Comparative evaluation of MALDI-ToF mass spectrometry and Sanger sequencing of the , , and genes for non tuberculous mycobacteria species identification.

Non tuberculous mycobacteria (NTM) infections are increasing globally, underscoring the critical importance of accurate species-level identification for effective clinical management. This study aimed to evaluate the use of three conserved markers in the mycobacterial family (, , and ) for NTM identification through Sanger sequencing, comparing the results to those obtained using MALDI-ToF MS. A total of 59 clinical NTM isolates from plastic surgery patients, previously characterized by MALDI-ToF MS, were analyzed. These isolates underwent DNA extraction, PCR amplification, and Sanger sequencing. Species identification was performed through phylogenetic analyses of each marker individually and concatenated as a multi locus sequencing approach. Concordance between MALDI-ToF MS and Sanger sequencing was assessed using Cohen's Kappa statistical analysis. Cohen's Kappa values indicated moderate concordance of 0.46 for , 0.51 for , and 0.69 for . Concatenated phylogenetic analysis yielded improved concordance values of 0.71 for ( + , 0.76 for ( + , 0.69 for ( + , and 0.72 for (16S + + ). Our results show that NTM identification is more accurate when employing a multi locus sequencing approach. Notably, the combination of outperformed the three-marker concatenation, offering the highest concordance for species-level identification. NTM identification is challenging, and concatenated phylogenetic analysis of two or more gene fragments should be used when MALDI-ToF MS or whole genome sequencing is not available.