Development of novel diagnostics for African non-falciparum malaria

项目总结 虽然非洲因恶性疟原虫导致的疟疾死亡人数在过去十年中有所下降，但 非恶性疟原虫引起的疟疾病例正在上升。需要改进诊断来对此作出反应 不断变化的流行病学。然而，目前非洲的“检测和治疗”战略依赖于快速诊断检测。 (RDT)不能可靠地检测到大多数非恶性疟原虫感染，并可能受到富含组氨酸的危害 蛋白2(PfHRP2-)阴性寄生虫。我们建议开发和比较新型的重组酶聚合酶 放大-(RPA-)和基于CRISPR的诊断平台，有可能推出下一代 RDT的产生，能够灵敏地多重检测所有疟原虫物种。我们已经这么做了 成功地采用了RPA和名为Sherlock(特定高灵敏酶)的新技术 记者解锁)对于疟疾，达到了与实时聚合酶链式反应一样的检测极限。我们现在建议 解决疟疾和卵形疟原虫更难检测的问题。我们在中国的经验 诊断发展和评估、非恶性疟和低密度疟疾感染方面的专门知识，以及 与多名非洲国内调查人员的合作关系使我们特别适合开创这一先河 新技术。在目标1中，我们将开发和优化疟原虫和卵形疟原虫rpa和Sherlock 更高的灵敏度和稳健性，利用一系列以前允许的策略 多种病原体的简化、多路检测。我们将使用一种 RPA的多路侧向流动装置和Sherlock的简单荧光仪。检测结果的敏感性和特异性 新的化验方法将使用大量来自民主党的存档分离物样本库进行基准测试。 刚果共和国和坦桑尼亚。在《目标2》中，我们将现场测试表现最好的《神探夏洛克》。 坦桑尼亚巴加莫约的化验结果。我们将确定它们的敏感度，并将它们的性能与当前 RDT，以及实时荧光聚合酶链式反应。这些研究将利用前景看好的病原体新技术。 为在低资源环境下研究非恶性疟疾提供一个低成本的平台。如果 如果成功，它们将为开发针对物种的、敏感的RDTs奠定基础 非洲恶性疟原虫和非恶性疟疾的多重检测。

PROJECT SUMMARY While malaria deaths in Africa due to Plasmodium falciparum have fallen over the last decade, the proportion of malaria cases due to non-falciparum species is rising. Improved diagnostics are needed to respond to this changing epidemiology. Yet current “test and treat” strategies in Africa rely upon rapid diagnostic tests (RDTs) that do not reliably detect most non-falciparum infections and may be compromised by histidine-rich protein 2- (PfHRP2-) negative parasites. We propose to develop and compare novel recombinase polymerase amplification- (RPA-) and CRISPR-based diagnostic platforms that have the potential to launch the next generation of RDTs, capable of sensitive multiplexed detection of all Plasmodium species. We have already successfully adapted RPA and a new technology called SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) for malaria, reaching limits of detection on par with real-time PCR. We now propose to tackle the more difficult detection of Plasmodium malariae and Plasmodium ovale. Our experience in diagnostic development and evaluation, expertise in non-falciparum and low-density malaria infections, and collaborative relationship with multiple in-country African investigators make us uniquely suited to pioneer this new technology. In Aim 1, we will develop and optimize P. malariae and P. ovale RPA and SHERLOCK assays with enhanced sensitivity and robustness, utilizing a range of strategies that have previously allowed streamlined, multiplexed detection of multiple pathogens. We will detect target nucleic acids using a multiplexed lateral flow device for RPA and simple fluorimeter for SHERLOCK. Sensitivity and specificity of the new assays will be benchmarked using a large sample bank of archived isolates from the Democratic Republic of the Congo and Tanzania. In Aim 2, we will field test the best performing RPA or SHERLOCK assays in Bagamoyo, Tanzania. We will determine their sensitivity and compare their performance to current RDTs, as well as real-time PCR. These studies will leverage promising new technology for pathogen detection to provide a low-cost platform for the study of non-falciparum malaria in low-resource settings. If successful, they will lay the groundwork for development of species-specific, sensitive RDTs capable of multiplexed detection of both P. falciparum and non-falciparum malaria in Africa.