Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus
Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus
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DOI：
10.1128/aac.01083-17
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发表时间：
2017-12-01
影响因子：
4.9
通讯作者：
Alastruey-Izquierdo, A.
中科院分区：
文献类型：
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作者：
Bernal-Martinez, L.;Gil, H.;Alastruey-Izquierdo, A.
唑类耐药烟曲霉菌株的全球出现是一个日益增长的公共卫生问题。不同的唑类耐药模式与cyp51A突变有关。因此，准确描述唑类耐药的机制对于指导曲霉病患者选择最合适的抗真菌药物至关重要。这项研究描述了一种新的无测序分子筛选工具，用于早期检测已知与A.烟熏。设计了靶向cyp51A在位置G54、Y121、G448和M220处的突变以及靶向启动子区中的不同串联重复（TR）的PCR。使用相同的循环条件同时进行所有PCR。然后使用高分辨率解链测定区分扩增子。为了标准化，30个充分表征的抗唑A.使用烟曲霉菌株，对于每种靶标产生不同抗性机制的解链曲线簇，并允许检测最常见的唑类抗性突变，即，G54 E、G54 V、G54 R、G54 W、Y121 F、M220 V、M220 I、M220 T、M220 K和G448 S，以及串联重复序列TR34、TR46和TR53。使用80 A盲板进行方法验证。烟曲霉唑敏感或唑抗性菌株。采用开发的方法将盲板中包含的所有菌株正确分类为敏感或耐药。该筛选方法的实施可以减少检测耐唑A的时间。烟曲霉分离株，因此有利于选择最佳的抗真菌治疗曲霉病患者。
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.