Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study

Manisha Dass; Simran Aittan; Rajagopalan Muthumohan; Divya Anthwal; Rakesh Kumar Gupta; Gargi Mahajan; Pooja Kumari; Neera Sharma; Rajesh S Taneja; Lokesh Kumar Sharma; Ritu Shree; Vivek Lal; Jaya Sivaswami Tyagi; Sagarika Haldar

doi:10.1016/j.tube.2022.102213

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study

Tuberculosis (Edinb). 2022 Jul:135:102213. doi: 10.1016/j.tube.2022.102213. Epub 2022 May 28.

Authors

Affiliations

¹ Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
² Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
³ Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
⁴ Department of Biochemistry, Dr. Ram Manohar Lohia Hospital, New Delhi, India.
⁵ Department of Medicine, Dr. Ram Manohar Lohia Hospital, New Delhi, India.
⁶ Department of Neurology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
⁷ Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India; Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
⁸ Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India; Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India. Electronic address: sagarikahaldar.pgimer@gmail.com.

PMID: 35696959
DOI: 10.1016/j.tube.2022.102213

Abstract

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as 'Definite' TBM (n = 8), 'Probable' TBM (n = 15), 'Possible' TBM (n = 21)] and 'Non-TBM' group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of 'Definite' TBM and 'Non-TBM' category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1-100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8-69.6) for EV-DNA and 77.3% (95%CI:62.1-88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2-91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Keywords: Cell-free DNA; Extracellular vesicles; Molecular diagnostics; Transrenal DNA; Tuberculous meningitis; Xpert MTB/RIF.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell-Free Nucleic Acids* / genetics
Humans
Molecular Diagnostic Techniques / methods
Mycobacterium tuberculosis* / genetics
Sensitivity and Specificity
Tuberculosis, Meningeal* / diagnosis
Tuberculosis, Meningeal* / genetics
Tuberculosis, Meningeal* / microbiology

Substances

Cell-Free Nucleic Acids