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Laboratory diagnosis of fungal infections depends on: (a) direct microscopy, (b) culture, (c) serological tests, (d ) non-culture methods, and (e) molecular methods.
Direct microscopic examination depends on demonstration of characteristic asexual spores, hyphae, or yeast in various clini-cal specimens by light microscopy. The commonly used clinical specimens are sputum, lung biopsy material, and skin scrapings.
The specimen is either treated with 10% KOH or stained with special fungal stains. Use of 10% KOH dissolves tissue material, leaving the alkali-resistant fungi intact.
Calcofluor dye is a fluorescent dye that combines with fungal cell wall and is useful in identification of fungi in tissue specimens. Methenamine silver stain is useful for demonstra-tion of fungi in tissues. India ink preparation of cerebrospinal fluid (CSF) is a useful method for demonstration of white cap-sule of C. neoformans in CSF. Gram staining is also useful to demonstrate Gram-positive Candida species in the specimen.
The disadvantages of microscopy are that it shows low sensitivity and requires an experienced microscopist for specific identification.
Fungal culture is a frequently used method for confirming the diagnosis of fungal infection. SDA is the most commonly used medium for fungal culture. Other media include CHROM agar, blood agar, etc. The low pH of the medium and addition of chloramphenicol and cycloheximide to the medium inhibit the growth of bacteria in the specimen and thereby facilitate the appearance of slow-growing fungi.
Fungal colony is identified by rapidity of growth, color, and morphology of the colony at the obverse and pigmentation at the reverse.
Microscopy of the fungal colony is carried out in lactophe-nol cotton blue (LPCB) mount to study the morphology of hyphae, spores, and other structures. The appearance of the mycelium and the nature of the asexual spores are very much helpful to identify the fungus.
Culture, however, is time-consuming in most cases and also the yield is not very good. Culture following lysis of the specimens, such as blood, obviates this problem. Blood lysed by addition of certain substances, followed by centrifuga-tion, increases yield of fungi by culture. Yield can be fur-ther increased with a shortening of time by combining with BACTEC systems.
Demonstration of the antibodies in patient’s serum or CSF is useful for diagnosis of fungal infections, especially in sys-temic fungal infections. A significant rise of antibody titer in a paired sera sample confirms the diagnosis. The complement fixation test was the earliest test used in fungal serology and is still used in the diagnosis of suspected cases of histoplasmo-sis, blastomycosis, or coccidiomycosis. Recently, newer tests like ELISA (enzyme-linked immunosorbent assay), Western blot, and radioimmunoassays are increasingly used for serodi-agnosis of fungal infections.
These methods include (a) detection of fungal antigen, (b) detection of fungal cell wall markers, and (c) detection of fungal metabolites.
Antigen detection: It is useful in immunocompromised hostswhere antibody detection is not as sensitive. Detection of fun-gal antigen in serum, CSF, and urine is increasingly used for diagnosis of many fungal infections. Demonstration of antigen indicates recent or active infection. Latex agglutination test is a frequently used test to demonstrate polysaccharide capsular antigen of C. neoformans in CSF for diagnosis of cryptococcal meningitis. False-positive reactions due to Trichosporon beigelli and Capnocytophaga canimorsus are known.
Detection of fungal cell wall markers: Mannan is a highlyimmunogenic component of the candidal cell wall. Mannan antigen detection, therefore, is most widely used method in the diagnosis of candidiasis.
Galactomannan is a heat-stable heteropolysaccharide found in the cell walls of all Aspergillus species. Production of the galactomannan antigen is proportional to fungal load in tis-sue, hence is being used as the prognostic marker for diagnosis of invasive aspergillosis. A sandwich ELISA using rat monoclo-nal antibody EB-A2 against galactomannan antigen is being currently used in Europe for diagnosis of invasive aspergillosis.
Most pathogenic fungi have 1, 3-beta-D-glucan in their cell walls and minute quantities are secreted into the circulation during the life cycle. Detection of this antigen can also be used as an indicator of invasive fungal infections. Detection of 1, 3-beta-D-glucan is based on its ability to activate a coagulation cascade within amebocytes derived from the hemolymph of horseshoe crabs. This uses a different cascade than endotoxin to cause coagulation, hence is specific for fungi. The test does not detect certain species, such as C. neoformans and Zygomycetes.
Detection of fungal metabolites: Detection of distinctivefungal metabolites is another approach for the diagnosis of fungal infections. Gas liquid chromatography is being used to quantify arabinitol for diagnosis of C. albicans infections.
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