Establishing surrogate markers for fluconazole resistance in Candida albicans

Azole-resistant Candida can be a confounding factor for clinical management of opportunistic infections in immunocompromised patients, but rapid identification of such resistant organisms can improve patient outcome. New target-based molecular diagnostic strategies have the potential to identify resistant organisms faster than current culture-based assays. It was the objective of this study to determine whether target site mutations and/or drug pump over-expression are suitable surrogate markers of drug resistance that could aid new molecular-based diagnostic assays. A collection of 59 clinical isolates displaying a range of azole susceptibilities were assayed for mutations within the target gene Erg11 and for over-expression of drug-efflux pumps Cdr1, Cdr2, Flu1, and Mdr1, as well as drug target gene Erg11 by quantitative real-time PCR with molecular beacons. A fluconazole-resistant (MIC ≥ 64 μg/ml) phenotype was closely associated with over-expression of Cdr1 (p = 0.005), Cdr2 (p = 0.01), and Mdr1 (p = 0.03) along with four mutations in Erg11 (T229A, Y132F, S405F, G464S). Changes in expression levels for Erg11 and Flu1 were not statistically correlated with resistance (p = 0.27 and p = 0.86, respectively). Overall, these findings provide a statistical basis to establish Erg11 mutations and drug pump over-expression as surrogate markers for phenotypic fluconazole resistance.