Mycology - 03 Methods of Examination for Mycoses

MYCOLOGY METHODS OF EXAMINATION FOR MYCOSES OUTLINE • Specimen Collection and Handling o Specimen Collection • Laboratory Tests o Specimen Preparation o Isolation and Culture o Chemical Test SPECIMEN COLLECTION AND HANDLING SPECIMEN COLLECTION • Specimen collected must be: o process immediately o if specimen is contaminated with normal flora antibacterial or antifungal must be added e.g. cycloheximide o do not refrigerate specimen (except for urine) o Collect 2 to 3 specimens, if needed, several days apart • Hair o Pluck hair from affected scalp area using tweezers o Place hair in sterile plastic dish o Include at least one medium with antibiotics o For detection Microsporum, Trichophyton • Skin o Cleanse with 70% alcohol o Gently scrape specimen into sterile plastic petridish using edge of clean glass slide, scalpel or 1-sided razor blade o For detection of Candida, Microsporum, Trichophyton, Epidermophyton • Nails o Collect soft material under the nails o Include at least one medium with antibiotics o For detection of Aspergillus, Epidermophyton, Trichophyton • Pus and Exudates o Collect from cleansed side with syringe o Include at least one medium with antibiotics; at least one without • Respiratory o 1st morning, 3x, over 1 to 2 weeks o Requires medium with antibiotics o For detection of Candida, Aspergillus, Rhizopus and Penicillium • CSF ( cerebrospinal fluid) o Large volume recommended, 1x o Include at least 1 antibiotic free medium o All CSF should be tested for Cryptococcal-specific antigens o For detection of Cryptococcus, Candida, Histoplasma, Coccidioides o 2 processing strategies are acceptable: LABORATORY TESTS • Most clinical specimens can be examined directly for the presence of fungal pathogen. SPECIMEN PREPARATION WET PREPARATION • Simplest method for direct exam of specimen • specimens that are dry and viscous add NSS TEMPORARY MOUNT • KOH 10-20% o rapid method of detecting fungal elements • Lactophenol Cotton Blue (LPCB) o Preserves and stains fungi o Excellent mounting media for fungi / most useful stain for fungi • India Ink/Nigrossin o Negative staining technique used to demonstrate capsule of C. neoformans • Calcofluor White Stain o Visualization of fungal elements using Fluorescence microscopy PERMANENT MOUNTS • Gram Stain o useful for the detection of yeast • Giemsa or Wright’s o Useful in suspected cases of Histoplasma o Demonstrates yeast cells within macrophage • Periodic Acid Schiff (PAS) o Useful in demonstrating internal details of fungal elements • Gomori’s Methenamine Silver (GMS) o provides high contrast with minimal background staining • Hematoxylin & Eosion (H&E) o Used to determine if a fungus is hyaline or dematiaceous • Mayer’s Mucicarmine o Used to determine mucoid capsule of C. neoformans • Fontana-Masson Stain o Demonstration of melanin or melanin-like substances • Acid Fast Stain/Kinyoun’s o Used to differentiated Nocardia from Actinomyces • Acridine Orange o Useful in detecting Tinea versicolor ISOLATION AND CULTURE • Inoculated at RT with ambient air • Grows at 2 week incubation period • Dimorphic fungi are isolated after 14 days • Culture is held for 30 days • Airborne fungi must never be inoculated with petri dish but with tubes with screw caps are used • Use at least 3: o 1 non selective o 1 selective o 1 with blood (blood may be added to selective or non selective media) • Brain Heart Infusion Agar - Primary recovery of saprobic and pathogenic fungi • BHI with antibiotics - Primary recovery of pathogenic fungi exclusive of dermatophytes

• BHI biphasic blood culture bottles - Recovery of fungi from blood • Chromogenic agar - Isolation and presumptive identification of yeast and filamentous fungi • Dermatophyte Test Medium - Primary recovery of dermatophyte, recommended as screening medium only • Inhibitory mold agar - Primary recovery of pathogenic fungi exclusive of dermatophytes • Potato flake agar - Primary recovery of saprobic and pathogenic fungi • Mycosel - Primary recovery of dermatophyte • SABHI agar - Primary recovery of saprobic and pathogenic fungi • Yeast extract phosphate agar - Primary recovery of pathogenic fungi exclusive of dermatophytes • Ascospore agar - Detection of ascospores ( Saccharomyces spp.) • Christensen’s urea agar - For identification of Cryptococcus, Trichosporon and Rhodotorula • Cornmeal agar with Tween 80 and trypan blue - Identification of Candida albicans by chlamydospore production • Cottonseed conversion agar - Conversion of dimorphic fungus Blastomyces dermatitidis from mold to yeast • Czapek’s agar - Differential identification for Aspergillus spp. • Niger seed agar ( Birdseed) - Identification of Cryptococcus neoformans • Nitrate Reduction Medium - Identification of Cryptococcus neoformans • Potato dextrose agar - Demonstration of pigment of Trichophyton rubrum and for sporulation of dermatophytes • Rice Medium - For Microsporum audouinii CHEMICAL TEST • Woods Lamp • Germ Tube – for Candida albicans and Candida dubliniensis • L-DOPA ferric citrate test • Urease Test – differentiate T. rubrum(-) from T. mentagrophytes(+) • Hair Baiting Test/ Hair Perforation - differentiate T. rubrum(-) from T. mentagrophytes(+) • Exoantigen test – double diffusion test • Carbohydrate Assimilation Test o For identification of yeast isolate which is free from carbohydrates •