Molecular Medicine

Dr Paras SinghSpecialist
  

Research and Diagnostics Interests:

 Identification of molecular markers associated with following disease:

  • Tuberculosis;
  • Covid-19;
  • Lung cancer;
  • HIV; COPD
  • Rapid molecular detection of Mycobacterium tuberculosis in human clinical specimens by various NAATs and LAMP assay

List of Equipments:

  • PCR
  • COBAS Taq Man 48
  • Refrigerated centrifuge
  • Class II Type B2 Biohazard Safety Cabinet
  • Laminar Air Flow
  • FACS Count
  • Deep Freezers
  • Gel electrophoresis
  • NanoDrop
  • Gel-Doc

Research and Diagnostic Activities:

The department of Molecular Medicine is currently focusing on identification of genetic polymorphisms in the Indian population and its association with tuberculosis pathology; as well as developing cost and time effective molecular assays for diagnosis of TB.

Tuberculosis (TB) continues to cause increasing worldwide morbidity; being one of the top 10 causes of mortality in the developing world. According to the World Health Organization (WHO), TB is a worldwide pandemic. While India accounted for 26% of TB cases in the world in 2020; a genetic predisposition may be one of the major etiological factors contributing to high susceptibility and burden of disease. Hence, we are investigating the association of genetic polymorphisms and incidence of pulmonary or extrapulmonary M. tuberculosis infection and drug resistant TB infections in the Indian population. We have identified genetic variants in different human cytokine and chemokine genes, such as, CCR2, CCL2, IP-10, TLR4, IL-4, IL-6, IL-10 etc., which enhance inflammation in disease. The CCL2 (−2518A/G) and TLR4 (D299G or 896A/G & T399I or 1196C/T) gene polymorphisms were studied by PCR-SSP/ PCR-RFLP and genome sequencing method, while the CCR2 (V64I or 190G/A) gene polymorphism was done by ARMS-PCR method. In addition to genetic analysis, the protein expression of various biomarkers was also assessed by ELISA, in collaboration with National Institute of Pathology, Safdarjung Hospital Campus, ICMR, New Delhi.

Our previous study suggested that the Indian population is susceptible towards developing tuberculosis due to the presence of the variant alleles of genes CCR2 (190 G to A) and TLR4 (896A/G & 1196C/T). We are performing the same analysis on a higher number of gene pools. The conclusive data from this research work may help in screening of biomarkers to aid in better prognosis of tuberculosis in the Indian population.

Subsequently, another research study published in ‘Journal of Microbiological Methods’ (2019), demonstrated the efficiency of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid diagnosis of Mycobacterium Tuberculosis in Indian patients. The results conclude that LAMP assay can serve as a cost effective and promising technique for diagnosis of EPTB in resource-limited regions.

Our department is also working on “Identifying new potent mycobacterial proteins biomarkers and novel antigenic epitopes to be employed in immunodiagnostic and point of care tests for tuberculosis” in collaboration with the Department of Biochemistry, University of Delhi.

Our research work on molecular profiling of “EGFR (Exon 18-21) mutations and EML4-ALK fusions in non-small cell lung carcinoma (NSCLC)” is under review in an international journal. The identification of such mutations is significant for defining and monitoring the treatment outcomes of the NSCLC patients.

The department of Molecular Medicine also evaluated the immunophenotype and absolute values of CD3+, CD4+ and CD8+ cells in different categories of TB patients and healthy controls PBMCs, to get a better understanding of the pathology of disease. The Molecular Medicine department is also facilitating the multiplex TB PCR and Nested TB PCR for cost effective diagnosis of tuberculosis for poor patients in hospital. Recently, the department has started WHO approved LAMP Assay for identification and screening of pulmonary and extra-pulmonary Tb patients, to contribute to the diagnostics of hospitals. The endorsement of this TB LAMP assay will further enhance the treatment and management of tuberculosis patients in our hospital.

The major focus of the Departmental research activities is identification of biomarkers for PTB, EPTB, Lung cancer and Aspergillus-PCR for species identification. We are currently looking for differentiating biomarkers,such as, chemokines or their receptors and ligands, different gene variants (EGFR, XRCC4), otherSNPs (CCR2, CCR5, MCP-1, ICAM, TNF, IFN-γ, ESAT6, DC-SIGN, IP10, TLR4 and TLR9 etc. for diseases, such as, TB, HIV, lung cancer. Since its establishment, the Department has published 16research articles in peer-reviewed international journals, such as, Infection, Genetics & Evolution,Journal of Microbiological Methods, Meta Gene, etc.

Our recent review article entitled “A review of Novel Corona infection: Covid 19” published in Health Science Journal 2021, has prompted us to expand our research in the area of understanding the association of genetic profiling and prognosis of Covid infection in Indian scenarios.

We have designed the following future research proposal: (a) Genetic association of polymorphism inACE2 and TMPRSS2.receptors with variable severity of Covid 19 disease in Indian patients.(b) Evaluation of antibody response and it’s persistence in naturally infected Covid 19 patients and vaccinated individuals. (c) Correlation of distinct cytokine signatures in Covid-19infection and different co-morbidities. (d) Determination of distinct cytokine signatures in TBand TB with Covid-co infection. (e) Interleukin 6 polymorphisms as an indicator of COVID-19 severity in Indian patient prospective. (f) Association of TNF-α G-308 aPromoter Polymorphism with the Course and Outcome of COVID-19 Patients admitted in hospital.

The Department is also running short term training program for Master and undergraduates students on “Molecular diagnostics tools and Molecular Biology Techniques”. We are consistently working on developing cost and time effective molecular diagnostic methods for

identification of MTB in clinical specimens, with major focus on early diagnosis and treatment of tuberculosis in pediatric cases. It is also our aim to develop strategies for an efficient molecular diagnosis of MDR/XDR tuberculosis cases.

List of recent publications:

 

  1.  Kumar N, Singh P, Dewan R K, Chuzho N, Chauhan S and Khayyam K U. A review of Novel Corona infection: Covid 19. Health Sci Journal. 2021 May 10; 3:011.
  2.  Singh P, Rajput R, Mehra NK, Vajpayee M. Analysis of HLA association among North Indian HIV positive individuals with and without tuberculosis. Meta Gene. 2020; 24:100673.
  3.  Singh P, Rajput R, Mehra NK, Vajpayee M, Sarin R. Cytokine gene polymorphisms among North Indians: Implications for genetic predisposition? Infection, Genetics and Evolution. 2019 June 3; 73:450-459.
  4.  Rajput R, Singh P, Sarin R, Sethi P, Sharma S. Diagnostic accuracy of loop-mediated isothermal amplification assay for extra-pulmonary tuberculosis in Indian population. Journal of Microbiological Methods. 2019 Jan 28;158: 59-65.
  5.  Singh P, Rajput R, Sarin R, Tayal D. Significance of CCL2 (−2518A/G), CCR2 (190G/A) and TLR4 polymorphisms (896 A/G and 1196C/T) in tuberculosis risk in Indian population. Meta Gene. 2018 Sep 27; 18:184–190.
  6.  Verma AK, Sarin R, Arora VK, Kumar G, Arora J, Singh P, Myneedu VP. Amplification of Hsp 65 gene and usage of restriction endonuclease for identification of non tuberculous rapid grower mycobacterium. Indian J Tuberc. 2018 Sep 29; 65:57- 62.
  7.  Singh P, Singh M, Bhalla M, Singhla R, Gumma PK, Tayal D, Myneedu VP, Sarin R. Synergetic Effect of TLR4 Gene (D299G and T399I) Polymorphisms in Susceptibility to Pulmonary Tuberculosis. Univ J Med Sci. 2015;3(1):19-27.
  8.  Singh P, Singh M, Tayal D, Myneedu VP, Bhalla M, Adlakha P, Sarin R. The Clinical Utility of Polymerase Chain Reaction and Adenosine Deaminase (ADA), for the Diagnosis of Pleural Tuberculosis: Indian Scenario. Immunology and Infectious Diseases 2014;2(2):13-21.
  9.  Verma AK, Kumar G, Arora J, Singh P, Arora VK, Myneedu VP, Sarin R. Identification of mycobacterial species by PCR restriction enzyme analysis of the hsp65 gene – an Indian experience. Can J Microbiol. 2015;61(4):293-6. doi: 10.1139/cjm-2014-0525. Epub 2015 Jan 21.
  10. Singh M, Singh P, Myneedu VP, Bhalla M, Adlakha P, Verma P, Sarin R. “Evaluation of Polymerase Chain Reaction and CobasTaqMan Real Time PCR in the Diagnosis of Tuberculosis: Indian Prospective. Immunology and Infectious Diseases 2013;1(1): 1-9.
  11. Singh P, Singh M, Adlakha P, Verma P, Myneedu V P, Sarin R. Comparative Evaluation of PCRwith Commercial Multiplex M. tuberculosis Detection Kit. Immunology and Infectious Diseases2013; (2): 19-26.
  12. Mehra NK, Singh P, Singh M, Najmi N, Kaur G, Sharma SK, Katoch K, Katoch VM – Shared and Distinct Variants of MHC and non MHC genes in pulmonary TB and leprosy NIH publisher 2009.In the 44th US-Japan cooperative Medical Science Program, Forty fourth Tuberculosis andLeprosy Research Conference, Fukoka, Japan, p145-149, July 2009.
  13. Singh P, Kaur G, Sharma G, Mehra NK. Immunogenetic basis of HIV-1 infection, transmission and disease progression. Vaccine. 2008; 26(24): 2966-80. Epub 2008 Feb 4.
  14. Kaur G, Singh P, Rapthap CC, Kumar N, Vajpayee M, Sharma SK, Wanchu A, Mehra NK. Polymorphism in the CCR5 gene Promoter and HIV-1 infection in North Indians. Human Immunol. 2007; 68(5): 454-461.
  15. Kaur G, Singh P, Rapthap CC, Kumar N, Vajpayee M, Sharma SK, Wanchu A, Mehra NK. Distribution of CCR2 polymorphism in HIV-1 infected and healthy subjects in North India. Internat J Immunogenet. 2007; 34(3):153-156.
  16. P Singh, G.Sharma, G.Kaur, M.Vajpayee, S Sharma, N.Mehra. Host immunogenetics and HIV-1 infection (O-16) Tissue Antigens 2008; 72, 227–313.
  17. Panigrahi A, Shiddqui J, Bhowmik D, Singh P, Tiwari S, Guleria S. Neutrophil gelatinase associated lipocalin (NGAL) as a biomarker to predict acute tubular necrosis (ATN) in renal transplant allografts (P-O16) Tissue Antigens 2008; 72, 227–313.