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

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45 Plasmodium vivax malaria is much less common in Africa than the rest of the world 46 because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) 47 to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, 48 there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a 49 high number of them being in Duffy negative individuals, potentially indicating P. vivax 50 has evolved an alternative invasion mechanism that can overcome Duffy negativity.51 Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation 52 (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from 53 symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and 54 Duffy negative individuals are found. A total of 236,351 SNPs were detected, of which 55 21.9% was nonsynonymous and 78.1% was synonymous mutations. The largest 56 number of SNPs were detected on chromosomes 9 (33,478 SNPs; 14% of total) and 10 57 (28,133 SNPs; 11.9%). There were particularly high levels of polymorphism in 58 erythrocyte binding gene candidates including reticulocyte binding protein 2c (RBP2c), 59 merozoite surface protein 1 (MSP1), and merozoite surface protein 3 (MSP3.5, 60 MSP3.85 and MSP3.9). Thirteen genes related to immunogenicity and erythrocyte 61 binding function were detected with significant signals of positive selection. Variation in 62 gene copy number was also concentrated in genes involved in host-parasite 63 interactions, including the expansion of the Duffy binding protein gene (PvDBP) on 64 chromosome 6 and several PIR genes. Based on the phylogeny constructed from the 65 whole genome sequences, the expansion of these genes was an independent process 66 among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. 67 vivax infections among study sites and showed various levels of gene flow at a small 68 geographical scale. The genomic features of P. vivax provided baseline data for future 69 comparison with those in Duffy-negative individuals, and allowed us to develop a panel 70 of informative Single Nucleotide Polymorphic markers diagnostic at a micro-71 geographical scale. 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 90 Vivax malaria is the most geographically widespread human malaria, causing over 130 91 million clinical cases per year worldwide [1]. Plasmodium vivax can produce dormant 92 liver-stage hypnozoites within infected hosts, giving rise to relapse infections from 93 months to years. This unique feature of P. vivax contributes to an increase in 94 transmission potential and increases the challenge of elimination [2]. Understanding P.95 vivax genome variation will advance our knowledge of parasite biology and host-96 parasite interactions, as well as identify potential drug resistance mechanisms [3, 4].97 Such data will also help identify molecular targets for vaccine development [5][6][7], and 98 provide new means to track the transmission and spread of ...

The identification and management of low parasitemia infections have become increasingly challenging for malaria control and elimination. Submicroscopic Plasmodium infections and G6PD deficiency among febrile patients require more sensitive diagnostic methods to improve detection and careful treatment regime of these infections. In Ethiopia, information on the low density submicroscopic malarial infections and frequency of G6PD deficiency (G6PDd) is scarce. In this study, 297 malaria suspected febrile patient samples were collected from health facilities of Bonga town in southwestern Ethiopia. The positivity rates of Plasmodium infection were determined by microscopy and quantitative PCR. G6PD activity level was determined by careSTART™ G6PD biosensor and the frequency of three common variants: G6PD*A (A376G), G6PD*A− (G202A) and Mediterranean (C563T) were investigated. G6PD gene sequencing was performed to detect mutations in exons 2–11 for both G6PD normal and deficient samples based on the phenotypic assay. More than twice Plasmodium infected samples was detected by qPCR (52/297; 17.4%) than microscopy (21/297; 7.0%). About 31 (10%) of the infections were submicroscopic. Bednet usage and age had a significant association with Plasmodium infection. Of the 271 participants who were tested for G6PD phenotype, 19 (7.0%) had low G6PD level. No mutations were observed in A376G, G202A, and C563T in the G6PDd samples, but three novel non-synonymous mutations in exon 2 including a C to T transition at position ChrX:6504 (Arg to Thr), G to T at ChrX:6369 (Ser to IIe), and G to C at ChrX:6664 (Gln to His) were detected. A high number of submicroscopic Plasmodium infections observed in this study pose a challenge for accurate and timely diagnosis, which could hinder malaria control efforts. G6PD deficiency in malaria patients pose danger when treating patients with primaquine. The three novel mutations detected in exon 2 of the G6PD gene merit further investigation on the hemolytic risk when exposed to oxidative antimalarials, their prevalence, and clinical significance.