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BackgroundThe interaction between thePlasmodium vivaxDuffy-binding protein and the corresponding Duffy Antigen Receptor for Chemokines (DARC) is primarily responsible for the invasion of reticulocytes byP. vivax. The Duffy-negative host phenotype, highly prevalent in sub-Saharan Africa, is caused by a single point mutation in the GATA-1 transcription factor binding site of the DARC gene promoter. The aim of this study was to assess the Duffy status of patients withP. vivaxinfection from different study sites in Ethiopia.MethodsA cross-sectional study was conducted from February 2021 to September 2022 at five varying eco-epidemiological malaria endemic sites in Ethiopia. Outpatients who were diagnosed withP. vivaxinfection (pure and mixedP. vivax/P. falciparum) by microscopy were subjected to qPCR genotyping at the DARC promoter. The associations betweenP. vivaxinfection, host genotypes and other factors were evaluated.ResultIn total, 361 patients withP. vivaxinfection were included in the study. Patients with pureP. vivaxinfections accounted for 89.8% (324/361), while the remaining 10.2% (37/361) had mixedP. vivax/P. falciparuminfections. About 95.6% (345/361) of the participants were Duffy-positives (21.2% homozygous and 78.8%, heterozygous) and 4.4% (16/361) were Duffy-negatives. The mean asexual parasite density in homozygous and heterozygous Duffy-positives was 12,165 p/µl (IQR25-75: 1,640-24,234 p/µl) and11,655 p/µl (IQR25-75: 1,676-14,065 p/µl), respectively, significantly higher than that in Duffy-negatives (1,227p/µl; IQR25-75: 539-1,732p/µl).ConclusionThis study confirms that Duffy-negativity does not provide complete protection againstP. vivaxinfection. The development ofP. vivax-specific elimination strategies, including alternative antimalarial vaccines should be facilitated by a better understanding of the epidemiological landscape ofvivaxmalaria in Africa. More importantly, low parasitemia associated withP. vivaxinfections in Duffy-negative patients may represent hidden reservoirs of transmission in Ethiopia.Author SummaryPlasmodium vivaxgenerally receives less attention thanP. falciparumand was neglected in sub-Saharan Africa. However, the characteristics ofP. vivaxinfection in Duffy-negative individuals, and the distribution of Duffy blood group in different eco-epidemiological zones and ethnic groups of Ethiopia are not well documented. Here, we determined the Duffy genotypes ofP. vivaxinfected patients across broad regions of Ethiopia. It is clear that Duffy negative individuals in Ethiopia are not fully protected againstP. vivaxinfection, and that these infections in Duffy negatives are often associated with low parasitemia. Our findings lend support to the notion thatP. vivaxmay have developed a Duffy-independent erythrocyte invasion pathway and/or evolution in host immune responses.

Challenges in understanding the origin of recurrentPlasmodium vivaxinfections constrains the surveillance of antimalarial efficacy and transmission of this neglected parasite. Recurrent infections within an individual may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or new inoculations (reinfection). Molecular inference of familial relatedness (identity-by-descent or IBD) based on whole genome sequence data, together with analysis of the intervals between parasitaemic episodes (“time-to-event” analysis), can help resolve the probable origin of recurrences. Whole genome sequencing of predominantly low-densityP. vivaxinfections is challenging, so an accurate and scalable genotyping method to determine the origins of recurrent parasitaemia would be of significant benefit. We have developed aP. vivaxgenome-wide informatics pipeline to select specific microhaplotype panels that can capture IBD within small, amplifiable segments of the genome. Using a global set of 615P. vivaxgenomes, we derived a panel of 100 microhaplotypes, each comprising 3-10 high frequency SNPs within HE= 0.70-0.81) and it captured 89% (273/307) of the polyclonal infections detected with genome-wide datasets. Using data simulations, we demonstrate lower error in estimating pairwise IBD using microhaplotypes, relative to traditional biallelic SNP barcodes. Our panel exhibited high accuracy in predicting the country of origin (median Matthew’s correlation coefficient >0.9 in 90% countries tested) and it also captured local infection outbreak and bottlenecking events. The informatics pipeline is available open-source and yields microhaplotypes that can be readily transferred to high-throughput amplicon sequencing assays for surveillance in malaria-endemic regions.

The emergence and spread of drug- and diagnostic-resistantPlasmodium falciparumare major impediments to malaria control and elimination. We deep sequenced known drug resistance mutations and other informative loci across the genome of 609 samples collected during a study across three regions of Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused byP. falciparumcarrying the candidate artemisinin partial-resistanceK13622I mutation, which occurred less commonly in diagnostic-resistantpfhrp2/3-deleted than normal non-deleted parasites (p=0.03). Identity-by-descent analysis showed that 622I parasites were significantly more related than wild-type (pPfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Parasites carrying bothpfhrp2/3deletion and 622I mutation were observed in some sites. These findings raise concern for future spread of combined drug- and diagnostic-resistant parasites and warrant close monitoring.

P. malariaeis found worldwide and causes chronic parasitism in its human hosts. We developed aP. malariae (Pm)diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies /µL (∼1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was 35 minutes. Combined with simplified DNA extraction methods, the assay has potential for future field-deployable point-of-care use to detect a parasite species that remains largely undiagnosed.

Histidine-rich protein 2- (HRP2-) based rapid diagnostic tests (RDTs) are widely used to detectPlasmodium falciparumin sub-Saharan Africa. Reports of parasites withpfhrp2and/orpfhrp3 (pfhrp2/3)gene deletions in Africa raise concerns about the long-term viability of HRP2-based RDTs. We evaluated changes inpfhrp2/3deletion prevalence over time using a 2018-2021 longitudinal study of 1,635 enrolled individuals in Kinshasa Province, Democratic Republic of the Congo (DRC). Samples collected during biannual household visits with ≥ 100 parasites/μL by quantitative real-time PCR were genotyped using a multiplex real-time PCR assay. Among 2,726 P.falciparumPCR-positive samples collected from 993 participants during the study period, 1,267 (46.5%) were genotyped. Nopfhrp2/3deletions or mixedpfhrp2/3-intact and -deleted infections were identified in our study.Pfhrp2/3-deleted parasites were not detected in Kinshasa Province; ongoing use of HRP2-based RDTs is appropriate.

Imported cases present a considerable challenge to the elimination of malaria. Traditionally, patient travel history has been used to identify imported cases, but the long-latency liver stages confound this approach in Plasmodium vivax. Molecular tools to identify and map imported cases offer a more robust approach, that can be combined with drug resistance and other surveillance markers in high-throughput, population-based genotyping frameworks. Using a machine learning approach incorporating hierarchical FST (HFST) and decision tree (DT) analysis applied to 831 P. vivax genomes from 20 countries, we identified a 28-Single Nucleotide Polymorphism (SNP) barcode with high capacity to predict the country of origin. The Matthews correlation coefficient (MCC), which provides a measure of the quality of the classifications, ranging from −1 (total disagreement) to 1 (perfect prediction), exceeded 0.9 in 15 countries in cross-validation evaluations. When combined with an existing 37-SNP P. vivax barcode, the 65-SNP panel exhibits MCC scores exceeding 0.9 in 17 countries with up to 30% missing data. As a secondary objective, several genes were identified with moderate MCC scores (median MCC range from 0.54-0.68), amenable as markers for rapid testing using low-throughput genotyping approaches. A likelihood-based classifier framework was established, that supports analysis of missing data and polyclonal infections. To facilitate investigator-lead analyses, the likelihood framework is provided as a web-based, open-access platform (vivaxGEN-geo) to support the analysis and interpretation of data produced either at the 28-SNP core or full 65-SNP barcode. These tools can be used by malaria control programs to identify the main reservoirs of infection so that resources can be focused to where they are needed most.