A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility
A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility
复制标题
DOI：
10.1093/jac/dkv366
复制
发表时间：
2016-02-01
影响因子：
5.2
通讯作者：
Whiley, David M.
中科院分区：
文献类型：
--
作者：
Buckley, Cameron;Trembizki, Ella;Whiley, David M.
本研究的目的是建立一种直接检测淋球菌GyrA基因91位点的实时荧光定量聚合酶链式反应方法，以预测淋球菌对环丙沙星的敏感性。使用淋病奈瑟氏菌和摩拉氏菌的共生菌种群(nEuroS=aEuroS55分离株)和临床样本(经商业淋病奈瑟氏菌核酸扩增试验(NAAT)方法检测结果为阴性)(nEuroS=aEuroS171)，初步评估了该方法的性能。然后将GyrA91-PCR直接应用于2014年以来NAAT阳性的淋球菌临床标本(NAEuroS=aEuroS210)，并获得相应的具有药敏结果的淋球菌分离株。GyrA91-PCR准确地表征了所有70株淋球菌的gyrA 91基因座(敏感度aEuroS=aEuroS100%，95%CIaEuroS=aEuroS94.9%-100%)，而所有非淋球菌分离株和NAAT阴性淋球菌的GyrA91-PCR结果均为阴性(特异性为aEuroS100%，95%的CIaEuroS=aEuroS98.4%-100%)。当应用于210份NAAT阳性的淋球菌临床样本时，GyrA91-PCR成功地鉴定了195份样本(92.9%，95%CIaEuroS=aEuroS88.5%-95.9%)。与相应的细菌培养结果相比较，GyrA91-PCRWT探针的阳性预测161例淋球菌对环丙沙星的敏感性(99.4%，95%CIaEuroS=aEuroS96.6%-99.9%)。
The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility.The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (naEuroS=aEuroS70), a panel of commensal Neisseria and Moraxella species (naEuroS=aEuroS55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (naEuroS=aEuroS171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (naEuroS=aEuroS210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available.The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivityaEuroS=aEuroS100%, 95% CIaEuroS=aEuroS94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificityaEuroS=aEuroS100%, 95% CIaEuroS=aEuroS98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CIaEuroS=aEuroS88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CIaEuroS=aEuroS96.6%-99.9%) samples.The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.