Ethiopia is one of the few African countries where Plasmodium vivax is co-endemic with P. falciparum. Malaria transmission is seasonal and transmission intensity varies mainly by landscape and climate. Although the recent emergence of drug resistant parasites presents a major issue to malaria control in Ethiopia, little is known about the transmission pathways of parasite species and prevalence of resistant markers. This study used microsatellites to determine population diversity and gene flow patterns of P. falciparum (N = 226) and P. vivax (N = 205), as well as prevalence of drug resistant markers to infer the impact of gene flow and existing malaria treatment regimes. Plasmodium falciparum indicated a higher rate of polyclonal infections than P. vivax. Both species revealed moderate genetic diversity and similar population structure. Populations in the northern highlands were closely related to the eastern Rift Valley, but slightly distinct from the southern basin area. Gene flow via human migrations between the northern and eastern populations were frequent and mostly bidirectional. Landscape genetic analyses indicated that environmental heterogeneity and geographical distance did not constrain parasite gene flow. This may partly explain similar patterns of resistant marker prevalence. In P. falciparum, a high prevalence of mutant alleles was detected in codons related to chloroquine (pfcrt and pfmdr1) and sulfadoxine-pyrimethamine (pfdhps and pfdhfr) resistance. Over 60% of the samples showed pfmdr1 duplications. Nevertheless, no mutation was detected in pfK13 that relates to artemisinin resistance. In P. vivax, while sequences of pvcrt-o were highly conserved and less than 5% of the samples showed pvmdr duplications, over 50% of the samples had pvmdr1 976F mutation. It remains to be tested if this mutation relates to chloroquine resistance. Monitoring the extent of malaria spread and markers of drug resistance is imperative to inform policy for evidence-based antimalarial choice and interventions. To effectively reduce malaria burden in Ethiopia, control efforts should focus on seasonal migrant populations. Sub-Saharan Africa is home to nearly 90% of malaria cases. In Ethiopia, two-thirds of the population lives in areas at risk of malaria infection. Malaria spread via human migrations and emergence of drug resistant parasites are major issues to malaria control in this country. Our study used microsatellite markers to determine gene flow patterns of P. falciparum and P. vivax in different parts of Ethiopia. We found that gene flow occurred across broad geographical distance and that environmental heterogeneity did not appear to influence gene flow. Unconstrained parasite gene flow may partly explain similar patterns of resistance marker prevalence across the country. While no mutation was detected in pfK13 that relates to artemisinin resistance in P. falciparum, over 50% of our P. vivax samples had pvmdr1 976F mutation that may relate to chloroquine resistance. This merits further clinical observations and/or in vitro testing. Our findings heighten the concern of chloroquine resistance for P. vivax malaria after more than a decade-application and suggests alternative treatment regime to alleviate the problem. Broadly, malaria control efforts should focus on seasonal migrant populations to effectively reduce malaria burden in Ethiopia.

Antimalarial drug resistance has threatened global malaria control since chloroquine (CQ)-resistant Plasmodium falciparum emerged in Asia in the 1950s. Understanding the impacts of changing antimalarial drug policy on resistance is critical for resistance management. Plasmodium falciparum isolates were collected from 2003 to 2015 in western Kenya and analyzed for genetic markers associated with resistance to CQ (Pfcrt), sulfadoxine–pyrimethamine (SP) (Pfdhfr/Pfdhps), and artemether–lumefantrine (AL) (PfKelch13/Pfmdr1) antimalarials. In addition, household antimalarial drug use surveys were administered. Pfcrt 76T prevalence decreased from 76% to 6% from 2003 to 2015. Pfdhfr/Pfdhps quintuple mutants decreased from 70% in 2003 to 14% in 2008, but increased to near fixation by 2015. SP “super resistant” alleles Pfdhps 581G and 613S/T were not detected in the 2015 samples that were assessed. The Pfmdr1 N86-184F-D1246 haplotype associated with decreased lumefantrine susceptibility increased significantly from 4% in 2005 to 51% in 2015. No PfKelch13 mutations that have been previously associated with artemisinin resistance were detected in the study populations. The increase in Pfdhfr/Pfdhps quintuple mutants that associates with SP resistance may have resulted from the increased usage of SP for intermittent preventative therapy in pregnancy (IPTp) and for malaria treatment in the community. Prevalent Pfdhfr/Pfdhps mutations call for careful monitoring of SP resistance and effectiveness of the current IPTp program in Kenya. In addition, the commonly occurring Pfmdr1 N86-184F-D1246 haplotype associated with increased lumefantrine tolerance calls for surveillance of AL efficacy in Kenya, as well as consideration for a rotating artemisinin-combination therapy regimen.

Background: Diversity in Plasmodium falciparum poses a major threat to malaria control and elimination interventions. This study utilized 12 polymorphic microsatellite (MS) markers and the Msp2 marker to examine diversity, multiplicity of infection (MOI) as well as the population structure of parasites circulating in two sites separated by about 92 km and with varying malaria transmission intensities within the Greater Accra Region of Ghana. Methods: The diversity and MOI of P. falciparum parasites in 160 non-symptomatic volunteers living in Obom (high malaria transmission intensity) and Asutsuare (low malaria transmission intensity) aged between 8 and 60 years was determined using Msp2 genotyping and microsatellite analysis. Results: The prevalence of asymptomatic P. falciparum carriers as well as the parasite density of infections was significantly higher in Obom than in Asutsuare. Samples from Asutsuare and Obom were 100% and 65% clonal, respectively, by Msp2 genotyping but decreased to 50% and 5%, respectively, when determined by MS analysis. The genetic composition of parasites from Obom and Asutsuare were highly distinct, with parasites from Obom being more diverse than those from Asutsuare. Conclusion: Plasmodium falciparum parasites circulating in Obom are genetically more diverse and distinct from those circulating in Asutsuare. The MOI in samples from both Obom and Asutsuare increased when assessed by MS analysis relative to MSP2 genotyping. The TA40 and TA87 loci are useful markers for estimating MOI in high and low parasite prevalence settings.

Plasmodium vivax malaria was thought to be rare in Africans who lack the Duffy blood group antigen expression. However, recent studies indicate that P. vivax can infect Duffy-negative individuals and has spread into areas of high Duffy negativity across Africa. Our study compared epidemiological and genetic features of P. vivax between African regions. A standardized approach was used to identify and quantify P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. The study involved sequencing the Duffy binding protein (DBP) gene and inferring genetic relationships among P. vivax populations across Africa. Among 1215 febrile patients, the proportions of Duffy negativity ranged from 20-36% in East Africa to 84% in southern Africa. Average P. vivax prevalence among Duffy-negative populations ranged from 9.2% in Sudan to 86% in Botswana. Parasite density in Duffy-negative infections was significantly lower than in Duffy-positive infections. P. vivax in Duffy-negative populations were not monophyletic, with P. vivax in Duffy-negative and Duffy-positive populations sharing similar DBP haplotypes and occurring in multiple, well-supported clades. Duffy-negative Africans are not resistant to P. vivax, and the public health significance of this should not be neglected. Our study highlights the need for a standardized approach and more resources/training directed towards the diagnosis of vivax malaria in Africa.

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 invasion of human erythrocytes relies on interaction between the Duffy antigen and P. vivax Duffy Binding Protein (PvDBP). Whole genome sequences from P. vivax field isolates in Madagascar identified a duplication of the PvDBP gene and PvDBP duplication has also been detected in non-African P. vivax-endemic countries. Two types of PvDBP duplications have been reported, termed Cambodian and Malagasy-type duplications. Our study used a combination of PCR-based diagnostic method, a novel quantitative real-time PCR assay, and whole genome sequencing to determine the prevalence and type of PvDBP duplications, as well as PvDBP copy number on a broad number of P. vivax samples in Ethiopia. We found that over 65% of P. vivax isolated from the symptomatic infections were detected with PvDBP duplications and PvDBP varied from 1 to >4 copies. The majority of PvDBP duplications belongs to the Cambodian-type while the Malagasy-type duplications was also detected. For the asymptomatic infections, despite a small sample size, the majority of P. vivax were detected with a single-copy based on both PCR and qPCR assays. There was no significant difference in PvDBP copy number between Duffy-null heterozygote and Duffy-positive homozygote/heterozygote. Further investigation is needed with expanded Duffy-null homozygotes to examine the functional significance of PvDBP expansion.

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 sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale. Plasmodium vivax is the most geographically widespread parasite species that causes malaria in humans. Although it occurs in Africa as a member of a mix of Plasmodium species, P. vivax is dominant in other parts of the world outside of Africa (e.g., Brazil). It was previously thought that most African populations were immune to P. vivax infections due to the absence of Duffy antigen chemokine receptor (DARC) gene expression required for erythrocyte invasion. However, several recent reports have indicated the emergence and potential spread of P. vivax across human populations in Africa. Compared to Southeast Asia and South America where P. vivax is highly endemic, data on polymorphisms in erythrocyte binding gene candidates of P. vivax from Africa is limited. Filling this knowlege gap is critical for identifying functional genes in erythrocyte invasion, biomarkers for tracking the P. vivax isolates from Africa, as well as potential gene targets for vaccine development. This paper examined the level of genetic polymorphisms in a panel of 43 potential erythrocyte binding protein genes based on whole genome sequences and described transmission patterns of P. vivax infections from different study sites in Ethiopia based on the genetic variants. Our analyses showed that chromosomes 9 and 10 of the P. vivax genomes isolated in Ethiopia had the most high-quality genetic polymorphisms. Among all erythrocyte binding protein gene candidates, the merozoite surface proteins 1 and merozoite surface protein 3 showed high levels of polymorphism. MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. The expansion of the Duffy binding protein and merozoite surface protein 3 gene copies was an independent process among the P. vivax lineages in Ethiopia. Various levels of gene flow were observed even at a smaller geographical scale. Our study provided baseline data for future comparison with P. vivax in Duffy negative individuals and help develop a panel of genetic markers that are informative at a micro-geographical scale.