{ "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.", "abstract_shown": true, "affiliations": [ "Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand", "Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand", "Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand", "Office of Disease Prevention and Control Region 10th, Ministry of Public Health, Ubon Ratchathani, Thailand", "Department of Medical Technology and Clinical Pathology, Surin Hospital, Surin, Thailand", "Department of Internal Medicine, Surin Hospital, Surin, Thailand", "Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand", "The Academy of Science, The Royal Society of Thailand, Bangkok, Thailand" ], "article_type": "Research Article", "arxiv": null, "author_total": 10, "authors": [ { "affs": [ 0 ], "name": "Hasyanee Binmaeil" }, { "affs": [ 1 ], "name": "Thanakon Bunsong" }, { "affs": [ 2 ], "name": "Janjira Thaipadungpanit" }, { "affs": [ 3 ], "name": "Wipawee Saenwongsa" }, { "affs": [ 4 ], "name": "Montira Ngoenpramual" }, { "affs": [ 5 ], "name": "Sutthinan Chawsorngkorn" }, { "affs": [ 6 ], "name": "Soawarat Deekae" }, { "affs": [ 7 ], "name": "Sunee Chayangsu" }, { "name": "Wang Nguitragool" }, { "name": "Narisara Chantratita" } ], "bibtex": "https://research.draconitic.com/paper/17fvm3bwjb7cw.bib", "cited_by": 0, "doi": "10.1371/journal.pntd.0014730", "fulltext_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014730", "id": "17fvm3bwjb7cw", "issns": [ "1935-2735" ], "landing_url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014730", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2713|Mycobacterium research and diagnosis" ], [ "plos", "/Biology and life sciences/Biochemistry/Enzymology/Enzymes/Hydrolases/Nucleases/Ribonucleases" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/DNA-binding proteins/Nucleases/Ribonucleases" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Enzymes/Hydrolases/Nucleases/Ribonucleases" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Bacterial pathogens/Burkholderia/Burkholderia pseudomallei" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Artificial gene amplification and extension/Polymerase chain reaction" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Actinobacteria/Mycobacterium avium complex" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Actinobacteria/Mycobacterium tuberculosis" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Burkholderia/Burkholderia pseudomallei" ], [ "plos", "/Engineering and technology/Signal processing/Signal amplification" ], [ "plos", "/Medicine and health sciences/Medical conditions/Infectious diseases/Bacterial diseases/Tuberculosis" ], [ "plos", "/Medicine and health sciences/Medical conditions/Infectious diseases/Co-infections" ], [ "plos", "/Medicine and health sciences/Medical conditions/Tropical diseases/Tuberculosis" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Bacterial pathogens/Burkholderia/Burkholderia pseudomallei" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Artificial gene amplification and extension/Polymerase chain reaction" ] ], "openalex": "W7214165893", "pdf_url": "https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0014730&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-24", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/17fvm3bwjb7cw.ris", "sources": [ { "first_seen": "2026-10-30T12:17:43Z", "native_id": "10.1371/journal.pntd.0014730", "source": "plos", "url": "https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014730" } ], "subjects": [ { "field": "biochemistry", "origin": "plos:enzymology", "subfield": "enzymology-metabolism" }, { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" }, { "field": "medicine", "origin": "plos:infectious diseases", "subfield": "infectious-disease" } ], "title": "Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens", "title_text": "Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:43Z", "url": "https://research.draconitic.com/paper/17fvm3bwjb7cw", "venue": "PLOS Neglected Tropical Diseases", "versions": [] }