Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
Issued Date
2026-09-01
Resource Type
eISSN
19352735
Scopus ID
2-s2.0-105051657041
Pubmed ID
42784601
Journal Title
Plos Neglected Tropical Diseases
Volume
20
Issue
9
Rights Holder(s)
SCOPUS
Bibliographic Citation
Plos Neglected Tropical Diseases Vol.20 No.9 (2026) , e0014730
Suggested Citation
Binmaeil H., Bunsong T., Thaipadungpanit J., Saenwongsa W., Ngoenpramual M., Chawsorngkorn S., Deekae S., Chayangsu S., Nguitragool W., Chantratita N. Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens. Plos Neglected Tropical Diseases Vol.20 No.9 (2026) , e0014730. doi:10.1371/journal.pntd.0014730 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/119388
Title
Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
Corresponding Author(s)
Other Contributor(s)
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.