‘Cell-free trans-renal DNA’: a novel tool for the diagnosis of Tuberculous Meningitis.
Manisha Dass 1, Simran 1, Rajagopalan Muthumohan 2, Divya Anthwal 1, Rakesh Kumar Gupta 1, Pooja Kumari 5, Neera Sharma 3, R.S. Taneja 4, Lokesh Kumar Sharma 3, Jaya Sivaswami Tyagi 2,5 and Sagarika Haldar 1,2
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 Biochemistry, Dr. Ram Manohar Lohia Hospital, New Delhi, India
4 Department of Medicine, Dr. Ram Manohar Lohia Hospital, New Delhi, India
5 Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
Abstract: Tuberculous Meningitis (TBM) is associated with a high mortality and morbidity in developing countries and its diagnosis is limited due to inadequate accuracy of existing tests. In this study, we assessed the utility of detecting cell-free trans-renal Mycobacterium tuberculosis DNA (cf-Tr-TB DNA) in urine samples for TBM diagnosis. We also endeavored to find the source of this cf-Tr-TB DNA. Firstly, a pilot study was performed, wherein urinary extracellular vesicles (EVs) were isolated from TBM and non-TBM samples. The presence of urinary exosomes and microvesicles was confirmed by immunoblotting and scanning electron microscopy. Further, we successfully isolated cf-Tr-TB DNA from the EV-fraction (EV-DNA) and EV-free fraction (EV-free DNA). We developed a ‘cf-Tr-TB DNA’ assay to detect a highly repetitive 36-bp fragment specific to Mycobacterium tuberculosis complex and used EV-DNA and EV-free DNA for assay development. The developed assay could successfully identify cf-Tr-TB DNA in either the EV and/or EV-free fraction. The developed assay was applied in a blinded study on suspected TBM (n=44) and Non-TBM samples (n=10). The suspected TBM samples were categorized in ‘Definite TBM’ (n=8), ‘Probable TBM’ (n= 15), ‘Possible TBM’ (n=21) category according to the composite reference standard developed by Marais et al., 2010. ROC curves were generated using qPCR results from ‘Definite’ TBM and ‘Non-TBM’ samples in both EV-DNA, EVF-DNA and cut-off values were generated to give a 100% (95% CI: 69.1% to 100%) specificity. In ‘Definite’ TBM, cf-Tr-TB DNA assay had a sensitivity of 100% (95% CI: 63.1% to 100%) for EV-DNA and 87.5% (95% CI: 47.3% to 99.6%) for EVF-DNA. When results of EV-DNA + EVF-DNA (urine DNA) were combined, the cf-Tr-TB DNA assay in TBM group (‘Definite + Probable + Possible’ TBM) gave a sensitivity of 81.8% (95% CI: 67.2% to 91.8%). Together, these findings confirmed that one of the sources of ‘cf-Tr-TB DNA’ as extracellular vesicles and that these vesicles play some role in the pathophysiology of TBM. The assay estimates exceed the Target-product-profile defined by WHO, and show promise to be evaluated in larger studies to accurately assess its utility for TBM diagnosis.
Reference: Marais S, Thwaites G, Schoeman JF, et al. Tuberculous meningitis: a uniform case definition for use in clinical research. Lancet Infect Dis. 2010;10(11):803-812. doi:10.1016/S1473-3099(10)70138-9.