Molecular epidemiological surveillance for non-tuberculous mycobacterial pulmonary disease: a single center prospective cohort study

Background: Bacteria species cultured from sputum often change during treatment or observation for non-tuberculous mycobacterial pulmonary disease (NTM-PD); however, strain-level changes remain unrecognized in clinical practice. Variable number tandem repeat (VNTR) typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. We aimed to elucidate species-subspecies and strain dynamics in NTM and to develop a simple sequence-based strain-level determination method.Methods: We performed a single-center prospective cohort study of 112 patients with NTM-PD. Whole genome sequencing was performed on two sputum samples collected at enrolment and at the end of follow-up at intervals of more than 6 months. VNTR typing was then performed. We developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy.Results: Core genome multi-locus sequencing typing (cgMLST) revealed species/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species/subspecies changes. Pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [-] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (-) patients. Clinical characteristics showed no significant differences. dVNTR results aligned with those of conventional VNTR typing.Interpretation: Pathogen shifts occurred in 25.9% of NTM-PD patients over 1.5 years of treatment or observation. Because susceptibility factors remain unclear, routine species/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. cgMLST with a dVNTR identified pathogen shifts, innovating NTM-PD management.