Effect of three years' seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali.

BACKGROUND: In 2012, seasonal malaria chemoprevention (SMC) was recommended as policy for malaria control by the World Health Organization (WHO) in areas of highly seasonal malaria transmission across the Sahel sub-region in Africa along with monitoring of drug resistance. We assessed the long-term impact of SMC on Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) over a 3-year period of SMC implementation in the health district of Ouelessebougou, Mali. METHODS: In 8 randomly selected sub-districts of Ouelessebougou, Mali, children aged 0-5 years were randomly selected during cross-sectional surveys at baseline (August 2014) and 1, 2 and 3 years post-SMC, at the beginning and end of the malaria transmission season. Blood smears and blood spots on filter paper were obtained and frequencies of mutation in P. falciparum genes related to resistance to SP and AQ (Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt) were assessed by PCR amplification on individual samples and PCR amplification followed by deep sequencing on pooled (by site and year) samples. RESULTS: At each survey, approximately 50-100 individual samples were analysed by PCR amplification and a total of 1,164 samples were analysed by deep sequencing with an average read depth of 18,018-36,918 after pooling by site and year. Most molecular markers of resistance did not increase in frequency over the period of study (2014-2016). After 3 years of SMC, the frequencies of Pfdhps 540E, Pfdhps 437G and Pfcrt K76T remained similar compared to baseline (4.0 vs 1.4%, p = 0.41; 74.5 vs 64.6%, p = 0.22; 71.3 vs 67.4%, p = 0.69). Nearly all samples tested carried Pfdhfr 59R, and this proportion remained similar 3 years after SMC implementation (98.8 vs 100%, p = 1). The frequency of Pfmdr1 N86Y increased significantly over time from 5.6% at baseline to 18.6% after 3 years of SMC (p = 0.016). Results of pooled analysis using deep sequencing were consistent with those by individual analysis with standard PCR, but also indicated for the first time the presence of mutations at the Pfdhps A581G allele at a frequency of 11.7% after 2 years of SMC, as well as the Pfdhps I431V allele at frequencies of 1.6-9.3% following 1 and 2 years of SMC, respectively. CONCLUSION: Two and 3 years of SMC implementation were associated with increased frequency of the Pfmdr1 N86Y mutation but not Pfdhps 540E, Pfdhps 437G and Pfcrt K76T. The first-time detection of the Pfdhps haplotype bearing the I431V and A581G mutations in Mali, even at low frequency, warrants further long-term surveillance.

Malaria Infection Is Common and Associated With Perinatal Mortality and Preterm Delivery Despite Widespread Use of Chemoprevention in Mali: An Observational Study 2010 to 2014.

BACKGROUND: In malaria-endemic areas, pregnant women and especially first-time mothers are more susceptible to Plasmodium falciparum. Malaria diagnosis is often missed during pregnancy, because many women with placental malaria remain asymptomatic or have submicroscopic parasitemia, masking the association between malaria and pregnancy outcomes. Severe maternal anemia and low birthweight deliveries are well-established sequelae, but few studies have confirmed the relationship between malaria infection and severe outcomes like perinatal mortality in high transmission zones. METHODS: Pregnant women of any gestational age enrolled at antenatal clinic into a longitudinal cohort study in Ouelessebougou, Mali, an area of high seasonal malaria transmission. Follow-up visits included scheduled and unscheduled visits throughout pregnancy. Blood smear microscopy and polymerase chain reaction (PCR) analysis were employed to detect both microscopic and submicroscopic infections, respectively. Intermittent preventative treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) was documented and prompt treatment regardless of symptoms given upon malaria diagnosis. RESULTS: Of the 1850 women followed through delivery, 72.6% of women received 2 or more IPTp-SP doses, 67.2% of women experienced at least 1 infection between enrollment up to and including delivery. Malaria infection increased the risks of stillbirth (adjusted hazard ratio [aHR] 3.87, 95% confidence interval [CI]: 1.18-12.71) and preterm delivery (aHR 2.41, 95% CI: 1.35-4.29) in primigravidae, and early neonatal death (death within 7 days) in secundigravidae and multigravidae (aHR 6.30, 95% CI: 1.41-28.15). CONCLUSIONS: Malaria treatment after diagnosis, alongside IPTp-SP, is insufficient to prevent malaria-related stillbirth, early neonatal death and preterm delivery (PTD). Although IPTp-SP was beneficial in Mali during the study period, new tools are needed to improve pregnancy outcomes. CLINICAL TRIALS REGISTRATION: NCT01168271.

Age-dependent increase in antibodies that inhibit Plasmodium falciparum adhesion to a subset of endothelial receptors.

BACKGROUND: Plasmodium falciparum-infected erythrocytes (IE) sequester in deep vascular beds where their adhesion is mediated by an array of endothelial surface receptors. Because parasite adhesion has been associated with disease, antibodies that block this activity may confer protective immunity. Here, levels of plasma anti-adhesion activity and surface reactivity against freshly collected IEs from malaria-infected children were measured in a Malian birth cohort and related to child age and malaria infection history. METHODS: Plasma samples from children enrolled at birth in a longitudinal cohort study of mother-infant pairs in Ouelessebougou, Mali were collected at multiple time points during follow-up visits. Anti-adhesion antibodies (i.e., inhibit IE binding to any of several endothelial receptors) and reactivity with surface IE proteins were measured using a binding inhibition assay and by flow cytometry, respectively. RESULTS: Levels of antibodies that inhibit the binding of children's IE to the receptors ICAM-1, integrin α3ß1 and laminin increased with age. The breadth of antibodies that inhibit ICAM-1 and laminin adhesion (defined as the proportion of IE isolates whose binding was reduced by ≥ 50%) also significantly increased with age. The number of malaria infections prior to plasma collection was associated with levels of plasma reactivity to IE surface proteins, but not levels of anti-adhesion activity. CONCLUSIONS: Age is associated with increased levels of antibodies that reduce adhesion of children's IE to three of the ten endothelial receptors evaluated here. These results suggest that anti-adhesion antibodies to some but not all endothelial receptors are acquired during the first few years of life.

Antibody levels to recombinant VAR2CSA domains vary with Plasmodium falciparum parasitaemia, gestational age, and gravidity, but do not predict pregnancy outcomes.

BACKGROUND: Maternal malaria is a tropical scourge associated with poor pregnancy outcomes. Women become resistant to Plasmodium falciparum pregnancy malaria as they acquire antibodies to the variant surface antigen VAR2CSA, a leading vaccine candidate. Because malaria infection may increase VAR2CSA antibody levels and thereby confound analyses of immune protection, gravidity-dependent changes in antibody levels during and after infection, and the effect of VAR2CSA antibodies on pregnancy outcomes were evaluated. METHODS: Pregnant women enrolled in a longitudinal cohort study of mother-infant pairs in Ouelessebougou, Mali provided plasma samples at enrollment, gestational week 30-32, and delivery. Antibody levels to VAR2CSA domains were measured using a multiplex bead-based assay. RESULTS: Antibody levels to VAR2CSA were higher in multigravidae than primigravidae. Malaria infection was associated with increased antibody levels to VAR2CSA domains. In primigravidae but not in secundigravidae or multigravidae, antibodies levels sharply declined after an infection. A relationship between any VAR2CSA antibody specificity and protection from adverse pregnancy outcomes was not detected. CONCLUSIONS: During malaria infection, primigravidae acquire short-lived antibodies. The lack of an association between VAR2CSA domain antibody reactivity and improved pregnancy outcomes suggests that the recombinant proteins may not present native epitopes targeted by protective antibodies.

Host factors that modify Plasmodium falciparum adhesion to endothelial receptors.

P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3ß1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5ß1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5ß1 significantly increased with age.

Systemic Inflammatory Response to Malaria During Pregnancy Is Associated With Pregnancy Loss and Preterm Delivery.

BACKGROUND: Pregnancy malaria (PM) is associated with a proinflammatory immune response characterized by increased levels of cytokines and chemokines such as tumor necrosis factor-α, interferon-γ, interleukin 10 (IL-10), and CXCL9. These changes are associated with poor outcomes including low birthweight delivery and maternal anemia. However, it is unknown if inflammatory pathways during malaria are related to pregnancy loss and preterm delivery (PTD). METHODS: Cytokine and chemokine levels were measured in maternal peripheral blood at enrollment, gestational week 30-32, and delivery, and in placental blood, of 638 women during a longitudinal cohort study in Ouelessebougou, Mali. Plasmodium falciparum infection was assessed by blood smear microscopy at all visits. RESULTS: PM was associated with increased levels of cytokines and chemokines including IL-10 and CXCL9. In a competing risks model adjusted for known covariates, high CXCL9 levels measured in the peripheral blood during pregnancy were associated with increased risk of pregnancy loss and PTD. At delivery, high IL-10 levels in maternal blood were associated with an increase in pregnancy loss, and increased IL-1ß levels in placental blood were associated with pregnancy loss and PTD. CONCLUSIONS: PM is associated with increased proinflammatory cytokine and chemokine levels in placental and maternal peripheral blood. Systemic inflammatory responses to malaria during pregnancy predict increased risk of pregnancy loss and PTD. CLINICAL TRIALS REGISTRATION: NCT01168271.

Effect of seasonal malaria chemoprevention on the acquisition of antibodies to Plasmodium falciparum antigens in Ouelessebougou, Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a new strategy to reduce malaria burden in young children in Sahelian countries. It consists of the administration of full treatment courses of sulfadoxine-pyrimethamine plus amodiaquine to children at monthly intervals during the malaria season. However, it is not clear if there is a cumulative effect of SMC over time on acquisition of antibodies to malaria antigens. METHODS: A cross-sectional serosurvey was carried out 1 month after the last dose of SMC in 2016. Children aged 3-4 years were randomly selected from areas where SMC was given for 1, 2 or 3 years during the malaria season. Children in the areas where SMC had been implemented for 1 year but who failed to receive SMC were used as comparison group. Antibody extracted from dry blood spots was used to measure IgG levels to CSP, MSP-142 and AMA1. RESULTS: The prevalence of antibodies to AMA-1 were high and similar in children who received SMC for 1, 2 or 3 years and also when compared to those who never received SMC (96.3 vs 97.5%, adjusted OR = 0.99, 95% CI 0.33-2.97, p = 0.99). The prevalence of antibodies to MSP-142 and to CSP were similar in children that received SMC for 1, 2 or 3 years, but were lower in these children compared to those who did not receive SMC (87.1 vs 91.2%, adjusted OR = 0.55, 95% CI 0.29-1.01, p = 0.05 for MSP-142; 79.8 vs 89.2%, adjusted OR = 0.52, 95% CI 0.30-0.90, p = 0.019 for CSP). CONCLUSIONS: SMC reduced seropositivity to MSP-142 and CSP, but the duration of SMC did not further reduce seropositivity. Exposure to SMC did not reduce the seropositivity to AMA1.