Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.
PLoS One. 2021 Jan 13;16(1):e0245160. doi: 10.1371/journal.pone.0245160. eCollection 2021.
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.
越来越多的关于热带假丝酵母菌唑类耐药的报告强调了快速耐药检测技术的发展。在 14α-去甲基羊毛甾醇 C （ERG11）基因中的非同义突变是热带假丝酵母菌唑类耐药的主要机制之一。我们评估了基于 ERG11 多态性的四引物扩增受阻突变系统-PCR（T-ARMS-PCR）、限制位点突变（RSM）和高分辨率熔解（HRM）分析方法在热带假丝酵母菌快速耐药检测中的应用。纳入了 12 株唑类耐药和 19 株敏感的热带假丝酵母菌分离株。对分离株进行 DNA 测序，以检查耐药和敏感分离株中 ERG11 多态性的状态。评估并验证了 T-ARMS-PCR、RSM 和 HRM 三种方法，用于快速检测 ERG11 突变。12 株耐药和 19 株敏感分离株的氟康唑 MIC 分别为 32-256mg/L 和 0.5-1mg/L。耐药分离株在 ERG11 基因中显示出 A339T 和 C461T 突变。T-ARMS-PCR 和 RSM 方法可区分所有耐药和敏感分离株，而 HRM 分析除了一个敏感分离株外，均可区分。这些方法的敏感性、特异性、分析敏感性、时间和分析成本表明，这三种方法可用于快速检测热带假丝酵母菌中的 ERG11 突变。此外，这三种方法均与 DNA 测序具有极好的一致性。T-ARMS-PCR、RSM 和 HRM 方法快速、敏感且廉价，适用于基于热带假丝酵母菌 ERG11 多态性的唑类耐药检测，可在临床环境中实施，以更好地管理患者。
