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

描述（由申请方提供）：靶向核糖体RNA（rRNA）的疟原虫属荧光原位杂交（P-Genus FISH）试验是一种检测风干血涂片上所有疟疾种属的方法。PFV-FISH检测是一种双探针检测方法，用于在单个空气干燥的薄血涂片上检测和区分恶性疟原虫（PF）和间日疟原虫（PV）。P-Genus检测试剂盒使用塔姆拉标记的P-Genus特异性探针。因此，所有疟原虫物种在德克萨斯红滤光片下将发出红色荧光。PFV-FISH检测使用分别用红色和绿色荧光染料标记的PF和PV特异性探针。因此，在特定的双通滤波器下，PF发红色荧光，PV发绿色荧光。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。该测定是简单且廉价的（< $3.00/测试，并且用于过滤器的一次性费用为~$1000）。该检测包括五个步骤：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。在第一阶段，我们优化了固定和杂交条件，并建立了FISH检测的质量控制程序。我们证明，检测限为1至9寄生虫/300场在1000倍，FISH测定是非常可重复的。根据对300多例患者涂片进行的临床研究，检测灵敏度优于Giemsa。与PCR相比，FISH测定灵敏度为83- 89%，而Giemsa灵敏度为54- 60%。与PCR相比，两种检测的FISH检测特异性均为100%。第二阶段的具体目标（1）购买临床研究用设备和试剂盒试剂制备用罩。(2)试剂盒生产和试剂有效期的确定。(3)对CDC从猴血液中制备的用于检测恶性疟原虫、间日疟原虫和三日疟原虫的涂片;从黑猩猩血液中制备的用于检测卵形疟原虫的涂片进行灵敏度分析。(4)临床试验-4a：临床研究中心进行试验的培训和能力。4b：在临床研究中心进行测定的重现性测定;以及4c：在临床研究中心对全血样本进行的血清学制备和测定性能。(5)廉价的显微镜和过滤器的评价。进入III期将基于完成临床试验，并证明特异性至少为95%，特异性和灵敏度等同于或优于Giemsa染色涂片（与PCR相比）。III期（1）申报510 K或PMA的具体目的。（2）在肯尼亚建立制造业。(3)：市场营销。(4)与世卫组织合作获得其批准。这将避免许多国家在批准测试方面的长期拖延。(5)开发PMO-FISH检测方法，用于在单个空气干燥的薄血涂片上检测和区分三日疟原虫和卵形疟原虫。 据估计，全世界每年有超过3.5亿至5亿人感染疟疾寄生虫。每年有70万至270万人死于疟疾，其中75%是非洲儿童。疟疾可能是危及生命的疾病，特别是在儿童中，如果不及时治疗，因此，快速准确的诊断非常重要。尽管疟疾是许多发展中国家经常遇到的疾病，但仅依靠临床体征很难做出正确的诊断。治疗疟疾的药物选择取决于存在的疟疾种类。恶性疟的延误或漏诊增加了并发症或严重疾病的风险，这可能是致命的脆弱人群。来自世界大部分地区的恶性疟原虫在很大程度上是氯喹抗性的，因此不能使用间日疟原虫的标准治疗。为了防止不必要的抗疟治疗和疟疾寄生虫未来的耐药性菌株，重要的是要通过良好的实验室检测来确认临床可疑。鉴于包括坦桑尼亚和肯尼亚在内的许多非洲国家不断变化的药物政策，昂贵的青蒿素综合疗法药物被作为一线治疗处方，良好的实验室确认也将对经济产生影响。吉姆萨染色很有用。然而，当寄生虫水平非常低或混合感染时，通过显微镜检查吉姆萨染色涂片获得的信息是有限的，在某些情况下，由于无法投入必要的时间来检查血涂片，因此存在偏见。PCR会有帮助。不幸的是，这是耗时的。因此，用于检测疟原虫和用于区分空气干燥血涂片中的恶性疟原虫和间日疟原虫的FISH测试（PFV-FISH）在这种疾病的快速诊断中具有潜力。 优势：P-Genus和PFV FISH检测 (1)P-属FISH检测可检测所有四种疟原虫，即恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫。因此，Genus FISH检测可用于筛选。如有必要，可使用PFV-FISH检测试剂盒对任何阳性样本进行进一步检测，以确定感染是否由恶性疟原虫、间日疟原虫、两者或两者均不引起。 (2)两种检测试剂的专属性与扩增检测试剂相当。 (3)当由“典型显微镜医师”进行检测时，检测的灵敏度应与由“专业显微镜医师”进行的姬姆萨染色涂片相同或更好（即，每5 l血液中寄生虫数量为5个或更少） (4)能够在风干全血涂片上进行检测。 (5)结果可以在一个小时或更短的时间内获得将是有价值的。 (6)提供寄生虫形态学，如姬姆萨染色涂片。 (7)易于执行。 (8)低成本

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 assay 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. Therefore, 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 (< $3.00/test and a one time expense of ~$1000 for filters). The assays 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. In Phase I, we optimized the fixation and hybridization conditions and set up Quality Control Procedures for the FISH assays. 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 compared 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 preparing 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 compared 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 PMO-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 falciparum 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 unnecessary anti-malarial treatment and future drug-resistance strains of malaria parasites, it is important to confirm clinical suspicious with a good laboratory test. In light of the changing drug policies of many African countries, including Tanzania and Kenya, where the expensive artemisinin combination therapy (ACT) drugs are prescribed as first-line treatment, a good laboratory confirmation will also have an impact on the economics. The Giemsa stain is helpful. However, when parasite levels are very low, or in mixed infections, the information obtained by examination of Giemsa stained smear by microscopy is limited, and in some cases, biased, by the inability to devote the necessary amount of time to the examination of blood smears. PCR would help. Unfortunately it is time-consuming. Thus, FISH Test for detection of Plasmodium and for differentiation of P. falciparum and P. vivax (PFV-FISH) in an air-dried blood smear has potential in the rapid diagnosis of this disease. Advantages: P-Genus and PFV FISH Assays (1) P- Genus FISH Assay detects all four species of Plasmodium, P. falciparum, P. vivax, P. malariae and P. ovale. Therefore the Genus FISH assay can be used for screening. Any sample that is positive can be tested further if necessary with the PFV- FISH Assay to determine whether the infection is due to P. falciparum, P. vivax, both or neither. (2) Specificity of both assays is equivalent to the amplified assays. (3) Sensitivity of the assays when performed by a "typical microscopists" should be the same as or better than Giemsa stained smear performed by an "expert microscopists" (i.e. 5 or less parasites per 5l of blood) (4) Ability to perform the tests on an air-dried whole blood smear. (5) Results can be obtained in one hour or less would be valuable. (6) Provides parasite morphology as the Giemsa stained smear. (7) Easy to perform. (8) Low Cost.