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

描述(申请人提供)：疟疾可能是一种危及生命的疾病，特别是在儿童中，如果不治疗的话。根据世卫组织的数据，每年有52.5万至200万非洲儿童死于疟疾。目前诊断的金标准是镜检Giemsa染色涂片。然而，当寄生虫水平很低时，或在混合感染中，通过显微镜检查Giemsa染色涂片获得的信息有限。在SBIR第二阶段，我们已经开发了3种试剂盒，P-Genus，PF和PV FISH检测试剂盒。这些试剂盒通过荧光原位杂交(FISH)技术，使用针对活寄生虫核糖体RNA的特定荧光标记DNA探针，在用特殊试剂制备的血液涂片上检测疟疾寄生虫。P-Genus试剂盒可检测所有种类的疟原虫。PF-FISH试剂盒和PV-FISH试剂盒在任何P-Genus筛查阳性血样上分别检测和区分恶性疟原虫和间日疟原虫与其他疟原虫。由于核糖体RNA是目标，因此也获得了形态信息。这些化验方法简单、半定量，可检测疟疾寄生虫的所有阶段。唯一的要求是一台荧光显微镜。我们已经验证了一个带有2个滤光片的LED光源单元，它可以连接到常规光学显微镜上，用于读取鱼类检测结果。检测包括六个步骤：用专有试剂制备涂片、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。总的分析时间约为90分钟。检测下限为每300个田野2-3个寄生虫，目标值为100倍。基于对来自肯尼亚、印度和秘鲁的500名有疟疾样症状的患者的研究，FISH检测的敏感度为95%，而显微镜下Giemsa染色涂片的敏感度在84-91%之间。在IIB阶段，我们将完成FDA要求提交510(K)的所有额外验证研究。具体目标是：(1)最终确定研究方案；(2)制造3批试剂盒并完成试剂盒和试剂的稳定性螺柱；(3)使用恶性疟原虫和间日疟原虫阳性的患者样本进行分析敏感性研究；(4)使用患者样本进行接近检测极限的重复性研究；(5)完成特异性研究(包括分析特异性和干扰物质；(6)在3个地点进行临床研究；(7)对所有临床研究样本进行聚合酶链式反应并对所有聚合酶链式反应阳性样本进行测序；(8)数据分析；以及(9)撰写510(K)提交报告。(10)在不需要FDA批准的国家开始营销。在第三阶段(1)为510(K)(2)建立营销基础设施；(3)增加市场份额(4)发展卵形疟原虫、疟疾原虫和诺氏原虫特异性鱼类检测。

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.