Trypanosomatid parasites of the genus Leishmania infect close to 15 million people world-wide. Diagnosis is difficult in endemic areas because of the currently avialbe detection schemes are inadequate because of lack of sensitivity, time intensiveness. Recent concern regarding possible link between the Gulfwar syndrome and Leishmaniasis has accentuated the lack of and need for better diagnostic modalities for both patients and blood products. A more reliable target for detection is the parasite's own mitochondrial DNA (kDNA)which remains constant through its life cycle and is present in thousands of copies per cell. We have characterized kDNA sequences from eight different Leishmania isolates and have used a PCR-based scheme to design detection systems for several species. This techinque can readily detect Leishmania kDNA from single cell in the presence of vast amounts of human DNA. The utility of these PCR schemes has been further shown by their ability to positively identify parasite isolates from kala-azar patients and individuals with cutaneous infections. Based on our experience with the PCR-based detection schemes, we want to expolite this techique to detect adventious agents in the parasite vaccines. Many species of Leishmania are persistently infected with double stranded RNA virus named as Leishmainavirus (LRV). The complete nucleotide sequences of two LRV isolates from both new world and old world strains have been reported. We plan to identify sequences which are conserved among the strains and develop primers which can be used in the PCR based detection assay. Using these primers we will develop PCR assay to detect the virus in wide range of isolates form different regions of the world which are available to us. We would also like to test various vaccine preprartions, obtained from WHO, to test the presence of viral sequences in them.
