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.