Influence of Test Conditions on Antifungal Time-Kill Curve Results: Proposal for Standardized Methods

Abstract

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°C. Prior to testing, the existence of antifungal carryover was evaluated at antifungal concentrations ranging from 1× to 16× MIC by four plating methods: direct plating of 10, 30, and 100 μl of test suspension and filtration of 30 μl of test suspension through a 0.45-μm-pore-size filter. Time-kill curves were performed with each isolate at drug concentrations equal to 2× MIC, using a starting inoculum of approximately 105 CFU/ml, and incubated with or without agitation. Last, inoculum experiments were conducted over three ranges of starting inocula: 5 × 102 to 1 × 104, >1 × 104 to 1 × 106, and >1 × 106 to 1 × 108 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.

Analysis of triclosan inclusion complexes with β-cyclodextrin and its water-soluble polymeric derivative

“…Klepser et al [34] demonstrated the ability of this methodology as an indispensable tool for assessing the activity of antimicrobials against bacteria. In fact this approach allows the estimation of drug antimicrobial activity as a function of time, which could be a more accurate determinant for clinical outcome of therapy than a simple numerical MIC or MMC [35].…”

Characterising the post-antifungal effects of micafungin against Candida albicans, Candida glabrata, Candida parapsilosis and Candida krusei isolates

“…Significant antifungal carry-over [defined as a reduction in colony-forming units (CFU) >25% of the control] was excluded using standard methods [6]. The starting inoculum was 0.5-2.5 × 10 3 cells/mL.…”

2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF); antimicrobial compound with cell cycle arrest in nosocomial pathogens

“…Additionally, log-phase fungal cells (2 × 10 3 CFU/ml) were incubated with 10 μg of DMHF or 5 μg of amphotericin B used as a positive control. The culture was obtained and spread on YPD agar plate, and then the CFUs were counted after incubation for 24 h at 28°C (Klepser et al, 1998). The values were the average of triplicate measurements in three independent assays.…”