honeycombs Giemsa Cyst wall doesn t t stain, but nucleus does 10x more trophs- more sensitive IFA- surface glycoproteins Can also be seen by calcofluor white 4
Pneumocystis jiroveci Aspergillus IFA GMS In soil; conidia in air (insulation, fireproofing materials) 95% of infections A. fumigatus A. flavus A. niger Granulocytopenic (<1000/mm 3 ) patients at risk Aspergillus A. fumigatus Chain of conidia produced by phialide- youngest at base In tissue: branching, hyaline, septate hyphae 45 degree branching Parallel walls (3-6um); uniform size Septa at regular intervals Angioinvasive Aspergillus Aspergillus H&E Galactomannan antigen detection a major constituent of Aspergillus cell walls released during growth of hyphae serum ELISA as a diagnostic test for invasive aspergillosis permits detection of antigenemia in some patients an average of 5-8 days before the presence of clinical signs, an abnormal chest x-ray, or positive cultures Sensitivity, 61-71% Specificity, 89-93% NPV, 95-98% PPV, 26-53% Piperacililn/tazobactam and amoxicillin-clavulanic acid causes false positives GMS 5
Fusarium Fusarium Ubiquitous in environment Inhalation, catheter contamination, breaks in skin or mucous membrane Uncommon; disseminated disease ~60% Risk Factors: hematologic malignancies, BM transplant, extensive burns Neutropenic patients often get localized infection of toes prior to hematogenous spread Very high mortality (50-80%) Fusarium Inhibited by cyclohexamide F. solani most common species Unlike Aspergillus, Fusarium disseminated disease has positive blood cultures (75%) In tissue, looks like Aspergillus,, but often has right angle branching Angioinvasive Resistant to many anti-fungals fungals: : I-R I R to AmB or Voriconazole Fusarium Other Hyaline Fungi Acremonium Paecilomyces Scedosporium apiospermum Chrysosporium Penicillium Scopulariopsis Sepedonium Zygomycetes Rhizopus arrhizus,, R. microsporus Absidia, Mucor, Rhizomucor, Cunninghamella Risk factors: diabetic ketoacidosis, lymphoma, leukemia, neutropenia,, long term suppressive therapy with iron chelator HIV not a significant risk factor Vascular invasion Zygomycetes In tissue, broad thin-walled pauciseptate hyphae (6-25um) Wide, non-parallel sides (ribbon-like) Random branching ~90 angle Stain weakly with GMS Clinical hallmark: rapid onset of necrosis and fever Rhinocerebral mucormycosis (67% mortality) Pulmonary (83% mortality) Disseminated (>99% mortality) Neutropenic patients w/ pulmonary involvement 6
Rhizopus Zygomycete H&E Dematiaceous Fungi Dark pigmentation (melanin) in cells walls and/or hyphae Alternaria, Bipolaris, Curvularia, Cladosporium, Exserohilum Eumycotic mycetoma (black grain)- P. boydii most common in U.S. (Madurella( worldwide) Chromoblastomycosis (chronic infection of skin and subcutaneous tissue)- finding of sclerotic bodies is diagnostic (Fonsecaea( most common) Dematiaceous Fungi Phaeohyphomycoses Superficial- tinea nigra (Phaeoannellomyces werneckii); black piedra (Piedraia hortae) Cutaneous Dermatomycosis- Alternaria Also keratitis- Curvularia, Alternaria, Bipolaris Subcutaneous- most frequent Exophiala jeanselmei, Wangiella dermatiditis, Phialophora, Bipolaris Systemic- cerebral; Poor prognosis Cladophialophora bantiana most common Also Wangiella, Fonsecaea, Curvularia Dematiaceous Fungi Endemic Mycoses Histoplasma capsulatum Blastomyces dermatiditis Coccidioides immitis Paracoccidioides brasiliensis Primary infection Reactivation in immunocompromised patients (HIV, transplant, chemotherapy, older age) 7
Endemic Mycoses Thermally dimorphic fungi Infectious conidia or arthrospores inhaled Primary pulmonary infection asymptomatic in competent hosts May progress to dissemination in immunocompromised hosts Histoplasma capsulatum Inhabits soil, esp. with bat and avian droppings (chicken houses, bat caves) Ohio and Mississippi River valleys, Latin America, some Caribbean islands Budding yeast in tissue Phagocytosis by macrophages- intracellular Mould in environment Tuberculate macroconidia Histoplasma capsulatum Cutaneous infection Primary inoculation Pulmonary infection >95% are asymptomatic Disseminated infection To RES via hematogenous spread Asymptomatic patients may have spleen and pulmonary calcifications Reactivation is common, esp. AIDS Histoplasma capsulatum Specimens: respiratory, bone marrow, blood, CSF, tissue Giemsa stain for yeast cells, esp. intracellular (T.( cruzi and Leishmania similar- look for kinetoplasts) Culture for mould at 25-30 30 C Also, var. duboisii E. Africa Larger yeast cells with thicker walls Greater risk for bone involvement H. capsulatum Histoplasma serology ID: immunodiffusion for H and M bands CF: complement fixation with yeast and mycelial antigens 8
Blastomyces dermatiditis Ohio, Mississippi River valleys, Africa Riverbank soil, beaver dams Not as common in AIDS patients as Histo, Cocci,, and P. marneffei Yeast in tissue Broad-based bud Culture for mould at 25-30 30 C Blastomyces dermatiditis Infections Asymptomatic Cutaneous (primary and chronic) Pulmonary Disseminated Skin, bone (33%), GI, CNS, spleen Specimens Respiratory, tissue, blood B. dermatiditis Coccidioides immitis Found in soil; growth enhanced by bat and rodent excreta Desert SW U.S., N. Mexico, Central America AIDS patients: usually primary infection not reactivation Darker skinned individuals may have higher risk of disseminated disease Pregnancy (3 rd trimester) also a risk factor Endospore release stimulated by hormones Coccidioides immitis C. immitis Saprobic phase- mould (25-30 30 C) Arthrocondidia infectious 1 of top 10 laboratory-acquired acquired infections Parasitic phase- spherules containing endospores (37 C) Seen only in tissue C. posadasii- non-california Cocci 9
Paracoccidioides brasilensis P. braziliensis S. America, esp. Brazil Natural reservoirs- soil, armadillos Primary pulmonary infections that disseminate often manifest as mucosal infections Predisposition for males (78:1) Yeast form has multiple buds- mariner s wheel Other Dimorphic Fungi Sporothrix schenckii Penicillium marneffei Sporothrix Global distribution, most common in Mexico Pleomorphic,, spherical to oval or cigar- shaped yeasts with single buds Often not seen in tissue Cutaneous or subcutaneous infection following direct inoculation Rose-gardener gardener s s disease Lymphatic spread is common S. schenckii Penicillium marneffei SE Asia, esp. Thailand Bamboo rat- carrier Route of transmission not established Prolonged latency (10+ yrs after travel) Yeast looks similar to Histo,, but does not bud Reproduces by fission Forms a single transverse septum 10
P. marneffei Dermatophytes Keratinophilic fungi: infections of skin, hair and nails Microsporum- hair, skin Trichophyton- hair, skin, nail Epidermophyton floccosum- skin, nail Most common (80-90%): T. mentagrophytes and T. rubrum Transmission by direct inoculation KOH important for direct exam Grow in the presence of cyclohexamide Trichophyton -Macroconidia rare -Microconidia numerous Dermatophytes T. rubrum Microsporum -Macroconidia numerous, thickwalled, rough -Microconidia usually present Anti-fungal Resistance Ampho B intrinsic resistance C. lusitaniae,, C. guilliermondii, Trichosporon Fusarium,, P. boydii, Scedosporium prolificans E. floccosum M. canis Epidermophyton -Macroconidia numerous, thin and thickwalled, smooth -Microconidia not formed Azole intrinsic resistance C. krusei,, C. inconspicua,, C. norvegensis, Rhodotorula rubra,, C. glabrata (SDD), C. dubliniensis (with pressure) Antifungal Susceptibilities- Candida MIC without interpretation Amphotericin B Ketoconazole Caspofungin 11