Evaluation of the clinical application of MALDI-TOF MS for identification of difficult-to-classify nontuberculous mycobacterial strains isolated in the laboratory

Objectives This study aims to evaluate the clinical application value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the verification and identification of difficult-to-classify nontuberculous mycobacterial (NTM) strains. Methods From December 2024 to June 2025, 106 suspected NTM isolates were collected from 10 districts in Shenzhen, China. Initial identification was performed using HRM and REBA commercial kits, with targeted nanopore sequencing and mNGS as the composite reference standard. MALDI-TOF MS was used to verify strains unresolved by the kits, and its diagnostic performance was evaluated. Results The HRM kit demonstrated concordance with the reference standard in 98 of 106 samples (concordance rate: 89.1%), whereas the REBA kit concorded in 88 samples (concordance rate: 80.0%). The REBA kit exhibited a tendency toward misidentification of NTM species as Mycobacterium tuberculosis . When the reference results were used as a baseline with tNanopore providing parallel validation (achieving ≥95% concordance with reference results), MALDI-TOF MS demonstrated poor performance in identifying difficult-to-classify NTM strains. Specifically, MALDI-TOF MS showed poor concordance in detecting M. abscessus (Kappa = 0.244), while Mycobacterium intracellulare , Mycobacterium kansasii , and Mycobacterium gordonae demonstrated kappa values of 0.543, 0.477, and 0.483, respectively, indicating low concordance overall. Furthermore, 13 species exceeded the detection range of MALDI-TOF MS, resulting in false-positive identifications or detection failures, with Mycobacterium abscessus exhibiting the highest rate of misidentification. Conclusion The limitations of MALDI-TOF MS in verifying difficult-to-classify NTM strains have been demonstrated. The findings emphasize that PCR-based molecular detection combined with gene sequence analysis remains the most reliable methodological approach for accurately identifying challenging NTM species in clinical practice.