P.falciparum and P.vivax Fluorescent In Situ Hybridization

描述(申请人提供)：疟原虫-双探针荧光原位杂交(PFV-FISH)是一种在单个风干的薄血涂片上检测和区分恶性疟原虫(PF)核糖体RNA(RRNA)和间日疟原虫(PV)rRNA的方法。该方法使用标记了不同荧光染料的PF和PV特异性探针。因此，在特定的双通滤光片下，PF和PV应发出不同颜色的荧光。这种化验方法简单且价格低廉(一次化验5.00美元，一次性滤镜费用约600美元，显微镜和滤镜一次性费用1700美元)。它包括固定、杂交、洗涤、反染色和在荧光显微镜下观察处理后的涂片五个步骤。总检测时间不到1小时。对恶性疟和间日疟的治疗是不同的。因此，在恶性疟原虫和间日疟原虫同时流行的地区，PFV-FISH检测是非常有用的。具体目的：开发一种简单、廉价的双探针FISH试剂盒，用于直接检测风干全血涂片上的PF和PV。该试剂盒有望提供相当于扩增的DMA探针分析的特异性和相当于Giemsa染色涂片的灵敏度。试剂盒应包含所有试剂和控制涂片。第一阶段：(1)PFV-FISH试验的可行性研究，以确定该方法检测和区分恶性疟原虫和间日疟原虫的敏感性。(2)双色PFV-FISH杂交条件的确定。(3)检测研究的局限性和特异性研究的局限性。(4)对100例临床标本的检测结果。临床样本通常不会在第一阶段进行测试，然而，这些是唯一可用于研究化验的样本。培养样本是可以获得的，但不是现成的。第二阶段：(1)分析发展--PFV-FISH的优化。根据对100个临床样本进行的研究结果。(2)建立质量控制程序和控制(3)建立制造设施。(4)对临床样本进行内部研究。(需要测试的样本数量将由生物统计学家斯塔布斯博士从统计上确定)(5)确定和确认印度和肯尼亚第三阶段的潜在临床地点。第三阶段：(1)在美国进行临床试验；同时在肯尼亚和印度进行临床试验。(2)获得FDA批准。(3)在美国和美国以外的市场营销。(4)在疟疾流行的欠发达国家(如肯尼亚或印度)建立生产工厂。(4)扩大检测范围，将卵形疟原虫和疟疾纳入检测范围。

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.