MLS Review - Mycology

MEDICAL LABORATORY SCIENCE REVIEW STRUCTURES OF FUNGI MYCOLOGY HYPHAE OUTLINE • • • • • • • • • General Characteristics of Fungi Yeast & Mold Structures of Fungi Diagnostic Mycology Superficial Mycoses Cutaneous Mycoses Subcutaneous Mycoses Systemic Mycoses Opportunistic Mycoses MYCOLOGY • • • • • Mycology - study of fungi and fungal infection There are a few clinically significant fungi but are not as many as clinically significant bacteria GENERAL CHARACTERISTICS OF FUNGI • • • Eukaryotic and lacks chlorophyll o Contains true nucleus enclosed with membrane o Has membrane-bound organelles Aerobes, gram positive o Most are obligate aerobes, some are facultative With chitin on cell wall YEAST & MOLD • • • • Yeast o Unicellular o Colonies are moist, creamy, opaque, and pasty o Grows at 37C o Zygomycetes Mold (filamentous fungi) o Multicellular, filamentous o Colonies are fluffy, cottony, wooly, or powdery o Mycelia - group of intertwined hyphae ▪ Parts: • Aerial - extend above the surface; for reproduction • Vegetative/thallus - for nutrition/absorption ▪ Hyphae - functional unit Monomorphic o Exist as yeast or mold only Dimorphic o Exist either as yeast or mold o Temperature dependent ▪ Yeast - grows at 37C ▪ Mold - grows at room temp (25-27-30C) o Examples ▪ Sporothrix schneckii - causes subcutaneous mycosis ▪ Those causing systemic mycosis • Penicllium marneffei • Histoplasma capsulatum • Blastomyces dermatitidis • Coccidioides immitis • Paracoccidioides braziliense • Basic structural unit of filamentous fungi (molds) According to presence or absence of septate o Septated - with cross walls ▪ Almost all fungi are septate except zygomycetes o Aseptate - without cross walls ▪ Sparsely septate - alternative nomenclature ▪ Zygomycetes - with aseptate hyphae (sparsely septate) • Mucor, Absidia, Physoid (rhizopus) According to appearance o With pectinate body - M. audouinii o With antler hyphae (swollen hyphae) - T. schoenleinii o Racquet hyphae (club-shaped) - Epidermophyton floccosum o Spiral hyphae (tightly coiled) - Trichophyton mentagrophytes o With nodular bodies - Microsporum canis According to pigmentation o Hyaline - non-pigmented or lightly pigmented o Dematiaceous - pigmented ▪ Pigmentation is due to presence of melanin SPORES • • Structure for reproduction Sexual spores are formed as result of nuclear fusion o Aka Teleomorph o Perfect fungi o Ascospores - spores formed within ascus ▪ Ascus - sac like structure o Basidiospores - spores formed within basidium ▪ Basidium - club shaped structure o Zygospores - formed from the union of 2 undifferentiated (identical) cells o Oospores - formed from the union of 2 differentiated (non-identical) cells • Asexual spores are formed without nuclear fusion o Aka anamorph o Imperfect fungi o Conidia ▪ According to size • Microconidia - spiriform • Macroconidia - spindle-shaped ▪ According to shape • Spiriform - microconidia • Spindle-shaped - macroconidia 1 • • o o o Blastoconidia - formed thru budding - small outgrowth of hyphae pinches off Arthroconidia - formed thru fragmentation of hyphal cell Chlamydoconidia - formed thru enlargement of hyphal cell • Tests that’ll differentiate T. mentagrophytes & T. rubrum o Urease test - (+) result is pink to red color o Hair Baiting or Hair Perforation Test - (+) result is V-shaped or conical hair perforation o T. mentagrophytes - Hair Baiting & Urease (+) o T. rubrum - negative in both bests Germ tube test o Presumptive test for Candida albicans & C. dubliniensis o Positive result - germ tube with no constriction ▪ Can be mistaken w/ pseudohyphae (+ constriction) o (+) constriction - false germ tube but is pseudohyphae o Suspension small inoculum of yeast in 0.5 mL Schiff serum or rabbit plasma; Incubate at 35-37C for no longer than 3 hrs; Suspension is examined microscopically L-Dopa ferric citrate test o Rapid identification of C. neoformans o (+) result: black TYPES OF MUCOSES ACCORDING TO GEOGRAPHICAL/ANATOMICAL DISTRIBUTION DIAGNOSTIC MYCOLOGY • • • Identifies/detects fungi and fungal infections Microscopic Examination o Uses various stains o Temporary Mount ▪ 10-20% KOH - detects fungal elements from skin, hair, and nails ▪ Lactophenol Cotton Blue (LCB) - preservative and stain ▪ India Ink/Nigrosin - to demonstrate C. neoformans capsule ▪ Calcofluor white stain - to demonstrate chitin in cell wall; best for direct microscopy o Permanent Mounts ▪ Periodic Acid Schiff (PAS) & Gomori’s Methenamine Silver • To detect fungi in histologic sections • Gomori’s - Best for permanent mounting ▪ Mayer’s Mucicarmine - demonstration of C. neoformans ▪ Fontana Masson & H&E - detects hyphal pigmentation ▪ Giemsa or Wright’s - detects yeast within mononuclear cells i.e. Histoplasma capsulatum ▪ Gram stain, Acid fast & Kinyoun stain - also used for fungal staining o Wood’s Lamp - demonstration of fluorescence of fungal elements usually in hair; based on fluorescence Culture examination o Incubation Temperature of Fungi - 25-30C or RT o Plates/tubes are incubated in ambient air o Most fungi grow within incubation time of 2 weeks o Antimicrobial agents in Fungal media ▪ Chloramphenicol - prevent bacterial overgrowth ▪ Cycloheximide - prevent saprophytic fungi o Culture media ▪ Czapek agar - Aspergillus ▪ Mycosel/Mycobiotic - for dermatophytes ▪ Cornmeal agar - stimulate chlamydospore formation of C. albicans ▪ Bird Seed, Niger seed, Staib’s medium - C. neoformans • • • • Superficial mycoses - found on outermost layer of skin, hair, hands, and feet Cutaneous mycoses - affect keratinized tissues (skin, hair, nails) Subcutaneous mycoses - affect deep layers of the skin Systemic mycoses - affect organ/s or system/s SUPERFICIAL MYCOSES • Pityriasis Versicolor o Chronic superficial fungal infection which leads to hypopigmented or hyperpigmented patches on skin o With sunlight exposure, the skin around the patches will tan, but patches will remain white o Caused by Malassezia furfur - causes Tinea or Pityriasis versicolor - superficial mycoses; a lipophilic yeast; spaghetti with meatballs appearance • Tinea nigra o Superficial fungal infection that causes dark brown to black painless patches on the soles of hands and feet o Caused by Exophiala werneckii (formerly Hortaea werneckii) Piedra o Nodular infection of the hair shaft o Axillary, pubic, beard, and scalp hair maybe infected o Black Piedra - caused by Pidraia hortai o White Piedra - caused by Trichosporon beigelii/cutaneum • 2 • Microsporum audouinii o Bizarre shaped macroconidia o Rice medium (-) SUBCUTANEOUS MYCOSES CUTANEOUS MYCOSES • • • • • • • Aka as dermatophytosis/dermatomycoses Aka as ring worm infection Affects keratinized tissues, skin, hair, & nails Agents: Trichophyton, Epidermophyton, Microsporum o Microsporum - skin and hair, rarely nails o Trichophyton - skin, nails, rarely hair o Epidermophyton - skin, hair, & nails Epidermophyton floccosum o Unable to produce microconifia o 2-4 smooth cells macroconidia o Macroconidia smooth walled, appearing like beaver’s tail o Produces Khaki colored colonies o Eg. Tinea cruris (hadhad) Trichophyton mentagrophytes o Microconidia are globose & arranged in grape-like clusters o Eg. Tinea unguium Trichophyton rubrum o Teardrop-shaped microconidia laterally on hyphae o May give the so-called “Birds on a fence” appearance o Eg. Tinea corporis (buni) • • • • • • • • • Type affecting deep layers of skin (usually muscles) Agents are usually found on soil, so feet is always affected Acquired through trauma Sporothricosis - S. schenckii o Most commonly encountered Chromoblastomycosis – usually caused by the following: Phialophora verrucosa; Cladosporium carrionii ; Fonsecaea pedrosoi o Most commonly encountered Phaeohyphomycosis – caused by the ff: genera: Alternaria, Bipolaris, Curvularia & Dreschella , Exophiala (ABCDE) Mycetoma – caused by Pseudoallescheria boydii, Exophiala jeanselmi Sporothrix schenckii o Cause Rose Gardener’s disease o Dimorphic fungi o Yeast: cigar-shaped yeast with single or multiple buds ▪ Elliptoid, budding cigar shaped yeast singly or multiple budding o Mold: conidia in flowerette arrangement ▪ Hyphae with pyriform conidia Yeast Mold Sclerotic bodies/ Muriform cells o May be diagnostic of Chromoblastomycoses/ chromomycosis or Mossy foot disease/ hematomycosis o Copper colored fungal cells SYSTEMIC MYCOSES • • Tricrophyton verrucosum o Produces rat tail or string bean appearing macroconidia Microsporum canis o Large spindle shaped macroconidia o Thick-walled spindle shaped multiseptated macroconidia with knobby projections o Rice medium (+) o Eg. Tinea capitis • • • • • • Caused by dimorphic fungi Acquired through inhalation, so lungs is usually affected Infectious form: Mold (mycelial) form Diagnostic form: Yeast Agents include: o B. dermatitidis o H. capsulatum o C. immitis o P. braziliensis Blastomyces dermatitidis o Blastomycosis/ North American Blastomycosis/ Chicago disease/ Gilchrist disease/ Missouri River Valley o Produces yeast with single bud on a broad base o With round or pyriform conidia appearing like lollipops o Mycelial phase ▪ Septate hyphae with round or pyriform conidia born singly on conidiospores or directly on hyphae 3 • Coccidioides immitis o Causes San Joaquin Valley fever o Produces a yeast appearing like thick-walled spherule with many endospores o Barrel-shaped arthroconidia alternating with empty disjuncter cells o Major biohazard in the lab and most virulent of all the mycotic agents o On media produces cobweb appearing colonies o Found in soil of many arid or desert regions in US o Virulence factor includes extracellular proteinases o Mycelial phase ▪ Coarse, septate, branched hyphae that produce thick, walled barrel shaped rectangular arthroconidia that alternate with empty disjuncter cells ▪ RT: thick-walled shaped arthroconidia that alternate with empty disjuncter cells (non-viable cells) which cause an uneven staining o Yeast form ▪ 37C: thick-walled spherules filled with endospores with double refractive cell wall • OPPORTUNISTIC MYCOSES • • • Histoplasma capsulatum o Darling’s disease/ Histoplasmosis/ Cave disease/ Spelunker disease/ Reticuloendothelial cytomycosis/ Ohio valley fever/ Central Mississippi Valley Fever o Produces a yeast within mononuclear cells o To demonstrate yeast, Giemsa or Wright’s may be used o Mycelial phase ▪ Septate hyphae with round pyriform microconidia on short branches or directly on hyphal stalk; later large round, thick-walled knobby tuberculate macroconidia forms ▪ RT: septate hyphae with round pyriform microconidia on short branches or directly on hyphal stalk on prolonged incubation, knobby tuberculate macroconidia • • • • o Yeast form ▪ 37C: Small Budding yest within mononuclear cell Paracoccidioides brasiliensis o South American Blastomycosis / Brazilian Blastomycosis/ Luts Splendore Almeide disease o Produces a yeast with multiple buds giving it a Ship’s wheel or mariner’s wheel or pilot’s wheel appearance o Yeast may resemble a mickey mouse cap o Mycelial phase ▪ RT: Presence of terminal chlamydoconidia o Yeast form ▪ 37C: yeast with multiple buds resembling a ship’s wheel or mariner’s wheel or parent cell surrounded by daughter cells or mickey mouse cap to the yeast cell appearance Cryptococcus neoformans o Major cause of opportunistic infection in those with AIDS o Causes European blastomycosis, Torulosis, Cryptococcosis, Torula Meningitis, Bussebuschke dse o Produces an encapsulated yeast that can be demonstrated using India ink/nigrosin o Phenol oxidase & urease test (+); nitrate (-) Candida albicans o Most common cause of yeast infection worldwide o Normal flora but can be opportunistic o Causes vulvovaginitis, oral thrush o Forms germ tubes, blastoconidia & chlamydosposres o (+) growth at 42C Germ tube test o Test to differentiate C. albicans from C. tropicalis o Presumptive test for C. albicans Aspergillus o Causes pulmonary disease, otomycosis o Agent of Fungus ball, aspergilloma, Farmer’s lung dse o Acquired thru inhalation o Aspergillus niger - black pepper effect on culture media, fungus ball in lungs o Aspergillus fumigatus - most common cause of pulmonary aspergillosis often found in compost piles and soil of potted plants; green color on culture media Talaromyces marneffei o Previously Penicillium marneffei o Causes Talaromycosis o Dimorphic fungi o Yeast - sausage shaped while mold with flask shaped phialides, paint brush like TB like symptoms in HIV patients in SEA Zygomycota o Zygomyctes o Includes clinically significant genera: Rhizopus, Absidia Mucor o Causes Rhinosinus, sinus orbital or craniofacial zygomycosis o Rapid growers, lid lifters, with aseptate hyphae o (+) growth on SDA without cycloheximide 4 • • Other Causes of Opportunistic Mycoses o Fusarium species (a hyaline mold) – classified as fungi imperfecti, with normal microflora of commodities (rice) o Geotrichum - cause wound infection and oral thrush ▪ forms Hockey stick shaped arthroconidia on corn meal agar o Penicillium (hyaline mold) – can cause keratitis, external ear infection o Acremonium – can cause mycetoma, corneal and nail infections Pneumocystis jiroveci o Previously Pneumocystis carinii o Previously a protozoa but now an atypical fungus o Formerly: Pneumocystis pneumonia (PCP) o Pneumonia in AIDS individual acquired from immunocompetent mammals o Preferred sample: bronchoalveolar lavage fluid (BALF) o Gomori methenamine silver is stain of choice for cyst but not trophozoite 5