Direct microscopic examination of clinical specimens can provide a presumptive diagnosis of fungal infection by revealing the presence of fungal elements. Different stains and techniques are used to visualize fungi depending on the suspected infection. KOH wet mounts are useful for superficial mycoses while GMS, H&E and fluorescent antibody stains aid in diagnosis of deep mycoses from tissue biopsies and body fluids. Proper specimen collection and rapid microscopic evaluation can help initiate appropriate antifungal treatment.

1. MYCOLOGY SPECIMEN COLLECTION & HANDLING

2. Specimen collection & transport Important considerations: Proper collection Rapid delivery to the laboratory Prompt and correct processing Inoculation into proper and appropriate medium Incubation at a suitable temperature

4. 100,000 units of Streptomycin

6. Dermatological: 15 – 30 OC

8. Specimen collection & transport BLOOD and BONE MARROW Transport medium: at 1:10 proportion TSB or TSA (biphasic agar or broth) BHI transport medium Thioglycollate broth Volume: 10 ml

9. CEREBROSPINAL: Transport immediately. Do NOT refrigerate. For suspected Cryptococcus, Coccidioides infections, containers must be leak proof and lab manipulations should be done under a hood Specimen collection & transport

10. Specimen collection & transport DERMATOLOGICAL SPECIMENS SKIN LESIONS Sterilized area with 70% alcohol or sterile water Collect at the the active border

11. Specimen collection & transport NAILS Clean with 70% alcohol If: Dorsal plate: scrape the deeper portion Nail plate: scrape beneath the nail plate Whole nail or clippings

12. Specimen collection & transport HAIR Collect from: Areas of scaling Alopecia Hair that fluoresce under Wood’s lamp

13. Specimen collection & transport EXUDATES & PUS Undrained or unruptured abscess Aspirate using sterile syringe, recap needle and transport to lab immediately Failed aspiration, do skin biopsy

14. Specimen collection & transport URINE First early morning Transport and perform test ASAP within 2 hours If not possible, refrigerate specimen.

17. MYCOLOGY METHODS OF IDENTIFICATION

18. A. DIRECT FUNGAL MICROSCOPY Clinical significance: Provide an immediate presumptive diagnosis Aid in the selection of appropriate culture media Aid in decision of what’s best inoculation technique to use It will provide evidence of infection despite negative culture

19. A. DIRECT FUNGAL MICROSCOPY Macroscopic Examination (physical exam): Note for: caseous material Purulent exudate Necrotic material Granules Punch biopsies Layers of skin that are broken vertically (fissures) Obtain specimens for microscopy and culture fro

20. Preparation for Microscopic examination: Mince or grind hard specimens Centrifuge for 3-5 minutes fluid specimens Pulvorize nail clippings Volume for fluid specimens: 0.5 ml Assemble a wet chamber for incubation A. DIRECT FUNGAL MICROSCOPY

21. REAGENTS used for DIRECT MICROSCOPIC STUDY KOH 10-20% Routinely used 10% = skin and soft tissues, body fluids 20% = nail and hard tissues Calcoflour white Green flourescense India ink “Dark field” microscopy for Cryptococcus neoformans A. DIRECT FUNGAL MICROSCOPY

22. A. DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Lactophenol Blue very popular for quick evaluation of fungal structures stains the chitin in cell walls of fungi blue Use for following up fungal culture growths Wright’s/Giemsa stain (Diff quick) For rapid staining of blood and bone marrow fungi (ex: Histoplasmacapsulatum) Modified Acid-Fast Stain used to differentiate the acid-fast Nocardia from other aerobic Actinomyces Gram Stain generally fungi are gram positive Actinomyces and Nocardia are gram variable

23. A. DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Stains for tissue mycoses: Periodic Acid - Schiff Stain (PAS) stains certain polysaccharide in the cell walls of fungi Fungi stain pink-red with blue nuclei. GomoriMethenamine Silver Stain silver nitrate outlines fungi in black due to the silver precipitating on the fungi cell wall. The internal parts of hyphae are deep rose to black, and the background is light green. Gridley Stain Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow.

24. Stains for tissue mycoses … Fluorescent Antibody Stain simple, sensitive, and extremely specific method of detecting fungi in tissues or fluids. Applications for many different fungal organisms. Mayer Mucicarmine Stain will stain capsules of Cryptococcus neoformansdeep rose. Papanicolaou Stain good for initial differentiation of dimorphic fungi Works well on sputum smears also A. DIRECT FUNGAL MICROSCOPY

25. KOH Wet Mounts Principle: KOH softens most tissues, dissolves fat droplets, bleaches many pigments and dissolves the “cement” that holds keratinized cells together; glycerine clears tissue debris, thus making it easier to demonstrate presence of fungal elements. Reagents: 10 – 20 % KOH: KOH pellets 10 – 20 grams Glycerine (optional) 10 ml Distilled water 90 ml

26. KOH Wet Mounts Procedure: Place a small amount of specimen on a clean glass slide place 1-2 drops of KOH on the specimen and overlay a cover slip Allow the preparation to stand for 10-30 minutes in a wet chamber. You can gently heat preparation to hasten the action of KOH Do not over heat for it may crystallize the KOH Examine preparation under low then high magnification. Take note for the presence of fungal elements (hyphae and/or spores)

27. INDIA INK PREPARATION aka: Nigrosin stain Principle: Specimen placed in a drop of India ink becomes darkly colored because of the carbon particle in the ink. Hyaline structures such as capsules and cell walls will be highlighted against a dark background of inked colored specimen creating an illusion of darkfield microscopy. Reagent: 1:1 dilution of the ink

28. India Ink Preparation Procedure: Place a drop of the specimen (body fluid or from culture) on a clean glass Put a drop of India Ink, mix and overlay a cover slip Examine under low power and high power with a bright field microscope Result: India ink creates a dark background against which hyaline fungal cell wall and capsules can se seen Limitation: wbc may be confused as fungi

29. Lactophenol Cotton Blue Principle: The morphology of fungal elements are preserved and stained better. Reagents: Lactic acid & Phenol Kills the organism Glycerin Prevents easy dehydration Cotton blue Dye or stain

30. DIAGNOSIS OF MYCOSES by UNSTAINED & STAINED MICROSCOPY

31. A. SKIN or DERMATOMYCOSIS KOH for superficial involvement, look for: Spaghetti & meat balls (lung aspirate) Malasseziafurfur Pseudohyphae and yeasts (vaginal secretions) Candida species

32. A. SKIN or DERMATOMYCOSIS KOH & LPCB for superficial involvement, look for: Hyaline septatehyphae ex: Dermatophytes Dematiaceousseptatehyphae ex: Tineanigra Alternaria

33. A. SKIN or DERMATOMYCOSIS H & E stain for Oral Candidiasis on Skin biopsy of tongue, look for: Pseudohyphae yeasts

34. B. DRAINING SINUS for MYCETOMAS & ACTINOMYCOSIS KOH, look for Various colored granules Actinomycosis/ Nocardiosis GMS stain, look for Various granules Mycetoma

35. C. EYE SCRAPINGS & ASPIRATE for KERATOMYCOSIS KOH & LPCB, look for Septate hyaline hyphae Aspergillus species Fusarium species Coenocytic hyaline hyphae Mucor species Pseudohyphae and yeasts Candida species

36. D. NASOPHARYGNEAL ASPIRATES f for RHINOSPORIDIOSIS KOH, look for Large sporangium with spores (lacrimal gland aspirate) Rhinosporidium species

37. E. HAIR for DERMATOMYCOSES & ALOPECIA KOH, look for Endothrix spores/hyphae Trichophyton Ectothrix spores/hyphae Trichophytonmentsgrophytes

38. E. HAIR for PIEDRA KOH, look for: Hard, brown, compact nodules (Black piedra) Piedraiahortae Soft, off-white, concretions/nodules (White piedra) Trichosporonbeigeli

39. F. NAILS for ONYCHOMYCOSIS KOH & LPCB, look for Septate, hyaline hyphae Dermatophytes Epidermophyton Trichophyton Microsporon Pseudohyphae and yeast cells Candida species

40. G. SYSTEMIC MYCOSESSpecimens: blood, CSF, sputum, other body fluids KOH & Mucicarmine stain systemic involvement, look for: Pseudohyphae and yeast cells (CSF) Candida species Broad based buds (brain and CSF) Blastomyces species

41. SYSTEMIC MYCOSES GMS for systemic involvement, look for: Spherules/sporangia (CSF) Coccidioidesimmitis PAS stain for systemic involvement, look for: Dematiaceousseptatehyphae (brain tissue)

42. SYSTEMIC MYCOSES H & E stain for systemic involvement, look for: Endospores (CSF brain tissue) Coccidioides species Fission/sclerotic bodies Chromomyces species

43. SYSTEMIC MYCOSES Wright’s/Giemsa stain (Diff quick) & LPCB for systemic involvement, look for: Small, intracellular budding yeast (CSF) Histoplasma species Small, intracellular yeast dividing by fission (CSF) Penicillin species

44. SYSTEMIC MYCOSES Mucicarmine stain & India Ink for systemic involvement, look for: Encapsulate yeast (CSF) Cryptococcus neoformans

45. SYSTEMIC MYCOSES LPCB & Fluorescent Antibody stain for systemic involvement, look for Large yeast with multiple buds called “mariner’s wheel” Paracoccidioidesbraziliensis

46. SYSTEMIC MYCOSES Calcoflour mounts for systemic mycoses , look for (flourescence) Pseudohyphae and yeasts (blood) Candida species Septate, hyaline at right degrees angle (bronchial lavage) Aspergillus species

47. Interpretation of Direct Microscopic Findings (Summary)