Abstract
Increasing reports of azole resistance in Candida 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 in C. 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 on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. 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 the ERG11 gene. 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 of ERG11 mutations in C. 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 on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.
Highlights
Candida species are common commensals residing on human skin, genitourinary, respiratory, and gastrointestinal tracts
In the present study, we have developed and evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM) and high-resolution melt (HRM) analysis approaches for rapid detection of ERG11 mutations in the clinical isolates of C. tropicalis
Twelve fluconazole-resistant isolates (MIC range: 32–256 mg/L) with A339T and C461T mutations and 19 susceptible isolates (MIC range: 0.5–1 mg/L) without these mutations were obtained from National Culture Collection of Pathogenic Fungi (NCCPF) to include in the present study (Table 2 and S2 Table in S1 File)
Summary
Candida species are common commensals residing on human skin, genitourinary, respiratory, and gastrointestinal tracts. They hold pathogenic potential causing a wide range of infections ranging from superficial to serious life-threatening systemic disease [1, 2].
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