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

BACKGROUND: (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.