Identifying drug-resistant Candida species using SuperSelective primer PCR

项目总结 金黄色念珠菌是一种新出现的泛多药耐药真菌病原体，对全球健康构成严重威胁 威胁。重要的是，金黄色葡萄球菌是第一个临床表现明显且有时无法治愈的真菌病原体。 对所有已知的抗真菌类药物，包括棘球菌素类和两性霉素B类的抗药性。 然而，用于早期检测金黄色葡萄球菌感染耐药性的诊断工具要么是不足的 或者不够敏感，无法识别耐药的临床菌株。因此，抗真菌药物的组合 耐药性，加上缺乏灵敏的诊断分析，削弱了有效的治疗策略 克服或预防金黄色葡萄球菌的耐药性。因此，总体目标是开发和验证 超选择性聚合酶链式反应检测棘球绦虫或唑类耐药性基因突变， 尽管有丰富的野生型金龟子DNA背景。我们假设超选择性的聚合酶链式反应分析 将检测灵敏度超过1：104的突变金黄色葡萄球菌突变：野生型单核苷酸多态 在与棘球菌素或唑类抗药性有关的基因座上。前提是我们的概念验证工作 Auris FKS1突变可产生高水平的棘球绦虫耐药。我们希望检验我们的假设 并通过追求两个具体目标来实现目标：1)开发和验证一种新的超选 聚合酶链式反应平台检测金黄色葡萄球菌对棘球菌素和唑类抗真菌药物耐药基因突变 毒品。2)验证超选择性聚合酶链式反应方法检测肾组织中棘球绦虫和唑类药物的异质性耐药 活动性念珠菌病小鼠的匀浆。这项提议的回报预计将是巨大的，因为 这项拟议的研究将开发一种新的超选择性引物聚合酶链式反应平台来鉴定突变， 临床分离的异抗或耐药金黄色葡萄球菌。它还将为机械性理解提供一个框架 金黄色葡萄球菌的多药耐药性。

PROJECT SUMMARY Candida auris is an emerging pan-multidrug-resistant fungal pathogen and presents a serious global health threat. Importantly, C. auris is the first fungal pathogen showing pronounced and sometimes untreatable clinical drug resistance to all known antifungal classes, including echinocandins azoles, and amphotericin B. However, the diagnostic tools for the early detection of drug resistance in C. auris infections are either inadequate or not sensitive enough for the identification of drug resistant clinical isolates. Thus, the combination of antifungal drug resistance, along with a lack of sensitive diagnostic assays impair efficient therapeutic strategies to overcome or prevent drug resistance in C. auris. Hence, the overall objective is to develop and validate SuperSelective PCR assays for the detection of mutations in genes encoding echinocandin or azole resistance, despite an abundant background of wild-type C. auris DNA. We hypothesize that SuperSelective PCR assays will detect mutant C. auris with a sensitivity exceeding 1:104 mutant:wild type for single nucleotide polymorphisms at genetic loci conferring echinocandin or azole resistance. The premise is based on our proof-of-concept work with an C. auris FKS1 mutation conferring high-level echinocandin resistance. We expect to test our hypothesis and to accomplish the objectives by pursuing two Specific Aims: 1) Develop and validate a novel SuperSelective PCR platform to detect mutations in C. auris genes conferring resistance to echinocandin and azole antifungal drugs. 2) Validate SuperSelective PCR assays to detect echinocandin and azole heteroresistance in the kidney homogenates of mice with active candidiasis. The payoffs of this proposal are expected to be significant, because the proposed study will develop a novel SuperSelective primer PCR platform to identify mutations, heteroresistant or tolerant clinical C. auris isolates. It will also provide a framework for mechanistic understanding of multidrug resistance in C. auris.