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Mycology i
- 1. DR V SVATKAR Asso Prof Microbiology Department D Y Patil Medical College, Kolhapur
- 2. Objectives Classification of fungi Infectionscaused by fungi like superficial mycoses, deep mycoses, opportunistic mycoses Mycotoxicoses Laboratory diagnosis of fungi
- 3. Mykos (Greek word):fungus, study of fungi Most of the fungi : saprophytes Human infections : mainly opportunistic Widespread use of modern advances in treatment: antibiotics, steroids & immunosuppressive agents, leads to opportunistic fungal infection Introduction
- 4. Eukaryotes Rigid cell wall:chitin, mannan & other polysaccharides Cytoplasmic membrane contains sterols True nuclei with nuclear membrane & paired chromosomes Divide asexually, sexually or by both May be unicellular/multicellular Characteristics of fungi
- 5. CLASSIFICATION MORPHOLOGICAL CLASSIFICATION YEAST YEAST-LIKEMOULDS DIMORPHIC FUNGI Cryptococcus Candida Aspergillus Mucor Penicillium Rhizopus Dermatophytes Histoplasma capsulatum Cocciidioides immitis Sporothrix schnkii Blastomyces dermatidis Paracoccidiodes brasiliensis Penicilliosis marneffei
- 6. Morphological Classification Yeasts: Unicellular Reproduce by budding Macroscopic : pasty colonies Microscopic: spherical/oval forms, filamentous forms: not seen e g Cryptococcus neofrmance Yeast like fungi: Unicellular Reproduce by budding & by fission Macroscopic : pasty colonies Microscopic : spherical/oval forms, filamentous forms seen e g Candida albicans
- 7. Morphological Classification Filamentousfungi/Moulds: Hyphae: septe (cross walls, multicellular) or aseptate (coenocytic) Reproduction: asexual: spore formation (some exhibit sexual reproduction) Macroscopic: cottony/woolly/velvety/granu lar, pigmentation on the reverse Microscopic: thread like filamentous hyphae aseptate or septate. Mycelium : vegetative/arial & shapes like racquet, nodular, pectinate , spiral, root-like etc e g Aspergillus fumigatus, Penicillium, Mucor etc
- 9. Thermally Dimorphic fungi: Grow as filaments in culture at 22 0 C & in environment yeast forms at 37 0 C e g Histoplasma capsulatum Morphological Classification
- 11. Phycomycets: lower fungi, Non septate hyphae Forms endogenous spores called sporangeospores : swollen sac like structures called SPORANGIA Sexual spores: oospores in some fungi & zygospores in others SYSTEMIC CLASSIFICATION based on their sexual spore formation
- 12. Ascomycets: Highre fungi Sexual spores (ascospores) within a sac Includes both filamentous & yeast forms
- 13. Basidiomycetes: Higher fungi Forms sexual spores (basidiospores) on the base/ basidium
- 14. Fungi imperfecti: Mostof the medically important fungi belongs to this group
- 17. Pathogenic classification Primary pathogenic:causes infection in healthy person e g diamorphic fungi Opportunistic pathogenic: causes infection in immunocompromised patients
- 18. Superficial mycoses Cutaneous mycoses Subcutaneousmycoses Deep/systemic mycoses Opportunistic mycoses Mycotoxicoses: disease produce due to fungal toxins Hypersensitivity (allergic reactions): inhalation of fungal spores Fungal infections (Mycoses)
- 19. Microscopy Culture Serology Skin test Antifungal susceptibilitytest Laboratory Diagnosis
- 20. Collect fromaffected site : skin scrapings, nail bed etc Disseminated infections: blood Specimens
- 21. Potassium hydroxide (KOH)preparation: Tissue specimens like skin scrapings : examined as wet mount (10% KOH) Tissue : kept in KOH for 20 min incubator at 37 0 C, for affected nail samples 40% KOH is used Alkali digest cells & other tissue materials, we can see fungus clearly Microscopy
- 22. Calcoflour white: Sensitivestaining technique, good visualization of fungal morphology Gram stain: yeast & Yeast like fungus
- 23. India ink preparations:Negative staining: to visualise the capsule of fungus
- 24. Methenamine silver stain& Periodic –acid- schiff (PAS) stain: used to demonstrate fungus in tissue Lactophenol cotton blue stain: widely used method to see morphology
- 25. Most ofthe fungi grow in media used to grow bacteria but at low pH Grow better at 25-30 0 C except fungi causing systemic mycoses(37 0 C). Aspergillus fumigatus : grow even at 50 0C Media : Specific medias Non- Specific medias Culture
- 26. SABOURAUD DEXTROSE agar, Sabouraud glucose neopeptone agar (antibiotics added) Common culture media pH 5.4, neutral Czapek-Dox medium, bird seed agar, Niger seed agar, corn meal agar Cyclohexamide : incorporated in it to prevent contamination by moulds Cultures : incubated at room temperature (22 0 C) for wk & at 37 0C for days
- 27. Growth characteristics Rapidityof growth Colour & morphology of the colony on obverse Pigmentation on the reverse Morphology of hypha: diameter, septa, conidia etc
- 28. Non specific media: Brain heart infusion agar Brain heart infusion broth Blood agar
- 29. Teased mount: Bitof fungal colony is teased out from culture plate, Place a drop of lactophenol cotton blue stain Put a cover slip View under low power & then focus under high power Proper morphology is difficult to view in teased mount Morphological studies can be studied by teased mount, slide culture & cellophane tape preparation
- 30. Proper morphologyof fungus is seen . Procedure: sterile glass slide , place it on a bent glass rod in a petri dish, a 1 cm square block of SDA is placed on it Test strain of fungus is inoculated at four sides of agar block Inoculated block is covered with sterile cover slip Incubate it at 25 0 C for 48 hrs Take a clean glass slide, put a drop of LCB stain over it, transfer a cover slip on this slide Observe under microscope Slide Culture
- 31. Cellophane tape mount Technically easy Serology: Antibody detection: agglutination, Complement Fixation, Immunodiffusion, CIEP, ELISA Antigen detection: Latex agglutination (Cryptococcus) Newer rapid diagnostic tests: Nucleic acid hybridization, PCR
- 32. Infection ofdead layer of skin & its appendages No inflammatory response Cosmetic effects Cutaneous Infections Dermatophytes mainly causative agents Candida Superficial Mycoses
- 33. Pityriasis versicolar (Tinea versicolar) Usually asymptomatic Does not beyond stratum corneum layer of skin World wide distribution Prevalent in torpics Seen in young adults mainly Causitive agent : Malassezia furfur C/F: confluent macular areas of discolouration or depigmentation of skin of chest, abdomen, upper limbs and back. Present on normal skin , but opportunistic pathogen Diagnosis: skin scrapings shows yeast like cells, short branched filaments, Grow on SDA covered with olive oil
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- 35. Tinea nigra Infectionof stratum corneum Palms Black/brownish macular lesions Seen in tropics Causative agent: Exophila werneckii, Exophila castellanii Diagnosis: Skin scraping Brownish, branched, septate hyphae and budding cells SDA colonies: grey or black colonies
- 36. Infection of hair Firm, irregular nodules along the hair shaft Nodules : composed of fungal elements, cemented together on the hair shaft Black piedra : caused by Piedraia hortae White piedra: caused by Trichosporon beigelii Piedra
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- 41. Commonly called ringwormor tinea infection Infection of keratinised structures like hair, nail & skin caused by keratophilic Dermatophytes acute or chronic Dermatophytes
- 42. Characteristics of dermatophytes: Hyaline filamentous fungi, digest keratin by producing enzyme keratinase Resistant to cyclohexamide Classified into 3 genera Microsporum Trichophyton Epidermophyton Microsporum: M gypseum, M cannis Trichophyton: T rubrum, T mentagrophytes , t verrucosum Epidermophyton: E floccosum
- 43. Twice commonin males than females Tinea barbae (barber’s itch): involves beared area of face & neck Tinea corporis: smooth or non-hairy skin of the body Tinea imbricata: found in tropics, extensive concentric rings papulosquamous scaly patches Tinea capitis: affects scalp Tinea cruris: affects groin & perineum Tinea pedis: (athlet’s foot) Tinea manuum: affects hands Tinea unguium: affects nails Clinical aspects
- 44. Tinea barbae Tinea corporis Tineacruris Tinea capitis
- 45. Tinea manuum Tinea pedis Tineaimbricata Tinea unguium
- 46. Clinical features Skin lesions:circular, dry, erythematous, scaly & itchy Hair lesions: kerion, sacrring & alopecia, a) Favus: chronic type of ringworm, dence crusts (scutula) develop in the hair follicles leading to alopecia & scarring b) Kerion: severe boggy lesions with marked inflammation sometimes develop on scalp Nail lesions: deformed, friable, discolouration Accumulation of debris under the nails
- 47. Mechanism :unclear Fungal products : responsible for local inflammation Hypersensitivity to fungal Ags: sterile vesicular lesions, sometimes seen in sites distant from ringworm lesions, These lesions are called as dermatophytidis Diagnosis : by clinical features, use of Wood’s lamp Pathogenicity
- 48. Specimen: skin scrapings(from edges of the lesions), nail & hair clippings(hair plucked from the scalp) Microscopy: wet preparation: scrapings from the lesion is placed in a drop of 10-20% KOH on a slide, put a cover slip, keep for 10-20 min to digest keratin. Then observe the slide under the microscope Laboratory diagnosis
- 49. Hair: pluckedhair examined under UV light (Woods lamp) Ectothrix : arthrospores seen surrounding the hair shaft Endothrix: spores : inside the hair shaft
- 50. Specimen :inoculated on SDA containing Chloramphenicol or SDA with chloramphenicol & cycloheximide Incubated aerobically at 25-30 0 C upto 21 days identification by macroscopic & microscopic appearance of fungal colonies Trichophyton: powdery, velvety or waxy with pigmentation Microsporum: cotton like, velvety or powdery with white to brown pigmenation Epidermophyton: powdery greenish or yellow colonies Culture
- 52. Trichophyton: Microconidia abundant,scanty Macroconidia: thin, elongated , blunt ends, spiral hyphae, racquet mycelium, affects hair, nail & skin Microsporum: scany microconidia, Macroconidia: spore form, large, arranged singly on the end of hyphae, affects hair & skin Epidermophyton: powdery & greenish yellow, microconidia : absent, Macroconidia: pear shaped, arranged in clusters, affects skin & nails Microscopic examination
- 56. Species identification: physiological tests Hydrolysesurea Grow on polished rice grains Special amino acids & vitamins can be tested Antigen detection: hypersensitivity test (trichophytin)
- 57. Mild infection :topical imidazole Severe infections: griseofulvin orally 4-6 wks Hair : 3-6 mnths treatment Nails: upto 1 year Alternative treatment: oral imedazole like ketoconazole 200 mg b d daily or itraconazole 100 mg od daily, fluconazole 150 mg od daily Treatment
- 58. Human : mainhost for Anthrophilic dermatophytes e g Tricophyton rubrum, Microsporum audouinii, Epidermophyton floccosum Zoophilic: affects animals e g T. verrucosum, M cannis in dogs Geophilic: naturally in soil, less pathogenic for human e g M gypseum Epidemiology & Prevention
- 59. Personal cleanliness avoidcontact with infectious material Proper treatment
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