Malaria Diagnosis with Focus in hrp2/3 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.