Research Article For reprint orders, please contact: reprints@futuremedicine.com Quantitative detection of a cocktail of mycobacterial MPT64 and PstS1 in tuberculosis patients by real-time immuno-PCR Suman Sharma 1 , Abhishek Sheoran 2 , Krishna B Gupta 3 , Aparna Yadav 4 , Mandira Varma-Basil 5 , Vishnubhatla Sreenivas 6 , Dhruva Chaudhary 7 & Promod K Mehta* ,1 1 Centre for Biotechnology, Maharshi Dayanand University, Rohtak-124001, India 2 Department of Statistics, Ramanujan College, University of Delhi, New Delhi-110019, India 3 Department of TB & Respiratory Medicine, University of Health Sciences, Rohtak-124001, India 4 Department of Microbiology, University of Health Sciences, Rohtak-124001, India 5 Microbiology Department, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi-110007, India 6 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi-110029, India 7 Department of Pulmonary & Critical Care Medicine, University of Health Sciences, Rohtak-124001, India *Author for correspondence: Tel.: +91 989 650 4193; Fax: +91 126 227 4640; pkmehta3@hotmail.com Aim: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). Methods: Real- time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacte- rial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. Results: A broad dynamic range of 0.95 pg/ml– 95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% speci- fcity, whereas a sensitivity of 77.9% and a specifcity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, signifcantly reduced MPT64+PstS1 concentrations (p < 0.001) were noticed in pa- tients on therapy by RT-I-PCR as compared with untreated patients. Conclusion: Our RT-I-PCR assay re- vealed high sensitivity especially for the rapid diagnosis of smear-negative pulmonary TB and paucibacil- lary extrapulmonary TB samples, which could also monitor the dynamics of disease in patients on therapy. First draft submitted: 19 October 2018; Accepted for publication: 18 December 2018; Published online: 21 January 2019 Keywords: diagnosis • EPTB • GeneXpert assay • MPT64+PstS1 • Mycobacterium tuberculosis • PTB • real-time immuno-PCR • sensitivity • specifcity • TB TB remains a global health problem. In 2017, approximately 10 million people developed TB throughout the world [1]. There were approximately 1.3 million deaths among HIV-negative individuals and 0.3 million deaths among HIV-positive co-infected TB individuals. Strikingly, India accounted for the highest TB burden (27%) worldwide followed by China and Indonesia [1]. Rapid and accurate diagnosis of TB is very essential to control the disease. Smear microscopy, culture, histopathology, IFN-γ release assays and nucleic acid amplification tests (NAATs) are the main modalities used for the diagnosis of TB, which have their own merits and demerits [2, 3]. Although culture is considered as the gold standard, the method is labor intensive with a turnaround time of approximately 4–6 weeks and has a limited utility in diagnosing paucibacillary extrapulmonary TB (EPTB) specimens [2, 3]. Among nucleic acid amplification tests, PCR tests targeting IS6110, mpt64, pstS1, hsp65 (Rv0440), etc. are widely employed but often lead to false-positive and false-negative results [2, 4]. GeneXpert MTB/RIF assay, a seminested real-time PCR targeting rpoB (Rv0664) has been a major breakthrough for the simultaneous detection of Mycobacterium tuberculosis and rifampin resistance [5, 6], which reveals promising results for pulmonary TB (PTB); however, its usefulness to diagnose paucibacillary EPTB specimens is uncertain [6]. Moreover, its wide application in developing countries is restricted primarily due to high cost. Immuno-PCR (I-PCR) method, which combines the versatility of ELISA with the exponential amplification capacity and sensitivity of PCR has been devised for the ultralow detection of several biomarkers, that is, cancer markers, toxins, cytokines and various microbial antigens such as Future Microbiol. (Epub ahead of print) ISSN 1746-0913 10.2217/fmb-2018-0284 C  2019 Future Medicine Ltd