Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures

Objectives: Nontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques.

Methods: Extrapulmonary mycobacteria growth indicator tube primary cultures collected retrospectively between 2019 and 2023, featuring unidentified mycobacteria species detected by GenoType Mycobacterium line probe assays, underwent multilocus targeted next-generation sequencing using Oxford Nanopore Technology, polymerase chain reaction amplicon Sanger sequencing, and Deeplex Myc-TB analysis. Previously collected clinical and laboratory data were reported.

Results: A total of 28 cultures, collected from extrapulmonary sites, each from different patients, were included. Mycobacterial mixtures were identified in 19 of 28 (68%) cultures, with four of 28 (14%) showing unidentified species based on sequencing of rpoB and hsp65 targets. Mycobacterium monacense was present in 13 of 28 (46%) of the cultures. Culturable Mycobacterium tuberculosis complex (MTBC) was identified in five extrapulmonary specimens that previously tested negative for MTBC using Xpert MTB/RIF Ultra. The comparative analysis between Sanger and targeted next-generation sequencing using Oxford Nanopore Technology sequencing (for hsp65) demonstrated 27 of 28 (96%) agreement on the predominant strain. Deeplex Myc-TB could not identify NTM-MTBC co-infections in minor subpopulations.

Conclusions: This study highlights the role of advanced sequencing in identifying NTM mixtures and mycobacterial co-infections. It calls for ongoing efforts to integrate next-generation sequencing into mycobacteria testing algorithms.