Summary

This study highlights the diagnostic gaps in current routine assays for non-tuberculous mycobacteria (NTM) and underscores the added value of combining targeted deep sequencing (Deeplex Myc-TB) with whole-genome sequencing (WGS) for the identification of rare and novel NTM. By analyzing previously unidentifiable isolates from Slovakia and the Czech Republic, the study reveals a high diversity of rare NTM species, including clinically relevant Mycobacterium chimaera variants that were missed by standard line probe assays. It also reports the discovery of two novel species, one of which was linked to pulmonary disease. These results emphasize that uncommon NTM can also be clinically relevant and highlights the need for improved surveillance and diagnostics.