Copy Number Variations of Plasmodium vivax DBP1, EBP/DBP2, and RBP2b in Ethiopians Who Are Duffy Positive and Duffy Negative.

Recent evidence challenges the belief that individuals who are Duffy-negative are resistant to Plasmodium vivax due to lacking the Duffy antigen receptor for chemokines. Erythrocyte-binding protein (EBP/DBP2) has shown moderate binding to Duffy-negative erythrocytes in vitro. Reticulocyte-binding protein 2b (RBP2b) interactions with transferrin receptor 1 suggest involvement in Duffy-negative infections. Gene copy number variations in PvDBP1, PvEBP/DBP2, and PvRBP2b were investigated in Duffy-positive and Duffy-negative P vivax infections from Ethiopia. Among Duffy-positive samples, 34% displayed PvDBP1 duplications (Cambodian type). In Duffy-negative infections, 30% showed duplications, mostly Cambodian type. For PvEBP/DBP2 and PvRBP2b, Duffy-positive samples exhibited higher duplication rates (1-8 copies for PvEBP/DBP2, 46%; 1-5 copies for PvRBP2b, 43%) as compared with Duffy-negative samples (20.8% and 26%, respectively). The range of copy number variations was lower in Duffy-negative infections. Demographic and clinical factors associated with gene multiplications in both Duffy types were explored, enhancing understanding of P vivax evolution in Africans who are Duffy negative.

Using serological diagnostics to characterize remaining high-incidence pockets of malaria in forest-fringe Cambodia.

BACKGROUND: Over the last decades, the number of malaria cases has drastically reduced in Cambodia. As the overall prevalence of malaria in Cambodia declines, residual malaria transmission becomes increasingly fragmented over smaller remote regions. The aim of this study was to get an insight into the burden and epidemiological parameters of Plasmodium infections on the forest-fringe of Cambodia. METHODS: 950 participants were recruited in the province of Mondulkiri in Cambodia and followed up from 2018 to 2020. Whole-blood samples were processed for Plasmodium spp. identification by PCR as well as for a serological immunoassay. A risk factor analysis was conducted for Plasmodium vivax PCR-detected infections throughout the study, and for P. vivax seropositivity at baseline. To evaluate the predictive effect of seropositivity at baseline on subsequent PCR-positivity, an analysis of P. vivax infection-free survival time stratified by serological status at baseline was performed. RESULTS: Living inside the forest significantly increased the odds of P. vivax PCR-positivity by a factor of 18.3 (95% C.I. 7.7-43.5). Being a male adult was also a significant predictor of PCR-positivity. Similar risk profiles were identified for P. vivax seropositivity. The survival analysis showed that serological status at baseline significantly correlated with subsequent infection. Serology is most informative outside of the forest, where 94.0% (95% C.I. 90.7-97.4%) of seronegative individuals survived infection-free, compared to 32.4% (95% C.I.: 22.6-46.6%) of seropositive individuals. CONCLUSION: This study justifies the need for serological diagnostic assays to target interventions in this region, particularly in demographic groups where a lot of risk heterogeneity persists, such as outside of the forest.

Human monoclonal antibodies inhibit invasion of transgenic Plasmodium knowlesi expressing Plasmodium vivax Duffy binding protein.

BACKGROUND: Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). METHODS: Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). RESULTS: The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. CONCLUSION: The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.

The enigmatic mechanisms by which Plasmodium vivax infects Duffy-negative individuals.

The absence of the Duffy protein at the surface of erythrocytes was considered for decades to confer full protection against Plasmodium vivax as this blood group is the receptor for the key parasite ligand P. vivax Duffy binding protein (PvDBP). However, it is now clear that the parasite is able to break through this protection and induce clinical malaria in Duffy-negative people, although the underlying mechanisms are still not understood. Here, we briefly review the evidence of Duffy-negative infections by P. vivax and summarize the current hypothesis at the basis of this invasion process. We discuss those in the perspective of malaria-elimination challenges, notably in African countries.

Choosing interventions to eliminate forest malaria: preliminary results of two operational research studies inside Cambodian forests.

BACKGROUND: Rapid elimination of Plasmodium falciparum malaria in Cambodia is a goal with both national and international significance. Transmission of malaria in Cambodia is limited to forest environments, and the main population at risk consists of forest-goers who rely on forest products for income or sustenance. The ideal interventions to eliminate malaria from this population are unknown. METHODS: In two forested regions of Cambodia, forest-goers were trained to become forest malaria workers (FMWs). In one region, FMWs performed mass screening and treatment, focal screening and treatment, and passive case detection inside the forest. In the other region, FMWs played an observational role for the first year, to inform the choice of intervention for the second year. In both forests, FMWs collected blood samples and questionnaire data from all forest-goers they encountered. Mosquito collections were performed in each forest. RESULTS: Malaria prevalence by PCR was high in the forest, with 2.3-5.0% positive for P. falciparum and 14.6-25.0% positive for Plasmodium vivax among forest-goers in each study site. In vectors, malaria prevalence ranged from 2.1% to 9.6%, but no P. falciparum was observed. Results showed poor performance of mass screening and treatment, with sensitivity of rapid diagnostic tests equal to 9.1% (95% CI 1.1%, 29.2%) for P. falciparum and 4.4% (95% CI 1.6%, 9.2%) for P. vivax. Malaria infections were observed in all demographics and throughout the studied forests, with no clear risk factors emerging. CONCLUSIONS: Malaria prevalence remains high among Cambodian forest-goers, but performance of rapid diagnostic tests is poor. More adapted strategies to this population, such as intermittent preventive treatment of forest goers, should be considered.

Identification and Characterization of Functional Human Monoclonal Antibodies to Plasmodium vivax Duffy-Binding Protein.

Plasmodium vivax invasion of reticulocytes relies on distinct receptor-ligand interactions between the parasite and host erythrocytes. Engagement of the highly polymorphic domain II of the P. vivax Duffy-binding protein (DBPII) with the erythrocyte's Duffy Ag receptor for chemokines (DARC) is essential. Some P. vivax-exposed individuals acquired Abs to DBPII that block DBPII-DARC interaction and inhibit P. vivax reticulocyte invasion, and Ab levels correlate with protection against P. vivax malaria. To better understand the functional characteristics and fine specificity of protective human Abs to DBPII, we sorted single DBPII-specific IgG+ memory B cells from three individuals with high blocking activity to DBPII. We identified 12 DBPII-specific human mAbs from distinct lineages that blocked DBPII-DARC binding. All mAbs were P. vivax strain transcending and targeted known binding motifs of DBPII with DARC. Eleven mAbs competed with each other for binding, indicating recognition of the same or overlapping epitopes. Naturally acquired blocking Abs to DBPII from individuals with high levels residing in different P. vivax-endemic areas worldwide competed with mAbs, suggesting broadly shared recognition sites. We also found that mAbs inhibited P. vivax entry into reticulocytes in vitro. These findings suggest that IgG+ memory B cell activity in individuals with P. vivax strain-transcending Abs to DBPII display a limited clonal response with inhibitory blocking directed against a distinct region of the molecule.

Impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on Plasmodium vivax drug susceptibility in Cambodia.

BACKGROUND: Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite's intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. OBJECTIVES: To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. METHODS: In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. RESULTS: Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4-22.3) chloroquine, 3.4 nM (IQR 2.1-5.0) mefloquine and 4.6 nM (IQR 2.7-7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. CONCLUSIONS: P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017-18.

Recrudescence, Reinfection, or Relapse? A More Rigorous Framework to Assess Chloroquine Efficacy for Plasmodium vivax Malaria.

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes. Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area. Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones. Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Therapeutic and Transmission-Blocking Efficacy of Dihydroartemisinin/Piperaquine and Chloroquine against Plasmodium vivax Malaria, Cambodia.

We assessed the efficacy of standard 3-day courses of chloroquine and dihydroartemisinin/piperaquine against Plasmodium vivax malaria. Compared with chloroquine, dihydroartemisinin/piperaquine was faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission. This drug combination was also more effective in preventing potential recurrences for >2 months.

Reduced polymorphism in the Kelch propeller domain in Plasmodium vivax isolates from Cambodia.

Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism.

Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay.

BACKGROUND: A major requirement for malaria elimination is the development of transmission-blocking interventions. In vitro transmission-blocking bioassays currently mostly rely on the use of very few Plasmodium falciparum reference laboratory strains isolated decades ago. To fill a piece of the gap between laboratory experimental models and natural systems, the purpose of this work was to determine if culture-adapted field isolates of P. falciparum are suitable for in vitro transmission-blocking bioassays targeting functional maturity of male gametocytes: exflagellation. METHODS: Plasmodium falciparum isolates were adapted to in vitro culture before being used for in vitro gametocyte production. Maturation was assessed by microscopic observation of gametocyte morphology over time of culture and the functional viability of male gametocytes was assessed by microscopic counting of exflagellating gametocytes. Suitability for in vitro exflagellation-blocking bioassays was determined using dihydroartemisinin and methylene blue. RESULTS: In vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia. Functional maturity of male gametocytes was assessed by exflagellation observations and all four isolates could be used in exflagellation-blocking bioassays with adequate response to methylene blue and dihydroartemisinin. CONCLUSION: This work shows that in vitro culture-adapted P. falciparum field isolates of different genetic background, from South America and Southeast Asia, can successfully be used for bioassays targeting the male gametocyte to gamete transition, exflagellation.

Effects of mefloquine use on Plasmodium vivax multidrug resistance.

Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non-P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites.

The toll and Imd pathways are not required for wolbachia-mediated dengue virus interference.

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virus-blocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system.

Unraveling the intricacies of host-pathogen interaction through single-cell genomics.

Single-cell genomics provide researchers with tools to assess host-pathogen interactions at a resolution previously inaccessible. Transcriptome analysis, epigenome analysis, and immune profiling techniques allow for a better comprehension of the heterogeneity underlying both the host response and infectious agents. Here, we highlight technological advancements and data analysis workflows that increase our understanding of host-pathogen interactions at the single-cell level. We review various studies that have used these tools to better understand host-pathogen dynamics in a variety of infectious disease contexts, including viral, bacterial, and parasitic diseases. We conclude by discussing how single-cell genomics can advance our understanding of host-pathogen interactions.

Comparative transcriptomics reveal differential gene expression among Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms.

The documentation of Plasmodium vivax malaria across Africa especially in regions where Duffy negatives are dominant suggests possibly alternative erythrocyte invasion mechanisms. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa is unclear. In this study, we examined the expression of 4,404 gene transcripts belong to 12 functional groups and 43 erythrocyte binding gene candidates in Ethiopian isolates and compared them with the Cambodian and Brazilian P. vivax transcriptomes. Overall, there were 10-26% differences in the gene expression profile amongst geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian isolates. Members of the reticulocyte binding protein PvRBP2a and PvRBP3 expressed six-fold higher than Duffy binding protein PvDBP1 and 60-fold higher than PvEBP/DBP2 in the Ethiopian isolates. Other genes including PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression patterns were observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, gametocyte genes including PvAP2-G, PvGAP (female gametocytes), and Pvs47 (male gametocytes) were highly expressed across geographical samples.

Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

Intermittent preventive treatment for forest goers by forest malaria workers: an observational study on a key intervention for malaria elimination in Cambodia.

Background: Cambodia targets P. falciparum malaria elimination by 2023 and all human malaria species by 2025, aligning with WHO's Mekong Malaria Elimination program. The Intermittent Preventive Treatment for Forest Goers (IPTf) project aimed at forest-specific malaria elimination. The study aims to pinpoint the main factors driving malaria transmission in Cambodian forests and evaluate the initial implementation and effectiveness of IPTf in accelerating the elimination of malaria by treating and preventing infections among at-risk populations in these areas. Methods: From March 11, 2019, to January 30, 2021, a malaria intervention program took place in isolated forests in Northeast Cambodia. The first phase focused on observing forest goers (FGs) within the forests, documenting their malaria risk. In the second phase, a monthly artesunate-mefloquine IPTf was implemented by trained forest malaria workers who were former FGs conducting interviews, blood collection, and IPTf administration. Findings: Throughout the two-year period, 2198 FGs were involved in 3579 interviews, with 284 in both the observation and intervention phases. Following IPTf implementation, PCR-confirmed malaria prevalence significantly decreased from 2.9% to 0.5% for P. falciparum and from 21.0% to 4.7% for P. vivax. Among the 284 participants tracked through both phases, malaria prevalence fell from 2.5% to 0.3% for P. falciparum and from 22.5% to 3.7% for P. vivax. The intervention phase demonstrated a rapid decline in P. falciparum prevalence among mobile and previously inaccessible populations, while also revealing a higher P. falciparum infection risk associated with activities inaccurately labelled as farming, underscoring the need for customized interventions. Interpretation: The successful implementation of IPTf in Cambodia's remote forests has markedly decreased malaria prevalence among high-risk groups. Cambodia's National Malaria Program has acknowledged this strategy as essential for malaria elimination intervention, endorsing forest-specific approaches to meet the 2025 goal of eradicating all human malaria species in Cambodia. Funding: The study received funding from the French 5% Initiative (Initiative Canal 2-17SANIN205).

Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial.

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study.

BACKGROUND: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. FINDINGS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. INTERPRETATION: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. FUNDING: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.