Dermatology Hospital, Southern Medical University, Guangzhou 510091, China.
Guangdong Dermatology Clinical College, Anhui Medical University, Hefei 230022, China.
J Antimicrob Chemother. 2022 Feb 23;77(3):656-664. doi: 10.1093/jac/dkab447.
Gonorrhoea, caused by Neisseria gonorrhoeae, has spread worldwide. Strains resistant to most antibiotics, including ceftriaxone and azithromycin, have emerged to an alarming level. Rapid testing for N. gonorrhoeae and its antimicrobial resistance will therefore contribute to clinical decision making for early diagnosis and rational drug use.
A Cas13a-based assay (specific high-sensitivity enzymatic reporter unlocking; SHERLOCK) was developed for N. gonorrhoeae detection (porA gene) and azithromycin resistance identification (A2059G, C2611T). Assays were evaluated for sensitivity with purified dsDNA and specificity with 17 non-gonococcal strains. Performance of SHERLOCK (porA) was compared with Roche Cobas 4800 using 43 urine samples. Identification of azithromycin resistance mutations (A2059G, C2611T) was evaluated using a total of 84 clinical isolates and 18 urine samples. Lateral flow was tested for this assay as a readout tool. Moreover, we directly assayed 27 urethral swabs from patients with urethritis to evaluate their status in terms of N. gonorrhoeae infection and azithromycin resistance.
The SHERLOCK assay was successfully developed with a sensitivity of 10 copies/reaction, except 100 copies/reaction for A2059G, and no cross-reaction with other species. Comparison of the SHERLOCK assay with the Cobas 4800 revealed 100% concordance within 18 positive and 25 negative urine samples. Of the 84 isolates, 21 strains with azithromycin resistance mutations were distinguished and further verified by sequencing and MIC determination. In addition, 62.96% (17/27) strains from swab samples were detected with no mutant strains confirmed by sequencing.
The SHERLOCK assay for rapid N. gonorrhoeae detection combined with azithromycin resistance testing is a promising method for application in clinical practice.
淋病是由淋病奈瑟菌引起的，已在全球范围内传播。对抗生素（包括头孢曲松和阿奇霉素）耐药的菌株已经大量出现。因此，快速检测淋病奈瑟菌及其对抗生素的耐药性将有助于临床决策，实现早期诊断和合理用药。
开发了一种基于 Cas13a 的检测方法（特定高灵敏度酶报告解锁；SHERLOCK），用于检测淋病奈瑟菌（porA 基因）和阿奇霉素耐药性（A2059G、C2611T）。使用纯化的 dsDNA 评估了检测方法的灵敏度，用 17 株非淋病奈瑟菌株评估了检测方法的特异性。使用 43 份尿液样本比较了 SHERLOCK（porA）与罗氏 Cobas 4800 的性能。使用总共 84 株临床分离株和 18 份尿液样本评估了阿奇霉素耐药突变（A2059G、C2611T）的鉴定。该检测方法使用侧向流动作为读出工具进行了测试。此外，我们直接检测了 27 份来自尿道炎患者的尿道拭子，以评估他们的淋病奈瑟菌感染和阿奇霉素耐药状况。
SHERLOCK 检测方法成功建立，灵敏度为 10 拷贝/反应，除了 A2059G 的灵敏度为 100 拷贝/反应，与其他物种无交叉反应。SHERLOCK 检测方法与 Cobas 4800 的比较显示，在 18 份阳性和 25 份阴性尿液样本中具有 100%的一致性。在 84 株分离株中，21 株具有阿奇霉素耐药突变的菌株通过测序和 MIC 测定得到区分和进一步验证。此外，从拭子样本中检测到 62.96%（17/27）的菌株，但通过测序未确认有突变菌株。
快速检测淋病奈瑟菌并结合阿奇霉素耐药性检测的 SHERLOCK 检测方法是一种很有前途的临床应用方法。
