Quantification of MPT-64 within pleural fluid extracellular vesicles of tuberculous pleurisy patients by real-time immuno-PCR

Diagnosis of tuberculous (TB) pleurisy is an exigent task owing to atypical clinical presentations and low bacillary content in clinical samples. Hence, there is a crucial need to deliberate a quick and consistent diagnostic test. We recently quantified Mycobacterium tuberculosis (Mtb)-specific MPT-64 (Rv1980c) within pleural fluid extracellular vesicles (pEVs) of TB pleurisy patients by SYBR Green real-time immuno-PCR (RT-I-PCR) assay and compared its diagnostic efficacy with respective ELISA and GeneXpert assay. The size of pEVs of TB pleurisy patients ranged between 47.7 and 170.2 nm as evaluated by Nanoparticle tracking analysis and Transmission electron microscopy. Noticeably, a dynamic range (0.7 pg/mL-9.7 ng/mL) of Mtb MPT-64 was quantitatively detected within pEVs of TB pleurisy individuals by RT-I-PCR, albeit ELISA exhibited a thin range (2.5 ng/mL-11.2 ng/mL). Our RT-I-PCR demonstrated sensitivity of 80 % and 80.9 % in clinically suspected/probable (n = 35) and total (n = 42) TB pleurisy individuals, respectively, with 97.3 % specificity in 38 non-TB controls, against a composite reference standard. Concurrently, MPT-64 detection within pEVs of clinically suspected/probable TB pleurisy cases by ELISA and GeneXpert displayed substantially lower sensitivities (p < 0.05-0.01) than RT-I-PCR. After further improving the sensitivity and authenticating these RT-I-PCR results with a larger sample size, this assay may yield a promising diagnostic kit.