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Year : 2004  |  Volume : 22  |  Issue : 2  |  Page : 134--135

Seroprevalence of leptospirosis in Delhi using indirect haemagglutination assay

N Gupta1, RS Rao2, P Bhalla3, SK Agarwal4,  
1 Department of Microbiology, Pt. B.D. Sharma Post Graduate Institute of Medical Sciences, Rohtak - 124 001, Haryana, India
2 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry - 605 006, India
3 Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002, India
4 Department of Medicine, Maulana Azad Medical College, New Delhi - 110 002, India

Correspondence Address:
N Gupta
Department of Microbiology, Pt. B.D. Sharma Post Graduate Institute of Medical Sciences, Rohtak - 124 001, Haryana

How to cite this article:
Gupta N, Rao R S, Bhalla P, Agarwal S K. Seroprevalence of leptospirosis in Delhi using indirect haemagglutination assay.Indian J Med Microbiol 2004;22:134-135

How to cite this URL:
Gupta N, Rao R S, Bhalla P, Agarwal S K. Seroprevalence of leptospirosis in Delhi using indirect haemagglutination assay. Indian J Med Microbiol [serial online] 2004 [cited 2021 Jan 23 ];22:134-135
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Dear Editor,

Serology remains the mainstay for the diagnosis of leptospirosis. Few laboratories have resources and expertise to perform microscopic agglutination test. Therefore, rapid and simple tests are required for early diagnosis and clinical management. A total of 140 serum samples were collected from patients attending Lok Nayak Hospital, Delhi. Of 140 samples 30 were jaundice cases in whom viral hepatitis A, B and C were ruled out by anti-HAV IgM ELISA, HBs Ag, anti-HBc IgM ELISA, and anti-HCV IgM ELISA. Thirty cases were pyrexia of unknown origin (>7d) patients in whom enteric fever had been excluded by widal test and blood culture and 30 were acute renal failure patients in whom streptococcal etiology had been ruled out by ASLO test. Another 50 serum samples were obtained from healthy blood donors which served as controls. All sera were stored at -20°C till tested by indirect haemagglutination test (IHA).

The IHA test was standardized in-house. Leptospira biflexa Patoc 1 strain was obtained from Regional Medical Research Centre, Port-Blair and cultures which were pure and attained density of 108-109 leptospires/ mL in Korthof's medium were used for preparing erythrocyte sensitizing substance (ESS). Subsequently, sensitization of formalinized sheep RBC was done using ESS.[1] The optimal dilution of ESS determined with positive control serum was found to be 1:8 by checker board titration.

Positive and negative control serum were included in each batch of IHA test and results were interpreted according to criteria described earlier.[1] Sera that were positive by the initial screening test were titrated by testing serial dilutions from 1:25 to 1:6400. Sera that had equivocal results in IHA test were subjected to absorption with 3% suspension of sheep RBC for 45 minutes and retested. Sera with equivocal results in IHA screening test were also tested for the presence of heterophile antibodies associated with infectious mononucleosis using latex agglutination test kit (Accutex™ infectious mononucleosis latex test).

Forty-eight (96%) out of 50 controls had titre of 25 was considered significant in IHA to diagnose leptospirosis. Screening test was done with 1:25 dilution of sera; 15 (16.6%) sera had positive result, 23 (25.5%) were equivocal and 52 (57.7%) were negative [Table].

Despite absorption with higher percentage of RBC and longer duration, there was no change in results of equivocal sera. It was further seen that out of 23 equivocal sera, 11 (47.82%) were positive for heterophile antibodies (for infectious mononucleosis) and 4 were rheumatoid factor positive (2 sera were positive for both RF and heterophile antibodies). Overall titres in positive cases ranged from 100 to 400 (except one case which had titre of 6400) with geometric mean titre 190. The occurrence of non specific agglutination in 12 sera could not be explained. Similar non specific haemagglutination has been reported previously in 3 of 27 dog sera.[2] No human cases have been reported so far, to the best of our knowledge.

IHA detects both IgM and IgG antibodies and sensitivity of IHA has been reported to be 100% and specificity 94% compared to MAT.[2] In comparison with IgM ELISA, IHA has been reported to detect 81% cases of disease.[3] Leptospirosis in 18% jaundice cases and 24% in PUO cases was reported in Madras.[4] Seroprevalence rate of 12% leptospirosis was reported from Pondicherry.[5]

Because of diverse clinical presentation, laboratory assistance is required for timely diagnosis and treatment of leptospirosis to ensure desirable clinical outcome. Conversely, a poorly performing test can have negative impact on diagnostic follow up and clinical management. Therefore, better screening test for diagnosis of leptospirosis are urgently needed in Delhi and physicians should verify a positive screening test with a confirmatory test.


1Faine S. In: Guidelines for the control of leptospirosis. Geneva WHO, 1982:67.
2Sulzer CR, Jones WL. Leptospirosis: methods in laboratory diagnosis. Centre for Disease Control, Atlanta Georgia, HEW Publication No. (CDC) 1978;79-8275.
3Levett PN, Whittington CU. Evaluation of indirect Haemagglutination Assay for diagnosis of acute leptospirosis. J Clin Microbiol 1998;36:11-14.
4Ratnam S, Sundararaj T, Thyagarajan SP, Rao RS, Madanagopalan N, Subramanian S. Serological evidence of leptospirosis in jaundice and pyrexia of unknown origin. Indian J Med Res 1983;77(4):427-430.
5Prabhakar PK, Harish BN, Rao RS. Seroprevalence of leptospirosis among febrile and jaundice patients. Indian J Med Microbiol 1995;13(4):189-91.