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首页> 外文期刊>Antimicrobial agents and chemotherapy. >Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus
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Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

机译：高分辨率熔化测定检测含吡咯的抗唑抗性的发展与验证

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The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.
机译：抗唑抗性的全球出现抗植物毛茛属菌株是越来越多的公共卫生问题。不同的唑抗性模式与CYP51A中的突变连接。因此，准确表征唑抗性抗原的基础机制至关重要，对曲霉病患者选择最合适的抗真菌剂。该研究描述了一种新的无序分子筛查工具，用于早期检测已知的最常见的突变与A. fumigatus中的唑抗性相关。设计了PCR靶向CYP51A在位置G54，Y121，G448和M220和靶向启动子区域中的不同串联重复（TRS）的突变。使用相同的循环条件同时进行所有PCR。然后使用高分辨率熔化测定来区分扩增子。为了标准化，使用30种良好的抗唑抗A. fumigatus菌株，用于每个靶标的不同抗性机制的熔化曲线簇，并允许检测最常见的唑抗突变，即G54E，G54V，G54R，G54W，Y121F ，M220V，M220I，M220T，M220K和G448S，以及串联重复TR34，TR46和TR53。使用80A的盲板进行验证来进行该方法。Fumigatus唑易溶或抗唑抗性菌株进行。盲板中包含的所有应变被适当地归类为易感或耐药方法。该筛选方法的实施可以减少检测抗唑抗性A. Fumigatus分离物所需的时间，从而促进曲霉病患者的最佳抗真菌治疗。

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