AUTHOR=Liu Aimei , Liu Sang , Lv Kangyan , Zhu Qingdong , Wen Jun , Li Jianpeng , Liang Chengyuan , Huang Xuegang , Gong Chunming , Sun Qingfeng , Gu Hongcang 

TITLE=Rapid detection of multidrug resistance in tuberculosis using nanopore-based targeted next-generation sequencing: a multicenter, double-blind study

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1349715

DOI=10.3389/fmicb.2024.1349715

ISSN=1664-302X

ABSTRACT=<sec id="sec1001"><title>Background</title><p>Resistance to anti-tuberculous drugs is a major challenge in the treatment of tuberculosis (TB). We aimed to evaluate the clinical availability of nanopore-based targeted next-generation sequencing (NanoTNGS) for the diagnosis of drug-resistant tuberculosis (DR-TB).</p></sec><sec id="sec2001"><title>Methods</title><p>This study enrolled 253 patients with suspected DR-TB from six hospitals. The diagnostic efficacy of NanoTNGS for detecting <italic>Mycobacterium tuberculosis</italic> and its susceptibility or resistance to first- and second-line anti-tuberculosis drugs was assessed by comparing conventional phenotypic drug susceptibility testing (pDST) and Xpert MTB/RIF assays. NanoTNGS can be performed within 12 hours from DNA extraction to the result delivery.</p></sec><sec id="sec3001"><title>Results</title><p>NanoTNGS showed a remarkable concordance rate of 99.44% (179/180) with the culture assay for identifying the <italic>Mycobacterium tuberculosis</italic> complex. The sensitivity of NanoTNGS for detecting drug resistance was 93.53% for rifampicin, 89.72% for isoniazid, 85.45% for ethambutol, 74.00% for streptomycin, and 88.89% for fluoroquinolones. Specificities ranged from 83.33% to 100% for all drugs tested. Sensitivity for rifampicin-resistant tuberculosis using NanoTNGS increased by 9.73% compared to Xpert MTB/RIF. The most common mutations were S531L (codon in E. coli) in the <italic>rpoB</italic> gene, S315T in the <italic>katG</italic> gene, and M306V in the <italic>embB</italic> gene, conferring resistance to rifampicin, isoniazid, and ethambutol, respectively. In addition, mutations in the pncA gene, potentially contributing to pyrazinamide resistance, were detected in 32 patients. Other prevalent variants, including D94G in the <italic>gyrA</italic> gene and K43R in the <italic>rpsL</italic> gene, conferred resistance to fluoroquinolones and streptomycin, respectively. Furthermore, the <italic>rv0678</italic> R94Q mutation was detected in one sample, indicating potential resistance to bedaquiline.</p></sec><sec id="sec4001"><title>Conclusion</title><p>NanoTNGS rapidly and accurately identifies resistance or susceptibility to anti-TB drugs, outperforming traditional methods. Clinical implementation of the technique can recognize DR-TB in time and provide guidance for choosing appropriate antituberculosis agents.</p></sec>