Multi-center evaluation of antifungal time-kill methods

We have previously developed and published methods for conducting antifungal time-kill studies. The purpose of this study was to evaluate the inter- and intra-laboratory reproducibility of results obtained using these methods. Seven sites were recruited for participation in this study. Each site conducted a series of time-kill experiments with fluconazole and amphotericin B against two isolates of Candida albicans and one isolate each of Candida glabrata and Candida tropicalis. Time-kill studies were conducted under the following conditions: RPMI 1640 served as growth medium; cultures were incubated at 35°C with agitation; sampling volume was 30 μl; and potato dextrose agar was used for solid media. A control, fluconazole at concentrations of 1, 10, and 20 μg/ml and amphotericin B at concentrations of 0.25, 1, and 2.5 μg/ml were run for each isolate. Experiments were conducted over 24 hours, with samples removed for colony determination at pre-determined time points. All experiments were performed in triplicate. Examination of intra- and inter-laboratory data suggest good reproducibility with respect to sampling methods and time-kill data for all four isolates. The percent agreement among centers with respect to classification of regimens as cither fungistatic, fungicidal, or no effect at the various test concentrations was 90% for fluconazole and 93% for amphotericin B. Methods for conducting antifungal time-kill studies are reproducible among different laboratories when examined using fluconazole and amphotericin B against Candida species.