Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy.

BACKGROUND: Prevention of reinfection and resurgence is an integral component of the goal to eradicate malaria. However, the adverse effects of malaria resurgences are not known. METHODS: We assessed the prevalence of Plasmodium falciparum infection among 1819 Mozambican women who delivered infants between 2003 and 2012. We used microscopic and histologic examination and a quantitative polymerase-chain-reaction (qPCR) assay, as well as flow-cytometric analysis of IgG antibody responses against two parasite lines. RESULTS: Positive qPCR tests for P. falciparum decreased from 33% in 2003 to 2% in 2010 and increased to 6% in 2012, with antimalarial IgG antibody responses mirroring these trends. Parasite densities in peripheral blood on qPCR assay were higher in 2010-2012 (geometric mean [±SD], 409±1569 genomes per microliter) than in 2003-2005 (44±169 genomes per microliter, P=0.02), as were parasite densities in placental blood on histologic assessment (50±39% of infected erythrocytes vs. 4±6%, P<0.001). The malaria-associated reduction in maternal hemoglobin levels was larger in 2010-2012 (10.1±1.8 g per deciliter in infected women vs. 10.9±1.7 g per deciliter in uninfected women; mean difference, -0.82 g per deciliter; 95% confidence interval [CI], -1.39 to -0.25) than in 2003-2005 (10.5±1.1 g per deciliter vs. 10.6±1.5 g per deciliter; difference, -0.12 g per deciliter; 95% CI, -0.67 to 0.43), as was the reduction in birth weight (2863±440 g in women with past or chronic infections vs. 3070±482 g in uninfected women in 2010-2012; mean difference, -164.5 g; 95% CI, -289.7 to -39.4; and 2994±487 g vs. 3117±455 g in 2003-2005; difference, -44.8 g; 95% CI, -139.1 to 49.5). CONCLUSIONS: Antimalarial antibodies were reduced and the adverse consequences of P. falciparum infections were increased in pregnant women after 5 years of a decline in the prevalence of malaria. (Funded by Malaria Eradication Scientific Alliance and others.)

Efficacy and safety of intermittent preventive treatment with sulfadoxine-pyrimethamine for malaria in African infants: a pooled analysis of six randomised, placebo-controlled trials.

BACKGROUND: Intermittent preventive treatment (IPT) is a promising strategy for malaria control in infants. We undertook a pooled analysis of the safety and efficacy of IPT in infants (IPTi) with sulfadoxine-pyrimethamine in Africa. METHODS: We pooled data from six double-blind, randomised, placebo-controlled trials (undertaken one each in Tanzania, Mozambique, and Gabon, and three in Ghana) that assessed the efficacy of IPTi with sulfadoxine-pyrimethamine. In all trials, IPTi or placebo was given to infants at the time of routine vaccinations delivered by WHO's Expanded Program on Immunization. Data from the trials for incidence of clinical malaria, risk of anaemia (packed-cell volume <25% or haemoglobin <80 g/L), and incidence of hospital admissions and adverse events in infants up to 12 months of age were reanalysed by use of standard outcome definitions and time periods. Analysis was by modified intention to treat, including all infants who received at least one dose of IPTi or placebo. FINDINGS: The six trials provided data for 7930 infants (IPTi, n=3958; placebo, n=3972). IPTi had a protective efficacy of 30.3% (95% CI 19.8-39.4, p<0.0001) against clinical malaria, 21.3% (8.2-32.5, p=0.002) against the risk of anaemia, 38.1% (12.5-56.2, p=0.007) against hospital admissions associated with malaria parasitaemia, and 22.9% (10.0-34.0, p=0.001) against all-cause hospital admissions. There were 56 deaths in the IPTi group compared with 53 in the placebo group (rate ratio 1.05, 95% CI 0.72-1.54, p=0.79). One death, judged as possibly related to IPTi because it occurred 19 days after a treatment dose, was subsequently attributed to probable sepsis. Four of 676 non-fatal hospital admissions in the IPTi group were deemed related to study treatment compared with five of 860 in the placebo group. None of three serious dermatological adverse events in the IPTi group were judged related to study treatment compared with one of 13 in the placebo group. INTERPRETATION: IPTi with sulfadoxine-pyrimethamine was safe and efficacious across a range of malaria transmission settings, suggesting that this intervention is a useful contribution to malaria control. FUNDING: Bill & Melinda Gates Foundation

Multiplexing detection of IgG against <i>Plasmodium falciparum</i> pregnancy-specific antigens

<div><p>Background</p><p>Pregnant women exposed to <i>Plasmodium falciparum</i> generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens.</p><p>Methods and findings</p><p>We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R² = 0.99 and peptide array: R² = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003–2005 than during 2010–2012, in accordance with the levels of malaria transmission reported for these years in Mozambique.</p><p>Conclusions</p><p>The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the level of exposure to malaria. This platform provides a high-throughput approach to investigating correlates of protection and identifying serological markers of exposure for malaria in pregnancy.</p></div

IgG antibodies measured in plasma from VAR2CSA-immune, pregnant Mozambican women compared with plasma from Spanish individuals and Mozambican men.

<p><b>A)</b> Ratio of nMFIs against the protein array (3 VAR2CSA recombinant domains + AMA1, MSP1₁₉, rCSP and tetanus toxin) and the peptide array (46 VAR2CSA-based peptides + pCSP) measured in VAR2CSA-immune pregnant Mozambican women and Spanish individuals (open circle) or Mozambican men (black circle), obtained by linear regression. T-bars correspond to the 95% confidence interval (CI). B) Bars represent the mean nMFIs from VAR2CSA-immune pregnant women. T-bars correspond to the 95% CI. Red dashed line represents the mean nMFI from BSA plus 3 standard deviations (SD) (BSA reactivity threshold). Asterisk indicates that IgG level from VAR2CSA-immune pregnant women was above the BSA reactivity threshold and statistically higher from both never exposed individuals and exposed men (p < 0.05 by linear regression).</p

Correlation of nMFIs measured in VAR2CSA hyperimmune plasma in singleplex and multiplex.

<p>A) Protein array (3 VAR2CSA recombinant domains + AMA1, MSP1₁₉, rCSP and tetanus toxin). B) Peptide array (46 VAR2CSA-based peptides + pCSP). Each dot corresponds to the nMFI value for each antigen of the array.</p

Characteristics of pregnant Mozambican women included in the study.

<p>Characteristics of pregnant Mozambican women included in the study.</p

Agreement of nMFIs measured in plasma and corresponding dried blood spots before and after discarding improperly eluted samples.

<p>Agreement of nMFIs measured in plasma and corresponding dried blood spots before and after discarding improperly eluted samples.</p

Antigens coupled on beads were highly recognized by IgGs in hyperimmune samples compared with negative controls.

<p>A) nMFIs obtained from 10 two-fold dilutions (1:400 to 1:20,000) of CSP hyperimmune serum measured against the CSP recombinant protein (rCSP) and peptide (pCSP). B) and C) nMFIs obtained from 15 two-fold dilutions (1:50 to 1:1.6x10⁶) of VAR2CSA hyperimmune plasma (HIP-VAR2CSA) measured against the protein array (3 VAR2CSA recombinant domains + AMA1, MSP1₁₉, rCSP, tetanus toxin) and the peptide array (46 VAR2CSA-based peptides + pCSP). D) nMFIs measured in HIP-VAR2CSA divided by mean nMFIs measured in 5 pregnant Spanish women never exposed to malaria (PW) (dilution 1:100 for peptides array and 1:400 for proteins array).</p

Agreement of seroprevalences defined by finite mixture models and pregnant women never exposed to malaria in periods of different malaria transmission intensity.

<p>Three epidemiological scenarios were considered to test the agreement of seroprevalences against the protein array (3 VAR2CSA recombinant domains + AMA1, MSP1₁₉, rCSP and tetanus toxin) and the peptide array (subset comprised by 15 VAR2CSA-based peptides + pCSP): A) high malaria intensity; B) low malaria intensity; C) both periods together (high & low). Black bars correspond to seroprevalence obtained by finite mixture models (FMM), grey bars to seroprevalence obtained by pregnant women never exposed to malaria and T-bars indicate the 95% confidence interval (CI). Dots are the Kappa statistic of agreement of seroprevalences obtained by both methods for each antigen, red line represents the mean of Kappa score, dashed line represents 0.7 Kappa score threshold for good agreement and T-bars represent standard errors of the mean (SEM).</p