Fungal infections diagnosis
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This document provides information on laboratory diagnosis of fungal infections. It begins with an introduction to mycology and classification of fungi. It then discusses various diagnostic methods for different types of fungal infections, including microscopic examination, culture techniques, biochemical methods, and molecular identification. Specific techniques are described for diagnosing candidiasis, including microscopic morphology, culture characteristics on different media, biochemical profiles, commercially available identification systems, and molecular methods. Sample collection and processing methods are also summarized.
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Fungal infections diagnosis
- 2. Contents Introduction Classification of fungal infections Diagnostic methods for various types of fungal infections References
- 3. Introduction • Mycology- Study of fungi • Fungi – eukaryotic microorganisms • Cell walls - chitin, mannan & other polysaccharides • Cytoplasmic membrane - sterols • Possess–true nuclei, nuclear membrane and paired chromosomes • Divides – Sexually, asexually or both
- 4. Classification Depending on cell morphology Yeasts • Unicellular – spherical or epllipsoidal – reproduce by simple budding Cryptococcus neoformans Yeast like fungi • Grows partly as yeast and partly as hyphae – Candida Albicans Moulds or filamentious fungi • Multicellular – form true mycelia – reproduce – formation of spores - Dermatophytes Dimorphic fungi
- 5. Identification Yeasts • Biochemical Reactions • Fermentation And AssimilationOf Carbohydrates • UtilizationOf Enzyme Substrates And • Other MetabolicActivities Moulds • Colour •Texture ColonialAnd • Microscopic Morphology The Specialised Asexual Reproductive Structures Of Moulds Are Useful In Useful In DifferentiatingVarious Species Of Moulds
- 6. Fungal infections of oral cavity • Candidiasis • Blastomycosis • Histoplasmosis • Coccidioidomycosis • Cryptococcosis • Geotrichosis • Phycomycosis • Sporotrichosis • Rhinosporidiosis • Aspergillosis
- 7. Specimen Selection & collection Acc to Epstein and Pearsall et al. guidelines for specimen collection are • Specimen should be collected from active lesion. Old burn out lesions don’t contain viable organisms • Specimen should be collected under aseptic conditions • Collect sufficient specimen • Use sterile collection devices and containers • Specimen should be labelled appropriately
- 8. Specimen transport, storage & processing • Specimen should be examined & cultured immediately & transported by normal saline / phosphate buffer saline.
- 9. Candidiasis C. Albicans C. Gulliermondi C. Intermedia C. Krusei C. Paraspilosis C. Pseudotropicalis C.Stellatoidea C. Tropicalis C. Glabrata C. Viswanathii Ovoid / spherical budding yeast cell, 3-5µm in diameter Produces pseudomyclia both in culture and in tissues Most common opportunistic agents causing infections in humans
- 10. Identification of Candida species A. Simple microscopic examination I. KOH preparation II. Calcoflour white stain III. Fluorescent antibody stains – Acridine Orange IV. Tissue stains – PAS, Methanamine Silver, Wright’s Stain B. Culture media C. Morphological criteria I. Germ tube test II. Cornmeal Tween 80 D. Physiological criteria I. Carbohydrate assimilation tests II. Carbohydrate fermentation tests
- 11. E. Commercially available identification system I. API – 20 C AUX and API – 32 C II. Minitek III. Candifast IV. Rap ID yeast plus V. Iatron candida check F. Instrumental based identification system I. AMS – YBC II. Abbott MS – 2 III. Abbott Quantum II G. Molecular methods I. Electrophoretic karyotype differences II. Restriction fragment length polymorphisms (RFLPs) using gel electrophoresis or DNA-DNA hybridisation III. Polymerase chain reaction IV. Microarrays
- 12. Simple microscopic examination KOH preparation: Wet Mount • Examines material from specimen directly • KOH - clear cellular debris and make pseudohyphae, and spores more apparent • Works well for skin and mucosa scrapings, pus material, sputum, hair and nails • Yeast cells, pseudohyphae and hyphae that were by cellular material or mucus will be visible after occurs
- 13. Calcoflour – white stain: • Calcofluor white - non-specific fluorochrome that binds with cellulose contained in the cell walls of fungi • Used for direct examination of specimens using fluorescent microscopy • Fungi stain bright green or blue-white depending upon the filters used
- 14. • FLUORESCENT ANTIBODY STAIN: • Specific reagents for fluorescent antibody stains are available for some of fungi • Specificity is very poor and cross-reactions are common
- 15. TISSUE STAINS PAS technique: Preparation of solutions: A) Periodic acid solution: •Periodic acid-1gm •Distilled water-100ml B) Schiff's reagent: Dissolve 1gm basic fuschin and 1.9gms of Sodium metabisulfite in 100ml of HCL Shake the solution at intervals for 2 hrs Solution should be clear and yellow to light brown Add 500mgs activated charcoal and shake for 1-2 mins Filter through a No.1 Whatman filter, the solution should be clear and colorless If the solution is yellow repeat charcoal decolorization Store at 4°c
- 16. Deparaffinize in xylene and rehydrate through graded ethanols to deionized water Oxidize with periodic acid for 5min Rinse with several changes of distilled water Cover with Schiff's solution for 15min Wash in running tap water for 5-10min Stain nuclei with Harris’ heamatoxylin, differentiate in acid-alcohol and blue the sections Dehydrate in graded ethanols Method
- 17. • Glycogen and other periodate reactive carbohydrates-magenta • Nuclei-blue Results
- 18. Grocott hexamine (methenamine) silver By Grocott, Glomori, 1946 Sections: Formalin-fixed Paraffin
- 19. • Tone in 0.1% gold chloride, 4min • Rinse in distilled water • Treat in 2% sodium thiosulphate, 1min • Counterstain in Arzac’s stain, or 0.1% light green in 0.1% acetic acid,15-30 seconds, or stain with H&E • Blot, dehydrate, clear and mount in DPX • Deparaffinize sections and hydrate to distil water • Oxidase in 5% aqueous chromium trioxide (chromic acid), 1hr • Wash in tap water • Rinse in 1% sodium metabisulphite • Wash in tap water, 5 min • Rinse in distilled water, then place in preheated incubating solution in the dark, up to 1hr Method
- 20. Culture media Sabouraud’s dextrose agar • Glucose 20gm • Peptone 10gm • Agar 15gm • Water 1 litre • Steam to dissolve • pH 5.4 • A freshly collected specimen is spread on plates and incubated at 28°c. • The colonies will be apparent with in 2-3 days. • Candida species appears as smooth, creamy, white and glistening colonies
- 21. C. Albicans Colonies are creamy, smooth, white and glistering and older colonies are cream colored waxy or soft & smooth C. Guillermondii Thin, flat, glossy, cream to pinkish colonies are C. Glabrata Cream colored soft glossy smooth colonies. cells. C. Krusei Colonies are flat, dull dirty, greenish yellow color. C. Colonies are yellowish, glistering smooth. C. Small creamy & smooth colonies (on blood agar – stellate colonies) C. Tropicalis Dull soft, & wrinkled colonies. C. Pseudotropic lis Creamy, reticulate or smooth colonies. C. Viswanathi Cream colored & soft glistering colonies
- 22. • Culture media:- • Pagano-Levin agar distinguishes between Candida species based on reduction triphenyltetrazolium chloride • The medium produces pale coloured – colonies of C.albicans, whilst colonies Candida’ species exhibit varying degrees of pink coloration. • Other commercially available chromogenic agars • CHROM agar, Albicans ID, Fluroplate and Candichrom albicans, Potato Agar
- 23. Chrom agar media • Novel differential culture for isolation. • A single colony is streaked on to chrom agar plates & incubated at 37°c with carbon dioxide in dark – 48 hrs. C. Albicans Light green C. Glabarata Purple C.Tropicalis Blue with pink hallow C. Parapsilosis Cream colored C. Krusei Pink
- 24. Corn meal agar (cornmeal+ agar+water) • Differential media for identification of species of candida. • In the corn meal agar plate load the test specimen by making a well in it. • Place a cover slip over it incubate at 22°c for 48 hrs & • Examine the plate under microscope.
- 25. MORPHOLOGICAL CRITERIAGERM TUBE TEST • It is a rapid screening procedure for differentiating C. from other species. • A germ tube is a filamentous, cylindrical outgrowth from yeast cell with no constriction present at the base.
- 26. PHYSIOLOGICAL CRITERIA • Carbohydrate assimilation profiles: • Assimilation tests detect whether the yeast can utilize the carbohydrate as a sole carbon without regard to whether acid or gas is • Can be obtained by examining zones of growth around discs or wells impregnated with various sugars on basal agars.
- 27. CARBOHYDRATE FERMENTATION TEST: • Fermentation tests detect whether yeast can produce gas from the carbohydrate under anaerobic conditions • Physiological tests are time consuming and laborious to perform
- 28. Commercially available identification system • I. API – 20 C AUX and API – 32 C : • Consist of identification plastic strips based on carbohydrate assimilation that provide identification after 24-72 hrs incubation at 300C • Permits identification of 24 and 38 types of Candida species • Identification - comparing the pattern of reactions with the manufacturer’s charts • 96-99% as accurate as • II. Minitek : • Minitek system examines the ability of yeast to grow in the presence of specific carbohydrates • The test uses sugar impregnated discs, placed on seeded basal agars and is therefore the same in principle as traditional assimilation tests
- 29. • III. Candifast : • Identifies Candida based on sugar fermentation reactions, urease production and resistance to • It is simple to use and provides results after 24-48 h incubation at 37°C • Interpretation of colour changes test wells is subjective • Sensitivity of Candida to seven agents is incorporated into the kit • IV. Rap ID : • The RapID is a 4-h micropanel system that utilizes single-substrate test reactions based on preformed enzymes in tested isolates to identify yeasts. • Reactions are determined by color changes of chromogenic substrates
- 30. V. Iatron candida check : • It identifies candida by slide agglutination of yeast cells with specific antisera • A battery of 10 antisera is available, enabling the identification of eight Candida species
- 31. INSTRUMENTAL BASED IDENTIFICATION SYSTEM • Semi automated approaches • Expensive – laboratories – large – specimens • Various systems available • AMS –YBC • Abbott Quantum II
- 32. AMS –YBC • TheVitek-AMS system - originally designed - automated identification of bacteria - use - extended to yeasts • The system is used in conjunction with a yeast biochemical card permitting the identification of 36 species of yeast including 16 species of Candida • Results are obtained after 4 – 8 hrs • based on the transmission of light through inoculated substrate pools
- 33. Abbott Quantum II •Semi-automated system •Identifies medically important yeasts based on 19 biochemical tests and germ-tube formation
- 34. MOLECULAR METHODS (Genetic analysis) Electrophoretic karyotyping • Well-established method - typing Candida spp • In particular • Pulsed field gel electrophoresis (PFGE) by contour clamped homogeneous electric field (CHEF) of C. albicans allows to species in several karyotypes and to distinguish it from close species like C. dubliniensis • Used to characterize strains of C. albicans responsible for recurrent oropharingeal candidiasis
- 35. Restriction fragment length polymorphisms (RFLPs) using gel electrophoresis or DNA-DNA hybridisation • RFLP - based - digestion - DNA Every organism possesses unique nucleotide sequences that distinguish it from every other organism on the basis of the number and size of the fragments • DNA is extracted from isolates • Cleaved into fragments by restriction endonucleases • separated by gel electrophoresis
- 36. • RFLP method - successfully applied - exact identification Candida species • A HaeIII digest - distinguishing C. albicans species from other non- C. albicans species • BfaI is - differentiation of C. parapsilosis and C. krusei • DdeI digestion - efficient to identify C. albicans species
- 37. Polymerase chain reaction • Used - identify candida • Based on detection of candidal genes encoding - chitin synthase, actin and cytochrome P450LIAI • Enables discrimination of C. Albicans, C. Glabrata, C.Tropicalis, C. Krusei and C. Dubliniensis
- 38. Microarray • Microarrays powerful genomics tool • illuminate differences in the expression of genes within cells • Studies - focused - specific issues - investigating how Candida albicans - human fungal pathogen - is able to protect itself from the toxic effects - nitric oxide produced by - immune system • Revealed - group - nine genes were over-expressed during exposure to nitric oxide • - nine genes yhb1 - produces a flavohemoglobin that detoxifies nitric oxide - most highly expressed
- 39. Serological tests • Assessment of cell-mediated immunity against Candida albicans and other antigens - severe chronic candidosis • Oral candidosis - moderately elevated antibody titers in serum and saliva against C. albicans Serologic tests are normally not a diagnostic tool for oral candidosis
- 40. Imprint Culture Technique Square , plastic foam pads Dipped – Sabouraud’s broth Placed–specific oral site–60sec Pagano – levin / Sabouraud agar –8 hrs - 37ºC Candidal density – determined– Gallenkamp colony counter- CFU/mm2
- 41. Advantages 1. Quantitative assesment – yeast growth – diff areas – oral mucosa. 2. Useful – localizing – site – infection & estimating candidal load – specific area either – health / disease Disadvantages : • Possible accidental risk – inhalation - foam pads - pt especially in children / elderly. • Colony counts > 30 CFU /mm2 – mucosa – dentate & >49 CFU/mm2 – denture wearers – suggest candidal infection.
- 42. Impression technique Maxillary & mandibular alginate impressions Transporting - lab Casted – 6% agar -Sabourauds dextrose broth wide necked, Sterile , screw topped jar at 37ºC CFU – estimated
- 43. Oral rinse technique • CFU per ml – using spiral plater • Advantage : simple – perform – does not involve – clinician – judgement – sampling site. Rinse - 10ml – phosphate buffered saline - 60 sec Expell – universal container Centrifuged at 1700º C – 10min Resuspended – 1ml – sterile PBS Inoculated – Appro media
- 44. Salivary Culture Technique 2ml – Mixed unstimulated saliva Sterile universal container Vibrated - 30 sec – Bench vibrator CFU / ml- resultant growth– Sabouraud agar • Carriers & pts – oral candida – be distinguished reliably. • Clinical candidosis harbor > 400 CFU / ml.
- 45. Paper points • Detected previously – high no’s – subgingival flora / gingival tissues – acute periodontal abscesses. • An absorbent sterile paper point – inserted – depth – pocket & then transffered - laboratory & plated on appropriate media
- 46. Histoplasmosis Histoplasma Capsulatum • DME – demonstrate yeasts 2 - 4 µm diameter • Culture - SDA • H/P - PAS or GMS • Serologic testing for antibody and antigen detection
- 47. • Granulomatous infection - chiefly affects - reticulo endothelial system • The mucosal epithelium – ulceration in non ulcerated areas, pseudoepitheliomatous hyperplasia - seen • Sub mucosa - dense infiltrate - granulocytes, lymphocytes, plasma cells and histiocytes • Multinucleated giant cells and caseous necrosis - seen • PAS and Grocott-Gomori methenamine silver methods • Demonstrate the characteristic 2-4 µm diameter yeasts of H.capsulatam
- 48. Blastomycosis Types 1.North American Blastomycosis 2. South American Blastomycosis • Culture - Sabouraud’s agar, growth is slow up to 6 weeks at 32°c. • H/P - PAS or Methanamine silver stain
- 49. North American blastomycosis Blastomyces Dermatitidis • Inflamed connective tissue -mixture of acute and granulomatous inflammation, doubly refractile cell wall, giant cells, macrophages •Microabscesses • Lesions are not ulcerated, overlying psuedoepitheliomatos hyperplasia - prominent
- 50. South American Blastomycosis Blastomyces Brasiliensis • Microscopy - pseudoepitheliomatous hyperplasia - ulceration - overlying surface epithelium • Collections - epithelioid macrophages multinucleated giant cells • Scattered large yeasts - identified after staining - tissue sections - grocott-gomori methenamine silver or PAS method • Organism - multiple daughter buds on the parent cell - resulting in an appearance - described as resembling “mickey mouse ears “ or spokes of a ship’s steering wheel (mariner’s wheel)
- 51. Coccidioidomycosis Coccidioides Immitis Types : • Primary non disseminated coccidioidomycosis • Progressive disseminated coccidioidomycosis • DME - Large 20-60 µm round spherules that may contain endospores • Culture • H/P - foci of coagulation necrosis with multinucleated giant Organisms seen with no budding, endospores with large • Animal inoculation - injected peritoneally, with in 5-6 days pus aspirated shows spherules
- 52. Cryptococcosis Cryptococus Neoforms • DME - shows round to oval budding cells with thick wall and refractile gelatinous capsule • Culture • H/P • Serum or CSF study
- 53. H/P – Gram positive, budding yeast like - 5-8µm - large clear halo – tissue microcyst, capsule intensely colored with PAS, GMS or mucicarmine due to mucopolysaccharide capsule
- 54. Zygomycosis Mucorales • Types – Superficial,Visceral • Culture • H/P
- 55. Necrosis with thrombi consisting of organism, predilection for blood vessels and penetrate their walls Organism - 6-30µm, large, non septate hyphae with branching out obtuse angle and round to ovoid sporangia is also seen This distrupts normal blood flow to the tissue, resulting - infarction and necrosis Neutrophilic infiltrate - predominates in the viable
- 56. Sporotrichosis Sporotrichum Schenckii • Ovoid branching organism - septate hyphae - budding forms • 3-5µm – diameter - small size - seldom recognized - routine tissue sections • Cultured on sabouraud’s medium
- 57. •The tissue reaction - granulomatous - epithelioid cells, multinucleated giant cells - langhans type , lymphocytes, surrounding a central area of purulent or caseous necrosis •Polymorphonuclear leukocytes - prominent •Asteroid bodies – seen •Pseudo epitheliomatous hyperplasia - overlying epithelium - skin or mucosal lesions
- 58. Rhinosporidiosis Rhinosporidium Seeberi • The organism appears - sporangia - containing - large number - round or ovoid endospores, 5-7 microns in diameter • Sporangia are characteristic in appearance • Focal abscess formation - occasional multinucleated giant cells • acute and chronic inflammatory cells
- 59. Aspergillosis aspergilli fumigatus • DME : Septate hyphae • culture • Histopathology :- septate hyphae branching at acute angles, of 3 - 4µm in diameter • Occlusion of vessels - results in the characteristic pattern of necrosis
- 60. References • Prof. C.P.Baveja.Text book of Medical Microbiology. 2nd edition:Arya publications; 2008. • Shafer, Hine, Levy.Textbook of Oral Pathology. 6th edition. Noida: Elsevier Publications; 2009. • Neville, Damm, Allen, Bouquot. Textbook of Oral & Maxillofacial Pathology. 3rd edition. Philadelphia: Saunders- An Imprint of Elsevier Publications; 2005. • Regezi, Sciubba, Jordan. Textbok of Oral Pathology- Clinical pathologic correlations. 4th edition. Missouri: Saunders- An Imprint of Elsevier Publications; 2003. • Pathogenesis and treatment of oral candidosis- journal of oral microbiology 2011 david w ,michaell • Fungal infections of the oral mucosa- indian journal of dental research, 23(5), 2012 anitha krishnanp • Laboratory manual for diagnosis of fungal opportunistic infections in HIV/AIDS patients: World Health Organization 2009 • Mohammad Shahid, Iqbal Ahmad. Laboratory Diagnosis of Fungal Infections:An Overview: Chapter 9.
Editor's Notes
- KOH preparation : It is used to examine material from specimen directly. The KOH will clear cellular debris and make pseudohyphae, hyphae and spores more apparent. This method works well for skin and mucosa scrapings, pus material, sputum, hairs and nails. Yeast cells, pseudohyphae and hyphae that were obliterated by cellular material or mucus will be visible after clearing occurs.
- II. Calcoflour – white stain : Calcofluor white is a non-specific fluorochrome that binds with cellulose and chitin contained in the cell walls of fungi. It is used for direct examination of most specimens using fluorescent microscopy. Fungi stain bright green or blue-white depending upon the filters used.
- III. Fluorescent antibody stain : Specific reagents for fluorescent antibody stains are available for some of fungi. However, specificity is very poor and cross-reactions are common.
- IV. Tissue stains : Although swabs and smears are essential for a microbiological diagnosis of a number of types of oral candidiasis when candidal leukoplakia (chronic hyperplastic candidiasis) is suspected, a biopsy specimen should be taken. Candida species stain poorly by hemotoxylin and eosin, staining with periodic acid Schiff (PAS) or Gridley’s or Gomori’s methenamine silver (GMS) stains are used. Fungi in these stains take up pinkish-red. The presence of blastopores and characteristic pseudohyphae or hyphae in the superficial epithelium tissues identifies the fungus as a species of Candida. Therefore, if Candida are seen in tissue sections of suspect patients, serial sections should be carefully searched for pseudohyphae or hyphae of Candida species.
- FUNGS COLOUR ***********
- B. Culture media : The most frequently used primary isolation medium for Candida is Sabouraud’s dextrose agar (SDA), although it permits growth of Candida, but it also suppresses the growth of many species of oral bacteria due to its low pH. Pagano-Levin agar distinguishes between Candida species based on reduction of triphenyltetrazolium chloride. The medium produces pale coloured-colonies of C. albicans, whilst colonies of other Candida’ species exhibit varying degrees of pink coloration. Other commercially available chromogenic agars are CHROM agar, Albicans ID, Fluroplate and Candichrom albicans.
- COLOUR **************
- Mycelium – thread like process, hyphae : filament composing of mycelium.
- C. Morphological criteria I. Germ tube test : It is a rapid screening procedure for differentiating C. albicans from other species. A germ tube is a filamentous, cylindrical outgrowth from the yeast cell with no constriction present at the base.
- D. Physiological criteria : Physiological properties need to be examined in conjunction with morphological criteria. I. Carbohydrate assimilation profiles : Assimilation tests simply detect whether the yeast can utilize the carbohydrate as a sole carbon source without regard to whether acid or gas is produced. Carbohydrate assimilation profiles for Candida species can be obtained by examining zones of Candida growth around discs or wells impregnated with various sugars on basal agars.
- II. Carbohydrate fermentation test : Fermentation tests are generally performed in liquid media and to detect whether or not the yeast can produce acid or gas from the carbohydrate under anaerobic conditions. Candidal species were identified based on their carbohydrate fermentation characteristics. Individually these physiological tests are time-consuming and laborious to perform.
- E. Commercially available identification system : The introduction of commercially available identification systems has greatly reduced laboratory time involved in the speciation of Candidal isolates. The commercial systems presently available are based on growth or enzyme production profiles. Growth systems are relatively slow, typically requiring 72 h incubation, whilst enzyme systems can potentially provide results within a few hours. I. API – 20 C AUX and API – 32 C : Consist of identification plastic strips based on carbohydrate assimilation that provide identification after 24-72 hrs incubation at 300C, and permit the identification of 24 and 38 types of Candida species, respectively. Identification is made by comparing the pattern of reactions with the manufacturer’s charts. They are 96-99% as accurate as conventional pro-cedures. II. Minitek : Minitek system examines the ability of yeast to grow in the presence of specific carbohydrates. The test uses sugar impregnated discs, placed on seeded basal agars and is therefore the same in principle as traditional assimilation tests.
- III. Candifast : Identifies Candida based on sugar fermentation reactions, urease pro-duction and resistance to actidione. It is simple to use and provides results after 24-48 h incubation at 37°C. Interpretation of colour changes within test wells is subjec-tive. Sensitivity of Candida to seven antifungal agents is incorporated into the kit. IV. Rap ID : The RapID is a 4-h micropanel system that utilizes single-substrate test reactions based on preformed enzymes in tested isolates to identify yeasts. Reactions are determined by color changes of chromogenic substrates within the sample wells, some of which require addition of reagents.
- V. Iatron candida check : It identifies candida by slide agglutination of yeast cells with specific antisera. A battery of 10 antisera is available, enabling the identification of eight Candida species. Serotyping of C. albicans into two antigenic groups, A and B, is also possible, although discrepancy with other antisera has been reported. Upto 24% of C. albicans cannot be typed due to autoagglutination in saline solution
- F. Instrumental based identification system : Semi-automated approaches are available for the identifi-cation of Candida species. Since these methods are largely instrument-based, they are expensive and therefore most suited to laboratories ·processing large numbers of specimens. The various systems available are, I. AMS – YBC II. Abbott Quantum II
- I. AMS – YBC : The Vitek-AMS system was originally designed for the automated identification of bacteria but its use has since been extended to yeasts. The system is used in conjunction with a yeast biochemical card permit-ting the identification of 36 species of yeast including 16 species of Candida. Results are obtained after 4 – 8 hrs, based on the transmission of light through inoculated substrate pools.
- II. Abbott Quantum II : It is a semi-automated system that identifies medically important yeasts based on 19 biochemical tests and germ-tube formation.
- I. Electrophoretic karyotype differences : Electrophoretic karyotyping (EK) is a well-established method for typing Candida spp. In particular, pulsed field gel electrophoresis (PFGE) by contour clamped homogeneous electric field (CHEF) of C. albicans allows to subdivide this species in several karyotypes and to distinguish it from phenotypically close species like C. dubliniensis. EK has been used to characterize strains of C. albicans responsible for recurrent oropharyngeal candidiasis and in epidemiological investigations to identify the sources of hospital acquired C. albicans infections
- II. Restriction fragment length polymorphisms (RFLPs) using gel electrophoresis or DNA-DNA hybridisation: RFLP is based on the digestion of DNA. Every organism possesses unique nucleotide sequences that distinguish it from every other organism on the basis of the number and size of the fragments. DNA is extracted from isolates and cleaved into fragments by restriction endonucleases, the fragments are separated by gel electrophoresis. RFLP method has been successfully applied for the exact identification of these Candida species. The differences in the restriction patterns for the rDNA regions of the various Candida species serve as a rapid means of differentiating among these organisms. A HaeIII digest is definitive for distinguishing C. albicans species from other non-C. albicans species. BfaI is found to be useful in the differentiation of C. parapsilosis and C. krusei. DdeI digestion also seems to be efficient to identify C. albicans species.
- III. Polymerase chain reaction : It has been used to identify Candida based on detection of candidal genes encoding for chitin synthase, actin and cytochrome P450LIAI. Ribosomal DNA (rDNA; genes encoding for ribosomal RNA) is also a frequent target in PCR systems since rDNA sequences exist in multiple copies within the genome, thereby offering greater sensitivity. A relatively small divergence of the small subunit rDNA (l6S-like) sequences has occurred during evolution and therefore con-served regions exist between distantly related species. However, spacer regions between rDNA conserved sequences evolve faster and variations in the primary struc-ture of these spacers can exist between genera and species. Primers that target conserved rDNA sequence adjacent to the spacer regions permit the incorporation of the spacers into the PCR product. Although rDNA sequences have been widely used in typing and phylogenetic determination of bacteria, there have been relatively few studies examining the rDNA of fungi. RFLP studies have shown that rDNA sequences differ between Candida species and PCR amplification of 18S rDNA, enables dis-crimination of C. albicans, C. glabrata, C. tropicalis, C. krusei and C. dubliniensis. Despite the advantages of PCR- - based identification systems over phenotypic methods, the application of such molecular techniques is still relatively limited due to constraints of either technical staff training or in laboratory financing. However, the PCR is being increasingly used within hospital laboratories for the rou-tine detection of human gene mutations and also HLA typing, and therefore it is possible that genetic analysis will be more widely used in the future .
- IV. Microarray : Microarrays are a powerful genomics tool, designed to illuminate differences in the expression of genes within cells. A number of studies have focused on specific issues such as investigating how Candida albicans, a human fungal pathogen, is able to protect itself from the toxic effects of nitric oxide produced by the immune system. Microarray analysis revealed a group of nine genes were over-expressed during exposure to nitric oxide. Of these nine genes yhb1, which produces a flavohemoglobin that detoxifies nitric oxide, was the most highly expressed.
- Imprint culture Sensitive & reliable method Sterile plastic foam pads (2.5x2.5cm) dipped in sabouraud’s plate left for first 8 hrs of 48hrs and counted by gallenkamp counter
- Impression culture technique Alginate impression & casting with 6% agar with incorporated sabouraud’s dextrose broth incubated for 48-72hrs at 37°c and estimated
- Oral rinse technique 60sec with 10ml of phosphate buffered saline , it is concentrated by spinning at 1700xg for 10mins & resuspending in 1ml of sterile PBS Inoculated on media
- Salivary culture technique Expectorate 2ml of mixed unstimulated saliva in sterile universal container and vibrated for 30sec and counted on sabouraud’s agar
- Histoplasmosis DEM- stained with giemsa or wright’s stain shows small oval yeast like cells,1-5µm with polymorphonuclear cells Culture- BHI with cyclohexamide & chloramphenicol incubated at 25c & one with out antibiotics – septate hyphae with 2-3 m smooth walled H/P- granulomatous infection affecting reticuloendothelial system Fluorescent antibody technique is useful in diagnosis