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Peer-reviewedCC BY 4.0Frontiers in Microbiology

A novel multiplex assay for species-level identification of non-tuberculous mycobacteria in culture-positive respiratory specimens based on capillary electrophoresis: development and clinical validation

  • Zhen Li1,2
  • Yaocheng Wang1,3
  • Li Lai1,3
  • Yi Huang1,3
  • Li Li1,2,4
4 affiliations
  1. 1Shengli Clinical Medical College of Fujian Medical University, Fujian Medical University
  2. 2Tuberculosis Laboratory, International Medical Examination Center, Fuzhou University Affiliated Provincial Hospital
  3. 3Department of Clinical Laboratory, Fuzhou University Affiliated Provincial Hospital
  4. 4Department of Health Management, Fuzhou University Affiliated Provincial Hospital
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Abstract

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is rising globally, driven predominantly by Mycobacterium avium complex (MAC), yet treatment is highly species-dependent. We developed a Multiplex assay targeting four clinically important non-tuberculous mycobacteria (NTM) species– M. avium , M. intracellulare , M. abscessus , and M. fortuitum –and compared it with metagenomic next-generation sequencing (mNGS), using colony-based multigene Sanger sequencing as the reference method. Among 101 specimens, 63 (62.4%) yielded polymicrobial results, of which mixed-species detection of M. avium – M. intracellulare accounted for 46 (73.0%). The Multiplex assay and mNGS showed no statistically significant difference in overall accuracy in this cohort (82.2% vs. 81.2%; raw agreement 83.2%, P = 1.000). The Multiplex assay achieved 100.0% sensitivity for all four species, with specificity of 87.8% and 87.5% for M. avium and M. intracellulare , respectively, and >98% for M. abscessus and M. fortuitum ; mNGS showed the converse pattern, with lower MAC sensitivity (88.5%, 92.2%) but higher MAC specificity (93.9%, 95.8%). Requiring only a single PCR reaction without sequencing or bioinformatic analysis, the Multiplex assay showed no statistically significant difference in diagnostic accuracy from mNGS in this culture-positive cohort, without requiring library preparation or sequencing, representing a promising candidate for further evaluation as a routine NTM species-identification tool.

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Cite

BibTeXRIS
BibTeX
@article{li2026novel,
  title = {{A novel multiplex assay for species-level identification of non-tuberculous mycobacteria in culture-positive respiratory specimens based on capillary electrophoresis: development and clinical validation}},
  author = {Zhen Li and Yaocheng Wang and Li Lai and Yi Huang and Li Li},
  journal = {Frontiers in Microbiology},
  year = {2026},
  month = oct,
  doi = {10.3389/fmicb.2026.1966137},
  url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1966137/full},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - A novel multiplex assay for species-level identification of non-tuberculous mycobacteria in culture-positive respiratory specimens based on capillary electrophoresis: development and clinical validation
AU  - Zhen Li
AU  - Yaocheng Wang
AU  - Li Lai
AU  - Yi Huang
AU  - Li Li
PY  - 2026
DA  - 2026/10/08
JO  - Frontiers in Microbiology
DO  - 10.3389/fmicb.2026.1966137
UR  - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1966137/full
AB  - Nontuberculous mycobacterial pulmonary disease (NTM-PD) is rising globally, driven predominantly by Mycobacterium avium complex (MAC), yet treatment is highly species-dependent. We developed a Multiplex assay targeting four clinically important non-tuberculous mycobacteria (NTM) species– M. avium , M. intracellulare , M. abscessus , and M. fortuitum –and compared it with metagenomic next-generation sequencing (mNGS), using colony-based multigene Sanger sequencing as the reference method. Among 101 specimens, 63 (62.4%) yielded polymicrobial results, of which mixed-species detection of M. avium – M. intracellulare accounted for 46 (73.0%). The Multiplex assay and mNGS showed no statistically significant difference in overall accuracy in this cohort (82.2% vs. 81.2%; raw agreement 83.2%, P = 1.000). The Multiplex assay achieved 100.0% sensitivity for all four species, with specificity of 87.8% and 87.5% for M. avium and M. intracellulare , respectively, and >98% for M. abscessus and M. fortuitum ; mNGS showed the converse pattern, with lower MAC sensitivity (88.5%, 92.2%) but higher MAC specificity (93.9%, 95.8%). Requiring only a single PCR reaction without sequencing or bioinformatic analysis, the Multiplex assay showed no statistically significant difference in diagnostic accuracy from mNGS in this culture-positive cohort, without requiring library preparation or sequencing, representing a promising candidate for further evaluation as a routine NTM species-identification tool.
SN  - 1664-302X
ER  -