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Background: During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles., Methods: Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement., Results: Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women., Conclusions: In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.

Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1(19)) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.

This study aimed to evaluate whether intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) had an effect on the acquisition of IgG against Plasmodium falciparum variant surface antigens (VSA) and growth-inhibitory antibodies in Manhiça, Mozambique. In addition, we assessed factors affecting the magnitude of these responses and the association between antibody levels and protection against malaria. IgG to VSA expressed by MOZ2, R29 and E8B parasite isolates were measured in plasma samples collected at 5, 9, 12 and 24 months of age by flow cytometry. Growth-inhibitory antibodies in dialyzed plasmas using GFP-D10 parasites were measured by flow cytometry at 12 and 24 months. IPTi-SP did not significantly modify the levels of IgG against VSA nor the growth-inhibitory capacity of antibodies up to 2 years of age. Age but not previous episodes of malaria influenced the magnitude of these responses. In addition, anti-VSA IgG levels were 7% higher in children with current P. falciparum infection and were associated with neighborhood of residence. Children aged 24 months had 10% less parasite growth than those aged 12 months (95% CI 0.88-0.93, P<0.0001). Growth-inhibitory antibodies correlated with levels of IgG against AMA-1, when evaluating the 10% (R(2)=0.444, P=0.049) and 20% (R(2)=0.230, P=0.037) highest inhibitory samples. None of the responses were associated with subsequent risk of malaria. In conclusion, IPTi-SP does not negatively affect the development of antibody responses thought to be major contributors to the acquisition of immunity to malaria in infancy., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Factors involved in the development of resistance to sulphadoxine-pyrimethamine (SP) by Plasmodium falciparum, particularly in the context of intermittent preventive treatment during pregnancy (IPTp), are not well known. We aimed to determine the impact of IPTp and human immunodeficiency virus (HIV) infection on molecular markers of SP resistance and the clinical relevance of resistant infections., Methods: SP resistance alleles were determined in peripheral (n = 125) and placental (n = 145) P. falciparum isolates obtained from pregnant women enrolled in a randomized, placebo-controlled trial of IPTp in Manhiça, Mozambique., Results: Prevalence of quintuple mutant infections was 12% (23 of 185 isolates) in pregnant women who received placebo and 24% (20 of 85 isolates) in those who received SP (P = .031). When the last IPTp dose was administered at late pregnancy, mutant infections at delivery were more prevalent in placental samples (7 [23%] of 30, samples) than in peripheral blood samples (2 [7%] of 30 samples; P = .025), more prevalent in women who received IPTp-SP than in those who received placebo (odds ratio [OR], 8.13; 95% confidence interval [CI], 1.69-39.08), and more prevalent in HIV-positive women than in HIV-negative women (OR, 5.17; 95% CI, 1.23-21.66). No association was found between mutant infections and increased parasite density or malaria-related morbidity in mothers and children., Conclusions: IPTp with SP increases the prevalence of resistance markers in the placenta and in HIV-infected women at delivery, which suggests that host immunity is key for the clearance of drug-resistant infections. However, this effect of IPTp is limited to the period when blood levels of SP are likely to be significant and does not translate into more-severe infections or adverse clinical outcomes.

Background: The RTS,S/AS02(D) vaccine has been shown to have a promising safety profile, to be immunogenic and to confer protection against malaria in children and infants., Methods and Findings: We did a randomized, controlled, phase I/IIb trial of RTS,S/AS02(D) given at 10, 14 and 18 weeks of age staggered with routine immunization vaccines in 214 Mozambican infants. The study was double-blind until the young child completed 6 months of follow-up over which period vaccine efficacy against new Plasmodium falciparum infections was estimated at 65.9% (95% CI 42.6-79.8, p<0.0001). We now report safety, immunogenicity and estimated efficacy against clinical malaria up to 14 months after study start. Vaccine efficacy was assessed using Cox regression models. The frequency of serious adverse events was 32.7% in the RTS,S/AS02(D) and 31.8% in the control group. The geometric mean titers of anti-circumsporozoite antibodies declined from 199.9 to 7.3 EU/mL from one to 12 months post dose three of RTS,S/AS02(D), remaining 15-fold higher than in the control group. Vaccine efficacy against clinical malaria was 33% (95% CI: -4.3-56.9, p = 0.076) over 14 months of follow-up. The hazard rate of disease per 2-fold increase in anti-CS titters was reduced by 84% (95% CI 35.1-88.2, p = 0.003)., Conclusion: The RTS,S/AS02(D) malaria vaccine administered to young infants has a good safety profile and remains efficacious over 14 months. A strong association between anti-CS antibodies and risk of clinical malaria has been described for the first time. The results also suggest a decrease of both anti-CS antibodies and vaccine efficacy over time., Trial Registration: ClinicalTrials.gov NCT00197028.

Results from clinical trials in areas where malaria is endemic have shown that immunization with RTS,S/AS02A malaria vaccine candidate induces partial protection in adults and children and cellular effector and memory responses in adults. For the first time in a malaria vaccine trial, we sought to assess the cell-mediated immune responses to RTS,S antigen components in infants under 1 year of age participating in a clinical phase I/IIb trial of RTS,S/AS02D in Mozambique. Circumsporozoite protein (CSP)-specific responses were detected in approximately half of RTS,S-immunized infants and included gamma interferon (IFN-gamma), interleukin-2 (IL-2), and combined IL-2/IL-4 responses. The median stimulation indices of cytokine-producing CD4(+) and CD8(+) cells were very low but significantly higher in RTS,S-immunized infants than in infants that received the comparator vaccine. Protection against subsequent malarial infection tended to be associated with a higher percentage of individuals with CSP-specific IL-2 in the supernatant (P = 0.053) and with higher CSP-specific IFN-gamma-producing CD8(+) T-cell responses (P = 0.07). These results report for the first time the detection of malaria-specific cellular immune responses after vaccination of infants less than 1 year of age and pave the way for future field studies of cellular immunity to malaria vaccine candidates.

Background: Control of malaria in pregnancy remains a public health challenge. Improvements in its correct diagnosis and the adequacy of protocols to evaluate anti-malarial drug efficacy in pregnancy, are essential to achieve this goal., Methods: The presence of Plasmodium falciparum was assessed by real-time (RT) PCR in 284 blood samples from pregnant women with clinical complaints suggestive of malaria, attending the maternity clinic of a Mozambican rural hospital. Parasite recrudescences in 33 consecutive paired episodes during the same pregnancy were identified by msp1 and msp2 genotyping., Results: Prevalence of parasitaemia by microscopy was 5.3% (15/284) and 23.2% (66/284) by RT-PCR. Sensitivity of microscopy, compared to RT-PCR detection, was 22.7%. Risk of maternal anaemia was higher in PCR-positive women than in PCR-negative women (odds ratio [OR] = 1.92, 95% confidence interval [CI] 1.09-3.36). Genotyping confirmed that recrudescence after malaria treatment occurred in 7 (21%) out of 33 pregnant women with consecutive episodes during the same pregnancy (time range between recrudescent episodes: 14 to 187 days)., Conclusion: More accurate and sensitive diagnostic indicators of malaria infection in pregnancy are needed to improve malaria control. Longer follow-up periods than the standard in vivo drug efficacy protocol should be used to assess anti-malarial drug efficacy in pregnancy.

We evaluated the impact of intermittent preventive treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP), which was given at ages 3, 4, and 9 months through the Expanded Program on Immunization (EPI), on the development of antibody responses to Plasmodium falciparum in Mozambique. Immunoglobulin M (IgM) and IgG subclass antibodies specific to whole asexual parasites and to recombinant MSP-1(19), AMA-1, and EBA-175 were measured at ages 5, 9, 12, and 24 months for 302 children by immunofluorescence antibody tests and by enzyme-linked immunosorbent assays. Antibody responses did not significantly differ between children receiving IPTi with SP and those receiving a placebo at any time point measured, with the exception of the responses of IgG and IgG1 to AMA-1 and/or MSP-1(19), which were significantly higher in the SP-treated group than in the placebo group at ages 5, 9, and/or 24 months. IPTi with SP given through the EPI reduces the frequency of malarial illness while allowing the development of naturally acquired antibody responses to P. falciparum antigens.

Background: Intermittent preventive treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a potential malaria control strategy. There is concern about the impact that increasing in vivo resistance to SP has on the efficacy of IPTi, as well as about the potential contribution of IPTi to increases in resistance., Methods: We compared the frequency of clinical episodes of malaria caused by P. falciparum parasites with mutations in dhfr and dhps among sick children who received SP or placebo in the context of a randomized, double-blind, placebo-controlled IPTi trial in Mozambique., Results: Half of the children who received placebo harbored quintuple-pure mutant parasites. Nevertheless, the protective efficacy of IPTi within the 35 days after the third dose was 70.8% (95% confidence interval [CI], 40.7%-85.6%). Between month 2 after the third IPTi dose and the end of the follow-up period, children receiving SP harbored more dhps codon 437 mixed infections (odds ratio [OR], 10.56 [95% CI, 1.30-86.14]) and fewer dhps double-pure mutant parasites (OR, 0.43 [95% CI, 0.22-0.84]) than did placebo recipients., Conclusions: IPTi appears to be associated with some changes in the prevalence of genotypes involved in SP resistance. In the face of a high prevalence of quintuple-mutant parasites, SP exhibited a high level of efficacy in the prevention of new episodes of malaria in infants.

Background: Malaria remains a leading global health problem that requires the improved use of existing interventions and the accelerated development of new control methods. We aimed to assess the safety, immunogenicity, and initial efficacy of the malaria vaccine RTS,S/AS02D in infants in Africa., Methods: We did a phase I/IIb double-blind randomised trial of 214 infants in Mozambique. Infants were randomly assigned to receive three doses either of RTS,S/AS02D or the hepatitis B vaccine Engerix-B at ages 10 weeks, 14 weeks, and 18 weeks of age, as well as routine immunisation vaccines given at 8, 12, and 16 weeks of age. The primary endpoint was safety of the RTS,S/AS02D during the first 6 months of the study, and analysis was by intention to treat. Secondary endpoints included immunogenicity and analysis of new Plasmodium falciparum infections during a 3-month follow up after the third dose. Time to new infections in the per-protocol cohort were compared between groups using Cox regression models. This study is registered with ClinicalTrials.gov, number NCT00197028., Findings: There were 17 children (15.9%; 95% CI 9.5-24.2) with serious adverse events in each group. In the follow-up which ended on March 6, 2007, there were 31 serious adverse events in the RTS,S/AS02D group and 30 serious adverse events in the Engerix-B group, none of which were reported as related to vaccination. There were four deaths during this same follow-up period; all of them after the active detection of infection period had finished at study month 6 (two in RTSS/AS02D group and two in the Engerix-B group). RTS,S/AS02D induced high titres of anti-circumsporozoite antibodies. 68 first or only P falciparum infections were documented: 22 in the RTS,S/AS02D group and 46 in the control group. The adjusted vaccine efficacy was 65.9% (95% CI 42.6-79.8%, p<0.0001)., Interpretation: The RTS,S/AS02D malaria vaccine was safe, well tolerated, and immunogenic in young infants. These findings set the stage for expanded phase III efficacy studies to confirm vaccine efficacy against clinical malaria disease.

Background: RTS,S/AS02A is a pre-erythrocytic stage malaria vaccine that provides partial protection against infection in malaria-naive adult volunteers and hyperimmune adults. A previous report showed that this vaccine reduced risk of clinical malaria, delayed time to new infection, and reduced episodes of severe malaria over 6 months in African children. An important remaining issue is the durability of protection against clinical disease in these children., Methods: We did a randomised, controlled, phase IIb trial of RTS,S/AS02A given at 0, 1, and 2 months in 2022 Mozambican children aged 1-4 years. We previously determined vaccine efficacy (VE) against clinical malaria in a double-blind phase that included study months 2.5-8.5 (VE(2.5-8.5)). We now report VE in a single-blind phase up to month 21 (VE(8.5-21)). The primary endpoint was time to first or only clinical episode of Plasmodium falciparum malaria (axillary temperature 37.5 degrees C and P falciparum asexual parasitaemia >2500 per microL) detected through a passive case detection system. We also determined VE for other case definitions and for episodes of severe malaria. This study is registered with the ClinicalTrials.gov identifier NCT00197041., Findings: During the single-blind phase, VE(8.5-21) was 28.9% (95% CI 8.4-44.8; p=0.008). At month 21, prevalence of P falciparum infection was 29% lower in the RTS,S/AS02A group than in the control (p=0.017). Considering the entire study period, VE(2.5-21) was 35.3% (95% CI 21.6-46.6; p<0.0001) and VE(2.5-21) for severe malaria was 48.6% (95% CI 12.3-71.0; p=0.02)., Interpretation: These results show that RTS,S/AS02A confers partial protection in African children aged 1-4 years living in rural endemic areas against a range of clinical disease caused by P falciparum for at least 18 months, and confirm the potential of malaria vaccines to become credible control tools for public-health use.

Background: The RTS,S/AS02D vaccine has been shown to have a promising safety profile, to be immunogenic and to confer protection against malaria in children and infants. Methods and Findings: We did a randomized, controlled, phase I/IIb trial of RTS,S/AS02D given at 10, 14 and 18 weeks of age staggered with routine immunization vaccines in 214 Mozambican infants. The study was double-blind until the young child completed 6 months of follow-up over which period vaccine efficacy against new Plasmodium falciparum infections was estimated at 65.9% (95% CI 42.6-79.8, p<0.0001). We now report safety, immunogenicity and estimated efficacy against clinical malaria up to 14 months after study start. Vaccine efficacy was assessed using Cox regression models. The frequency of serious adverse events was 32.7% in the RTS,S/AS02D and 31.8% in the control group. The geometric mean titers of anti-circumsporozoite antibodies declined from 199.9 to 7.3 EU/mL from one to 12 months post dose three of RTS,S/AS02D, remaining 15-fold higher than in the control group. Vaccine efficacy against clinical malaria was 33% (95% CI: 24.3-56.9, p = 0.076) over 14 months of follow-up. The hazard rate of disease per 2-fold increase in anti-CS titters was reduced by 84% (95% CI 35.1-88.2, p = 0.003). Conclusion: The RTS,S/AS02D malaria vaccine administered to young infants has a good safety profile and remains efficacious over 14 months. A strong association between anti-CS antibodies and risk of clinical malaria has been described for the first time. The results also suggest a decrease of both anti-CS antibodies and vaccine efficacy over time. [ABSTRACT FROM AUTHOR]

Background: There is a widespread notion, based on limited information, that in areas of stable malaria transmission most pregnant women with Plasmodium falciparum infection are asymptomatic. This study aim to characterize the clinical presentation of malaria in African pregnant women and to evaluate the adequacy of case management based on clinical complaints. Methods: A hospital-based descriptive study between August 2003 and November 2005 was conducted at the maternity clinic of a rural hospital in Mozambique. All women attending the maternity clinic were invited to participate. A total of 2,330 women made 3,437 eligible visits, 3129 were analysed, the remainder were excluded because diagnostic results were unavailable or they were repeat visits. Women gave a standardized clinical history and had a medical exam. Malaria parasitaemia and haematocrit in capillary blood was determined for all women with signs or symptoms compatible with malaria including: presence and history of fever, arthromyalgias, headache, history of convulsions and pallor. Outcome measure was association of malaria symptoms or signs with positive blood slide for malaria parasitaemia. Results: In 77.4% of visits pregnant women had symptoms suggestive of malaria; 23% (708/3129) were in the first trimester. Malaria parasitaemia was confirmed in 26.9% (842/3129) of visits. Headache, arthromyalgias and history of fever were the most common symptoms (86.5%, 74.8% and 65.4%) presented, but their positive predictive values for malaria parasitaemia were low [28% (27-30), 29% (28-31), and 33% (31-35), respectively]. Conclusion: Symptoms suggestive of malaria were very frequent among pregnant women attending a rural maternity clinic in an area of stable malaria transmission. However, less than a third of them were parasitaemic. In the absence of microscopy or rapid diagnostic tests, a large proportion of women, including those in the first trimester of gestation, would be unnecessarily receiving antimalarial drugs, often those with unknown safety profiles for pregnancy. Accessibility to malaria diagnostic tools needs to be improved for pregnant women and drugs with a safety profile in all gestational ages are urgently needed. [ABSTRACT FROM AUTHOR]

Background Development of an effective malaria vaccine could greatly contribute to disease control. RTS,S/AS02A is a pre-erythrocytic vaccine candidate based on Plasmodium falciparum circumsporozoite surface antigen. We aimed to assess vaccine efficacy, immunogenicity, and safety in young African children. Methods We did a double-blind, phase IIb, randomised controlled trial in Mozambique in 2022 children aged 1--4 years. The study included two cohorts of children living in two separate areas which underwent different follow-up schemes. Participants were randomly allocated three doses of either RTS,S/AS02A candidate malaria vaccine or control vaccines. The primary endpoint, determined in cohort 1 (n=1605), was time to first clinical episode of P falciparum malaria (axillary temperature 37.5°C and P falciparum asexual parasitaemia >2500 per L) over a 6-month surveillance period. Efficacy for prevention of new infections was determined in cohort 2 (n=417). Analysis was per protocol. Findings 115 children in cohort 1 and 50 in cohort 2 did not receive all three doses and were excluded from the perprotocol analysis. Vaccine efficacy for the first clinical episodes was 29.9% (95% CI 11.0--44.8; p=0.004). At the end of the 6-month observation period, prevalence of P falciparum infection was 37% lower in the RTS,S/AS02A group compared with the control group (11.9% vs 18.9%; p=0.0003). Vaccine efficacy for severe malaria was 57.7% (95% CI 16.2--80.6; p=0.019). In cohort 2, vaccine efficacy for extending time to first infection was 45.0% (31.4--55.9; p<0.0001). Interpretation The RTS,S/AS02A vaccine was safe, well tolerated, and immunogenic. Our results show development of an effective vaccine against malaria is feasible. [ABSTRACT FROM AUTHOR]