Rapid detection of ERG11 polymorphism associated azole resistance in Candida tropicalis

Abstract

Increasing reports of azole resistance inCandida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance inC.tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based onERG11polymorphism inC.tropicalis. Twelve azole-resistant and 19 susceptible isolates ofC.tropicaliswere included. DNA sequencing of the isolates was performed to check theERG11polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection ofERG11mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32–256 mg/L and 0.5–1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in theERG11gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection ofERG11mutations inC.tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based onERG11polymorphism inC.tropicalisand can be implemented in clinical setups for batter patient management.