Nanopore-based targeted next-generation sequencing (tNGS): A versatile technology specialized in detecting low bacterial load clinical specimens

Abstract

Introduction 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. Methods 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. Results 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. Conclusion It is anticipated that tNGS will play a crucial clinical role in the early prevention and control of tuberculosis in the future.