Abstract
Tuberculous pleuritis (TBP) is the second most common extra-pulmonary manifestation of tuberculosis. Histopathology using pleural biopsy is the most sensitive diagnostic procedure for TBP. Since biopsy acquisition is invasive, better biomarkers for diagnosis using pleural fluid are needed. The current study was designed to identify mycobacterial RNA biomarkers in TBP and to assess their diagnostic utility in pleural fluid. 261 TBP suspects were recruited for the study. 45 suspects were excluded and remaining (n = 216) were divided into TBP (n = 54) and non-TBP (n = 162) groups based on composite reference standard (CRS). Whole genome microarray was carried using M.tb RNA from pleural biopsies of TBP patients. The data was validated using qRT-PCR and the diagnostic utility of top two highly expressed genes was evaluated in pleural fluid, using qualitative real time RT-PCR. 1856 genes were differentially expressed in microarray with 1365 upregulated and 491 downregulated genes. The diagnostic utility of transcripts corresponding to two genes, Rv1586c and Rv2819c was assessed post microarray validation. The presence of either of two genes led to pleural TB identification with 79.6% sensitivity and 93.28% specificity. Thus, the transcripts of genes Rv1586c and Rv2819c holds potential for development of RNA based diagnostic assay for TBP using pleural fluid.
Data availability
The gene expression data obtained in the current study has been submitted to Gene Expression Omnibus (GEO) vide accession no. GSE231472. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE231472.
References
WHO. Global TB report 2021. (Accessed Dec 2021) http://www.who.int (WHO, 2021).
Cascio, C., Kaul, V., Dhooria, S., Agrawal, A. & Chaddha, U. Diagnosis of tuberculous pleural effusions: A review. Respir Med. 188, 106607 (2021).
Bielsa, S., Acosta, C., Pardina, M., Civit, C. & Porcel, J. M. Tuberculous pleural effusion: Clinical characteristics of 320 patients. Arch. Bronconeumol. (Engl Ed). 55, 17–22 (2019).
Wang, Z. et al. Diagnostic value and safety of medical thoracoscopy in tuberculous pleural effusion. Respir Med. 109, 1188–1192 (2015).
Ruan, S. et al. Revisiting tuberculous pleurisy: pleural fluid characteristics and diagnostic yield of mycobacterial culture in an endemic area. Thorax 67, 822–827 (2012).
Bibby, A. & Maskell, N. Pleural biopsies in undiagnosed pleural effusions; Abrams vs image-guided vs thoracoscopic biopsies. Curr. Opin. Pulm Med. 22, 392–398 (2016).
Porcel, J. Tuberculous pleural effusion. Lung 187, 263–270 (2009).
Trajman, A. & Luiz, R. McNemar chi2 test revisited: comparing sensitivity and specificity of diagnostic examinations. Scand. J. Clin. Lab. Invest. 68, 77–80 (2008).
Aggarwal, A. N. et al. Unstimulated pleural fluid interferon gamma for diagnosis of tuberculous pleural effusion: A systematic review and meta-analysis. J. Clin. Microbiol. 59, 2112–2132 (2021).
Meldau, R. et al. Same-Day Tools, including Xpert Ultra and IRISA-TB, for rapid diagnosis of pleural tuberculosis: a prospective observational study. J. Clin. Microbiol. 57, e00614–e00619 (2019).
Wang, G. et al. Accuracy of Xpert MTB/RIF Ultra for the diagnosis of pleural TB in a multicenter cohort study. Chest 157, 268–275 (2020).
Gao, S. et al. Xpert MTB/RIF Ultra enhanced tuberculous pleurisy diagnosis for patients with unexplained exudative pleural effusion who underwent a pleural biopsy via thoracoscopy: A prospective cohort study. Int. J. Infect. Dis. 106, 370–375 (2021).
Theron, G. et al. Xpert MTB/RIF results in patients with previous tuberculosis: can we distinguish true from false positive results? Clin. infect. Dis. 62, 995–1001 (2016).
Sharma, S. et al. Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients. PLoS One. 12, e0173508 (2017).
Abhishek, S. et al. Transcriptional profile of mycobacterium tuberculosis in an in vitro model of intraocular tuberculosis. Front. Cell. Infect. Microbiol. 8, 1–20 (2018).
Schnappinger, D. et al. Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment. J. Exp. Med. 198, 693–704 (2003).
Rachman, H. et al. Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infect. Immun. 74, 1233–1242 (2006).
Lew, J., Kapopoulou, A., Jones, L. & Cole, S. TubercuList–10 years after. Tuberculosis (Edinb). 91, 1–7 (2011).
Karp, P. D. et al. The BioCyc collection of microbial genomes and metabolic pathways. Brief. Bioinform. 20, 1085–1093 (2019).
Gogia & Gupta, P. Diagnosing pleural tuberculosis is simple, yet not so simple! Curr. Med. Res. Pract. 14, 137–139 (2024).
Ryndak, M., Singh, K., Peng, Z. & Laal, S. Transcriptional profile of mycobacterium tuberculosis replicating in type II alveolar epithelial cells. PLoS One. 10, e0123745 (2015).
Garton, N. J. et al. Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med. 5, e75 (2008).
Warsinske, H., Vashisht, R. & Khatri, P. Host-response-based gene signatures for tuberculosis diagnosis: A systematic comparison of 16 signatures. PLoS Med. 16, e1002786 (2019).
Hoang, L. et al. Transcriptomic signatures for diagnosing tuberculosis in clinical practice: a prospective, multicentre cohort study. Lancet Infect. Dis. 21, 366–375 (2021).
Morey, J., Ryan, J. & Van Dolah, F. Microarray validation: Factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol. Proced. Online. 8, 175–193 (2006).
Bayés-Genis, A. et al. Prognostic value of circulating microRNAs on heart failure-related morbidity and mortality in two large diverse cohorts of general heart failure patients. Eur. J. Hear. Fail. 20, 67–75 (2018).
Bustin, S., Coward, A., Sadler, G., Teare, L. & Nolan, T. CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19. Sci. Rep. 10, 22214 (2020).
Lusiba, J. et al. Evaluation of Cepheid’s Xpert MTB/Rif test on pleural fluid in the diagnosis of pleural tuberculosis in a high prevalence HIV/TB setting. PLoS One. 9, e102702 (2014).
Denkinger, C. et al. Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis. Eur. Respir J. 44, 435–446 (2014).
Aggarwal, A. N. et al. Xpert MTB/RIF Ultra versus Xpert MTB/RIF for diagnosis of tuberculous pleural effusion: A systematic review and comparative meta-analysis. PLoS One. 17, e0268483 (2022).
Arend, S. M. & van Soolingen, D. Performance of Xpert MTB/RIF Ultra: a matter of dead or alive. Lancet Infect. Dis. 18, 8–10 (2018).
Brennan, M. J. The enigmatic PE/PPE multigene family of mycobacteria and tuberculosis vaccination. Infect. Immun. 85, e00969–e00916 (2017).
Akhter, Y., Ehebauer, M., Mukhopadhyay, S. & Hasnain, S. The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation. Perhaps more? Biochimie. 94, 110–116 (2012).
Muñoz-Elías, E. & McKinney, J. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11, 638–644 (2005).
Gago, G., Diacovich, L. & Gramajo, H. Lipid metabolism and its implication in mycobacteria-host interaction. Curr. Opin. Microbiol. 41, 36–42 (2018).
Wilburn, K., Fieweger, R. & VanderVen, B. Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis. Pathog Dis. 76, fty021 (2018).
Kantardjieff, K. et al. Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target. Acta Crystallogr. D Biol. Crystallogr. 61, 355–364 (2005).
Rosenberg, A., Sinai, L., Smith, Y. & Ben-Yehuda, S. Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level. PLoS One. 7, e41921 (2012).
Wang, G. et al. Accuracy of Xpert MTB/RIF Ultra for the diagnosis of pleural TB in a multicenter cohort study. Chest 157, 268–275 (2020).
Meldau, R. et al. Same-Day Tools, including Xpert Ultra and IRISA-TB, for rapid diagnosis of pleural tuberculosis: A prospective observational study. J. Clin. Microbiol. 57, e00614–e00619 (2019).
Kaur, K. et al. Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis. J. Infect. 86, 134–146 (2023).
Sharma, S., Aggarwal, A. N., Kaur, P., Yadav, R., Sethi, S. & Verma, I. In-vivo expressed mycobacterial transcripts as diagnostic targets for pulmonary tuberculosis. Tuberculosis (Edinb). 144, 102431 (2024).
Livak, K. & Schmittgen, T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).
Laurell, H. et al. Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Res. 40, e51 (2012).
Funding
The current research was funded by Department of Biotechnology, New Delhi, India vide Grant No. TB.BT/PR20863/MED/30/1889/2017.
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Conceptualization: PK, SS, IV; Methodology: PK, SS and IV; Investigation: PK, SA, KK; Data Curation: PK, SS, ANA, RY, SSethi, AB; Formal Analysis: PK, SS, ANA and IV; Funding Acquisition: IV Project Supervision: IV Visualization: PK, SS, ANA, IV; Writing (Original Draft): PK; Writing (Review And Editing): SS and IV.
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Kaur, P., Sharma, S., Abhishek, S. et al. A proof of concept study on the diagnostic utility of in vivo expressed mycobacterial transcripts in tuberculous pleuritis. Sci Rep (2026). https://doi.org/10.1038/s41598-026-42637-2
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DOI: https://doi.org/10.1038/s41598-026-42637-2
