Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations
Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations
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DOI：
10.1007/s10096-018-3350-3
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发表时间：
2018-11-01
影响因子：
4.5
通讯作者：
Kusters, Johannes G.
中科院分区：
文献类型：
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作者：
Braam, Joyce F.;van Marm, Sebastian;Kusters, Johannes G.
生殖支原体感染的患者通常使用大环内酯类阿奇霉素进行经验性治疗。大环内酯类耐药性正变得相当普遍；经验性治疗受到了损害。测序最初用于检测阿奇霉素耐药相关突变。由于这是费时费力的，已经开发了用于检测它们的qPCRs。在本研究中，我们描述了一种快速、灵敏和特异的定量聚合酶链式反应方法，它可以在一次检测中对生殖支原体和大环内酯类抗生素耐药相关突变进行常规检测。这些研究用于比较(I)检测生殖支原体感染的常用MGPA法(MGPA QPCR)，(Ii)用于检测大环内酯类抗生素耐药突变的23S rRNA基因聚合酶链式反应/测序法(mg23S qPCR/测序)，以及(Iii)我们新开发的用于同时检测生殖支原体和大环内酯类耐药突变的基于探针的熔融曲线qPCR(Macrolide-R/MG Elite MGB Kit，Elitech Bothel USA简称mg Macrolide(R)qPCR)。使用对一系列其他微生物检测呈阳性的泌尿生殖道样本来测试qPCR的特异性。MGPA qPCR196/236(83.1%)标本中检出生殖支原体，而联合测序法检测172/236(72.9%)，MG大环内酯(R)qPCR202/236(85.6%)。在检测大环内酯类耐药突变方面，镁大环内酯(R)qPCR与镁23S qPCR/测序试验(分别可分型201个和202个)具有很高的一致性。在镁大环内酯(R)qPCR检测中，没有其他泌尿生殖道病原体呈阳性，表明具有特异性。镁大环内酯(注册商标)定量聚合酶链式反应快速、灵敏、特异，在常规诊断中很容易实施。
Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg Macrolide(R) qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg Macrolide(R) qPCR. The Mg Macrolide(R) qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg Macrolide(R) qPCR, indicating specificity. The Mg Macrolide(R) qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.