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Antimicrob Agents Chemother. 1998 May;42(5):1207-12.

Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods.

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College of Pharmacy, The University of Iowa, Iowa City 52242, USA.


This study was designed to examine the effects of antifungal carryover, agitation, and starting inoculum on the results of time-kill tests conducted with various Candida species. Two isolates each of Candida albicans, Candida tropicalis, and Candida glabrata were utilized. Test antifungal agents included fluconazole, amphotericin B, and LY303366. Time-kill tests were conducted in RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to a pH of 7.0 and incubated at 35 degrees C. Prior to testing, the existence of antifungal carryover was evaluated at antifungal concentrations ranging from 1x to 16x MIC by four plating methods: direct plating of 10, 30, and 100 microl of test suspension and filtration of 30 microl of test suspension through a 0.45-microm-pore-size filter. Time-kill curves were performed with each isolate at drug concentrations equal to 2 x MIC, using a starting inoculum of approximately 10(5) CFU/ml, and incubated with or without agitation. Last, inoculum experiments were conducted over three ranges of starting inocula: 5 x 10(2) to 1 x 10(4), >1 x 10(4) to 1 x 10(6), and >1 x 10(6) to 1 x 10(8) CFU/ml. Significant antifungal carryover (>25% reduction in CFU/milliliter from the control value) was observed with amphotericin B and fluconazole; however, carryover was eliminated with filtration. Agitation did not appreciably affect results. The starting inoculum did not significantly affect the activity of fluconazole or amphotericin B; however, the activity of LY303366 may be influenced by the starting inoculum. Before antifungal time-kill curve methods are routinely employed by investigators, methodology should be scrutinized and standardized procedures should be developed.

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