Using protein-DNA chimeras for HAT diagnosis

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DESCRIPTION (provided by applicant): Trypanosoma brucei gambiense and T. b. rhodesiense are pathogens responsible for human African trypanosomiasis (HAT). Death from HAT is inevitable if untreated. In the rural areas where patients are typically seen, failure to microscopically observe trypanosomes in blood smears and/or CSF in the critical early stages of the disease is probably the single most important factor in failed treatment. We propose a new technology that we believe will be important for the detection of parasites in the CSF during Stage 2 disease (CNS involvement), particularly in the field setting. The procedure uses protein-DNA chimeras termed 'Tadpoles'. To create 'Tadpoles', a 'DNA barcode' is added to a single site on a protein. Amplification of the 'Tadpoles' DNA by PCR creates assays with great dynamic range and specificity. However, as an alternative to PCR we propose to amplify the DNA-barcode associated with Tadpoles by loop-mediated isothermal amplification (LAMP). The LAMP reaction amplifies DNA with high specificity, efficiency and speed under isothermal conditions and allows for easy visual positive identification of the target DNA. We propose to combine the technology of LAMP with 'Tadpoles' to create LAMP-based 'LAMPole' assays for the diagnosis of HAT suitable for diagnostic applications in the field. There are two Specific Aims. These are 1) to design oligonucleotides and create "LAMPoles" for the highly sensitive detection of trypanosome signature proteins, and 2) to define analytical/clinical sensitivity and specificity for LAMPoles in the diagnosis of Stage 2 HAT. Success in the completion of the Specific Aims of this application will provide novel diagnostic tests for the early detection of Stage 2 HAT applicable in African health care centers and in the field. PUBLIC HEALTH RELEVANCE: Human African trypanosomiasis or sleeping sickness is 100% fatal if not treated. At least 40,000 people die every year in Africa because good tests for diagnosis in the more rural areas do not exist. Using a simple water bath, we believe that a new technology we will develop called 'LAMPoles' will provide rapid, accurate, evidence for infection in field conditions.