Malaria is more common in pregnant than in non-pregnant Nigerian women, and is associated with small birth size and the attendant short- and long-term health risks. The influence of malaria on maternal metabolic status in pregnancy and in cord blood and how this relates to birth size has not been studied. The study objective was to define relationships between maternal and cord serum metabolic markers, maternal malaria status and birth size.During pregnancy, anthropometric measurements, blood film for malaria parasites and assays for lipids, glucose, insulin and TNF were obtained from 467 mothers and these analytes and insulin-like growth factor-I (IGF-I) were obtained from cord blood of 187 babies.versus no malaria: 76.5 μg/L, p = 0.03. On regression analysis, the key determinants of birth weight included maternal total cholesterol, malarial status and cord insulin and IGF-I.Overall prevalence of maternal malaria was 52%, associated with younger age, anaemia and smaller infant birth size. Mothers with malaria had significantly lower cholesterol  and higher TNF, but no difference in triglyceride. In contrast, there was no effect of maternal malaria on cord blood lipids, but the median (range) cord IGF-I was significantly lower in babies whose mothers had malaria: 60.4 μg/L, Malaria in pregnancy was common and associated with reduced birth size, lower maternal lipids and higher TNF. In the setting of endemic malaria, maternal total cholesterol during pregnancy and cord blood insulin and IGF-I levels are potential biomarkers of foetal growth and birth size. In Nigeria, increasing mortality from stroke and end-stage renal failure is associated with a high prevalence of hypertension, obesity, diabetes and high serum triglycerides especially in women . NumerouThe biologic mechanisms underlying this hypothesis are poorly understood and mediators of the relationships between LBW, malaria in pregnancy and later cardiovascular and metabolic morbidity have not been clearly identified. Few studies have examined the relationships between maternal metabolic markers, foetal hormones and birth weight, nor has the effect of malaria in pregnancy on these relationships been explored. Maternal and cord blood levels of lipids, glucose and insulin and cord insulin-like growth factor-I (IGF-I) levels have been investigated as possible determinants of birth weight -12. MateThis study sought to explore the hypothesis that malaria parasitaemia in pregnancy would induce changes in maternal metabolic markers, which would be associated with reduced birth size. The relationships between maternal and cord blood metabolic profiles and birth size in the setting of endemic malaria was examined and potential pregnancy biomarkers of LBW were identified using an established cohort of mothers and infants born in Nigeria. The study was carried out in Yemetu-Adeoyo, a semi-urban community in Ibadan in south-west Nigeria where transmission of malaria is perennial. The hospital in this community, Adeoyo Maternity Hospital (AMH), is the oldest maternity hospital in Nigeria, dating from 1927. There are over 4,000 deliveries annually. Ethical approval for the study was obtained from the joint University of Ibadan/University College Hospital ethical committee and the University of Manchester Ethics committee.Healthy pregnant women aged 18-45 years presenting at AMH before 36 weeks' gestation and all babies born ≥ 37 weeks' gestation were eligible. Women who were HIV positive or had sexually transmitted infections at booking, those with pre-term deliveries, as well as those with multiple pregnancies or with chronic diseases, such as hypertension and diabetes were excluded. Babies with known syndromes, metabolic defects, major congenital abnormalities or severe birth trauma were also excluded.Standard operating procedures (SOP) were developed and women were enrolled over one year to cover both wet and dry seasons. The study protocol and the rationale for the study were explained carefully in appropriate language, most commonly Yoruba or English, with questions answered as needed and written informed consent was obtained from all participants. After the delivery of their babies, another written informed consent was also obtained for the participation of the babies in the study.Information on socio-demographic, obstetric, family, and health history including malarial frequency and use of anti-malarial drugs, was collected. All participants were issued prescriptions of sulphadoxine-pyrimethamine for intermittent preventive therapy (IPT) for malaria according to standard hospital practice.Antenatal visits followed routine practice with frequency of attendance determined by gestational age. Standardized measures of anthropometry were carried out on all women at every visit until delivery. Maternal weight was measured to the nearest 0.1 kg on a SECA scale, and height on a stadiometer, both without shoes according to the SOP.At booking, 2 ml of blood was collected into an EDTA tube for full blood count and blood films for malaria parasites (MP) were prepared, stained with 3% Giemsa at pH 7.2. Repeat thick blood films for MP were obtained at every subsequent visit and at delivery. In addition, the placenta was weighed, turned to the maternal surface, cotyledons exposed and 1 ml of blood obtained from the inter-villous space for a placental malarial blood smear.Blood films were examined for MP under light microscopy and recorded as negative only after 100 or more high-power microscope fields had been scanned. In those with malaria, parasite densities were determined by counting the number of parasites (np) among 200 leucocytes on the thick film using the following equation: Absolute parasite counts = (np x TLC)/200 where TLC = subject's total leucocyte count. For quality control, 30% of negative samples and 40% of positive samples were re-examined by two different, trained microscopists.At the second antenatal visit, of 624 healthy pregnant women enrolled, 467 women gave their consent for fasting blood to be taken for biochemical markers while 396 of them had samples for malaria parasites. At delivery, 27 were excluded due to four maternal deaths (0.9%), 11 still births (2.4%), five miscarriages (1.1%) and seven neonatal deaths (1.5%), leaving 436 mother-baby pairs.Cord blood was collected from the umbilical vein on the foetal surface of the placenta. Plasma was separated by centrifugation at 3,000 rpm and 4°C for 10 minutes and aliquoted into microtubes and stored at -80°C prior to lipid, glucose, insulin, TNF and IGF-I (cord blood only) assays. Cord glucose could not be measured immediately and so was not included in the assays.Based on availability of complete maternal and cord blood samples, 187 mother-baby pairs were selected. There were no significant differences in the socio-demographic and clinical data of excluded women.Plasmodium falciparum in peripheral blood. There were two definitions of malaria:Malaria was defined as the presence of asexual blood stages of a) 'Malaria at recruitment' = Malaria at second antenatal visit when blood for biochemical markers was also obtained. This was only used in analyses of effects on maternal biochemical markers at the same time-point. There were 72 blood smears positive for malaria parasites out of the 396 samples giving the prevalence of malaria as 18%.b) 'Maternal malaria' = Malaria parasitaemia present at least once during pregnancy and/or at delivery (n = 211). These included (i) malaria parasitaemia in peripheral blood at least once during pregnancy (n = 138); and (ii) at delivery and/or in the placenta (n = 73). This was used in analyses of relationships with cord parameters and effects on birth indices.Out of 187 mother-baby pairs with complete maternal and cord blood samples, 97 had malaria giving a prevalence of 52%.Anaemia was defined as a packed cell volume (PCV) < 30%.Total cholesterol (TC), High-density lipoprotein-cholesterol (HDL-C) and triglyceride concentrations were determined by using standard enzymatic procedures on an automatic analyser . The inter- and intra-assay coefficients of variation (CVs) for all parameters were < 5%. Low-density lipoprotein- cholesterol (LDL-C) was calculated using the Friedewald formula .The normal values of lipids (mmol/L) in adult women are TC 3-5, HDL-C 1.2-2.2, LDL-C 2-3 and TG 0.6-1.68.Maternal glucose was measured by the glucose oxidase method using a commercial kit  on a YSI 2300 stat plus analyser . The intra-assay CV was 1.5% at 4.1 mmol/L, and inter-assay CVs were 2.8% and 1.7% at 4.1 and 14.1 mmol/L respectively. Normal fasting glucose values are 3.9-6 mmol/L.Insulin was measured by ELISA, using a commercial kit . Assay sensitivity was 1 mU/L. Intra-assay CVs were 3.4% and 3.2% at 11 and 154 mU/L, and equivalent inter-assay CVs were 3.6% and 2.9%.IGF-I and TNF were measured using Immulite 2000 assays . Respective assay sensitivities were 25 μg/L and < 0.09 ng/L. Inter-assay CV values for IGF-1 at 48.9 and 158.5 μg/L were 7.6 and 9.2%. For TNF, intra-assay CVs were 6.7% and 5.3% at 6.3 and 19 ng/L, and the inter-assay CVs at 6.1 and 18.6 ng/L were 8.2 and 9.7% respectively.Babies were measured within 72 hours of birth. They were weighed naked to the nearest 0.1 kg and length measured on an infant stadiometer from crown to heel to the nearest 0.1 cm. Occipito-frontal circumference (OFC) was taken around the widest circumference of the head using a non-stretchable tape. Skinfold thicknesses  were measured using Holtain calipers on the left side to the nearest 0.1 mm. Measurements were obtained in duplicate or triplicate if disagreeing by > 15%.Based on the WHO Manual (1995), three nurses, already proficient in paediatric venipuncture, were trained in anthropometry methods. They carried out all measurements on the same equipment throughout the study. They also had three-monthly protocol-refresher training sessions and used training videos to minimize inter-observer and within-observer errors.Data were analysed using SPSS version 14 . Results were expressed as mean (SD) or median (IQ range), using Student t/Mann Whitney and Chi square tests for associations between maternal and infant clinical characteristics and malaria. Levels of insulin, IGF-I and TNF were skewed and, therefore, tested non-parametrically, while lipids and glucose levels were normally distributed and tested parametrically. Correlations were assessed by Spearman's test. All tests were two-sided and p values < 0.05 were considered significant.p < 0.001, p < 0.01 and < 0.05. Due to high co-linearity between biochemical parameters, a stepwise method of model selection was used to identify the key variables.Simple linear regression was used to assess the determinants of infant size, indicating those with The age and BMI of mothers were similar irrespective of trimester at recruitment. Mean (SD) gestational ages at booking and at delivery were 27.1 (5.1) and 39.3 (1.4) weeks respectively . Primigravidity was associated with 2.5-fold increase in risk of having malaria .The prevalence of malaria among the women at recruitment was 18%. Among all women recruited, 28% were primigravid, and this was associated with MP  and lower packed cell volume .Most women were asymptomatic and only seven had fever. Malaria was also associated with younger maternal age   and biceps skinfold thicknesses of infants born to women with malaria were globally smaller than those of women without malaria. Mean birth weight was significantly lower by 160 gms , biceps skinfold thickness by 1.5 mm (-4.1%) and MUAC by 1.8 mm (-1.8%), while birth length and OFC were lower by only 6.2 cm (-1.3%) and by 3.5 mm (-1%) respectively , such that for every 0.15 mmol/L increase in TC, there was a 100 g increase in birth weight.1 In the first model, the influence of malaria at recruitment and all maternal biochemical markers on birth weight was evaluated. Maternal TC was a powerful, independent determinant of birth weight (p = 0.007).2 In the second model, the influence of maternal malaria through pregnancy and at delivery and all cord biochemical markers on birth weight was examined. Maternal malaria was the only significant determinant of birth weight (3 In the final model, maternal malaria, and maternal and cord biochemical markers that had shown significant correlations to birth weight were included. Maternal malaria with cord blood insulin and IGF-I were significantly related to birth weight.et al reported no significant changes in HDL-C during pregnancy while others report an elevation [In agreement with other studies in various populations, including Nigerian pregnant women, this study showed an overall elevation in serum lipids, except HDL-C, during pregnancy. The elevation was highest in the third trimester -27. Jimelevation ,29. Theslevation ,27 and alevation  and to tlevation ,31,32. Clevation ,34.In vitro experiments have shown a selective uptake of HDL-C by Plasmodium falciparum indicating that the HDL-C fraction appears to be a major lipid source for parasite growth [P. falciparum is unable to synthesize lipids required during its erythrocytic cycle. Hence there is lipid transport through membrane flux into the parasite. Therefore, malaria in pregnancy results in demands for cholesterol from three sources including: the parasite for its growth and probably its attachment properties; the placenta for production of progesterone to maintain the pregnancy, and the fetus itself for growth [In these pregnant women, there is a marked cholesterol-lowering impact of malaria, including HDL and LDL, but triglyceride levels were unaffected. These findings corroborate older reports of lower TC and HDL associated with acute malaria but in that setting higher TG . A studye growth  and incre growth . P. falcr growth ,38. ThisMaternal plasma glucose and insulin were unaltered by malaria; the result contrasts with a previous study in pregnant women with acute malaria, who had hypoglycaemia associated with increased glucose turnover, attributed to enhanced pancreatic β-cell function . MaternaIn contrast this study indicated that malaria had a less pronounced effect on cord blood parameters: cord lipid profile, insulin and TNF levels were not altered, findings not previously described in an endemic malaria area. However cord IGF-I levels were lower.In this study, malaria in pregnancy was associated with younger age, first pregnancy and maternal anaemia, as well as smaller, shorter, thinner babies with smaller head circumferences as previously reported ,42.et al [There was no significant difference between the placental weight in infants of mothers with malaria and those without as previously reported by Mcgregor et al . The finet al ,45. Plac. The inadequate uteroplacental and umbilical blood flow, and alterations in the transplacental transfer of glucose or production of foetal insulin result in LBW babies due to foetal growth restriction [In babies of mothers with malaria, mean birth weight was lower by 5.3% compared to babies of mothers without malaria, biceps skinfold thickness by 4.1% and MUAC by 1.8%, while birth length and OFC were lower by only 1.3% and by 1% respectively (Table triction . It has triction .et al documented significantly lower mean birth weight, length, head circumference and mid upper arm circumference in babies of mothers with malaria [Very few studies have assessed the impact of malaria in pregnancy on other growth parameters at birth apart from birth weight. In agreement with our findings, Kalanda  malaria .Birth weight remains the single most important determinant of neonatal and infant survival and health. It is therefore important to understand factors that may influence birth weight. In this study, birth weight was strongly related to maternal total cholesterol, LDL-C, and less so to maternal triglyceride, glucose and insulin. This is in contrast to findings in Nigerian newborns about 15 years ago when maternal lipid samples were obtained at delivery but not during pregnancy ,33. ThisThe regression models highlight the most significant factors that determine birth weight, including maternal total cholesterol, malaria status and cord insulin and IGF-I levels. These findings are corroborated by a recent report that showed that maternal and cord IGF-I were reduced in women with placental malaria compared to those without .In the setting of endemic malaria, maternal total cholesterol during pregnancy and cord blood insulin and IGF-I levels are important markers of foetal growth restriction and reduced birth size likely mediated through placental insufficiency.The authors declare that they have no competing interests.OA participated in overall study conception and design, data collection, analysis, interpretation and manuscript preparation. AW was involved in laboratory assay and data analysis. WB was involved in data collection. OJ participated in data collection. JC and PC both participated in overall study conception and design, data interpretation and manuscript preparation. All authors read and approved the final manuscript.


Malaria is the second highest contributor to the disease burden in Africa and there is a need to identify low cost prevention strategies. The objectives of this study were to estimate the prevalence of malaria parasitaemia among infants and to measure the association between peer counselling for exclusive breastfeeding (EBF), vitamin A supplementation, anthropometric status (weight and length) and malaria parasitaemia.A cluster randomized intervention trial was conducted between 2006 and 2008 where 12 of 24 clusters, each comprising one or two villages, in Eastern Uganda were allocated to receive peer counselling for EBF. Women in their third trimester of pregnancy  were recruited in all 24 clusters and followed up until their children's first birthday. Blood was drawn from 483 infants between 3 and 12 months of age, to test for malaria parasitaemia.The prevalence of malaria parasitaemia was 11% in the intervention areas and 10% in the control areas. The intervention did not seem to decrease the prevalence of malaria . After controlling for potential confounders, infants not supplemented with Vitamin A had a higher prevalence for malaria compared to those who had been supplemented . Among children supplemented with vitamin A, every unit increase in length-for-age Z (LAZ) scores was associated with a reduced prevalence in malaria . There was no association between LAZ scores and malaria among children that had not been supplemented.Peer counselling for exclusive breastfeeding did not decrease the prevalence of malaria parasitaemia. Children that had not received Vitamin A supplementation had a higher prevalence of malaria compared to children that had been supplemented.NCT00397150.Clinicaltrials.gov:  It was responsible for 2.9% of the global disability-adjusted life years (DALYs) lost and 10.1% of the total DALYs lost in Africa Populations in these malaria endemic areas often live under conditions that predispose to malnutrition. Moreover, children, who have the highest risk of severe malaria often have the highest risk of poor nutrition The protocol for this trial and supporting CONSORT checklist are available as supporting information; see We obtained written informed consent from each study participant. Ethical approval was obtained from the Makerere University Research and Ethics Committee, the Uganda National Council for Science and Technology, and from the Regional Committee for Medical and Research Ethics for Western Norway .This was a sub-study undertaken during a cluster-randomized behaviour intervention trial , in which exclusive breastfeeding for the first six months of life by individual peer counselling in the intervention areas was promoted  2. There are two rain seasons: mid-February to end of May and August to December. Malaria is holoendemic in this area, mostly caused by plasmodium falciparum. The area is served by Mbale hospital, which doubles as the district and regional referral hospital. HIV prevalence among pregnant women in antenatal clinics in Mbale was approximately 5–6% during the study period. Most of the people living in the area were subsistence farmers.The study was conducted in Mbale district which had an estimated population of 720,000 st or 2nd branch off the main road, had a population of at least 1,000 inhabitants and represented a social and administrative unit. Efforts were made to avoid contamination between clusters. Twelve clusters were randomly designated to receive the intervention and 12 were left as control clusters.Mbale district comprised of seven counties; the study was conducted in the two biggest counties, namely Bungokho County  and Mbale Municipality (urban). Twenty four clusters were included in the study, 18 rural and 6 urban. The six urban clusters in Mbale municipality were selected from all its three municipal divisions. Most of the urban areas were large informal settlements. Eighteen clusters in Bungokho County were chosen from eight of its eleven sub-counties. Clusters were included if they neighboured the main road out from Mbale Municipality or were on the 1Assuming a parasitaemia prevalence of 50% in the control group, an alpha error of 0.05, and power of 80%, a total sample size of 368 infants was required to find a prevalence ratio of 0.7. In the PROMISE EBF trial all pregnant women in the selected clusters were approached by the study team. They were eligible if they resided in the study area, were at least seven months pregnant  or visibly pregnant, opted to breastfeed their infants and consented to participate in the study. In addition, women were excluded if they had an intention to leave the area during the study period, gave birth to twins or to infants with serious illnesses or deformities like cleft lip/palate that could interfere with breastfeeding. A total of 886 pregnant women were identified and approached. Of these, 875 women (99%) accepted to participate in the study. Out of the 875 women, 12 (1%) did not meet the eligibility criteria and 28 (3%) relocated out of the study area after recruitment but before delivery and were lost to follow-up. There were 835 deliveries. These children were followed up and blood was drawn from a subsample of 483 infants between 3 and 12 months. Children were enrolled into this sub sample consecutively, starting with the oldest until a sufficient sample size was attained. Children older than one year at the time of sample collection were excluded from the study. Because of the cluster design of the Promise-EBF study, once a cluster was visited, all eligible children were approached during the visit. All clusters were visited a couple of times until an adequate sample size was achieved. The intervention was peer counselling for exclusive breastfeeding in the first six months of life. Peer counsellors were identified from the local community and with the help of the local community. They attended a one week training course in which simplified materials based on the WHO breastfeeding counselling course were used The control clusters received only the routine government breastfeeding counselling services available at the time and this was mainly counselling for exclusive breastfeeding during antenatal care visits at the health centres.Residence in the intervention clusters, vitamin A supplementation and nutritional status were the main exposures in this study. Pre-tested structured questionnaires in the local language (Lumasaaba) were used to collect information on these exposures as well as on potential confounders such as socio-demographic characteristics, current pregnancy, immunization status, water and sanitation, previous pregnancies  and use of mosquito bed nets. Current breastfeeding was assessed at 3, 6, 12 and 24 week postpartum visits using a 24-hour and last 7-day recall. In addition, dietary recalls included 22 specified items and one open option. Breastfeeding mothers who did not give any other food or liquids other than breast milk disregarding medicines were categorised as exclusively breastfed, even if their infant had received medications P.falciparum malaria parasites per 200 white blood cells was determined on the thick film by a trained laboratory technologist who was blinded to the intervention allocation, vitamin A status and anthropometric values. Thin smears were used to identify the parasite species while thick smears were used to estimate parasite densities. Parasite density per microlitre (µl) was calculated assuming a white blood count of 8×103/µl.The outcome measure in this study was malaria parasitaemia. A blood sample for parasitaemia (thick and thin blood film) was collected from a finger prick by a trained laboratory technologist from all children in the sub sample. Blood slides were air dried and the thin smears were fixed in 99% methanol. The slides were stained with 10% Giemsa solution for 10 minutes. The number of asexual Data collectors and the laboratory technologist were blinded to the random allocation of the clusters. However, this being a behaviour change community trial, it was difficult to fully blind the data collectors and participants.www.openXdata.org, version 165.528-142 RC). Data analysis was undertaken using Stata version 9 . Exposures were residence in an intervention cluster, exclusive breastfeeding, vitamin A supplementation and nutritional status. Weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ) Z-scores were calculated with the software WHO Anthro for personal computers, version 2 Data was directly entered into hand held computers in the field using EpiHandy software  using multiple correspondence analysis (MCA). We used MCA on possession of a TV, radio, mobile phone, chair, cupboard, refrigerator, type of toilet, type of house walls as well as presence of electricity and water in the home.Crude prevalence ratios (PR) and prevalence differences (PD) and 95% confidence intervals were estimated for the exposure variables. We used multivariable generalized linear model (GLM) regression analysis with a log link to estimate the adjusted PR of the independent variables (exposures) on malaria parasitaemia. All final estimates were adjusted for bed net use, age of the infant, wealth, residence and the design effect. We tested for effect measure modification (interaction) on both additive and multiplicative scales between vitamin A supplementation and anthropometric status. Cross product terms were used to test for interaction on the multiplicative scale.Blood samples were collected from a total of 483 infants. The mean age at blood collection was 6.5 months . Half of the infants (242) were girls. The mean age of the mothers was 25 years, (range 14 to 44 years) and they had an average of 6 years of formal education (range 0 to 16 years). At 24 weeks, 205 (42%) of the children had received vitamin A supplementation. There were no baseline differences between the infants and mothers of infants in the intervention and control clusters in terms of age and sex of the children, mother's age, education, religion, or parity  and 2. BP. falciparum malaria was 11% (51 out of 483 children). Malaria prevalence was highest in October  which was also the month that registered the highest amount of rainfall (928.6 mm). In the period between July and October 2007, we registered the highest amounts of rainfall and the highest prevalence of malaria parasitaemia. The period between November 2007 and March 2008 had the lowest amount of rainfall and the lowest prevalence of parasitaemia.Prevalence of P. falciparum malaria was 11% in the intervention area and 10% in the control areas . At 12 weeks, infants in intervention areas were more likely to be exclusively breastfed than infants in control areas . Detailed results on the effect of the peer counselling intervention on breastfeeding have been presented elsewhere In a crude analysis, children in rural areas were 12 times more likely to have malaria than were children in the urban areas. After adjusting for use of bed nets, age of the child and intervention allocation, children in rural areas were still more likely to have malaria than children in urban areas . Infants whose mothers did not use bed nets were more likely to have malaria compared to those whose mothers' used bed nets. There was no association between maternal education, gender, antenatal care attendance or place of delivery and malaria .P. falciparum compared to 14% (40 out of 278) of the children that had not received Vitamin A supplementation. In a crude analysis, children who had not received vitamin A were more likely to have malaria parasitaemia than those who had received it . The association with vitamin A strengthened after controlling for use of bed nets, age and residence   had positive blood slides for 1, 17.6) .The mean weight for length Z score (WLZ) was 0.12±1.3, inter-quartile range (IQR): −0.81, 1.0. Mean length for age Z score (LAZ) was −0.7±1.1 (IQR: −1.5 to 0.1) and the mean weight for age (WAZ) was −0.5±1.2 (IQR: −1.2 to 0.2) at 12 weeks. Further, 7% of the children were wasted, 11% were stunted and 9% were underweight. There was no association between length for age Z scores (LAZ) and malaria parasitaemia. However, there was effect measure modification on both the additive and multiplicative scales between LAZ and vitamin A supplementation as indicated by a significant cross product term . Among cThe excess prevalence of malaria attributable to stunting was substantially greater among those who had not been supplemented with Vitamin A; prevalence difference 38/1,000  than among those children who had received Vitamin A supplementation; prevalence difference −6/1,000 , P-value for homogeneity of prevalence differences = 0.013.Though the relationship between malaria and nutrition is complex, increasingly, evidence suggests an association between the two In this study, peer counselling for exclusive breastfeeding did not decrease the prevalence of malaria parasitaemia. Similarly, exclusive breastfeeding for the same period did not have a significant impact on the prevalence of malaria parasitaemia. It has been hypothesized that the possible protective effect of breastfeeding is through immuno-protective mechanisms or alternately related to lactoferrin. Lactoferrin is a protein found in breast milk and neutrophils, with antibacterial properties as well as lipoprotein remnants P. falciparum malaria than those who had received them. In Uganda, the national expanded programme on immunization recommends that children between six and twelve months of age receive 100,000 IU of vitamin A while children less than six months whose mothers' did not receive postpartum vitamin A supplementation or who are not breastfeeding receive 50,000 IU. Vitamin A is necessary for normal immune function and although earlier animal studies showed vitamin A deficiency to be protective, it is now hypothesized that vitamin A could protect against malaria. Cross-sectional studies in humans have shown vitamin A to be associated with malaria, but causality has been uncertain P. falciparum malaria by 30% among preschool children Children who had not received Vitamin A supplements were about six times more likely to have plasmodium infection and nutritional indicators. However, a Gambian case control study among children admitted in hospital found mean admission weights to be lower for children admitted with malaria than for controls Though the weight for length Z scores were in line with the WHO growth reference standards, the prevalence of stunting (length for age Z score<−2) was quite high (11%) at 12 weeks. There was no association between nutritional status and prevalence of malaria parasitaemia. This is in line with findings from the Democratic Republic of Congo Children living in rural areas were more likely to have malaria parasitaemia than those in urban areas. This is comparable to national statistics that show a higher prevalence in rural areas The strength of this study lies in its prospective nature. In addition, data was collected from children in the community which enabled us to obtain malaria prevalence and make comparisons from the general population rather than from hospital cases as is often the case. Generalizability in this study is limited by the fact that clusters included in this study were close to main roads. We believe these findings can be applicable to regions with similar socio-demographic characteristics. Diagnosis of malaria in this study was based on microscopy. Microscopy has been shown to have a sensitivity of 30.5% and a specificity of 100% in comparison to PCR In conclusion, literature on the relationship between malaria and nutrition is controversial. This study shows no protective effect of peer counselling for exclusive breastfeeding against malaria. Children that had not received Vitamin A supplementation had a higher prevalence of malaria compared to children that had been supplemented. Among children supplemented with vitamin A, every unit increase in LAZ scores was associated with a reduced prevalence in malaria. There was no association between LAZ scores and malaria among children that had not been supplemented. We recommend further studies to explore the role of vitamin A supplementation in malaria endemic areas as an adjunct malaria prevention strategy.Protocol S1The PROMISE- EBF protocol and amendment of the protocol to include prevalence of malaria as one of the outcome measures.(DOC)Click here for additional data file.Checklist S1CONSORT 2010 checklist of information to include when reporting a randomised trial.(DOC)Click here for additional data file.


P. falciparum during different periods in infancy and explore the effect of age in the build-up of NAI, measured as risk of clinical malaria.The rate of acquisition of naturally acquired immunity (NAI) against malaria predominantly depends on transmission intensity and age, although disentangling the effects of these is difficult. We used chemoprophylaxis to selectively control exposure to A three-arm double-blind randomized placebo-controlled trial was conducted in 349 infants born to Mozambican HIV-negative women. The late exposure group (LEG) received monthly Sulfadoxine-Pyrimethamine (SP) plus Artesunate (AS) from 2.5–4.5 months of age and monthly placebo from 5.5–9.5 months; the early exposure group (EEG) received placebo from 2.5–4.5 months and SP+AS from 5.5–9.5 months; and the control group (CG) received placebo from 2.5–9.5 months. Active and passive case detection (PCD) were conducted from birth to 10.5 and 24 months respectively. The primary endpoint was time to first or only episode of malaria in the second year detected by PCD. The incidence of malaria during the second year was of 0.50, 0.51 and 0.35 episodes/PYAR in the LEG, EEG and CG respectively (p = 0.379 for the adjusted comparison of the 3 groups). The hazard ratio of the adjusted comparison between the LEG and the CG was 1.38  and that between the EEG and the CG was 1.35 .After considerably interfering with exposure during the first year of life, there was a trend towards a higher risk of malaria in the second year in children who had received chemoprophylaxis, but there was no significant rebound. No evidence was found that the age of first exposure to malaria affects the rate of acquisition of NAI. Thus, the timing of administration of antimalarial interventions like malaria vaccines during infancy does not appear to be a critical determinant.ClinicalTrials.govNCT00231452 Plasmodium falciparum infections from birth leads to the development of naturally acquired immunity (NAI), which is rapidly acquired against the most severe forms of malaria, takes longer against milder forms and is never sterilizing P. falciparum have not yet been identified in young children, the only surrogate of NAI being the risk of clinical malaria. We know the rate of acquisition of immunity predominantly depends on the intensity of malaria transmission and age of exposure, however it is difficult to disentangle the effects of these two factors and there is no clear evidence of the age interactions in the development of NAI In endemic areas, malaria affects primarily children younger than 5 years of age and pregnant women. Exposure to repetitive Studies in malaria-naïve transmigrants in Indonesia P. falciparum might play an important role in the development of NAI. Children who received chemoprophylaxis with Deltaprim® (Pyrimethamine plus Dapsone) from 8 to 48 weeks of age had a lower incidence of severe anaemia and clinical malaria during the first year of life compared to placebo recipients Previous trials of malaria chemoprophylaxis and vaccines conducted in Ifakara, Tanzania, suggested that the age of first exposure to P. falciparum from 4 to 9 months of age and infants in the continuous chemoprophylaxis trial were not. This difference in the age of exposure to the parasite led us to hypothesise that exposure to erythrocytic-stage antigens of P. falciparum during the first months of life did not contribute to the development of NAI, whereas exposure during the second half of infancy was crucial for the acquisition of NAI.The efficacy provided by continuous and intermittent prophylaxis during the first year of life in these two trials was similar; what differed was the development of NAI, which was impaired in children receiving continuous chemoprophylaxis for most of the first year but was not affected in children receiving IPTi. Given that there is almost no clinical malaria in children younger than 2 months Two trials of the candidate malaria vaccine Spf66 in the same area in two different age groups further supported this hypothesis. Spf66 had an estimated vaccine efficacy (VE) of 31%  in children aged 1 to 4 years P. falciparum enhances adequate development of NAI whereas exposure outside this window does not? If immune responses during the first months of life are impaired, would it be possible to enhance them? This has major implications for infant immunization with malaria vaccines, regarding both the timing and type of immunization, and the implementation of other malaria control tools to be administered during infancy. Accordingly we used monthly chemoprophylaxis to selectively control exposure to P. falciparum erythrocytic stage antigens during different periods in infancy and explored the effect of age of exposure in the build-up of NAI. The specific objective was to determine whether exposure to P. falciparum erythrocytic stage antigens between 0 to 5 months of age, or between 5 to 10 months of age, affected NAI, measured as the subsequent risk of clinical malaria between 12 and 24 months of age, as compared to infants with continuous exposure.Taken together, data from these four trials posed a fundamental question: is there a critical time window in infancy during which exposure to The protocol for this trial and supporting CONSORT checklist are available as supporting information; see P. falciparum is perennial with marked seasonality and of moderate intensity, with an entomological inoculation rate of 38 infective bites/person/year The study was conducted at the Centro de Investigação em Saúde de Manhiça (CISM), located in Manhiça, Maputo Province, southern Mozambique. The area has been described in detail elsewhere CISM runs a demographic surveillance system in its study area and a morbidity surveillance system at Manhiça District Hospital (MDH) and other health posts in the area through which standardized information on all paediatric outpatient visits and admissions to hospital is collected. Recruitment and follow up of study participants were done at the Maragra Health Post (MHP), in the south of the study area, from September 2005 to March 2009.The protocol was approved by the National Mozambican Ethics Review Committee and the Hospital Clinic of Barcelona Ethics Review Committee. The trial was conducted according to the ICH Good Clinical Practice guidelines and reviewed by a Local Safety Monitor (LSM) and a Data and Safety Monitoring Board (DSMB). The trial was registered in ClinicalTrials.gov .The study was designed as a double-blind randomized placebo-controlled trial . HIV-negP. falciparum erythrocytic stage antigens from 5.5 months onwards. In contrast, those in the early exposure group were considered exposed from 2.5 to 5.5 months and protected from 5.5 months to 10.5 months.Monthly chemoprophylaxis with SP (Fansidar® 500/25 mg) plus Artesunate  or placebo (provided by Roche and Sanofi-Aventis) was administered during different periods of the first year of life according to the randomization group . Group 1Drugs were administered by a field worker according to the following age-based dosing schedule: ½ tablet of SP or placebo and ½ tablet of AS or placebo on the first day and ½ tablet of AS or placebo on the second and third days. Tablets were crushed, mixed with water and administered to the child. If the child vomited, a replacement dose was administered.During the study, Mozambique's national first line-treatment policy for malaria was SP plus Amodiaquine, which was replaced by SP plus AS in July 2006. Prior to the administration of study drugs the field worker asked the mother/guardian and checked the health card to determine whether the child had received antimalarials during the previous 2 weeks and if so, the study drugs were not administered to avoid repeating a treatment with SP.Plasmodium parasitaemia. During the monthly visits the mothers/guardians of the participants were asked about the occurrence of any serious adverse events (SAEs) since the previous visit and the residence status was confirmed.Study participants were followed up until age 24 months. Weekly active case detection (ACD) was conducted from birth to approximately age 10.5 months (4 weeks after intervention H) and monthly home visits from 10.5 to 24 months of age. ACD visits were performed at home by field workers, who measured the axillary temperature with a digital thermometer and asked the mother/guardian about history of fever. Children presenting fever (axillary temperature ≥37.5°C) or whose guardians reported history of fever in the preceding 24 h were taken to MHP, where they were examined and parasitaemia and haematocrit were determined. Antimalarials were administered according to procedures explained below if the slide reading was positive for asexual Additionally, passive case detection (PCD) was carried out at the MHP and MDH through the morbidity surveillance system to monitor attendances to the outpatient clinics and admissions to hospital. Parents/guardians of study participants were asked to take their children to the MHP or MDH should they have any health problem at any time, where they were managed and treated according to Mozambican national guidelines by the attending medical personnel. Standard procedures included identification of the child, measurement of axillary temperature, completion of a standard morbidity questionnaire and physical examination. If the temperature was ≥37.5°C or there was a reported fever in the preceding 24 hours, haematocrit and malaria parasitaemia were assessed. Antimalarials were administered only if the blood slide was positive. As first line antimalarial treatment included SP, study participants who presented with malaria and had received a study intervention within the previous 2 weeks received oral quinine (10 mg/kg/8 h for a minimum of 5 days) for the treatment of their episode. Children seen at the MHP presenting severity criteria were transferred to MDH, where they were reassessed and admitted if necessary. Severe malaria in admitted patients was treated with parenteral quinine for a minimum of 6 doses, followed by SP or SP+AS when the first line treatment changed or continued up to 21 doses if given as monotherapy. Children presenting a haemoglobin (Hb) <10 g/dL on the full blood counts performed during the study cross-sectional samplings, were treated with oral iron for one month.Safety surveillance of SAEs, including a detailed evaluation of any skin reactions potentially related to SP, was done through the PCD and during study visits. When a child presented with a skin reaction, a specific questionnaire was filled out and pictures of the lesion were taken and assessed by a study physician. SAEs were followed up by a study physician and the LSM and the DSMB reviewed all skin reaction and SAE data. Axillary temperature, weight and length were measured during five cross-sectional surveys at approximately 2.5 months of age , 5.5 months , 10.5 months , 15 months  and 24 months .Blood samples were collected at delivery, during each cross-sectional survey, at the first clinical malaria episode and one month later. At delivery, the following samples were collected: 10 mL of peripheral venous blood (into an EDTA vacutainer), two blood slides and two drops of blood on filter paper from the mother, 8 mL of blood (into an EDTA vacutainer) and two drops of blood on filter paper from the cord, and a sample of placental tissue. When the delivery occurred outside the maternity, only the mother's sample was collected during the enrolment visit. During the cross-sectional visits, a 1 mL blood sample was collected into EDTA microtainers by finger-prick, blood spots were also collected on filter paper at cross-sectional visits 1 and 5 to determine parasitaemia by PCR and two blood smears were collected at visit 5.P. falciparum was conducted with filter paper samples as previously described Blood slides were read to quantify parasitaemia following standard quality-controlled procedures at the CISM laboratory. Blood films were air-dried, Giemsa-stained, and examined using a light microscope fitted with a 100× oil immersion lens and a 10× eyepiece. Parasite density was assessed by counting the number of asexual stage parasites until 500 leukocytes or parasites had been counted. Slides were declared negative only after 2000 leukocytes had been counted. Parasite numbers were converted to a count/µL by assuming a standard leukocyte count of 8000/µL. All sides were read by two independent microscopists and a third reading was performed if there was discrepancy in positivity or the ratio of densities from the two readings was more than 1.5 or the absolute difference was >10 parasites/µL. The final result was based on the definitive verdict for positivity or on the geometric mean of the positive densities for positive slides. Haematocrit was measured in heparinised microcapillary tubes with a Hawksley haematocrit reader  after centrifugation with a microhaematocrit centrifuge. Full blood counts were performed using a Sysmex KX-21N cell counter . Tissue samples were collected from the maternal side of the placenta and placed into 10% neutral buffered formalin. Biopsies were processed and analyzed following standard procedures to evaluate presence of parasites or pigment in the placenta Based on results from a previous chemoprophylaxis trial Randomization was done at CISM using Stata software 7  by a statistician who was not involved in other study procedures. The code was kept by the DSMB and released to the investigators once databases had been cleaned and locked after completion of follow-up. Questionnaires were double entered into databases using a program written in Fox Pro  at CISM.P. falciparum asexual stage parasites of any density. Secondary case definitions included different parasitaemia cut-offs. Moderate and severe anaemia was defined as Hb <8 g/dL or haematocrit <25% if Hb results were not available. An intervention was defined as completed if the first day and the second and/or third day of the intervention were completed without vomiting and the interval since the previous intervention was of 3–6 weeks. The intervention was also considered complete if the study intervention was not administered because the child had received documented antimalarials during the previous 2 weeks.The primary case definition of a clinical malaria episode was measured axillary temperature ≥37.5°C or history of fever within the prior 24 hours plus the presence of The according-to-protocol (ATP) cohort included children who completed > = 75% of the interventions (≥6 of 8 interventions completed), and should there be 2 missing interventions these should not be consecutive and should not be both in the first 3 months (interventions A to C). The Total cohort included children who had received at least one intervention and the intention-to-treat (ITT) cohort included all randomized children.Analyses were performed according to a predefined analytical plan using Stata/SE 10.1 . The primary endpoint of the study was the time to first or only episode of clinical malaria (according to the primary case definition) in the second year of follow up detected by PCD in the ATP cohort. The global comparison between the 3 groups and pairwise comparisons of the 3 groups are presented. Secondary endpoints included different case definitions, including multiple episodes of malaria, time to first or only episode of anaemia, total hospital visits and prevalence of parasitaemia and anaemia at different time points. Analyses were also performed in the Total and ITT cohorts and exploratory endpoints included time to first or only episode of clinical malaria during the different intervention periods of the first year of life detected by ACD or PCD in the ATP cohort.Time at risk was calculated as the number of PYAR from the date or expected date of intervention A until the end of follow-up, migration, death or withdrawal of consent, whichever occurred first. An arbitrary lag period of 28 days was applied after a case of clinical malaria, during which children did not contribute to the time at risk. In the ATP cohort, children who were absent from the study area for >3 months did not contribute to the time at risk after the migration date.Cox regression was used to evaluate the effect of the study intervention on the risk of first or only clinical malaria or anaemia episode. Negative binomial regression was used to evaluate the effect of the intervention on the incidence of multiple malaria episodes and total hospital visits. Logistic regression was used to compare prevalences. In the ATP cohort, the intervention effect was adjusted for use of insecticide treated nets (ITNs), indoor residual spraying (IRS), exclusive breastfeeding at 5.5 months, birth weight (Kg) and gender. Missing values in continuous covariates were imputed using the prediction from the best available subset of present data (determined by best-subset regression) in order to avoid losing observations and to compare the crude and adjusted models Continuous variables were compared by ANOVA and categorical variables were compared by Chi-squared test or Fisher's exact test, as appropriate. The global comparison of the interventions was evaluated using a likelihood ratio test and a global p-value for significance. To control for family-wise error, the Bonferroni correction for multiple testing was applied to the p-values from pairwise comparisons between intervention groups. Transformation of child anthropometric data to z-scores were performed using the LMS method Three hundred and forty-nine children were recruited and randomized into 3 groups . Controlling the age of first exposure was successfully achieved with highly efficacious SP+AS chemoprophylaxis, as shown by the incidence of malaria during the intervention periods. The incidence of malaria in the late exposure group during the first intervention period (from intervention A to one month after intervention C) was 0 (0 episodes/23.97 PYAR) and in the early exposure group during the second intervention period (from intervention D to one month after intervention H) was 0.06 (2 episodes/35.72 PYAR). There were 98 first episodes of clinical malaria meeting the primary case definition during the second year of life in the ATP cohort , yielding incidences of 0.50, 0.51 and 0.35 episodes per PYAR respectively, which were not significantly different between the 3 groups  also showed no differences between the three groups  in the total cohort, but only one (urticaria with pruritus) was assessed to be possibly related to the intervention and all resolved without sequelae. The occurrence of SAEs was similarly distributed among the groups, and none was deemed related to study drugs. There were 5 deaths during the complete follow up, none considered related to the intervention and 3 occurring in children who had not yet received an intervention.Plasmodium parasite. By selectively administering chemoprophylaxis to children during different periods of their first year of life, we evaluated the importance of age of first exposure to malaria in the development of NAI during infancy. NAI was measured as incidence of clinical malaria during the second year of life, as there are no surrogate markers of protection for P. falciparum, and comparing clinical malaria between groups is the only way to evaluate acquisition of immunity. Our hypothesis was that exposure to malaria during the first months of life would not lead to acquisition of NAI, while exposure during the second half of infancy would lead to and play a crucial role in the development of NAI.This study used monthly antimalarial chemoprophylaxis to control exposure to the erythrocytic-stage antigens of the Compliance with study drugs among participants was high, and chemoprophylaxis was well tolerated and highly efficacious, as proven by the near zero incidence of malaria during the different time periods in which children received SP+AS. Therefore, from two to twelve months of age the time of exposure to the parasite was decreased by 50% in the early exposure group and by 30% in the late exposure group compared to the control group. However, we did not find any significant differences in the outcomes between the three different study groups. Indeed, there were no significant differences between the groups in the incidence of clinical malaria, anaemia, total outpatient visits or hospital admissions during the second year of life. Also, there were no differences in the prevalence of parasitaemia or anaemia at the end of follow up.Although the incidence of malaria during the second year was higher by about 40% in the late exposure and early exposure groups when compared to the control group, and the risk of malaria was highest in the early exposure group for the more specific case definitions, none of these differences reached statistical significance. The sample size had been calculated to detect a relative risk of 2 between the groups, based on results from a previous continuous chemoprophylaxis trial Our hypothesis was based on results from previous malaria chemoprophylaxis and vaccine trials conducted in Tanzania in infants and young children. Data showed that infants receiving IPTi Taking together the results of the three trials, data suggest that contact with the parasite is needed at some point during infancy to be able to develop NAI, but that the timing of the exposure does not seem to be a major contributing factor. Clearly, no differences in the incidence of malaria during the second year were seen between the early and late exposure groups, and young infants with relatively immature immune systems and low exposure were able to build NAI as well as older infants. With available data it is not possible to establish the amount of exposure that is needed for the development of NAI, although exposure in our trial and in the Tanzanian IPTi trial varied from 5 to 7 months (from 2 to 12 months of age). Infants in these two trials received comparable challenge during the first year  and 0.43 in the latter Overall, no evidence was found that the age of first exposure to the parasite during the first year of life affects the rate of acquisition of NAI against malaria. Thus the timing of administration of antimalarial interventions like malaria vaccines during infancy does not appear to be a critical determinant, as infants should be equally able to mount an adequate immune response both during the initial months of life and later. Indeed, recent results from phase I/IIb trials of the RTS,S/AS0 candidate malaria vaccine conducted in endemic areas of Africa support this. In the RTS,S/AS0 trials performed after our trial had started, VE was similar in three trials in which the vaccine was administered to infants at different ages: 10, 14 and 18 weeks of age, staggered with EPI vaccines  Secondary objectives of the study included assessment of type and quality of immune responses, oxidative stress markers and host genetic factors, aiming at describing these outcomes according to age of first exposure and correlating them with clinical malaria during the second year of life. Results of these analyses will be presented in other specific papers and will hopefully shed light on the age-dependency of the immune responses and how those correlate with protection or susceptibility against malaria. Any step towards better understanding the development of NAI in infancy will enhance our chances of improving the current preventive tools and strategies against this deadly parasite.Therefore, from this study we can reasonably conclude that no evidence was found that the age at which the first encounter with the parasite occurs during infancy is a key determinant in the build up of immunity. Secondly, considerable reductions in exposure to parasite antigens in the first year of life do not result in large rebound in the incidence of malaria during the second year and massive reductions in exposure are needed to see a significant impact on the acquisition of NAI.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial protocol.(PDF)Click here for additional data file.


Hans Verhoef and colleagues report findings from a randomized trial conducted among Tanzanian children at high risk for malaria. Children in the trial received either daily oral supplementation with either zinc alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. The investigators did not find evidence from this study that zinc or multi-nutrients protected against malaria episodes. It is uncertain to what extent oral supplementation with zinc can reduce episodes of malaria in endemic areas. Protection may depend on other nutrients. We measured the effect of supplementation with zinc and other nutrients on malaria rates.Plasmodium infection at baseline were treated with artemether-lumefantrine. The primary outcome, an episode of malaria, was assessed among children reported sick at a primary care clinic, and pre-defined as current Plasmodium infection with an inflammatory response, shown by axillary temperature ≥37.5°C or whole blood C-reactive protein concentration ≥8 mg/L. Nutritional indicators were assessed at baseline and at 251 days . In the primary intention-to-treat analysis, we adjusted for pre-specified baseline factors, using Cox regression models that accounted for multiple episodes per child. 592 children completed the study. The primary analysis included 1,572 malaria episodes during 526 child-years of observation (median follow-up: 331 days). Malaria incidence in groups receiving zinc, multi-nutrients without zinc, multi-nutrients with zinc and placebo was 2.89/child-year, 2.95/child-year, 3.26/child-year, and 2.87/child-year, respectively. There was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Neither zinc nor multi-nutrients influenced malaria rates . The prevalence of zinc deficiency (plasma zinc concentration <9.9 µmol/L) was high at baseline  and strongly reduced by zinc supplementation.In a 2×2 factorial trial, 612 rural Tanzanian children aged 6–60 months in an area with intense malaria transmission and with height-for-age z-score≤−1.5 SD were randomized to receive daily oral supplementation with either zinc alone (10 mg), multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Intervention group was indicated by colour code, but neither participants, researchers, nor field staff knew who received what intervention. Those with We found no evidence from this trial that zinc supplementation protected against malaria.NCT00623857ClinicalTrials.gov Please see later in the article for the Editors' Summary. Malaria is a serious global public-health problem. Half of the world's population is at risk of this parasitic disease, which kills a million people (mainly children living in sub-Saharan Africa) every year. Malaria is transmitted to people through the bites of infected night-flying mosquitoes. Soon after entering the human body, the parasite begins to replicate in red blood cells, bursting out every 2–3 days and infecting more red blood cells. The presence of the parasite in the blood stream (parasitemia) causes malaria's characteristic recurring fever and can cause life-threatening organ damage and anemia (insufficient quantity of red blood cells). Malaria transmission can be reduced by using insecticide sprays to control the mosquitoes that spread the parasite and by avoiding mosquito bites by sleeping under insecticide-treated bed nets. Effective treatment with antimalarial drugs can also reduce malaria transmission.One reason why malaria kills so many children in Africa is poverty. Many children in Africa are malnourished, and malnutrition—in particular, insufficient micronutrients in the diet—impairs the immune system, which increases the frequency and severity of many childhood diseases. Micronutrients are vitamins and minerals that everyone needs in small quantities for good health. Zinc is one of the micronutrients that helps to maintain a healthy immune system, but zinc deficiency is very common among African children. Zinc supplementation has been shown to reduce the burden of diarrhea in developing countries, so might it also reduce the burden of malaria? Unfortunately, the existing evidence is confusing—some trials show that zinc supplementation protects against malaria but others show no evidence of protection. One possibility for these conflicting results could be that zinc supplementation alone is not sufficient—supplementation with other micronutrients might be needed for zinc to have an effect. In this randomized trial , the researchers investigate the effect of supplementation with zinc alone and in combination with other micronutrients on the rate of uncomplicated (mild) malaria among children living in Tanzania.The researchers enrolled 612 children aged 6–60 months who were living in a rural area of Tanzania with intense malaria transmission and randomly assigned them to receive daily oral supplements containing zinc alone, multi-nutrients (including iron) without zinc, multi-nutrients with zinc, or a placebo (no micronutrients). Nutritional indicators (including zinc concentrations in blood plasma) were assessed at baseline and 6–10 months after starting the intervention. During the study period, there were 1,572 malaria episodes. The incidence of malaria in all four intervention groups was very similar (about three episodes per child-year), and there was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Moreover, none of the supplements had any effect on malaria rates when compared to the placebo, even though the occurrence of zinc deficiency was strongly reduced by zinc supplementation. In a secondary analysis in which they analyzed their data by iron status at baseline, the researchers found that multi-nutrient supplementation increased the overall number of malaria episodes in children with iron deficiency by 41%, whereas multi-nutrient supplementation had no effect on the number of malaria episodes among children who were iron-replete at baseline.In this study, the researchers found no evidence that zinc supplementation protected against malaria among young children living in Tanzania when given alone or in combination with other multi-nutrients. However, the researchers did find some evidence that multi-nutrient supplementation may increase the risk of malaria in children with iron deficiency. Because this finding came out of a secondary analysis of the data, it needs to be confirmed in a trial specifically designed to assess the effect of multi-nutrient supplements on malaria risk in iron-deficient children. Nevertheless, it is a potentially worrying result because, on the basis of evidence from a single study, the World Health Organization currently recommends that regular iron supplements be given to iron-deficient children in settings where there is adequate access to anti-malarial treatment. This recommendation should be reconsidered, suggest the researchers, and the safety of multi-nutrient mixes that contain iron and that are dispensed in countries affected by malaria should also be carefully evaluated.http://dx.doi.org/10.1371/journal.pmed.1001125.Please access these Web sites via the online version of this summary at malaria , on micronutrients, and on zinc deficiency; the 2010 World Malaria Report provides details of the current global malaria situationInformation is available from the World Health Organization on malaria (in English and Spanish), including a selection of personal stories about malariaThe US Centers for Disease Control and Prevention provide information on global control of malaria and on malaria in AfricaInformation is available from the Roll Back Malaria Partnership on the Malaria Centre at the UK London School of Hygiene & Tropical Medicine develops tools, techniques, and knowledge about malaria, and has a strong emphasis on teaching, training, and translating research outcomes into practiceThe Micronutrient Initiative, the Global Alliance for Improved Nutrition, and the Flour Fortification Initiative are not-for-profit organizations dedicated to ensuring that people in developing countries get the minerals and vitamins they need to survive and thriveThe International Zinc Nutrition Consultative Group (iZiNCG) is a non-profit organization that aims to promote and assist efforts to reduce zinc deficiency worldwide, through advocacy efforts, education, and technical assistanceThe malaria (in English and Spanish)MedlinePlus provides links to additional information on  Plasmodium falciparum by 38% Preventive zinc supplementation among children in low-income countries can reduce the burden of diarrhoea and respiratory tract infections In these trials, malaria was detected among children self-reporting at clinics in Papua New Guinea and The Gambia, whereas cases were detected through regular home visits in Burkina Faso and Peru. This could indicate that zinc supplementation is more efficacious in preventing severe malaria than mild episodes, for which mothers do not immediately seek treatment. Alternatively, the response to zinc may be suppressed when children are also deficient in other nutrients There are concerns, however, about the safety of supplementation with micronutrients in malaria-endemic areas. In a large trial among children aged 1–35 months in Pemba Plasmodium infection at two surveys seemed more pronounced among those receiving iron Although randomised trials in malaria-endemic areas have consistently shown that preventive iron supplementation (with or without other micronutrients) reduces the prevalence of anaemia The primary aim of our study was to assess the effect of supplementation with zinc, alone or in combination with other nutrients, on rates of uncomplicated malaria. Secondary aims were to compare intervention effects in subgroups pre-defined by age, presence of parasitaemia, and zinc and iron status at baseline. We also assessed the effect of multi-nutrient supplementation on haemoglobin concentrations during febrile malaria episodes.We conducted the study between February 2008 and March 2009, in a rural area in Handeni District, Tanzania 12 (1.17 µg), vitamin B1 (0.625 mg as thiamine nitrate), vitamin B2 (0.55 mg riboflavin), copper (340 µg), and vitamin E (6.6 mg as RRR-α-tocopherol), all of which may have haematinic effects. For practical reasons, children participating in the study daily received one standard dose of supplements, regardless of age class. The amount of iron was set to supply on average 2 mg/kg body weight/day, as recommended to prevent iron deficiency anaemia In this trial with a parallel design, children were randomized to receive daily supplements with: (a) zinc; (b) both zinc and multi-nutrients; (c) multi-nutrients without zinc; or (d) placebo. Such a 2×2 factorial design allows the investigation of interaction between interventions; in the absence of interaction , data can be analysed ‘at the margins’ Our aim was to measure a malaria rate reduction by 30% unambiguously In four villages, resident children aged 6–60 months were listed, screened, and enrolled in daily batches until attaining the target number (n = 600) in August 2008 . AnthropP. falciparum or other human Plasmodium species. This test has a sensitivity of 96% for samples with >50 P. falciparum parasites/mL, and detects only current parasitaemia Plasmodium infection, as indicated by a positive result for a dipstick test, received a therapeutic course of artemether-lumefantrine upon enrolment so that they were at risk of malaria after a post-treatment prophylactic period Malaria dipstick tests  were used to detect lactate dehydrogenase produced by live Children with height-for-age z-scores>−1.5 SD (who are at lower risk of zinc deficiency) Plasmodium infection (yes/no infected) and age class . Interventions were indicated by colour code on paper slips in opaque, consecutively numbered envelopes that were prepared in advance, in excess of the expected number required. A simple colour code (one colour to distinguish each of the four supplement types) was used to minimize the possibility of children receiving the wrong supplements. This code was not revealed to researchers, field staff, or participants, who therefore did not know who received what intervention. At the end of each screening day, the names of eligible children were listed by screening number and each name was randomly allocated to an intervention by drawing the next envelope from a box that corresponded to the age- and malaria-specific stratum for that child.We used stratified block randomisation to allocate interventions. A colleague not otherwise involved in the trial used tables with random numbers to generate the allocation sequence consisting of randomly permuted blocks with random size (4 or 8) within each of six strata defined by Supplements, as powder in colour-coded capsules, were contained in blister packs, and administered orally after suspending capsule contents in clean water or breast milk. All types of powder had similar appearance, smell, and taste. At the end of each screening day, when eligibility had been fully established, children were individually allocated in order of their screening number to intervention groups by drawing successive envelopes from a box corresponding to the infection- and age-specific stratum for that child. The number of the envelope was then recorded on a list before the envelope was opened. The randomisation code was not revealed to researchers, field workers, or participants until data collection was completed and the database had been finalised and sent to the Trial Oversight Committee. The colour of the supplements received by each child was known to participants and field workers but not by the clinical outcome assessors.Community volunteers administered supplements 7 days per week close to the homes of participating children and reported daily to field staff, who followed up the same day in cases of non-compliance. Field staff made regular, unannounced spot checks to ensure adherence to procedures. Supplementation and follow-up continued for all children until 12 March 2009, when the trial was stopped . BecauseParents were requested to bring study children to the clinic if their child developed a fever or became unwell. A clinical officer was on 24-hour duty and collected medical information on standardised forms. Axillary temperature was measured using an electronic thermometer and dipstick tests administered for children with guardian-reported fever; for those with positive test results, we prepared two blood films and measured whole-blood C-reactive protein concentrations using a point-of-care test .In accordance with national guidelines, we treated uncomplicated malaria with artemether-lumefantrine . This drug combination is highly efficacious A second survey, at 251 days  after enrolment, followed similar procedures.Plasmodium parasites were counted against at least 200 leukocytes, and density, expressed per µL of blood, was estimated using an assumed leukocyte density of 8,000/µL. For children with very high densities, parasites were counted per 2,000 erythrocytes, in which case we used the estimated erythrocyte count at the time of the episode to determine the number of parasites per µL. The erythrocyte density was estimated based on haemoglobin concentration measured by HemoCue meter, using a linear model describing the relationship between haemoglobin concentrations and erythrocyte counts as assessed during surveys. Plasma concentrations of C-reactive protein and ferritin were measured  on a Beckman Coulter Unicel DxC880i system according to the manufacturer's instructions. Plasma zinc concentrations were determined by inductively-coupled plasma-mass spectrometry .Peripheral blood parasite density was determined by microscopy; slides with results that were inconsistent with those from the dipstick test were read twice. Asexual Data were analysed following a pre-specified plan, by intention-to-treat, using SPSS , CIA (v2.1.2) Plasmodium-infected participants were treated at baseline to clear parasitaemia before the start of surveillance. Episodes with pre-defined parasitaemia thresholds  were considered as secondary outcomes. We also assessed the effect of the intervention on relatively severe episodes .The primary outcome, an episode of malaria, was pre-defined as a positive result for the malaria dipstick test in children with guardian-reported fever in the previous 24 hours and either: (a) confirmed fever (axillary temperature ≥37.5°C), or (b) unconfirmed fever with inflammation (whole blood C-reactive protein concentrations ≥8 mg/L), separated by at least 14 days from a previous malaria episode. It has been recommended that only measured fever should be used to identify malaria cases, and to exclude cases of unconfirmed fever from the analysis a priori a reduction in overall malaria disease burden of primary public health importance Plasmodium infection, mosquito net use, distance between homestead and clinic, height-for-age z-score). We evaluated possible interaction between zinc and multi-nutrients by including an interaction term in the Cox regression model. We also conducted a pre-specified secondary analysis to assess the influence on effect estimates of excluding observations in a 14-day post-treatment prophylactic period. To assess changes in intervention effect over time, we explored effects on all malaria episodes within the first 100 days of supplementation versus the subsequent period. We arbitrarily defined a cut-point of 100 days because this period covered almost half of all episodes, and adjusted for baseline factors as described above. We similarly explored intervention effects within the first 50 days.Because we considered In a secondary analysis we assessed intervention effects on time-to-first malaria episode using Kaplan-Meier analysis, and compared hazard rates of first episodes using Cox regression.After we concluded that there was no evidence for interaction between zinc and multi-nutrients on malaria rates, we conducted pre-specified subgroup analyses  to explore to what extent the magnitude of marginal intervention effects on malaria frequency depended on age class, presence of parasitaemia, and zinc and iron status at baseline, by including (for each factor in turn) interaction terms in the Cox regression models. Lastly, we explored whether differences in intervention effects between subgroups were consistent when using higher parasite density cut-offs.Of 1,029 screened children, 662 had height-for-age z-scores≤−1.5 SD; of these, 612 were eligible and randomised. Twenty children (3%) did not complete the trial: three died, two were withdrawn by parents, and 15 emigrated from the area . AnotherGroups were similar in baseline characteristics except that there were slightly more boys and zinc-deficient children in the multi-nutrient group . The prePlasmodium infection as detected by dipstick test; for 46 children parasitaemia was not accompanied by confirmed fever or inflammation. Hence, 1,572 episodes classified as malaria: 1,499 (95%) due to P. falciparum and 73 (5%) due to other Plasmodium species. The incidence of malaria was 3.0 events/child-year. There were 1,408, 1,314, 1,248, 1,119, and 263 episodes with densities exceeding 1,000, 3,000, 5,000, 10,000, and 100,000 parasites/µL, respectively. Of all hospital admissions (68), almost half (30 cases occurring in 27 children) were due to malaria: five were admitted for life-threatening disease ; 25 lived far from the dispensary, and the clinician referred them as a precaution (18 for haemoglobin concentrations <60 g/L without respiratory distress or dehydration and seven for other reasons). There were no episodes of cerebral malaria.There were 3,268 clinic visits during the study period, of which 2,462 (75%) were accompanied by guardian-reported fever. Of 2,462 episodes of reported fever, 1,618 (66%) were accompanied by P. falciparum cases did not change our conclusions (unpublished data).Compared to placebo, there was no evidence of a meaningful effect of either zinc or multi-nutrients alone on the incidence of malaria. The hazard ratio for all episodes of malaria by zinc was 0.99; 95% CI 0.82–1.18 . Thus for zinc, the lower limit of the confidence interval is compatible with a reduction in incidence of all episodes of malaria of 18% only. The hazard ratio for all episodes of malaria by multi-nutrients (as compared to placebo) was 1.04, 95% CI 0.87–1.23 , 3,000 parasites/µL  or 5,000 parasites/µL , or that zinc influenced the incidence or time-to-first episode of malaria . Nor wasPlasmodium infection, height-for-age z-score or zinc status) or by sex (unpublished data).Lastly, we found no evidence that zinc protected against malaria after 100 days of supplementation , or that the intervention effect depended on the duration of supplementation , or by any of the pre-specified baseline factors , and in the first 50 days 1.23 (95% CI 1.02–1.50), but the statistical evidence for a change in the effect of multi-nutrients over time was weak . When analysis was limited to the first or only episode, malaria rates were greater in those who received multi-nutrients , and the median time to the first episode of malaria was 54 days for children receiving multi-nutrients, compared to 72 days for those without  in children with iron deficiency, whereas there was no evident effect in their iron-replete peers .The prevalence of iron deficiency at baseline decreased rapidly with age . As with initial iron status, age strongly modified the effect of multi-nutrients on malaria incidence . ContrasAnalysis of haemoglobin concentrations at the second survey  showed that the youngest children and those with iron deficiency at baseline responded most to multi-nutrient supplementation .One limitation of our study is that supplements were colour-coded and not in packs labelled with individual identity numbers. During the field work, however, we received no indication that participants or field staff discovered who received what intervention, or that participants favoured a particular type of supplement. It should be noted also that the clinical outcome assessors were blinded to what intervention had been assigned to individual children. HIV infection was not assessed in our study but is unlikely to have confounded its results: the prevalence of HIV infection in this paediatric population is probably below 5% and, due to the randomisation process, marked imbalances in HIV infection at baseline are unlikely.P. falciparum malaria in Papua New Guinea, as compared to 1,499 in our study). It seems unlikely that discrepancies with our findings are explained by differences in access to health care and a differential effect on rates of severe malaria: even with episodes with parasitaemia ≥100,000/µL, our confidence intervals , and hence falsely indicate iron deficiency. Plasma ferritin concentration below 12 µg/L as indicator of iron deficiency may fail to detect deficient children in the presence of inflammation, but it is a highly specific marker of iron deficiency, probably more so than the ZPP∶H ratio.In their report of the findings from the Pemba sub-study Plasmodium parasites We speculate that iron may have enhanced parasite proliferation specifically in children with iron deficiency, because iron absorption in this subgroup is more efficient and thus may lead to transient production of non-transferrin bound iron We found no support for our initial hypothesis that, at the time of malaria episodes, haemoglobin concentrations would be lower in children receiving multi-nutrients. In fact, young children with iron deficiency (who were most vulnerable to declines in haemoglobin) were better able to maintain their haemoglobin concentration when they received multi-nutrients. The critical issue remains whether the benefits of higher haemoglobin concentrations during acute malaria and thus the expected reduced risk of severe malarial anaemia, on one hand, outweigh the potential risk of severe disease manifestations that can be associated with an increased frequency of malaria, on the other hand. Access to primary care facilities may be a critical factor determining this balance, and in the absence of early treatment, the youngest children with iron deficiency may be a vulnerable subgroup.The estimated amount of iron absorbed from our supplement is close to that absorbed from multi-nutrient mixes used for home fortification In conclusion, when results from all trials are considered together, there is no evidence that zinc interventions can reduce the burden of malaria in African children. We have presented evidence that multi-nutrient supplementation may increase the risk of malaria in children with iron deficiency, strengthening earlier concerns about the safety of multi-nutrient supplementation in malaria-endemic areas, even in settings with good access to health care and appropriate treatment. Similar risks may apply to home fortification in such settings. Although safety may be improved by modifying the formulation of supplements (in particular by reducing the iron dose) implementing multi-nutrient supplementation or home fortification as a public health measure should be carefully monitored until this has been demonstrated. The World Health Organization recommendation that iron should be supplemented to iron-deficient children needs reconsideration, not because we claim that our study provides conclusive evidence that targeting this group is unsafe, but because the current guideline is based on weak evidence from a single study that is contradicted by our findings using highly specific markers of iron deficiency.Figure S1Participant flow over time. The black line indicates the cumulative number of children in the trial. Recruitment started in February 2008 (day 0) and was completed on 1 August 2008 (day 178) upon recruitment of the 612th participant. The dashed, red line indicates the date  that the trial was stopped for all participants. The blue line indicates the cumulative children who had been included in the second survey. This survey took place between 9 October 2008 (day 247) and 12 March 2009.(TIF)Click here for additional data file.Figure S2Effect of multi-nutrient supplementation on malaria rates with various case definitions, by initial iron status. For explanation, see Plasmodium infection at baseline led to similar estimates (not shown).(TIF)Click here for additional data file.Figure S3Effect of multi-nutrient supplementation on malaria rates with various case definitions, by age class. For explanation, see (TIF)Click here for additional data file.Table S1Target dose and form of multi-nutrient supplement (including zinc).(DOCX)Click here for additional data file.Text S1Study protocol.(DOC)Click here for additional data file.Text S2CONSORT checklist.(DOC)Click here for additional data file.