Improved medium for fluconazole susceptibility testing of Candida albicans

Abstract

We have compared fluconazole susceptibilities of 92 clinical isolates of Candida albicans by broth microdilution in two different media: standard RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per liter (RPMI-glucose). Preparation of media, drugs, and inocula, as well as incubation conditions, followed the preliminary recommendations of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) antifungal agent working group for broth macrodilution tests with antifungal agents, adapted to microdilution. Microtiter plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader set at 405 mm. The MIC endpoint was defined as an inhibitory concentration calculated from the turbidimetric data as a function of the turbidity in the drug-free control wells. The mean absorbances in the drug-free wells in RPMI and RPMI-glucose were, respectively, 0.38 (41.6% transmission) and 0.99 (10.2% transmission) (P < 0.001; Student's t test). Despite the increased growth in RPMI-glucose, 98.9% of the C. albicans strains tested for fluconazole susceptibility yielded similar MICs (+/- 1 dilution) in both media. Moreover, strains with decreased susceptibility to fluconazole displaying similar MICs in both media are easier to detect in RPMI-glucose because of the greater differences between turbidimetric readings in wells with grown or fluconazole-inhibited cultures. This objective turbidimetric method, with an easy-to-read improved medium (RPMI with glucose), together with previous experience of the National Committee for Clinical Laboratory Standards antifungal agent subcommittee, could overcome some of the present problems associated with lack of reproducibility of azole susceptibility testing.