Malaria Diagnosis with Focus in <em>hrp2/3 </em>Deletion

Malaria diagnosis plays a crucial role in the control and eventual elimination of the disease. In many malaria-endemic areas, diagnosis relies on microscopy and rapid diagnostic tests (RDTs). RDTs can yield false-negative results due to various factors, including the presence of parasites with deletions in the hrp2/3 genes which encode the HRP2 and HRP3 proteins detected by most RDTs. This dissertation aimed to develop and test a novel hrp2/3 deletion detection method that can be used not only in the laboratory but also in the field; additionally, the characterization of samples that carry the deletion of hrp2/3 from Kenya, Ghana, Ethiopia, Brazil, and Ecuador was performed.
First, I validated a novel assay for molecular surveillance of hrp2/hrp3 deletions based on droplet digital PCR (ddPCR). The assay quantifies hrp2, hrp3, and a control gene with very high accuracy. I compared side-by-side the performance of the conventional nested PCR (nPCR) assay and ddPCR assay. Then, I apply the ddPCR assay to screen 739 samples from Kenya, Ghana, Ethiopia, Brazil, and Ecuador.
Second, I optimized hrp2/3 protocols for typing on a portable dPCR platform (LOAA), including simplified DNA extraction protocols and extraction from archived RDTs. I adapted a more efficient method for DNA recovery with RDTs as a sample source. In a pilot study I applied my method to archived RDT samples from Gondar, Ethiopia.
Third, using the hrp2/3 ddPCR data from Brazilian and Ecuadorian samples, I characterized wild-type parasites and hrp2 deleted parasites by 6 microsatellite markers located in the flanking regions of hrp2. I found that it was impossible to analyze the samples because of the abundant presence of stutter peaks in almost all the markers. However, the marker MS -41, which was present in all the samples, suggested that the hrp2 deleted samples from Brazil were the product of at least one hrp2 deletion event.
In conclusion, this dissertation highlights the importance of molecular diagnosis in halting the transmission of hrp2/3-deletion-carrying parasites for effective malaria control.

疟疾诊断在该疾病的防控与最终消除中发挥着至关重要的作用。在诸多疟疾流行区，诊断主要依赖显微镜检查与快速诊断检测（rapid diagnostic tests, RDTs）。受多种因素影响，快速诊断检测可能出现假阴性结果，其中包括寄生虫的hrp2/3基因（hrp2/3 genes）发生缺失——大多数快速诊断检测正是通过检测该基因编码的HRP2和HRP3蛋白（HRP2 and HRP3 proteins）来实现诊断的。本论文旨在开发并验证一种新型hrp2/3基因缺失检测方法，该方法既可用于实验室场景，也可应用于现场检测；此外，本研究还对来自肯尼亚、加纳、埃塞俄比亚、巴西以及厄瓜多尔的hrp2/3基因缺失样本进行了特征分析。
首先，本研究验证了一种基于液滴数字PCR（droplet digital PCR, ddPCR）的新型hrp2/hrp3缺失分子监测检测方法。该检测方法可对hrp2、hrp3以及内参基因进行高精度定量。本研究对传统巢式PCR（nested PCR, nPCR）检测方法与液滴数字PCR检测方法的性能进行了平行对比。随后，本研究采用液滴数字PCR检测方法，对来自肯尼亚、加纳、埃塞俄比亚、巴西以及厄瓜多尔的739份样本进行了筛选。
其次，本研究优化了适用于便携式数字PCR平台（portable dPCR platform, LOAA）分型的hrp2/3实验流程，其中包括简化的DNA提取流程以及针对存档快速诊断检测样本的提取方法。本研究还改良了一种以快速诊断检测样本为起始材料的高效DNA回收方法。在一项预实验中，本研究将该方法应用于来自埃塞俄比亚贡达尔的存档快速诊断检测样本。
第三，本研究借助来自巴西与厄瓜多尔样本的hrp2/3液滴数字PCR数据，利用位于hrp2侧翼区域的6个微卫星标记（microsatellite markers）对野生型寄生虫与hrp2缺失型寄生虫进行了特征分析。研究发现，由于几乎所有标记均出现大量滑动峰（stutter peaks），无法对样本进行有效分析。不过，所有样本均携带的MS-41标记提示，来自巴西的hrp2缺失样本至少经历了一次hrp2基因缺失事件。
综上，本论文强调了分子诊断在阻断携带hrp2/3基因缺失的寄生虫传播、实现疟疾有效防控中的重要意义。