Diagnostic Utility of in Vivo Expressed Mycobacterial RNA Transcripts in Pleural Fluid for the Differential Diagnosis of Tuberculous Pleuritis

Background Tuberculous 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. Methods TBP 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 using M.tb RNA 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. Results Overall, 1856 genes were differentially expressed in microarray of which 1365 were upregulated and 491 were downregulated. After validation of microarray gene expression, two genes namely Rv1586 and Rv2543 were 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. Conclusion The transcripts of genes Rv1586 and Rv2543 holds potential for the development of a RNA based molecular diagnostic assay in pleural fluid of TBP patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The current research was funded by Department of Biotechnology, New Delhi, India vide Grant No. TB.BT/PR20863/MED/30/1889/2017. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Ethics Committee (IEC), Postgraduate Institute of Medical Education and Research, Chandigarh, India gave ethical approval (Ref no. INT/IEC/2016/2264) for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The gene expression data obtained in the current study has been submitted to Gene Expression Omnibus (GEO) vide accession no. GSE231472.