OBJECTIVES: While the reported incidence of non-tuberculous mycobacterial (NTM) infections is increasing, the true prevalence remains uncertain due to limitations in diagnostics and surveillance. The emergence of rare and novel species underscores the need for characterization to improve surveillance, detection, and management. METHODS: We performed whole-genome sequencing (WGS) and/or targeted deep-sequencing using the Deeplex Myc-TB assay on all NTM isolates collected in Slovakia and the Czech Republic between the years 2019 to 2023 that were unidentifiable at the species level by the routine diagnostic line probe assays (LPA) GenoType CM/AS and NTM-DR. Minimal inhibitory concentrations against amikacin, ciprofloxacin, moxifloxacin, clarithromycin, and linezolid were determined, and clinical data were collected. RESULTS: Twenty-eight cultures from different patients were included, of which 9 (32.1%) met the clinically relevant NTM disease criteria. The majority of those had pulmonary involvement, while two children presented with lymphadenitis. Antimycobacterial resistance rates were low. In total, 15 different NTM species were identified, predominantly rare NTM like M. neoaurum, M. kumamotonense and M. arupense. Notably, clinically relevant M. chimaera variants were also identified with WGS and Deeplex-Myc TB, which, unlike other M. chimaera strains, appeared to be undetectable by LPA assays. Deeplex detected four mixed infections that were missed by WGS analysis. In contrast, WGS identified two novel species, M. celatum and M. branderi, which were not detected by Deeplex-Myc TB. Importantly, one of these novel species strains was associated with clinically relevant pulmonary disease. DISCUSSION: Our study demonstrates the clinical relevance of uncommon NTM and the effectiveness of targeted deep-sequencing combined with WGS in identifying rare and novel NTM species.

Biomedical Centre Martin Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Clinic of Pediatric Respiratory Diseases and Tuberculosis National Institute of Pediatric Tuberculosis and Respiratory Diseases Dolny Smokovec Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Department of Genetics and Microbiology Faculty of Science Charles University Prague Czech Republic

Department of Internal Medicine Infectious Diseases Goethe University University Hospital Frankfurt Germany

Department of Pathological Physiology Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Department of Pharmacology Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Faculty of Health Catholic University Ružomberok Slovakia

German Center for Infection Research Partner Site Hamburg Lübeck Borstel Riems Borstel Germany

International Reference Laboratory of Mycobacteriology Statens Serum Institut Copenhagen Denmark

Molecular and Experimental Mycobacteriology Research Center Borstel Borstel Germany

National Institute of Public Health Prague Czech Republic

National Institute of Tuberculosis Lung Diseases and Thoracic Surgery Vyšné Hágy Slovakia

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