Development of novel diagnostics for African non-falciparum malaria

非洲非恶性疟疾新型诊断方法的开发

• 批准号: 10206017
• 负责人: Jessica Lin
• 金额: $ 23.08万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206017
• 关键词: Address; Affect; Africa; Africa South of the Sahara; African; Antigens; Archives; Benchmarking; Biological Assay; Blood; Cause of Death; Cessation of life; Characteristics; Clinical; Clinical Sensitivity; Clustered Regularly Interspaced Short Palindromic Repeats; Country; Democratic Republic of the Congo; Dengue Virus; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Drops; Epidemiology; Evaluation; Falciparum Malaria; Fingers; Fluorescence; Generations; HRP-2 protein; Human; Infection; Laboratories; Lactate Dehydrogenase; Lateral; Malaria; Methodology; Methods; Modernization; Mutation; Nucleic Acids; Parasites; Pathogen detection; Performance; Plasmodium; Plasmodium falciparum; Plasmodium malariae; Plasmodium ovale; Plasmodium vivax; Point of Care Technology; Polymerase; Prevalence; Rapid diagnostics; Reaction; Reporter; Reporting; Research Personnel; Resources; Ribonucleases; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Tanzania; Testing; Therapeutic; Time; Training; Translating; Work; World Health Organization; amplification detection; base; cost; density; detection limit; detection method; diagnostic platform; diagnostic technologies; experience; field study; handheld equipment; improved; isothermal amplification; malaria infection; mortality; multiplex detection; mutant; new technology; next generation; novel; novel diagnostics; nucleic acid detection; pathogen; point of care; point of care testing; programs; rapid test; recombinase; sample archive; trend

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.

项目概要 尽管过去十年中非洲因恶性疟原虫导致的疟疾死亡人数有所下降，但该比例 由非恶性疟原虫引起的疟疾病例正在上升。需要改进诊断来应对这一问题 改变流行病学。然而，非洲目前的“检测和治疗”策略依赖于快速诊断检测 （RDT）不能可靠地检测大多数非恶性疟原虫感染，并且可能受到富含组氨酸的影响 蛋白 2- (PfHRP2-) 阴性寄生虫。我们建议开发和比较新型重组酶聚合酶 基于扩增（RPA）和 CRISPR 的诊断平台有可能推出下一个 生成 RDT，能够对所有疟原虫物种进行灵敏的多重检测。我们已经 成功采用 RPA 和名为 SHERLOCK（特定高灵敏度酶促 Reporter Unlocking）针对疟疾，达到了与实时 PCR 相当的检测限。我们现在建议 解决更困难的三日疟原虫和卵形疟原虫检测问题。我们的经验 诊断开发和评估、非恶性疟和低密度疟疾感染方面的专业知识，以及 与多个非洲国内调查人员的合作关系使我们非常适合开拓这一领域 新技术。在目标 1 中，我们将开发和优化 P. malariae 和 P. ovale RPA 和 SHERLOCK 利用一系列以前允许的策略，提高了灵敏度和稳健性的测定 多种病原体的简化、多重检测。我们将使用 用于 RPA 的多重侧流装置和用于 SHERLOCK 的简单荧光计。敏感性和特异性 新的检测方法将使用来自民主党的存档分离株的大型样本库进行基准测试 刚果共和国和坦桑尼亚。在目标 2 中，我们将现场测试性能最佳的 RPA 或 SHERLOCK 坦桑尼亚巴加莫约的化验。我们将确定它们的灵敏度并将其性能与当前的性能进行比较 RDT 以及实时 PCR。这些研究将利用有前景的病原体新技术 检测，为资源匮乏地区的非恶性疟疾研究提供低成本平台。如果 如果成功的话，他们将为开发物种特异性、敏感的 RDT 奠定基础，该 RDT 能够 非洲恶性疟原虫和非恶性疟疾的多重检测。

项目成果

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

使用新型快速等温扩增侧流测定法检测三日疟原虫。

• DOI: 10.1101/2023.02.26.23286371
• 发表时间: 2023
• 期刊: medRxiv : the preprint server for health sciences
• 作者: Assefa,Ashenafi;Wamae,KevinK;Hennelly,ChrisM;Ngasala,Billy;Muller,Meredith;Kalonji,Albert;Phanzu,Fernandine;Cunningham,ClarkH;Lin,JessicaT;Parr,JonathanB
• 通讯作者: Parr,JonathanB