Polymorphism in two merozoite surface proteins of Plasmodium falciparum isolates from Gabon

BACKGROUND: Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine. METHODS: Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp-1 block 2, and msp-2 block 3, to analyze both parasite population polymorphism and clone fluctuations. RESULTS: Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence. CONCLUSION: P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity

Etude différentielle des protéines membranaires exprimées à la surface de l'hématie infectée par Plasmodium falciparum, en fonction de la symptomatologie

La virulence de Plasmodium falciparum est fonction de sa capacité à séquestrer les érythrocytes infectés (iEs) dans les organes profonds de l hôte. Elle est liée à la mise en place d un trafic protéique conséquent au niveau membranaire et sub-membranaire de l iE. Cet adressage protéique aboutit à l expression d antigènes variant de surface, dont P. falciparum Erythrocyte Membrane Protein-1, PfEMP-1. Cet adhésineprésente une affinité pour divers récepteurs de l'hôte impliqués dans la physiopathologie des formes graves, telles que le paludisme associé à la grossesse (PAM) et le paludisme cérébral (CM). La diversité de liaison de PfEMP-1 est associée à la variabilité de séquence de son domaine extracellulaire. Ce domaine est constitué d une succession de domaines DBL et CIDR en nombre variable. PfEMP-1 est codée par les gènes var classifiés en groupes Ups A, B, C, E et B/A, B/C. Des régions de PfEMP-1 constituées d une succession établie de 2 à 4 domaines, nommées Domain Cassette, DC sont retrouvées dans différents isolats. L obtention de données exhaustives par LC-MS/MS a permis d établir un comparatif du protéome membranaire et hypothétique à partir des échantillons PAM et CM en comparaison à des accès simples de paludisme (UM). L identification protéique des PfEMP-1 a montré la singularité de l expression d une PfEMP-1 particulière, VAR2CSA chez les PAM. Ces résultats corroborent les analyses des transcrits du gène var2csa comme surexprimé chez les PAM, transcrit qu on retrouve également dans les infections de début et de fin de grossesse. Les PfEMP-1 identifiés au sein des CM étaient significativement associés aux groupes Ups A et B/A et la plupart des peptides identifiés étaient associés à la cassette DC8 dont le transcrit était également surexprimé. L analyse de filter-based feature selection a permis d identifier un sous ensemble de 13 et 14 protéines assignées comme protéines membranaires ou hypothétiques, prédictives des formes de paludisme PAM et CM, respectivement. Parmi ces protéines surexprimées chez les PAM, la présence de VAR2CSA valide l approche d analyse. PFI1785w a été identifiée et associée au PAM, quatre autres protéines apparaissent prédictives de cette forme clinique PFB0115w, PFF0325c, PFA_0410w et PF14_0018. Pour les CM, on distingue les protéines PfEMP-2 et antigen-332 comme spécifiques de cette forme clinique. Les autres protéines qui émergent de ce groupe sont des protéines de fonction inconnue, dont trois sont assignées comme des protéines exportées. L obtention et l analyse en LC-MS/MS d extraits protéiques issus d isolats de terrain font l originalité de ce travail et permettent la corrélation entre les données cliniques et biologiques. Cette étude confirme les données de transcrits sur la famille des gènes var et suggère de nouvelles intéractions protéiques dans le cadre du paludisme associé à la grossesse et cérébral.Plasmodium falciparum is responsible for severe malaria (cerebral malaria, CM and pregnancy associated malaria, PAM). During the intra-erythrocytic maturation of P. falciparum, parasite-derived proteins are expressed, exported and presented at the surface of the infected erythrocyte (iE) membrane. These include Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1). PfEMP-1 is a highly polymorphic adherence receptor, variants of which have been assigned to four groups (A-E) based on their sequence homology. Semi-conserved types, defined by tandem runs of specific domains ( domain cassettes (DC)), are also recognized. The PfEMP-1 type expressed determines the iE adherence phenotype, and is associated with the clinical outcome of infection. Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of membrane and hypothetical proteins of Plasmodium by LC-MS/MS. Obtaining LC-MS/MS data enabled a comparison of hypothetical and membrane proteome samples from PAM and CM for comparison with UM samples. The proteomics-based identification of PfEMP-1 showed the expression of a particular PfEMP-1, VAR2CSA, in PAM isolates. These results corroborate the analysis of gene transcripts showing that var2csa is overexpressed in PAM, with transcripts found in isolates from infections both early and late in pregnancy. The PfEMP-1 variants identified in CM samples were predominantly from the Ups groups A and B/A, and most of the peptides identified by LC-MS/MS were associated with the DC8 cassette for which the transcripts were also overexpressed. Analysis using filter-based feature selection identified subsets of 13 and 14 proteins, assigned either as hypothetical or membrane proteins that were predictive of PAM and CM syndromes respectively. The presence of VAR2CSA amongst the proteins overexpressed in PAM samples validates the analytical approach. PFI1785w has previously been identified and associated with the PAM, whilst four other proteins, PFB0115w, PFF0325c, PFA_0410w and PF14_0018, appear to be also predictive of the syndrome. PfEMP-2 protein and antigen-332 were found to be specifically expressed in CM samples. Three other proteins, assigned as exported but with unknown function, were also associated with CM samples. Obtaining and analyzing LC-MS/MS-derived data from protein extracts of field isolates represents the originality of this work and it allowed the identification of correlations between clinical and biological data. This study confirms the transcriptional data relating to the var gene family and provides evidence of new protein interactions in the context both of malaria associated with pregnancy and of cerebral malaria.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Consequences of Gestational Malaria on Birth Weight: Finding the Best Timeframe for Intermittent Preventive Treatment Administration

To investigate the consequences of intermittent preventive treatment (IPTp) timing on birth weight, we pooled data from two studies conducted in Benin between 2005 and 2010: a prospective cohort of 1037 pregnant women and a randomised trial comparing sulfadoxine-pyrimethamine (SP) to mefloquine in 1601 women. A total of 1439 women (752 in the cohort and 687 in the SP arm of the randomised trial) who delivered live singletons were analysed. We showed that an early intake of the first SP dose (4 months of gestation) was associated with a lower risk of LBW compared to a late intake (6–7 months of gestation) (aOR = 0.5 p = 0.01). We also found a borderline increased risk of placental infection when the first SP dose was administered early in pregnancy (aOR = 1.7 p = 0.1). This study is the first to investigate the timing of SP administration during pregnancy. We clearly demonstrated that women who had an early intake of the first SP dose were less at risk of LBW compared to those who had a late intake. Pregnant women should be encouraged to attend antenatal visits early to get their first SP dose and a third dose of SP could be recommended to cover the whole duration of pregnancy and to avoid late infections of the placenta

Can treatment of malaria be restricted to parasitologically confirmed malaria? A school-based study in Benin in children with and without fever

<p>Abstract</p> <p>Background</p> <p>Applying the switch from presumptive treatment of malaria to new policies of anti-malarial prescriptions restricted to parasitologically-confirmed cases is a still unsolved challenge. Pragmatic studies can provide data on consequences of such a switch. In order to assess whether restricting anti-malarials to rapid diagnostic test (RDT)-confirmed cases in children of between five and 15 years of age is consistent with an adequate management of fevers, a school-based study was performed in Allada, Benin.</p> <p>Methods</p> <p>Children in the index group (with fever and a negative RDT) and the matched control group (without fever and a negative RDT) were not prescribed anti-malarials and actively followed-up during 14 days. Blood smears were collected at each assessment. Self-medication with chloroquine and quinine was assessed with blood spots. Malaria attacks during the follow-up were defined by persistent or recurrent fever concomitant to a positive malaria test.</p> <p>Results</p> <p>484 children were followed-up (242 in each group). At day 3, fever had disappeared in 94% of children from the index group. The incidence of malaria was similar (five cases in the index group and seven cases in the control group) between groups. Self-medication with chloroquine and quinine in this cohort was uncommon.</p> <p>Conclusions</p> <p>Applying a policy of restricting anti-malarials to RDT-confirmed cases is consistent with an adequate management of fevers in this population. Further studies on the management of fever in younger children are of upmost importance.</p

Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates

BACKGROUND: Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA) that can mediate adhesion to chondroitin sulfate A (CSA) in vitro. IgG antibodies with specificity for the VSA expressed by these parasites (VSA(PAM)) are associated with protection from maternal anaemia, prematurity and low birth weight, which is the greatest risk factor for death in the first month of life. METHODS: In this study, the development of anti-VSA(PAM )antibodies in a group of 151 women who presented to the maternity ward of Albert Schweitzer Hospital in Lambaréné, Gabon for delivery was analysed using flow cytometry assays. Plasma samples from placenta infected primiparous women were also investigated for their capacity to inhibit parasite binding to CSA in vitro. RESULTS: In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSA(PAM)-specific IgG compared to women who had no malaria infections at delivery. Placental isolates of Gabonese and Senegalese origin tested on plasma samples from Gabon showed parity dependency and gender specificity patterns. There was a significant correlation of plasma reactivity as measured by flow cytometry between different placental isolates. In the plasma of infected primiparous women, VSA(PAM)-specific IgG measured by flow cytometry could be correlated with anti-adhesion antibodies measured by the inhibition of CSA binding. CONCLUSION: Recognition of placental parasites shows a parity- and sex- dependent pattern, like that previously observed in laboratory strains selected to bind to CSA. Placental infections at delivery in primiparous women appear to be sufficient to induce functional antibodies which can both recognize the surface of the infected erythrocytes as well as block their binding to CSA. The correlation between serum reactivities of placental field isolates from different geographic locations and collected at different times is indicative of the conserved nature of the antigen(s) mediating PAM

Computational fluid dynamic simulations of maternal circulation : wall shear stress in the human placenta and its biological implications

Introduction In the human placenta the maternal blood circulates in the intervillous space (IVS). The syncytiotrophoblast (STB) is in direct contact with maternal blood. The wall shear stress (WSS) exerted by the maternal blood flow on the STB has not been evaluated. Our objective was to determine the physiological WSS exerted on the surface of the STB during the third trimester of pregnancy. Material and Methods To gain insight into the shear stress levels that the STB is expected to experience in vivo, we have formulated three different computational models of varying levels of complexity that reflect different physical representations of the IVS. Computations of the flow fields in all models were performed using the CFD module of the finite element code COMSOL Multi-physics 4.4. The mean velocity of maternal blood in the IVS during the third trimester was measured in vivo with dynamic MRI (0.94 +/- 0.14 mm.s(-1)). To investigate if the in silico results are consistent with physiological observations, we studied the cytoadhesion of human parasitized (Plasmodium falciparum) erythrocytes to primary human STB cultures, in flow conditions with different WSS values. Results The WSS applied to the STB is highly heterogeneous in the IVS. The estimated average values are relatively low (0.5 +/- 0.2 to 2.3 +/- 1.1 dyn.cm(-2)). The increase of WSS from 0.15 to 5 dyn.cm(-2) was associated with a significant decrease of infected erythrocyte cytoadhesion. No cytoadhesion of infected erythrocytes was observed above 5 dyn.cm(-2) applied for one hour. Conclusion Our study provides for the first time a WSS estimation in the maternal placental circulation. In spite of high maternal blood flow rates, the average WSS applied at the surface of the chorionic villi is low (<5 dyn.cm(-2)). These results provide the basis for future physiologicallyrelevant in vitro studies of the biological effects of WSS on the STB

Cellular immune response to Plasmodium falciparum after pregnancy is related to previous placental infection and parity

BACKGROUND: Malaria in pregnancy is characterised by the sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous spaces. Placental parasites express a specific phenotype, which allows them to cytoadhere to chondroitin sulfate A expressed by syncytiotrophoblasts. Malaria infection during pregnancy allows the acquisition of antibodies against placental parasites, these antibodies are thought to be involved in protection during subsequent pregnancies. METHODS: To investigate the development of a cellular response to placental parasites during pregnancy, peripheral blood mononuclear cells were collected from women at the time of their confinement. The study was performed in Cameroon where malaria transmission is perennial. In vitro cell proliferation and cytokine production were measured in response to non-malarial activators (concanavalin A and PPD), a recombinant protein from P. falciparum MSP-1, and erythrocytes infected by two P. falciparum lines, RP5 and W2. Like placental parasites, the RP5 line, but not W2, adheres to chondroitin sulfate A and to syncytiotrophoblasts. RESULTS: The proliferative response to all antigens was lower for cells obtained at delivery than 3 months later. Most interestingly, the cellular response to the RP5 line of P. falciparum was closely related to parity. The prevalence rate and the levels of response gradually increased with the number of previous pregnancies. No such relationship was observed with W2 line, or MSP-1 antigen. CONCLUSIONS: This suggests the occurrence of an immune response more specific for the RP5 line in women having had multiple pregnancies, and who are likely to develop immunity to pregnancy-associated parasites. Both humoral and cellular mechanisms may account for the lower susceptibility of multigravidae to malaria