Yang Chen, Gao Weiwei, Guo Yicheng, Zeng Yi
Department of Tuberculosis, The School of Public Health of Nanjing Medical University, The Second Hospital of Nanjing, Nanjing, China.
Department of Tuberculosis, The Second Hospital of Nanjing, Nanjing, China.
PLoS One. 2025 May 27;20(5):e0324003. doi: 10.1371/journal.pone.0324003. eCollection 2025.
The 2024 global tuberculosis report indicated that the epidemiological situation of tuberculosis remains concerning. Current tuberculosis detection methods have limitations, highlighting the urgent need to develop more convenient, effective, and widely utilized detection technologies in clinical settings to facilitate early diagnosis and treatment guidance for tuberculosis. Nanopore-based targeted next-generation sequencing (tNGS) offers advantages such as convenience, efficiency, and long-read sequencing, making it a commonly used method for mycobacteria identification.
This study compared the detection efficiency of tNGS with Xpert MTB/RIF, MTB culture, and AFB smear in sputum samples, BALF samples, and pathological tissue samples to evaluate the clinical applicability of tNGS in diagnosing tuberculosis and low bacterial load tuberculosis, including extrapulmonary and smear-negative cases.
Among the four detection methods, tNGS demonstrated the highest efficiency, sensitivity, specificity, and AUC values, which are 93.4%, 94.7%, and 0.94, respectively. This method was particularly advantageous for detecting tuberculosis in patients with low bacterial loads, as evidenced by a significantly different positive detection rate in histopathological specimens compared to other methods (P < 0.001). Furthermore, tNGS achieved overall positive detection rates of 93.8% for smear-negative tuberculosis patients and 89.1% for culture-negative tuberculosis patients, both of which were significantly higher than those of other detection methods (P < 0.05). Additionally, tNGS could directly identify bacterial strains and detect mutations associated with drug resistance. In this study, the agreement rate between tNGS strain identification of NTM and the final diagnosis was 94.1%. Among the 21 identified mutation sites associated with rifampicin resistance, one (Pro454His) was located outside RRDR.
It is anticipated that tNGS will play a crucial clinical role in the early prevention and control of tuberculosis in the future.
《2024年全球结核病报告》指出，结核病的流行病学形势依然令人担忧。当前的结核病检测方法存在局限性，凸显出迫切需要在临床环境中开发更便捷、有效且广泛应用的检测技术，以促进结核病的早期诊断和治疗指导。基于纳米孔的靶向新一代测序（tNGS）具有便捷、高效和长读长测序等优势，使其成为分枝杆菌鉴定的常用方法。
本研究比较了tNGS与Xpert MTB/RIF、MTB培养和AFB涂片在痰液样本、BALF样本及病理组织样本中的检测效率，以评估tNGS在诊断结核病及低菌量结核病（包括肺外和涂片阴性病例）中的临床适用性。
在这四种检测方法中，tNGS表现出最高的效率、灵敏度、特异性和AUC值，分别为93.4%、94.7%和0.94。该方法在检测低菌量患者的结核病方面具有特别优势，组织病理学标本中的阳性检出率与其他方法相比有显著差异（P < 0.001）。此外，tNGS对涂片阴性结核病患者的总体阳性检出率为93.8%，对培养阴性结核病患者的总体阳性检出率为89.1%，均显著高于其他检测方法（P < 0.05）。此外，tNGS可直接鉴定菌株并检测与耐药相关的突变。在本研究中，tNGS对非结核分枝杆菌的菌株鉴定与最终诊断的符合率为94.1%。在鉴定出的21个与利福平耐药相关的突变位点中，有一个（Pro454His）位于RRDR之外。
预计tNGS未来将在结核病的早期防控中发挥关键的临床作用。
