Investigation of compatibility of mass spectrometry and sequence analysis methods in identification of rarely isolated and difficult to identify microorganisms from clinical samples

AIM: The aim of this study was to evaluate the analytical and practical performance of mass spectrometry (MS) in the identification of rarely isolated and difficult-to-identify microorganisms from clinical samples, and to investigate its concordance with 16S rRNA sequencing analysis. MATERIALS AND METHODS: The study included 126 bacterial strains from clinical samples, 116 identified by MS and 10 unidentified strains, with rare and unusual morphological/biochemical properties. Strains stored at -80°C underwent nucleic acid extraction and 16S rRNA Sanger sequencing. Based on the sequencing and BLAST analysis similarity percentages, three different identification criteria (IC) were established, and MS results were compared accordingly. RESULTS: The concordance rates of strains at genus and species levels varied according to the criteria. For species-level identification with ≥99% and genus-level with ≥97% similarity cut-off values were applied (IC1), concordance was 73.8% at genus level and 32.5% at species level. Using cut-off values of ≥97% for species and ≥95% for genus (IC2), concordance increased to 77.7% (genus) and 59.5% (species). Without any similarity cut-off values (IC3), concordance rates were 65.1% (species) and 80.1% (genus). Misidentification rate at genus level was 10.3% across all criteria. CONCLUSION: In our study, results were evaluated by creating three different criteria, and it's the first study in the literature in this respect. Sequencing concordance rates with MS were found to be low in IC1, moderately higher in IC2, and highest in IC3. To effectively compare MS and sequencing methods, it's necessary to determine standard definition criteria. The rapid bacterial identification capability of MS method provides great convenience. Given the drawbacks of sequencing—such as high cost, time consumption, intensive workload, and limited feasibility for routine use—MS provides substantial advantages not only for frequently isolated bacteria but also for rarely detected and difficult-to-identify strains. More comprehensive research is needed on this subject