Plasmodium Genus and PFV Fluorescent In Situ Hybridization (FISH) Assay Kit for d

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DESCRIPTION (provided by applicant): Malaria can be a life-threatening disease, especially in children, if left untreated. According to WHO, 525,000 to 2.0 million African children die from Malaria every year. The current gold standard for diagnosis is examination of Giemsa stained smear by microscopy. However, when parasite levels are very low, or in mixed infections, the information obtained by examination of Giemsa stained smear by microscopy is limited. Under SBIR Phase II we have developed 3 kits, P-Genus, PF and PV FISH assay kits. These kits detect malaria parasites on blood smears prepared with a special reagent by Fluorescent in Situ Hybridization (FISH) technique, using specific fluorescent labeled DNA probes targeted to ribosomal RNA of viable parasites. P- Genus kit detects all species of Plasmodium. PF-FISH kit and PV-FISH kit detect and differentiate P. falciparum and P. vivax from other species of Plasmodium respectively, on any P-Genus screen positive blood sample. Since ribosomal RNA is the target, morphological information too is obtained. The assays are simple, semi-quantitative and detect all stages of the malaria parasites. The only requirement is a fluorescent microscope. We have validated a LED light unit with 2 filters that can be attached to a regular light microscope for reading the FISH assay results. The assays consist of six steps: smear preparation using proprietary reagent, fixation, hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total assay time is approximately 90 minutes. The limit of detection is between 2-3 parasites per 300 fields with 100X objective. Based on a study on 500 patients from Kenya, India and Peru with malaria- like symptoms, the sensitivity of the FISH assays was >95% whereas the sensitivity of Giemsa stained smear by microscopy was between 84 - 91%. In Phase IIB we will complete all the additional validation studies requested by FDA to file for 510(K). The specific aims are: (1) Finalization of study protocols; (2) Manufacture of 3 lots of kits and completion of stability stud of kits and reagents; (3) Analytical Sensitivity Study using patient samples positive for P. falciparum and P. vivax; (4) Reproducibility Study near limit of detection using patient samples; (5) Completion of Specificity Study (including Analytical Specificity and Interference Substances; (6) Clinical studies at 3 sites; (7) PCR on all clinical study samples and sequencing on all PCR positive samples (8) Analysis of data; and (9) Write 510(K) submission report. (10) Start marketing in countries that do not require FDA clearance. In Phase III (1) File for 510(K) (2) Set up infrastructure for marketing; (3) Increase the market share (4) Develop P. ovale, P. malariae and P. knowlesi specific FISH assays.