Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
8 affiliations
- 1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
- 2Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
- 3Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
- 4Office of Disease Prevention and Control Region 10th, Ministry of Public Health, Ubon Ratchathani, Thailand
- 5Department of Medical Technology and Clinical Pathology, Surin Hospital, Surin, Thailand
- 6Department of Internal Medicine, Surin Hospital, Surin, Thailand
- 7Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
- 8The Academy of Science, The Royal Society of Thailand, Bangkok, Thailand
Abstract
Effective control of tuberculosis, nontuberculous mycobacterial (NTM) disease, and melioidosis depends on rapid and accurate laboratory diagnosis. Mycobacterium tuberculosis complex (MTBC) remains a health priority, whereas NTM infections, particularly Mycobacterium avium complex (MAC), are increasingly recognized and may be misdiagnosed as tuberculosis. Melioidosis, caused by Burkholderia pseudomallei, is underdiagnosed in endemic regions. These infections have overlapping clinical presentations, and conventional diagnostics methods may not accurately differentiate among these diseases. We developed and evaluated a quadruplex real-time PCR assay (TAM-mPCR) for simultaneous detection of MTBC, MAC, and B. pseudomallei, targeting IS6110, rrs, and BPSS1187. A total of 607 pulmonary and extrapulmonary specimens from patients with suspected tuberculosis were collected at two sites in Thailand between 2024 and 2025. The limits of detection were 2 pg/reaction for MTBC, 0.1 ng/reaction for MAC, and 50 fg/reaction for B. pseudomallei, with 100% analytical specificity. For MTBC detection, TAM-mPCR demonstrated 100% sensitivity (330/330) and 99% (189/191) specificity. All three culture-confirmed MAC specimens were detected. For B. pseudomallei, the assay demonstrated 73.2% sensitivity (30/41) and 100% specificity (79/79). For MTBC, TAM-mPCR showed high concordance with NAAT and culture results, but differed significantly from AFB microscopy (P < 0.001). The assay also detected additional MTBC, MAC, and B. pseudomallei infections that were missed by routine diagnostics. TAM-mPCR enables rapid simultaneous pathogen detection and differentiation and may improve case detection and patient management in setting where tuberculosis and melioidosis co-endemic. Author summary: Accurate diagnosis of tuberculosis, nontuberculous mycobacterial (NTM) disease, and melioidosis is challenging due to overlapping clinical presentations and limitations of routine diagnostic methods. We developed a multiplex real-time PCR assay (TAM-mPCR; Tuberculosis–Avium–Melioidosis-mPCR) for simultaneous detection of Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium complex (MAC), and B. pseudomallei directly from clinical specimens. The assay was evaluated using 607 pulmonary and extrapulmonary specimens from patients with suspected tuberculosis in northeastern Thailand. The assay demonstrated high sensitivity and specificity across specimen types and showed strong concordance with nucleic acid amplification tests (NAAT) and bacterial culture. TAM-mPCR detected all three culture-confirmed MAC specimens and showed moderate sensitivity and 100% specificity for B. pseudomallei. Importantly, the assay identified additional MTBC, MAC, and B. pseudomallei infections, including MTBC–B. pseudomallei co-infections, among specimens with negative routine diagnostic results. These finding highlight the potential of TAM-mPCR to improve detection of clinically significant infections and support more accurate diagnosis in co-endemic settings.
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BibTeX
@article{binmaeil2026development,
title = {{Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens}},
author = {Hasyanee Binmaeil and Thanakon Bunsong and Janjira Thaipadungpanit and Wipawee Saenwongsa and Montira Ngoenpramual and Sutthinan Chawsorngkorn and Soawarat Deekae and Sunee Chayangsu and Wang Nguitragool and Narisara Chantratita},
journal = {PLOS Neglected Tropical Diseases},
year = {2026},
month = sep,
doi = {10.1371/journal.pntd.0014730},
url = {https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014730},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
AU - Hasyanee Binmaeil
AU - Thanakon Bunsong
AU - Janjira Thaipadungpanit
AU - Wipawee Saenwongsa
AU - Montira Ngoenpramual
AU - Sutthinan Chawsorngkorn
AU - Soawarat Deekae
AU - Sunee Chayangsu
AU - Wang Nguitragool
AU - Narisara Chantratita
PY - 2026
DA - 2026/09/24
JO - PLOS Neglected Tropical Diseases
DO - 10.1371/journal.pntd.0014730
UR - https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014730
AB - Effective control of tuberculosis, nontuberculous mycobacterial (NTM) disease, and melioidosis depends on rapid and accurate laboratory diagnosis. Mycobacterium tuberculosis complex (MTBC) remains a health priority, whereas NTM infections, particularly Mycobacterium avium complex (MAC), are increasingly recognized and may be misdiagnosed as tuberculosis. Melioidosis, caused by Burkholderia pseudomallei, is underdiagnosed in endemic regions. These infections have overlapping clinical presentations, and conventional diagnostics methods may not accurately differentiate among these diseases. We developed and evaluated a quadruplex real-time PCR assay (TAM-mPCR) for simultaneous detection of MTBC, MAC, and B. pseudomallei, targeting IS6110, rrs, and BPSS1187. A total of 607 pulmonary and extrapulmonary specimens from patients with suspected tuberculosis were collected at two sites in Thailand between 2024 and 2025. The limits of detection were 2 pg/reaction for MTBC, 0.1 ng/reaction for MAC, and 50 fg/reaction for B. pseudomallei, with 100% analytical specificity. For MTBC detection, TAM-mPCR demonstrated 100% sensitivity (330/330) and 99% (189/191) specificity. All three culture-confirmed MAC specimens were detected. For B. pseudomallei, the assay demonstrated 73.2% sensitivity (30/41) and 100% specificity (79/79). For MTBC, TAM-mPCR showed high concordance with NAAT and culture results, but differed significantly from AFB microscopy (P < 0.001). The assay also detected additional MTBC, MAC, and B. pseudomallei infections that were missed by routine diagnostics. TAM-mPCR enables rapid simultaneous pathogen detection and differentiation and may improve case detection and patient management in setting where tuberculosis and melioidosis co-endemic. Author summary: Accurate diagnosis of tuberculosis, nontuberculous mycobacterial (NTM) disease, and melioidosis is challenging due to overlapping clinical presentations and limitations of routine diagnostic methods. We developed a multiplex real-time PCR assay (TAM-mPCR; Tuberculosis–Avium–Melioidosis-mPCR) for simultaneous detection of Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium complex (MAC), and B. pseudomallei directly from clinical specimens. The assay was evaluated using 607 pulmonary and extrapulmonary specimens from patients with suspected tuberculosis in northeastern Thailand. The assay demonstrated high sensitivity and specificity across specimen types and showed strong concordance with nucleic acid amplification tests (NAAT) and bacterial culture. TAM-mPCR detected all three culture-confirmed MAC specimens and showed moderate sensitivity and 100% specificity for B. pseudomallei. Importantly, the assay identified additional MTBC, MAC, and B. pseudomallei infections, including MTBC–B. pseudomallei co-infections, among specimens with negative routine diagnostic results. These finding highlight the potential of TAM-mPCR to improve detection of clinically significant infections and support more accurate diagnosis in co-endemic settings.
SN - 1935-2735
ER -