Utility of pleural fluid-derived extracellular vesicles as a source of Mycobacterium tuberculosis antigens MPT51 and MPT64 for pleural TB diagnosis: a proof-of-concept study.

Extracellular vesicles (EVs) have recently emerged as a source of microbe-specific biomarkers for disease diagnosis. In the present study, we evaluated the utility of pleural fluid-derived extracellular vesicles (pEVs) as a source of Mycobacterium tuberculosis (M. tb.) antigens for pleural TB (pTB) diagnosis. EVs were isolated from pleural fluid (PF) samples and were characterized by scanning electron microscopy, and immunoblotting by targeting CD63 and LAMP2 markers. Antigen-detection ELISAs were developed for 2 M.tb.-specific antigens, MPT51 and MPT64 in pEVs (pEV-ELISA) and direct PF samples (PF-ELISA), and were evaluated on n = 86 samples in a blinded manner. Cut-off values were calculated by ROC-curve analysis to achieve 90 % (95%CI:73.47-97.89) and 86.67 % (95%CI:69.28-96.24) specificity for MPT51 and MPT64 pEV-ELISA respectively. The sensitivity of pEV-ELISA was 71.43 % (95%CI; 29.04-96.33) for MPT51 antigen and 57.14 % (95%CI; 18.41-90.1) for MPT64 antigen in the 'Definite' pTB group, while in the 'Definite and Probable' pTB group, the sensitivity was 62.86 % (95%CI:44.92-78.53) for MPT51 and 65.71 % (95%CI:47.79-80.87) for MPT64. The performance of PF-ELISA was sub-optimal, with 28.57 % (95%CI:3.67-70.96) and 14.29 % (95%CI:0.36-57.87) sensitivity for MPT51 and MPT64 in 'Definite' pTB group respectively. We conclude that M. tb.-antigens are concentrated in the EV-fraction of PF samples and EVs can be utilized for antigen-detection assays for pTB diagnosis.