Azizath Moussiliou

Asymptomatic Carriage of Plasmodium in Urban Dakar: The Risk of Malaria Should Not Be Underestimated

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<0.001) and a travel out of Dakar during last 3 months (OR = 2.27, P = 0.023) were associated with plasmodium carriage. Among the positive TBS, 95.8% (93/97) were positive by plasmodium PCR. Among the negative TBS, 13.9% (41/293) were positive by PCR. In blood spots, 15.2% (76/500) were positive by PCR. We estimated at 16.5% the parasite prevalence if PCR were performed in 4658 TBS. Conclusion Parasite prevalence in Dakar area seemed to be higher than the rate found by microscopy. PCR may be the best tool for measuring plasmodium prevalence in the context of low transmission. Environmental conditions play a major role in the heterogeneity of parasite prevalence within sites.

Submicroscopic Plasmodium falciparum Infections Are Associated With Maternal Anemia, Premature Births, and Low Birth Weight

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 document the 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.

First-Trimester Plasmodium falciparum Infections Display a Typical “Placental” Phenotype

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.

Structure-Guided Identification of a Family of Dual Receptor-Binding PfEMP1 that Is Associated with Cerebral Malaria

Summary Cerebral malaria is a deadly outcome of infection by Plasmodium falciparum, occurring when parasite-infected erythrocytes accumulate in the brain. These erythrocytes display parasite proteins of the PfEMP1 family that bind various endothelial receptors. Despite the importance of cerebral malaria, a binding phenotype linked to its symptoms has not been identified. Here, we used structural biology to determine how a group of PfEMP1 proteins interacts with intercellular adhesion molecule 1 (ICAM-1), allowing us to predict binders from a specific sequence motif alone. Analysis of multiple Plasmodium falciparum genomes showed that ICAM-1-binding PfEMP1s also interact with endothelial protein C receptor (EPCR), allowing infected erythrocytes to synergistically bind both receptors. Expression of these PfEMP1s, predicted to bind both ICAM-1 and EPCR, is associated with increased risk of developing cerebral malaria. This study therefore reveals an important PfEMP1-binding phenotype that could be targeted as part of a strategy to prevent cerebral malaria.

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.

Dynamics in the Cytoadherence Phenotypes of Plasmodium falciparum Infected Erythrocytes Isolated during Pregnancy

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. Co-existence 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.

High Plasma Levels of Soluble Endothelial Protein C Receptor Are Associated With Increased Mortality Among Children With Cerebral Malaria in Benin

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.

Parasites Causing Cerebral Falciparum Malaria Bind Multiple Endothelial Receptors and Express EPCR and ICAM-1-Binding PfEMP1

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 CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-β3 domains than parasites, which only bound CD36. Conclusions These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.

Low prevalence of the molecular markers of Plasmodium falciparum resistance to chloroquine and sulphadoxine/pyrimethamine in asymptomatic children in Northern Benin

BackgroundIn 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.MethodsThe 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.ResultsGenotyping 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 (Pfdhfr I51, R59, N108, and Pfdhps G437): 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.ConclusionsThis 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.

Electroporation-mediated genetic vaccination for antigen mapping: application to Plasmodium falciparum VAR2CSA protein.

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.