Membrane Filtration Test for Rapid Presumptive Differentiation of Four Candida Species

Abstract

ABSTRACT A rapid enzymatic two-step test for the presumptive differentiation of four Candida species commonly occurring in various clinical samples is described. The technique involves membrane filtration of a liquid sample, followed by preincubation of the membrane filter on Sabouraud glucose agar supplemented with ticarcillin-clavulanic acid to yield microcolonies. In a separate assay step, parts of the filter are placed on absorbent pads impregnated with fluorogenic 4-methylumbelliferyl (4-MU) enzyme substrates (4-MU- N -acetyl-β- d -galactosaminide, 4-MU-phosphate, 4-MU-pyrophosphate, and 4-MU-β- d -galactoside) in combination with 0.1% digitonin acting as a membrane permeabilizer. The membrane filter in contact with the assay medium is incubated to allow cleavage of the enzyme substrate, resulting in fluorescent microcolonies under long-wavelength UV light. This approach, tested on 301 clinical samples, is able to presumptively differentiate C. albicans , C. glabrata , C. krusei , and C. tropicalis and to distinguish them from other Candida spp. in about 9 to 11 h. Overall agreement with the conventional methods of 94.4% (one Candida species present in the sample) to 83.8% (multiple Candida spp. present) was obtained. The false-negative rates with reference to identification by traditional methods were 1.3% (single species) and 3.8% (multiple species).