Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

作者：Beth A. Arthington‐Skaggs, Hoda Jradi, Tejal Ashwin Desai, Christine J. Morrison · 发表于：Journal of Clinical Microbiology · 年份：1999 · DOI：10.1128/jcm.37.10.3332-3337.1999 · 被引用次数：369 · 研究领域：Antifungal resistance and susceptibility、Analytical Methods in Pharmaceuticals、Pharmacogenetics and Drug Metabolism

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 microg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of </=8 microg/ml (susceptible), 16 to 32 microg/ml (susceptible dose-dependent [SDD]), or >/=64 microg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 microg of fluconazole/ml, an 84% reduction after exposure to 4 microg/ml, and 95 and 100% reductions after...