Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites

从海地消除疟疾。重新发明DNA以根除地方性寄生虫 火鸟生物分子科学有限责任公司 佛罗里达大学 史蒂文·A·本纳 约翰·B·达姆 摘要 根除恶性疟原虫是导致最多疟疾死亡的寄生虫，是治疗疟疾的唯一途径 最后，在任何特定的地理区域治疗这种疾病。海地就是这样一个地区，那里的人口很少 (1100万)，地理上的隔绝和特殊的生态使根除成为可能。因此，盖茨夫妇 基金会刚刚向疾控中心基金会承诺提供2940万美元，以尝试根除这种疾病。 根除寄生虫的关键是一种能识别人类携带者的化验方法，即带有活跃寄生虫的无症状个体。 感染。这种分析必须：(A)只需要一小部分血液样本，可能是100uL；(B)检测到非常少的血液 活体寄生虫的数量，也许每个样本只有10种寄生虫；(C)操作简单，就像 常规疟疾“快速诊断测试”(RDT)；和(D)运行费用不到1美元。 用经典的分子诊断学，这些规格几乎是不可能的。然而， 火鸟在过去三年里开发了许多创新，使这种规格成为可能，包括：(A) 消除生物危害的样品制备过程，(B)无需离心即可捕获整个核酸，(C) 用自回避分子识别系统(SAMRS)和人工免疫技术进行恒温核酸扩增 实际扩展的遗传信息系统(宙斯盾)和(D)宙斯盾分子信标。火鸟已经展示了 例如，这些支持在一具蚊子身体中检测到22种蚊媒RNA病毒的测试。 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，目前在 用RT-PCR在疟疾感染的血液中可以检测到每条寄生虫大约104个拷贝。聚合酶链式反应检测不能 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，如果血液样本只包含一种微生物，则检测是可靠的。 该项目将结合这些创新来开发一种检测恶性疟原虫rRNA的方法，它可以像 RDT，但敏感度要高得多。这种方法的一个优点是它使用了真实的现场样本 在真正的血液中做恶性疟原虫做标杆。该检测的成本不到1.00美元，如果 存在可直接读取或通过手机摄像头传输以进行远程确认的恶性疟原虫。 我们将对(I)释放(Ii)捕获的恶性疟原虫rRNA的样本制备工作流进行基准测试 在固体载体上，(Iii)捕获的rRNA用带有SAMRS和Aegis的引物等温扩增 核苷酸，带有宙斯盾信标检测到的(Iv)扩增产物。结果将与分析的样本进行比较 RT-PCR法。成功的衡量标准包括在一个样本中检测10种寄生虫的能力。在第二阶段， 测试将在一项小型现场试验中进行，血液来自海地疟疾流行地区的个人， 再次与RT-PCR进行比较。这也将支持携带者身份识别背后的流行病学科学- 由于我们仍然不知道可以稳定保持在一个国家的最低寄生虫病水平 个人的。该产品应获得世卫组织和盖茨基金会的支持，并为其他试剂提供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.