Evaluation of TaqMan based real-time PCR assay targeting LipL32 gene for leptospirosis in serologically positive human urine samples from north India.

PURPOSE: Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In India, especially in north India; leptospirosis is a grossly underreported disease, probably due to the lack of diagnostic modalities and lack of awareness of the disease among physicians. In this study, we aimed to identify leptospires in urine samples from serologically positive patients of leptospirosis through TaqMan based Real-time PCR using the LipL32 gene. Although environmental conditions are suitable in north India nowadays, very few studies were there and none by real-time PCR.

METHOD: A total of 50 urine and blood samples (40 seropositive and 10 seronegative by IgM ELISA) were recruited in the study. Urine samples were tested by real-time PCR to detect Leptospira DNA and blood samples were used for microscopic agglutination test (MAT) evaluation.

RESULT: We were able to detect leptospires in 20% (10/50) cases using our real-time PCR assay. In all 10 PCR positive urine samples, the LipL32 gene was detected and the lower limit of detection in L. interrogans DNA was found to be 20 GE/μL with a 95% cutoff value.

CONCLUSION

We conclude that this real-time PCR assay with high specificity and sensitivity less prone to contamination may prove to be a promising approach for diagnosing acute leptospires in urine samples in the second week of acute febrile illness due to leptospiruria starts approximately in the second week of illness.