More than 50 per cent of the suspected as well as serologically diagnosed cases were registered during these four months ((32 %) and (20%). E were also seen. Since leptospirosis is a treatable disease, correct and rapid diagnosis may help in effective management of patients. spp. A total of 192 IgM ELISA positive blood and urine samples were subjected to culture for leptospires. Culture was performed by injecting one drop of blood and urine in separate tubes containing the Ellinghausen, McCullough, Johnson, and Harris (EMJH) medium supplemented with 3 per cent rabbit serum enriched with L-asparagin (3%), calcium chloride (1%), magnesium chloride (1%), pyruvate sodium (1%) and 0.1% agarose/agar (w/v). This Exicorilant culture medium (in house) was prepared in two formulations, one without antibiotics (non-selective) and the other with addition of 5-fluoruracil (300 mg/l) and nalidixic acid (20 mg/l), named as the selective medium. The medium, after inoculation, was incubated aerobically at 28C for a maximum period of 6 wk. A drop from each culture medium was examined by dark field microscopy from second week onwards to a maximum period of up to six weeks. Of the IgM ELISA positive samples (n=391), MAT was performed in 138 serum samples, whereas PCR was performed Exicorilant in 115 blood and 38 urine samples. MAT was performed using the following eight serovars – sensu lato (Australis, Autumnalis, Pomona, Sejroe, Tarassovi, Icterohaemorrhagica, Hebdomadis) and serovar Patoc, and an agglutination titre of 100 was considered reactive, where the end point was defined as the highest dilution of serum which agglutinated 50 per cent of the leptospires. DNA was extracted from blood and urine samples by Qiagen DNA extraction Kit (Gmbh, Hilden, Germany). PCR was performed on blood and urine samples by the procedure described by Merien F 16S gene, respectively. Amplification of DNA was performed in a total volume of 25 l. The reaction mixture consisted of 1.5 mM MgCl2, 20 pmole each oligonucleotide primer, 200 M each dATP, dTTP, dCTP, and dGTP, one unit of Taq DNA polymerase and 50-100 ng of template. The 331 base pair PCR product from a standard culture strain ((Advantage MAL CARD, J. Mitra & Co. Pvt. Ltd, New Delhi, India) and the Quantitative Buffy Coat (QBC) assay (QBC Diagnostic Inc., Philipsburg, USA). Dengue virus, hepatitis A virus (HAV) and hepatitis E virus (HEV) infections were diagnosed serologically using commercial ELISA kits (Dengue Duo cassette, PanBio, France; Anti-HAV total, Diasorin; HEV IgM Beijing Wantai Biological Enterprise Ltd., China). Clinical data of the patients were collected and analyzed using a structured questionnaire, which included the detailed clinical history and laboratory data from the hospital records. Results This study was conducted from April 2000 to March 2010 Gpr124 (10 years study period) and it was observed that the number of cases with suspected leptospirosis started to rise in the month of July and peaked in August. The number almost plateaued in the next two months and was finally declined after October. More than 50 per cent of the suspected as well as serologically diagnosed cases were registered Exicorilant during these four months ((32 %) and (20%). All samples could not be tested by MAT due to technical limitations. Amongst the IgM ELISA positive cases, PCR was performed on 115 blood and 38 urine samples. Ten samples each of blood (8.7%) and urine (26.31%) were positive by PCR. Of these, two cases were those in whom both blood and urine were positive for spp. by PCR. Positivity of PCR in urine was found to be higher than that in blood. Of the IgM ELISA negative cases, PCR was performed in selected blood and urine samples. PCR from blood was positive in 14 of 136 (10.29 %) cases, whereas PCR from urine was positive in 12 of 46 (26%) cases. Of these, three were positive for both blood and urine for leptospires by PCR. Again, positivity of PCR in urine was found to be better than that in blood. Of the total cases (1,453) included in this study, overall 414 (391 ELISA positive and 23 PCR positive) were positive. The year-wise seropositivity in the initial three year was constant (30%) but gradually decreased to 10 per cent in the last four years of the study (Fig.). There was clustering of cases in the monsoon season. This increases the likelihood of co-infections by various other pathogens which.