UC Irvine

In the last two decades’ tremendous progress has been made towards global goals of malaria control and elimination, yet there were still an estimated 227 million cases in 20191. While the vast majority of these cases occur via the malaria species Plasmodium falciparum on the African continent, Plasmodium vivax remains the most geographically widespread. It is endemic to large swaths of Asia and South America, and remains among the most challenging of tropical diseases to control and eradicate due to its unique biology and global presence2. Additionally, while Plasmodium vivax was once considered to be a benign infection, it is now recognized as a significant global health threat3. Latent liver-stage hypnozoites can cause relapse weeks or months after initial infection, and difficulties in hypnoizoite treatment related to G6PD deficiency based primaquine toxicity4-6 significantly hinder control and intervention efforts. In spite of its global dispersal, P. vivax is relatively absent in Sub-Saharan Africa. The lack of P. vivax endemicity in Africa has long been attributed to the near fixation of the Duffy negative phenotype on the continent7. The Duffy antigen receptor for chemokines (DARC) was established as the required binding antigen for P. vivax merozoites over four decades ago8, yet recent work has highlighted the occurrence of P. vivax in confirmed Duffy negatives in several countries throughout Africa9-13. New attention has been given to these rare cases that challenge historical dogma and epidemiology; questioning how these infections are occurring, if there are alternate erythrocyte invasion mechanisms at play, if these infections are novel or merely historically undetected, and importantly what public health significance do they pose to Duffy negative individuals throughout Africa. The presented dissertation explores these infections through multiple lenses; investigating immunology, epidemiology and population genetics of unconventional Plasmodium vivax infections in Duffy negative people in Ethiopia. In our first chapter we assess the impact Duffy genotype has on naturally acquired immunity. We calculated seroreactivity to a suite of Plasmodium falciparum and P. vivax target antigens and sequenced two sections of the DARC gene to interpret Duffy genotype in humans. We found that Duffy negative individuals developed significantly reduced immune responses to P. vivax antigens compared to P. falciparum antigens across almost all antigenic targets. Additionally, we assessed seroprevalence and found that overall immune responses to P. vivax were greatly diminished in Duffy negative individuals highlighting the need for consideration of Duffy heterogeneity in control efforts and vaccine development. In chapter 2 we first optimized a pooling strategy in order to process copious numbers of Dried Blood Spots (DBS), then assessed malaria prevalence and parasitemia in individuals of different Duffy phenotypes. Out of over 15 thousand samples a mere 15 were both P. vivax positive and Duffy negative, highlighting the rarity of these infections in Ethiopia. Interestingly we found that these infections were most commonly found as mixed co-infections with Plasmodium falciparum, and that parasitemia did not vary between Duffy phenotype. Our third chapter is a literature review which sought to explore the best population genetic approaches currently available for malaria epidemiology. We found that despite microsatellite, SNP-barcode, and whole genome sequencing (WGS) all being commonly used with great success, each individually falls short in some aspect, making intensive population genetic and genomic analyses of malaria epidemiology challenging. Highly multiplexed amplicon deep sequencing for microhaplotypes is a novel genetic approach that poses great promise for garnering high-resolution genetic data and in this chapter we present recent developments of this technology, highlight its novel use in P. falciparum genomic exploration, and argue for its superiority as a population genetics approach and tool for malaria and specifically P. vivax malaria.