BRIEF RESEARCH REPORT article
Front. Cell. Infect. Microbiol.
Sec. Clinical Infectious Diseases
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1612459
This article is part of the Research TopicOne Health Approach to Mycobacterial Infections in Veterinary ScienceView all 11 articles
Comparative evaluation of MALDI-ToF mass spectrometry and Sanger sequencing of the 16S, hsp65, and rpoB genes for non tuberculous mycobacteria species identification
Provisionally accepted
Angel Sebastian Rodriguez Pazmino1Darwin Santiago Paredes1Elsy Carvajal1Melissa Carvajal Capa2Joselyn Calderon3Solon Alberto Orlando3Henry Parra Vera4
Miguel Angel Garcia Bereguiain1*- 1University of the Americas, Quito, Pichincha, Ecuador
- 2Instituto de Investigación Salud Integral, Universidad Católica de Santiago de Guayaquil, Guayaquil, Guayas, Ecuador
- 3National Institute of Public Health and Research (Ecuador), Quito, Pichincha, Ecuador
- 4Centro de Investigacion Microbiologica., Guayaquil, Guayas, Ecuador
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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 (16S, hsp65, and rpoB) 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 16S, 0.51 for hsp65, and 0.69 for rpoB. Concatenated phylogenetic analysis yielded improved concordance values of 0.71 for (16S + hsp65), 0.76 for (16S + rpoB), 0.69 for (rpoB + hsp65), and 0.72 for (16S + hsp65 + rpoB). Our results show that NTM identification is more accurate when employing a multi locus sequencing approach. Notably, the combination of 16S + rpoB 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.
Keywords: Sanger sequencing, MALDI-TOF MS, Non-tuberculous mycobacteria, Ecuador, Clinical performance
Received: 15 Apr 2025; Accepted: 02 Jul 2025.
Copyright: © 2025 Rodriguez Pazmino, Paredes, Carvajal, Carvajal Capa, Calderon, Orlando, Parra Vera and Garcia Bereguiain. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Miguel Angel Garcia Bereguiain, University of the Americas, Quito, 170137, Pichincha, Ecuador
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