Widespread incidence of Leishmania infections, increased global AIDS problems, with an increased incidence of co-infections, has made Leishmaniasis the world's seventh largest public health problem. Current diagnostic procedures require either long-term cultures or serological assays which, at times, are not reliable. Consequently, a DNA probe-based assay is a preferable choice. In Phase l, we developed a simple Specimen preparation procedure which renders minicircle kDNA more available for PCR amplification, and provides a 10,000-fold increase in sensitivity. This was established with consensus primers and probes to detect all Leishmania species, albeit with different levels of sensitivity. This increase in sensitivity from our lysis buffer should allow the development of a direct hybridization kit to test the majority of field.specimens, In Phase II, we propose to develop a PCR-based test kit for detection and species identification of Leishmania from clinical specimens, containing an extremely small number of parasites. A less expensive direct hybridization-based kit is proposed for the majority of the specimens. We will incorporate a simplified specimen handling system to meet the needs of the endemic regions of the world. In addition to clinical diagnostics, the test kits will be designed for large throughput, so that they can be used by public health officials of various government agencies for eradication purposes.
We proposed to develop a rapid, economical, and reliable DNA probe test for the detection of Leishmania, both Old and New World species, in clinical samples. The test will rely on methodologies which are practical for application in developing nations, and will provide significant improvement In relIability over existing tests.
