Description

Circulating cell-free M. tuberculosis DNA: a novel tool for the diagnosis of abdominal tuberculosis.
Author(s): Pratibha Sharma1, Divya Anthwal1, Pooja Kumari2, Rakesh Kumar Gupta1, Surabhi Lavania2, Neera Sharma3, Lokesh Kumar Sharma3, R L Taneja4, Jaya Sivaswami Tyagi2,5 and Sagarika Haldar1,5
Institutes-
1Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India,
3Department of Biochemistry, Dr. Ram Manohar Lohia Hospital, New Delhi, India, 4Department of Medicine, Dr. Ram Manohar Lohia Hospital, New Delhi, India,
5Center for Bio-design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India,

Background: Abdominal TB (ATB) contributes to 3% of all EPTB cases in India.  ATB is a major diagnostic challenge due to its non-specific clinical features and limited yield of standard diagnostic tests. In spite of the development of several molecular assays till date; no single test is suitable for ATB diagnosis. Here, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis DNA (cMTB-DNA) in ascitic fluid (AF) and its utility in ATB diagnosis.
Methods: A composite reference standard (CRS) comprising of biochemical, cytological, microbiological, radiological parameters and response to therapy was developed to categorize the prospectively enrolled patients (n=67) into ‘Definite ATB’ (culture positive n=2), ‘Probable ATB’ (n=15), ‘Possible ATB’ (n=13) and ‘Non-TB’ category (n=37). All samples were subjected to liquid culture, cytology, biochemical and molecular assays which included the devR-based cfMTB-DNA qPCR assay and Xpert MTB/RIF assay (Xpert). The diagnostic accuracy of the molecular assays was assessed using CRS as reference standard.
 Results: Fever, weight loss, alcoholism and positive mantoux findings were found to be associated with ATB disease (p<0.05). ROC curves were generated from cMTB-DNA qPCR data of ‘Definite+Probable’ ATB and ‘Non-TB’ group and cut-off was set to achieve a specificity of ≥ 95%. The cfMTB-DNA qPCR assay had a sensitivity of ~ 66.7% (95% CI: 40.9,86.7) in ‘Definite ATB’ and ‘Probable ATB’ category collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined ‘Definite’, ‘Probable’ and ‘Possible’ ATB group with similar specificity. Xpert had a poor sensitivity of ≤ 16.7% with 100% specificity (95% CI: 89.7,100).
Conclusions: We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.

Author(s)

• Pratibha Sharma, MSc , Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India