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

d 疟原虫属和 PFV 荧光原位杂交 (FISH) 检测试剂盒

• 批准号: 8648805
• 负责人: JYOTSNA S SHAH
• 金额: $ 88.53万
• 依托单位: ID FISH TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648805
• 关键词: Accounting; Affect; Africa; African; Air; Antimalarials; Artemisinins; Biological Assay; Blood; Blood specimen; Cessation of life; Child; Chloroquine resistance; Clinical; Clinical Research; Combined Modality Therapy; Country; DNA Probes; Data Analyses; Democratic Republic of the Congo; Detection; Developing Countries; Diagnosis; Disease; Drug Prescriptions; Drug resistance; Economics; Erythrocytes; Falciparum Malaria; Far East; Fluorescent in Situ Hybridization; Future; Giemsa stain; Gold; Grant; Hour; India; Indonesia; Infection; Ivory Coast; Kenya; Label; Laboratories; Left; Life; Light; Light Microscope; Malaria; Marketing; Microscopy; Morphology; Mozambique; Nigeria; Parasites; Parasitic Diseases; Patients; Peru; Pharmaceutical Preparations; Phase; Plasmodium; Plasmodium falciparum; Policies; Preparation; Principal Investigator; Process; Protocols documentation; Reading; Reagent; Reporting; Reproducibility; Research Infrastructure; Ribosomal RNA; Risk; Sampling; Selection for Treatments; Site; Small Business Innovation Research Grant; Specificity; Staging; Symptoms; Tanzania; Techniques; Testing; Time; Uganda; Vulnerable Populations; Whole Blood; Writing; artemisinine; base; cost; diagnosis standard; fluorescence microscope; prevent; programs; public health relevance; rapid diagnosis; resistant strain; sample fixation; screening; standard care; validation studies

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.

描述（由申请人提供）：如果不及时治疗，疟疾可能是一种危及生命的疾病，特别是对儿童而言。据世卫组织统计，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吉姆萨染色涂片。然而，当寄生虫水平很低或混合感染时，通过显微镜检查吉姆萨染色涂片获得的信息是有限的。在SBIR II阶段，我们开发了3个试剂盒，P-Genus， PF和PV FISH检测试剂盒。这些试剂盒采用荧光原位杂交（FISH）技术制备了一种特殊试剂，使用针对活寄生虫核糖体RNA的特异性荧光标记DNA探针，在血液涂片上检测疟疾寄生虫。P-属试剂盒检测所有种类的疟原虫。PF-FISH试剂盒和PV-FISH试剂盒分别在任何p属筛选阳性血液样本上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是靶标，因此也获得了形态信息。这些检测方法简单、半定量，可检测疟疾寄生虫的所有阶段。唯一需要的是一台荧光显微镜。我们已经验证了一个带有2个滤光片的LED灯单元，可以连接到常规光学显微镜上，用于读取FISH检测结果。检测包括六个步骤：使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。总分析时间约为90分钟。检测限为每300田2-3只寄生虫，物镜为100倍。根据一项对来自肯尼亚、印度和秘鲁的500名疟疾样症状患者的研究，FISH检测的灵敏度为95%，而显微镜下吉姆萨染色涂片的灵敏度在84% - 91%之间。在IIB期，我们将完成FDA要求的所有额外验证研究，以提交510(K)申请。具体目标是：(1)最终确定研究方案；(2)制作3批试剂盒，完成试剂盒和试剂的稳定性螺柱；(3)恶性疟原虫和间日疟原虫阳性患者样本的分析敏感性研究；(4)使用患者样本进行接近检出限的重复性研究；(5)完成特异性研究（包括分析特异性和干扰物质）；(6)3个地点的临床研究；(7)对所有临床研究样本进行PCR检测，并对所有PCR阳性样本进行测序(8)数据分析；(9)撰写510(K)提交报告。（10）开始在不需要FDA许可的国家进行营销。在第三阶段(1)申请510(K)(2)建立营销基础设施；(3)增加市场份额(4)开发卵形疟原虫、疟疾疟原虫和诺氏疟原虫特异性FISH检测方法。