Fungal diagnosis

Outline of fungal diagnosis

1. LABORATORY DIAGNOSIS OF FUNGAL DISEASES - Dr. ANKUR KUMAR

2. TOPICS for discussion - SPECIMEN COLLECTION MICROSCOPY CULTURE IMMUNOLOGICAL METHODS TESTS FOR METABOLITES TESTS TO DEMONSTRATE DELAYED HYPERSENSITIVITY MOLECULAR METHODS

3. Depends on the site of infection such as Skin scrapping Hair Nail Sputum Blood sample- For systemic mycoses may also be collected. Cerebrospinal fluid (CSF) is collected for cryptococcal meningitis Specimen Collection

4. Fungal diagnosis Hair Sputum & other Blood Urine CSF Nail respi . secretion Skin G/S India ink G/S KOH KOH Bac T SDA Mannual cult. on Biphasic media Identification culture SDA SDA s/c to check G/S Identification s/c to check latex agglut . LPCB of growth

5. Identification yeast mould G/S, India ink LPCB colony creamy white, smooth, and cottony, powdery, wolly , waxy, velvety pasty with typical yeasty odor mucoid creamy white GTT Dalmau CHROM agar Assimilation

6. Microscopy Direct microscopic examination of material from the lesion. Potassium hydroxide (KOH) preparation : Gram stain India ink and Nigrosin stains Lactophenol cotton blue (LPCB) Calcofluor white stain Histopathological stains

7. Potassium hydroxide (KOH) preparation Skin scrapings and plucked hair samples ( Keratinized tissue specimens) Treated with 10% KOH which digests the keratin material release fungal hyphae clearly seen under the microscope. 1O% KOH used. 20-40 % KOH - for Nail and Biopsy tissues that take longer time to dissolve. Glycerol (10%) is added to prevent drying DMSO ( dimethyl sulfoxide )- can be added which helps in tissue digestion.

8. India ink and nigrosin stains : Negative stains for demonstration of capsule of Cryptococcus neoformans . Gram stain : identifying the yeasts (e.g. Cryptococcus) and yeast like fungi (e.g. Candida). They appear as gram-positive budding yeast cells. Pseudohyphae - In yeast cell, the bud remains attached to the mother cell, elongates and undergoes repeated budding to form chains of elongated cells known as pseudohyphae .

9. Calcofluor white stain : more sensitive than other stains; binds to cellulose and chitin of fungal cell wall and fluoresce under UV light

10. Demonstrate the fungal elements from biopsy tissues (deep mycoses) Periodic acid schiff (PAS)stain : fungi appear magenta/deep pink , whereas the nuclei stain blue . G omori methenamine silver ( G MS ) stain : Alternative to PAS. Stains both live and dead fungi , as compared to PAS which stains only the live fungi . Stains the polysaccharide component of the cell wall. Fungi appear black whereas the background tissue takes pale green color . Mucicarmine stain : staining tie carminophilic cell wall of Cryptococcus and Rhinosporidium . Masson fontana stain : used for pigmemed (or pheoid ) fungi. Hematoxylin and Eosin (Hand E) stain. Histopathological stains

11. Culture Fungal culture - performed for isolation and correct identification of the fungi . Culture Media Basic media Sabouraud's dextrose agar (SDA ): Neutral SDA (Emmons' modification ) SDA with antibiotics

12. Culture SDA: It is the most commonly used media in diagnostic mycology. It contains Pepone 1% Dextrose 4% Agar 2% pH of 5.6. may not support some pathogenic fungi. Neutral SDA (Emmons' modification): lt differs from original SDA in having Neopeptone ( 1%) Dextrose (2%) and pH of 7.2

13. 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 : Culture plates should be incubated for 2-3 weeks Antibiotics added to the culture media to inhibit bacterial growth. Cycloheximide ( Actidione ), chloramphenicol and gentamicin

14. Culture Identification Macroscopic and microscopic appearances of the colonies grown on culture. Macroscopic Appearance of the Colony Rate of growth: Rapid growth (<5 days): in saprophytes, yeasts and agents of opportunistic mycoses. Slow growth ( 1-4 weeks): in agents of subcutaneous and systemic mycoses. Pigmentation: seen on obverse and reverse of the culture media. Texture: glabrous (waxy/leathery), velvety, yeast like or cottony or granular/powdery. Colony topography : Colony surface may be rugose (radial grooves), folded or verrucose or cerebriform (brain-Like).

15. Microscopic Appearance of Fungi Teased mount: A bit of fungal colony is teased out from the culture tube and LPCB mount is made on a slide viewed under microscope. ldentification is based on the following: Nature of hyphae -such as septate or aseptate , hyaline or phaeoid , narrow or wide) and Type of sporularion (conidia or sporangia). Slide culture: this is a tedious procedure, It gives most accurate in situ microscopic appearance of the fungal colony. A sterile slide is placed on a bent glass rod in a sterile petri dish. 2 square agar blocks measuring around 1 cm 2 (smaller than the coverslip ) are placed on the slide. Bits of fungal colony are inoculated onto the margins of the agar block. Then the coverslip is placed on the agar block and the petri dish is incubated ac 25 C. LPCB mounts are made both from the coverslip and the underneath slide

16. Lactophenolcottonblue (LPCB): Used to Study the microscopic appearance of the fungal isolates grown in culture. It contains: Phenol- acts as disinfectant Lactic acid- preserves the morphology of fungi. Glycerol- prevents drying. Cotton blue- stains the fungal elements blue.

17. Cellophane tape mount: This is easy to perform than slide culture in-situ fungal morphology is also maintained. placing the cellophane tape on the colonies present on the surface of SDA plate, then LPCB mount is made from the cellophane tape.

18. Other Methods of Identification For Candida: Germ tube rest Dalmau plate culture Sugar fermentation and sugar assimilation For dermatophytes : Hair perforation tests Dermacophyte identification medium For Cryptococcus Urease test

19. Germ tube test: Also called Reynolds Braude phenomenon . Rapid method of identifying C. albicans . It is a specific test for C. albicans but also be positive for C. dubliniensis Procedure- Few colonies are mixed with human or sheep serum and incubated for 2 hours. Wet mount preparation is examined under microscope. Germ tubes are formed, described as long tube like projections extending from the yeast cell. It is differentiated from pseudohyphae as there is no constriction at the origin.

20. Dalmau plate culture: Culture on cornmeal agar / Rice starch agar at 20 c can provide clue for species identification. C. albicans produces refractile , terminal thick walled chlamydospores .

21. Identification Nutritionally deficient media Corn meal agar and rice starch agar: Enriched media Brain heart infusion (BHI) agar and blood agar: Niger seed agar and bird seed agar: Differential/Indicator media CHROM agar Cadida medium:

22. Identification Corn 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 : for selective growth of Cryptococcus

23. Identification CHROM agar Candida medium: selective as well as differential media for speciation of Candida . C. albicans - Light green C. dubliniensis - Dark green C. glabrata - Pink to purple C. krusei - Pink

24. Tests for species Identification • CHROM agar: Different Candida species produce different colored colonies on CHROM agar… C. albicans - Light green C. dubliniensis - Dark green • Growth at 45 C : It differentiates C. albicans (grows) from C. dubliniensis (does not grow at 45°C). • Sugar fermentation test and sugar assimilation test differentiate between various Candida species. • Molecular methods – PCR using species specific primers are useful for species identificacion .

25. Immunological Methods Detect the antibody or antigen from serum and/or other body fluids. Antibody detection by ELISA, immunodiffusion test, agglutination test, and complement fixation test (CFT). Antigen detection: latex agglutination test for detecting cryptococcal antigen from CSF lmmunohistochemistry : useful in deep mycoses detect antigens ( e.g proteins) on the cells of a tissue section by using fluorescent tagged antibodies that bind specifically to the antigens.

26. Tests for Metabolites Alternate approach for the diagnosis of fungal infections is detection of specific fungal metabolites in body fluids By gas liquid chromatography. Tests to Demonstrate Delayed Hypersensitivity Skin tests hypersensitivity for Histoplasma , Blastomyces , Coccidioides , Paracoccidioides , Dermatophyte , Sporothrix and Candida.

27. Molecular Methods Polymerase chain reaction (PCR) Multiplex PCR, nested PCR and the most advanced real time PCR and DNA sequencing methods accurate identification of fungi from culture as well as from the specimens.

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