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. MYCOLOGYSPECIMEN COLLECTION & HANDLING

2. Specimen collection & transportImportant considerations:Proper collectionRapid delivery to the laboratoryPrompt and correct processingInoculation into proper and appropriate mediumIncubation at a suitable temperature

3. Specimen collection & transportTransport of Specimen:Antibiotics may be incorporated in body fluid specimens to prevent proliferation of bacteria:50, 000 units of Penicillin

4. 100,000 units of Streptomycin

5. 0.2 mg of ChloramphenicolSpecimen collection & transportTransport of Specimen:Storage temperature of specimen for fungal culture:Blood & CSF: 30 – 37 OC

6. Dermatological: 15 – 30 OC

7. Others: 4 OCSPUTUMfirst early morning sampleDeep cough specimen; may be induced by:Aqueous aerosol Bronchial tapVolume: 5 – 10 mlSpecimen collection & transport

8. Specimen collection & transportBLOOD and BONE MARROW Transport medium: at 1:10 proportionTSB or TSA (biphasic agar or broth)BHI transport mediumThioglycollate brothVolume: 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 hoodSpecimen collection & transport

10. Specimen collection & transportDERMATOLOGICAL SPECIMENSSKIN LESIONSSterilized area with 70% alcohol or sterile waterCollect at the the active border

11. Specimen collection & transportNAILSClean with 70% alcoholIf:Dorsal plate:scrape the deeper portionNail plate: scrape beneath the nail plateWhole nail or clippings

12. Specimen collection & transportHAIR Collect from:Areas of scalingAlopeciaHair that fluoresce under Wood’s lamp

13. Specimen collection & transportEXUDATES & PUS Undrained or unruptured abscessAspirate using sterile syringe, recap needle and transport to lab immediatelyFailed aspiration, do skin biopsy

14. Specimen collection & transportURINEFirst early morningTransport and perform test ASAP within 2 hoursIf not possible, refrigerate specimen.

15. Specimen collection & transportVAGINAL SECRETIONSSterile swabs

16. Put in transport medium or primary isolation broth immediately (ex: TSB)Specimen collection & transportTISSUES & Biopsy specimens:Collect aseptically at the center and edge of the lesionPlace in between sterile gauze wet with sterile NSS or transport medium.

17. MYCOLOGYMETHODS OF IDENTIFICATION

18. A. DIRECT FUNGAL MICROSCOPYClinical significance:Provide an immediate presumptive diagnosisAid in the selection of appropriate culture mediaAid in decision of what’s best inoculation technique to useIt will provide evidence of infection despite negative culture

19. A. DIRECT FUNGAL MICROSCOPYMacroscopic Examination (physical exam):Note for:caseous materialPurulent exudateNecrotic materialGranulesPunch biopsiesLayers of skin that are broken vertically (fissures)Obtain specimens for microscopy and culture fro

20. Preparation for Microscopic examination:Mince or grind hard specimensCentrifuge for 3-5 minutes fluid specimensPulvorize nail clippingsVolume for fluid specimens: 0.5 mlAssemble a wet chamber for incubationA. DIRECT FUNGAL MICROSCOPY

21. REAGENTS used for DIRECT MICROSCOPIC STUDYKOH 10-20%Routinely used10% = skin and soft tissues, body fluids20% = nail and hard tissuesCalcoflour whiteGreen flourescenseIndia ink“Dark field” microscopy for Cryptococcus neoformansA. DIRECT FUNGAL MICROSCOPY

22. A. DIRECT FUNGAL MICROSCOPYSTAINS for MICROSCOPIC STUDIES:Lactophenol Bluevery popular for quick evaluation of fungal structuresstains the chitin in cell walls of fungi blueUse for following up fungal culture growthsWright’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 ActinomycesGram Stain generally fungi are gram positiveActinomyces and Nocardia are gram variable

23. A. DIRECT FUNGAL MICROSCOPYSTAINS for MICROSCOPIC STUDIES:Stains for tissue mycoses:Periodic Acid - Schiff Stain (PAS) stains certain polysaccharide in the cell walls of fungiFungi 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 fungiWorks well on sputum smears alsoA. DIRECT FUNGAL MICROSCOPY

25. KOH Wet MountsPrinciple: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 gramsGlycerine (optional) 10 mlDistilled water 90 ml

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

27. INDIA INK PREPARATIONaka: Nigrosin stainPrinciple: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 PreparationProcedure:Place a drop of the specimen (body fluid or from culture) on a clean glassPut a drop of India Ink, mix and overlay a cover slipExamine under low power and high power with a bright field microscopeResult:India ink creates a dark background against which hyaline fungal cell wall and capsules can se seenLimitation: wbc may be confused as fungi

29. Lactophenol Cotton Blue Principle:The morphology of fungal elements are preserved and stained better.Reagents:Lactic acid & PhenolKills the organism GlycerinPrevents easy dehydrationCotton blueDye or stain

30. DIAGNOSIS OF MYCOSES by UNSTAINED & STAINED MICROSCOPY

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

32. A. SKIN or DERMATOMYCOSISKOH & LPCB for superficial involvement, look for:Hyaline septatehyphae ex: DermatophytesDematiaceousseptatehyphae ex: TineanigraAlternaria

33. A. SKIN or DERMATOMYCOSISH & E stain for Oral Candidiasis on Skin biopsy of tongue, look for:Pseudohyphaeyeasts

34. B. DRAINING SINUS for MYCETOMAS & ACTINOMYCOSISKOH, look forVarious colored granulesActinomycosis/ NocardiosisGMS stain, look for Various granulesMycetoma

35. C. EYE SCRAPINGS & ASPIRATE for KERATOMYCOSISKOH & LPCB, look forSeptate hyaline hyphaeAspergillus speciesFusarium speciesCoenocytic hyaline hyphaeMucor speciesPseudohyphae and yeastsCandida species

36. D. NASOPHARYGNEAL ASPIRATES f for RHINOSPORIDIOSISKOH, look forLarge sporangium with spores (lacrimal gland aspirate)Rhinosporidium species

37. E. HAIR for DERMATOMYCOSES & ALOPECIAKOH, look forEndothrix spores/hyphaeTrichophytonEctothrix spores/hyphaeTrichophytonmentsgrophytes

38. E. HAIR for PIEDRAKOH, look for:Hard, brown, compact nodules (Black piedra)PiedraiahortaeSoft, off-white, concretions/nodules (White piedra) Trichosporonbeigeli

39. F. NAILS for ONYCHOMYCOSISKOH & LPCB, look forSeptate, hyaline hyphaeDermatophytesEpidermophytonTrichophytonMicrosporonPseudohyphae and yeast cellsCandida species

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

41. SYSTEMIC MYCOSESGMS for systemic involvement, look for:Spherules/sporangia (CSF)CoccidioidesimmitisPAS stain for systemic involvement, look for:Dematiaceousseptatehyphae (brain tissue)

42. SYSTEMIC MYCOSESH & E stain for systemic involvement, look for:Endospores (CSF brain tissue)Coccidioides speciesFission/sclerotic bodiesChromomyces species

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

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

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

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

47. Interpretation of Direct Microscopic Findings (Summary)