P.falciparum and P.vivax Fluorescent In Situ Hybridization

描述（由申请方提供）：疟原虫-双探针荧光原位杂交（PFV-FISH）是一种检测和区分单个风干薄血涂片上的恶性疟原虫（PF）核糖体RNA（rRNA）和间日疟原虫（PV）rRNA的方法。该测定使用用不同荧光染料标记的PF和PV特异性探针。因此，PF和PV应在特定的双通滤光片下发出不同颜色的荧光。该测定法简单且便宜（< $5.00/测试和用于过滤器的约$600或用于显微镜和过滤器的<$1700的一次性费用）。它包括五个步骤-固定，杂交，洗涤，复染和在荧光显微镜下观察处理后的涂片。总测定时间小于1小时。恶性疟原虫和间日疟原虫的治疗方法不同。因此，PFV-FISH检测在恶性疟原虫和间日疟原虫同时流行的地区是非常有用的。具体目标：开发一种简单、廉价的双探针FISH检测试剂盒（PFV-FISH），用于直接检测空气干燥全血涂片上的PF和PV。该试剂盒预期提供与扩增DMA探针检测相当的特异性和与吉姆萨染色涂片相当的灵敏度。试剂盒应包含所有试剂和质控涂片。第一阶段：（1）PFV-FISH检测方法的可行性研究，以确定PFV-FISH检测方法检测和区分恶性疟原虫和间日疟原虫的灵敏度。(2)双色PFV-FISH检测杂交条件的确定。(3)有限专属性研究和检测限研究。(4)100份临床样本的检测性能。一般来说，临床样本在I期不进行检测。然而，这些是可用于研究测定的唯一样品。可获得P. faciparum的培养样品，但不容易获得。第II阶段：（1）试验开发-PFV-FISH的优化。基于对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）建立生产设施。(4)对临床样本进行内部研究。（待测样本数量应由生物统计学家Dr. Stubbs统计确定。）(5)确定和验证印度和肯尼亚的III期潜在临床研究中心。III期：（1）在美国进行临床试验;同时在肯尼亚和印度进行。(2)得到食品药物管理局的批准。(3)在美国境内和境外销售。(4)在疟疾流行的不发达国家（如肯尼亚或印度）建立制造工厂。(4)将检测试剂扩展至包括卵形疟原虫和疟疾疟原虫。

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.