Comparison of molecular testing methods for diagnosing non-tuberculous mycobacterial infections

Purposes: Rapid and accurate identification of non-tuberculous mycobacteria (NTM) is crucial yet challenging, promoting the development of novel molecular techniques such as amplification-based targeted high-throughput sequencing and metagenomic unbiased high-throughput sequencing. We aimed to evaluate the diagnostic value of these molecular techniques for NTM infection.

Methods: A total of 115 clinical specimens from patients with confirmed NTM infection were subjected to multiplex polymerase chain reaction detection techniques (multi-PCR), metagenomic Next-Generation Sequencing (mNGS), targeted Next-Generation Sequencing (tNGS), and targeted Nanopore sequencing (tNanopore). Positivity rates and species identification were compared among these techniques.

Results: The sensitivity of mNGS, tNGS, and multi-PCR in NTM-infection diagnosis was 44.3%, 42.6%, and 36.5%, respectively, while the sensitivity of the three methods in combination increased to 54.8%. The pathogen identification results of mNGS, tNGS and multi-PCR were matched in 80.6% (25/31) samples at the species level, among which 14 samples (45.2%) was completely matched at the subspecies level. The results of tNanopore, tNGS and mNGS at the species level were completely matched in 73.3% (22/30) samples.

Conclusions: These molecular assays demonstrated comparable performance in precisely identifying NTM species in clinical specimens, showing their promising potential as efficient and alternative tools for the rapid diagnosis of NTM disease.