Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand.

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe. The duplex-PCR allowed detection of two products of 279 bp for LipL32 gene and 430 bp for 16S rRNA gene. In water samples artificially seeded with serovar bratislava, at least 10(3) cells could be analyzed by PCR-agarose gel electrophoresis and 1-10 cells by PCR-Southern blot hybridization. The protocol was applied to the detection of pathogenic Leptospira in environmental waters collected from endemic areas in the northeast region of Thailand. Of 100 water samples analyzed, 23 samples were positive for pathogenic Leptospira with PCR performed with Southern blot hybridization only, but none was detected by PCR-agarose gel-electrophoresis. However, PCR performed with the chemiluminescent LipL32 probe using the Fluorescein ULS labeling facilitated the detection of low numbers of pathogenic Leptospira in water. This method should prove useful for monitoring of pathogenic Leptospira pollution in environmental waters, and has the potential to become a valuable tool to the surveillance of leptospirosis in endemic areas, thus leading to enhanced public health protection.