Diagnostic utility of in vivo expressed mycobacterial RNA transcripts in pleural fluid for the differential diagnosis of tuberculous pleuritis

Abstract

ABSTRACTBackgroundTuberculous pleuritis (TBP), the extra-pulmonary manifestation of tuberculosis, is the second most common after TB lymphadenitis. Histopathology using biopsy samples is the most sensitive diagnostic procedure for TBP, however the biopsy acquisition is invasive. Therefore, better screening markers for diagnosis using pleural fluid are required. The pathogen biomarkers expressed at the site of infection may play a potential role in designing a newer diagnostic assay. Thus, the current study was planned to look for mycobacterial RNA biomarkers in TBP and to assess their diagnostic utility in pleural fluid.MethodsTBP suspects (n=261) were recruited in the current study. Out of these 45 suspects were excluded and the remaining (n=216) were divided into TBP (n=54) and non-TBP (n=162) groups based on composite reference standard. A whole genome microarray was carried usingM.tbRNA from pleural biopsies of TB patients. The data was validated using qRT-PCR and the diagnostic utility of top two highly expressed genes was assessed in pleural fluid of using a real time RT-PCR assay.ResultsOverall, 1856 genes were differentially expressed in microarray of which 1365 were upregulated and 491 were downregulated. After validation of microarray gene expression, two genes namelyRv1586andRv2543were selected for assessment of their diagnostic utility in TBP. The combined analysis for the presence of either of genes in the pleural fluid led to identification of pleural TB patients with 79.6% sensitivity and 93.28% specificity.ConclusionThe transcripts of genesRv1586andRv2543holds potential for the development of a RNA based molecular diagnostic assay in pleural fluid of TBP patients.