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Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome PLOS NEGLECTED TROPICAL DISEASES

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly.

Plasmodium vivax malaria was thought to be rare in Africa, but an increasing number of P. vivax cases reported across Africa and in Duffy-negative individuals challenges this conventional dogma. The genetic characteristics of P. vivax in Duffy-negative infections, the transmission of P. vivax in East Africa, and the impact of environments on transmission remain largely unknown. This study examined genetic and transmission features of P. vivax from 107 Duffy-negative and 305 Duffy-positive individuals in Ethiopia and Sudan. No clear genetic differentiation was found in P. vivax between the two Duffy groups, indicating between-host transmission. P. vivax from Ethiopia and Sudan showed similar genetic clusters, except samples from Khartoum, possibly due to distance and road density that inhibited parasite gene flow. This study is the first to show that P. vivax can transmit to and from Duffy-negative individuals and provides critical insights into the spread of P. vivax in sub-Saharan Africa.

Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria. The documentation of P. vivax infections in different parts of Africa where Duffy-negative individuals are predominant suggested that there are alternative pathways for P. vivax to invade human erythrocytes. Duffy-negative individuals may be just as fit as Duffy-positive individuals and are no longer resistant to P.vivax malaria. In this review, we describe the complexity of P. vivax malaria, characterize pathogenesis and candidate invasion genes of P. vivax, and host immune responses to P. vivax infections. We provide a comprehensive review on parasite ligands in several Plasmodium species that further justify candidate genes in P. vivax. We also summarize previous genomic and transcriptomic studies related to the identification of ligand and receptor proteins in P. vivax erythrocyte invasion. Finally, we identify topics that remain unclear and propose future studies that will greatly contribute to our knowledge of P. vivax.