Plasmodium Genus and P. falciparum - P. vivax FISH Assays

DESCRIPTION (provided by applicant): Plasmodium Genus Fluorescent In Situ Hybridization (P-Genus FISH) assay targeted to ribosomal RNA (rRNA) is a method that detects all the species of malaria on an air-dried blood smear. PFV-FISH assay is a dual probe as say for detecting and differentiating P. falciparum (PF) and P. vivax (PV) on a SINGLE air-dried thin blood smear. The P-Genus assay uses P-Genus specific probe labeled with Tamra. Thus all the Plasmodium species will fluoresce red under Texas Red filter. The PFV-FISH assay uses PF and PV specific probes labeled with red and green fluorescent dyes, respectively. Thus, PF fluoresces red and PV fluoresces green under specific dual pass filters. The treatment for P. falciparum and P. vivax is different. Theref ore, PFV-FISH assay would be very useful in areas where both P. falciparum and P. vivax are endemic. The assays are simple and in-expensive (lt 3.00/test and a one time expense of ~ 1000 for filters). The assays consists of five steps; fixation, hybridiza tion, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total assay time is less than 1 hour. In Phase I, we optimized the fixation and hybridization conditions and set up Quality Control Procedures for the FISH a ssays. We demonstrated that the limit of detection was 1 to 9 parasites/300 fields at 1000X and that the FISH assays are very reproducible. Based on a clinical study performed on over 300 patient smears, the assay sensitivity was better than Giemsa. As com pared to PCR the FISH assay sensitivity was 83-89%, whereas Giemsa sensitivity was between 54-60%. FISH assay specificity as compared to PCR was 100% for both assays. Specific Aims for Phase II (1) Purchase of equipment for Clinical Study and hoods for pre paring reagents for kits. (2) Kit manufacturing and determination of shelf-life of reagents. (3) Analytical Sensitivity to be performed on smears prepared by CDC from in monkey blood for P. falciparum, P. vivax and P. malariae; from chimpanzee blood with P . ovale. (4) Clinical Trials - 4a: Training and competency of Clinical Sites to perform assay. 4b: Reproducibility determination at Clinical Sites to perform assay; and 4c: Smear preparation and assay performance on whole blood samples at Clinical Sites. ( 5) Evaluations of inexpensive Microscopes and Filters. Proceeding to Phase III will be based on completing the clinical trials and demonstrating specificity of at least 95% specificity and sensitivity equivalent to or better than Giemsa stained smear as co mpared to PCR. Specific Aims for Phase III (1) Filing 510K or PMA. (2)Set up manufacturing in Kenya. (3): Marketing. (4) Work with WHO to obtain their stamp of approval. This will avoid long delays in approval of the tests in many countries. (5) Develop PM O-FISH assay for detecting and differentiating P. malariae and P. ovale on a SINGLE air-dried thin blood smear. It is estimated that worldwide there are more than 350-500 million people infected with malaria parasites each year. Between 700,000 to 2 .7 million people die of malaria every year, of which 75% are African children. Malaria can be life-threatening disease, especially in children, if left untreated and therefore, it is important to have a quick and accurate diagnosis. Even though malaria is a frequently encountered disease in many developing countries, it is difficult to make the right diagnosis relying on clinical signs only. Drug selection for the treatment of malaria depends on species of malaria present. Delayed or missed diagnosis of fa lciparum malaria increases the risk of complicated or severe disease, which may be fatal vulnerable populations. P. falciparum from much of the world is largely chloroquine resistant and thus the standard treatment for P. vivax cannot be used. To prevent u nnecessary anti-malarial treatment and future drug-resistance strains of malaria parasites, it is important to confirm clinical suspicious with a good labora