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. 2025 Feb 17;24(1):14.
doi: 10.1186/s12941-025-00781-z.

Diagnostics, resistance and clinical relevance of non-tuberculous mycobacteria unidentified at the species level by line probe assays: a bi-national study

Matúš Dohál  1 , Nils Wetzstein  2   3   4 , Michaela Hromádková  5 , Simona Mäsiarová  6 , Erik M Rasmussen  7 , Peter Kunč  8   9 , Mária Škereňová  10 , Igor Porvazník  11   12 , Ivan Solovič  11   12 , Stefan Niemann  2   3 , Jarmila Hnilicová  13 , Juraj Mokrý  6 , Věra Dvořáková #  14 , Margo Diricks #  2   3
Affiliations

Diagnostics, resistance and clinical relevance of non-tuberculous mycobacteria unidentified at the species level by line probe assays: a bi-national study

Matúš Dohál et al. Ann Clin Microbiol Antimicrob. .

Abstract

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.

Keywords: Diagnostics; Non-tuberculous mycobacteria; Novel species; Targeted next-generation sequencing; Whole-genome sequencing.

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Conflict of interest statement

Declarations. Ethical approval: This study was approved by the Ethical Committee of the Jessenius Faculty of Medicine in Martin, Comenius University Bratislava (EK65/2021). All methods were carried out following relevant guidelines and regulations. Consent for publication: Not applicable. Content of the publication: During the preparation of this work the author(s) used chatgpt in order to enhance the clarity and readability of the text. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
A summary of the initial diagnostic assessment upon which sample collection was based
Fig. 2
Fig. 2
NTM species identified by Deeplex Myc-TB
Fig. 3
Fig. 3
Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudobootstrap support values > 60% from 100 replications, with an average branch support of 81.5%. The tree was rooted at the midpoint. Red crosses indicate novel NTM species. Three highly contaminated samples (CZ11223, CZ61421 and CZ45720) were not included. User strain refers to the isolates obtained in this study. The blue plus (+) symbol represents previously identified NTM species, while the red plus (+) symbol indicates novel NTM species
See this image and copyright information in PMC

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