The data obtained indicate that zinc deficiency is associated with an increase in TNF response by 37%; 95% CI:14% to 118% and IFN-γ response by 74%; 95% CI: 24% to 297%. Magnesium deficiency, on the other hand, was associated with an increase in production of IL-13 by 80%; 95% CI: 31% to 371% and a reduction in IFN-γ production. These results reflect a shift in cytokine profile to a more type I cytokine profile and cell-cell mediated responses in zinc deficiency and a type II response in magnesium deficiency. The data also reveal a non-specific decrease in cytokine production in children due to iron deficiency anaemia that is largely associated with malaria infection status. Figure An appropriate balance between pro-inflammatory and anti-inflammatory cytokines that mediate innate and adaptive immune responses is required for effective protection against human malaria and to avoid immunopathology. In malaria endemic countries, this immunological balance may be influenced by micronutrient deficiencies. Peripheral blood mononuclear cells from Tanzanian preschool children were stimulated The pathological sequels of malaria potentially depend more on the balance between type I and type II cytokine responses than on absolute suppression of these cytokines and this balance may be influenced by a combination of micronutrient deficiencies and malaria status.


Despite low endemicity, malaria remains a major health problem in urban areas where a high proportion of fevers are presumptively treated using anti-malarial drugs. Low acquired malaria immunity, behaviour of city-dwellers, access to health care and preventive interventions, and heterogenic suitability of urban ecosystems for malaria transmission contribute to the complexity of the malaria epidemiology in urban areas.Plasmodium falciparum infection, and the prevalence of malarial disease in areas of Ouagadougou, Burkina-Faso. Thick blood smears, dried blood spots and clinical status have been collected from 3,354 randomly chosen children aged 6 months to 12 years using two cross-sectional surveys (during the dry and rainy seasons) in eight areas from four ecological strata defined according to building density and land tenure (regular versus irregular). Demographic characteristics, socio-economic information, and sanitary and environmental data concerning the children or their households were simultaneously collected. Dependent variables were analysed using mixed multivariable models with random effects, taking into account the clustering of participants within compounds and areas.The study was designed to identify the determinants of malaria transmission estimated by the prevalence of anti-circumsporozoite (CSP) antibodies, the prevalence and density of P. falciparum infections were 7.7% and 16.6% during the dry season, and 12.4% and 26.1% during the rainy season, respectively, with significant differences according to ecological strata. Malaria risk was significantly higher among children who i) lived in households with lower economic or education levels, iii) near the hydrographic network, iv) in sparsely built-up areas, v) in irregularly built areas, vi) who did not use a bed net, vii) were sampled during the rainy season or ii) had traveled outside of Ouagadougou.Overall prevalences of CSP-antibodies and Malaria control should be focused in areas which are irregularly or sparsely built-up or near the hydrographic network. Furthermore, urban children would benefit from preventive interventions  aimed at reducing malaria risk during and after travel in rural areas. According to the UN Population Fund's State of World Population 2007 report, Africa had an urbanization level of 38% in 2005, and 72% of sub-Saharan Africa's urban population lived in slum conditions. By 2040, over half of the population of Africa is expected to live in urban areas. It has been shown that the level of malaria endemicity in sub-Saharan Africa is generally lower in these areas than in rural areas . It is gMoreover there could be major heterogeneities in malaria transmission ,9-18 andPlasmodium falciparum infection, and the prevalence of malaria disease, in different areas of Ouagadougou.The aim of the present study is to identify the determinants of malaria transmission, the prevalence and density of et al [et al [Previous studies have investigated the prevalence of malaria in Ouagadougou, Burkina Faso. A study conducted by Sabatinelli et al  in 0 to et al  also repl [et al  showed tOuagadougou is the capital of Burkina Faso (lat. 12°22'N and long. 1°31'W), and had a population of around 1,086,000 inhabitants in 2006. The annual rainfall is 750 to 900 mm. The rainy season is between June and October, the cold and dry season is between November and January, and the hot and dry season is between February and May. Three artificial lakes are located within the city intended to supply water to the population.There is a variety of ecological situations and urbanization processes in Ouagadougou. The first step in the present study was to identify these different situations and processes. A two-stage stratified sampling approach was used. The stratification was carried out using a SPOT 5 panchromatic satellite image of Ouagadougou along with cadastral data. These data were completed and validated by field observations. ERDAS software was used for analysis of the satellite image, and ArcView software was used to compute building density level.The first stratification criterion was the allotment status which opposed regular to irregular areas. The regular city is characterized by a network of hierarchical streets, and the presence of basic services like electricity, running water, health structures and schools. In the regular area, land is allotted by administration. The irregular areas are composed of spatially-disorganized districts, that lack services. In the irregular city, land is given from informal delivery systems. The regularity of the area was chosen as a marker of vulnerability of the population, habitants of the irregular city being more vulnerable. The second stratification criterion was the building density considered as a proxy of the population density that could be a risk factor for infectious diseases and malaria transmission. The density of building was calculated at a cadastral unit scale called sections (survey areas were constituted by a pool of sections having the same building density). The units have been categorized in non built-up, sparsely built-up and densely built-up units, according to the density of building. Non buit-up units were discarded from the sampling.Therefore, four strata representative of four different ecological situations in Ouagadougou were defined according to the regularity and density criteria. In each stratum, two survey areas were selected for the study: two densely built-up irregular areas (Somgande and Yamtenga), two sparsely built-up irregular areas (Burundi and Zongo), two densely built-up regular areas (Dapoya and Patte d'oie) and two sparsely built-up regular areas (Gounghin and Tanghin) Figure . In reguTo ensure a sufficient exposure to urban lifestyle, only households whose head had at least five years of residence in Ouagadougou were eligible. Consequently, in each selected yard, the household was eligible only if the head of household was born in Ouagadougou or had been living in Ouagadougou for more than five years, as we supposed that anyone having lived five years in a town would have acquired a urban way of life. In an eligible household, all children aged 6 months to 12 years and adults aged over 35 years were invited to participate in the study.The health determinants studied were the following: anaemia, malaria and dengue fever in children, nutritional status in children and adults, dental status in children up to 6 years old and adults, hypertension in adults. This paper focuses only on malaria in children between 6 months and 12 years of age.Two cross-sectional surveys were implemented to take seasonal variations in health into account (April-May and September-October 2004). A mobile health post was established in each area at the time of the survey, with a team including two sociologists to interview participants, and a medical doctor for basic clinical examination and blood sampling. A questionnaire was administered to mothers (or carers) on socio-economic information , demographic characteristics , and sanitary data . Environmental data – geographic coordinates of the compound, distance from the compound to the closest drinking fountain, hydrographic network or artificial lake, and urban variables (land tenure and building density of the area) – were also recorded for each compound. Each child was clinically examined and his/her axillary temperature was measured. A temperature ≥ 37.5°C was considered to be a fever. Blood samples were collected by fingerprick, both for thick blood smears and spotting on filter paper. The team was trained prior to data collection, and then supervised by the multidisciplinary research team composed of epidemiologists and medical geography specialists. Sick children were treated free-of-charge according to the clinical diagnosis made by the medical doctor.The research protocol was validated by the Ethics Committee for Research in Health of Burkina Faso. The mother, the father or the legal guardian of each child formally agreed that he/she takes part in the investigation after being well informed of the study and before his/her inclusion in the study.Plasmodium species. Plasmodium falciparum was the predominant species found. Asexual stages of P. falciparum were counted in the blood volume occupied by 200 leukocytes and parasite density was calculated by assuming 8,000 leukocytes/μL of blood. Thick blood smears from each area were read by the same experienced technician, under the supervision of a parasitologist. The technicians were also compared on the same set of blood samples. The rate of parasite detection and parasite density estimates did not differ significantly between technicians. Cross-check quality control was regularly conducted on a randomly selected sample representing 10% of all thick smears. Blood samples for detection of anti-circumsporozoite antibodies (anti-CSP antibodies) were collected on filter paper . Blood spots were dried and stored with silica gel at ambient temperature. Antibodies (IgG) to the P. falciparum circumsporozoïte protein were detected by the ELISA test by using a technique described elsewhere [Thick blood smears were Giemsa-stained in the field and examined at the Centre Muraz in Bobo-Dioulasso to identify lsewhere ,38 at thP. falciparum anti-CSP antibodies representing exposure to malaria transmission [P. falciparum infection, iii) P. falciparum parasite density  and iv) the prevalence of P. falciparum malaria attacks. Two definitions were used for a malaria attack: presence of fever with a parasitaemia > 1,000 P. falciparum asexual stages/μL of blood (definition A), and presence of fever with a parasitaemia > 0 (definition B). To analyse these four dependent variables, socio-economic, demographic, sanitary and environmental information concerning the children or their families and the households or the compounds was used. The age variable was broken into two classes of equivalent sizes: 0–6 years and 7–12 years. An equipment index and an education level index were calculated for each family. The equipment index, determined by possession of assets, is a proxy for socio-economic status. Telephone (mobile or not), television, refrigerator, ventilator, bed, living room, and motorcycle were considered in the same component. Two categories, high and low equipment index, were determined. Education level was estimated by whether the head of the household had attended school, whatever the duration.Four dependent variables were analysed: i) the prevalence of smission ,37,39,40R software package, version 2.5.1, along with lme4 and survey. Significance was chosen as p-values less than 0.05.First, a descriptive analysis of the dependent and independent variables was carried out, taking into account the design of the study for the calculation of confidence intervals for dependent variables. The spatial distribution of the dependent variables in each area was also analysed. The index proposed by Moran was used to test for spatial autocorrelations . Moran'sP. falciparum asexual parasites/μL of blood (95%CI: 545–665). The number of children, households and compounds enrolled in the study, the dependent variables, the qualitative independent variables reported as potential risk factors are shown in Table This study enrolled a total of 3,354 children, with at least 214 children in each selected area. Over the year of data collection, no seasonal fluctuation was observed in patient attendance. The number of children enrolled in each compound varied from one to 16, with a median of three children. Parasitaemia was detected in 728 children (22%), the presence of anti-CSP antibodies measuring the exposure to malaria transmission was detected in 343 children (10%), and 31 children (1%) were undergoing a clinical malaria attack during the data collection. The mean of the parasitaemia among positive children was 602 Anti-CSP antibodies are considered to be a sero-epidemiological marker of exposure to malaria transmission. The age of child, whether the child had traveled out of Ouagadougou during the preceding month, the season, the building density of the living area and the distance of the compound to the hydrographic network were significantly associated with the prevalence of anti-CSP antibodies in bivariate analysis Table . AccordiThe age of a child, the use of a bed net, equipment and education levels, season, the land tenure of the living area, and the distance of the compound to the drinking fountain were significantly associated with the prevalence of infection in bivariate analysis Table . AccordiThe age of a child, the season, and travel outside of Ouagadougou during the preceding month were significantly associated with parasite density in bivariate analysis Table . AccordiThe number of fevers with a parasitaemia > 1,000 μL (definition A) varied from one attack per area  to seven attacks per area . The number of fevers with a parasitaemia > 0 (definition B) varied from three attacks per area (Patte d'Oie) to 16 attacks per area (Somgande). There were no significant differences between areas, whatever the clinical case definition. There were three clinical malaria attacks (0.19%) in the dry season and 28 (1.56%) in the rainy season according to definition A , and 18 clinical malaria attacks (1.15%) in the dry season and 50 (2.79%) in the rainy season according to definition B . There was no significant difference in the rate of clinical malaria attacks according to age. No multivariate analysis was performed because the number of attacks was too small.Before taking explanatory variables into account, the p-values of the global Moran tests for the prevalence of infection and parasite density were 0.02 and 0.001, respectively; hence, these variables were spatially correlated. Testing the random-effects of the fitted model, including explanatory variables using the Moran tests, indicated that there was no residual spatial correlation when the explanatory variables were taken into account (p = 0.29 and p = 0.63 respectively). For the prevalence of anti-CSP antibodies, the p-value of the global Moran test was 0.10, so the prevalence of anti-CSP antibodies was not significantly spatially correlated.Anopheles gambiae s. l. larval breeding sites [Variations in the intensity of malaria transmission can be important in different areas of a town in sub-Saharan Africa ,9-18. Inng sites ,35,36,42ng sites ,39,40.i.e. high transmission season, only had an effect  when children did not use a bed net. This may be explained by very low vector densities during the dry season and a corresponding lack of contrast in exposure according to bed net use. The use of a bed net was also significantly associated with a lower prevalence of infection , as expected [The prevalence and density of infection, and the prevalence of anti-CSP antibodies varied according to the season, as already shown by others in Ouagadougou . Concernexpected , withoutChildren between seven and 12 years of age had a higher risk of having anti-CSP antibodies or an infection than younger children . Young children obviously have less skin surface exposed to mosquito bites and are consequently less exposed to malaria transmission . Moreovei.e. the exposure to malaria transmission. CSP is an antigen that is only expressed at the pre-erythrocytic stages of the parasite. These stages are not sensible to the drugs used in Burkina-Faso (i.e. amino-8-quinolines are not used). Therefore, the antibody responses against CSP depend on exposure to malaria transmission but not on anti-malarial treatments  or resistance of the parasite to these drugs. On the other hand, the prevalence of parasitaemia depends both on malaria transmission, access to anti-malarial treatments and drug efficacy. Higher socioeconomic levels are supposed to be associated with easier access to prevention and treatment as described in other African cities [i.e. anti-malarial treatment, more than to anti-vector measures independent of bed net use.Independent of bed net use, higher socio-economic level (according to equipment and education levels) was associated with a significantly lower risk of infection , without significant associations with the prevalence of anti-CSP antibodies n cities ,44. ThenEven if the risk of having anti-CSP antibodies was not significantly associated with socio-economic variables, it was associated with the two urban variables . The prevalence of infection was also significantly higher in irregular areas  and in sparsely built-up areas . Then, malaria risk was higher in irregular areas, where its transmission is known to be higher in Ouagadougou . In thesAn. gambiae s.l. [et al [An. gambiae s.l. were collected within a distance of 300 meters from the breeding sites. The distance to the artificial lakes was not identified as a risk factor in the present study. Indeed, only 1% of the surveyed households were close to any artificial lake (less than 200 m), and most of the studied areas were several hundreds of meters from artificial lakes , the lower their risk of having anti-CSP antibodies was . It is known that the hydrographic network and the artificial lakes are favourable breeding sites for iae s.l. ,42. Saba. [et al  showed tgadougou . Due to et al [P. falciparum populations found in urban areas [Children who traveled out of Ouagadougou during the preceding month had a higher risk for having anti-CSP antibodies  and higher parasite densities . Robert an areas .A difference between malaria attacks in different areas or age groups could not been shown due to insufficient statistical power for a multivariate analysis. However, a difference in the number of attacks between the dry and the rainy season  was found, as expected, and as reported by others ,25.The prevalence of infection and parasite density were spatially correlated before taking the explanative variables into account. According to the Moran test, these spatial correlations could be explained by the variables included in the mixed models. A similar lack of spatial correlation has also been shown in another study of urban malaria . Moreoveet al who conducted a rapid malaria appraisal in Ouagadougou in 2002, two years before the present study, and published their results in 2005 [et al).The results of the present study are similar to those of Wang  in 2005 . They al in 2005 ,24. It iet al [et al), a different period of recruitment or lower reading effort for the blood films .Wang et al  found anIn conclusion, the present results contribute to a better understanding of malaria epidemiology in Ouagadougou and confirm the results of previous studies ,35,36. UThe authors declare that they have no competing interests.MB carried out statistical analysis and interpretation of the data, drafted and revised the manuscript. FF was the principal scientific investigator of the study and revised the manuscript. MCH was the investigator responsible of the malaria part of the study, participated in the study design, coordinated the malaria field and laboratory work in Burkina Faso, interpreted the data and revised the manuscript. SA conducted immunological work in the research department of CR. CR participated in the design of the malaria part of the study, supervised the immunological and statistical analysis, interpreted the data, drafted and revised the manuscript. HO took part in the field work and study design. GS conceived the research programme. All authors read and approved the final manuscript. None declared conflict of interest.Description of quantitative independent variables in the areas. The data provided for each area are the number of compounds, the number of households and the number of children aged 6 months-12 years that have been investigated. It presents also the age of children , the distances from the compounds to the closest hydrographic network, to the closest drinking fountain and to the closest artificial lake .Click here for file


Placental malaria (PM) is associated with poor foetal development, but the pathophysiological processes involved are poorly understood. Cyclooxygenase (COX) and lipoxygenase (LOX) which convert fatty acids to prostaglandins and leukotrienes, play important roles in pregnancy and foetal development. COX-2, currently targeted by specific drugs, plays a dual role as it associates with both pre-eclampsia pathology and recovery during infection. The role of COX during PM was questioned by quantifying at delivery COX-1, COX-2, 15-LOX, and IL-10 expression in two groups of malaria infected and uninfected placenta.Placental biopsies were collected at delivery for mRNA isolation and quantification, using real time PCR.COX-2 and IL-10 mRNAs increased mainly during chronic infections , whereas COX-1 transcripts remained constant. COX-2 over-expression was associated with a higher birth weight of the baby, but with a lower rate of haemoglobin of the mother. It was associated with a macrophage infiltration of the placenta and with a low haemozoin infiltration. In the opposite way, placental infection was associated with lower expression of 15-LOX mRNA. A high degree of haemozoin deposition correlates with low birth weight and decreased expression of COX-2.These data provide evidence that COX-2 and IL-10 are highly induced during chronic infection of the placenta, but were not associated with preterm delivery or low birth weight. The data support the involvement of COX-2 in the recovery phase of the placental infection. In Africa, most of the malaria mortality and morbidity is borne by children and pregnant women with an estimate 200,000 infant deaths each year as a result of malaria infection in pregnancy . During et al published conflicting results describing a reduction of COX-2 and of the amount of NHE-1 in pre-eclamptic placentas with an unaltered level of COX-1 [Decreased blood flow in the placenta is the major factor triggering LBW, which had previously been linked to a modulation of placental cytokine expression . In thisof COX-1 .During placental malaria, modulation of these PGs pathway could play a role in preterm delivery and low birth weight as observed during pre-eclampsia. A foetal COX-2 gene polymorphism was associated with placental malperfusion during placental malaria . In the Two other arachidonic acid pathways, the epoxide and the lipoxygenase (LOX) pathway , are invP. falciparum compared to uninfected placenta. Although delivery is only an instant in the whole pregnancy, it is however a good snapshot of the placenta statement. Studies conducted at delivery also give information on the late stage of the mother-baby interaction.Eicosanoids are thus powerful regulators of placenta perfusion and overall of the pregnancy outcome. New analgesic and anti-inflammatory drugs, inhibitors of COX activity, are often used in malaria endemic areas to reduce fever, pain, and inflammation . They caCollection of placentas was done at delivery, at the maternity ward the Centre de Santé Roi Baudoin, Guediawaye, Dakar Sénégal. Details regarding enrolment and standard laboratory procedures have been reported elsewhere . It invoThe HIV infection status of the participants was not established at the time of the study however, HIV sero-prevalence in Senegalese population was low (0.8% of parturients in Dakar) . All ICTAfter collection, whole placentas were refrigerated until assessment (less than two hours after delivery). For each placenta, four biopsies were sampled , rinsed Immediately after collection, samples for histology and immunohistochemistry were fixed in 4% buffered paraformaldehyde and processed as previously described . 5 μm crAcute infections (AI) were defined by the presence of parasites on sections without malaria pigment, chronic infections (CI) by presence of parasites and pigment, and past infections (PI) by the presence of pigment alone. PfHRP2+ placentas with no evidence of parasites or pigment were classified as "no-parasite" (NP). For the control group all PfHRP2 rapid diagnosis test, parasites and pigment detection were negative in placenta and in the venous blood.For immunochemistry, after blocking endogenous peroxidases and non-specific binding, tissue sections were incubated for 1 h at room temperature with primary antibodies or control isotypes and revealed using LSAB detection kit (DakoCytomation). Antibodies used were CD68 (PG-M1), and HLA-DR (TAL.1B5) from DakoCytomation mouse antibodies against. The number of cells in the intervillous space was counted over 100 fields (magnitude ×400).et al [Frozen biopsies were ground into powder in liquid nitrogen before homogenization in 4 M guanidium buffer. RNAs were extracted using standard phenol-chloroform procedure, precipitated with isopropanol and washed before quantification. Integrity of total RNA was assessed on an ethidium bromide (EtBr) stained denaturing agarose gel. For reverse transcription 2 μg of RNA was denatured at 65°C for 10 min, cooled on ice-water, and reverse-transcribed for 60 min at 43°C with 40 U/μl RNase inhibitor and oligo-dT. Amplification was performed in an ABI Prism 7000 Sequence Detection System using Taqman Universal PCR Master Mix (PE Biosystems), containing 300 nM of primers and 200 nM of fluorogenic probes. Each measurement was carried out in duplicate and amplification of Peptidyl Propyl Isomerase A  was conducted on the same plate. Others housekeeping genes such as beta-2-microglobulin and beta-actin were run together with PPIA, but were not selected for standardization as less constant between infected and uninfected placentas. Probes and primers  for two groups or with Kruskal-Wallis test (KW) and Median test for more than two. Correlations were calculated using a Spearman R test (SP). Alpha risk was set at 0.05.Plasmodium falciparum histidine rich protein positive-RDT (PfHRP2+) (10.3%). For these PfHRP2+ women, 25% of the babies had LBW and 67% of the women were anaemic [During this study, 692 pregnant women were examined among whom 71 had a  anaemic . CompareCOX-2 mRNA increased during PM especially during late stage of the infection: 9.0, 5.4, 3.8 and 3-time higher in placentas with CI, AI, PI and NP, respectively, than in the control group (p < 0.01 MW for CI and AI). The expression of COX-1 remained low in all the placentas without significant differences between PfHRP2- (Ctrl) and PfHRP2+  groups, but trends to increase during past infection Figure .Because IL-10 has been shown to exert direct regulatory effects on prostaglandin production, we also measured IL-10 mRNA in our placental samples. The levels of IL-10 transcripts were higher in PfHRP2+-placentas than in controls . It was also lower during AI and CI than in control group. However, women who delivered babies with normal birth weight had higher level of COX-2 transcripts compared to others  Figure . HaemoglHigh level of pigment in the placenta was associated with significantly lower birth weight  COX-2 seems mainly expressed by placenta and not by PBMC and as ii) the effect of IL10 on COX-2 and PGE2 seems to be different in preterm and in term placentas . In the Interestingly, 15-LOX expression correlated with the presence of parasites in the intervillous space, which is consistent with study stating that 15-HETE and 15-HPETE stimulated red blood cell adhesion to endothelium . HoweverIn summary, this study showed that COX-2 and IL-10 are highly expressed in active PM. COX-2 correlates with macrophage infiltration and is related to a better outcome of the pregnancy with higher birth weight. All these data support the increase of COX-2 as a witness of placental defence against infection and draw attention on uncontrolled use of anti-COX2 during placental malaria.Plasmodium falciparum histidine rich protein.PM: placental malaria; COX: cyclooxygenase; LOX: lipooxygenase; LBW: low birth weight; ICT: immunochromatographic test; RDT: rapid diagnosis test; HZ: hemozoin; PBMC: peripheral blood mononuclear cell; CI: Chronic infection; AI: acute infection; PI: past infection; NP: No parasite and pigment (PfHRP2+); PfHRP: The authors declare that they have no competing interests.RJ conceived the study. DS and EF performed the experiments. DS, LM, JMD and RJ analysed the data. DS and RJ wrote the manuscript. DA and LM helped revise it. All authors read and approved the final manuscript.


Although maternal common mental disorder (CMD) appears to be a risk factor for infant undernutrition in South Asian countries, the position in sub-Saharan Africa (SSA) is unclearA population-based cohort of 1065 women, in the third trimester of pregnancy, was identified from the demographic surveillance site (DSS) in Butajira, to investigate the effect of maternal CMD on infant undernutrition in a predominantly rural Ethiopian population. Participants were interviewed at recruitment and at two months post-partum. Maternal CMD was measured using the locally validated Self-Reported Questionnaire (score of ≥ six indicating high levels of CMD). Infant anthropometry was recorded at six and twelve months of age.The prevalence of CMD was 12% during pregnancy and 5% at the two month postnatal time-point. In bivariate analysis antenatal CMD which had resolved after delivery predicted underweight at twelve months . There were no other statistically significant differences in the prevalence of underweight or stunted infants in mothers with high levels of CMD compared to those with low levels. The associations between CMD and infant nutritional status were not significant after adjusting for pre-specified potential confounders.Our negative finding adds to the inconsistent picture emerging from SSA. The association between CMD and infant undernutrition might be modified by study methodology as well as degree of shared parenting among family members, making it difficult to extrapolate across low- and middle-income countries. Infant undernutrition is a well recognised public health problem in low and middle income countries (LAMIC) -3, the cStudies from South Asia ,8,10,11 Methodological issues may explain some of the conflicting findings across studies. Variation in the age of children at nutritional assessment, homogeneity of study participants across studies, rural versus urban setting, cultural validity of instruments used to ascertain maternal CMD, use of different nutritional indices as outcomes, as well as different scales of measurement (binary or continuous), the frequencies of exposure and outcomes investigated, the timing at which the effect of exposure on the outcome was evaluated, and the quality of study design may all play a part ,27. FurtWe now report results from a population based cohort, the Perinatal Maternal Mental Disorder in Ethiopia (P-MaMiE) study , with th(a) does maternal CMD in pregnancy significantly contribute to infant undernutrition at six and twelve months of age?(b) does postnatal CMD significantly contribute to infant undernutrition at six and twelve months of age?,(c) compared to infants whose mothers had no experience of CMD either in pregnancy or the postnatal period, are infants whose mothers had CMD (i) in pregnancy only, resolving after giving birth, (ii) postnatally, but not in pregnancy , and (iii) persistently from pregnancy to the postnatal period , at a higher risk of being undernourished at six and twelve months of age?Ensete ventricosun) for subsistence and produce chilli-peppers and khat  as cash crops. In recent years, however, the population has been affected by periodic food insecurity. There is a primary health service and primary schools for residents within a maximum distance of 5-6 km. Butajira town is the capital of the district within which the DSS is located. It has basic infrastructure including an all-weather road that runs to the bordering districts, a hospital, a health centre, drug stores, electricity, and digital telephone services.A population based prospective cohort of pregnant women was established  within tGrowth measurements were taken by project data collectors, DSS enumerators and community health agents (CHAs). In six sub-districts  CHAs who lived and worked in the same sub-district were trained to measure birth weight. During recruitment, participating women were requested to inform the CHA immediately after giving birth to enable the neonate to be weighed ideally within 24 to 48 hours of birth. The remaining four sub-districts had no suitable health worker to measure birth weight and that information was not collected. Infant weight, including birth weight, was measured using SECA 725 scales measuring to an accuracy of 10 g. Infant length was obtained using a locally adapted measuring board. First authors (GM and CH) and one of the collaborators (FT) trained all individuals involved in growth measurements to minimise inter-individual variability.CMD was measured during the third trimester of pregnancy and at two months postnatal using the locally validated Self-Reporting Questionnaire (SRQ-20) . The SRQPotential confounding variables were grouped into domains as shown below:Household characteristics: residential area , number of children aged under five years, age of husband and three composite scores:(1) a. Poverty index including the following variables: non-literate wife, non-literate husband, do not own radio, do not own bed, do not possess valuable goods like gold and jewellery, own home, possess large animals, possess small animals, animals spend night within the living room, house has a window. Individual items of this scale were identified through a rigorous process including exploratory and confirmatory factor analysis. The final scale score was obtained by adding individual items with equal weight. The resulting scale had a Cronbach alpha value of 0.73, indicating an acceptable level of internal consistency.b. Poor sanitary conditions scale including: not having a toilet facility, not having safe water and disposing of rubbish on the field. We aggregated these three variables as all of them are known risk factors of undernutrition in Ethiopia even though the internal consistency of the resulting scale was relatively low: Cronbach alpha = 0.49.c. Support to the mother, including: able to visit friends, enough help at home, enough help with looking after children, enough help from husband, no experience of violence. The resulting scale had a Cronbach alpha value of 0.47 which is relatively low; however, these items measure quite different sources of support and we would not expect them to correlate highly.Child characteristics: gender, vaccination status at two months of age, history of severe illness before the age of two months and birth weight .(2) Maternal characteristics: Age, height, mid upper arm circumference, type of marriage (polygamous versus non-polygamous), substance use , at least one obstetric complication during current delivery (prolonged labour (>24 hours) or assisted delivery  or self reported post-partum haemorrhage or post-partum fever) and 'autonomy' scale. The degree of household autonomy was assessed by asking whether the participant had to ask her husband before she was able to sell crops (yes/no), spend household money (yes/no), attend women's groups or other meetings(yes/no), purchase medications for herself or her children (yes/no), attend a health facility(yes/no). Responses to the five categories were summed with equal weights resulting in a scale with a Cronbach alpha value of 0.93.(3) Early infant feeding practices: no pre-lacteal feed, given colostrums, initiation of breast-feeding within one hour of delivery.(4) Standardized z-scores (height-for-age and weight-for-age) were generated using the new WHO reference population . These sWe hypothesised that the infants born to women with high levels of CMD (SRQ20 ≥ 6) during their third trimester would have a 1.5 times higher risk of being stunted at six months of age compared to infants of mothers with a low SRQ score. Based on the demographic and health survey data  we assumData were checked in the field by supervisors and usually double-entered on the same day using Epidata . Women wPrior to the first interview the women were informed about the objective of the study. Written, informed consent was obtained in keeping with requirements of the Ethiopia ethics committee. As the majority of women were non-literate, the form was read out and participants were asked to give a finger-print to signify willingness to participate. Arrangements were made within locally existing public health institutions for the study project to pay all health-related expenses of the women and children participating in the study. The study was granted ethical approval from the National Ethics Review Committee in Ethiopia and the Research Ethics Committee of King's College London in the UK.Data analysis was restricted to singleton infants who had growth measurements at six or twelve month follow-up. Means and proportions were used to describe continuous and categorical characteristics, respectively. Independent sample t-tests were used to compare mean score of nutritional indices of infants born to mothers with and without a high level of CMD. The proportions of undernourished infants among those born to mothers with and without high levels of CMD were compared using Fisher's exact test. The independent effect of CMD on infant nutritional status was evaluated by defining three main exposure variables: (a) antenatal prevalent case, (b) postnatal prevalent case, and (c) four level categorical exposure variable ("no exposure at both time points" (reference), only antenatal exposure, incident postnatal, and "chronic" or persistent exposure) of CMD. Taking each of the three CMD exposures in turn, the association with infant nutritional status was investigated with logistic regression for binary outcomes (undernourished versus well-nourished) and linear regression for continuous outcomes (weight-for-age and height-for-age z scores). In the process of modelling each outcome (weight-for-age and height-for-age) at each time point (six month and twelve month) three steps were followed: (1) bivariate regression taking one of the three CMD exposure variables, (2) multivariable regression adjusting for the effect of CMD on an outcome for a given domain of covariates , (3) multivariable regression fully adjusting the effect of CMD for all covariates. Unadjusted and adjusted odds ratios from logistic regression and unstandardised regression coefficients from linear regression with corresponding 95% confidence intervals were used to assess statistical significance and the magnitude of effects. All data analysis was done using STATA  with theRecruitment and attrition at every stage of follow-up are detailed in Figure Selected characteristics of the whole cohort at recruitment are presented in Table A descriptive summary of infant nutritional status (standardised weight and height/stunting and underweight) stratified by infant age and level of CMD is presented in Table Odds ratios and corresponding 95% confidence intervals from bivariate and multivariable logistic regressions assessing the association between the course of CMD from pregnancy to two months postnatally  and infant undernutrition are presented in Table The results of bivariate and multivariable logistic regression with antenatal CMD  and postnatal CMD  as the main exposures for infant undernutrition are presented in Table Modelling of the association between CMD and infant nutritional status was repeated using linear regression For this purpose, weight and length of infants in standard deviation units were kept as continuous outcome variables and CMD as the main exposure was defined as in the methods section . None of the findings showed statistically significant effect of CMD on nutritional status of infants (result not shown). Use of the SRQ score as a continuous exposure variable did not alter our finding of no association between CMD and height-for-age z score or weight-for-age z score, either at six months and at twelve months of age.In this population-based prospective study from rural Ethiopia we evaluated the effect of maternal CMD in pregnancy and at two months postnatal upon infant nutritional status assessed at six and twelve months of age. The prevalence of infant undernutrition, indicated by stunting (length for age z score less than -2) and being underweight (weight for age z score less than -2), was high at both time points; however, the prevalence of maternal CMD was relatively low, particularly at the two month postnatal time-point. In fully adjusted multivariable analyses, infant exposure to maternal CMD in pregnancy, at two months postnatal, or at both perinatal time-points was not significantly associated with infant nutritional status at six months or at one year of age. When maternal CMD was considered as a four level categorical variable  CMD in pregnancy that resolved following delivery was associated with the infant being underweight at one year. However, this association became non-significant after adjusting for household and maternal characteristics. Neither this nor any other effects of maternal CMD were significant in the fully adjusted model, whether we considered nutritional indices as dichotomous or as continuous outcomes.The credibility of the current results is based on the strengths of the study which include: (a) a large population-based sample from an area with a high prevalence of infant undernutrition and low levels of loss to follow-up over 12 months, (b) the first study from sub-Saharan Africa and the second from a LAMIC setting to ascertain CMD during pregnancy as well as at two months postnatally and to assess their effects on infant outcomes prospectively, (c) assessment of infant nutritional status at both six and twelve months of age, and (d) adjustment for a large number of potentially confounding variables. However, the study has some limitations. The SRQ-20 is a scale-based measure of maternal CMD symptoms, rather than providing a definitive diagnostic assessment of mental disorder. In three,9,12 outThe possible association between maternal CMD and child undernutrition in LAMIC has captured the attention of researchers in recent years, and has been tested using epidemiological studies of varying methodological quality that may have contributed to the different findings across settings. However, consistent and significant associations have been observed in south Asia independent of these and other heterogeneities.The two previously published population-based cohort studies ,26, bothThe timing of measurement of infant undernutrition could have relevance, with the two previously negative studies from sub-Saharan Africa evaluating children at an older age: 18 months and 6 toMost previous studies reported categorical indicators of infant nutritional status. Where the analyses were repeated for both categorical and continuous nutritional indices, only the categorical measure was associated with maternal CMD in Brazil,39, and Although our study adjusted for a broader range of potential confounding variables than most other studies, there is little evidence that over-adjustment occurred as no positive associations were observed in the univariate analyses.Persistent perinatal CMD could impact on nutrition during pregnancy and after birth. The nature of any interaction between CMD in pregnancy and the postnatal period to cause under-nutrition is unclear. Contrary to the current findings there is strong evidence in Pakistan  showing Including the current study, five other studies, from Ethiopia , South AOur population-based study from rural Ethiopia found no significant association between maternal perinatal CMD  and infant undernutrition at six or twelve months of age. This result, in the context of other research, questions the universality of the proposed causal link between CMD and impaired infant growth across LAMIC.The authors declare that they have no competing interests.CH, MP, AA conceived the idea and CH designed the study. GM and CH developed data collection instruments, coordinated data collection and have full access to the raw data. GM analysed the data and drafted the manuscript. CH, MD, and MP contributed to data analysis. CH, MD, MP, VP, MH, MT, FT critically commented on the draft manuscript. All authors contributed to and have approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/10/32/prepub


Plasmodium falciparum.Vitamin A and zinc are crucial for normal immune function, and may play a synergistic role for reducing the risk of infection including malaria caused by A randomized, double-blind, placebo-controlled trial of a single dose of 200 000 IU of vitamin A with daily zinc supplementation was done in children of Sourkoudougou village, Burkina Faso. Children aged from 6 to 72 months were randomized to receive a single dose of 200 000 IU of vitamin A plus 10 mg elemental zinc, six days a week (n = 74) or placebo (n = 74) for a period of six months. Cross-sectional surveys were conducted at the beginning and the end of the study, and children were evaluated daily for fever. Microscopic examination of blood smear was done in the case of fever (temperature ≥37.5°C) for malaria parasite detection.At the end of the study we observed a significant decrease in the prevalence malaria in the supplemented group (34%) compared to the placebo group (3.5%) (p < 0.001). Malaria episodes were lower in the supplemented group (p = 0.029), with a 30.2% reduction of malaria cases (p = 0.025). Time to first malaria episode was longer in the supplemented group (p = 0.015). The supplemented group also had 22% fewer fever episodes than the placebo group (p = 0.030).These results suggest that combined vitamin A plus zinc supplementation reduces the risk of fever and clinical malaria episodes among children, and thus may play a key role in malaria control strategies for children in Africa. Plasmodium falciparum remains a major cause of morbidity and mortality among African children . There was no difference in mean parasite density, spleen enlargement, or mean hemoglobin concentrations at baseline although the prevalence of severe anemia was higher in the vitamin A and zinc supplemented group.One hundred fifty children were randomized figure ; 2 childAt the end of the study, the prevalence of malaria was significantly lower in the supplemented group (34% vs. 3.5%), respectively p < 0.001). The mean parasite density was higher in the placebo group compared to the supplemented group at the end of the study (p = 0.048). Considering the variation of the parasite density within each group, we observed a significant decrease in the supplemented group from 1589 [1026–2459] to 1011 [647–1579] (p < 0.001) and a significant increase in the placebo group from 1444 [923–2259] to 1945 [1155–3275] (p = 0.023). At the end of the study, there was no difference in the proportion of children with splenomegaly between the two groups. Regarding nutritional parameters we observed that wasting and stunting continued to be severe in both groups  declare that they have no competing interests.ANZ coordinated the data collection entry, analysis and drafted the paper. NR, HS, IZ, and JR were the assistant of ANZ in the study, for data collection and laboratory analyses. NM from the International atomic energy agency (IAEA) was the technical advisor for the study and contributed to reviews of the paper. DHH provided technical input to the analysis and revisions of the manuscript. JBO, the director of the institute, designed the study with NM, coordinated the manuscript draft, and supervised the whole activities of the study. All authors approved the final manuscript.


Malaria has traditionally been a major endemic disease in Equatorial Guinea. Although parasitaemia prevalence on the insular region has been substantially reduced by vector control in the past few years, the prevalence in the mainland remains over 50% in children younger than five years. The aim of this study is to investigate the risk factors for parasitaemia and treatment seeking behaviour for febrile illness at country level, in order to provide evidence that will reinforce the EG National Malaria Control Programme.Plasmodium infection. A multivariate logistic regression model was used to determine risk factors for parasitaemia, taking into account the cluster design.The study was a cross-sectional survey of children 0 to 5 years old, using a multistaged, stratified, cluster-selected sample at the national level. It included a socio-demographic, health and dietary questionnaires, anthropometric measurements, and thick and thin blood smears to determine the The overall prevalence of parasitemia was 50.9%; it was higher in rural (58.8%) compared to urban areas . Age was positively associated with parasitemia (p < 0.0001). In rural areas, risk factors included longer distance to health facilities (p = 0.01) and a low proportion of households with access to protected water in the community (p = 0.02). Having had an episode of cough in the 15 days prior to the survey was inversely related to parasitemia (p = 0.04). In urban areas, the risk factors were stunting (p = 0.005), not having taken colostrum (p = 0.01), and that someone in the household slept under a bed net (p = 0.002); maternal antimalarial medication intake during pregnancy (p = 0.003) and the household socio-economic status (p = 0.0002) were negatively associated with parasitemia. Only 55% of children with fever were taken outside their homes for care, and treatment seeking behaviour differed substantially between rural and urban populations.Results suggest that a national programme to fight malaria in Equatorial Guinea should take into account the differences between rural and urban communities in relation to risk factors for parasitaemia and treatment seeking behaviour, integrate nutrition programmes, incorporate campaigns on the importance of early treatment, and target appropriately for bed nets to reach the under-fives. Equatorial Guinea is a small West Central African country divided into a mainland region (Río Muni) and an insular one (Bioko as the main island). Traditionally, malaria has been a major endemic disease in the country ,2, but nIn the past, the country's economy was based on forestry and agriculture, but since oil was discovered in the mid 90's on the coastline, oil extraction has become the main economic activity of the country. The development of the oil industry has led to rapid urbanization and to a growing availability of resources to address important public health problems through public and private initiatives .One of these initiatives has been the Bioko Island Malaria Control Project (BIMCP) mainly consisting of the indoor residual spraying (IRS) programme, that was established in 2004 to eliminate malaria infection in the island  and thatThe identification of risk factors for parasitaemia at the country level can contribute to the elimination efforts in Bioko island. In addition, it will help improve the program as it is extended to the mainland region, where geographic and socio-demograhic conditions, including urbanization (27% in the mainland region versus 85% in Bioko island ), differCase management through early diagnosis and prompt treatment of all fever cases in malaria endemic areas is a cornerstone of the global strategy for malaria control . Prompt Plasmodium falciparum infection, and to describe treatment seeking behaviour for febrile illness in preschool children from Equatorial Guinea, using data from a national survey carried out in February 2004, before the BIMCP was implemented.The aims of this study were to identify the social, behavioural, and nutritional factors that are associated with 2 and a population of ≈ 500,000 inhabitants. The proportion of the population living in urban areas has increased from 27.1% in 1975 to 48.3% in 2003 . In rural areas, distance to the closest health facility was positively associated with infection [odds ratios (95% CI): 2.9 ], whereas the proportion of households with protected water, and having had an episode of cough in the previous 15 days were inversely associated with it, .Table P. falciparum infection in the child . On the other hand, children who had taken colostrum, those whose mothers had taken anti-malarial drugs during pregnancy and those from higher SES families had lower prevalence of P. falciparum infection .In the urban settings; stunting, and the fact that someone in the house slept under a bed net were each positively related with P. falciparum infection and the sex of the child; wasting, underweight or other morbidity indicators of the child; age, ethnicity, or education of the mother; breastfeeding; immunization status; household owning a domestic animal or a garden; child sleeping under a bed net or other community indicators, such as proportion of households with access to electricity in the community or the community endowment index.When introduced in the multivariate models, no significant association was found between Table P. falciparum parasitaemia on the mainland region remains very high, 59% for children under 5 years [P. falciparum infection for the whole country differ substantially for rural and urban populations.The prevalence of malaria parasitaemia in this study was 50.9%. This is consistent with results from previous years when the estimates of community prevalence of infection in children younger than 10 years of age exceeded 50% [ 5 years . The socP. falciparum infection in both urban and rural areas. It has already been established that, during the first months of life, the risk of infection is lower because there is still a degree of immunity from the mother. The risk of infection first increases with age and starts decreasing when the individual himself reaches a degree of immunity due to repeated exposure to the parasite [Increasing age was a risk factor for parasite .P. falciparum infection might be associated with the common practice in the African Region to give anti-malarials presumptively to all patients who present with fever [In the rural settings, results show that the characteristics of the community have an important role on the individual risk of infection, including distance to health facility and the proportion of households with access to protected water. In previous results of this same survey the absence of high quality health services in the community was a risk factor for under nutrition only in rural areas, whereas a low community endowment index was a risk factor for anaemia in these same areas. Both these variables were associated with the negative effects of the process of rapid urbanization in the deterioration of rural communities . The facth fever .P. falciparum parasitaemia. The relation between under-nutrition and malaria has been controversial for many years, but recent meta-analyses suggest that under nutrition is an important underlying risk factor for infectious diseases in general [P. falciparum IgG Ab response [Plasmodium is warranted.Among urban children, risk factors for parasitaemia differ substantially from the ones found in rural areas. From the nutritional perspective, stunting and not having taken colostrum are positively associated with  general  and for  general . More spresponse . The facresponse ,22, and response . Althougresponse . The facresponse . HoweverThe usage of mosquito nets is more frequent among the urban population than among the rural . However, no significant association was found between infection and children sleeping under a bed net in any of the two settings. This result does not coincide with the analyses of only the Bioko island population for this same survey  , and goeFurthermore, recent studies in the region point out that the individual protection that children receive from the bed net density of a household is because children sleep with their parents, as bed net allocation is not prioritised to children less than five years of age but to the individual adults ,30. The The protective association of maternal intake of anti-malarial medication during pregnancy can be related to the shown effects on reducing placental malaria, maternal anaemia and low birth weight in other African countries -33, factThe relation between poverty and malaria has been widely described  and specOnly 55% of the febrile children were taken outside their homes for care. In urban areas more children were taken to private drug stores than to health facilities and in rural areas around one fifth sought care only after two days or more of illness.To reduce mortality from febrile illnesses and to prevent transmission, sick children not only need to get efficacious and appropriate drugs, but also need to get them in time; which WHO recommends being within 24 hours from illness onset ,38, and Results suggest that a national programme to fight malaria in Equatorial Guinea should a) integrate nutrition programmes specially designed for rural and urban populations, b) incorporate information, communication and education (ICE) campaigns on the importance of correct diagnosis and early treatment in rural and urban communities, c) include the reinforcement of the health system and improve the access to health facilities in rural areas or the provision of early diagnoses and treatment made through a programme of home-based management of malaria and d) design an appropriate targeting for bed nets to reach the under-fives and consider the importance of distributing long-lasting insecticide nets (LLINS).The authors declare that they have no competing interests.EC was involved in survey design and data collection, performed the statistical analysis and interpretation of the data and drafted the manuscript. MAD was involved in the survey design, the interpretation of the statistical analysis and helped to draft the manuscript. EV was involved in the performance and interpretation of the statistical analysis and revised critically the manuscript. LM was involved in the data collection and helped to draft the manuscript. IS participated in the survey design, the data collection and helped draft the manuscript. ML was involved in the data collection and helped to draft the manuscript. CB participated in the survey design, the interpretation of statistical analysis and helped draft the manuscript. AB participated in the survey design, the interpretation of statistical analysis and helped draft the manuscript. JR participated in the survey design and data collection, the performance and interpretation of statistical analysis and coordinated the draft of the manuscript. All authors read and approved the final manuscript.


The Nias district of the North Sumatra Province of Indonesia has long been known to be endemic for malaria. Following the economic crisis at the end of 1998 and the subsequent tsunami and earthquake, in December 2004 and March 2005, respectively, the malaria control programme in the area deteriorated. The present study aims to provide baseline data for the establishment of a suitable malaria control programme in the area and to analyse the frequency distribution of drug resistance alleles associated with resistance to chloroquine and sulphadoxine-pyrimethamine.band 3 (SAO), pfcrt, pfmdr1, dhfr, and dhps. In addition, haemoglobin measurement was performed in the second and third surveys for the subjects less than 10 years old.Malariometric and entomology surveys were performed in three subdistricts. Thin and thick blood smears were stained with Giemsa and examined under binocular light microscopy. Blood blots on filter paper were also prepared for isolation of parasite and host DNA to be used for molecular analysis of Plasmodium falciparum isolates from three sub-districts were successfully analysed. Alleles of the dhfr and dhps genes associated with resistance to sulphadoxine-pyrimethamine, dhfr C59R and S108N, and dhps A437G and K540E, were present at frequencies of 52.2%, 82.5%, 1.18% and 1.18%, respectively. The pfmdr1 alleles N86Y and N1042D, putatively associated with mefloquine resistance, were present at 31.4% and 2%, respectively. All but one sample carried the pfcrt 76T allele associated with chloroquine resistance. Entomologic surveys identified three potential anopheline vectors in the area, Anopheles barbirostris, Anopheles kochi and Anopheles sundaicus.Results of the three surveys revealed an average slide positivity rate of 8.13%, with a relatively higher rate in certain foci. Host genetic analysis, to identify the Band 3 deletion associated with Southeast Asian Ovalocytosis (SAO), revealed an overall frequency of 1.0% among the 1,484 samples examined. One hundred six The cross sectional surveys in three different sub-districts of Nias District clearly demonstrated the presence of relatively stable endemic foci of malaria in Nias District, North Sumatra Province, Indonesia. Molecular analysis of the malaria parasite isolates collected from this area strongly indicates resistance to chloroquine and a growing threat of resistance to sulphadoxine-pyrimethamine. This situation highlights the need to develop sustainable malaria control measures through regular surveillance and proper antimalarial drug deployment. Nias dies kochi ,7, but oThe tsunami and the earthquake have badly affected areas along the north and west coast of the Nias archipelago. Many primary health centers and their supporting facilities were damaged. In addition, some health professionals were either killed or displaced during the two natural disasters, impairing disease control efforts in the area. In relation to malaria surveillance specifically, no primary health center in Nias District currently has any adequately trained microscopists. The present study is an effort to provide baseline data for the establishment of an appropriate malaria control programme in the area, by reviving the malaria surveillance programme in the tsunami-areas in Nanggroe Aceh Darussalam (NAD) Province and Nias District of the North Sumatra Province, using conventional and molecular tools. Results of three cross sectional malariometric surveys, host genetic analysis and entomologic surveys in Nias District, North Sumatra Province Indonesia are presented here.2 with a total population of approximately 641,832 in Nias district alone. Rainfall is very high, with over three meters annually and 270 rainy days per year. The relative humidity is about 90% all year round with average temperature between 14–31°C from January to June and 22–30°C from July to December. The rainy season is from October to January, whereas the drier season starts from February to July. The main occupation of the inhabitants is farming.Nias archipelago is located off the western coast of the island of Sumatra, between 0.5°–1.5° North latitude and 96° 59'–97°58' East longitude, and comprises one main island and several smaller islands . In all cases, persons who were parasite positive were given appropriate drug treatment. This study received ethical clearance for the use of human subject from the Eijkman Institute Research Ethics Committee, Jakarta, IndonesiaMalariometric surveillance was performed in three selected sub-districts, Lahewa, Sirombu and Mandrehe. This activity involved active and passive detection of malaria cases, using thick and thin blood smears, rapid diagnostic tests using immunochromatography test (ICT), and molecular detection using polymerase chain reaction (PCR) amplification of parasite DNA isolated from filter paper blood spots prepared from finger-pricks. For field surveys, selection of the village was based on the previous available information on malaria endemicity. All of the village inhabitants were invited to undergo parasitologic screening for malaria by submitting a drop of blood for blood smears and blot. The blood samples were considered positive if either the blood smear or the PCR was positive. Because PCR is demonstrably more sensitive than microscopy, a positive finding by PCR being consistent with a negative finding microscopy was regarded positive. During such surveys, basic 7-day morbidity history, including axial temperature and spleen grading, was recorded along with nutritional status and haemoglobin levels using a portable digital haemocytometer , according to the procedure described previously . The DNAdhfr, dhps, pfmdr1, pfcrt and of the host genes such as a band-3 gene deletion, indicative of Southeast Asian Ovalocytosis (SAO). The PCR reactions were carried out as previously described [Molecular analyses were performed using PCR amplification and restriction fragment length polymorphism (RFLP) on several genes of the parasite, including escribed .Plasmodium falciparum and P. vivax were found, mostly as single infections except in one case in Sirombu village. PCR analysis of all samples positive by microscopy and 10% randomly-chosen slide-negative samples revealed no discordance between the diagnosis by microscopy and PCR. The overall slide positivity rates were 9.9%, 6.4% and 8.1% in October 2005, December 2005 and February 2006, respectively. The vast majority of the malaria positive cases were in children less than 10 years old  level was also measured in the 448 children of less than 10 years of age sampled during the second and third surveys. The relationship between Hb status and malaria infection is shown in Table The frequency distribution of SAO, a genetic disorder of red blood cells that is associated with malaria morbidity, was also checked among the samples examined. The results indicated that the overall frequency of this genetic disorder in the three selected sub-district was 1.0% Table . HoweverAn. barbirostris and An. kochi in Sirombu village of Sirombu sub-district. These two species were captured using light trap installed outside of houses in the village. The larval stage of both species was also found in grassy ground pools and grassy rice fields in the village. In Lahewa village, larvae of An. sundaicus and some other unidentified anopheline species were found in ground pools and concrete tanks, respectively. No adult anopheline mosquitoes were captured inside houses.Entomologic surveys to identify the potential mosquito vector in the surveyed area identified two anopheline species, P. falciparum isolates were collected during three malariometric surveys in Nias District, Indonesia. Some samples failed to amplify using certain oligos, hence the gene polymorphism information obtained was incomplete, as described below.In total, 109 pfcrt among the 106 P. falciparum DNA isolates from Nias District was successful in 101 isolates. RFLP analysis of the amplicons revealed that all carried the 76T allele in pfcrt, except one isolate collected from Marfala village, which carried the wild-type allele  as the first-line antimalarial treatment in the area.In support of the finding, the results of the molecular analysis also indicated that virtually all of the falciparum isolates examined carried the dhfr indicated a high proportion of isolates carrying mutant alleles associated with resistance to pyrimethamine. However, the frequency of mutant alleles of dhfr is slightly lower than that reported in a previous study done in the southern part of the island, but the difference is probably due to the larger sample size that was examined in this study. In the previous survey, the mutant alleles of dhps that associated with sulphadoxine were not found in any of the isolates examined. In this survey, two isolates that carry the 437G and 540E alleles respectively were detected. In other parts of Indonesia, mutations associated with resistance to sulphadoxine in the dhps gene are still relatively rare and the 437G polymorphism is the most common and earliest selected mutant allele of dhps [Analysis of  of dhps . In this of dhps ,12.400-Ala408 in the band 3 gene [P. falciparum but that the condition is associated with protection against severe malaria in children [Analysis of the frequency distribution of SAO indicated that this genetic disorder is also present in Nias population, although its frequency is much lower than that of the population in the eastern part of Indonesia where malaria is highly endemic ,14. Soutd 3 gene . The higchildren ,17. The children ,17. In tThe results of haemoglobin measurements showed a high prevalence of anaemia among children in Nias District. However, there was no significant association between anemia and parasitemia. This finding indicates that anaemia alone cannot be used as a good predictor for malaria in this area. Other possibilities including nutritional anaemia or hookworm infection should be further explored ,19.An. sundaicus was the predominant species along the coastal areas of Sumatra and Nias. Although in this survey only three anopheline species were detected, An. barbirostris that was found to breed in lowland rice field has never been reported previously [The results of the mosquito survey are also very similar to the previous reports that eviously . The relIn conclusion, the cross-sectional surveys in three different sub-districts of Nias District clearly demonstrate the presence of relatively stable malaria endemic foci in Nias District, North Sumatra Province Indonesia. Molecular analysis of the malaria parasite isolates collected from this area strongly indicate resistance to chloroquine and a growing threat of resistance to sulphadoxine-pyrimethamine. This situation highlights the need of developing a sustainable malaria control measures through regular surveillance and proper antimalarial drug deployment.DS designed the study and was responsible for data collection, management, fund raising for this study and the manuscript writing. PBSA performed the molecular analysis, data analysis, and also was involved in manuscript preparation. IW, RMD, ST, IL, WH and PZ collected field samples and performed data analysis. FL and AHS contributed to the experimental design, data analysis and the manuscript writing. All authors read and approved the manuscript.


Plasmodium falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion. In distinct malaria pathologies, such as placental malaria, specific antibody response against VSA provides protection. This study investigated the antibody response specifically directed against VSA expressed by parasites isolated from individuals presenting a given type of clinical presentation.Variant surface antigens (VSA) expressed on the surface of Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults, patients presenting uncomplicated malaria (UM), cerebral malaria (CM), or pregnancy-associated malaria (PAM). The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group.UM were predominant in CM, UM and HA plasmas. When analysed according to age in all plasma groups, anti-VSACM and -VSAUM antibody levels were similar until six years of age. In older groups (6-18 and >19 years of age), VSAUM antibody levels were higher than VSACM antibody levels . Mean MFI values, measured in all plasmas groups except the PAM plasmas, remained low for anti-VSAPAM antibodies and did not vary with age. One month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar, both during the infection and one month later.Antibody responses against VSACM and VSAUM. CM isolates were shown to share common epitopes. Specific antibody response to VSAUM was predominant, suggesting a relative low diversity of VSAUM in the study area.In conclusion, this study suggests the existence of serologically distinct VSA Plasmodium falciparum transmission, protective immunity to malaria is gradually acquired during childhood, leading to decreased susceptibility at adulthood. Immunological protection against parasite blood stages is mainly antibody mediated [P. falciparum-infected red blood cells (IRBC) [var genes was shown, although evidence of geographic structuring of variation emerged in isolates causing pregnancy-associated malaria (PAM) [SM) were described as more commonly recognized than VSA expressed by parasites isolated from children with uncomplicated malaria (VSAUM), suggesting that distinct serological groups are related to the clinical status of the infection [In areas of intense mediated ,2. Amongs (IRBC) . Their ls (IRBC) . Clinicas (IRBC) -6. Unforia (PAM) . PAM mosia (PAM) ,9. Similnfection -12.CM, as a subgroup of VSASM, is likely to be relatively conserved due to restricted functional specialization for high binding capacities in brain capillaries. In the current study, the antibody response specifically directed against VSA expressed by parasites was investigated. Parasites were isolated from a given type of clinical presentation, cerebral, uncomplicated, or pregnancy-associated malaria.VSAs contribute to the sequestration of IRBC in deep organs via the binding to endothelial receptors. This mechanism enables the parasites to avoid splenic clearance . In PAM,et al., submitted). The transmission level in Cotonou was characterized in 1992 by 33 infecting bites per man per year [Patients were enrolled in Cotonou  and Allada  in southern Benin over one malaria season (April to September 2008). Allada is about 40 km north of Cotonou. In southern Benin, malaria is mesoendemic and transmission is perennial . A questionnaire was administered to the children' guardian, or the pregnant women to collect social data, clinical signs and treatment received before hospital admission.Three groups of patients presenting distinct clinical malaria status were enrolled: uncomplicated malaria (UM), cerebral malaria (CM) and pregnancy-associated malaria (PAM). Briefly, UM was defined as the combination of fever (tympanic temperature ≥ 37.8°C) or a history of fever in the preceding 24 hours, confirmed presence of  the WHO . CM was Plasmodium falciparum infections were confirmed by Giemsa-stained thick blood films, and parasitaemia was quantified by counting against 200 leucocytes assuming a mean of 8,000 leucocytes per millimeter of blood. Blood group was determined for each blood sample. Plasmas were separated and frozen from all subjects. Parasite isolates from all malaria-infected patients with O blood group were frozen. Blood count and lumbar puncture for meningitis diagnostic were achieved for patients from the CM group.Two additional groups of subjects were added as plasma controls, healthy male adults (HA) living in Cotonou, and non-immune healthy French volunteers (NI) living in France and having never been exposed to malaria. Peripheral venous blood sample (5 ml) was collected from all study individuals in vacutainer tubes containing EDTA. P. falciparum isolates from patients  were frozen at -150°C. Cryopreserved IE were subsequently thawed and matured in vitro for 24-36 hrs [msp1) and merozoite surface protein-2 (msp2), as described previously [Infected red blood cells obtained from patients with O blood group were snap-frozen in a glycerolyte solution, as described . Twenty-eviously . The MOIA total of 256 plasma samples were collected as follows: 127 samples were collected at the time of malaria diagnosis (D0), 41 from CM patients, 62 from UM patients, and 24 from women with PAM. CM and UM patients were also seen at convalescence (D30), 17 plasma samples were obtained in the CM group, and 62 in the UM group. The high number of lost to follow up in the CM group is mainly explained by the urban localization of the children enrolled, and the difficulty to get them back one month after a severe malaria attack. Two sets of control plasmas were obtained from 44 healthy adults (HA) living in Cotonou, and from six French volunteers never exposed to malaria (NI).5 IRBC in PBS-FCS 2% were labeled with ethidium bromide (0.002 mg/ml) to allow flow cytometric exclusion of remaining uninfected erythrocytes, and with 100 μl of goat anti-human IgG - FITC . Labeled IRBC were sequentially exposed to 5 μl of plasma. Samples were analysed from a minimum acquisition of 5,000 IRBC using a Facscalibur cytometer (Becton Dickinson). The level of IgG recognizing VSAs was expressed as mean fluorescence intensity (MFI) in the channel for ethidium bromide-labeled IRBC. Nonspecific labeling was evaluated by analysis of uninfected (ethidium bromide-negative) erythrocytes. All plasma samples tested against a particular parasite isolate were processed and analysed in a single assay. NI plasmas samples were tested against all isolates. However, due to limitations in amounts of IRBC available, not all plasma samples were tested against all parasite isolates (Table The fluorescence-activated cell sorter (FACS) analysis was used for measurement of specific anti-VSA antibody, as described before . Briefly2 test. VSA Ab recognition was evaluated by analysis of covariance (ANCOVA), two-factor ANOVA, t-test and Wilcoxon rank test for paired data. Clonality and msp family prevalence were compared between clinical groups by Kruskall-Wallis and Mann-Whitney U-tests. Spearman correlation test was used to estimate MOI and parasite density correlation. Values of P < 0.05 were considered statistically significant.Data were analysed by using the Statview software programme. For analyses performed with data obtained the day of diagnosis, data obtained with homologous plasma/isolate pairs were excluded. The levels of antibody responders were compared using the χA total of 127 malaria-patients were enrolled. Three groups of patients were identified as CM (n = 41), UM (n = 62), and PAM (n = 24) patients, as well as one group of 34 malaria-exposed healthy male adults (HA). CM patients were between six months and seven years of age with a mean (± SD) of 3.3 (± 1.7) years. UM patients were aged 9.3 (± 2.1) years, PAM women were aged 24.9 (± 6.4) years, and HA were aged 35.1 (± 8.9) years. In PAM women, gravidity varied between 1 and 5 (mean 1.9 ± 1.2), 13 were primigravidae (PG) and 11 were multigravidae (MG). Parasitaemia was highly diverse between and within groups: CM, median (interquartile range) of 32,653  per μl of blood; UM, 3,555 /μl; PAM, 17,391 /μl. Mean tympanic temperature (± SD) at inclusion was of 38.8 (± 1.2) for the CM patients, 37.2 (± .7) in UM patients and 38.0 (± .9) in pregnant women.The CM patients were all comatose, admitted at hospital for at least two hours and presented a mean Blantyre score (± SD) of 1.8 ± 0.46 . The coma lasted 2.5 days on average . Meningitis was excluded for all children. Anaemia characterized the CM group with a mean haemoglobin level of 6.6 g/L (± 1.5) and a mean hematocrit of 20.8 (± 4.5). Erythrocytes count was low with 2.7 (± .6) T/L although the mean globular volume and the mean haemoglobin corpuscular content were normal. Respiratory distress occurred in 4/39 (10.3%) children of the CM group. Among the children suffering of CM, six died and five came out of hospital without medical consent. At that time among these five children, two presented a deep coma, two a normal consciousness and the data was missing for one.P = .73 by Kruskall-Wallis test). However, MOI increased with parasitaemia . The prevalence rates of the allelic families for all isolates were 65%, 27% and 40% for the K1, MAD20 and RO33 families of the msp1 locus, respectively, and 94% and 7% for the 3D7 and FC27 families of the msp2 locus, respectively. Interestingly, PAM isolates presented a higher frequency for MAD20 and FC27 alleles , and a lower frequency for 3D7 alleles  than other clinical groups of isolates. RO33 alleles were more frequent in UM isolates than in other groups (P = .05).Means (CI95%) for MOI were respectively 1.49 (0.6 - 3.6), 1.61 (0.6 - 3.1) and 1.83 (0.5 - 4.7) for CM, UM and PAM isolates . In HA plasmas, antibody responses against VSAUM were higher than VSAPAM. The primigravidae (PG) plasmas contained similar antibody responses to the different VSA types. In multigravidae (MG) plasmas, responses were higher to VSAUM than to VSACM. The levels of anti-VSAPAM antibody responders in MG and PG plasmas were similar. The quantitative results are also available . Anti-VSACM antibodies gradually increased until 18 years then decreased, although values did not vary statistically. Mean MFI values remained low for anti-VSAPAM Abs (max. 1.5) and did not vary with age. VSAUM and VSACM antibody levels were then compared according to three age groups: until 6 years old, between 6 and 18, older than 18. Until 6 years old, levels were similar (t-test). In both other age groups, VSAUM antibody levels were higher than VSACM antibody levels .The level of anti-VSA antibodies was analysed at different ages in all plasma groups, except for anti-VSAs Figure . Anti-VSUM antibodies had increased at D30 . For CM plasmas and CM isolates, only two pairs with both MFI at D0 and D30 for the homologous isolate were available and both showed increased D30 values (see Table UM antibodies at D0 and one month later (panel A), whereas CM plasmas exhibited higher anti-VSACM antibodies levels at D30 . The same analysis was performed after matching ages in UM and CM groups, i.e. with children aged between five and seven years of age. Results were similar as before, anti-VSA antibodies significantly increased at D30 in the CM plasmas (P = .033), but not in the UM plasmas (P = .206). The same type of analysis was performed to test the potential cross-acquisition of anti-VSACM by UM patients after an UM infection, as well as anti-VSAUM by CM patients after a CM infection. No difference was obtained between the anti-VSACM antibody levels displayed at D0 and D30 by UM plasmas (panel B). Conversely, anti-VSAUM antibodies were higher in CM plasmas at D30 than at D0 . These results show that infection with an isolate of the CM group induced the acquisition of antibodies directed against other CM isolates, but also against UM isolates. No such phenomenon was observed following infection with an isolate of the UM group.First, MFI values obtained in plasma samples the day of diagnosis and one month later were compared. Plasmas from the same patients were tested against the isolate infecting these patients. For UM plasmas and UM isolates, 10 pairs of results were obtained with both D0 and D30 plasma samples for the homologous isolate. As expected, the levels of anti-VSAThis study describes the levels of anti-VSA antibodies displayed by plasmas from different clinical malaria status, uncomplicated, cerebral, or pregnancy-associated malaria, and from healthy adults. The study took place in Benin where malaria transmission level is mesoendemic. Falciparum malaria protective immunity is acquired slowly, according to infections by new parasite variants, until the repertoire of all parasite variants from the area is recognized. Protective antibodies are partly directed against parasite variant surface antigens (VSAs), known to contribute to the cytoadherence of infected erythrocytes to vascular endothelium. In CM and PAM, cerebral capillaries and placenta respectively both concentrate preferentially the IRBC sequestration. In PAM, the existence of a conserved adhesion phenotype in selected parasite variants was proven ,15. HoweSM than VSAUM, and proposed the existence of rare and frequent falciparum variants [UM antibody responses were higher than VSACM antibody responses, although acquisition of both anti-VSA antibody types occurred at about 6 years old, as shown in Figure UM antibodies increased and reached higher levels than anti-VSACM. A high anti-VSA antibody response may reflect three options: i) VSA epitopes are conserved, ii) VSA epitopes are highly prevalent in the area, or iii) VSA epitopes are highly antigenic. The previously cited studies took place in Kenya and Ghana, where transmission intensity is much higher than in southern Benin. These groups proposed that P. falciparum favours VSASM variants presenting optimal binding ability. However, whether high virulence is advantageous for parasite population survival is questionable. Indeed, the foundations of parasitism involve the search for equilibrium between parasite and host, i.e. the survival of the host to ensure long-term parasite proliferation. In our understanding, the transmission level of the area allows a certain level for parasite clonal diversity. The level of anti-P. falciparum specific immunity certainly constitutes another factor influencing the parasite clonal diversity, as already suggested [UM antibody response compared to VSACM may be explained by the balance between a relative conservation of VSAUM, or relative low clonal diversity, and the level of immunity of the patient groups tested. Epidemiological, entomological and socio-cultural factors that are area-specific are also likely to interfere with clonal composition of P. falciparum parasite population.This study includes patient groups categorized by their malaria disease, uncomplicated, cerebral or associated to pregnancy. As the transmission level of the study area is mesoendemic and perennial, these patient classes were also different in age, particularly the CM and younger group. Consequently, anti-VSA antibody responses measured in the CM group were lower compared to the other plasma groups, due to their lesser exposition to malaria. Previous studies showed a more frequent recognition of VSAvariants ,12. In tuggested . In the SM and VSAPAM are thought to be more conserved than VSAUM, raising the hope for specific therapies targeting severe types of malaria. This hypothesis can be argued as one month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants had increased in CM patients , UM isolates , and PAM isolates . UM: uncomplicated malaria, CM: cerebral malaria, PAM: pregnancy-associated malaria, PG: primigravidae, MG: multigravidae.Click here for file