ObjectivesTuberculous pleurisy is one of the most common types of extra-pulmonary tuberculosis, but the sensitivity of conventional mycobacterial culture (Culture) or Xpert MTB/RIF assay (Xpert) is not satisfying. This multicentre cohort study evaluated the accuracy of a new cell-free DNA droplet digital PCR assay (cf-ddPCR) for diagnosing tuberculous pleurisy. MethodsPatients with suspected tuberculosis (≥5 years of age) with pleural effusion were consecutively recruited from nine research sites across six provinces in China between September 2020 to May 2022. Culture, Xpert, Xpert MTB/RIF Ultra assay (Ultra), real-time PCR, and cf-ddPCR were performed simultaneously for all specimens. ResultsA total of 321 participants were enrolled, and data from 281 (87.5%) participants were available, including 105 definite tuberculous pleurisy, 113 possible tuberculous pleurisy and 63 non-tuberculous pleurisy according to the composite reference standard. The sensitivity of cf-ddPCR was 90.5% (95/105, 95% CI, 82.8–95.1%) in the definite tuberculous pleurisy group, which was significantly higher than those of Culture (57.1%, 60/105, 95% CI, 47.1–66.6%, p < 0.001), Xpert (46.7%, 49/105, 95% CI, 37.0–56.6%, p < 0.001), Ultra (69.5%, 73/105, 95% CI, 59.7–77.9%, p < 0.001) and real-time PCR (75.2%, 79/105, 95% CI, 65.7–82.9%, p < 0.001). In possible tuberculous pleurisy, whose results of Culture and Xpert were both negative, the sensitivity of cf-ddPCR was 61.1% (69/113, 95% CI, 51.4–70.0%), which was still significantly higher than that of Ultra (27.4%, 31/113, 95% CI, 19.7–36.8%, p < 0.001) and real-time PCR (38.9%, 44/113, 95% CI, 30.0–48.6%, p < 0.001). DiscussionThe performance of cf-ddPCR is superior to Culture, Xpert, Ultra, and real-time PCR, indicating that improved diagnostic accuracy can be anticipated by incorporating this new assay.