Centre for HIV and STIs, National Institute for Communicable Diseases, National Health Laboratory Service, 1 Modderfontein Road, Sandringham 2192, South Africa.
Int J Antimicrob Agents. 2013 Jul;42(1):63-7. doi: 10.1016/j.ijantimicag.2013.02.026. Epub 2013 Apr 24.
A real-time PCR (RT-PCR) assay was modified to simultaneously detect Neisseria gonorrhoeae and to determine gonococcal susceptibility to ciprofloxacin using clinical samples. The modified RT-PCR assay was validated using DNA extracted from 40 linked isolates and urethral swabs, 24 of which had linked first-pass urine samples, obtained from men presenting with urethral gonorrhoea. The RT-PCR assay enabled amplification of N. gonorrhoeae dcmH, gyrA and parC genes. The quinolone resistance-determining regions (QRDRs) of the isolates' gyrA and parC genes were sequenced. Following successful validation, 33 first-pass urine-derived DNA extracts, obtained from men with gonorrhoea, were tested with the assay and results were compared with blinded ciprofloxacin susceptibility data. Gonococcal susceptibility to ciprofloxacin correlated perfectly with gyrA amplicon generation. No gyrA amplicons were detected for gonococcal infections due to ciprofloxacin-intermediate/resistant organisms. Amplification of parC correlated less well with ciprofloxacin susceptibility phenotypes. Simultaneous non-generation of gyrA and parC amplicons consistently predicted the presence of ciprofloxacin-resistant gonococci. Characteristic point mutations in the gyrA/parC QRDRs were found in DNA amplified from those extracts that failed to produce gyrA/parC amplicons. The RT-PCR assay performed well with DNA extracted from first-pass urine specimens and results correlated perfectly with ciprofloxacin susceptibility phenotypes. In conclusion, the modified RT-PCR assay can detect N. gonorrhoeae in DNA extracted from first-pass urine specimens of men with urethral gonorrhoea and accurately predicts gonococcal susceptibility to ciprofloxacin. This molecular assay provides a useful tool for surveillance and patient management in settings where fluoroquinolones can still be used for treatment of gonorrhoea.
我们修改了实时 PCR (RT-PCR) 检测方法，以便同时检测淋病奈瑟菌，并使用临床样本确定淋球菌对环丙沙星的敏感性。我们使用从 40 个相关分离株和尿道拭子中提取的 DNA 验证了改良的 RT-PCR 检测方法，其中 24 个分离株具有相关的首次尿液样本，这些样本来自患有尿道淋病的男性。RT-PCR 检测方法能够扩增淋病奈瑟菌的 dcmH、gyrA 和 parC 基因。对分离株 gyrA 和 parC 基因的喹诺酮耐药决定区 (QRDR) 进行了测序。成功验证后，我们使用该检测方法检测了 33 份来自淋病男性的首次尿液衍生 DNA 提取物，并将结果与盲法环丙沙星药敏数据进行了比较。淋球菌对环丙沙星的敏感性与 gyrA 扩增子的产生完全相关。对于由环丙沙星中介/耐药菌引起的淋病感染，未检测到 gyrA 扩增子。parC 扩增与环丙沙星药敏表型的相关性较差。gyrA 和 parC 扩增子同时不能扩增，始终可以预测存在环丙沙星耐药淋病奈瑟菌。在未能产生 gyrA/parC 扩增子的那些提取物中，在 DNA 中发现了 gyrA/parC QRDR 中的特征点突变。该 RT-PCR 检测方法在从患有尿道淋病的男性的首次尿液标本中提取的 DNA 中性能良好，与环丙沙星药敏表型的相关性非常好。总之，改良的 RT-PCR 检测方法可用于检测男性尿道淋病患者首次尿液标本中的淋病奈瑟菌，并能准确预测淋球菌对环丙沙星的敏感性。该分子检测方法为仍可用于治疗淋病的氟喹诺酮类药物的监测和患者管理提供了有用的工具。
