Enhanced diagnosis of tuberculous pleurisy using a multiplex droplet digital PCR assay targeting circulating mycobacterial DNA

Background 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 Mycobacterium tuberculosis (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. Methods 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 (49–59 bp) 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. Results MTB cfDNA exhibited significantly higher detection rates than gDNA in definite TBP cases (p=0.0006), with dominant fragment lengths of 60–80 bp. 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<0.0001) and liquid culture (35.3%, p<0.0001). Specificity remained high at 97.0% among cases of non-TB effusion (n=208). Conclusions 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.