Development and Validation of TaqMan Chemistry Probe-Based Rapid Assay for the Detection of Echinocandin-Resistance in Candida auris.
Development and Validation of TaqMan Chemistry Probe-Based Rapid Assay for the Detection of Echinocandin-Resistance in Candida auris.
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基于 TaqMan 化学探针的快速检测法的开发和验证，用于检测耳念珠菌中的棘白菌素耐药性。
DOI：
10.1128/jcm.01767-22
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发表时间：
2023
影响因子：
9.4
通讯作者：
Chaturvedi,Sudha
中科院分区：
文献类型：
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作者：
Zhu,YanChun;Hager,KelliM;Manjari,SwatiR;Banavali,NileshK;Chaturvedi,Vishnu;Chaturvedi,Sudha
耳念珠菌是一种多重耐药酵母病原体，在世界各地的医疗机构中引起疫情爆发，并且抗棘白菌素的耳念珠菌的出现令人担忧。目前使用的临床和实验室标准研究所 (CLSI) 和商业抗真菌药敏试验 (AFST) 是基于表型的、缓慢且不可扩展的，限制了它们在监测抗棘白菌素耳念珠菌方面的有效性。对准确、快速评估棘白菌素耐药性的方法的迫切需要怎么强调也不为过，因为此类抗真菌药物是患者管理的首选。我们报告了基于 TaqMan 化学探针的荧光熔解曲线分析 (FMCA) 的开发和验证，该分析遵循不对称聚合酶链式反应 (PCR)，以评估 FKS1 热点 1 (HS1) 区域内的突变，FKS1 是负责编码棘白菌素靶标 1,3-β-d-葡聚糖合酶的基因。该测定正确鉴定了 F635C、F635Y、F635del、F635S、S639F 或 S639Y、S639P 和 D642H/R645T 突变。 AFST 证实，在这些突变中，F635S 和 D642H/R645T 不涉及棘白菌素耐药性，而其余突变则涉及。在31例临床病例中，棘白菌素耐药的主要突变是S639F/Y（20例），其次是S639P（4例）、F635del（4例）、F635Y（2例）和F635C（1例）。 FMCA 测定具有高度特异性，不会与密切相关和远缘相关的念珠菌以及其他酵母和霉菌物种发生交叉反应。 Fks1 蛋白、其突变体和三种棘白菌素药物的对接构象的结构模型表明 Fks1 与棘白菌素的结合方向似乎合理。这些发现为未来评估额外的 FKS1 突变及其对耐药性发展的影响奠定了基础。基于 TaqMan 化学探针的 FMCA 将能够快速、高通量且准确地检测耳念珠菌中导致棘白菌素抗性的 FKS1 突变。
Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in health care facilities worldwide, and the emergence of echinocandin-resistant C. auris is a concern. Currently used Clinical and Laboratory Standards Institute (CLSI) and commercial antifungal susceptibility tests (AFST) are phenotype-based, slow, and not scalable, limiting their effectiveness in the surveillance of echinocandin-resistant C. auris. The urgent need for accurate and rapid methods of assessment of echinocandin resistance cannot be overstated, as this class of antifungal drugs is preferred for patient management. We report the development and validation of a TaqMan chemistry probe-based fluorescence melt curve analysis (FMCA) following asymmetric polymerase chain reaction (PCR) to assess mutations within the hot spot one (HS1) region ofFKS1,the gene responsible for encoding 1,3-β-d-glucan synthase that is a target for echinocandins. The assay correctly identified F635C, F635Y, F635del, F635S, S639F or S639Y, S639P, and D642H/R645T mutations. Of these mutations, F635S and D642H/R645T were not involved in echinocandin resistance, while the rest were, as confirmed by AFST. Of 31 clinical cases, the predominant mutation conferring echinocandin resistance was S639F/Y (20 cases) followed by S639P (4 cases), F635del (4 cases), F635Y (2 cases), and F635C (1 case). The FMCA assay was highly specific and did not cross-react with closely and distantly relatedCandidaand other yeast and mold species. Structural modeling of the Fks1 protein, its mutants, and docked conformations of three echinocandin drugs suggest a plausible Fks1 binding orientation for echinocandins. These findings lay the groundwork for future evaluations of additionalFKS1mutations and their impact on the development of drug resistance. The TaqMan chemistry probe-based FMCA would allow rapid, high throughput, and accurate detection ofFKS1mutations conferring echinocandin resistance in C. auris.