The University of Osaka achieves rapid identification of tuberculosis and pulmonary NTM species without culture

A research group including Graduate Student Kazuki Hashimoto and Invited Lecturer Kiyoharu Fukushima of the Graduate School of Medicine, along with Specially Appointed Assistant Professor Yuki Matsumoto and Associate Professor Shota Nakamura of the Department of Infection Metagenomics, Research Institute for Microbial Diseases, at the University of Osaka, announced on April 1 that they have developed a new rapid diagnostic method for mycobacteria (NaLC-Seq method) in collaboration with Osaka Toneyama Medical Center. The method enables species identification down to the subspecies level by the day after sample collection. While conventional identification methods require one to six weeks for culture alone, this technique bypasses the culture process entirely. Its practical application is awaited as it is expected to lead to earlier diagnosis and treatment. The results were published in the Journal of Clinical Microbiology on February 27.

Non-tuberculous mycobacteria (NTM) is a general term for approximately 200 species of bacteria, excluding Mycobacterium tuberculosis, which causes pulmonary tuberculosis, and Mycobacterium leprae, which causes leprosy. It causes pulmonary NTM disease, cases of which have been increasing globally in recent years; this increase is partly due to detection thanks to advancements in diagnostic methods such as CT scans.

Pulmonary NTM disease is a chronic, progressive, and refractory respiratory disease with a current incidence rate in Japan of approximately 19 per 100,000 people, surpassing pulmonary tuberculosis as a public health challenge. Because effective medications and prognoses vary by species and subspecies of mycobacteria, identification down to the subspecies level is necessary for appropriate treatment. While standard identification methods can comprehensively examine species, they require significant time for culture and involve multi-stage analysis, taking approximately one to two months for results. In contrast, methods like PCR or TRC are rapid but can only identify a few species.

To address this, the research group developed "NaLC-Seq," an identification method that combines DNA extraction from pre-treated sputum samples with gene amplification using a mycobacteria-specific panel and analysis via next-generation sequencing.

To verify the utility of the developed method, a clinical trial was conducted on 125 patients with pulmonary NTM at Osaka Toneyama Medical Center.

The results showed that for smear-positive samples with pre-treatment, an accuracy of 90.5% was achieved. The researchers confirmed that identification at the subspecies level is possible, and the identifiable species cover a comprehensive range of 186 species and 389 subspecies, including M. tuberculosis. The time required for results was 19 hours, allowing for species determination within a single day. This is expected to enable rapid treatment, particularly for patients with severe symptoms such as extensive lung lesions, cavity formation, or hemoptysis.

The group also confirmed that determinations can be made using "MinION," a portable next-generation sequencer. Furthermore, to solve the challenges of requiring significant computational resources and specialized technicians for analysis, they built a website that accesses the University of Osaka's supercomputer via the cloud, allowing for one-click analysis.

Currently, knowledge is being shared among multiple research institutions, including the University of Osaka and Osaka Toneyama Medical Center. The protocol will be improved based on feedback to make it easier to use.

Hashimoto stated: "At this point, we are distributing kits to interested researchers for expansion, but in the future, we aim to obtain approval as an in vitro diagnostic pharmaceutical. For practical application, there are additional necessary tests, so we will continue our research while consulting with the PMDA and relevant medical institutions to contribute to better medical care."

Journal Information
Publication: Journal of Clinical Microbiology
Title: Comparison of culture and culture-free methods for comprehensive identification of mycobacteria: a single-center prospective study
DOI: 10.1128/jcm.01128-25