Context: Leptospirosis is a zoonotic infection with worldwide significance. It is caused by a spirochete Leptospira interrogans, which has many serogroups and a large number of serovars. Leptospirosis is an emerging public health problem in India. Aims: Leptospirosis is an emerging public health problem and keeping this in mind 216 clinically suspected cases of leptospirosis have been considered for serological study. Failure to recognize these cases leads to serious morbidity and mortality. Hence, the present study was undertaken to identify these cases by simple laboratory techniques so that timely management could be undertaken. Settings and Design: Hospital-based cross-sectional case control study. Subjects and Methods: Patients with unexplained fever for more than seven days with or without jaundice or renal failure were taken up for this study. IgM ELISA tests were performed with the serum samples of the patients. Statistical Analysis Used: Simple statistical methods applying Epi info. Results: Total 77 patients were found to be reactive for IgM ELISA. Total 100% of seropositive patients had fever and 40% had jaundice. Peak incidence of the disease was found in the period from July to November (monsoon and post-monsoon period). Male preponderance was seen and mainly urban population was found to be seropositive in our study. Conclusions: Seroprevalence of leptospirosis was found to be remarkable in our study; 35% of the clinically suspected patients showed positive IgM ELISA tests.
Keywords: Fever, IgM ELISA, leptospirosis
Leptospirosis as found in our study is now a emerging public health problem and our study shows that serologically reactive leptospirosis cases are on a increase in this part of the world.
Leptospirosis is a zoonosis of global distribution.  It is primarily a contagious disease of animals and occasionally infects humans. Genus Leptospira is classified into two species – L. interrogans and L. biflexa comprising pathogenic and non-pathogenic strains, respectively. Within each species, large number of serovars have been identified using polyclonal agglutinating antibodies.  Human infection occurs by direct contact with infected urine of carrier animals or indirectly by contact with damp soil. Peak incidence is in rainy season in tropical regions. Outbreaks may follow periods of excessive rainfall.  Organisms enter the body through cuts and abrasions, mucous membrane and conjunctiva. Systemic vasculitis facilitates migration of spirochetes into organs and tissues. Severe vascular injury leads to pulmonary hemorrhage, renal tubular cell necrosis and destruction of hepatic architecture. Virulence factors include attachment and toxin production.  Subclinical infection is followed by seroconversion to two recognizable syndromes – one is a self-limiting systemic illness in 90% of infections and the other is a combination of severe potentially fatal illness accompanied by combination of renal failure, liver failure, pneumonitis with hemorrhagic diathesis. The most distinctive form of disease that may occur is Weils disease (impaired hepatic and renal function). Mortality rate in patients developing severe disease is 5%-40%.  Leptospira can be directly visualized in blood and urine but the method has low sensitivity (40.2%) and specificity (61.5%).  Blood, urine and CSF culture can be performed. IgM antibodies are detectable after about the fifth day of illness. 
The study was conducted in the Microbiology Dept. of School of Tropical Medicine, Kolkata for three years (2008-2011). Blood samples from patients with the following clinical parameters were sent to the laboratory for ELISA test to detect IgM antibodies against Leptospira.
The criteria for selection of patients to be included in the study were as follows:
Serological test was performed using IVD Leptospira IgM Microwell ELISA test kit (USA). Microwells were coated with purified Leptospira Patoc 1 antigen; 140 μl of patient’s serum (1:40 dilution) was transferred to test well and incubated at room temperature for 10 minutes. After washing two times with wash buffer, 2 drops of enzyme conjugate were added to each well and then incubated at room temperature for 10 minutes again. Then, after washing three times with wash buffer, two drops of chromogen were added and incubated again for 5 minutes. Two drops of stop solution were then added and final reading was taken in an ELISA reader. Positive and negative controls were kept in each test. Samples with O.D. -0 to 0.3 O.D. was considered negative. Samples having O.D. of >1 were considered as strongly reactive. Patients with weak reaction (0.5-<1) were tested 2-3 weeks later to detect seroconversion.
Thirty asymptomatic subjects were taken as control in this study.
Out of 216 blood samples sent from patients with the above-mentioned clinical parameters, 77 samples were found to be reactive for IgM Leptospira.
All of the 77 cases had history of fever; 31 cases had jaundice. Hepatomegaly was there in 7 cases and 4 patients presented with renal failure. Generalized edema was found in 8 cases and skin rashes in 9 cases [Table 1].
Out of 216 cases, 19 cases were in the age group of 0-5 years, 13 in the age group of 5-15 years and 45 cases were adults.
Male:female ratio was 7:4. All the 30 asymptomatic subjects taken as control in our study showed negative IgM ELISA. Forty-five patients (58.44%) were more than 15 years of age and 32 (41.4%) were of less than 15 years of age [Table 2]. Population engaged in outdoor activities like laborers and farmers showed 76.61% serological reactivity to leptospira IgM ELISA in our study [Table 3].
IgM ELISA, which uses Leptospira Patoc 1 strain, is a standard serological test for early diagnosis of leptospirosis. Antibodies do not reach detectable levels until the 2 nd week of illness.  In our study, IgM ELISA shows a positivity of 35%. An outbreak of Leptospirosis in Mumbai in 2002 showed a positivity of IgM ELISA of 36.27%.  In the studies of Chandrasekhar et al.  and Babu et al.  IgM ELISA positivity were 41.77% and 88.9%, respectively.
Dark ground microscopy from plasma is a simple economical method for early diagnosis.  Sensitivity of the test varies from 27% to 40%. MAT is a widely used reference test for leptospirosis but it has its limitations. It is inadequate for rapid case identification as it requires analysis of paired sera and can be performed in few reference laboratories.  Moreover, the prevalent serovars in a particular geographic area must be known as it is cumbersome to test for all 200 serovars of L. interrogans. Several rapid assays have come up that are used for screening acutely ill patients. The sensitivity of these tests varies from 88% to 100% and specificity from 95% to 99%.  The seroprevalence in our study has been found to be 35.64%. In a study conducted in Chennai in 1988 during the peak monsoon season, out of 40 patients 33 (82.5%) had specific leptospiral antibodies.  In 1987, a seroprevalence of 25% was reported in patients hospitalized in Karachi, Pakistan.  Studies from different parts of India have revealed a seroprevalence ranging from 17.8% to 40.5% (Ratnam et al.).  Fever was the commonest presenting symptom in our study. All 77 (100%) seropositive patients had fever and of whom 31 (40.2%) cases had jaundice. A study conducted by Muthusethupati et al. in 1995,  fever and jaundice were the most common presentation. Male preponderance (63.6%) has been found in our study and 58.4% of the cases has been found in adults >15 years of age. The higher prevalence in males can be attributed to more frequent outdoor activities;  62 (80.5%) reactive cases were noted during the months of July-November (monsoon and post-monsoon season) in our study. Children had history of fever with generalized edema in few cases and respiratory symptoms in few cases. As regard to therapy, penicillin and doxycycline are still useful agents in treatment.  Further epidemiological studies should be carried out for proper evaluation of the scenario of endemicity of leptospirosis in this part of the country.
The authors are grateful to Dr. Krishnangshu Ray, Director, School of Tropical Medicine, Kolkata, for providing support and encouragement for conducting this study.
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]