International Journal of Pharma and Bio Sciences
    ISSN 0975-6299

Int J Pharm Bio Sci Volume 12 Issue 3, 2021 (July-September), Pages:86-93

Molecular and Immunological Surveillance of Leptospira Incidence at Gwalior Region of Central India

Bharti Mankere, Preeti Gupta, Kundan Tandel, Urmil Tuteja , Mahendra K Gupta and Kulanthaivel Thavachelvum

Leptospirosis is an infection caused by the spirochete bacteria Leptospira.  It is ubiquitous around the world and in India it has been reported from several parts of the country during the rainfall season. In our present work, a detailed cross sectional study with the serum samples from pyrexia of unknown origin (PUO) patients around the poorly studied Gwalior district of the central India region was undertaken to assess the Leptospirosis incidence. Immunological methods such as MAT (Microscopic agglutination test), ELISA (Enzyme linked immune sorbent assay), and WB (Western blotting), along with molecular methods such as PCR and 2D protein analysis were employed and it was found that about 2.82% (n=854) of samples tested were positive for Leptospirosis. The antibody response was obtained against Leptospira interrogans serovars Bataviae, Ballum, Cyanoptera, Grippotyphosa and Ictero-haemorrhagiae copenhageni. The 2-D Gel electrophoresis of the serovar Copenhageni when probed with positive sera in WB identified a major antigen that provoked the immune response in patients. The reaction corresponding to the 32-kDa size protein and an isoelectric point pI value of 6.5 indicated that the immune response was against the outer membrane protein lipL32 of Leptospira spp. Further confirmation with the MALDI TOF analysis of the excised sample from the parallel gel confirmed the tryptic digested products of lipL32. Isolation of leptospires could not be successful; however, the detection of leptospiral DNA by PCR using lipL32 primers in the MAT positive samples confirmed the etiology of the disease. The present study suggests that Leptospirosis could be considered as one of the prime suspects, in the PUO cases, around Gwalior region of Central India.

Keywords: Leptospirosis, PUO cases, MAT, PCR, MALDI –TOF, LipL32.
Full HTML:


  1. Ido Y, Hoki R, Ito H, Wani H. The rat as a carrier of spirochaetaicterrohaemorrhagiae the causative agent of spirochaetosisicterrohaemorrhagica. J Exp Med. 1917 Feb;26(3):341-53. doi: 10.1084/jem.26.3.341, PMID 19868153.
  2. Borkakoty B, Jakharia A, Biswas D, Mahanta J. Co- infection of scrub typhus and leptospirosis in patients with pyrexia of unknown origin in longding district of Arunachal Pradesh in 2013. Indian J Med Microbiol. 2016 Jan;34(1):88-91. doi: 10.4103/0255-0857.174116, PMID 26776126.
  3. Padma V, Javed S M. Leptospirosis: A cross sectional study. World App SciJou. 2014 Jan; 29(7):879-883. Doi: 10.5829/idosi.2014.29.07.8274.
  4. Shah LJ, Mulla S, Patel MG, Vaghela G. Outbreak surveillance report on pulmonary leptospirosis after a heavy flood during 2006 in south Gujarat. Natl J Med Res. 2012 Mar;2(1):42-6.ISSN 2249 4995.
  5. Narayanan R, Sumathi G, Prabhakaran SG, Shanmughapriya S, Natarajaseenivasan K. Paediatric leptospirosis: A population based case-control study from Chennai, India. Indian J Med Microbiol. 2016 Jan 09;34(2):228-32. doi: 10.4103/0255-0857.180353, PMID 27080780.
  6. Adler B. History of leptospirosis and leptospira. Curr Top MicrobiolImmunol. 2015 Jan;387:1-9. doi: 10.1007/978-3-662-45059-8_1, PMID 25388129.
  7. Chandy S, Kirubanandhan L, Hemavathy P, Khadeeja AM, Kurian SJ, Venkataraman K, Mørch K, Mathai D, Manoharan A. Serovar prevalence of Leptospira in semirural India and the development of an IgM- based indirect ELISA. J Infect DevCtries. 2017 Mar 31;11(3):234-41. doi: 10.3855/jidc.8067, PMID 28368857.
  8. Sethi S, Sood A, Pooja SS, Sengupta C, Sharma M. Leptospirosis in north India: a clinical and serological study. Southeast Asian J Trop Pub Health. 2003 Dec;34(4):822-5. PMID:15115094.
  9. Jena AB, Mohanty KC, Devadasan N. An outbreak of leptospirosis in Orissa, India: the Importance of surveillance. Trop Med Int Health. 2004 Sep;9(9):1016-21.doi:10.1111/j.13653156.2004.01293.x,PMID 15361116.
  10. Karande S, Kulkarni H, Kulkarni M, De A, Varaiya A. Leptospirosis in children in Mumbai Slums. Indian J Pediatr. 2002 Oct;69(10):855-8. doi: 10.1007/BF02723705, PMID 12450293.
  11. Sethi S, Sharma N, Kakkar N, Taneja J, Chatterjee SS, Banga SS, Sharma M. Increasing trends of leptospirosis in northern India: a clinico-epidemiological study. PLOS Negl Trop Dis. 2010 Jan 12;4(1):e579. doi: 10.1371/journal.pntd.0000579, PMID 20084097.
  12. Ko AI, Galvão Reis M, RibeiroDourado CM, Johnson WD Jr, Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador leptospirosis study group. Lancet. 1999 Sep 4;354(9181):820-5. doi: 10.1016/s0140-6736(99)80012-9, PMID 10485724.
  13. Johnson MAS, Smith H, Joeph P, Gilman RH, Bautista CT, Campos KJ, Cespedes M, Klatsky P, Vidal C, Terry H, Calderon MM, Coral C, Cabrera L, Parmar PS, Vinetz JM. Environmental exposure and Leptospirosis, Peru. Emerg Infect Dis. 2004 Jun;10(6):1016-22. doi: 10.3201/eid1006.030660, PMID 15207052.
  14. Zhang Y, Li S, Dai B. Amplified 23S rRNA gene of 52 strains of Leptospira and detection of leptospiral DNA in 55 patients by PCR. Hua Xi Yi Ke Da XueXueBao. 1993 Sep;24(3):262-7. PMID 8288193.
  15. Faine S. Guidelines for the control of leptospirosis/edited by S. Faine. Vol. 67. World Health Organization; 1982. p. 1-171.
  16. Wolf JW. The Laboratory diagnosis of leptospirosis. Charles C. Thomas, Blackwell Scientific Publishers; 1954; Record No. : 19552700312, USA; UK.
  17. Carbrey EA. The relative importance of variable factors in the agglutination-lysis test. 64th Annual. Proceedings of the United States Livestock Sanitary Association; 1960.
  18. Cole JR, Sulzer CR, Pursell AR. Improved microtechnique for the leptospiral microscopic agglutination test. ApplMicrobiol. 1973 Jun;25(6):976-80. doi: 10.1128/AM.25.6.976-980.1973, PMID 4736794.
  19. Brown JA, LeFebvre RB, Pan MJ. Protein and antigen profiles of prevalent serovars of Leptospirainterrogans. Infect Immun. 1991 May;59(5):1772-7. doi: 10.1128/IAI.59.5.1772-1777.1991, PMID 2019441.
  20. Perwez K, Suman VB, Kumar NA, Hussain MA. Leptospirosis in febrile patients: diagnosis by serology and polymerase chain reaction. J Biol Life Sci2011;2(1):E2.Doi:10.5296/jbls.v2il.535. 
  21. Görg A, Obermaier C, Boguth G, Weiss W. Recent developments in two – dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis. 1999 May;20(4-5):712-7. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<712::AID-ELPS712>3.0.CO;2-Y, PMID 10344237.
  22. Haake DA, Matsunaga J. Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins. Infect Immun. 2002 Sep;70(9):4936-45. doi: 10.1128/iai.70.9.4936-4945.2002, PMID 12183539.
  23. Calderaro A, Piccolo G, Gorrini C, Montecchini S, Buttrini M, Rossi S, Piergianni M, De Conto F, Arcangeletti MC, Chezzi C, Medici MC. Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation. BMC Res Notes. 2014;7:330. doi: 10.1186/1756-0500-7-330, PMID 24890024.
  24. Murray CK, Gray MR, Mende K, Parker TM, Samir A, Rahman BA, Habashy EE, Hospenthal DR, Pimentel G. Use of patient-specific Leptospira isolates in the diagnosis of leptospirosis employing microscopic agglutination testing (MAT). Trans R Soc Trop Med Hyg. 2011 Apr;105(4):209-13. doi: 10.1016/j.trstmh.2010.12.004, PMID 21334705.
  25. Bourhy P, collet L, Lernout T, Zinini F, Hartskeerl RA, van der Linden HVD, Thiberge JM, Diancourt L, Brisse S, Giry C, Pettinelli F, Picardeau M. Human Leptospira isolates circulating in Mayotte (Indian Ocean) have unique serological and molecular features. J ClinMicrobiol. 2012 Feb;50(2):307-11. doi: 10.1128/JCM.05931-11, PMID 22162544.
  26. Guerreiro H, Croda JL, Flannery B, Mazel M, Matsunaga J, Galvão Reis M, Levett PN, Ko AI, Haake DA. Leptospiral proteins recognized during the humoral immune response to Leptospirosis in humans. Infect Immun. 2001 Aug;69(8):4958-68. doi: 10.1128/IAI.69.8.4958-4968.2001, PMID 11447174.
  27. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Memish ZA. Aboyans Vet al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systemic analysis for the global burden of disease study 2010. Lancet. 2012 Dec 15;380(9859):2095-128. doi: 10.1016/S0140-6736(12)61728-0, PMID 23245604.
  28. Niloofa R, Fernando N, De silva NL, Karunanayake L, Wickramasinghe H, Dikmadugoda N, Premawansa G, Wickramasinghe R, de Silva HJ, Premawansa S, Rajapakse S, Handunnetti S. Diagnosis of Leptospirosis: comparison between microscopic agglutination test, IgM-ELISA and IgMrpidimmunochromatography Test. PLOS ONE. 2015 Jun 18;10(6):e0129236. doi: 10.1371/journal.pone.0129236, PMID 26086800.
  29. Cumberland P, Everard CO, Levett PN. Assessment of the efficacy of an IgM-ELISA and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis. Am J Trop Med Hyg. 1999 Nov;61(5):731-4. doi: 10.4269/ajtmh.1999.61.731, PMID 10586903.
  30. Vijayachari P, Sugunan AP, Shriram AN. Leptospirosis: an emerging global public health problem. J Biosci. 2008 Nov;33(4):557-69. doi: 10.1007/s12038-008-0074-z, PMID 19208981.
  31. Calderaro A, Piccolo G, Gorrini C, Montecchini S, Buttrini M, Rossi S, Piergianni M, De Conto F, Arcangeletti MC, Chezzi C, Medici MC. Leptospira species and serovars identified by MALDI-TOF mass spectrometry after databaseimplementation. BMC Res Notes. 2014;7:330. doi: 10.1186/1756-0500-7-330, PMID 24890024.
  32. Hsu S, Hung C, Chang M, ChingKo Y, Yang H, Hsu HH et al. Active components of Leptospira outer membrane protein LipL32 to toll like receptor 2 [sci rep:2017] Aug 21; 7. Vol. 8363.
  33. Goris MGA, Hartskeerl RA. Leptospirosis serodiagnosis by the Microscopic agglutination test. Curr Proto Microbiol. 2014 Feb 6;32(1): 1E:7.1-12E.5.18. doi: 10.1002/9780471729259.mc12e05s32, PMID 24510846.
  34. Evangelista KV, Coburn J. Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses. Future Microbiol. Sep 2010;5(9):1413-25. doi: 10.2217/fmb.10.102, PMID 20860485.
  35. Guernier V, Lagadec E, Cordonin C, Le Minter GL, Gomard Y, Pagès F, Jaffar-Bandjee MC, Michault A, Tortosa P, Dellagi K. Human leptospirosis on Reunion Island, Indian Ocean: are rodents the (only) ones to blame? PLOS Negl Trop Dis. 2016 Jun 13;10(6):e0004733. doi: 10.1371/journal.pntd.0004733, PMID 27294677.
  36. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM, Peru-United States Leptospirosis Consortium. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis. 2003 Dec;3(12):757-71. doi: 10.1016/s1473-3099(03)00830-2, PMID 14652202.
  37. Kuriakose M, Eapen CK, Paul R. Leptospirosis in kolenchery, Kerela, India: epidemiology, prevalent local serogroups and a new serovar. Eur J Epidemiol. 1997 Sep;13(6):691-7. doi: 10.1023/a:1007300729615, PMID 9324217.
  38. Patil DY, Dahake RV, Chowdhary  A S, Deshmukh RA. Clinico-epide miological observations of human leptospirosis from Mumbai, India. J Infect Public Health.2017Apr;10(2):2478.doi:10.1016/j.jiph.2016.05.009, PMID 27312818.
  39. Swapna RN, Tuteja U, Nair L, Sudarsana J. Seroprevalence of leptospirosis of high-risk groups in Calicut, North Kerala, India. Indian J Med Microbiol. 2006 Oct;24(4):349-52. doi: 10.4103/0255-0857.29418, PMID 17185876.
  40. Mansoor T, Fomda BA, Ahmed T, Altaf I. Prevalence of leptospirosis among cases of undifferentiated febrile illness reported to a tertiary care teaching hospital of Kashmir Valley. Journal of Med Sci Med Research. 2020May05;8(5):385390.Doi:
  41. Pandey D, Saroshe S, Dixit S, Jain C. A case of leptospirosis reported from metropolitan city of central India. Indian J Com Family Med. 2015 Jan 28;1(1):55-7. doi: 10.4103/2395-2113.251608.
[Download PDF]
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy
Pharmaceutical Fields
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy Pharmaceutics
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy Novel drug delivery system
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy Nanotechnology
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy Pharmacology
Welcome to IJPBS,Pharmaceutics, Novel, drug, delivery, system, Nanotechnology, Pharmacology, Pharmacognosy Pharmacognosy
© Copyright 2009-2015 IJPBS, India. All rights reserved. Specialized online journals by ubijournal. Website by Ubitech Solutions
         Home I Contact I Terms & Conditions