Abstract

Background

The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens.

Methods

Multiplex real-time PCR (RT–PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR −35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method.

Results

SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT–PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively.

Conclusions

We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

中文翻译：

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多重实时PCR分析法可预测淋病奈瑟氏菌阳性核酸扩增测试样品的头孢菌素，环丙沙星和阿奇霉素的抗菌药敏性。

摘要

背景

在加拿大，耐药性淋病奈瑟氏球菌（GC）的发病率正在上升。但是，对于通过核酸扩增试验（NAAT）直接从临床标本中诊断出的感染，没有抗菌药耐药性（AMR）监测数据，占诊断的80％以上。我们开发了一套10种改进的分子检测方法，用于监测GC-AMR和预测NAAT标本的药敏性。

方法

多重实时被开发PCR（RT-PCR）测定来检测与头孢菌素（相关的SNPs PONA，PORB，mtrR基因-35delA，尼亚A311V，尼亚A501，N513Y，G545S），环丙沙星（的gyrA S91，的parC D86 / S87 / S88 ）和阿奇霉素[23S（A2059G，C2611T），mtrR基因脑膜炎样启动子]的电阻。该方法在127株淋球菌，51株非淋球菌和50株NAATs中进行了验证。将通过测定确定的SNP与通过WGS数据的计算机分析确定的SNP进行比较。使用琼脂稀释法确定培养分离物的MIC。

结果

50个NAAT标本的SNP分析与匹配的培养物RT-PCR分析具有96％的一致性。与MICs相比，penA A311V或penA A501以及两个或多个其他SNP的存在与敏感性降低相关，而三个或三个以上其他S​​NP的存在与对头孢菌素的中等敏感性相关；任何与环丙沙星或阿奇霉素耐药相关的相关SNP的存在。NAAT-AMR预测与匹配培养的头孢菌素，环丙沙星和阿奇霉素的MIC分别相关，分别为94％，100％和98％。

结论

我们通过添加新的基因座和多重反应来扩大对淋病奈瑟菌AMR预测的分子测试，以改善无法获得培养分离物的监测。