Influence of Glucose Supplementation and Inoculum Size on Growth Kinetics and Antifungal Susceptibility Testing of Candida spp

Abstract

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates ( Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei , and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h ( P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A ₅₄₀ values at 24 h of incubation with medium containing glucose and an inoculum of 10 ⁵ CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis ( A ₅₄₀ = 0.26 ± 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10 ⁵ CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10 ⁵ CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.