P.falciparum and P.vivax Fluorescent In Situ Hybridization

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DESCRIPTION (provided by applicant): Plasmodium -Dual Probe Fluorescent In-Situ Hybridization (PFV- FISH) is a method of detecting and differentiating Plasmodium falciparum (PF) ribosomal RNA (rRNA) and P. vivax (PV) rRNA on a SINGLE air- dried thin blood smear. The assay uses PF and PV specific probes labeled with different fluorescent dyes. Thus, PF and PV shall fluoresce with different colors under specific dual pass filters. The assay is simple and inexpensive (< $5.00/test and a one time expense of about $600 for filters or <$1700 for the microscope and filters). It consists of five steps -fixation, hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total assay time is less than 1 hour. The treatment for P. falciparum and P. vivax is different. Therefore, PFV-FISH assay would be very useful in areas where both P. falciparum and P. vivax are endemic. Specific Aims: Develop a simple and inexpensive Dual Probe FISH assay (PFV-FISH) kit, for direct detection of PF and PV on an air-dried whole blood smear. The kit is expected to provide the specificity equivalent to amplified DMA probe assays, and sensitivity equivalent to giemsa stained smear. The kit shall contain all the reagents and control smears. Phase I: (1) The feasibility study of the PFV - FISH Test to determine the sensitivity of the assay to detect and differentiate P. falciparum and P. vivax. (2) Determination of the Hybridization Conditions for a Dual Color PFV-FISH Assay. (3) Limited Specificity Study and Limit of Detection Study. (4) Assay Performance on 100 Clinical Samples. Generally clinical samples are not tested in Phase I. However, these are the only samples that can be used to study the assay. Culture samples are available for P. faciparum, but are not readily available. Phase II: (1) Assay Development - Optimization of the PFV-FISH. based on the results of the study performed on 100 clinical samples. (2) Set up Quality Control Procedures and Controls (3) Set up manufacturing facilities. (4) Perform an in-house study on clinical samples. (Number of samples to be tested shall be determined statistically by the Biostatistician, Dr. Stubbs.) (5) Identification and validation of Potential Clinical Sites India and Kenya for Phase III. Phase III: (1) Perform clinical trials in the US; and concurrently in Kenya and India. (2) Get FDA approval. (3) Marketing within US and Outside US. (4) Set up manufacturing plant in an underdeveloped country where malaria is endemic, e.g. Kenya or India.(4) Extend the assay to include P. ovate and P. malaria.