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Variable number tandem repeat typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. Therefore, we aimed to elucidate species-subspecies and strain dynamics in non-tuberculous mycobacteria and develop a simple sequence-based strain-level determination method. We performed a single-center prospective cohort study of 112 patients with non-tuberculous mycobacterial pulmonary disease. Whole-genome sequencing was performed on two sputum samples collected at enrollment and at the end of follow-up, followed by variable number tandem repeat (VNTR) typing. We also developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy. Our results demonstrate that core genome multi-locus sequencing typing revealed species\/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species\/subspecies changes. Overall, pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [\u2212] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (\u2212) patients. dVNTR results aligned with those of conventional VNTR typing. In conclusion, since susceptibility factors remain unclear, routine species\/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. Core genome multi-locus sequencing typing with a dVNTR identified pathogen shifts, innovating non-tuberculous mycobacterial pulmonary disease management.<\/jats:p>\n                    <jats:p\/>\n                    <jats:sec>\n                      <jats:title>\n                        <jats:bold>Clinical Trials<\/jats:bold>\n                      <\/jats:title>\n                      <jats:p>\n                        This study is registered with UMIN as\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/center6.umin.ac.jp\/cgi-open-bin\/ctr_e\/ctr_view.cgi?recptno=R000064060\" xlink:type=\"simple\">UMIN 000056067<\/jats:ext-link>\n                        .\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Pulmonary non-tuberculous mycobacterial disease is a chronic infection in which the causative pathogens may change at the species, subspecies, or strain level over time. Accurate tracking of these changes is essential for optimizing treatment; however, conventional clinical practice lacks efficient methods for monitoring such dynamics. Our study revealed pathogen changes in approximately one-quarter of patients over 1.5 years, prompting the development of a novel surveillance system that integrates next-generation sequencing for both species-subspecies identification and strain-level molecular epidemiology. This innovation enables real-time monitoring of pathogen dynamics, allowing clinicians to promptly adjust treatment strategies and improve patient care through more informed decision-making.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00436-25","type":"journal-article","created":{"date-parts":[[2025,8,21]],"date-time":"2025-08-21T14:10:54Z","timestamp":1755785454000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Molecular epidemiological surveillance for non-tuberculous mycobacterial pulmonary disease: a single-center prospective cohort study"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-8838-6674","authenticated-orcid":false,"given":"Kazuki","family":"Hashimoto","sequence":"first","affiliation":[{"name":"Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine","place":["Suita, 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