Severe malaria risk varies between individuals, and most of this variation remains unexplained. Here, we examined the hypothesis that cytokine profiles at birth reflect inter-individual differences that persist and influence malaria parasite density and disease severity throughout early childhood.Cytokine levels  were measured at birth  and during subsequent routine follow-up visits  for children enrolled between 2002 and 2006 into a birth cohort in Muheza, Tanzania. Children underwent blood smear and clinical assessments every 2-4 weeks, and at the time of any illness. Cord blood levels of all cytokines were positively correlated with each other (Spearman’s rank correlation). Cord levels of IL-1β and TNF-α (but not other cytokines) correlated with levels of the same cytokine measured at routine visits during early life (P < 0.05). Higher cord levels of IL-1β but not TNF-α were associated with lower parasite densities during infancy  method), with an average ~40% reduction versus children with low cord IL-1β levels, and with decreased risk of severe malaria during follow-up (Cox regression): adjusted hazard ratio (95% CI) 0.60 (0.39-0.92), P = 0.02.IL-1β levels at birth are related to future IL-1β levels as well as the risk of severe malaria in early life. The effect on severe malaria risk may be due in part to the effect of inflammatory cytokines to control parasite density. Despite renewed efforts at control and elimination, malaria remains a major cause of morbidity and mortality in Africa, where 174 million clinical cases occur annually, resulting in an estimated 596,000 deaths , most ofDuring infection, cytokines play a dual role by controlling parasite growth on the one hand while exacerbating pathology on the other. These opposing effects have been attributed to the timing of cytokine expression  as well Cytokine levels at birth might reflect inter-individual differences that persist and influence malaria outcomes during childhood. In malaria endemic areas, fetal sensitization to malaria antigens is common: cord blood lymphocytes often respond to stimulation with malaria antigens by proliferating ,18 and pin utero immune profiles will persist during early childhood and influence malaria outcomes, we measured plasma cytokine levels at birth and analysed their relationship with cytokine levels and the risk of severe P. falciparum malaria during early life. We report for the first time that high levels of IL-1β in cord blood persist, and are associated with both improved control of parasite density and with decreased risk of severe malaria during infancy. To test the hypothesis that Mothers and newborns were enrolled in a birth cohort study known locally as the Mother-Offspring Malaria Studies (MOMS) Project at Muheza Designated District Hospital, Muheza, Tanzania. Clinical procedures for the MOMS Project have been previously described . ChildreChildren were examined and blood smears obtained by finger or heel prick every 2 weeks during infancy and every 4 weeks post-infancy. Peripheral blood was collected into CPD anticoagulant during routine visits at roughly 3 months of age and then at ~6 month age intervals thereafter. Whenever children developed symptoms, they were examined by a study clinician. Children were classified as having severe malaria according to WHO criteria . Written informed consent was obtained from mothers prior to enrollment. Protocols for procedures used in this study were approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle BioMed and the National Medical Research Coordinating Committee in Tanzania.Cord blood samples were obtained by clamping the cord and cannulating umbilical vessels immediately after delivery. After removal of the umbilical cord and fetal membranes, placental blood samples were obtained by manual compression of the placental tissue in a grinder. Placental and cord blood samples were anticoagulated with EDTA, and stored on ice until processing the same day. Malaria parasitemia diagnosis: Parasitemia was defined as identification of any parasites in a Giemsa-stained blood smear by microscopy, after counting at least 200 white blood cells at a magnification of 100x.Determination of red blood cell disorders: Hemoglobin type  was determined by cellulose acetate paper electrophoresis according to the manufacturer’s instructions . Genotyping for α-thalassemia was done according to the protocol described by Chong et al .g for 3 minutes and was stored frozen at -70°C until it was thawed on the day that cytokine assays were performed. Each plasma sample was analysed using a multiplex, bead-based platform  and custom-made assay kits as previously described [Cytokine assays: Plasma was obtained by centrifugation at 3,000 * escribed . For eacNon-parametric tests  were used to compare cord blood cytokine levels by baseline variables . Malaria transmission season was defined as high between May and October, based on the peak incidence of parasitemia observed in our cohort. Correlations between cytokines at birth and at routine uninfected healthy visits were evaluated by Spearman’s rank correlation coefficient. Bonferroni correction was used in the pairwise correlations between cytokines at birth. Cox regression models were fitted to evaluate the relationship between cord blood cytokine levels and the time to first episode of severe malaria. Schoenfeld residuals were used to test the proportional hazards assumption. Kaplan-Meier curves were used to display severe malaria rates in children with high versus low levels of cord cytokines IL-1β and TNF-α. To account for the correlation between visits of the same child, the association between cord blood cytokines and parasite density (log-transformed) was assessed by Generalized Estimating Equation (GEE) method. In this analysis, each positive blood smear was included as an observation. Exchangeable correlation structure and identity link function were used; and robust standard errors were estimated. Cytokine levels were included in Cox and GEE models as binary variables, determined by the median: values above the median were considered high and values below the median, low. Median values (in pg/ml) for cord blood cytokines were: TNF-α, 120.8; IL-1β, 6.0; IL-5, 2.6; IL-6, 7.0; IL-10, 3.5. Models were adjusted for sickle cell trait status, alpha-thalassemia, transmission season, bed net use, and village of residence. Since parity and placental malaria interact to influence malaria infection and clinical malaria risk ,25, thesAverage parasite densities during infections in infants, children aged 1 year or less, were estimated for children with high and low cord IL-1β levels: geometric mean parasite density during visits with infection was calculated for each child; the geometric mean of these values was then estimated for groups with different IL-1β levels at birth .Data analyses were conducted using STATA version 11.1 105 (13.4%) newborns were delivered by mothers with placental malaria (PM+), and this was most common in first and second time mothers  1. Mean Cord blood levels of some cytokines differed according to malaria transmission season, placental malaria status and maternal parity 2. Cord Pro-inflammatory and anti-inflammatory cytokines can be counter-regulatory. We analysed pairwise relationships between and within these two groups of cytokines at birth 3. The pWe examined the relationship between cord levels of cytokines at birth, and levels measured in peripheral blood of these children during follow-up 4. We inDuring a malaria infection, pro-inflammatory cytokines are rapidly released from innate and adaptive immune cells, and may contribute to control of parasite density . We analFinally, we analysed the influence of cord blood TNF-α and IL-1β levels on severe malaria risk. Kaplan-Meier curves  indicatein utero sensitization or constitutive expression levels of cytokines, reflected by cord blood cytokine levels, contributes to the risk of severe malaria during childhood. In this study, we investigated whether cord blood cytokines of children living in a malaria endemic area are related to cytokine levels during early life and whether they predict severe malaria risk. Our results show for the first time that higher levels of the pro-inflammatory cytokine IL-1β at birth are significantly associated with improved control of parasite density and with decreased risk of severe malaria during early life. These findings support the idea that Previous studies have shown that cord blood cytokine responses might influence the risk of common childhood diseases. In the US, greater IFN-γ secretion by cord blood mononuclear cells (CBMC) reduces the risk of acute lower respiratory illness in the first year of life . In a prP. falciparum infection were at lower risk of subsequent P. falciparum clinical episodes [In our birth cohort, infants with high levels of TNF-α and IL-1β at birth persist in this pattern during infancy, and these intrinsically higher levels might act early during an infection to control parasite density. TNF-α is a major effector cytokine and is implicated in both protection and pathogenicity during malaria infection. Several studies have observed that plasma levels of TNF–α are significantly higher in infected humans presenting with severe malaria ,13. In oepisodes , which iepisodes .P. yoelii, an effect partly mediated by IL-6 secretion [P. berghei [IL-1 acts synergistically with TNF-α to enhance NO and IFN-γ production in murine models of malaria . PBMC IFecretion , and con berghei . Our fin berghei . In our Malaria infection during pregnancy modifies the risk of malaria infection and disease for the offspring ,42. In Tin utero imprinting, and these differences could influence parasite density and severe malaria risk during early life. We have found that all cytokines tested are positively correlated at birth, suggesting that cytokine levels in cord blood might have been influenced by a common mechanism (such as inflammation) more so than by individual genetic polymorphisms.Future studies should also determine the mechanisms that control or modify cord blood cytokine profiles. Other maternal infections endemic in the study area such as lymphatic filariasis could be modifying cord blood cytokine production, but were not studied in this cohort. Alternatively, children may have intrinsic differences in cytokine expression, owing to, for example, genetic polymorphisms or Severe malaria represents a heterogeneous group of clinical presentations, including severe anemia, cerebral malaria, and respiratory distress, among others. These different syndromes might be associated with specific immune responses: for example, children with severe anemia have lower IL-10 levels compared to children with cerebral malaria ,43. FutuIn conclusion, IL-1β levels in cord blood predict IL-1β levels, parasite density, and severe malaria risk throughout infancy. Therefore, factors that influence the cytokine profile or pro-inflammatory bias of the unborn child may have a significant impact on the outcome of malaria infections throughout early life. Placental malaria also increases parasite density and severe malaria risk in offspring, but it does not influence cord cytokine levels and therefore acts independently of the cord cytokine effect. Further study is needed to identify the factors that influence the fetal cytokine profile, as well as interventions that might target these factors to improve malaria outcomes during early childhood.


Plasmodium infection in Ontario travellers. Our retrospective study included 4,551 confirmed cases of imported malaria reported throughout Ontario, with additional analysis at the local health unit level . During the 20-year period, Plasmodium vivax accounted for 50.6% of all cases, P. falciparum (38.6%), Plasmodium sp. (6.0%), P. ovale (3.1%), and P. malariae (1.8%). During the first ten years of the study (1990–1999), P. vivax  was the dominant agent, followed by P. falciparum (28%); however, during the second ten years (2000–2009) the situation reversed and P. falciparum (55%) dominated, followed by P. vivax (30%). The prevalence of P. falciparum and P. vivax cases varied spatially , temporally , and demographically . Infection rates per 100,000 international travellers were estimated: rates of infection were 2× higher in males compared to females; rates associated with travel to Africa were 37× higher compared to travel to Asia and 126× higher compared to travel to the Americas; rates of infection were 2.3–3.5× higher in June and July compared to October through March; and rates of infection were highest in those 65–69 years old. Where exposure country was reported, 71% of P. falciparum cases reported exposure in Ghana or Nigeria and 63% of P. vivax cases reported exposure in India. Our study provides insights toward improving pre-travel programs for Ontarians visiting malaria-endemic regions and underscores the changing epidemiology of imported malaria in the province.We examined malaria cases reported to Ontario’s public health surveillance systems from 1990 through 2009 to determine how temporal scale , spatial scale , and demography  contribute to  Plasmodium vivax) and 4% of those infected died . Si. Si18]. P. falciparum and P. vivax in various jurisdictions, including Ontario  compared with Western Australia where only 24% of all cases are due to P. falciparum. In Ontario, 39% of malaria cases were caused by P. falciparum (1990–2009). Although P. vivax dominated cases for the entire study period (and from 1990–1999), there were more P. falciparum cases compared to P. vivax cases from 2000 through 2009 . In Peel, there were a significantly higher mean number of cases per year of P. vivax than P. falciparum .Thirty-five out of Ontario’s 36 health units reported at least one imported malaria case during the surveillance period . Torontolciparum . In Otta in Peel . The frePlasmodium infection and renewed attention to established guidelines for Canadian travellers to malaria-endemic regions [We report 4,551 cases of imported malaria and an average infection rate of 10.7 per 100,000 international travellers during the 20-year surveillance period. The incidence of cases has remained stable since 1998; however, with increasing international travel, the rates of infection have been decreasing since 2000. The increase in cases in 1996 and 1997 has been noted previously in other non-malaria-endemic jurisdictions  and, while exposure information for Ontario cases is lacking for these years, the increase is likely due to malaria epidemics in several northwestern states of India such as Haryana and Rajasthan ,23,30. I regions .P. vivax was the dominant malarial agent during the first half of the surveillance period (1990–1999); however, since 2000, P. falciparum has supplanted P. vivax as the dominant agent. An increase in P. falciparum cases and associated decrease in P. vivax was similarly noted in a UK study (1987–2006) [P. falciparum cases in the UK was attributed to increased travel to West Africa, a region with high P. falciparum-transmission rates. The purpose or reason for travel to West Africa was not captured in our passive surveillance systems; however, jurisdictions with similar imported malaria etiology  have identified that most travellers to West Africa are classified as visiting friends and relatives (VFRs) [Plasmodium infection due to several factors, including 1) visiting malaria-endemic countries for longer periods, 2) travel to areas of higher transmission intensity, 3) belief that they are immune, and 4) a lack appropriate pre-travel advice [P. vivax decrease trend in the UK was attributed to an increase in the proportion of travellers to South Asia that visited urban areas  [In Ontario, 87–2006) . The incs (VFRs) ,23. Travl advice –23. The Plasmodium species in Ontario was associated with diagnosing health unit. Patterns seen in a jurisdiction’s Plasmodium composition mirror its population’s indigenous background, language, and colonial ties to malaria-endemic countries. For example, France’s population maintains a close relationship to former French colonies, with a greater number of its cases having originated in Cameroon and Côte d’Ivoire [Plasmodium species. Relative to provincial percentages, Peel has a greater percentage (44%) of Asian-born residents and a greater prevalence of P. vivax [P. falciparum. One limitation to our study is that travel data for patients at the health unit level were not available, but further investigation of prospective cases (with travel history) will provide further insight into risk or rate of infection for travellers at this scale. In recent research, Ontario’s imported malaria cases clustered at the neighborhood level, with clustering particularly evident in low-income areas [Plasmodium species; therefore, tailoring pre-travel education  at the local level should prove beneficial to higher-risk populations.Distribution of d’Ivoire . In the d’Ivoire . Ontariod’Ivoire ; howeverP. vivax . In contme areas . The indPlasmodium infection in males was twice that of females [Plasmodium species. Toronto and Ottawa had a relatively higher male to female case ratio and a greater percentage of the province’s P. falciparum infections; however, Peel had a relatively lower male to female case ratio and a greater percentage of the province’s P. vivax infections. Infection rates vary by gender in malaria-endemic regions, where cultural traditions and occupation contribute to a gender disparity, providing a basis for the gender-specific patterns seen for imported malaria. Risk of infection is often greater in men because they are more likely to migrate to malaria-endemic areas for agricultural or mining work and more likely to sleep outdoors during peak biting times of vectors; however, gender-specific behaviours of Ontario cases are unknown [Plasmodium species) in Ontario is similar to other jurisdictions such as the UK (31 years old), Calgary, Alberta (32 years old), and Barcelona, Spain (33 years old) [Men accounted for the majority of imported malaria cases in Ontario and most of cases occurred in those 20–54 years old; however, estimated rates of infection per 100,000 international travellers were greatest in those over 50 years old. In Ontario, females make up the majority of international travellers (57%), yet our analysis indicated that the rate of  females . Predomi females ,23,29. T unknown ,37. The ars old) ,23,25. WP. falciparum, June–August and, to a lesser extent, December–January; P. vivax, June–July). For all Plasmodium species, rates of infection were approximately 2.3-3.5× higher in June and July compared to October through March. The number of international travellers was not useful in explaining the monthly incidence of cases in Ontario, revealing that risk of infection is not based solely on volume of travellers to endemic regions, rather that other factors contribute more to risk . Similarly, in the UK, P. falciparum cases peaked in September and again in January; P. vivax cases peaked in July [P. vivax, case increases are because of greater travel volume in the summer, a period when there is also increased risk in destination countries (e.g. summer monsoon season in India). Pre-travel health care providers consider seasonality (dry versus rainy season of destination), as a part of an overall exposure assessment prior to a patient’s travel [Imported malaria cases in Ontario occurred more often in the summer ; therefore, our estimates for rates of infection are likely lower than the true rates for travellers to malaria-endemic countries [More than 50% of all imported malaria cases in Ontario, where country of exposure was reported (2006–2009), originated in Ghana or Nigeria and more than 19% of all cases originated in India. Travel to Ghana and Nigeria accounted for 34% of all cases in the UK , 32% of ountries . While rountries . Where dPlasmodium abroad; however, assessing risk should consider health unit-, spatiotemporal-, and demographic-specific dynamics. Despite being a rare event in North America, local transmission of Plasmodium will continue to occur, leaving surveillance as an important component of protecting public health [P. vivax malaria in Greece where locally-acquired infections were epidemiologically linked to imported cases [Travel destination for Ontario travellers represents the main risk factor for acquiring c health ,39. Surved cases . Surveil


Malaria prevention and iron supplementation are associated with improved maternal and infant outcomes. However, evidence from studies in children suggests iron may adversely modify the risk of malaria. We reviewed the evidence in pregnancy of the association between malaria and markers of iron status, iron supplementation or parenteral treatment.P. falciparum malaria during pregnancy or delivery in Africa  0.66–1.20, I2 = 78.8%, 5 studies). One study in Asia reported an increased risk of P. vivax within 30 days of iron supplementation , but not after 60 days. Iron deficiency (based on ferritin and C-reactive protein) was associated with lower odds for malaria infection . With the exception of the acute phase protein ferritin, biomarkers of iron deficiency were generally not associated with malaria infection.We searched MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, the Global Health Library, and the Malaria in Pregnancy library to identify studies that investigated the association between iron status, iron treatment or supplementation during pregnancy and malaria. Thirty one studies contributed to the analysis; 3 experimental and 28 observational studies. Iron supplementation was not associated with an increased risk of P vivax, but not with an increased risk of P. falciparum; however, data are insufficient to rule out the potential for an increased risk of P. falciparum. Iron deficiency was associated with a decreased malaria risk in pregnancy only when measured with ferritin. Until there is more evidence, it is prudent to provide iron in combination with malaria prevention during pregnancy.Iron supplementation was associated with a temporal increase in  Anemia affects the lives of more than 500 million women in developing countries. The consequences of anemia during pregnancy include maternal mortality and stillbirth A recently updated Cochrane review showed evidence that pregnant women taking iron supplements reduced their risk on anemia by 70%  and iron-deficiency at term by 57%  The benefits of maternal iron supplementation in malaria-endemic areas are less clear. An analysis of 101,636 singleton live-born infants conducted using data from Demographic and Health Surveys of 19 malaria-endemic countries in sub-Saharan Africa found that infants whose mothers received any iron/folic acid supplements and intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) for malaria during pregnancy were less likely to have a neonatal death compared to women who did not receive either iron/folic acid or malaria prophylaxis (hazard ratio (HR): 0.76; 95% CI 0.58–0.99) In summary, malaria prevention and iron supplementation are each associated with improved maternal and infant outcomes. However, the benefits of iron supplementation in pregnancy must be carefully weighed against the possibility of adverse consequences caused by this intervention in certain settings. Evidence from several studies among children suggests iron supplementation and iron status may adversely modify the risk of malaria, complicating a universal policy of routine iron supplementation in children in malaria endemic areas Despite the universal recommendation for iron supplementation in many malaria endemic countries, this question has yet to be examined in pregnancy, thereby creating uncertainty among public health programs regarding the use of iron supplementation during pregnancy in malaria endemic areas. To address this gap, we review the evidence in pregnancy of the association between the risk of malaria and iron status, iron treatment or iron supplementation.Studies investigating the association between iron status, iron treatment or iron supplementation during pregnancy and malaria risk were identified by searching MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, the Global Health Library from the World Health Organization (WHO) and the Malaria in Pregnancy library from their inception to January 2013 inclusive without language restrictions Using the search criteria defined above, 299 publications were identified. Each manuscript was reviewed by two authors (LS and AVE) and included if it met the following criteria: (1) study population included pregnant women; (2) data were available to assess the association between iron status, iron treatment, or iron supplementation and malaria; (3) for longitudinal studies and trials: an appropriate control group was included that did not receive iron; and (4) iron biomarkers were limited to serum ferritin, serum iron, serum transferrin, transferrin saturation (TS%), soluble transferrin receptor (sTfR), total iron binding capacity (TIBC), and erythrocyte protoporphyrin (EP). Discrepancies between reviewers were resolved through discussions until consensus was reached. For articles where it could be assumed that information was available but not presented in the format needed , the authors were contacted for additional information.Details of the methods used to assess validity are included in the supplemental appendix. In summary, the Cochrane Collaboration’s tool for assessing the risk of bias among randomized trials was used to determine the quality of included trials as ‘low’ (high risk of bias), ‘high’ (low risk of bias), or ‘unclear’ A standardized data abstraction form was used to collect the following data elements: year of publication, geographic location, population, design, number of subjects enrolled, malaria endemicity, duration, frequency and dose of iron, concurrent malaria treatment or prevention, incidence or prevalence of malaria parasitemia or placental malaria infection and biomarkers for iron status.Studies focusing on iron status, iron treatment, and iron supplementation were considered separately. Iron status studies were further stratified into iron deficiency and iron biomarkers studies. Study design was classified as randomized-controlled trial (RCT), prospective cohort study, retrospective cohort study, case-control study, cross-sectional study, or before-after study.The primary outcomes of interest were peripheral malaria parasitemia during pregnancy or peripheral or placental malaria infection at the time of delivery by blood smear. Information from additional malaria tests were included where available .Data from studies of iron deficiency status and individual iron biomarkers were summarized using forest plots and meta-analysis was conducted where possible. If malaria was assessed by more than one method, the most common test was utilized for the pooled analysis to decrease heterogeneity. Iron supplementation data were transformed into summary risk ratios (RR) and 95% confidence intervals (CI) estimated as the ratio of the proportion of women with malaria infection among those receiving and not-receiving iron supplementation during pregnancy. Available information in included studies allowed three subgroup analyses: by HIV status, by duration of iron supplementation, and by sickle cell genotype. Causes of anemia among HIV-infected pregnant women, iron status and effect of iron supplementation differ from HIV-negative pregnant women Iron deficiency data was described as ratio of the odds of malaria among iron deficient vs. non-deficient pregnant women in cross-sectional studies and case-control studies. Pooled analyses of iron deficiency and malaria were stratified by timing of malaria test (during pregnancy or at delivery) and the definition of iron deficiency. We used three groups; 1) an iron-deficiency definition based on ferritin and C-reactive protein , 2) a definition based on ferritin alone or in another combination (e.g. sTfR/log ferritin ratio), and 3) all other definitions that did not use ferritin (e.g. erythrocyte protoporphyrin). The summary odds ratio (OR) and 95% CI were computed as the pooled ratio of the odds of malaria among iron deficient and iron replete women; where available, adjusted odds ratios were used.p<0.10 was evaluated. The extent of heterogeneity was measured using I2, a measure of the proportion of total variability explained by heterogeneity; this is expressed as a percentage, with 0–25% indicating no or little heterogeneity Mean differences (MD) were computed to compare the overall effect for all individual biomarkers by transforming biomarkers to the same unit . Because of skewed distributions for most of the ferritin data, we used geometric means for ferritin; studies which did not present geometric means were transformed and geometric mean difference and 95% confidence interval calculated using the Taylor series approximation Of 299 published studies identified, 31 met the inclusion criteria . IncludeThe two trials, both published before the more widespread adoption of the CONSORT guidelines, were assessed as low quality, mainly because allocation concealment was not described. Overall, there were ten observational studies among the 25 evaluated which we marked as good quality. None of the observational studies reported a justification for the sample size of the study. For 16 studies, the association between iron and malaria was reported as one of the primary purposes of the analysis. The lack of a description of methods used to adjust for potential confounding was the most common deficit in the reporting of observational studies .The effect of iron supplementation on malaria risk in pregnancy was assessed in seven studies which included the two trials pooled 0.89, 95% CI 0.66–1.20; I2 78.8%, pooled 0.58, 95% CI 0.20–1.73; I2 89.9%) or at the time of delivery  1.30, 95% CI 0.67–2.50, p = 0.4, The results of further sub-group analysis can be seen in the supplement . No signP. vivax malaria, there was an increase in risk of first episode of P. vixax parasitemia associated with iron supplementation, but only during the first 30 days  and 16–30 days , after which the effect declined, and reversed for women who had taken iron supplementation for >60 days  and serum transferrin . StudiesThe same pattern was seen in four studies which could not be included in the meta-analysis . No signP. falciparum malaria among the participants who received oral iron supplementation, whereas one cohort study in Asia showed an increased risk of P. vivax in the first 30 days of oral iron (and folate) supplementation. None of the studies however, assessed the individual’s baseline iron status which may modify the effect of iron supplementation on malaria risk, complicating the interpretation of these data. Except for the studies using parenteral iron treatment, all included studies used oral iron supplementation for the prevention of iron deficiency anemia during pregnancy; oral iron was not given for the treatment of established anemia and/or iron-deficiency in these studies. This difference can be important because the treatment of anemia in pregnancy recommends using temporarily higher doses of iron, specific policies for iron dose and duration vary by country To our knowledge, this systematic review represents the first effort to assess the association of iron status and use on the risk of malaria in pregnancy. Pooled analyses of iron deficiency status, using a definition based on ferritin and adjusting for inflammation with CRP, indicated pregnant women with iron deficiency had a reduced risk of malaria infection during pregnancy. However, pooled analyses from the remaining biomarkers of iron deficiency were not associated with malaria. Data from randomized and observational studies did not show an increased risk of Only two studies evaluated parenteral treatment with iron dextran and the study qualities were insufficient to make conclusions. In addition, new parenteral iron treatment options using complex iron-polysaccharides are available which may not have a similar risk profile with regards to infections such as malaria P. falciparum and because iron can down-regulate the formation of nitric oxide within macrophages Several hypotheses have been provided as to why malaria infection may be less prevalent among people with iron deficiency. Iron is vital for the survival of the malaria parasite and the parasite is unable to thrive in iron-deficient environments Evidence from iron chelation studies using desferrioxamine (DFO) provides support that iron-deficient environments are unfavorable to foster malaria infections. In one randomized double-blind placebo cross-over study, DFO among asymptomatic parasitemic adults was associated with enhanced parasite clearance compared to placebo Multiple iron biomarkers have been associated with malaria infection among various populations. The most widely reported is serum ferritin, a known acute-phase reactant protein that can increase in response to underlying infection or inflammation regardless of underlying iron stores Despite the evidence that serum ferritin increases as a result of pregnancy per se, infection or inflammation, it remains the international standard for defining iron deficiency While the evidence of iron supplementation related to malaria risk in pregnancy is not of strong quality, the available data suggest iron supplementation does not increase the risk of malaria. Among the seven studies included in this review, only two of them represented primary data analyses from randomized trials, both of which failed to see an association between iron supplementation in pregnancy and malaria risk. No routine medical malaria prevention was used in these trials among the women included in this review, and they were conducted before the wide-scale introduction of insecticide treated nets Prevalence of use of malaria prevention strategies varied greatly between the two surveys included for iron supplementation, and these showed the greatest variety in the association between iron supplementation and malaria pooled 0.79, 95% CI 0.73–0.85, four studies, I2 31.7%, Figure S5.1 in 2 15·6%, Figure S5.2 in The higher risk of malaria among women that used iron for >30 days in the subgroup analysis  and the prevalence of malaria varied greatly across these studies, as did the use of malaria prevention strategies, and the dose and duration of iron supplementation . Although IPTp and insecticide treated nets are currently the main policies to prevent malaria in pregnant women in sub-Saharan Africa, their coverage is less than optimal and it would be important to ensure that individual women receiving iron supplementation are indeed protected by an insecticide treated net and receive IPTp Our review suggests iron supplementation may be safe during pregnancy in malaria endemic areas. However, the available data on iron supplementation in pregnancy and malaria risk are limited and insufficient to rule out any potential for an increased risk of malaria in malaria endemic settings. While iron deficiency (based on a definition with ferritin and CRP) is associated with a marked decreased risk of malaria in pregnancy, this association was not present among any of the other biomarkers of iron deficiency. Therefore, we find the results of iron deficiency and malaria risk to be inconclusive. Until more data are available from pregnant women, it would be prudent, based on the available evidence among children, to recommend that the provision of universal iron supplementation in pregnant women should always occur in conjunction with malaria prevention strategies during pregnancy in malaria endemic areas.File S1Supporting Information(DOCX)Click here for additional data file.Checklist S1PRISMA Checklist(DOCX)Click here for additional data file.


Plasmodium falciparum malaria.Volume substitution remains subject of controversy in the light of effusions and oedema potentially complicating this highly febrile disease. Understanding the role of myocardial and circulatory function appears to be essential for clinical management. In the present study, cardiac function and cardiac proteins have been assessed and correlated with parasitological and immunologic parameters in patients with imported P. falciparum malaria were included and findings were compared with 26 healthy controls. Cardiac function parameters were assessed by an innovative non-invasive method based on the re-breathing technique. In addition, cardiac enzymes and pro- and anti-inflammatory cytokines were measured and assessed with respect to clinical symptoms and conditions of malaria.In a prospective case-control study, 28 patients with uncomplicated and complicated 2 versus 3.4 l/min/m2; P < 0.001). In contrast, systemic vascular resistance index (SVRI) was increased by 29% (32.6 mmHg⋅m2/(l/min) versus 23.2 mmHg⋅m2/(l/min); P < 0.001). This correlated with increased cardiac proteins in patients versus controls: pro-BNP 139.3 pg/ml versus 60.4 pg/ml (P = 0.03), myoglobin 43.6 μg/l versus 27.8 μg/l (P = < 0.001). All measured cytokines were significantly increased in patients with malaria. CI, SVRI as well as cytokine levels did not correlate with blood parasite density.Cardiac index (CI) as a measurement of cardiac output (CO) was 21% lower in malaria patients than in healthy controls (2.7 l/min/mP. falciparum malaria. Findings may be relevant for fluid management and should be further explored in endemic regions.The results support previous reports suggesting impaired cardiac function contributing to clinical manifestations in  Plasmodium falciparum malaria ) were related to the body surface [m2] and respective cardiac-, stroke- and systemic vascular resistance indices  were included into statistical analyses. Patients did not receive volume infusions shortly before or during the re-breathing measurements.In addition to clinical symptoms and conditions defining severe malaria ,13, card® (BD) Biosciences, Heidelberg, Germany). The following cytokines were measured: IL-2, IL-6, TNF-α, IFN-γ (Th-1 response), IL-4, IL-10 (Th2-response), and IL-17A (Th-17 response). Briefly, phycoerythrin (PE)-conjugated  beads with distinct PE fluorescence intensities which have been coated with capture antibodies specific for the various cytokines were mixed with serum enhancement buffer and recombinant cytokine standards (20-5000 pg/ml) or patients serum samples. After centrifugation at 200 g for 5 min the supernatants were aspirated and discarded. After incubation of the beads for 3 h with a PE detection reagent at room temperature, wash buffer was added and the suspension centrifuged as before. After discarding the supernatant the beads were resuspended in wash buffer for acquiring and analysis of the samples in a flow cytometer  applying BD CellQuest® software.Sera of the patients were analysed for pro- and anti-inflammatory cytokines with a Cytokine Bead Array following manufacturer instructions  was used for statistical analysis. Normal distribution was assessed by the Kolmogorov-Smirnov test. The independent sample t-test or the Mann-Whitney U-test were used to assess differences between cases and controls, as appropriate. Proportions were assessed by chi-square test or Fisher's exact test (two-tailed) as applicable. For correlation of cardiac function tests and cytokines Pearson correlation test was used. Statistical significance was considered a P < 0.05.The SPSS 17.0 software package , acute renal insufficiency , hyperparasitaemia , and jaundice (1 case). Geometric mean parasite density (GMPD) was 4,449/μl in all 28 patients and 2,673/μl and 22,315/μl in uncomplicated and complicated malaria cases, respectively (p = 0.01). Demographic, clinical and biochemical characteristics of malaria cases and healthy controls are shown in Table Cardiac function, serological and parasitological parameters were investigated in 28 patients with imported 2 versus 3.4 l/min/m2, P < 0.001; SI 31.3 ml/m2 versus 43.6 ml/m2, P < 0.001, respectively). The systemic vascular resistance index (SVRI), in contrast, was significantly elevated in malaria patients . Cardiac function parameters did not differ significantly between uncomplicated and complicated malaria cases . Heart rate was significantly higher in malaria patients than in controls  and systemic vascular resistance (SVRI) nor cardiac enzymes were associated with parasite levels (data not shown). Cytokine levels of malaria cases and controls as well as patients with uncomplicated and complicated malaria are summarized in Table P. falciparum malaria. Cardiac output (CO) representing the blood volume pumped per minute was significantly decreased in malaria patients compared to healthy controls. The increase in heart rate, however, was not sufficient to compensate for the lower stroke volume in malaria patients. This finding is in line with recent publications supporting evidence for cardiac dysfunction in malaria patients [The results presented here show impaired myocardial function in patients with imported patients ,12.Reasons for impaired cardiac function may be related to impaired pre- or postcardial circulatory parameters or result from myocardial dysfunction itself. In recent years, intravascular fluid depletion has been shown to be present in patients with severe malaria contributing to at least those pathophysiological processes related to impaired microcirculation . In patiP. falciparum malaria are the parasite adhesion to the endothelium, rosetting, the sequestration of parasitized and unparasitized red blood cells (RBC) in peripheral small vessels, and the decreased deformability of RBCs resulting in impaired microcirculation and lactic acidosis [By the methodological approach used in this study, it was possible to calculate peripheral vascular resistance which was significantly increased in patients with malaria. This may indicate that in this patient group increased after-load might contribute to the decreased CO. Major pathophysiological processes typical for acidosis ,17-21. Tacidosis . It was acidosis . InteresFinally, malaria may affect cardiac function itself. NT-proBNP is a sensitive marker for impaired left ventricular (LV) function and is significantly elevated predominantly in severe malaria patients ,3. ParasTo assess the influence of the host immune reaction, a series of pro- and anti-inflammatory cytokines was assessed. As anticipated, all cytokines assessed were significantly elevated in malaria patients compared to controls, especially IL-17A in complicated malaria Table . Their aThe present study has some limitations. A rather small patient population was assessed which increases the risk of type I errors. Furthermore, the study population was rather heterogeneous in that pathophysiological processes may differ in German travellers and African migrants. All African migrants, however, had lived in Germany for at least two years and persistent semi-immunity was unlikely to have a major influence on the findings presented here. Finally, one substantial limitation results from the unmatched control group despite the fact that respective cardiac function parameters were expressed as indices to correct for differences in body weight and height.The innovative non-invasive methodological approach allowed the assessment of several dynamic functional parameters. As it is usually not indicated or possible to assess cardiovascular function by the gold standard of invasive thermodilution techniques our findings may contribute to the existing evidence on cardiovascular function in malaria patients. To obtain further information on the cardiovascular system in patients with malaria a larger scale trial on cardiac function in children with severe malaria is currently being completed in hyper-endemic regions of Ghana.P. falciparum malaria. Reduced cardiac output is paralleled by increased systemic vascular resistance. Findings may be relevant for fluid management and should be further explored in endemic regions.Findings presented here support previous reports suggesting impaired cardiac function contributing to clinical manifestations in The authors declare that they have no competing interests.Subjects were mainly recruited by JH with contributions by JPC, DW and SS. JPC and JH drafted the manuscript. NWB carried out the immunoassays. JPC and JH participated in the design of the study, PM and JPC performed the statistical analyses with major contributions from the other authors. JPC and GDB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Roll Back Malaria (RBM) interventions such as insecticide-treated mosquito nets (ITN) and artemisinin-based combination therapy (ACT) have become implemented with different velocities in the endemic countries of sub-Saharan Africa (SSA) in recent years. There is conflicting evidence on how much can be achieved under real life conditions with the current interventions in the highly endemic savannah areas of SSA.The study took place in a rural area of north-western Burkina Faso, which was defined as holoendemic in 1999. Clinical and parasitological data were compared in two cohorts of young children of the same age range from eight villages. Surveys took place in June and December of the year 1999 and 2009 respectively.P. falciparum prevalence was significantly lower compared to 1999 .Prevalence of mosquito net use increased from 22% in 1999 to 73% in 2009, with the majority of nets being ITNs in 2009. In 2009, The reduction in malaria prevalence in young children observed between 1999 and 2009 in a rural and formerly malaria holoendemic area of Burkina Faso is likely attributable to the increase in ITN availability and utilization over time. Roll Back Malaria (RBM) initiative coordinating global malaria control and elimination efforts [Global Health Initiatives (GHI), malaria control interventions - in particular insecticide-treated mosquito nets (ITN) and artemisinin-based combination therapy (ACT) - are currently rolled out in SSA on a large scale. However, the impact of these new developments on the malaria burden in the highly endemic areas of SSA continues to be hotly debated [Malaria remains a major cause of global morbidity and mortality, with most of the burden being in sub-Saharan Africa (SSA) ,2. Inter efforts . ConsequHere a systematic comparison of malaria survey data is presented, which were collected in 1999 and in 2009 in cohorts of young children of the same age range, from an endemic area of rural Burkina Faso.Burkina Faso, a landlocked country in the heart of West Africa, is composed of 45 provinces divided into 13 administrative regions. The Kossi Province belongs to the Mouhoun Region and borders the Republic of Mali. Its administrative centre, Nouna town, is located 300 km north-west from Ouagadougou, the capital of the country.2 and a population of about 320.000 in the year 2009 [The Kossi Province corresponds geographically to the Nouna Health District area, which has a size of 7464.44 kmear 2009 . The dryP. falciparum [Formal health services in the district are restricted to the hospital in Nouna town and 29 village-based health facilities . Malarialciparum . The perlciparum ,7, but tlciparum ,8, with The fieldwork for this study was conducted in collaboration with the Nouna Health Research Center (CRSN), which is situated in the Kossi Province. The study area of the CRSN consists of Nouna town and 58 of the surrounding villages. A health and demographic surveillance system (HDSS) has been operating in the area since 1992 .This paper is part of a PhD project which compares systematically child health data from the year 1999 with corresponding data from the year 2009 in the Nouna area. The data from 1999 was collected within the frame of a large placebo-controlled community-based intervention trial, which looked at the efficacy of a zinc supplementation scheme on malaria morbidity and malnutrition ,10,11. IIn June 2009, children from the same eight villages and the same age range as in the zinc trial of 1999 were enrolled. They were randomly chosen from the HDSS register of the CRSN. Data collection in this children cohort took place through two cross-sectional surveys, conducted in June and December 2009. During surveys, a team consisting of two study physicians, study nurses, laboratory technicians and specifically trained field workers visited the villages. The study villages were Bourasso, Cisse, Kodougou, Koro, Nokui, Seriba, Sikoro and Solimana. The sample size calculation, which resulted in 460 children to be recruited in 2009, was based on expected differences between 1999 and 2009 in the primary outcomes (anthropometric variables) of the project. The chosen sample size allows for a detection of a reduction in malaria parasite prevalence of 15% with 80% power and at a significance level of 0.05.For both study cohorts (1999 and 2009) data on various malaria parameters such as temperature, species-specific parasite rates and densities, malaria prevalence rates, as well as mosquito net utilization rates were collected. Data collection procedures for 1999 are described in detail elsewhere ,11,12. FP. falciparum trophozoites [Plasmodium falciparum prevalence) and a general linear model (SAS PROC GLM) was used for the continuous outcome variable . The analyses were adjusted for age, sex, and village. Significance level was 0.05%.Data analysis was done using SAS version 9.2 . Malaria was defined as ≥ 37.5°Celsius axillary temperature + ≥ 5000 Ethical approval was obtained by the Ethical Committee of the Heidelberg University Medical School and the local authorities . Prior to the study implementation, the trial was always explained in detail to and discussed with all relevant district authorities and the concerned communities. Community consent was sought. During the surveys, individual oral (in 1999) and written (in 2009) informed consent was sought from the respective caretakers of study children. The study children benefited from being treated free of charge in case of disease.For the 1999 study, data were analysed for 179 and 197 children who took part in the June and December survey respectively. In 2009, data were analysed for 460 and 409 children who took part in the June and December survey respectively.vs. 17.6 months, p ≤.0001). Moreover, there were small differences between 1999 and 2009 in the proportion of study children recruited from the eight study villages as well as in the reported ethnicity of participating children.Table During 1999, reported mosquito net use of study children during the last night was rather low and averaged 20% . In 2009, the corresponding figure was 73% . While in 1999, none of the study children slept under an ITN, in 2009 the great majority of study children's mosquito nets (72%) were ITNs.P. falciparum parasites/μl) prevalence between the corresponding survey months in 1999 and 2009, but P. falciparum prevalence was significantly lower in the 2009 compared to the 1999 surveys. There was an overall 22.8% reduction in P. falciparum prevalence between 1999 and 2009.Additional file P. falciparum parasite prevalence in 2009 compared to 1999, although overall parasite prevalence remained above 50% in this population of young children in rural SSA. This development is most likely attributable to an increase in mosquito net and in particular ITN utilization by young children in the area. Major reductions in the rates of malaria disease and all-cause childhood mortality have consistently been associated with the use of ITNs in the endemic areas of SSA, and this has recently been shown to be of comparable magnitude under programme conditions [The main finding from this study is a significant reduction in nditions . Howevernditions . Recentlnditions . In thesHowever, in 2009 ITN coverage was still below the RBM target of 80% in this population of rural Burkina Faso. It remains to be seen how malaria parameters will develop after universal coverage has been achieved ,16. At tThis study has several strengths and limitations. First, it systematically compares malaria parameters in a large sample of young children over a period of ten years, before and after the roll-out of ITN programme in the country. However, it only compares data from two years and the findings may thus be influenced by unknown confounding variables such as weather conditions. Second, the two study cohorts were carefully matched for key socio-demographic parameters such as age, sex and village. However, they still differed slightly in a number of parameters, and even after adjustment in the analysis for known confounders, there might have remained some residual confounding. Third, there might also have been slight differences in the performance of examinations during the 1999 and 2009 surveys, as different study teams were in place. Although an influence of this cannot be ruled out, the parasitological results are likely not to have been affected much as there are standard operation procedures for malaria diagnosis in the laboratory of the CRSN. It also has to be considered that the study only shows malaria prevalence developments in eight purposively selected villages which can't be generalised to the whole Nouna Health District or to the whole country. Finally, there were many developments in the study area over the last 10 years, such as improvements in access to health services and in nutrition indicators, which may all have some influence on the local malaria burden.The findings from this study show a significant reduction in malaria parasite prevalence in young children between 1999 and 2009 in a rural and formerly malaria holoendemic area of Burkina Faso, which is likely attributable to the increase in ITN utilization over time.The authors declare that they have no competing interests.CB contributed to the conception and design of the study, developed the questionnaire, collected the data, analysed the data and wrote the paper. MB contributed to the conception and design of the study, developed the questionnaire, collected the data, and contributed to writing the paper. JT contributed to developing the questionnaire, collected the data and contributed to writing the paper. MA contributed to the conception and design of the study, developing the questionnaire, contributed to analysis and interpretation of the data and to writing the paper. VL contributed to designing the study and to writing the paper. BC contributed to designing the study and to writing the paper. MY contributed to designing the study and to writing the paper. OM initiated the conception and design of the study and contributed to interpretation of the data and to writing the paper. All authors read and approved the final manuscript.Table S1: Comparison of clinical and parasitological survey data in 1999 and in 2009. Additional file Click here for file


Despite increased control activities, malaria did not substantially decline. Plasmodium falciparum–positive slides among febrile children at the hospital declined early in the decade, but no further reductions were observed after 2005. The number of admissions for cerebral malaria did not differ significantly by year. Continued surveillance for malaria is needed to evaluate the effects of the increased malaria control efforts.In some areas of Africa, health facility data have indicated declines in malaria that might have resulted from increasingly effective control programs. Most such reports have been from countries where malaria transmission is highly seasonal or of modest intensity. In Malawi, perennial malaria transmission is intense, and malaria control measures have been scaled up during the past decade. We examined health facility data for children seen as outpatients and parasitemia-positive children hospitalized with cerebral malaria in a large national hospital. The proportion of  QECH serves a population of ≈1 million in Blantyre, surrounding townships, and outlying villages. Most hospitalizations are for community-acquired illnesses, and patients admitted are largely either self-referred or referred from a primary health center within Blantyre. Malaria diagnosis in both the PAEU and research ward was performed by the same personnel, who received regular refresher training throughout the study period. BCIH is a 66-bed teaching hospital that specializes in treating the orthopedic needs of children and adults. It is a referral hospital and therefore covers a population from both urban and rural areas in the same region. Health care is provided free to children at both facilities.From every child in the PAEU who had a febrile illness, blood was obtained for a thick blood film malaria parasite examination and for measurement of hematocrit. These criteria did not change during the study period. Total monthly numbers of outpatient visits to QECH by children, malaria slides taken, and results of thick-film microscopy were available from the PAEU laboratory records. To obtain estimates of parasite density and blood hematocrit levels, we abstracted individual-level records from January 2001 through December 2010. Because of the large volume of data recorded since 2001, we abstracted data from only 2 days (Monday and Thursday) of the first and last weeks of each month.Plasmodium falciparum parasitemia, negative cerebrospinal fluid, and no other cause of coma identified on examination or investigation. These children were a subset of children admitted to QECH and represent virtually all children admitted with cerebral malaria each year during the rainy seasons. All the diagnoses were reviewed daily by an experienced clinician.The criteria for admission into the research ward with cerebral malaria were consistent with World Health Organization guidelines , moderate-to-severe anemia (PCV 15%–24%), and mild (PCV 24%–33%) anemia also were obtained for the same period. Approximate parasite densities were based on numbers of asexual-stage P. falciparum parasites per oil-immersion field on the thick blood film  for descriptive and statistical analyses. Monthly slide positivity rates (SPRs) were calculated as the proportion of positive slides of all slides in a given month. Monthly figures for the prevalence of severe anemia  visits, and 61,320 (25.2%) of these were parasite positive for 001–2003 . The pat001–2003 . SPRs pe001–2003 .To analyze parasite densities and hematocrits of children in the PAEU, we abstracted 22,397 records  from the PAEU laboratory books. Books were available for the entire period, except for 2 quarters for which books were missing (October–December 2004 and January–March 2010). No clear differences were observed in parasite density levels over time , panel AThe overall prevalences of mild, moderate, and severe anemia were 49.7 (95% CI 49.1–50.4), 12.9 (95% CI 12.5–13.4), and 3.2 (95% CI 2.9–3.4), respectively. Each anemia category peaked in 2001 and significantly fell by 23%, 26%, and 39%, respectively, in 2002 (p<0.001). In 2003, the prevalences of all anemia categories significantly decreased by an additional 13%, 29%, and 23%, respectively (p<0.001). No further reductions occurred after 2004 , panel B<15%). Data for 2005 were not included because the research ward was not fully functional during that year.During January–June in 2001–2010, a total of 1,048 children with cerebral malaria were admitted to the research ward at QECH; 433 (41%) also had severe malarial anemia .The mean age of children with cerebral malaria was significantly higher after 2007 than in 2001. A shift of susceptibility to severe malaria toward older children is theoretically possible in areas where malaria control interventions are being rapidly scaled up and has been reported elsewhere  as a proxy for indicators likely to prevail in the community surrounding both hospitals. These data showed no substantial reductions in SPR during 2003–2009 but some evidence of a reduction (p<0.05) in 2010 from 2003. The SPR obtained from these trauma patients is a good proxy for the malaria prevalence in the community , and the number of slides taken remained roughly constant over time. This trend could represent a decrease in the proportion of attendees with a malaria-like illness; alternatively, the facility might have reached its capacity to conduct malaria tests, and when that capacity was exceeded, patients were treated for malaria on the basis of a presumptive diagnosis. If the latter occurred, we would have underestimated malaria in the later years of our study period. This potential underestimation is an intrinsic problem of retrospective health facility–based studies that might have resulted in a systematic bias in observed trends. Although a systematic bias could have occurred in the PAEU, we are confident that it did not affect the high-dependency ward because all cerebral malaria patients are admitted to this ward during the rainy season. However, previous studies reporting declining trends of hospital admissions for malaria (As malaria control efforts increase in Malawi and in similar areas, continued surveillance is needed to describe changes in malaria among a population exposed to lower intensities of malaria transmission. Significant increases in the mean age of outpatient and hospitalized children with positive slides and a rise in the proportion of cerebral malaria among all those admitted for severe malaria are among changes that might occur (


Plasmodium falciparum can protect against disease, although the precise mechanisms remain unclear. Although antibody levels can be measured by ELISA, few studies have investigated functional antibody assays in relation to clinical outcomes. In this study we applied a recently developed functional assay of antibody-mediated opsonisation of merozoites, to plasma samples from a longitudinal cohort study conducted in a malaria endemic region of Papua New Guinea (PNG). Phagocytic activity was quantified by flow cytometry using a standardized and high-throughput protocol, and was subsequently evaluated for association with protection from clinical malaria and high-density parasitemia. Opsonising antibody responses were found to: i) increase with age, ii) be enhanced by concurrent infection, and iii) correlate with protection from clinical episodes and high-density parasitemia. Stronger protective associations were observed in individuals with no detectable parasitemia at baseline. This study presents the first evidence for merozoite phagocytosis as a correlate of acquired immunity and clinical protection against P. falciparum malaria.Naturally acquired humoral immunity to the malarial parasite  Plasmodium spp., of which Plasmodium falciparum is responsible for most of the mortality and morbidity associated with the disease worldwide. There have been major gains in malaria control in endemic countries since 2000, due to measures such as long-lasting insecticidal nets, indoor residual spraying, rapid diagnostic testing, and access to artemisinin combination therapy. While incidence and mortality have declined, malaria still causes 216 million cases and 655,000 deaths per year, predominantly in young children and pregnant women (WHO 2011 report). In addition there are increasing reports of emerging artemisinin drug and insecticide resistance in some regions Malaria is caused by the protozoan parasites P. falciparum malaria.The rationale for vaccine development arose from observations of immunity acquired through natural exposure to malaria. Individuals living in malaria-endemic areas can develop both humoral and cell mediated immunity over time and with exposure , respiratory burst and phagocytosis assays Opsonising antibodies against merozoites require interactions with neutrophils or monocytes to trigger an anti-parasitic response. Functional in vitro in the presence of monocytes To date, merozoite opsonisation assays have been inadequately applied to the study of naturally acquired immunity and disease risk. The ADCI assay has not been rigorously validated for associations with clinical protection in longitudinal cohorts or otherwise, nor has the soluble factor responsible for parasite killing been identified. Antibody-dependent respiratory burst against merozoites has recently been shown to correlate with protection from clinical episodes P. falciparum malaria.To further investigate the contribution of merozoite opsonising antibodies to naturally acquired immunity to malaria, we applied a recently developed merozoite phagocytosis assay P. falciparum infection was determined by post-polymerase chain reaction (PCR) ligase detection reaction-florescent microsphere assay (LDR-FMA) and light microscopy (LM), with detection thresholds of approximately >0.3 parasite/µL and >40parasite/µL, respectively Details of the cohort were described elsewhere A venous blood sample was collected at enrolment into heparinised collection tubes, and plasma collected and stored at −80°C. Positive control sera was collected from adults within the Madang province, and negative control sera from malaria naïve anonymous Australian blood donors. For use in phagocytosis assays, plasma samples were serially diluted to a 1/2000 dilution in THP-1 medium.5 cells/mL.The human monocytic cell line THP-1  was maintained in RPMI-1640 supplemented with 10% foetal bovine serum (FBS) and 55 µM 2-mercapthoethanol (THP-1 medium), and maintained below a density of 5×10P. falciparum 3D7 parasites were cultured at 4% hematocrit in RPMI-1640 supplemented with 25 mg/mL HEPES, 2 mg/mL sodium bicarbonate, and 10% pooled human serum (parasite medium). Cultures were maintained at 37°C in an atmosphere of 5% CO2, 1% O2 and 94% N2, and synchronized using 5% sorbitol. Late-stage parasites (36–40 hr) were isolated (>95% purity) from uninfected RBCs with a MAC magnet separation column (Miltenyi Biotech). Schizonts were treated with 10 µM E64 (Sigma-Aldrich) for up to 10 hours, and merozoites isolated as previously described 6 EtBr-stained merozoites in 150 µL were mixed with 10 µL of diluted plasma samples, and aliquots of 50 µL were transferred to FBS-coated 96-well U-bottom plates containing 1×105 THP-1 cells to make a final volume of 200 µL per well. Each sample was tested in triplicate. Plates were incubated for 40 minutes at 37°C in 5% CO2 humidified incubator, followed by two washes in ice-cold PBS to arrest phagocytosis. Samples were fixed in 2% paraformaldehyde (PFA), and 20,000 events were acquired using a FACSCalibur flow cytometer (BD Bioscience). Data analysis was performed using FlowJo software. Viable cells were gated by forward and side scatter, and phagocytosis positive gates were determined using THP-1 cells alone. Percentage phagocytosis refers to the percentage of EtBr positive cells with immune plasma minus the percentage of positive cells with non-immune plasma.Phagocytosis assays were performed as previously described P. falciparum parasitemia by PCR or LM during follow up were regarded as non-exposed, and as such 5% of children were excluded from analysis. In addition, children that failed to clear infections following treatment at baseline(n = 12) as determined by msp2 genotyping, were also excluded from analysis. A range of demographic and clinical variables were assessed as potential confounders. The median age of individuals was 9.3 years . A range of age groups was investigated, and two groups (<9 years and ≥9 years) were most informative in concordance with previous investigations with this cohort Statistical analysis was performed using STATA 9.2 . Differences in %phagocytosis between categorical variables were assessed using two-sample Wilcoxon rank sum test or Kruskal-Wallis tests. For analysis of associations with clinical outcomes, children with no detectable P. falciparum infection and clinical episodes, phagocytosis responses were rescaled (%phagocytosis20) to generate 4 groups reflecting 20% increases . Poisson regression modeling produced incidence rate ratios (IRR) for the incidence of clinical malaria episodes or high-density parasitemia (>5000parasites/µL). Clinical malaria was defined as a measured fever (axillary temperature ≤37.5°C) or history of febrile illness during the 48hours preceding examination in conjunction with P. falciparum infection of any density as measured by light microscopy. Eighty percent of the 100 individuals that experienced at least 1 clinical episode had an infection density of greater than 5000 parasites/µL. Seventeen percent of the 92 individuals with an infection of >5000 parasites/µL did not present with clinical symptoms. Cox proportional hazards modeling was used to calculate hazard ratios (HR) for time to first P. falciparum infection by PCR, light microscopy, and time to first infection of >500 and >5000 parasites/µL. Although some children had multiple episodes of parasitemia or malaria, HR analysis included time to first re-infection or first clinical episode only.To describe optimally associations between opsonising antibody responses and subsequent episodes of s  = 0.91, p<0.0001; expt1 vs. expt3 rs  = 0.93 p<0.0001) and highly concordant data . Parasite positive individuals at baseline displayed increased opsonising responses  than those without concurrent infection   . When in 0.0001) . SimilarAs highly heterogeneous opsonising antibody responses were observed we investigated whether responses were associated with clinical outcomes and risk of clinical malaria. Higher opsonising antibody responses were observed in individuals who did not experience a clinical episode  than in individuals that experienced any number of clinical episodes . As up to 3 clinical episodes were observed for some individuals, we investigated whether the level of opsonising antibodies differed between those that experienced none, 1, 2 or 3 clinical episodes during follow-up. In addition, we assessed whether baseline parasitaemia influenced the level of opsonising antibody within these clinical groups. When individuals with no detectable parasitemia at baseline were investigated, lower phagocytic responses were observed in individuals who experienced multiple clinical episodes, than in individuals who experienced none or one clinical episode during follow-up . Individ20 was associated with a reduced risk of clinical malaria . Interestingly, a significant and stronger association with reduced risk of clinical disease was observed for individuals with no detectable parasitemia at baseline , while parasite PCR+ individuals showed no significant association . The lack of protective association for infection at baseline was maintained when individuals were stratified into PCR+LM− or PCR+LM+ infections at baseline (data not shown). Incidence rate ratios in PCR- individuals remained significant after adjustment for age and location of residence . However, a reduced risk of acquiring a high-density infection (>5000 parasites/µL) was observed for opsonising responses . This association was observed without stratification by PCR+ infection, and was unaltered by adjustment for age and location  (We then investigated whether opsonising responses were associated with reduced risk of acquiring new  = 0.04) . This eqMerozoite invasion has been proposed to occur within seconds following schizont rupture P. falciparum antibodies in children, or boosting of immune responses in response to infection. We found that individuals infected with P. falciparum at baseline showed increased opsonising antibody levels, an effect observed more strongly in younger children. Fifty per cent of the baseline PCR+ individuals were below the detection limits of light microscopy, which indicates low-density infections (<40 parasites/µL) are enough to boost antibody responses. However, antibody responses to P. falciparum antigens are believed to have a dynamic and short-lived profile, particularly in young children or non-immune young adults that are unable to mount an efficient anti-malaria B cell memory response Limited data exists on the maintenance of P. falciparum, as detectible on thick smear by light microscopy, has been reported to confer some protection from clinical episodes Interestingly, no association with reduced incidence of clinical episodes and opsonising antibody responses was observed in children who were parasitaemic at baseline. Asymptomatic carriage of in vivoP. falciparum MSP119 failed to transfer immunity to mice unless recipients were transgenic for the human FcγRI receptor P. falciparum trophozoites Monocytes purified from immune individuals have been shown to phagocytose merozoites more efficiently than monocytes from non-immune individuals; indicating that monocytes may also be primed for merozoite phagocytosis in vitro IFNγ output from peripheral blood mononuclear cells in response to malaria parasites provides a correlate of cellular immunity to P. falciparumIn this same cohort, we have previously shown that P. falciparum merozoites with protection from clinical disease and high-density parasitemia, measured in an FcγR-dependent in vitro assay. ADCI and respiratory burst assays require the use of primary cells, which may be a factor responsible for the inter-experimental variability and reproducibility issues associated with such assays. The use of a monocytic cell line in this study enabled a highly reproducible readout, and the associations with clinical outcomes supports the application of this functional assay of anti-merozoite immunity to larger cohorts and vaccine trials. The clinical protection observed in this study suggests merozoite phagocytosis may be an important anti-parasitic effector mechanism of natural acquired immunity to P. falciparum malaria, thus further investigation into the protective antigenic targets of this response is warranted.Currently, there are vaccines under development that are designed to induce a cytophilic antibody profile and promote FcγR-mediated phagocyte responses


Intermittent preventive treatment (IPT), the main strategy to prevent malaria and reduce anaemia and low birthweight, focuses on the second half of pregnancy. However, intrauterine growth restriction may occur earlier in pregnancy. The aim of this study was to measure the effects of malaria in the first half of pregnancy by comparing the fetal biparietal diameter (BPD) of infected and uninfected women whose pregnancies had been accurately dated by crown rump length (CRL) before 14 weeks of gestation.Plasmodium (P) falciparum and/or P.vivax malaria infections were significantly lower than in non-infected pregnancies; −0.57 (1.13) versus −0.10 (1.17), p<0.001. Even a single or an asymptomatic malaria episode resulted in a significantly lower z-score. Fetal female sex (p<0.001) and low body mass index (p = 0.01) were also independently associated with a smaller BPD in multivariate analysis.In 3,779 women living on the Thai-Myanmar border who delivered a normal singleton live born baby between 2001–10 and who had gestational age estimated by CRL measurement <14 weeks, the observed and expected BPD z-scores (<24 weeks) in pregnancies that were (n = 336) and were not  complicated by malaria between the two scans were compared. The mean (standard deviation) fetal BPD z-scores in women with P.falciparum or P.vivax malaria infections in the first half of pregnancy result in a smaller than expected mid-trimester fetal head diameter. Strategies to prevent malaria in pregnancy should include early pregnancy.Despite early treatment in all positive women, one or more (a)symptomatic  Plasmodium (P.) falciparum or P.vivax malaria during pregnancy has a negative effect on birthweight P.vivax infected pregnancies where placental sequestration is probably limited Malaria remains one of the most common parasitic infection of human pregnancy P.falciparumP.vivaxThe Shoklo Malaria Research Unit (SMRU) is located on the border between Thailand and Burma in Tak province where the majority of people belongs to the Karen ethnic group st scan occurs at the booking visit (between 8–14 weeks gestation) where ultrasound is used to determine viability, identify multiple pregnancy and estimate GA by CRL measurement. Based on this GA estimate women then return for a 2nd scan performed at 18–24 weeks to re-assess viability, measure fetal biometry, identify major fetal abnormalities and determine placental location. Fetal biometry is routinely measured twice in each woman at each scan, as part of an existing quality control Locally trained health workers (10 sonographers at 3 clinics) obtain all ultrasound scans using Toshiba Powervision 7000 (since 2006), Dynamic Imaging (since 2001), Fukuda Denshi UF 4100 (since 2002) ultrasound machines. Their practice is supervised by doctors certified in fetal ultrasonography. All women are offered two scans in pregnancy. The 1The training manual and protocol for obtaining trans-abdominal CRL and biometry measurements were from the British Medical Ultrasound Society recommendation The BPD is measured at the cross-sectional view of the fetal head at the level of the ventricles by placing the calipers on the outer border of the upper and the inner border of the lower parietal bones  across the widest part of the skull. Importantly for this study, operators taking fetal measurements were not aware of the maternal malaria status.All women are encouraged to deliver in the SMRU facilities under supervision of locally trained midwives who speak their language. Women requiring Caesarean section are referred to the nearest Thai hospital; in this study the Caesarean section rate was 3.4% . Each baby is weighed within 24 hours on electronic SECA  digital newborn scales.This is a retrospective hospital record analysis. For those patients in trials a written informed consent was obtained including storing of data and samples. For the women seen in the ANC the routine clinical records were anonymized and these pregnancy records have been routinely entered into a database since 1987. Permission was granted by Oxford Tropical Research Ethics Committee (reference: OXTREC 28–09) to use these records for analysis.P. vivax or P. falciparum malaria infection is defined by the presence of asexual stages of the respective parasite in the peripheral blood.Malaria is diagnosed by Giemsa stained thick and thin blood films; 200 fields on the thick film are read before being declared negative. All parasite densities are counted per 500 white blood cells or per 1000 red blood cells. st and the 2nd scan she was classified as symptomatic. Mid Upper Arm Circumference (MUAC) was measured at the first ANC consultation on an unclothed left arm with a SECA measuring tape (model 212) accurate to one mm and low MUAC is defined as <21.0 cm Severe malaria is defined as per WHO treatment guidelines st scan) and BPD measured <24 weeks (2nd scan), were included in the analysis. Twin pregnancies, pregnancies that were complicated by miscarriage, stillbirth or fetal structural abnormalities and pregnancies with an unknown outcome were excluded .All women who had GA estimated by CRL measurement <14 weeks  was computed from all pregnancies in SMRU with accurate ultrasound dating. Only newborns who were weighed within the first 24 hours after delivery were included In order to assess the impact of malaria infection on fetal growth during early pregnancy, the mean BPD z-scores were compared in women who did and did not have peripheral parasitaemia in the window between the 1st (CRL measurement <14 weeks) and a 2nd scan (BPD measurement <24 weeks). Women with an unknown pregnancy outcome  were excluded from further analysis; they were more likely to be younger and primigravid, to book at a lower GA and have malaria (data not shown). A further 391 women (8.5%) were excluded for miscarriage/stillbirth (n = 40), congenital abnormality (n = 52), twin pregnancy (n = 12), first malaria before the CRL dating scan (n = 96) or first malaria infection after the BPD scan (n = 191)  . There wThe median number of antenatal visits was 23 [range 3–38]. At each visit a malaria smear was obtained and malaria parasites were detected in 930 (1.1%) of the available 86,416 blood slides; these were from 336/3,779 (8.9%) women. The median number of malaria episodes per woman was 2 [range 1–8] for the duration of pregnancy.P.vivax infections and 96 (28.6%) had P.falciparum or both infections . There were only six women (1.8%) with a hyperparasitaemic P.falciparum infection in the window and no severe malaria infections  (n = 96) and P.vivax (n = 240) were associated with a lower mean z-score than uninfected women (−0.45 (1.2), p = 0.006 and −0.62 (1.1) respectively, p<0.001). There was no significant difference in mean (SD) z-scores between women with symptomatic (n = 165) or asymptomatic malaria (n = 169) episodes: −0.59 (1.1) versus −0.54 (1.2) respectively, p = 0.68. Women with multiple malaria infections (n = 103) had the lowest mean (SD) z-scores (−0.72 (1.0), p<0.001) but even a single malaria episode between the 2 scans (n = 233) resulted in a significantly lower mean (SD) BPD z-score: −0.51 (1.2), p<0.001). There was no significant difference in mean z-scores between women with uncomplicated and hyperparasitaemic malaria, but the number of hyperparasitaemic women was too small for analysis , p<0.001. Seventy two of the 114 women (63.2%) who had all their malaria infections between the two scans only, had their baby weighted within 24 hours. Neonates from these pregnancies had similar birth outcomes as the ones from uninfected pregnancies : mean (SD) birthweight 2919 (SD 533) g versus 2973 (SD 433) g, p = 0.40, mean GA 272 (SD 13) days (or 38.9 (SD 1.9) weeks) versus 273 (SD 11) days (or 39.0 (SD 1.6) weeks), p = 0.22 and birthweight centile 0.00 (SD 1.2) versus 0.03 (SD 1.0) respectively. Within this small malaria sub-group (n = 72) there was no association between BPD z-score and birthweight centile.in-utero. Even a single infection of treated P.vivax or P.falciparum was associated with reduced BPD irrespective of whether the woman was symptomatic or not. The mechanisms underlying the adverse effects of malaria in pregnancy are not fully understood P.falciparum is thought to sequester in the placenta and interfere with materno-fetal exchanges but other mechanisms may also be involved P. vivax related growth restriction, as there is little evidence that P.vivax sequesters in the placenta, like P.falciparum does In this study a significantly smaller fetal BPD was observed by ultrasound when malaria infection occurred in the first half of pregnancy, compared to pregnancies unaffected by malaria. Previous studies have shown that malaria early in pregnancy has an impact on birthweight, but they were limited by small numbers of first trimester exposures Studies on the impact of malaria in pregnancy have almost always focused on birthweight. However, for infections that occur in early pregnancy, the size of the fetal head may be a more appropriate indicator of growth restriction. It has been shown that the growth velocity of the fetal head (in mm/day) is maximal during the second trimester P.vivax are most likely from liver stage relapses instead of newly acquired infections. There is no treatment available in pregnancy for liver stages. Furthermore, in this setting of multidrug resistant parasites there are no safe and effective drugs available to prevent malaria from the start of pregnancy. To protect the developing fetus from growth restriction from both symptomatic and asymptomatic P.vivax and P.falciparum infections, prevention strategies from early pregnancy onwards or even pre-pregnancy interventions should be considered The reduction in BPD size occurred despite prompt treatment with effective antimalarials in this setting, which highlights the importance of prevention in pregnancy. Multiple episodes of There are some limitations to this analysis. Firstly, although dating by CRL is generally considered to be the most accurate method of estimating GA, some factors that may have had an impact on CRL dating, for example maternal age Intermittent preventive treatment, one of the main WHO recommended strategies for malaria prevention and control during pregnancy in areas of stable malaria transmission, aims to provide two or three treatment doses after quickening (around 20 weeks GA) at least one month apart File S1Calculations derived from the equations based on a Chinese population.(DOC)Click here for additional data file.Table S1Factors associated with mean BPD z-score when calculated from the Chinese equation.(DOC)Click here for additional data file.


Severe falciparum malaria is associated with considerable rates of mortality, despite the administration of appropriate anti-malarial treatment. Since overall survival is associated with total parasite biomass, blood exchange transfusion has been proposed as a potential method to rapidly reduce peripheral parasitaemia. However, current evidence suggests that this treatment modality may not improve outcome. Automated red blood cell exchange  has been advocated as an alternative method to rapidly remove parasites from circulating blood without affecting patients’ volume and electrolyte status. However, only limited evidence from case reports and case series is available for this adjunctive treatment. This retrospective cohort study describes the use of automated red blood cell exchange for the treatment of severe malaria at the Medical University of Vienna.Epidemiologic data for imported malaria cases in Austria are reported and data of patients treated for malaria at the General Hospital/Medical University of Vienna were extracted from electronic hospital records.Between 2000 and 2010, 146 patients were hospitalized at the Medical University of Vienna due to malaria and 16 of those were classified as severe malaria cases. Eleven patients of this cohort were potentially eligible for an adjunctive treatment with automated red blood cell exchange. Five patients eventually underwent this procedure within a period of seven hours (range: 3–19 hours) after hospital admission. Six patients did not undergo this adjunctive treatment following the decision of the treating physician. The procedure was well tolerated in all cases and rapid reduction in parasite counts was achieved without occurrence of haemodynamic complications. One patient died within seven days, whereas four patients survived without any sequelae.Automated red blood cell exchange was a safe and efficient procedure to rapidly clear peripheral parasitaemia. Whether the fast reduction in parasite biomass may ultimately improve patient survival remains however unclear. Randomized controlled trials are needed to conclusively appreciate the value of this adjunctive treatment. Plasmodium falciparum, is associated with high mortality despite appropriate management at intensive care facilities. Few therapeutic interventions have been shown effective to improve survival, including parenteral artesunate, mechanical ventilation and renal replacement therapy [Severe malaria, caused by  therapy -6. Other therapy -9.Whole blood exchange transfusion has been proposed as a method to rapidly reduce parasite biomass . A meta-Automated red blood cell (RBC) exchange is a technique that may potentially overcome problems associated with whole blood exchange -16. WithEpidemiologic data on the number of malaria cases in Austria were obtained from mandatory reports of notifiable infectious diseases. According to Austrian law, all suspected, confirmed or fatal cases of malaria have to be reported to public authorities and the federal ministry of health, respectively. A list of all patients treated for malaria at the Medical University of Vienna was obtained from an automated database search of the institution’s electronic patient record system. Individual patient data were extracted from electronic hospital records. All clinical and laboratory data associated with the automated RBC exchange were obtained from patient record forms at the Department of Bloodgroup Serology and Transfusion Medicine. Records were entered into an electronic database. The study was approved by the Ethics Committee of the Medical University of Vienna. The World Health Organization (WHO) criteria for severe malaria  were empP. falciparum malaria demonstrating hyperparasitaemia defined as >10 % infected erythrocytes and single or multi-organ failure were eligible for RBC exchange in addition to routine anti-malarial drug administration. For further classification, subsequent assessment of treatment responses and parasitaemia, Giemsa-stained thick and thin blood smears were prepared using standard protocols at admission, before and after RBC exchange, and regularly until specimen were free of parasites.Automated RBC exchange was conducted by dedicated personnel of the Department of Bloodgroup Serology and Transfusion Medicine as an adjunctive treatment to anti-malarial chemotherapy. According to institutional consensus guidelines, patients diagnosed with acute P. falciparum infection in 107 cases (73.3 %), four of which were mixed infections with Plasmodium vivax (n = 2) or Plasmodium ovale (n = 2). Sixteen (11 %) patients suffered from severe malaria according to WHO criteria [Six hundred fifteen cases of malaria were reported to federal agencies in Austria between January 2000 and December 2010. Almost half of them  occurred in the capital Vienna [P. falciparum mono-infection and one patient showed mixed infection of P. falciparum and P. vivax. Intravenous anti-malarial chemotherapy  was initiated immediately after diagnosis. Electrocardiography, continuous blood glucose measurements and frequent clinical assessments during infusion ensured adequate monitoring for quinine related side effects. Eleven patients qualified for adjunctive RBC exchange according to the institution’s guidelines. Eventually, five adult patients received automated RBC exchange   who were potentially eligible according to institution guidelines . This diAutomated RBC exchange was commenced within an acceptable time period and the procedure was well tolerated without any signs and symptoms of clinical deterioration, electrolyte disturbances or bleeding complications. The efficacy of physical removal of parasites was shown by rapid clearance of peripheral parasitaemia. Despite the exchange volume of approximately 1–1.5 times of patients total RBC pool, apheresis did not lead to problems in fluid overload and haemodynamic distress, a phenomenon which was more frequently observed in patients undergoing whole blood exchange transfusion -19. MoreIn summary, one out of five patients treated with automated RBC exchange died whereas all eight patients in the control group survived in this retrospective cohort study Table . HoweverAutomated RBC exchange has been shown to be a safe method to rapidly reduce parasitaemia in critical ill patients. Since high-quality evidence from randomized controlled trials is missing, a final judgement on the risk benefit ratio of this adjunctive treatment for severe malaria is not possible to date. Although total biomass of parasites has been shown to correlate with severity of falciparum malaria ,29, it rLimitations of this report include most importantly the retrospective study design since this does not guarantee comparability of patient groups in the absence of randomization and unified consistent definitions in the malaria patients’ general care . Improving the level of evidence is therefore important; however the conduction of prospective studies evaluating this intervention is inherently difficult since high transmission regions often lack resources for purchasing and maintenance of equipment and the availability of safe blood products. Resource rich regions on the other hand generally do not have adequate numbers of patients with this condition. It is, therefore, likely that cohort studies will remain the main source of evidence in the near future.Whereas the reported experience with automated RBC exchange is comparable to previous case reports -17, the The authors declare that they have no competing interests.LAH, NW, SW and MR have designed the study, were responsible for data acquisition, performed data analysis and drafted the manuscript. All authors have reviewed and approved the final version of the manuscript.