ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Clinical Infectious Diseases
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1584237
This article is part of the Research TopicUnveiling Distinctions: Active Tuberculosis versus Latent Tuberculosis Infection - Immunological Insights, Biomarkers, and Innovative ApproachesView all 12 articles
A comprehensive evaluation of a novel targeted-sequencing workflow for Mycobacterium species identification and anti-tuberculosis drug resistance detection
Provisionally accepted
Xichao Ou1
Shaojun Pei1
Hongru Li2
Zhonghua Qin3
Richard Anthony4Jichun Wang1
Zexuan Song1
Ruida Xing1
Lixia Zhang3Chong Teng5
Hui Xia1
Yang Zhou1Yuanyuan Song1Yang Zheng1
Shengfen Wang1Bing Zhao1*
Yanlin Zhao1*- 1Chinese Center For Disease Control and Prevention, Beijing, China
- 2First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China
- 3Tianjin Haihe Hospital, Tianjin, China
- 4National Institute for Public Health and the Environment (Netherlands), Bilthoven, Utrecht, Netherlands
- 5Dongcheng Center for Disease Control and Prevention, Beijing, China
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Background: Although several WHO-endorsed targeted next-generation sequencing (tNGS) assays exist for tuberculosis drug-resistance detection, their target selection and diagnostic accuracy vary widely. In this study, we developed a novel tNGS workflow (the TB Pro assay) and evaluated its performance in identifying Mycobacterium species and predicting drug resistance. Methods: The TB Pro assay was validated for identifying 10 Mycobacterium tuberculosis complex (MTBC) and 39 nontuberculous mycobacterial (NTM) species, as well as predicting resistance to 4 first-line and 13 second-line anti-TB drugs. The limit of detection (LOD) was determined using 11 reference strains spiked in sputum. The prediction of resistance to anti-tuberculous drugs/drug classes was compared with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) using 435 clinical isolates. Results: The assay demonstrated high sensitivity with a calculated LOD of 3.0 CFU/mL for M. Tuberculosis and 1.4-16.2 CFU/mL for most NTMs, except for Mycobacterium intracellulare with 117.9 CFU/mL. Using pDST as the reference standard, the sensitivity of the TB Pro assay for the detection of resistance ranged from 74.3% (ethambutol) to 94.4% (rifampicin), with specificity values >98% for all drugs. Compared with WGS, the sensitivity of the TB Pro assay was over 98.0% for all drugs except pyrazinamide (66.7%), and the specificity values were all nearly 100.0%. Directly on sputum, TB Pro assay showed 100% agreement with smear and culture positive sputum specimens. Conclusions: The TB Pro assay represents a sensitive and specific solution for simultaneous mycobacterial identification and comprehensive drug-resistance profiling, performing robustly on both cultured isolates and direct clinical specimens.
Keywords: Mycobacterium tuberculosis, Non-tuberculous mycobacteriosis, Drug Resistance, Multiplex PCR (polymerase chain reaction), targeted next generation sequencing (tNGS)
Received: 27 Feb 2025; Accepted: 14 May 2025.
Copyright: © 2025 Ou, Pei, Li, Qin, Anthony, Wang, Song, Xing, Zhang, Teng, Xia, Zhou, Song, Zheng, Wang, Zhao and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Bing Zhao, Chinese Center For Disease Control and Prevention, Beijing, China
Yanlin Zhao, Chinese Center For Disease Control and Prevention, Beijing, China
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.