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.

项目摘要 虽然由于恶性疟原虫造成的疟疾死亡在过去十年中下降了 需要改进的诊断来应对此事 改变流行病学。然而，非洲当前的“测试和治疗”策略依赖于快速诊断测试 （RDTS）无法可靠地检测到大多数非falciparum感染，并且可能会因富含组氨酸而损害 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，有可能启动下一个平台 RDT的产生，能够对所有疟原虫种类的敏感多路复用检测。我们已经 成功调整了RPA和一种称为Sherlock的新技术（特定的高灵敏度酶促性 疟疾的记者解锁），与实时PCR达到检测的限制。我们现在建议 解决对疟原虫和卵子疟原虫的更难检测。我们的经验 诊断开发和评估，非钙质和低密度疟疾感染的专业知识以及 与多个国家非洲调查人员的合作关系使我们非常适合此事 新技术。在AIM 1中，我们将开发和优化P.疟疾和P. ovale RPA和Sherlock 使用以前允许的一系列策略，具有增强的灵敏度和鲁棒性的测定 流线型的多重病原体的多路复用检测。我们将使用A检测到目标核酸 用于RPA的多路复用侧流设备，简单的荧光计用于夏洛克。灵敏度和特异性 新的测定将使用民主党的大量存档隔离物进行基准测试 刚果共和国和坦桑尼亚。在AIM 2中，我们将测试表现最好的RPA或Sherlock 在坦桑尼亚的Bagamoyo进行测定。我们将确定他们的敏感性，并将其表现与当前的表现进行比较 RDT以及实时PCR。这些研究将利用有希望的新技术为病原体 检测提供一个低成本平台，用于研究低资源环境中非fal骨疟疾。如果 成功，他们将为开发规格特定，敏感的RDT的开发奠定基础 非洲的恶性疟原虫和非falciparum疟疾的多路复用检测。

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