Nicaise Tuikue Ndam

High Level of var2csa Transcription by Plasmodium falciparum Isolated from the Placenta

Plasmodium falciparum parasites that bind to chondroitin sulphate A (CSA) express unique variant surface antigens that are involved in the placental sequestration that precipitates pregnancy-associated malaria (PAM). Two var gene subfamilies, var1csa and var2csa, have been associated with CSA binding. We show here that placental P. falciparum isolates highly transcribed var2csa but not var1csa. var2csa was not transcribed or was only minimally transcribed by parasites isolated from nonpregnant women. Placental parasites that effectively bound to placental chondroitin sulphate proteoglycans transcribed higher levels of var2csa. In pregnant women, levels of var2csa transcription and plasma anti-VAR2CSA immunoglobulin G were associated. These findings support the idea that VAR2CSA plays a crucial role in PAM and strengthen the rationale for the development of VAR2CSA-based vaccines.

Epitope Mapping and Topographic Analysis of VAR2CSA DBL3X Involved in P. falciparum Placental Sequestration

Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an α-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.

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.

Influence of the Timing of Malaria Infection during Pregnancy on Birth Weight and on Maternal Anemia in Benin

Abstract. Although consequences of malaria in pregnancy are well known, the period of pregnancy in which infection has the highest impact is still unclear. In Benin, we followed up a cohort of 1,037 women through pregnancy until delivery. The objective was to evaluate the relationship between the timing of infection and birth weight, and maternal anemia at delivery. At the beginning of pregnancy, peripheral infections were associated with a decrease in mean birth weight (-98.5 g; P = 0.03) and an increase in the risk of anemia at delivery (adjusted odds ratio [aOR] = 1.6; P = 0.03). Infections in late pregnancy were related to a higher risk of maternal anemia at delivery (aOR = 1.7; P = 0.001). To fully protect the women during the whole pregnancy, already implemented measures (insecticide-treated nets and intermittent preventive treatment) should be reinforced. In the future, a vaccine against pregnancy-associated malaria parasites could protect the women in early pregnancy, which seems to be a high-risk period.

Variable Adhesion Abilities and Overlapping Antigenic Properties in Placental Plasmodium falciparum Isolates

BACKGROUND Pregnancy-associated malaria is characterized by selection and multiplication, in the placenta, of a distinct population of Plasmodium falciparum expressing particular variant surface antigens (VSAs) that adhere to chondroitin sulfate A (CSA). METHODS The adhesion of 40 freshly collected placental parasite isolates to bovine CSA and human placental low-sulfated chondroitin proteoglycans (CSPGs) was investigated. Plasma samples from 30 pregnant women were used to test, by flow cytometry, their recognition of and their adhesion-inhibition capacity toward 6 of these isolates. RESULTS Adhesion to CSA and CSPGs varied between isolates but was strongly correlated between receptors (P<.001). Adhesion of isolates to receptors strongly and negatively correlated with low birth weight (LBW) of the neonate (odds ratio [95% confidence interval], 5.2 [1.1-25.1]). In plasma samples from pregnant women, the level of specific immunoglobulin G against each placental isolate (anti-VSA(PAP)) strongly correlated with the level of anti-VSA(PAP) antibodies against all other isolates (P<.05) and increased with parity in all isolates (P<.01). Conversely, adhesion-inhibitory antibodies did not correlate with isolates or with the level of anti-VSA(PAP) antibodies. CONCLUSION The level of adhesion of placental parasites to chondroitin sulfate receptors is an important risk factor for LBW. Parasite heterogeneity suggests that they are composed of mixed adhesion phenotypes capable of inducing immune responses to a range of different and overlapping targets.

Multiple var2csa-type PfEMP1 genes located at different chromosomal loci occur in many Plasmodium falciparum isolates

Background The var2csa gene encodes a Plasmodium falciparum adhesion receptor which binds chondroitin sulfate A (CSA). This var gene is more conserved than other PfEMP1/var genes and is found in all P. falciparum isolates. In isolates 3D7, FCR3/It4 and HB3, var2csa is transcribed from a sub-telomeric position on the left arm of chromosome 12, but it is not known if this location is conserved in all parasites. Genome sequencing indicates that the var2csa gene is duplicated in HB3, but whether this is true in natural populations is uncertain. Methodology/Principal Findings To assess global variation in the VAR2CSA protein, sequence variation in the DBL2X region of var2csa genes in 54 P.falciparum samples was analyzed. Chromosome mapping of var2csa loci was carried out and a quantitative PCR assay was developed to estimate the number of var2csa genes in P.falciparum isolates from the placenta of pregnant women and from the peripheral circulation of other malaria patients. Sequence analysis, gene mapping and copy number quantitation in P.falciparum isolates indicate that there are at least two loci and that both var2csa-like genes can be transcribed. All VAR2CSA DBL2X domains fall into one of two distinct phylogenetic groups possessing one or the other variant of a large (∼26 amino acid) dimorphic motif, but whether either motif variant is linked to a specific locus is not known. Conclusions/Significance Two or more related but distinct var2csa-type PfEMP1/var genes exist in many P. falciparum isolates. One gene is on chromosome 12 but additional var2csa-type genes are on different chromosomes in different isolates. Multiplicity of var2csa genes appears more common in infected placentae than in samples from non-pregnant donors indicating a possible advantage of this genotype in pregnancy associated malaria.

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.

VAR2CSA Expression on the Surface of Placenta-Derived Plasmodium falciparum-Infected Erythrocytes

Malaria remains a major threat, in sub-Saharan Africa primarily, and the most deadly infections are those with Plasmodium falciparum. Pregnancy-associated malaria is a clinically important complication of infection; it results from a unique interaction between proteoglycans in the placental intervillous space and parasite antigens. Both placental and chondroitin sulphate A-selected parasites have high-level transcripts of a unique var gene named var2csa. However, VAR2CSA has not been consistently found by proteomic analysis of placental parasites. Contrary to this, we found VAR2CSA expressed on the surface of infected erythrocytes from placenta. Importantly, this was achieved with cross-reactive antibodies against VAR2CSA.

The NTS-DBL2X Region of VAR2CSA Induces Cross-Reactive Antibodies That Inhibit Adhesion of Several Plasmodium falciparum Isolates to Chondroitin Sulfate A

BACKGROUND Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS This study highlights an important step achieved toward development of a protective vaccine against placental malaria.

Insecticide Resistance Alleles Affect Vector Competence of Anopheles gambiae s.s. for Plasmodium falciparum Field Isolates

The widespread insecticide resistance raises concerns for vector control implementation and sustainability particularly for the control of the main vector of human malaria, Anopheles gambiae sensu stricto. However, the extent to which insecticide resistance mechanisms interfere with the development of the malignant malaria parasite in its vector and their impact on overall malaria transmission remains unknown. We explore the impact of insecticide resistance on the outcome of Plasmodium falciparum infection in its natural vector using three An. gambiae strains sharing a common genetic background, one susceptible to insecticides and two resistant, one homozygous for the ace-1R mutation and one for the kdr mutation. Experimental infections of the three strains were conducted in parallel with field isolates of P. falciparum from Burkina Faso (West Africa) by direct membrane feeding assays. Both insecticide resistant mutations influence the outcome of malaria infection by increasing the prevalence of infection. In contrast, the kdr resistant allele is associated with reduced parasite burden in infected individuals at the oocyst stage, when compared to the susceptible strain, while the ace-1 R resistant allele showing no such association. Thus insecticide resistance, which is particularly problematic for malaria control efforts, impacts vector competence towards P. falciparum and probably parasite transmission through increased sporozoite prevalence in kdr resistant mosquitoes. These results are of great concern for the epidemiology of malaria considering the widespread pyrethroid resistance currently observed in Sub-Saharan Africa and the efforts deployed to control the disease.