Xiao Guohui, Liu Houming, Xu Hui, Shi Hongyu, Liu Dongxin, Ou Min, Liu Peng, Zhang Guoliang
National Clinical Research Center for Infectious Diseases, Guangdong Provincial Clinical Research Center for Tuberculosis, Shenzhen Third People's Hospital, Southern University of Science and Technology, Shenzhen, 518112, China.
Department of Clinical Laboratory, Shenzhen Third People's Hospital, Southern University of Science and Technology, Shenzhen, 518112, China.
BMC Microbiol. 2025 Apr 2;25(1):188. doi: 10.1186/s12866-025-03899-4.
The increasing prevalence of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for an efficient approach to identify Mycobacterium tuberculosis complex (MTBC) strains resistant to rifampicin (RIF) and isoniazid (INH). In response, we developed a CRISPR-Cas14a MTB RIF/INH platform that can detect the most common mutations associated with RIF and INH resistance. To evaluate the sensitivity and specificity of our CRISPR-Cas14a MTB RIF/INH platform, we carried out a comprehensive assessment using clinical isolates of M. tuberculosis and sputum samples from TB patients, making direct comparisons with phenotypic drug susceptibility testing (pDST). A total of 60 clinical isolates from TB patients were utilized, consisting of 18 RIF mono-resistant, 15 INH mono-resistant, 24 MDR isolates, and 3 fully susceptible isolates. Among the 42 RIF-resistant isolates, our platform accurately identified 39, achieving a sensitivity of 93.3% (95% CI, 80.0-98.5) and a specificity of 100% (95% CI, 81.6-100). Similarly, out of the 39 INH-resistant isolates, the platform successfully identified 38, demonstrating a sensitivity of 97.5% (95% CI, 86.5-99.9) and a specificity of 100% (95% CI, 83.8-100) when compared with pDST. Moreover, in the analysis of 55 sputum samples, our platform accurately identified RIF resistance in 10 out of 12 samples (85.7%) and INH resistance in all 11 samples (100%). Notably, excluding the nucleic acid extraction step, the entire testing procedure can be completed in approximately 1.5 h. These results suggest that the CRISPR-Cas14a MTB RIF/INH platform is a reliable and promising novel tool for detecting RIF and INH resistance in isolates or directly from sputum samples.
耐多药结核病（MDR-TB）的日益流行凸显了迫切需要一种有效的方法来鉴定对利福平（RIF）和异烟肼（INH）耐药的结核分枝杆菌复合群（MTBC）菌株。作为回应，我们开发了一种CRISPR-Cas14a MTB RIF/INH平台，该平台可以检测与RIF和INH耐药性相关的最常见突变。为了评估我们的CRISPR-Cas14a MTB RIF/INH平台的敏感性和特异性，我们使用结核分枝杆菌临床分离株和结核病患者的痰液样本进行了全面评估，并与表型药物敏感性试验（pDST）进行了直接比较。共使用了60株来自结核病患者的临床分离株，包括18株对RIF单耐药、15株对INH单耐药、24株耐多药分离株和3株完全敏感分离株。在42株对RIF耐药的分离株中，我们的平台准确鉴定出39株，敏感性为93.3%（95%CI，80.0-98.5），特异性为100%（95%CI，81.6-100）。同样，在39株对INH耐药的分离株中，该平台成功鉴定出38株，与pDST相比，敏感性为97.5%（95%CI，86.5-99.9），特异性为100%（95%CI，83.8-。此外，在对55份痰液样本的分析中，我们的平台准确鉴定出12份样本中的10份（85.7%）对RIF耐药，以及所有11份样本（100%）对INH耐药。值得注意的是，排除核酸提取步骤后，整个检测过程大约可在1.5小时内完成。这些结果表明，CRISPR-Cas14a MTB RIF/INH平台是一种可靠且有前景的新型工具，可用于检测分离株或直接从痰液样本中检测RIF和INH耐药性。
