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

描述（由申请人提供）：针对核糖体RNA （rRNA）的疟原虫属荧光原位杂交（p属FISH）测定是一种在风干血液涂片上检测所有疟疾种类的方法。PFV-FISH检测是一种双探针检测和区分恶性疟原虫（PF）和间日疟原虫（PV）单风干薄血涂片。p属测定采用Tamra标记的p属特异性探针。因此，所有种类的疟原虫在德州红滤光器下都会发出红色荧光。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发出红色荧光，PV发出绿色荧光。恶性疟原虫和间日疟原虫的治疗是不同的。因此，PFV-FISH检测在恶性疟原虫和间日疟原虫都流行的地区非常有用。检测方法简单且价格昂贵（< 3.00美元/次测试，一次性费用约为1000美元的过滤器）。检测包括五个步骤；固定，杂交，洗涤，反染色，并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，在1000倍的条件下，检测限为1 ~ 9个寄生虫/300场，并且FISH分析具有很高的可重复性。根据对300多名患者涂片进行的临床研究，该检测的敏感性优于吉姆萨。与PCR相比，FISH检测灵敏度为83-89%，而吉姆萨检测灵敏度为54-60%。与PCR相比，FISH检测的特异性为100%。II期的具体目标(1)购买临床研究设备和用于制备试剂盒试剂的罩。(2)试剂盒制作及试剂保质期测定。(3) CDC制备的猴血涂片对恶性疟原虫、间日疟原虫和疟疾疟原虫的分析敏感性；来自黑猩猩的卵形疟原虫。(4)临床试验- 4a：临床现场进行化验的培训和能力。4b：在临床试验现场进行重复性测定；和4c：临床站点全血样本的涂片制备和检测性能。(5)廉价显微镜和滤镜的评价。进入III期将基于完成临床试验，并证明至少95%的特异性和敏感性相当于或优于Giemsa染色涂片与PCR。III(1)期申请510K或PMA的具体目标。(2)在肯尼亚设厂。(3):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这些试验方面的长时间拖延。(5)建立PMO-FISH检测方法，在单张风干薄血涂片上检测和区分疟疾疟原虫和卵形疟原虫。

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.