Time-Kill Studies of Voriconazole and Fluconazole against Candida Species Determined by Flow Cytometry and by Colony Forming Units

Rama Ramani, Vishnu Chaturvedi


Background: Time-kill studies allow determination of the rate and extent of antifungal activity, relationship between concentration of drug,pharmacodynamic characteristics. In this study, we used flow cytometry susceptibility testing to perform time-kill studies, as it provides shorter incubation time,greater precision due to individual cell analysis. Methods: Voriconazole MIC for C. albicans ATCC 24433 (flu - S, 0.03 µg/ml) and forC. krusei ATCC 6258 (flu - R, 0.25 µg/ml) were determined by NCCLS broth microdilution method (M-27A)and by flow cytometry susceptibility testing (FC) method recently published from our lab. Time-kill studies were done using antifungal concentrations 0.25 - 8 times the MIC value. Samples were drawn at 0 hour and at hourly intervals for 8 hours. Colony forming units (CFU) were determined ensuring that there was no antifungal carryover. Mean channel fluorescence (MCF) was determined by flow cytometry using FACScan with Cell Quest software. Dose response plots (inhibition versus multiple of MIC), 50% effective concentration (EC50) was determined using sigmoidal modified Hill 4 - parameter model. Results: Time-kill plots revealed that at higher drug concentrations (2XMIC - 8XMIC), 1% reduction in CFU and 80% increase in MCF was seen against both drugs for flu-S strain. However, no such effect was evident with flu-R strain in interaction with fluconazole. The dose-response curves by CFU method illustrated the transition of antifungal activity over a narrow range of concentrations (MIC to 2XMIC). On the contrary FC method showed a wider range (0.5XMIC - 4XMIC) for both isolates. Voriconazole EC50 values for C. albicans was 0.40(FC) and 0.28(CFU), and for C. krusei was 0.31(FC) and 0.61(CFU). Fluconazole EC50 values for C. albicans was 0.21 (FC) and 0.18(CFU) and for C. krusei was 1.8 (FC) and 0.81(CFU). Conclusions: Flow cytometry provided a rapid assessment of drug - yeast interactions under constant antifungal concentrations. The concentration - effect relationships against Candida species were similar for both voriconazole and fluconazole. Voriconazole was equally effective against flu-S and flu-R isolates.

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American Society for Microbiology General Meeting
    • ASM 102nd (2002)