Membrane filtration test for rapid presumptive differentiation of four Candida species

A rapid enzymatic two-step test for the presumptive differentiation of four Candida species commonly occurring in various clinical samples is describe d. The technique involves membrane filtration of a liquid sample, followed by preincubation of the membrane filter on Sabouraud glucose agar supplemen ted with ticarcillin-clavulanic acid to yield microcolonies. In a separate assay step, parts of the filter are placed on absorbent pads impregnated;vi th fluorogenic 4-methylumbelliferyl ( 4-MU) enzyme substrates (4-MU-N-acety l-beta-D-galactosaminide, 4-MU-phosphate, 4-MU-pyrophosphate. and 4-MU-beta -D-galactoside) in combination with 0.1% digitonin acting as a membrane per meabilizer. The membrane Biter in contact with the assay medium is incubate d to allow cleavage of the enzyme substrate, resulting in fluorescent micro colonies under long-wavelength UV light This approach, tested on 301 clinic al samples, is able to presumptively differentiate C. albicans, C. glabrata , C. krusei, and C. tropicalis and to distinguish them from other Candida s pp. in about 9 to 11 h. Overall agreement with the conventional methods of 94.4% tone Candida species present in the sample to 83.8% (multiple Candida spp. present) aas obtained, The false-negative rates, with reference to id entification by traditional methods were 1.39 (single species) and 3.8% (mu ltiple species).