Amplicon-based nanopore sequencing for Mycobacterium spp. identification in clinical isolates and sputum

Objectives This study developed a mycobacterial amplicon-based nanopore sequencing (Myco-ANS) assay for differentiating eight Mycobacterium spp. and evaluated its diagnostic accuracy in clinical isolates and sputum samples. Methods PCR primers targeting the rpoB gene were optimized for sequencing with Oxford Nanopore Technology. Eight American Type Culture Collection (ATCC) reference strains were used for assay development. Clinical isolates and sputum samples were obtained from King Chulalongkorn Memorial Hospital. Results were compared with those from the line probe assay (LPA). Results A total of 125 clinical isolates and 46 sputum samples were analyzed. Myco-ANS correctly identified eight species: M. tuberculosis , M. bovis BCG, M. abscessus , M. avium , M. fortuitum group, M. kansasii , M. gordonae , and M. intracellulare . For isolates, sensitivity ranged from 89.5–100.0%, specificity from 99.2–100.0%, and diagnostic accuracy from 98.4–100.0%. In sputum, species-level identification showed substantial agreement with LPA (kappa = 0.70). Conclusions Myco-ANS provides rapid, accurate identification of clinically relevant mycobacteria from both isolates and sputum. With results available within three days, this assay shortens turnaround time compared to conventional methods, supporting faster treatment decisions and reducing transmission risk.