Laboratory Diagnosis of Fungi.pdf

Mycology Laboratory Diagnosis of Fungi NAILS Scrapings or cuttings Cleaned with 70% isopropyl alcohol Specim...

Mycology Laboratory Diagnosis of Fungi NAILS Scrapings or cuttings Cleaned with 70% isopropyl alcohol Specimen Collection and Transport in Mycology BLOOD AND BONE MARROW Lysis centrifugation system SPECIMENS Collected into ○ BHI (Brain Heart Infusion) broth Hair ○ Dupont Isolator tubes for transport ○Superficial mycoses and cutaneous and processing mycosis Systemic specimen Skin Nails Blood and bone marrow CSF Respiratory specimen Subcutaneous specimen Abscess fluid, wound exudates, and tissues Urogenital and fecal specimens CEREBROSPINAL FLUID Collection of Specimens for Mycologic Cultures Collect by Doctor must include Aseptically collected and transported IMMEDIATELY to the lab Sterile technique Should be concentrated by centrifugation Adequate amount before inoculation Sample from the area most likely affected ○ 1 drop of concrete →(microscopic examination) India ink (for capsule) HAIR or latex agglutination (for capsule) for Cryptococcus ○ Remainder → inoculated onto media Sterile forceps should be used to pull affected hair Cutting the hairs close to the scalp with ABSCESS FLUID , WOUND EXUDATES, AND sterile scissors TISSUES Placed directly into a sterile petri dish Aseptically aspirated from undrained Inoculated onto fungal medium and abscess with sterile needle and syringe inoculated at 22°C to 30°C (room Tissue should be gently minced before temperature) inoculation ○ Fungi 5-10 days slow grower RESPIRATORY SPECIMEN SKIN Collected early in the morning Put into sterile container for transport Must be cleaned with isopropyl alcohol Specimen before sampling ○ Sputum Skin scraped from the other edge of a ○ Transtracheal aspirates surface lesion ○ Pleural lavage fluid  UROGENITAL SPECIMENS Urine specimens Should collected in the morning ; clean catch Urine or catheterized ○ First-voided morning urine specimen ○ Centrifuged and inoculate the sediment Vaginal and cervical specimens BODY SITE AND POSSIBLE FUNGAL ○ Collected on sterile swabs PATHOGENS ○ Placed into transport media or broth Blood Histoplasma capsulatum Candida spp Blastomyces dermatitidis Coccidioides immitis Cerebros Histoplasma capsulatum pinal fluid Candida spp Coccidioides immitis Cryptococcus neoformans Hairs Trichophyton Microsporum Nails Epidermophyton Aspergillus Trichophyton SPECIMEN TRANSPORT Skin Microsporum Blood and bone Collected in Brain heart Candida marrow infusion and Dupont Blastomyces Isolator Tubes Epidermophyton Trichophyton Cerebrospinal fluid Aseptically collected and transported Lungs Penicillium immediately to the lab Coccidioides immitis Candida albicans Hairs, Nails, and Skin Cleaned w/ 70% Histoplasma capsulatum isopropyl alcohol; Aspergillus placed in a sterile petri Rhizopus plate for transport Blastomyces dermatitidis Respiratory Tract Collected in the Throat Geotrichum candidum Specimen morning, put into sterile Candida albicans container for transport Urine Candida albicans Tissues and Biopsy Aseptically collected; Candida glabrata Specimens kept moist w/ saline for transport Genital Candida albicans tract Vaginal and Cervical Collected into sterile Specimen swabs; placed into transport media or broth Scrapings from Placed into sterile wound and lesions saline for transport  Sensitive stain Binds to chitin MICROSCOPY OF CLINICAL SPECIMENS Fluorescent dye→ yellow green KOH - calcofluor white stain Direct Microscopic Examination Methods ○ KOH → dissolve keratin in specimen Saline wet mounts Potassium hydroxide Calcofluor white stain Lactophenol Cotton Blue stain (LPCB) Gram stain Acid Fast stain India Ink LACTOPHENOL COTTON BLUE STAIN Histologic Stains Periodic acid-Schiff Used to stain and preserve fungal elements GMS in culture isolates Giemsa stain Common stain Hematoxylin & Eosin stain Component of the reagent Masson-fontana ○ Lactic acid- preservative Mucicarmine stain ○ Phenol- kill organism Acridine orange ○ Cotton blue- dye ○ Glycerine- fixative Direct Microscopic Examination Methods GRAM STAIN SALINE WET MOUNT Used primarily to observe yeast and pseudohyphae Reagent KOH or Normal Saline Solution G+ →purple Allow the observation of fungal element ○ Budding yeast, hyphae, conidia ACID FAST STAIN Commonly used for observation of vaginal specimens Blastomyces and Histoplasma can be appeared red POTASSIUM HYDROXIDE (KOH) Ideal for observation of skin, hair, nail samples Serves as dissolving and clearing agent Dissolve non fungal materials and keratin; enhances visibility of fungi INDIA INK 2 Concentration ○ 10% KOH- for soft tissue (skin,hair) Nigrosin ○ 20% KOH- for hard tissue (nails) Negative stain Screening CSF samples of patients with CALCOFLUOR WHITE STAIN suspected Cryptococcal meningitis  HEMATOXYLIN AND EOSIN STAIN (H&E stain) Visualizes host tissue response to the fungus Aspergillus spp. and Zygomycetes stain well Difficult to distinguish some types of fungi , HISTOLOGIC STAINS as well as locate fungi in tissue if they are present in small numbers PERIODIC ACID SCHIFF-STAIN (PAS) MASSON-FONTANA Stains fungi magenta against a light pink or green background Used to detect melanin of dematiaceous Stains the polysaccharides components fungi GROCOTT METHENAMINE SILVER NITRATE (GMS) STAIN SOUTHGATE'S MUCICARMINE STAIN Gomori Methylene stain Useful for staining encapsulated fungi Fungal cell walls → dark brown or black e.g. Cryptococcus neoformans Cryptococcal organism ACRIDINE ORANGE GIEMSA STAIN Fluorescent stain Detect Stain fungi in tissue sections ○ Aspirates: ○ Greenish red fluorescence H. capsulatum in blood or BM ○ Pneumocystis jirovecii cysts in BAL or sputum  CULTURE MEDIA General Selective Differential Purpose Media Media Media Sabouraud Mycosel agar Potato Dextrose Dextrose Agar Agar (SDA) Modified Inhibitory mold Birdseed agar Sabouraud agar (Caffeic Acid Dextrose Agar Agar) Modifies SDA SDA w/ Dermatophyte Corn meal Tween cycloheximide and Test Medium 80 agar (CMT 80) chloramphenicol (DTM) Chester Emmons in 1977 Sabouraud Brain Trichophyton Test Adjusted the pH into neutral and lowered Heart Infusion Agar the dextrose concentration Agar Cons: ○ Bacterial contaminants can grow Brain-Heart Urea agar slant Infusion Agar SDA w/ Cycloheximide and Chloramphenicol Reserved for skin, hair, nail specimen GENERAL SUPPORTIVE MEDIA Selective form Cycloheximide Sabouraud Dextrose Agar (SDA) ○ Antifungal Chloramphenicol 5.6 pH ○ Broad spectrum antibiotic Inhibits bacterial growth Raymond Sabouraud in 1892 Sabouraud-Brain Heart Infusion Agar (SABHI) Cons: ○ Fungal contaminants can grow A nonselective medium for isolation of all fungi Contains dextrose, peptone , and brain heart infusion Can be made selective for dimorphic fungi by addition of cycloheximide , chloramphenicol, and gentamicin NOTES: Cycloheximide ○ Inhibits the saprophytic fungi Chloramphenicol ○ Inhibits gram + and gram - bacteria Gentamicin ○ Inhibits gram + and gram -bacteria Brain Heart Infusion Agar (BHI) Brain heart infusion Non-selective fungal culture medium BHI agar with blood BHIA with blood , cyclohexylamine, chloramphenicol  Guizotia abyssinica seeds SELECTIVE MEDIA contains caffeic detect production of Mycosel agar phenol oxidase C. neoformans Mycobiotic agar ○ Utilizes caffie acid to produce Highly selective medium melanin) Black to brown colonies ○ Cycloheximide ,chloramphenicol Used to recover dermatophytes ○ 3 genus Microsporum Epidermophyton Trichophyton Inhibitory Mold Agar (IMA) Used to grow most fungal pathogens Corn meal Tween 80 Agar (CMT 80) Contains gentamicin and chloramphenicol Recommended for isolation of Histoplasma capsulatum and Dermatophytes Used to differentiate Candida spp. Used for demonstration of blastoconidia, Dermatophytes Test Medium pseudohyphae, arthroconidia and chlamydospore Screening medium Indicator Used for the isolation of dermatophytes ○ 1% glucose in corn meal Selective and differential ○ Selective: Trycophyton Test Agar Cycloheximide and Gentamicin ○ Differential: Used to differentiate Trichophyton spp Indicator: phenol red ○ Rice medium Dermatophytes ○ Casein medium ○ Red (Alkaline) ○ Thiamine (enriched medium) Non-dermatophytes Trichophyton tonsurans ○ No color change Trichophyton violaceum Positive is alkaline result must be color red Trichophyton verrucosum Urea Agar Slant DIFFERENTIAL MEDIA Used to demonstrate urease production Potato Dextrose Agar (PDA) pH indicator : phenol red Amine product Used to enhance population and Urea positive (alkaline red/pink) pigmentation ○ Trichosporon, Cryptococcus ○ Enhances pigment development of Urea Negative (acid yellow) Trichophyton rubrum ○ Geotrichum , Saccharomyces, and Usually used as subculture medium most Candida Indicator: Potato dextrose Birdseed agar (Caffeic Acid Agar) Used to grow C. neoformans Selective and differential media ○ Selective agent: Chloramphenicol ○ Differential agent  Cellophane / Scotch Tape Method CULTURE CONSIDERATIONS Used to transfer aerial hyphae from the Fungal cultures are incubated at 30° C colony to a microscope slide for examination Growth requires from several days to several weeks Culture should be maintained in a moist environment Several techniques are used to obtain culture material for slide preparation Observe daily COLONIAL MORPHOLOGY Culture Slide Method Visualization of cultured fungi (25°C and 37°C) A block of agar overlaid with coverslip Growth rate Fungal colonies are grown on the slide of the ○ Contaminants generally grow faster agar block than pathogens Cover slip is removed and used for Pigment microscopic examination ○ Forward and reverse Prevents damage to the fungal structure Colony shape Need incubate Margins Texture ○ Fluffy, cottony, glabrous (skin like)leathery, powdery SLIDE PREPARATIONS TECHNIQUES FROM CULTURE Tease mount method Cellophane /Scotch Tape method Culture Slide Method Tease Mount Method Dissecting needle is used to pull-apart a CELLULAR MORPHOLOGY fungal colony on a slide Cons: ○ May damage fungal structure Hyphal morphology ○ It may take several attempts to obtain ○ Aseptate a specimen with intact conidia ○ Septate ○ Other types of hyphae Spore morphology IDENTIFICATION OF YEAST Biochemical test Behavior in broth and serum ○ (germ tube formation) Behavior on cornmeal agar ○ (pseudohyphae formation)