P015

1:National Reference Laboratory for Mycobacteria, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal; 2:Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal

Nontuberculous mycobacteria (NTM) are increasing worldwide and becoming a major public health problem. Among over 180 known NTM species, Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABC) are the most common cause of human disease, with some subspecies being associated with worse clinical outcomes. Accurate subspecies identification is crucial for guiding effective treatment. However, current laboratory diagnostics are culture-based, delaying diagnosis, and the available commercial kits often lack specificity as they do not take into account the genetic diversity of MAC and MABC subspecies. Here, we developed a culture-independent methodology based on targeted sequencing for correct subspeciation of both MAC and MABC directly from clinical samples.

Based on the current state-of-the-art, we selected 16 genetic regions that allow the identification of key subspecies: M. bolletii, M. massiliensis, M. abscessus, M. avium spp hominissuis, M. intracellulare and M. chimaera. A set of 21 primer pairs, with amplicon sizes ranging from 160-500 bp, were first validated individually with DNA from isolates of each subspecies, and then optimized into two multiplex-PCR reactions under the same cycling conditions. Amplicons were sequenced on an ONT MinION device and sequencing data analysed using the user-friendly bioinformatics platform INSaFLU. Final protocol optimization is ongoing using a set of 30 clinical samples from previously diagnosed patients.

Overall, we present a simple and straightforward approach that enables accurate identification of six clinically relevant NTM subspecies with a rapid turnaround time (~24 hours), offering a practical tool for diagnostic laboratories.