Université Paris-Descartes, Faculté de Médecine, APHP, Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France.
Laboratoire de Parasitologie-Mycologie, Hôpital Pellegrin, Bordeaux, France.
J Clin Microbiol. 2017 Nov;55(11):3210-3218. doi: 10.1128/JCM.01032-17. Epub 2017 Aug 16.
is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in is worrisome. The aim of this study was to validate the new MycoGENIE real-time PCR kit and to evaluate its performance on clinical samples for the detection of and its azole resistance. This multiplex assay detects DNA from the species complex by targeting the multicopy 28S rRNA gene and specific TR and L98H mutations in the single-copy-number gene of The specificity of mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the 28S rRNA gene and 6 copies for the gene harboring the TR and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of DNA and azole resistance due to TR and L98H mutations in clinical samples.
是导致人类曲霉病的主要物种。曲霉病的诊断仍然很困难，而 对唑类药物的耐药性迅速出现令人担忧。本研究旨在验证新型 MycoGENIE 实时 PCR 试剂盒，并评估其在检测 和其唑类耐药性的临床样本中的性能。该多重检测通过靶向多拷贝 28S rRNA 基因和单拷贝数 基因中的特定 TR 和 L98H 突变来检测 种复合体的 DNA。通过测试来自 25 个野生型或突变临床 的 DNA 样本评估 突变检测的特异性。对 62 名患者的 88 份呼吸道样本和 16 名确诊或可能患有曲霉病的患者的 69 份血清样本以及 13 名无曲霉病的患者进行了临床验证。 28S rRNA 基因的检测限<1 拷贝，携带 TR 和 L98H 改变的 基因的检测限为 6 拷贝。与各种真菌和细菌无交叉反应。通过定量 PCR（qPCR）分析，准确检测到携带 TR 和 L98H 突变的所有分离株。对于呼吸道样本，qPCR 结果的敏感性和特异性分别为 92.9%和 90.1%，而对于血清样本，敏感性和特异性分别为 100%和 84.6%。我们的研究表明，这种新型实时 PCR 试剂盒能够在临床样本中灵敏、快速地检测 DNA 和由于 TR 和 L98H 突变导致的唑类耐药性。
