{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T18:43:38Z","timestamp":1789670618860,"version":"4.0.1"},"reference-count":33,"publisher":"BMJ","issue":"8","funder":[{"name":"the Fuzhou Municipal Science and Technology Program","award":["2023-S-042"],"award-info":[{"award-number":["2023-S-042"]}]},{"name":"Major Science and Technology Project of Xiamen","award":["3502Z20231025"],"award-info":[{"award-number":["3502Z20231025"]}]},{"name":"the Natural Science and Technology Major Project of the Xiamen","award":["3502Z20231034"],"award-info":[{"award-number":["3502Z20231034"]}]},{"name":"the key technological innovation and industrialization projects in Fujian Province in 2023","award":["2023G051"],"award-info":[{"award-number":["2023G051"]}]},{"name":"the Guiding Project of the Fujian Scientifc and Technological Program","award":["2023D012"],"award-info":[{"award-number":["2023D012"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["Thorax"],"accepted":{"date-parts":[[2025,11,12]]},"published-print":{"date-parts":[[2026,8]]},"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Tuberculous pleurisy (TBP) is a leading aetiology of exudative pleural effusion in tuberculosis (TB)-endemic regions and poses significant diagnostic challenges due to its paucibacillary nature of\n                      <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                      (MTB) in pleural fluid. This study aims to characterise MTB-derived cell-free DNA (cfDNA) in pleural effusion and to develop an optimised molecular assay to improve TBP diagnostic accuracy.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We quantified MTB cfDNA\/genomic DNA (gDNA) concentrations and characterised cfDNA fragment profiles in pleural effusion specimens using ddPCR. A multiplex droplet digital PCR (ddPCR) assay was developed, targeting two insertion sequences (IS6110 and IS1081) using ultra-short amplicons (49\u201359\u2009bp) to enhance detection efficiency. Diagnostic performance was prospectively evaluated in 356 consecutive adults with radiologically confirmed pleural effusion, using composite microbiological criteria as the reference standard.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>MTB cfDNA exhibited significantly higher detection rates than gDNA in definite TBP cases (p=0.0006), with dominant fragment lengths of 60\u201380\u2009bp. The multiplex ddPCR assay demonstrated a limit of detection of 0.2 genome equivalents per reaction. Among microbiologically confirmed TBP cases (n=69), the assay achieved a sensitivity of 94.2%, significantly outperforming Xpert MTB\/RIF (52.0%, p&lt;0.0001) and liquid culture (35.3%, p&lt;0.0001). Specificity remained high at 97.0% among cases of non-TB effusion (n=208).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Our findings establish MTB cfDNA as the predominant nucleic acid form in tuberculous pleural effusion. The optimised multiplex ddPCR platform, leveraging multicopy targets and fragment-length-adapted amplification, achieves improved diagnostic sensitivity without compromising specificity in a high-prevalence TB setting. This approach may help address key limitations of TBP diagnostics and shows promise for clinical implementation.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/thorax-2025-223553","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T11:13:44Z","timestamp":1765278824000},"page":"738-748","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":5,"title":["Enhanced diagnosis of tuberculous pleurisy using a multiplex droplet digital PCR assay targeting circulating mycobacterial DNA"],"prefix":"10.1136","volume":"81","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7833-0069","authenticated-orcid":false,"given":"Siqi","family":"Zhang","sequence":"first","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of China"},{"name":"National Clinical Research Center for Infectious Diseases, Shenzhen Clinical Research Center for Tuberculosis, Shenzhen Third People's Hospital, Shenzhen, Guangdong, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5066-4698","authenticated-orcid":false,"given":"Ye","family":"Xu","sequence":"additional","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5824-5372","authenticated-orcid":false,"given":"Mingxiang","family":"Huang","sequence":"additional","affiliation":[{"name":"Pulmonary Hospital of Fuzhou in Fujian Province, Fuzhou, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuying","family":"Pan","sequence":"additional","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jichang","family":"Shangguan","sequence":"additional","affiliation":[{"name":"Pulmonary Hospital of Fuzhou in Fujian Province, Fuzhou, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Peng","sequence":"additional","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoman","family":"Hu","sequence":"additional","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Zheng","sequence":"additional","affiliation":[{"name":"Engineering Research Centre of Molecular Diagnostics of Ministry of Education, State Key Laboratory of Cellular Stress Biology, Xiamen Key Laboratory of Multi-Target and Automated Genetic Testing, School of Life Sciences, Xiamen University, Xiamen, Fujian, People's Republic of 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