MEDICAL MYCOLOGY GENERAL MYCOLOGY • Medical mycology - branch of medical science thatdeals with the study of medically important fungi • ‘fungus’ is derived from Greek ‘mykes’ meaning mushroom (a type of edible fungus) Fungi differ from bacteria & other eukaryotes • Eukaryotic and possess eukaryotic cell organelles • Possess a rigid cell wall, composed of chitin, βglucans and other polysaccharides • Cell membrane contains ergosterol instead of cholesterol • May be unicellular or multicellular • Lack chlorophyll and divide by asexual and/or sexual means by producing spores Morphological Classification of Fungi 1. Yeast: Round to oval cells that reproduce by budding - Cryptococcus neoformans (pathogenic) - Saccharomyces cerevisiae (non-pathogenic) 2. Yeast-like: Yeasts forming pseudohyphae (e.g. Candida) - Differentiated from true hyphae as they have constrictions at septa 3. Molds: long branching filaments called hyphae - Hyphae - septate or nonseptate Morphological forms of fungi Classification of Fungi • Based on the growth pattern in culture medium • Aerial mycelium: It is the part of the mycelium which projects above the surface of culture medium • Vegetative mycelium: It is the part of the mycelium that grows on the surface of the culture medium Morphological Classification of Fungi 4. Dimorphic fungi: exist as molds (hyphal form) in the environment at ambient temperature (25°C) and as yeasts in human tissues at body temperature (37°C) - Histoplasma capsulatum - Blastomyces dermatitidis - Coccidioides immitis - Paracoccidioides brasiliensis - Penicillium marneffei - Sporothrix schenckii. Taxonomical Classification • Based on the production of sexual spores 1. Phylum zygomycota: sexual spores – zygospores, and possess aseptate hyphae, e.g. Rhizopus and Mucor. 2. Phylum ascomycota: Sexual spores - ascospores and possess septate hyphae, e.g. Aspergillus. 3. Phylum basidiomycota: Sexual spores - basidiospore e.g. Cryptococcus 4. Phylum deuteromycota (Fungi imperfecti): sexual state is either absent or unidentified yet Types of fungal spores Sexual Spore Observed in- Zygospores Zygomycetes Ascospores Aspergillus Basidiospores Cryptococcus Asexual Spore Observed in Vegetative asexual spore Arthrospore Coccidioides Trichosporon Blastospore Candida Chlamydospore Candida albicans Types of fungal spores Aerial asexual spore Conidiospore or conidia Aspergillus Sporangiospore Zygomycetes Microconidia Dermatophytes Macroconidia Dermatophytes CLASSIFICATION OF FUNGAL DISEASES Superficial mycoses Tinea versicolor Tinea nigra Piedra Dermatophytosis Agents Malassezia furfur Hortaea werneckii Trichosporon beigelii, Piedraia hortae Trichophyton, Microsporum, Epidermophyton Subcutaneous mycoses Mycetoma Sporotrichosis Chromoblastomycosis Rhinosporidiosis Madurella mycetomatis, Pseudallescheria boydii, etc Sporothrix schenckii Phialophora verrucosa Fonsecaea pedrosoi CLASSIFICATION OF FUNGAL DISEASES Systemic mycoses Histoplasmosis Histoplasma capsulatum Blastomycosis Blastomyces dermatitidis Coccidioidomycosis Coccidioides immitis Paracoccidioidomycosis Paracoccidioides brasiliensis CLASSIFICATION OF FUNGAL DISEASES Opportunistic mycoses Candidiasis Candida albicans, Other Candida species Cryptococcosis Cryptococcus neoformans Zygomycosis Rhizopus, Mucor, Absidia Aspergillosis Penicilliosis Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger Penicillium marneffei, Other Penicillium species pneumocystosis Pneumocystis jirovecii Mycotoxicoses LABORATORY DIAGNOSIS OF FUNGAL DISEASES • Specimen Collection - Depends on site of infection - skin scraping, hair, nail, sputum, etc. - Systemic mycoses - blood sample, Cerebrospinal fluid (CSF), etc Microscopy • Demonstration of Fungal elements in the specimen • Potassium hydroxide (KOH) preparation: Keratinized tissue specimens treated with 10% KOH  digests keratin  fungal hyphae clearly seen • 20–40% KOH - nail & hair Microscopy  KOH preparation Biopsy specimens - dissolved in 10% KOH in a test tube and • examined after overnight incubation • Glycerol (10%) can be added to prevent drying • DMSO (dimethyl sulfoxide) - help in tissue digestion  Lactophenol cotton blue (LPCB): - Phenol acts as disinfectant - Lactic acid preserves the morphology of fungi - Glycerol prevents drying - Cotton blue stains the fungal elements blue Microscopy • Gram stain: yeasts & yeast like fungi appear as gram-positive budding yeast • India ink and nigrosin stains: negative stains for demonstration of capsule of Cryptococcus neoformans • Calcofluor white stain: more sensitive - Binds to cellulose and chitin of fungal cell wall and fluoresce under UV light Histopathological stains • For demonstrating fungal elements from biopsy tissues Periodic acid schiff (PAS) stain: • - PAS positive fungi appear magenta/deep pink, whereas the nuclei stain blue - PAS which stains only the live fungi Masson fontana stain: for pigmented (or pheoid) fungi • • Hematoxylin and Eosin stain • Mucicarmine stain: for staining the carminophilic cell wall of Cryptococcus and Rhinosporidium Histopathological stains • Gomori methenamine silver (GMS) stain: - Alternative to PAS - Stains both live and dead fungi - Stains the polysaccharide component of the cell wall - Fungi appear black & background pale green color Culture Media • Sabouraud’s dextrose agar (SDA): - Most commonly used medium - Contains peptone (1%), dextrose (4%) and pH of 5.6 - May not support some pathogenic fungi • Neutral SDA (Emmons’ modification): - Differs from original SDA in having neopeptone (1%) and dextrose (2%) and pH of 7.2 Culture Media • C orn meal agar and rice starch agar: Nutritionally deficient media used for stimulation of chlamydospore production • Brain heart infusion (BHI) agar and blood agar: Enriched media, used for growing fastidious fungi like Cryptococcus and Histoplasma • Niger seed agar and bird seed agar: selective growth of Cryptococcus • CHROMagar Candida medium: selective as well as differential medium for speciation of Candida Culture Condition • Temperature: Most of the fungi grow well at 25–30°C except the dimorphic fungi that grow at both 25°C and 37°C • BOD incubators (biological oxygen demand): capable of maintaining low temperature • Incubation time: 2–3 weeks • Antibiotics - cycloheximide (actidione), chloramphenicol and gentamicin - added to the culture media to inhibit bacterial growth Culture Identification • Based on macroscopic appearance of the colonies grown on culture and microscopic appearance • Macroscopic Appearance of the Colony • Rate of growth: - Rapid growth (<5 days) - saprophytes, yeasts and agents of opportunistic mycoses - Slow growth (1–4 weeks): dermatophytes, agents of subcutaneous and systemic mycoses Culture Identification • P igmentation: reverse side of the culture media • Texture: Refers to how the colony would have felt if allowed to touch - glabrous (waxy/leathery), velvety, yeast like, cottony or granular/powdery • Colony topography: rugose (radial grooves), folded, verrucose or cerebriform (brain-like) Microscopic Appearance of Fungi • - Teased mount: LPCB tease mount Identification is based on : Nature of hyphae (septate or aseptate, hyaline or phaeoid, narrow or wide) and Type of sporulation (conidia or sporangia) • Slide culture: in situ microscopic appearance of the fungal colony Microscopic Appearance of Fungi • Cellophane tape mount: - Impressions taken by placing the cellophane tape on the colonies  LPCB mount is made - Easy to perform than slide culture and in-situ fungal morphology maintained Other Methods of Identification • For Candida: Germ tube test, Dalmau plate culture, carbohydrate fermentation and carbohydrate assimilation tests are done • For dermatophytes: Hair perforation test, dermatophyte test medium and dermatophyte identification medium are used • U rease test can be done for the fungi that produce urease enzyme, e.g. Cryptococcus Other Methods of Identification • Immunological Methods - to detect antibody or antigen from serum and/or other body fluids • Antibody detection - ELISA, immunodiffusion test, agglutination test, and complement fixation test (CFT) • Antigen detection - latex agglutination test for cryptococcal antigen from CSF • Immunohistochemistry: detecting antigens (e.g. proteins) on cells of a tissue Other Methods of Identification • Tests for Metabolites - in body fluids by gas liquid chromatography • Tests to Demonstrate Delayed Hypersensitivity - Skin tests - Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Dermatophyte, Sporothrix and Candida. • Molecular Methods - Polymerase chain reaction (PCR) and its modifications - DNA sequencing methods Antifungal Agents Class Drug Antifungal Antibiotics Amphotericin B Polyenes (AMB) Nystatin, Hamycin Echinocandins Caspofungin, micafungin Mechanisms Use Bind to ergosterol and disrupts fungal cell membranes Inhibits β-glucan synthesis in fungal cell wall Systemic mycoses Topical use (skin infection) Systemic mycoses Synthetic Antifungals & their uses • Azoles - Inhibit ergosterol synthesis of fungi Imidazoles: Clotrimazole, miconazole, ketoconazole, oxiconazole Topical use (except, ketoconazole can be used for both topical and systemic use) Triazoles Systemic mycoses (invasive fungal infections) Itraconazole Sporothrix, chromoblastomycosis, dermatophytes, Cryptococcus, Candida (except C. krusei, C. glabrata) and Trichosporon Fluconazole Cryptococcus & Candida (ext C. krusei, C. glabrata) Voriconazole, osaconazole, isavuconazole All invasive fungal infections except mucormycosis Synthetic Antifungals & their uses Class Antimetabolite Allylamines Other topical agents Drug Mechanisms Use Systemic mycoses Flucytosine (5-FC) Inhibits DNA synthesis in fungi; Used in 5-FC is converted association with AMB to fluorouracil, which inhibits thymidylate synthetase Terbinafine Inhibits ergosterol Topical use synthesis of fungi Tolnaftate, Benzoic acid, Whitfield’s ointment, Essentials of Medical © 2018, Jaypee Undecylenic acid,Microbiology Ciclopirox olamine