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The endothelial protein C receptor (EPCR)–rs867186 G allele has been linked to high plasma levels of soluble EPCR (sEPCR) and controversially associated with either susceptibility or resistance to severe and cerebral malaria. In this study, quantitative enzyme-linked immunosorbent assay and sequencing were used to assess sEPCR levels and EPCR-rs867186 polymorphism in blood samples from Beninese children with different clinical presentations of malaria. Our findings show that sEPCR levels were higher at hospital admission than during convalescence and that EPCR-rs867186 G allele was associated with increased sEPCR plasma levels, malaria severity, and mortality rate (P < .001, P = .03, and P = .04, respectively), suggesting a role of sEPCR in the pathogenesis of severe malaria.

Symptomatic and asymptomatic malaria patients are considered as the reservoirs of human Plasmodium. In the present study, we have evaluated the Plasmodium falciparum merozoite surface protein-1 (Pfmsp1) and protein-2 (Pfmsp2) genetic diversity among the symptomatic and asymptomatic malaria infection from health facilities in Cotonou, Benin Republic. A cross-sectional study recruited 158 individuals, including 77 from the asymptomatic and 81 from the symptomatic groups. The parasites were genotyped using Nested Polymerase Chain Reaction. Samples identified as Plasmodium falciparum were genotyped for their genetic diversity. No significant difference was observed in the overall multiplicity of infection (MOI) between the asymptomatic and symptomatic groups. In the symptomatic group, the overall frequency of K1, MAD20, and RO33 allelic family was more predominant (98.5%) followed by 3D7 (87.3%) and FC27 (83.1%). However, in asymptomatic group, the K1 alleles were the most prevalent (100%) followed by FC27 (89.9%), 3D7 (76.8%), MAD20 (60.5%), and RO33 (35.5%). The frequency of multiple allelic types (K1+MAD20+RO33) at the Pfmsp1 loci in the symptomatic infections was significantly higher when compared to that of the asymptomatic ones (97% vs. 34%, p 0.05), whereas no difference was observed in the frequency of multiple allelic types (3D7 and FC27) at the Pfmsp2 loci between the two groups. The high presence of msp1 multiple infections in the symptomatic group compared to asymptomatic ones suggests an association between the genetic diversity and the onset of malaria symptoms. These data can provide valuable information in the development of a vaccine that could reduce the symptomatic disease.

The EPCR-rs867186-G allele has been linked to high plasma level of soluble-EPCR and controversially associated with either susceptibility or resistance to severe and cerebral malaria. In this study, quantitative-ELISA and sequencing were used to assess sEPCR levels and EPCR-rs867186 polymorphism in blood samples from Beninese children with different clinical presentations of malaria. Our findings show that sEPCR levels were higher at admission to hospital than during convalescence and that EPCR-rs867186-G-allele was associated with increased sEPCR plasma levels, severe malaria and mortality (p-values .0001, = .03 and = .04, respectively), suggesting a role of sEPCR in the pathogenesis of severe malaria.

Background: Malaria-related deaths could be prevented if prompt diagnosis and prognostic biomarkers are available to allow rapid adequate treatment. Understanding of the mechanisms implicated in the progression from P. falciparum asymptomatic and uncomplicated malaria infections to severe life-threatening disease is necessary to identify such indicators. Methods: Using quantitative ELISA, we assessed the plasma concentrations of Procalcitonin, Pentraxine 3, Ang-2, sTie-2, suPAR, sEPCR, and sICAM-1 in a cohort of 337 Beninese children who presented with pediatric malaria to investigate the potential association with clinical manifestations and outcomes. Results: The molecules showed higher levels in children with severe or cerebral malaria compared to those with uncomplicated malaria. Plasmas concentrations of PTX3, PCT and the soluble receptors suPAR, sTie-2, sICAM-1 were significantly higher in children with deep coma as defined by a Blantyre Coma Score < 3 (P p=0.037, p=0.035, and p=0.002 respectively).Conclusion: Our findings indicate the ability of these seven bioactive molecules to discriminate among the clinical manifestations of malaria and therefore, given their potential utility as prognostic biomarkers for severe and fatal malaria, they might be useful to improve severe cases management.

Background. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods. In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results. IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDR alpha 1.4 and CIDR alpha 1.5 domain subclasses was higher in patients with severe disease (P

Additional file 2. Antibodies raised against VAR19-NTS-DBLγ6 inhibit its interaction with EPCR.

Background Among the Plasmodium species that infect humans, adverse effects of P. falciparum and P. vivax have been extensively studied and reported with respect to poor outcomes particularly in first time mothers and in pregnant women living in areas with unstable malaria transmission. Although, other non-falciparum malaria infections during pregnancy have sometimes been reported, little is known about the dynamics of these infections during pregnancy. Methods and findings Using a quantitative PCR approach, blood samples collected from Beninese pregnant women during the first antenatal visit (ANV) and at delivery including placental blood were screened for Plasmodium spp. Risk factors associated with Plasmodium spp. infection during pregnancy were assessed as well as the relationships with pregnancy outcomes. P. falciparum was the most prevalent Plasmodium species detected during pregnancy, irrespective either of parity, of age or of season during which the infection occurred. Although no P. vivax infections were detected in this cohort, P. malariae (9.2%) and P. ovale (5.8%) infections were observed in samples collected during the first ANV. These non-falciparum infections were also detected in maternal peripheral blood (1.3% for P. malariae and 1.2% for P. ovale) at delivery. Importantly, higher prevalence of P. malariae (5.5%) was observed in placental than peripheral blood while that of P. ovale was similar (1.8% in placental blood). Among the non-falciparum infected pregnant women with paired peripheral and placental samples, P. malariae infections in the placental blood was significantly higher than in the peripheral blood, suggesting a possible affinity of P. malariae for the placenta. However, no assoctiation of non-falciparum infections and the pregnancy outcomes was observed Conclusions Overall this study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, indicating placental infection by non-falciparum Plasmodium and the lack of association of these infections with adverse pregnancy outcomes., Author summary P. falciparum and P. vivax infections during pregnancy have been extensively studied. Meanwhile, the dynamics of other non-falciparum malaria infections during pregnancy is not well understood. We investigated the prevalence of Plasmodium spp. in samples collected from Beninese women early in pregnancy and at delivery using a quantitative PCR approach. Factors associated with Plasmodium spp. infection during pregnancy were assessed. P. falciparum was the most prevalent Plasmodium species detected. P. malariae and P. ovale infections were also detected early in pregnancy, and in the maternal peripheral and placental blood at delivery. Noteworthily, the high prevalence of P. malariae in the placenta of pregnant women with paired peripheral and placental samples, suggests a placental affinity of P. malariae during pregnancy. However, association of non-falciparum infections with pregnancy outcomes was not observed. This study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, and highlighted the underestimated prevalence of non-falciparum infections in pregnancy-related malaria.

Genetic vaccination, consisting in delivering a genetically engineered plasmid DNA by a non-viral vector or technique into a tissue, is currently of great interest. New delivery technique including DNA transfer by electroporation recently greatly improved the potency of this concept. Because it avoids the step of producing a recombinant protein, it is particularly of use in studying the immunogenic properties of large proteins. Here we describe the use of electroporation mediated DNA immunization to identify important protective epitopes from the large VAR2CSA protein from Plasmodium falciparum implicated in the pathology of placental malaria. Immunizing mice and rabbit with DNA plasmids encoding different fragments of VAR2CSA leads to high titer antisera. Moreover an N-terminal region of the protein was found to induce protective functional antibodies.

Malaria is still one of the most prevalent infectious diseases in the world. Sequestration of infected erythrocytes (IEs) is the prime mediator of disease. Cytoadhesion of IEs is mediated by members of the highly diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). A restricted sub-set of var genes encoding for PfEMP1s possessing the domain cassettes DC8 and DC13 were found to bind to the endothelial protein C receptor (EPCR). These var genes were shown to be highly expressed by parasites from patients with severe malaria clinical outcomes compared to those from patients with uncomplicated symptoms.In order to further study the molecular mechanisms underlying DC8/DC13 expressing IEs adhesion to EPCR, a method was developed to produce highly pure recombinant EPCR. The IT4 parasite strain was selected on either anti-IT4-VAR19 purified IgG, EPCR or human brain endothelial cell line and their var gene expression profiles as well as their binding phenotypes were compared. The N-terminal region of IT4-VAR19 comprising a full-length DC8 cassette as well as the single EPCR binding CIDRα1.1 domain were also produced, and their immune recognition (IgG) was assessed using plasma samples from Beninese children presenting acute mild malaria, severe malaria or cerebral malaria at the time of their admission to the clinic, and from convalescent-phase plasma collected 30 days after anti-malarial treatment.The multi-domain VAR19-NTS-DBLγ6 binds to EPCR with a greater affinity than the CIDRα1.1 domain alone and this study also demonstrates that VAR19-NTS-DBLγ6 binding to the EPCR-expressing endothelial cell line (HBEC5i) is more pronounced than that of the CIDRα1.1 domain alone. IT4-VAR19 represents the preferentially expressed-PfEMP1 when FCR3-IEs are selected based on their capability to bind EPCR. Notably, no significant difference in the levels of antibodies towards IT4-VAR19 antigens was observed within all clinical groups between plasma samples collected during the acute malaria phase compared to samples collected 30 days after anti-malaria treatment.These data indicate that even being the preferentially selected IT4-EPCR-binding variant, the IT4-VAR19-DC8 region does not appear to be associated with the acquisition of antibodies during a single severe paediatric malaria episode in Benin.

Background. Molecular, as opposed to microscopic, detection measures the real prevalence of Plasmodium falciparum infections. Such occult infections are common during pregnancy but their impact on pregnancy outcomes is unclear. We performed a longitudinal study to describe that impact. Methods. In a cohort of 1037 Beninese pregnant women, we used ultrasound to accurately estimate gestational ages. Infection with P. falciparum, hemoglobin concentration, use of intermittent preventive treatment during pregnancy (IPTp) for malaria, and other parameters were recorded during pregnancy. Using multivariate analyses, we evaluated the impact of submicroscopic infections on maternal anemia, premature birth, and low birth weight. Results. At inclusion, polymerase chain reaction (PCR) and microscopy detected infection in 40% and 16% of women, respectively. The proportion infected declined markedly after 2 doses of IPTp but rebounded to 34% (by PCR) at delivery. Submicroscopic infections during pregnancy were associated with lower mean hemoglobin irrespective of gravidity, and with increased anemia risk in primigravidae (odds ratio [OR], 2.23; 95% confidence interval [CI], .98–5.07). Prospectively, submicroscopic infections at inclusion were associated with significantly increased risks of low birth weight in primigravidae (OR, 6.09; 95% CI, 1.16–31.95) and premature births in multigravidae (OR, 2.25; 95% CI, 1.13–4.46). Conclusions. In this detailed longitudinal study, we documen tt he deleterious impact of submicroscopic P. falciparum parasitemia during pregnancy on multiple pregnancy outcomes. Parasitemia occurs frequently during pregnancy, but routine microscopic and rapid diagnostic tests fail to detect the vast majority of episodes. Our findings imply caution in any revision of the current strategies for prevention of pregnancy-associated malaria.

Loss of endothelial protein C receptor (EPCR) occurs at the sites of Plasmodium falciparum-infected erythrocyte sequestration in patients with or who died from cerebral malaria. In children presenting with different clinical syndromes of malaria, we assessed the relationships between endogenous plasma soluble EPCR (sEPCR) levels and clinical presentation or mortality. After adjustment for age, for treatment before admission, and for a known genetic factor, sEPCR level at admission was positively associated with cerebral malaria (P = .011) and with malaria-related mortality (P = .0003). Measuring sEPCR levels at admission could provide an early biological marker of the outcome of cerebral malaria.

International audience; Pregnant women become susceptible to malaria infection despite their acquired immunity to this disease from childhood. The placental sequestration of Plasmodium falciparum infected erythrocytes (IE) is the major feature of malaria during pregnancy, due to ability of these parasites to bind chondroitin sulfate A (CSA) in the placenta through the VAR2CSA protein that parasites express on the surface of IE. We collected parasites at different times of pregnancy and investigated the adhesion pattern of freshly collected isolates on the three well described host receptors (CSPG, CD36 and ICAM-1). Var genes transcription profile and VAR2CSA surface-expression were assessed in these isolates. Although adhesion of IE to CD36 and ICAM-1 was observed in some isolates, CSA-adhesion was the predominant binding feature in all isolates analyzed. Coexistence in the peripheral blood of several adhesion phenotypes in early pregnancy isolates was observed, a diversity that gradually tightens with gestational age in favour of the CSA-adhesion phenotype. Infections occurring in primigravidae were often by parasites that adhered more to CSA than those from multigravidae. Data from this study further emphasize the specificity of CSA adhesion and VAR2CSA expression by parasites responsible for pregnancy malaria, while drawing attention to the phenotypic complexity of infections occurring early in pregnancy as well as in multigravidae.

Background. Plasmodium falciparum–infected erythrocytes (IEs) adhere to host cell receptors, allowing parasites to sequester into deep vascular beds of various organs. This defining phenomenon of malaria pathogenesis is key to the severe clinical complications associated with cerebral and placental malaria. The principal ligand associated with the binding to chondroitin sulfate A (CSA) that allows placental sequestration of IEs is a P. falciparum erythrocyte membrane protein 1 (PfEMP1) family member encoded by the var2csa gene. Methods. Here, we investigated the transcription pattern of var genes by real-time polymerase chain reaction, the expression of VAR2CSA, protein by flow cytometry, and the CSA-binding ability of IEs collected at different stages of pregnancy using a static-based Petri dish assay. Results. Through comparison with the profiles of isolates from nonpregnant hosts, we report several lines of evidence showing that parasites infecting women during pregnancy preferentially express VAR2CSA protein, and that selection for the capacity to adhere to CSA via VAR2CSA expression occurs early in pregnancy. Conclusions. Our data suggest that the placental tropism of P. falciparum is already established in the first trimester of pregnancy, with consequent implications for the development of the pathology associated with placental malaria.

International audience; Introduction The objective of this study was to measure the rate of asymptomatic carriage of plasmodium in the Dakar region two years after the implementation of new strategies in clinical malaria management. Methodology Between October and December 2008, 2952 households selected in 50 sites of Dakar area, were visited for interviews and blood sampling. Giemsa-stained thick blood smears (TBS) were performed for microscopy in asymptomatic adult women and children aged 2 to 10 years. To ensure the quality of the microscopy, we performed a polymerase chain reaction (PCR) with real time qPCR in all positive TBS by microscopy and in a sample of negative TBS and filter paper blood spots. Results The analysis has concerned 2427 women and 2231 children. The mean age of the women was 35.6 years. The mean age of the children was 5.4 years. The parasite prevalence was 2.01% (49/2427) in women and 2.15% (48/2231) in children. Parasite prevalence varied from one study site to another, ranging from 0 to 7.41%. In multivariate analysis, reporting a malaria episode in 2008 was associated with plasmodium carriage (OR = 2.57, P = 0.002) in women; in children, a malaria episode (OR = 6.19, P

Background Despite widespread parasite resistance to sulphadoxine-pyrimethamine (SP) its use for intermittent preventative treatment during pregnancy remains the policy in Benin and throughout most of sub-Saharan Africa. Methods In a prospective study, 982 pregnant women were recruited in Benin and followed until delivery. The prevalence of point mutations in the pfdhfr and pfdhps genes associated with Plasmodium falciparum resistance to SP during consecutive antenatal visits was determined. Parasites clearance among women infected at SP intake was assessed by microscopy and PCR. Association between the persistence of parasites and malaria consequences, were investigated. Recurrent parasites were genotyped to identify recrudescences from re-infections. Results The prevalence of pfdhfr/pfdhps quadruple mutants (triple pfdhfr + single pfdhps) was consistently above 80% while quintuple and sextuple mutants remained low. Importantly the higly mutated parasites apparently never included the two key mutations, pfdhfr 164 L or pfdhps 540E. Based on PCR results, SP failed to clear existing parasitaemia in half (48%) of the women who were infected at IPTp schedule. The frequency of recrudescence reached 76% after the second dose. Women with persistent parasitaemia had an increased prevalence of anaemia (P = 0.03). Conclusion The data presented here, highlight the inability of SP to ensure optimal antiplasmodial protection in late pregnancy, and invite urgent consideration of an alternative drug or strategy.

Background: In Benin, very few studies have been done on the genetics of Plasmodium falciparum and the resistance markers of anti-malarial drugs, while malaria treatment policy changed in 2004. Chloroquine (CQ) and sulphadoxine pyrimethamine (SP) have been removed and replaced by artemisinin-combination therapy (ACT). The objective of this study was to determine the genetic diversity of P. falciparum and the prevalence of P. falciparum molecular markers that are associated with resistance to CQ and SP in northern Benin seven years after the new policy was instituted. Methods: The study was conducted in northern Benin, a region characterized by a seasonal malaria transmission. Blood samples were collected in 2012 from children presenting with asymptomatic P. falciparum infections. Samples collected in filter paper were genotyped by primary and nested PCR in block 2 of msp-1 and block 3 of msp-2 to analyse the diversity of P. falciparum. The prevalence of critical point mutations in the genes of Pfcrt (codon 76), Pfmdr1 (codon 86), Pfdhfr (codons, 51, 59 and 108) and Pfdhps (codons 437, 540) was examined in parasite isolates by mutation-specific restriction enzyme digestion. Results: Genotyping of 195 isolates from asymptomatic children showed 34 msp-1 and 38 msp-2 genotypes. The multiplicity of infection was 4.51 +/- 0.35 for msp-1 and 4.84 +/- 0.30 for msp-2. Only the codon 51 of Pfdhfr and codon 437 of Pfdhps showed a high mutation rate: I51: 64.4% (57.3; 71.2); G437: 47.4% (40.2; 54.7), respectively. The prevalence of Pfdhfr triple mutant IRN (I51, R59 and N108) was 1.5% (0.3; 3.9), and Pfdhfr/Pfdhps quadruple mutant IRNG (PfdhfrI51, R59, N108, and PfdhpsG437): 0. 5% (0; 2.5). No mutation was found with codon 540 of Pfdhps. Analysis of mutation according to age (younger or older than ten years) showed similar frequencies in each category without significant difference between the two groups. Conclusions: This study showed a high diversity of P. falciparum in northern Benin with a very low prevalence of resistance markers to CQ and SP that dramatically contrasted with the pattern observed in southern Benin. No influence of age on genetic diversity of P. falciparum and on distribution of the mutations was observed.