Intermittent preventive treatment with sulfadoxine-pyrimethamine does not modify plasma cytokines and chemokines or intracellular cytokine responses to Plasmodium falciparum in Mozambican children

Cytokines and chemokines are key mediators of anti-malarial immunity. We evaluated whether Intermittent Preventive Treatment in infants with Sulfadoxine-Pyrimethamine (IPTi-SP) had an effect on the acquisition of these cellular immune responses in Mozambican children. Multiple cytokines and chemokines were quantified in plasma by luminex, and antigen-specific cytokine production in whole blood was determined by intracellular cytokine staining and flow cytometry, at ages 5, 9, 12 and 24 months. Results: IPTi-SP did not significantly affect the proportion of CD3+ cells producing IFN-γ, IL-4 or IL-10. Overall, plasma cytokine or chemokine concentrations did not differ between treatment groups. Th1 and pro-inflammatory responses were higher than Th2 and anti-inflammatory responses, respectively, and IFN-γ:IL-4 ratios were higher for placebo than for SP recipients. Levels of cytokines and chemokines varied according to age, declining from 5 to 9 months. Plasma concentrations of IL-10, IL-12 and IL-13 were associated with current infection or prior malaria episodes. Higher frequencies of IFN-γ and IL-10 producing CD3+ cells and elevated IL-10, IFN-γ, MCP-1 and IL-13 in plasma were individually associated with increased malaria incidence, at different time points. When all markers were analyzed together, only higher IL-17 at 12 months was associated with lower incidence of malaria up to 24 months. Conclusions: Our work has confirmed that IPTi-SP does not negatively affect the development of cellular immune response during early childhood. This study has also provided new insights as to how these cytokine responses are acquired upon age and exposure to P. falciparum, as well as their associations with malaria susceptibility.

IgG against Plasmodium falciparum variant surface antigens and growth inhibitory antibodies in Mozambican children receiving intermittent preventive treatment with sulfadoxine-pyrimethamine

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.

Sub-microscopic infections and long-term recrudescence of Plasmodium falciparum in Mozambican pregnant women

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

Impact of intermittent preventive treatment with sulfadoxine-pyrimethamine on antibody responses to erythrocytic-stage Plasmodium falciparum antigens in infants in Mozambique

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