Antifungal susceptibility testing of Candida species by flow cytometry

The feasibility of flow cytometric antifungal susceptibility testing has been studied using the fluorescent anionic membrane potential probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The in vitro antifungal susceptibility testing of amphotericin B was performed on 8 Candida isolates from clinical specimens and 2 ATCC strains by flow cytometry with the results compared to those of the National Committee of Clinical Laboratory Standards (NCCLS) M27-T, broth macrodilution method. The flow cytometric method is based on an increase of fluorescence given out by DiBAC4(3) in fungi when they are killed by antifungal agents. Minimum inhibitory concentration (MIC) of amphotericin B ranged from 0.25 to 1 microg/mL. All results agreed within +/-2 dilution between the flow cytometric method and the M27-T method. MIC with ATCC strains were within recommended ranges of M27-T. The new flow cytometric method revealed a clear and distinct reproducible test end point. A four hr of incubation was sufficient for the test. In conclusion, flow cytometry using DiBAC4(3) is a rapid and accurate in vitro antifungal susceptibility testing method.