Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites

消灭海地的疟疾。重塑DNA以根除地方性寄生虫 Firebird Biomolecular Sciences LLC 佛罗里达大学 Steven A. Benner 约翰B。Dame 摘要 根除恶性疟原虫是造成最多疟疾死亡的寄生虫， 最终在任何特定的地理区域治疗疾病。海地就是这样一个地区， (11百万），地理隔离和特殊的生态使根除成为真实的可能。因此， 基金会刚刚承诺向疾病预防控制中心基金会提供2940万美元，以尝试根除这种疾病。 根除的关键是一种鉴定人类携带者，即携带活性寄生虫的无症状个体的检测方法 感染.该分析必须：（a）仅需要少量血液样本，可能为100微升;（B）检测非常小的 数量的活寄生虫，每个样本可能只有10个寄生虫;（c）操作简单， 传统的疟疾“快速诊断测试”（RDT）;（d）运行成本低于1美元。 这些规格几乎是不可能的经典分子诊断。但是，在这方面， 在过去的三年里，Firebird开发了许多创新，使这种规格成为可能，包括（a） 去除生物危害的样品制备过程，（B）无需离心的全核酸捕获，（c） 使用自避免分子识别系统（SAMRS）和人工免疫的等温核酸扩增 （d）AEGIS分子信标。Firebird显示 这些支持的检测，例如，检测22蚊子携带的RNA病毒在一个蚊子尸体。 这项工作是由Dame实验室的工作指导的，该工作表明，恶性疟原虫核糖体RNA，存在于 每种寄生虫约104个拷贝，可以使用RT-PCR在疟疾感染的血液中检测到。PCR检测不 满足LRE所需的成本规格，并且不像RDT那样容易运行，但它显示了 针对恶性疟原虫rRNA的检测，如果血液样品仅含有一种生物体，则检测是稳健的。 该项目将联合收割机结合这些创新来开发一种检测恶性疟原虫rRNA的方法，该方法可以像 一个RDT，但它的灵敏度更高。这种方法的一个优点是它使用了真实的生活样本， 恶性疟原虫在真实的血液中做标杆。成本低于1.00美元，如果 可以直接读取或通过手机摄像头传输以进行远程确认。 我们将对（i）释放恶性疟原虫rRNA的样品制备工作流程进行基准测试， 在固体支持物上，其中（iii）使用具有SAMRS和AEGIS的引物等温扩增捕获的rRNA 核苷酸，（iv）AEGIS信标检测的扩增子。将结果与分析的样品进行比较， RT-PCR法成功的条件包括能够在一个样本中检测出10种寄生虫。在第二阶段， 该测试将在一个小型现场试验中进行验证，试验对象是来自海地疟疾流行地区的个人， 再次与RT-PCR进行比较。这也将支持载体识别背后的流行病学科学， 我们仍然不知道在一个国家中可以保持稳定的寄生虫血症的最低水平， 单独的.该产品应获得世卫组织和盖茨基金会的支持，并对其他制剂进行LRE测试。

Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites Firebird Biomolecular Sciences LLC University of Florida Steven A. Benner John B. Dame Abstract Eradication of Plasmodium falciparum, the parasite that causes the most malarial deaths, is the only way to finally treat the disease in any specific geographical region. Haiti is one such region, where a small population (11 million), geographical isolation, and special ecology make eradication a real possibility. Thus, the Gates Foundation has just committed $29.4 MM to the CDC Foundation to attempt this eradication. Key to eradication is an assay that identifies human carriers, asymptomatic individuals with active parasite infections. That assay must: (a) Require only a small sample of blood, perhaps 100 µL; (b) Detect very small numbers of living parasites, perhaps as few as 10 parasites per sample; (c) Be as simple to operate as a conventional malarial “rapid diagnostics test” (RDT); and (d) cost less than $1.00 to run. These specifications are virtually impossible with classical molecular diagnostics. However, Firebird has developed many innovations over the past three years that make such specs possible, including (a) sample prep processes that remove biohazard, (b) whole nucleic acid capture without centrifugation, (c) isothermal nucleic acid amplification using self-avoiding molecular recognition systems (SAMRS) and artifi- cially expanded genetic information systems (AEGIS), and (d) AEGIS molecular beacons. Firebird has shown that these support assays that, for example, detect 22 mosquito-borne RNA viruses in one mosquito carcass. The work is guided by work in the Dame lab showing that P. falciparum ribosomal RNA, present at ~104 copies per parasite, can be detected in malaria-infected blood using RT-PCR. The PCR assay does not meet the cost specs necessary for a LRE, and is not as easy to run as an RDT, but it shows the sensitivity of an assay directed at falciparum rRNA, where detection is robust if a sample of blood contains just one organism. This project will combine these innovations to develop an assay that detects falciparum rRNA as easily as an RDT, but which much higher sensitivity. One strength of the approach is its use of realistic samples of live falciparum in real blood to do benchmarking. Costing less than $1.00, the assay will generate fluorescence if falciparum is present that can be read directly or transmitted by cell phone camera for remote confirmation. We will benchmark (i) a sample preparation work-flow that releases P. falciparum rRNA that is (ii) captured on a solid support, where (iii) captured rRNA is amplified isothermally using primers with SAMRS and AEGIS nucleotides, with (iv) amplicons detected by AEGIS beacons. Results will be compared to samples analyzed by RT-PCR. Metrics for success include an ability to detect 10 parasites in a sample. In Phase 2, the efficacy of the test will be validated in a small field trial with blood from individuals from in a malaria-endemic area of Haiti, again comparing with RT-PCR. This will, as well, support the epidemiological science behind carrier identifica- tion and eradication, as we still do not know the lowest level of parasitemia that can remain stably in an individual. The product should gain WHO and Gates Foundation support, and yield LRE tests for other agents.