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fungal diagnosis.pptx
- 1. Laboratory Diagnosis of FungalInfections
- 2. Classification Based on molecularevidence: base sequences from ribosomal RNA (Patterson & Sogi
- 3. Comparison of fungiand bacteria Feature fungi bacteria Diameter 4um 1um Nucleus Eukaryotic prokaryotic Cytoplasm Mitochondria and endoplasmic reticulum present Mitochondria and endoplasmic reticulum absent Cell membrane Sterols present Sterols absent Cell wall chitin peptidoglycan Spores Sexual and asexual spores for reproduction Endospores for survival, not for reproduction Thermal dimorphism yes No Metabolism Require organic carbon; no obligate anaerobes May do not require organic carbon; many obligate anaerobes
- 4. It's advisable tocollect as much material as possible due to scarcity of many fungi in many specimens. The type of the sample collected is determined according to the suspected site of infection such as:-skin scrapping – nail clipping – hair epilation – sputum – blood, C.S.F. Sample
- 7. Sampling using bluntscalpel
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- 11. Skin, nail andhair: They are collected in air-tight containers such as small glass bottles. The fungi in test samples can remain viable for weeks or even months. They shouldn't be refrigerated as the viability of some species of dermatophytes is affected. Storage and transport
- 12. Other specimens shouldbe collected, stored and transported in a manner similar to that employed for bacteriological investigation. If specific fungus is suspected, the lab should be notified as special media and culture procedures may be needed and that also will be helpful for the safety of lab personnel.
- 13. Laboratory Diagnosis Direct examination:Very decisive in the diagnosis of fungal infections Fungal culture Serological tests Skin tests PCR & other molecular methods
- 14. Give anearly indication of the presence of yeast especially if from normally sterile tissue provided the sample collected aseptically, The presence of hyphea helps in establishing differential diagnosis. The observation of capsule surrounding yeast gives a presumptive diagnosis of cryptococcosis. It provides physician with early information regarding the possible need of treatment. Guide the microbiologist about the type of culture media used. Direct examination
- 15. A) Unstained preparations(wet mount) : KOH preparation for digestion of organic substances and keratinized material giving a clear background without affecting the fungus. DMSO can be added to KOH to hasten clearing in skin scrapings & nail clippings. India ink preparation to detect capsulated fungi i.e: Cryptococcous neoformans. Methods
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- 17. 27.05.09 Phase I/ ModuleVII Dr Ekta CFW – yeast form of Blastomyces KOH - Aspergillus India ink - Cryptococcus
- 18. B) Stained preparations: lactophenol cotton blue. Calcoflour white fluorescent stain. Gram stain for detection of aerobic actinomycetes. Modified kinyoun acid fast stain for detected of partially acid fast filaments of Nocardia species. Wright's stain and Giemsa stain to detect intracellular yeast forms of Histoplasma capsulatum in blood and bone marrow. PAS and Gomori's methanamine silver stain for demonstrating fungi in tissue.
- 19. Fluorescent- antibodystaining To detect fungal Ag in clinical specimen such as pus, blood, CSF, tissue sections Adv – can detect fungus even when few organisms are present 27.05.09
- 20. Gram stain– fungi are Gram positive
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- 36. Sabouraud DextroseAgar (SDA) Cyclohexamide should be added to sabauraud's for isolation of primary pathogens and inhibit opportunistic pathogens. Chloramphenicol or gentamicin to inhibit bacterial contamination. Selective media Brain Heart Infusion (BHI) agar :dimorphic & other fastidious fungi Fungal Culture
- 37. Phase I/ ModuleVII Dr Ekta Corn Meal Agar Bird Seed Agar
- 38. All fungiare aerobic Temperature requirement : temperature:25-27°C (for filamentous fungi) or at 37°C (for yeast). Majority of fungi – 37°C Superficial mycosis – 30°C Dimorphic fungi – 25°C & 37°C Incubation time:, At least 4 weeks Usually positive cultures are obtained in 7-10 days Candida & Aspergillus - 24 to 72 hrs
- 39. Specimens shouldbe cultured on agar slants: Safe Require less space More resistant to drying during prolonged incubation Blood cultures should be inoculated in to biphasic blood culture bottles
- 40. Interpretation of FungalCulture Isolation of an established pathogen like H. capsulatum or C. neoformans ; evidence of infection Isolation of commensal or opportunistic fungi like Candida or Aspergillus ; consider following points: 1. Isolation of same strain in all culture tubes 2. Repeated isolation of same strain in multiple specimens 3. Isolation of same strain from different sites 4. Immune status 5. Serological evidence
- 41. Identification of fungalcultures Colony character: Macroscopic examination of isolated colonies for the texture aspect, colour of the colony surface and the reverse of it, any pigment that diffuse in the medium.
- 42. Microscopic examination: Fungal morphologyunder microscope using Lactophenol Cotton Blue (LPCB) stain 27.05.09 Phase I/ Module VII Dr Ekta
- 43. Biochemical reactions : Fermentationof sugars Assimilation of sugars. Assimilation of N2. Other tests as urease test.
- 44. Identification of Candidaalbicans: The colonies are rounded white creamy in colour and creamy in texture. Film stained by lactophenol blue show budding yeast and filaments (pseudo-mycelium). If stained by Gram stain appears Gram positive. Germ tube test: culture of the yeast on serum causes rapid formation of filaments when incubated at 37°C for 4 hours. Fermentation of sugars.
- 45. Serology Detection of Agor Ab in serum or body fluids Ab detection: Diagnosis of systemic & subcutaneous mycoses Assess prognosis of the disease Assess response to treatment Ag detection: Early stages of infection In patients with impaired immunity 27.05.09
- 46. Experimental Animal infection Experimentalanimal inoculation is sometimes useful for the isolation of the causative fungus especially in deep mycoses..
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- 49. Guinea pig &Rabbit
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- 51. Serological tests usedin Medical Mycology Agglutination Immunodiffusion – most widely used Counter immunoelectrophoresis (CIEP) Indirect fluorescent Ab detection ELISA, RIA Delayed hypersensitivity reaction (Fungal skin test) : It has no value in diagnosis. Mainly used for epidemiological study
- 52. Other Methods Nucleic aciddetection: PCR RFLP Protein electrophoresis DNA hybridization (Nucleic acid probes) Woods light: it is an UVR produce fluorecent color when come in contact with fungal infection of skin and hair
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