SBIR-STTR Award

Our research will develop reagents and procedures for detection of nucleic acids from Leishmania. Leishmania are grouped broadly into the New and Old World species. Throughout tropical and subtropical regions of the world, leishmaniasis is a serious public health concern. Effective treatments exist; yet, diagnosis is difficult, owing to low abundance of parasites in the peripheral blood. Consensus probes and primers will be designed, based upon published mini circle kinetoplast sequences. Primers will incorporate inosines and mixed base positions to support amplification of Old and New World species. Non-radioactive microliter plate assays have been developed for performing hybridization analysis of amplified DNA. These methods, together with simplified DNA extraction and amplification techniques will be adapted to a consensus DNA probe assay for Leishmania using laboratory strains. In Phase II, these procedures will be refined and extended to detection in clinical specimens.Awardee's statement of the potential commercialapplications of the research: We will develop a rapid, economical, and reliable DNA probe test for the detection of Lelshmania, 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.National Institute of Allergy and Infectious Diseases (NIAID)

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. Proposed commercial applications: 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.Thesaurus termsLeishmania, communicable disease diagnosis, diagnosis design /evaluation, diagnosis quality /standard, method development, nucleic acid hybridization, nucleic acid probe, serotyping leishmaniasis, mass screening, microorganism classification, rapid diagnosis clinical research, human subject, polymerase chain reactionNational Institute of Allergy and Infectious Diseases (NIAID)