After successfully reducing the malaria burden to pre-elimination levels over the past two decades, the national malaria programme in Vietnam has recently switched from control to elimination. However, in forested areas of Central Vietnam malaria elimination is likely to be jeopardized by the high occurrence of asymptomatic and submicroscopic infections as shown by previous reports. This paper presents the results of a malaria survey carried out in a remote forested area of Central Vietnam where we evaluated malaria prevalence and risk factors for infection.After a full census , the study population was screened for malaria infections by standard microscopy and, if needed, treated according to national guidelines. An additional blood sample on filter paper was also taken in a random sample of the population for later polymerase chain reaction (PCR) and more accurate estimation of the actual burden of malaria infections. The risk factor analysis for malaria infections was done using survey multivariate logistic regression as well as the classification and regression tree method (CART).Plasmodium falciparum (81.4%) or Plasmodium vivax (17.7%) mono-infections; a large majority (69.9%) was asymptomatic. By PCR, the prevalence was estimated at 22.6% (ranging from 16.4 to 42.5%) with a higher proportion of P. vivax mono-infections (43.2%). The proportion of sub-patent infections increased with increasing age and with decreasing prevalence across villages. The main risk factors were young age, village, house structure, and absence of bed net.A total of 1,450 individuals were screened. Malaria prevalence by microscopy was 7.8% (ranging from 3.9 to 10.9% across villages) mostly This study confirmed that in Central Vietnam a substantial part of the human malaria reservoir is hidden. Additional studies are urgently needed to assess the contribution of this hidden reservoir to the maintenance of malaria transmission. Such evidence will be crucial for guiding elimination strategies. The past 20 years of continued malaria control efforts have resulted in the elimination of this disease in several provinces of Northern and Southern Vietnam . In 2011Plasmodium falciparum resistance to artemisinin derivatives has been reported [Plasmodium vivax is particularly high. A recent survey carried out in Ninh Thuan Province , in which filter paper blood samples were analysed by molecular techniques, showed the presence of a largely hidden human reservoir of malaria infections with numerous sub-patent infections (detected only by PCR but not by microscopy) including mixed infections with Plasmodium malariae and Plasmodium ovale [P. vivax and P. ovale may have dormant liver forms (hypnozoites) that can reactivate at varying times after the primary infection. Vietnamese treatment guidelines recommend the use of both a three-day course of chloroquine (0.25 mg/kg) and a 14-day course of primaquine (0.25 mg/kg/day) to clear both peripheral blood and liver stages of infection. However, a 14-day course of primaquine is rarely followed due to fears of haemolysis in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals [Currently, most malaria morbidity  and malaria deaths (eight in 2012) occur in 21 out of 58 provinces  and are located in Central and Central-Southern Vietnam [reported . Asymptoividuals . In addiThe study was carried out in four villages located in two communes (Tra Leng and Tra Don) situated in Nam Tra My district in Quang Nam Province  Figure . Study vAnopheles dirus sensu stricto and Anopheles minimus sensu stricto [The population mainly belonged to the M’nong and Ca Dong ethnic groups living in very poor socio-economic conditions, mainly subsistence farming, practising slash-and-burn agriculture in forest fields with maize, manioc and rice. Malaria transmission is perennial with two peaks, one in May-June and the other in October-November, with the two main vectors species being  stricto ,11. MalaThe Commune Health Centre (CHC) located in the centre of the commune (Village 1) was hardly accessible for Village 4 during the rainy season because of heavy rains and flooding. The local health staff  provided free-of-charge health care with the support of village health workers (VHWs).In February 2009 a full census of the study population  was carried out to collect household as well as individual socio-demographic data . Each resident in the study area was allocated a unique ID code. Each house was mapped using a geography position system device  .P.vivax infections to identify potential study participants for a cohort to be followed prospectively. This started by informing first all commune, village and household leaders on the objectives and study procedures and then the individual study subjects, who were all invited to be screened after oral informed consent. During the screening, each participant was interviewed for previous malaria symptoms during the previous 48 hours, the axillary temperature was checked and a blood slide collected for light microscopy (LM). Confirmed malaria infections were treated according to the national treatment guidelines.In April 2009, the entire study population was screened for In addition to blood slides taken during the screening, an additional blood sample was taken for haemoglobin measurement and for later molecular analysis (PCR) in a random sample of study participants (n = 327). This was done by randomly choosing one individual in each household after blindly drawing an ID number among those allocated to the house during the census. If the selected subject was temporarily absent, the survey team would return later; however, if the subject was absent for a long time or not willing to participate, a second drawing would be done. Survey participants  were asked to give their written informed consent (parent/guardian for children) after being explained the purpose of the additional sampling and investigations. Among these subjects, a face-to-face interview was done to collect data on the different outdoor activities in and outside the community, sleeping habits, as well as malaria prevention measures. For children under 12 years old, the parent/guardian would answer the questions.Thick and thin films were stained with a 3% Giemsa solution for 45 minutes. The number of asexual forms per 200 white blood cells (WBCs) was counted and parasite densities were computed assuming a mean WBCs count of 8,000/μl. Gametocytes were also counted. A slide was declared negative when no parasite was seen after counting 1,000 WBCs. All slides were read independently by two expert microscopists. In case of discrepant results, they re-examined the slide together until agreement was reached. Quality control of blood slides was done on all positives and 10% of negative blood slides by a senior laboratory technician at the National Institute of Malariology, Parasitology and Entomology (NIMPE), Hanoi; in case of disagreement, a second senior technician would re-read the slide until an agreement was reached.P. falciparum, P. vivax, P. malariae, and P. ovale [Plasmodium species. Cross-contamination during handling was checked for by implementing negative controls in each step from extraction to the nested PCR step. Quality control was done on 10% of the samples for which the SnM-PCR was repeated blindly by a senior technician. In case of discrepancy, the sample was re-analysed until agreement was reached.Haemoglobin concentration was measured with the HemoCue Hb 301 device following the manufacturer’s instructions . Filter P. ovale . The PCRet al. [All survey samples were analysed for G6PD deficiency at the Shoklo Malaria Research Unit, by genotyping for the Viangchan mutation following a modified protocol published by Nuchprayoon et al. . DNA waset al. . Genotypet al.  and MyTAP. vivax at 9%. The sample size was calculated by assuming a minimal prevalence of 9%, with 3% precision at 5% significance level and adding 10% security margin; a total of 330 individuals were needed for the survey (“CSample” command/EpiInfo6). Therefore, to simplify sampling procedures, one individual in each house visited during screening was randomly selected to be included in the survey.Sample size: According to the provincial malaria station data on surveys carried out in April-May, the overall parasite rate was around 16% (ranging from 5 to 39% across hamlets) and the prevalence of t-test as required, and a p-value <0.05 was used as cut-off for significance.Data were double-entered and cleaned using Epidata version 3.1 free software . The datThree different variables for livestock ownership (number of i) buffaloes, ii) cows, and iii) pigs) were considered as the best proxy (after discussion with household leaders) for the economic status of the households as all inhabitants were subsistence farmers and generally poor. In order to aggregate multiple variables to a single measure of economic status, a principal component analysis was performed . Using tstrata, and household sizes as p-weights. A survey logistic regression (“svy” command in STATA) was used to carry out a multivariate adjusted analysis for the risk of malaria infection (determined by PCR). Moreover, a classification tree analysis  was performed to explore the relationship and rank the relative importance of risk factors for all malaria infections identified by PCR, as well as for patent infections only (detected both by PCR and microscopy). Sub-patent (or sub-microscopic) malaria infections are detected by PCR only. The CART analysis is a non-parametric method enabling more direct and flexible analyses since, unlike logistic regression models, it allows for co-linearity and multiple interactions between different independent variables [The survey design (survey dataset) was taken into account using the svy- command in STATA, with villages as ariables . BrieflyEthical clearance was obtained from both the ethical committee of NIMPE in Hanoi and the University of Antwerp. The fundamental principles of ethics in research on human participants were upheld throughout the project. All study participants gave their informed consent after being explained the study procedures as well as their right to withdraw without prejudice for themselves or their families.A total of 1,810 individuals were identified during the census, representing 352 households distributed in 20 clusters within the four study villages  were screened for malaria by LM Table  and theiP. falciparum mono-infections represented the majority of LM detected infections (81.4%) although such proportion varied significantly between villages, i.e., from 59% in Village 1 to 96% in Village 3. The mean parasite density was significantly higher in P. falciparum  than in P. vivax (559.7/μl) infections, and increased with increasing prevalence of infection. Most infections were asymptomatic (69.9%), and approximately one-third (36.3%) carried gametocytes, with significant differences between villages (p <0.001). The prevalence of gametocyte carriage by village was significantly correlated to the prevalence of P. vivax infections . Overall, the risk of gametocyte carriage was significantly higher in asymptomatic (46.8%) than in symptomatic (11.8%) infections (p <0.001), and in P. vivax (95.0%) compared to P. falciparum (22.8%) infections (p <0.001). In addition, the risk of asymptomatic infection was significantly higher among P. vivax than P. falciparum infections .Overall versus 7.8%), ranging by village between 16.4 and 42.5% , while mixed species infections remained scarce (n = 1). More than half (58.7%) of all infections were sub-patent, i.e., negative by LM, and this proportion increased with decreasing village prevalence of infection as shown by the evolution of the ratio between sub-patent and patent infections .A total of 327 individuals were randomly selected to estimate malaria prevalence by PCR; their socio-demographic characteristics were similar to those of the whole population. Malaria prevalence estimated by PCR was about three-fold higher than by microscopy   and asymptomatic  compared to P. falciparum.Malaria prevalence significantly decreased with increasing age (p = 0.007), from 32.3% in children < ten years to 15.4% in adults ≥20 years, mainly because of a four-fold reduction of the prevalence of patent infections  with increasing age. Sub-patent infections did not decrease significantly with age , resulting in higher ratio sub-patent/patent infections in adults  among males and 1.4% (4/143) among females, and this was similar in Cadong  and M’nong ethnic groups .Village, age, ethnicity, bed net ownership, occupation, and wall type were risk factors for malaria infection identified by the univariate analysis Table ; the mulIn order to overcome the difficulty of handling the interaction (age/villages) and multiple collinearities , the CART method was also used to identify and rank the main risk factors for malaria infections Figure . The resThe CART analysis for the risk of patent infections (n = 113) using the screening dataset N = 1,450) showed that the first splitter was village, i.e. Villages 3 and 4 with the highest prevalence, and in these villages children had the highest risk of infection  and the interaction (village*age).vs 13.8%, respectively) while the difference was less important for sub-patents infections (12.1 vs 16.6%).Finally, assessing simultaneously the risk of patent and sub-patent malaria infections with CART, age was the only splitter, as adults had a lower risk than children particularly for patent  was extremely heterogeneous, both by LM and PCR, although the latter was performed only in a subset of the population, resulting in a lower precision. Heterogeneity of malaria transmission within short distances is a well-known, albeit not fully understood, phenomenon  but receSubpatent infections in this area represented an important proportion of all detected infections, with the difference between LM and PCR estimates similar to other reports ,22-24 anGametocyte carriage as determined by LM was similar to that found in other surveys carried out in Central Vietnam ,25. WhenP. vivax infections, possibly due to the ability of this species to produce gametocytes at very early stage of its erythrocytic schizogony, well before the occurrence of symptoms [P. vivax infections were also asymptomatic and subpatent can explain the association between gametocyte carriage and asymptomatic infections.The proportion of gametocyte carriers as determined by LM varied by village, and overall was highly correlated with the proportion of symptoms -30. The P. malariae, P. ovale or mixed infection were not common, reflecting the high heterogeneity of malaria transmission and species distribution. In this case, the absence of these species could be explained by the higher isolation and remoteness of the four study villages compared to those in Ninh Thuan Province where some of the villages were located near the district town or along the main district road, favouring parasite strains and species circulation through population movements to different endemic areas.Unlike a previous study carried out in the neighbouring Ninh Thuan Province , P. malaMalaria risk was the highest in people living in Village 4; because of co-linearity, such risk could not be dissociated in the logistic regression model from Cadong ethnicity since these were all living in Village 4. Indeed ethnicity has been repeatedly reported as being associated with malaria infection mainly due to socio-cultural -34 or toFurther analysis by CART showed that village but not ethnicity was the most important risk factor both for PCR detected malaria infections as well as for patent infections. Age was ranked second in importance for the risk of patent infections, but not for the risk of all infections detected by PCR; this may reflect the progressive build-up of partial immunity with age as illustrated in Figure Occupants of wooden houses were at higher risk of malaria infection. These were the most common type of houses and had been built by the government, thus not reflecting the actual socio-economic status of respective dwellers. Bamboo houses, usually with smaller doors and windows compared to wooden houses, are traditional for these ethnic minorities and may result in a lower risk of exposure to mosquito bites, though sampling variation cannot be excluded given the very small number of bamboo houses. Conversely, availability of bed nets, even non-treated, was protective, particularly in wooden houses, and was ranked as the second most important risk factor for malaria infection.P. vivax. The remoteness of the area together with the difficulty of detecting and treating both sub-patent and patent infections, particularly those carrying gametocytes, and P. vivax liver forms that keep releasing parasites in the blood stream, represent huge challenges for any malaria elimination programme. Until new approaches for dealing with these issues are available, eliminating malaria in these type of settings will remain extremely challenging.The malaria situation in this study area reflects the difficulties related to the goal of malaria elimination in Vietnam. There was a substantial and hidden human reservoir of malaria infection, largely represented by


Both iron deficiency (ID) and malaria are common among African children. Studies show that the iron-regulatory hormone hepcidin is induced by malaria, but few studies have investigated this relationship longitudinally. We measured hepcidin concentrations, markers of iron status, and antibodies to malaria antigens during two cross-sectional surveys within a cohort of 324 Kenyan children ≤ 8 years old who were under intensive surveillance for malaria and other febrile illnesses. Hepcidin concentrations were the highest in the youngest, and female infants, declined rapidly in infancy and more gradually thereafter. Asymptomatic malaria and malaria antibody titres were positively associated with hepcidin concentrations. Recent episodes of febrile malaria were associated with high hepcidin concentrations that fell over time. Hepcidin concentrations were not associated with the subsequent risk of either malaria or other febrile illnesses. Given that iron absorption is impaired by hepcidin, our data suggest that asymptomatic and febrile malaria contribute to the high burden of ID seen in African children. Further, the effectiveness of iron supplementation may be sub-optimal in the presence of asymptomatic malaria. Thus, strategies to prevent and eliminate malaria may have the added benefit of addressing an important cause of ID for African children. •Hepcidin fluctuates with age with the highest levels in female infants.•Asymptomatic and febrile malaria increase hepcidin levels, but hepcidin does not predict the risk of malaria or febrile illness.•Strategies to control malaria may have the added benefit of reducing iron deficiency in childrenIron deficiency and malaria are common among children living in Africa. Hepcidin is a hormone that inhibits dietary iron absorption. Hepcidin levels are increased during malaria infection and this protects against infection in mice. In Kenyan children we found that hepcidin levels varied by age and in infants by sex. Malaria increased hepcidin levels in well children and for a time after treatment in sick children. Hepcidin levels increased as antibodies to malaria increased, but hepcidin did not protect children from getting malaria. Our study suggests that malaria may be one of the causes of iron deficiency in African children. Malaria caused an estimated 437,000 deaths in young African children in 2013  and > 7022.1Individual written informed consent was obtained from the parents of all study participants and ethical permission for the study was granted by the Kenya Medical Research Institute (KEMRI)/National Ethical Review Committee.2.2The current study was nested within an ongoing, longitudinal cohort study evaluating the history and acquisition of natural immunity to malaria in children living in Kilifi District on the Kenyan coast . The cur2.3Plasmodium falciparum parasitaemia was determined as previously described  and merozoite surface protein 2 (MSP2) were assayed by enzyme linked immunosorbent assay (ELISA) (escribed . Haemoglescribed . Plasma escribed . IgG ant (ELISA) .Plasma hepcidin was quantified by competitive ELISA (Hepcidin-25 (human) EIA Kit, Bachem) . Standar2.4P. falciparum parasites at any density for children age < 1 year or at a density of > 2500 parasites/μl for children age ≥ 1 year  in conjunction with a positive blood smear for ≥ 1 year . Asympto≥ 5 mg/l . ID was ≥ 5 mg/l . The fer≥ 5 mg/l .2.5All analyses were conducted using STATA v.12.0 . Associations between hepcidin concentration (or other variables such as iron status) and independent parameters were evaluated using generalized estimating equation (GEE)-based linear regression models that included an exchangeable correlation structure and a robust variance estimator to account for correlation between measurements at two time points from the same child. Analyses were age-adjusted as appropriate. We did not restrict fitting independent parameters, such as age, to linear effects. We allowed for nonlinear effects by fitting and significance testing multivariable fractional polynomials with use of the Royston and Altman algorithm entering hepcidin concentration and other variables simultaneously in the model. This allowed the model to optimize the model fit using power and log functions to approximate the shape of the relationship of the parameter with hepcidin . The assMultivariable models included covariates with a significance of p ≤ 0·1 in univariable models. We used p < 0·05 to interpret the findings in the final multivariable model. For clinical malaria hazards ratios were adjusted for age, ethnicity, sickle cell trait and period of monitoring and for non-malarial fever hazards ratios were adjusted for age in years and period of monitoring.2.6Oxford University Clinical Academic Graduate School; The Academy of Medical Sciences with The Wellcome Trust, The British Heart Foundation, Arthritis Research UK (to SHA); a Beit Memorial Fellowship for Medical Research; an MRC New Investigator award; the National Institute for Health Research Oxford Biomedical Research Centre (to HD); a Senior Wellcome Trust Fellowship (grant number 091758 to TNW); a Senior Research Fellowship to JGB; and the European Union Framework Programme Seven European Virtual Institute of Malaria Research Consortium (grant number 242095 to TNW). The sponsors had no role in study design, data collection, data analyses, data interpretation, or writing of the report. The corresponding author had full access to all data in the study and the final responsibility for the decision to submit for publication.This work was funded by the 3P. falciparum with a mean parasite density of 909 parasites/μl . We found no significant association between geometric mean hepcidin concentrations and either sickle cell trait  or α-thalassaemia .A total of 324 children were included in the study with an average followup time of 7·4 months. 3.1Plasma hepcidin concentration varied markedly by age , while the best-fit fractional polynomial for the age profile of hepcidin suggested that levels were the highest in the youngest children, decreased very rapidly in infancy and then declined slowly in childhood A. These 3.2Ferritin, sTfR, and CRP levels explained 37.5%, 37.3%, and 10.2% of the variance in hepcidin concentrations respectively (p < 0.0005 for each). Hepcidin concentrations were very low in children with ID  in comparison to those without . The best-fit fractional polynomials suggested that hepcidin concentrations increased with increasing ferritin and CRP and decreased with increasing sTfR and ferritin index B–E. Infl3.3P. falciparum parasitaemia  and malaria parasitaemia explained 7% of the variation in hepcidin (p < 0·0005). Parasite density, ID, inflammation, and age modified the effects of parasitaemia on hepcidin . We found a significant interaction between age and antibody titres to AMA1 in predicting hepcidin concentrations (p = 0·01) and a trend towards an interaction with MSP2 (p = 0·08), so that in younger children a smaller unit change in AMA1 or MSP2 was associated with a much larger unit change in hepcidin compared with older children. We found no interaction between age and antibody titres to schizont extract in predicting hepcidin concentrations.We then assessed whether hepcidin concentrations correlated with antibodies to 3.5We next evaluated whether hepcidin concentrations varied with time after a clinical malaria or non-malarial fever episode. The best-fit fractional polynomial of the time profile of hepcidin after treatment for clinical malaria suggested that hepcidin concentrations declined steeply in the first week, then more slowly over the subsequent weeks A. Howeve3.6Finally, we tested the hypothesis that hepcidin concentrations influence the subsequent risk of malaria and non-malarial fever. Overall, we observed 148 first or only episodes of clinical malaria and 130 first or only episodes of non-malarial fever during 2402 months of monitoring. Survival plots for clinical malaria and non-malarial fever by hepcidin tertile are shown in 4P. falciparum antigens. Concentrations fell rapidly and then slowly after treatment of clinical malaria, but were not altered by non-malarial febrile illnesses. Nevertheless, hepcidin concentrations did not predict the subsequent risk of malaria or other febrile illnesses.Both malaria and ID are important public health problems in African children. In the current study 12% of children were parasitaemic and 46% had ID. Hepcidin concentrations fell rapidly in infancy and then more slowly with increasing age, while female infants had higher concentrations than males. We found that asymptomatic malaria was associated with significantly elevated hepcidin concentrations, which were proportional to parasite density and modified by age, inflammation, and the presence of ID. Furthermore, hepcidin was positively associated with antibody titres to We found a non-linear association between age and hepcidin. In agreement with a study in 3–12-month old Zimbabwean infants , hepcidiFerritin, sTfR and CRP explained 37·5%, 37·3%, and 10·2% of the variance in hepcidin respectively (p < 0·0005 for each). In agreement with a study in Indonesian school children , asymptoP. falciparum antigens in age-adjusted models. For AMA1 and MSP2 we found interactions between age and antibody titres so that in younger children a small increase in antibody titres was associated with a large increase in hepcidin concentrations, while in older children increased antibody titres had little effect on hepcidin. This agrees with our study showing that these antibodies act as measures of exposure in younger children with lower immunity and as measures of protective immunity in older children with higher antibody titres (Antibodies to malaria antigens are sensitive biomarkers of population-level malaria exposure and may be useful surveillance tools for malaria control and elimination . We invey titres . By conty titres . Taken tWe next evaluated the profile of hepcidin concentrations over a 3-month period after exposure to and treatment of febrile clinical malaria. In agreement with other studies in African children we found that concentrations were initially very high ; but felP. falciparum malaria or non-malarial fever. A number of possible explanations can be put forward for this negative finding. It is possible that, in contrast to mouse studies (Finally we hypothesized that hepcidin concentrations might influence the subsequent risk of malaria. Intriguing data from mouse studies suggest that hepcidin might play a role in modulating clinical malaria , but we  studies , hepcidi studies . However studies , hepcidi studies . However studies  may counIn conclusion, we have shown that asymptomatic and recent febrile malaria both significantly increase hepcidin in a population of children where ID is an important cause of morbidity. Given that dietary iron absorption is impaired by elevated hepcidin , our datIDiron deficiencyGEEgeneralized estimating equationAMA1apical membrane antigen 1MSP2merozoite surface protein 2CRPC-reactive proteinHRhazard ratioSHA, HD, AMP, JGB, TNW, SK, SMU, CKM, AEA, and PB wrote the manuscript. SHA, HD, AEA, and TNW conceived the study. All authors contributed to the design of the study. SK, SMU, and CKM performed laboratory analyses and data collection, SK performed hepcidin and iron measurements and SHA and PB performed all statistical analyses.All authors declare no conflicts of interest.


The association between malaria during pregnancy and low birth weight (LBW) is well described. This manuscript aims to quantify the relative contribution of malaria to small-for-gestational-age (SGA) infants and preterm birth (PTB) in pregnancies accurately dated by ultrasound on the Thai-Myanmar border at the Shoklo Malaria Research Unit.From 2001 to 2010 in a population cohort of prospectively followed pregnancies, we analyzed all singleton newborns who were live born, normal, weighed in the first hour of life and with a gestational age (GA) between 28+0 and 41+6 weeks. Fractional polynomial regression was used to determine the mean birthweight and standard deviation as functions of GA. Risk differences and factors of LBW and SGA were studied across the range of GA for malaria and non-malaria pregnancies. From 10,264 newborns records, population centiles were created.Women were screened for malaria by microscopy a median of 22 [range 1–38] times and it was detected and treated in 12.6%  of pregnancies. Malaria was associated with LBW, PTB, and SGA compared to those without malaria. Nearly two-thirds of PTB were classified as LBW (68% (539/789)), most of which 83% (447/539) were not SGA. After GA 39 weeks, 5%  of non-LBW births were identified as SGA. Low body mass index, primigravida, hypertension, smoking and female sex of the newborn were also significantly and independently associated with LBW and SGA consistent with previous publications.Treated malaria in pregnancy was associated with an increased risk for LBW, PTB, and SGA, of which the latter are most important for infant survival. Using LBW as an endpoint without adjusting for GA incorrectly estimated the effects of malaria in pregnancy. Ultrasound should be used for dating pregnancies and birth weights should be expressed as a function (or adjusted for GA) of GA in future malaria in pregnancy studies. Plasmodium falciparum and P.vivax malaria have an increased risk to deliver an infant with low birthweight  thcentile for the gestational age), or both Pregnant women infected with To distinguish between PTB and SGA accurate pregnancy dating is required When large numbers of pregnancies are dated precisely, and birth weights measured accurately This is a retrospective hospital record analysis. For those patients in trials a written informed consent was obtained including storing of data and samples. For the women seen in the ANC the routine clinical records were anonymized and these pregnancy records have been routinely entered into a database since 1987. Permission was granted by Oxford Tropical Research Ethics Committee (reference: OXTREC 28–09) to use these records for analysis.P.falciparum and P.vivax transmission is low and seasonal. Since the inception of the antenatal care program in 1986, all pregnant women have been encouraged to attend in the first trimester and return for weekly malaria screening and treatment and routine obstetric care. In 2001 antenatal ultrasound was introduced to improve GA estimation in this population with low literacy rates The Shoklo Malaria Research Unit (SMRU) is located on the border between Thailand and Myanmar, a malaria endemic area where All women were encouraged to deliver in the SMRU facilities under supervision of trained skilled birth attendants and midwives from the same population. Each labor was monitored using a WHO partogram. All medical and obstetric problems were investigated and treated by locally trained health workers in SMRU facilities and supported by doctors. Women requiring Caesarean section were referred to the nearest Thai hospital. Each baby was weighed within 1 hour on an electronic SECA  digital newborn scale. All infants born in SMRU delivery rooms have a standardized newborn surface examination done by formally trained staff. Abnormalities that were obvious by ultrasound  were verified by an obstetric doctor and, when conformed, were excluded from analysis.P.vivax or P. falciparum) in the peripheral blood irrespective of maternal symptoms. There was no presumptive treatment of malaria or chemoprophylaxis. Women were treated according to the WHO malaria guidelines. In this analysis, women were counted as malaria positive when they had at least one documented malaria infection in pregnancy.Malaria was diagnosed by Giemsa stained thick and thin blood films; 200 fields on the thick film were read before being declared negative. Malaria infection was defined by the presence of asexual stages of Plasmodium  using an established formula +6 weeks) using an Asian formula thcentile for the gestational age (or birth weight z-score less than 1.28 for a given GA using a modeled BW/relationship as described below).GA was expressed in weeks (X) and number of days (+y): X+0 to 41+6 weeks, and whose newborn was weighed within the first hour after birth.The cohort included women who attended the SMRU ANC between 2001 and 2010, had a CRL or BPD measurement before 24 weeks, delivered a live-born, congenitally normal singleton infant at SMRU delivery rooms, with a gestation within 28Clinical data and the results of the ultrasound scans were entered into a Microsoft Access database. Using R software version 2·15·2, fractional polynomial regression analysis The z-scores obtained this way should be approximately normally distributed and this was assessed by visual inspection of normal probability plots. As these z-scores have been adjusted for GA, a plot of z-scores versus gestational age should be randomly scattered around zero with approximately 95% of the values lying between the limits ±1·96.Centile curves were obtained using the equation:+6 weeks , and 51·4%  of the babies were male. Women were screened for malaria by microscopy a median of 22 (IQR 16–28) [range 1–38] times. Malaria infection was detected in 12·6%  of pregnancies. Overall, 7·7%  of women in this birth cohort had a PTB. The overall mean birthweight was 2953 (SD 454) g; 2906 (SD 434) g for girls and 2998 (SD 468) g for boys.10,264 pregnant women attended the SMRU ANC between 2001 and 2010 and met the inclusion criteria . They haIn 65% (6672/10264) of the women the GA was determined by CRL and in the remaining 35%  of the cohort by BPD. The percentage of women with malaria was similar in these groups . The difference in the mean GA at delivery between the two groups was small (273 days in the CRL group and 274 days in the BPD group) so all women were included in the analysis. The best fitting curves for the birthweight mean and standard deviation were found to be a two-term fractional polynomial with powers  and a one-term fractional polynomial with power 1  respectively. The equations to obtain the fitted values (GA in days) were as follows:The fitted values for mean birthweight and standard deviation were substituted into th centile so is not SGA, whereas the same birthweight (2500 g) at 41 weeks gestation has a z-score of −1·846 lying on the 3·2th centile and therefore is classified as SGA. The importance of accurate estimation of GA is highlighted by the following example. A baby born at week 35 weighing 2100 gram is premature but not SGA. In contrast a baby born with the same weight (2100 g) at week 38 is not premature but is SGA.For a new observation within the range of the GA of this population, the expected mean birthweight and standard deviation can be calculated using We used these fitted models to identify SGA births in the current population. The proportions of LBW, PTB, and SGA were significantly higher for pregnancies with malaria compared to those without malaria, although the risk difference for PTB was low . The risThe percentages of SGA and LBW by malaria infection in pregnancy for each week of GA are summarized in Regression models were used to explore the association of birthweight and SGA births with malaria, primigravida status, hypertension, smoking, age, body mass index (BMI) of the mother and the sex of the baby. Malaria during pregnancy, primigravida, hypertension, smoking and female sex of the newborn were found to be associated with lower birthweight  and SGA In this large cohort of pregnant women living in a malarious area, GA was obtained by ultrasound and malaria episodes were detected and treated by frequent screening. As expected malaria infection was a significant risk factor for LBW but also for SGA and to a much lesser extent for PTB . Other kInfants born preterm and SGA rather than only LBW have the highest risk of mortality in low-income and middle income countries The marginally higher risk difference (www.interbio21.org.uk/) A limitation of using SGA is that it still encompasses neonates that are constitutionally small and those that are pathologically small due to fetal growth restriction P.falciparum and P.vivax), GA at time of infection, symptoms and severity at the time of treatment and repeated episodes of malaria infections is in preparation. In this analysis most of the recommendations for birthweight analysis This prospective observational cohort study has some limitations. Due to the long study period subsequent pregnancies of a woman may have been included in the database. While the birth outcomes of a single woman  may have biased the results the sample size of this cohort is large and the bias small. Despite the overall size of the cohort the number of severely premature infants remained small and significant effects of malaria on the length of gestation prior to 34 weeks were not observed. More detailed analysis and modeling of data to describe the relative contribution to SGA from the two dominant malaria species  percentiles per completed gestational week for the refugee and migrant population on the Thai-Myanmar border.(DOCX)Click here for additional data file.


In Kinshasa, malaria remains a major public health problem but its spatial epidemiology has not been assessed for decades now. The city’s growth and transformation, as well as recent control measures, call for an update. To identify highly exposed communities and areas where control measures are less critically needed, detailed risk maps are required to target control and optimize resource allocation.In 2009 (end of the dry season) and 2011 (end of the rainy season), two cross-sectional surveys were conducted in Kinshasa to determine malaria prevalence, anaemia, history of fever, bed net ownership and use among children 6–59 months. Geo-referenced data for key parameters were mapped at the level of the health area (HA) by means of a geographic information system (GIS).Among 7517 children aged 6–59 months from 33 health zones (HZs), 6661 (3319 in 2009 and 3342 in 2011) were tested for both malaria (by Rapid Diagnostic Tests) and anaemia, and 856 (845 in 2009 and 11 in 2011) were tested for anaemia only. Fifteen HZs were sampled in 2009, 25 in 2011, with seven HZs sampled in both surveys. Mean prevalence for malaria and anaemia was 6.4 % (5.6–7.4) and 65.1 % (63.7–66.6) in 2009, and 17.0 % (15.7–18.3) and 64.2 % (62.6–65.9) in 2011. In two HZs sampled in both surveys, malaria prevalence was 14.1 % and 26.8 % in Selembao (peri-urban), in the 2009 dry season and 2011 rainy season respectively, and it was 1.0 % and 0.8 % in Ngiri Ngiri (urban). History of fever during the preceding two weeks was 13.2 % (12.5–14.3) and 22.3 % (20.8–23.4) in 2009 and 2011. Household ownership of at least one insecticide-treated net (ITN) was 78.7 % (77.4–80.0) and 65.0 % (63.7–66.3) at both time points, while use was 57.7 % (56.0–59.9) and 45.0 % (43.6–46.8), respectively.This study presents the first malaria risk map of Kinshasa, a mega city of roughly 10 million inhabitants and located in a highly endemic malaria zone. Prevalence of malaria, anaemia and reported fever was lower in urban areas, whereas low coverage of ITN and sub-optimal net use were frequent in peri-urban areas. Anopheles gambiae, which was less present in the city centre than in the periphery [Malaria is the leading cause of morbidity and death in children under 5 years in the Democratic Republic of Congo (DRC), accounting for an estimated 40 % of outpatient visits and 40 % of overall mortality . Malariaeriphery , 7. The eriphery .Programme d’Urgence de Réhabilitation Urbaine et Sociale). The National Malaria Control Programme (NMCP), along with technical and logistic support from Population Services International (PSI), distributed two ITNs per household. Eight months after that distribution, the Kinshasa School of Public Health (KSPH) conducted a survey on basic malaria indicators to assess the impact of the intervention in 15 health zones (HZs) of the city. In 2011, the Swiss Tropical and Public Health Institute (Swiss TPH), in collaboration with the KSPH, conducted a second survey to evaluate the coverage and use of key malaria indicators, prevalence of malaria by Rapid Diagnostic Test (RDT), anaemia and fever in the 23 HZs excluded from the 2009 survey. Kinshasa has expanded very rapidly in the past 20 years, thus updating and consolidating these data was urgently required for general malaria control purposes and for planning specific further research projects. Using geo-referenced prevalence data, this study aimed to generate the first map of malaria risk among children 6–59 months in Greater Kinshasa, down to the lowest level of the health system in DRC, the health area (HA). These maps will enable researchers and implementers to identify HZs of high priority for malaria control in Kinshasa.A first insecticide-treated net (ITN) distribution campaign in 2007 achieved a 15.9 % rate of ITN ownership and a 12.6 % rate of use among children under five [ville), some of which represent the most industrialized and commercial areas. To the south lies the cité, consisting of more recent, large, residential districts. The land use pattern is heterogeneous, with densely populated areas separated by large semi-rural areas where urban agriculture is practiced. The most heavily inhabited area of Kinshasa covers 583 square km [The study was conducted in Kinshasa, the capital of the DRC. The city is located along the southern bank of the Congo River, directly opposite the city of Brazzaville, capital of the Republic of the Congo. The climate is hot and humid (AW4 according to the Koppen classification), with a rainy season lasting from October to May . Charactquare km , of whiccommunes . The organization of the health system differs from the administrative system and comprises six health districts, divided into 35 health zones (HZs). These represent the primary operational units of the health system in DRC. An HZ usually covers a population of 100,000–150,000 inhabitants in rural areas and 200,000–250,000 in urban centres. They include a general referral hospital, some health centres and a dozen lower-level health facilities. Each HZ is further divided into 15 health areas (HAs), on average, which represent the lowest level of the health system. Each HA is clearly delimited and defined by the Ministry of Health and usually includes a population of 10,000–15,000 inhabitants. In Kinshasa Province, the three most eastern HZs are completely rural in nature, while the remaining 32 HZs are semi-rural or urbanized [Administratively, Kinshasa has the status of a province, divided into four districts, which are further divided into 24 rbanized . The stuTwo cross-sectional household surveys were conducted. The first survey was carried out at the end of the dry season between mid-September and end of October 2009 in 15 HZs, eight months after the first large ITN distribution campaign. The second survey was conducted at the end of the rainy season from mid-April to early June 2011 and covered 25 non-rural HZs. Seven HZs were sampled in both studies, including five HZs for which malaria prevalence was not measured in 2009 and two HZs for which prevalence was measured previously in 2009. The detailed list of HZs surveyed in 2009 and 2011 is presented in Table Fifteen HZs were selected using a probability proportional to size (PPS) sampling method, so that more populated HZs had a higher probability of being selected. Of these 15 HZs, 10 were selected by simple random sampling for the determination of malaria by rapid diagnostic test (RDT). In the remaining five HZs, only haemoglobin (Hb) was measured and malaria preventive measures were investigated using a pre-tested, structured questionnaire. In each HZ, data collection took place in half of the HAs, selected again with PPS. In case of an odd number of HAs per HZ, (n + 1)/2 HAs were selected. In a third stage, a list of all streets with their approximate population number was obtained for each selected HA. Streets with fewer than 200 inhabitants were excluded and three streets were selected by simple random sampling. Households with at least one child aged 6–59 months were listed by community health workers (CHW) for each of the three streets. From this list, 25 eligible households were randomly selected, proportional to the size of each street. The target sample size of 325 children was calculated based on an estimated prevalence of anaemia of 69.2 % in children aged 6–59 months. Assuming 1.3 children aged 6–59 months per household, a sample size of 260 households was set as the target per health zone [From mid-April to early June 2011, the remaining 23 HZs were sampled, including the five HZs for which malaria prevalence had not been measured in the 2009 survey. In all 23 HZs, a questionnaire was administered to households and malaria parasite prevalence and the Hb concentration were measured in children aged 6–59 months. Two additional HZs already investigated in 2009 were re-sampled in 2011 among children 6–59 months for both malaria and anaemia. To obtain the epidemiological age profile for all age groups in these latter HZs only, individuals older than five were also included. In all, 25 HZs were sampled in the 2011 survey. The primary outcome measure was documented malaria in study children, as measured by RDT. The sample size was calculated based on the prevalence estimate for 2009 survey (6.4 %) during the dry season, and increased to 10 % to take into account the seasonal variation. In each HZ, the aim was to measure children’s malaria with a precision of ±8 absolute percent. The sample size calculation indicated the need for 55 children in each HZ. With a design effect accounting for clustering of two, this number increased to 110. With an average 1.3 children under 5 years in households in Kinshasa, 87 households needed to be selected [lingua franca in Kinshasa) and field tested prior to the survey.In 2009, survey data were collected using a paper-based questionnaire. In 2011, survey data were collected using smartphone technology. For the 2011 survey, a validated electronic semi-quantitative questionnaire was developed on an HTC smartphone running Google’s Android operating system. Eight teams of three field workers  were trained in using the electronic questionnaire, in general interviewing skills and in administering informed consent during simulated interviews sessions. Each of the eight teams visited, on average, 25 households per day in each selected HA. The 2011 questionnaire was a simplified version of the one used in 2009, which was adapted from the standard Malaria Indicator Survey Household Questionnaire from the Roll Back Malaria Partnership. All questions retained from the 2009 survey form were kept as they were in 2011 to ensure comparability between both surveys. The questionnaire was developed in French with oral translation into Lingala , use of mosquito bed net and ITN in the night prior to the survey, history of fever (past 2 weeks), whether fever was present on the day of the survey and health seeking behaviour in case of a fever episode. During the 2011 survey, the coordinates (longitude and latitude) of all investigated households were recorded on-site using the integrated Global Positioning System (GPS) of the data collection devices. Households were revisited if no one was available for interview on the first attempt; if no one was available after two attempts, the interviewer continued to the next randomly selected household on the list until the desired number of households was obtained.Plasmodium falciparum-specific histidine rich protein 2 (HRP2) and other Plasmodium species   and assessing Hb level using a blood haemoglobin photometer . In two HZs in 2011, Selembao and Ngiri Ngiri, individuals of all ages (not only children) were surveyed. RDTs were used for on–site diagnosis of malaria and treatment with artesunate-amodiaquine, the official first-line malaria treatment at the time of the survey, was offered as needed. The HemoCue was validated by running a weekly high and low Hb liquid control (HemoCue–HemoTrol).For each selected participant who gave signed informed consent, the same laboratory procedures as in 2009 were adopted during the 2011 survey. They included measuring axillary temperature with a digital thermometer, collecting peripheral blood by standard finger prick to test for malaria parasites with an RDT for To ensure consistency and integrity of data collected during the 2009 survey, all paper forms were rechecked by team supervisors in the field at the end of each day. Incomplete entries were sent back to be filled the next day. Questionnaires were first checked for completeness, and the information was manually coded and entered using EpiData and crosschecked using EpiInfo (v. 6.04). Statistical analyses were performed using SPSS software for Windows (version 16.0), NCSS, and STATA (version 10).Data collection devices used in the 2011 survey (HTC phones) were equipped with Open Data Kit (ODK) software (University of Washington and Google Foundation) to allow for data entry in the field. ODK programming also allowed for systematic range and consistency checks. Data in xml format were downloaded every evening from the HTC smartphones and then converted on the ODK Aggregate Server into tabular format (ODK aggregate).Institut géographique du Congo (IGC), it was possible to develop shape files at the level of the HA. The most eastern rural HZs  were excluded from the final map due to the great effort that drawing boundaries in remote HAs would have entailed. This was beyond the means and the scope of this study.Statistical analyses were performed using Stata version 12.1 . Analysis and mapping for the 2011 survey were based on geo-referenced prevalence data at the level of the HA. Since households could not be georeferenced in 2009, HA spatial coordinates were assigned to the HA’s mean malaria prevalence. Maps were produced using ArcGIS version 10.0 . A centroid for every HA was first generated . The standardized prevalence data were then assigned to the centroids of the surveyed health zone. The next step involved using the IDW interpolation to get prevalence estimates at un-surveyed HZs. Lastly, the interpolated prevalence estimates were extracted using the centroids (points data) of the HZs. These estimates were subsequently used to map out the prevalence at HZs level (polygons data). Boundaries (shape files) were initially available at the level of the HZ only, from the Health Monitoring Information System Unit of the Ministry of Health (MoH). By using images developed by the Japan International Cooperation Agency (JICA) and through collaborating with a team of experts from the For both surveys, ethical clearance was obtained from the Ethics Committee of the KSPH, at the University of Kinshasa. In addition, the 2011 survey received authorization from the ethical committees in Basel  as well as clearance from Swiss TPH’s internal research commission. Signed informed consent to participate was obtained from parents or guardians on behalf of the enrolled children or by the adult participants themselves. Precautions to minimize the risk of secondary infection during blood collection were taken. All tested participants with a positive RDT but no evidence of severe illness were diagnosed as having uncomplicated malaria and given a voucher for treatment, free of charge, as per the DRC national malaria treatment policy (artesunate-amodiaquine or artemether-lumefantrine), at the nearest health facility. Drugs were provided to the relevant facilities one day before the household visits started in the area, to ensure drug availability for treatment. Participants diagnosed with severe anaemia and those with severe illnesses were excluded from the study and immediately referred to the nearest health facility for diagnosis and management, as recommended by national guidelines.Household and individual characteristics of the study populations in 2009 and 2011 are shown in Table Plasmodium falciparum accounted for 52 % (95 % CI 47.4–55.8) of infections in 2011 survey, non-falciparum infections for 0.3 % (95 % CI 0.0–1.3) while mixed infection (were not distinguished) prevalence was 48 % (95 % CI 43.9–52.3).Table In the two HZs sampled in both 2009 and 2011, prevalence of malaria in children aged 6–59 months was 1.0 % (0.3–2.6) and 0.8 % (0.0–4.2) in Ngiri Ngiri, and 14.1 % (10.6–18.2) and 26.8 % (19.9–34.7) in Selembao. Age-specific rates , p2 is the prevalence rate in survey 2 (2011), n1 is the number of study participants in survey 1, n2 is the number of study participants in survey 2, To prepare the data for mapping, direct standardization was used to make prevalence rates of malaria comparable between surveys, accounting for the two different years and seasons. The standardization was done according to the following formula:P. falciparum from the 2009 and 2011 surveys, at the level of the HA. Interpolated standardized prevalence rates are presented in Fig. Results from the two surveys were used to produce a representative and standardized map of risk for malaria in children aged 6–59 months. Figure A total of 4164 and 3353 children aged 6–59 months were tested for anaemia in the 2009 and 2011 surveys, respectively. The mean prevalence of anaemia (Hb < 11 g/dl) was similar between surveys: 65.1 % (63.7–66.6) in 2009 and 64.2 % (62.6–65.9) in 2011. Results also show that the prevalence of moderate (7.0–9.9 g/dl) and severe (<7.0 g/dl) anaemia was 34.2 % and 1.9 % in 2009, and 30.1 % and 1.9 % in 2011 (Table The proportion of children aged 6–59 months reporting a history of fever in the 2 weeks preceding the survey was 13.2 % (12.5–14.3) in 2009 and 22.3 % (20.8–23.4) in 2011. On the day of the 2011 survey (data not available for 2009), 3.2 % (106/3348) were febrile (defined as temperature >37.5 °C). The positive predictive value (PPV) of history of fever among children with a positive RDT was 29.7 % (26.3–32.7) in 2011. Health seeking behaviour in case of fever was high in 2011 (data not available for 2009): overall, 91.4 % (770/842) of children sought some type of care. In all, 53.9 % sought modern treatment at home by a family member, whereas 36.1 % were taken to a health facility. Very few (0.5 %) made use of traditional medicine. Private facilities were the most common provider of treatment among those who sought care outside the home, covering 65.4 % of the cases, whereas 22.9 % consulted a public facility and 11.8 % consulted a confessional structure. In case of home treatment, drug outlets represented the principal source of treatment (96.3 %). Unfortunately, only 4.3 % of the anti-malarials purchased were the recommended combination of artesunate-amodiaquine. As a result, only 3.6 % received the recommended treatment at home within the 24 h. In 66.5 % of fever cases, treatment was sought within 24 h regardless of whether treatment was recommended or not.Eight months after the 2009 distribution campaign, ITN coverage (measured by the possession of at least one ITN per household) reached 78.7 % 77.4–80.0). In 2011, it was 57.7 % (56.0–59.9). In 2009, coverage ranged from 51.7 % in Biyela (peri-urban) to 92.7 % in Maluku II (peri-urban) , either because they were absent  during the campaign or because the stock had been sold out (3.8 and 18.8 %). A high proportion of respondents reported having discarded or destroyed their ITN because of rumours (7.4 and 23.8 %). Other reasons given were heat (2.4 and 10.6 %) and the absence of mosquitoes at home (9.4 % in 2009).The proportion of respondents who reported that their child slept under an ITN the night before the survey decreased from 65.8 % (63.5–66.0) in 2009 to 45.0 % (43.6–46.8) in 2011. Figures A higher proportion of pregnant women, 83.1 % (77.5–87.7), reported using an ITN in 2009, than in 2011, were reported use decreased to 43.1 % (37.5–48.9). Again, the HZs on the outskirts of the city showed the lowest use rates.P.falciparum entomological inoculation rates (APfEIR) has been observed in the more urbanized central areas, with a tendency to increase gradually towards the peri-urban areas [Kinshasa falls within the perennial transmission in the classification of the Mapping Malaria Risk in Africa (MARA) project . This stan areas –19. The an areas , 7, 20. an areas .Anopheles gambiae, the main local vector [Anopheles breeding sites through elimination and pollution. However, exceptions exist, especially where urban agriculture and gardens persist. In particular, the districts extending towards the south-east and west maintain a semi-rural character. Various studies have documented the presence of higher prevalence or transmission rates in areas close to agriculture fields [boulevard Lumumba, encompassing the health zones of Kingasani, Biyela, Kimbanseke and Kikimi. The areas favour Anopheles sp. breeding sites and are consistent with the more homogeneous transmission pattern observed in the areas on the outskirts of the city, compared to the more urbanized zones.In this study, the overall standardized malaria prevalence was 11.9 % in children 6–59 months, ranging from 0.5 % in the downtown health zones of Kinshasa and Lingwala, to 31.7 % in Biyela, a semi-rural peripheral district extending south east. Results also show that spatial heterogeneity is high in the central and northern urbanized HZs, whereas in the western and south-eastern HZs, more homogeneous levels of high risk can be found Fig. . It is ll vector . In a see fields , 25–29. Additional factors, such as the use of personal protection against mosquitoes or socio-economic status, should also be considered as important determinants explaining the distribution of disease prevalence. A spatial regression analysis linking malaria prevalence to risk factors for malaria in Kinshasa will be published separately .Not surprisingly, the age groups with the highest prevalence, independent from the level of endemicity in both urban and semi-rural areas (Ngiri Ngiri and Selembao), were those aged 5–9 and 15–19. Hence, in Kinshasa, malaria infections seem to occur more frequently late in childhood. This could be in part explained by the age specific ITN usage across age groups, with highest use in younger children in the low endemicity setting (percent of usage in Ngiri Ngiri 49 % compared to 28 % in Selembao) as compared to lower and similar utilization rates among age groups in the high transmission setting . Higher malaria prevalence rates in older children were also found in school surveys carried out in the 1980s in Kinshasa and in Brazzaville. At that time, the finding was attributed to the increased use of anti-malarials in early childhood , 30.A concerted effort to scale-up ITN coverage through a free distribution in Kinshasa led to an ITN ownership rate of 78.7 % of households in 2009. This represented a 395 % increase in household possession of ≥1 ITN over the 2007 estimate of only 15.9 % .The prevalence of anaemia was high in 2009 (65.1 % in children 6–59 months) and in 2011 64.2 %). This is consistent with the 69.2 % prevalence reported by the DHS 2007 .2 %. Thi. In KinsThe maps we present for different variables reflect survey results from two distinct time periods and seasons. Since malaria transmission is neither constant throughout the year nor between years, this has likely introduced some mistakes. To account for these differences, we tried standardizing prevalence rates, but that is an imperfect means of accounting for such differences. Moreover, surveys were based on detection of cases of uncomplicated malaria and, therefore, it is not possible to draw strong conclusions about the prevalence of anaemia, which is more often related to severe malaria.This study provides the first comprehensive risk map of malaria at the level of the health areas in Kinshasa, a mega-city in a highly endemic malarious zone. Overall malaria prevalence has undoubtedly decreased over the last 30 years, but it is impossible to quantify the effect given the lack of representative historical data. As expected, prevalence rates were lower in the central urban districts compared to the more peripheral and more rural districts . The pen


Plasmodium falciparum parasites and the sequestration of parasite-infected RBCs in the microvasculature are mediated in part by complement receptor one (CR1). RBC surface CR1 level can vary between individuals by more than 20-fold and may be associated with the risk of severe malaria. The factors that influence RBC CR1 level variation are poorly understood, particularly in African populations. We studied 3535 child residents of a malaria-endemic region of coastal Kenya and report, for the first time, that the CR1 Knops blood group alleles Sl2 and McCb, and homozygous HbSS are positively associated with RBC CR1 level. Sickle cell trait and ABO blood group did not influence RBC CR1 level. We also confirm the previous observation that α+thalassaemia is associated with reduced RBC CR1 level, possibly due to small RBC volume, and that age-related changes in RBC CR1 expression occur throughout childhood. RBC CR1 level in malaria-endemic African populations is a complex phenotype influenced by multiple factors that should be taken into account in the design and interpretation of future studies on CR1 and malaria susceptibility.Both the invasion of red blood cells (RBCs) by  Plasmodium falciparum is the most common cause of malaria world wide and is responsible for the vast majority of malaria deaths.1P. falciparum exploits multiple host receptors during the process of red blood cell (RBC) invasion and in the subsequent sequestration of P. falciparum parasitised RBCs in the microvasculature.2 Understanding these host–parasite receptor–ligand interactions may help in the development of new methods for preventing and treating malaria.2 Complement receptor one (CR1) is a single-chain trans-membrane glycoprotein that is expressed on the surface of RBCs and leukocytes. The normal function of RBC CR1 is to regulate complement activation and to transfer immune complexes to phagocytic cells in the liver and spleen.3 In malaria, CR1 acts as a receptor on RBCs for both P. falciparum invasion7 and rosette formation, in which two or more uninfected RBCs bind to a P. falciparum parasitised RBC in vitro.9 Rosetting has been associated with severe malaria in multiple African study sites , potentially because it leads to enhanced parasitised RBC sequestration and decreased blood flow in post-capillary venules.12 CR1 acts as a receptor for P. falciparum RBC invasion via its interaction with the parasite ligand P. falciparum reticulocyte-binding-like homologue protein 4.78 and negative or weak reactions with CR1 antibodies,13 being most marked at <100 CR1 molecules per cell. Low RBC CR1 level has also been associated with reduced P. falciparum RBC invasion in vitro.6 Individuals with low RBC CR1 are common in some malaria-endemic regions,15 with up to 80% of the population in coastal Papua New Guinea having fewer than 200 CR1 molecules per RBC.14 Variation in RBC CR1 level may contribute to individual differences in susceptibility to severe malaria. Low RBC CR1 might protect against cerebral malaria through the mechanism of reduced rosetting,14 whereas, in contrast, increasing the risk of immune-complex-mediated tissue damage and severe malarial anaemia.17The mean number of CR1 molecules expressed on the RBC surface varies between individuals, typically between 50 and 1500 CR1 molecules/RBC. RBC with <200 CR1 molecules per cell show reduced rosetting18 In Caucasian and Asian populations, variation in RBC CR1 level is genetically determined and is associated with a HindIII restriction fragment length polymorphism that is in tight linkage disequilibrium with several other single-nucleotide polymorphisms (SNPs) in exons 19, 22 and 33 of the CR1 gene.22 However, in African populations, although RBC CR1 levels vary over the same range, there is no association between CR1 level and any of the above SNPs, and the causal mutations that determine RBC CR1 levels are unknown.23CR1 is also the site for SNPs giving rise to the Knops blood group system of antigens that include the allelic pairs Swain-Langley 1 and 2 (Sl1 and Sl2), McCoy a and b (McCa and McCb) and Knops a and b (Kna and Knb).27Sl2 and McCb occur at higher frequencies in African populations than non-African populations.28 To the best of our knowledge, the effect of the Knops Sl and McC genotypes on RBC CR1 level has not been studied previously.Factors determining the variation in RBC CR1 expression are not fully understood, but are proposed to be both genetic and acquired.+thalassaemia,14 an association that might exist in African populations where α+thalassaemia is also common. It is unknown whether other high-frequency African polymorphisms associated with malaria resistance such as HbS and blood group O might influence RBC CR1 level. Two previous studies have shown that RBC CR1 expression level varies with age in both malaria-endemic and non-endemic populations, being lowest during early childhood.29One study in Papua New Guinea found a dose-dependent decrease in RBC CR1 level with αIn the current study, we investigated RBC CR1 level and the factors that influence it in children living within a malaria-endemic region on the Kenyan coast.29 and this relationship was confirmed in the Kilifi population (29 and found that mean RBC CR1 level was higher in newborns (0–3 months) than in other age groups, and significantly declined during each age interval in the first two years of life =323–345), both heterozygous and homozygous α+thalassaemia were associated with significantly lower RBC CR1 levels:  and , respectively  compared with normal cells. We therefore examined whether reduced MCV could explain the lower CR1 levels seen with α+thalassaemia. One hundred and forty-seven RBC samples from the Mild Disease Cohort included in the above analyses had MCV measurements available. As expected, α+thalassaemia was associated with reduced MCV in both the heterozygous  and homozygous state  when compared with normal RBCs . As seen in the larger data set, unadjusted for MCV, RBC CR1 levels declined with α+thalassaemia in the 147 samples: CR1 level in heterozygous  and homozygous  α+thalassaemic RBCs compared with normal RBCs  and homozygous α+thalassaemic RBCs  was no longer statistically significant , homozygous sickle cell disease (HbSS) was associated with significantly elevated RBC CR1 level  in comparison to normal HbAA RBCs it HbAS;  or ABO bit HbAS; . AlthougSl2 and McCb alleles are present at high frequencies in African populations,28 but it is unknown whether they influence RBC CR1 level. We found that homozygous Sl2 genotype was associated with an increased RBC CR1 level  and , respectively, in comparison to homozygous McCa , when compared with the homozygous Sl1-McCa , CR1-B (250 kDa), CR1-C (190 kDa) and CR1-D (280 kDa). These differences are a result of unequal crossing-over leading to duplications or deletions of a long homologous repeat (LHR) unit, with CR1-A, B, C and D consisting of 4, 5, 3 and 6 LHR's, respectively. CR1-A and B are the most common, occurring at frequencies of between 75–98% and 2–13%, respectively, whereas CR1-C and D are rare.P. falciparum shows a significant positive correlation between RBC CR1 level and binding of the parasite invasion ligand P. falciparum reticulocyte-binding-like homologue protein 4ref.  and parasite invasion rate,5 suggesting that relatively small increases in RBC CR1 level may have the potential to increase invasion efficiency and increase overall parasite burden. Previous work on P. falciparum rosetting suggests that very low levels of RBC CR1 (less than 100 molecules per cell) reduce rosetting,9 but there is no evidence for a direct positive correlation between increasing amounts of CR1 and increasing rosetting. Instead, it seems more likely that there is a threshold effect, such that cells with less than 100–200 molecules per cell show reduced rosetting, whereas those with more than 100–200 molecules per cell show similar rosetting capacity over the full range of CR1 levels (200–1500 molecules per cell). The overall levels of CR1 measured in this study are relatively low, with mean values between 250 and 350 molecules per RBC for most age groups and genotypes .One unexplained contradiction in our data is that low CR1 is thought to protect against severe malaria,1 levels  and 6. T+thalassaemia and age are associated with RBC CR1 level in Kenyan children. The exact mechanisms by which levels of RBC CR1 are controlled remain unknown, but variation in RBC CR1 might be partly explained by differences in haematological indices such as MCV or by other complex processes including susceptibility to proteases and RBC vesiculation that are potential areas for future study.In summary, we have shown that a number of host factors including Knops blood group genotype, α60 The study involved a total of 3535 individuals aged 0 months to 13 years of age recruited into three different study cohorts, each of which have been previously described: the Kilifi Genetic Birth Cohort ,61 Birth Cohort Study 2 63 and Mild Disease Cohort .65 Following informed consent from parents or guardians, blood samples were collected by either venipuncture or by capillary sampling into sterile tubes containing EDTA . Because CR1 levels are known to decline during malaria infections,16 samples were only collected from malaria negative individuals, as tested by a rapid diagnostic test  and by light microscopy of thick and thin Giemsa-stained blood smears.All samples used in this study were collected from residents of the area served by the Kilifi Health and Demographic Surveillance System (KHDSS) on the coast of Kenya. This area and the resident population have been described in detail previously.M HEPES, 2 mM  l-glutamine (Invitrogen), 25 μg ml−1 gentamicin, 20 mMd-glucose (Sigma) and 6 mM NaOH) by centrifugation and plasma and white blood cells removed from the RBC pellet by aspiration. RBC surface CR1 level was quantified by flow cytometry on formaldehyde-fixed RBC samples, as described in detail previously.66 Previous data indicated that CR1 levels determined on formaldehyde-fixed RBCs are equivalent to those determined using fresh RBCs, as long as the standards (with known CR1 levels) are fixed and stored in an identical way.66 Fixed cells were stored at 4 °C and CR1 quantified within 8 weeks. Briefly, fixed RBC pellets were stained in duplicate with 0.5 μg ml−1 of mouse anti-CR1 monoclonal antibody J3D3  followed by a secondary staining with 5 μg ml−1 of Alexa Fluor 488-conjugated goat anti-mouse IgG  and the mean fluorescence intensity determined on a FC500 flow cytometer (Beckman-Coulter Inc.). CR1 levels of unknown samples were then extrapolated from a standard curve generated using three RBC samples of known CR1 levels  included in each experiment, and prepared and stored in the same way as the test samples.66 Thirteen samples were collected but were not included in the study due aberrant forward/side scatter plots or readings that were >2 standard deviations above the highest point on the standard curve.Whole blood was washed thrice in supplemented RPMI-1640 medium . Genotyping for the Sl and McC blood group genotypes was carried out as described previously using the SEQUENOM MassARRAY platform  multiplex system following DNA amplification by whole-genome amplification.67 ABO blood groups were determined by either standard haemagglutination methods or were inferred from A, B and O SNP haplotypes determine by Sequenom typing as detailed previously.67 Genotyping for HbAS and α+thalassaemia was by PCR as described in detail elsewhere.69DNA was extracted either from fresh whole blood samples using the semi-automated ABI PRISM 6100 Nucleic acid prep station  or from EDTA blood samples, previously stored at −80 Full blood counts for the determination of RBC MCV were performed on fresh EDTA blood samples from the mild disease cohort using a Coulter MDII-18 machine (Beckman-Coulter Inc.).+thalassaemia genotypes, whereas ABO and the Sl and McC Knops genotypes were available for all samples except the 460 samples from the Birth Cohort Study 2. MCV data were only available for the 147 samples collected from members of the Mild Disease Cohort Study.A total of 3535 samples were successfully typed for RBC CR1 level, HbS and αpost-hoc test to correct for multiple comparisons. We also analysed the relationship between RBC CR1 level and age as a continuous variable using piecewise-linear regression analysis allowing for adjustment for confounding by α+thalassaemia and HbS. Unlike conventional linear models that assume linearity over the whole range of a continuous independent variable, this model assumes a linear effect within defined ranges of the continuous variable that are connected. The age categories and ranges chosen were 0–3 months, 4–6 months, 7–12 months, 13–24 months, >2–4 years, >4–8 years and >8–13 years, as used in previous studies.29 The relationship between RBC CR1 level and α+thalassaemia, HbS, ABO blood group and the Sl and McC genotypes was tested by multivariate linear regression analysis. Each variable was first tested in a univariate regression model and all explanatory variables displaying a P value of <0.05  were included in a final multivariate model. Age as a categorical variable was also included in the multivariate model, using the age intervals described above. ABO blood group and Sl and McC genotypes were not available for the 460 children from Birth Cohort Study 2, therefore the multivariate analyses are restricted to the 3075 samples with full genotype data. For ease of interpretation, predicted mean CR1 levels and 95% CIs estimated from the multivariate regression results are presented in figures, rather than regression coefficients, using the ‘margins' post-estimation command in Stata. A two-sided significance level of <0.05 was considered statistically significant for all analyses.Statistical analyses were conducted using STATA v11  and Graph Pad Prism v5  and all graphs were generated using GraphPad Prism. The relationship between RBC CR1 level and age as a categorical variable was assessed using one-way analysis of variance with Tukey's Individual informed written consent to collect blood was obtained from all participants' parents or legal guardians. The study was approved by the Kenya Medical Research Institute National Ethical Review Committee in Nairobi and was conducted in accordance with the Declaration of Helsinki.


ICAM-1). Polymorphisms in the gene encoding ICAM-1 influence the rate of progression or confer protection from or susceptibility to malaria parasite infection. The objective of our study was to evaluate the frequency of ICAM-1 SNP in exon 6  in the Indian population and compare it with the global population influenced by the parasitic infection.Plasmodium falciparum malaria is the most severe form of malaria causing morbidity and mortality worldwide. The key event in P. falciparum pathogenesis is the sequestration of the infected erythrocytes to the capillary endothelium, mediated by the receptor, Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 has distinct characteristics of binding to varied host ligands including Inter Cellular Adhesion Molecule (ICAM-1 SNP rs5498 was performed by Restriction Fragment Length Polymorphism (RFLP) PCR. Analysis of ICAM-1 variants was performed in 200 healthy subjects.The genotyping for The allelic frequency for the variant ICAM1-Aallele was 59% in healthy control subjects. Chi square analysis reveals that it does not follow the Hardy Weinberg Equilibrium. Comparison with literature data indicates that frequency distribution in Indian population is similar to German, Swedish and Danish populations while it is significantly different from Finnish and Japanese populations. The genotypic frequency of AA was 35%, AG was 49% and GG was 16% in healthy control subjects.ICAM-1 adhesion molecule in the falciparum malaria pathogenesis in the Indian population. Upon comparison, a noteworthy difference in the genotype frequency of Nigerian children (p<0.001) was evinced. This data can be useful for predicting the potential risk of malaria in world population.The frequency data from the above study will help in understanding the role of genetic variations in


Plasmodium vivax malaria from across the globe have raised concerns among the scientific community. However, the risk of P. vivax resulting in complicated malaria and mortality is not as firmly established as it is with Plasmodium falciparum. This study was conducted to determine the severity proportion and factors associated with severity in cases of vivax and falciparum malaria.Mounting reports on severe P. vivax/P. falciparum infections from the year 2007-2011 were evaluated based on their hospital records. Severe malaria was defined as per the World Health Organization’s guidelines. Comparison was made across species and binary logistic regression was used to determine risk factors of severity.Adult patients microscopically diagnosed to have P. vivax was the largest  60.3-66.5%) infecting species, followed by P. falciparum  and their mixed infection . Severity in P. vivax and P. falciparum was noted to be 16.9% (95% CI 13.9-19.9%) and 36.3% (95% CI 31.0-41.6%) respectively. Plasmodium falciparum had significantly higher odds  of severe malaria than P. vivax. Rising respiratory rate [1.29 (1.15-1.46)], falling systolic blood pressure [0.96 (0.93-0.99)], leucocytosis [12.87 (1.43-115.93)] and haematuria [59.36 (13.51-260.81)] were the independent predictors of severity in P. vivax. Increasing parasite index [2.97 (1.11-7.98)] alone was the independent predictor of severity in P. falciparum. Mortality in vivax and falciparum malaria was 0.34% (95% CI -0.13-0.81%) and 2.21% (95% CI 0.59-3.83%), respectively. Except hyperparasitaemia and shock, other complications were associated (P < 0.05) with mortality in falciparum malaria. Pulmonary oedema/acute respiratory distress syndrome was associated (P = 0.003) with mortality in vivax malaria. Retrospective design of this study possesses inherent limitations.Of 922 malaria cases included in the study, Plasmodium vivax does cause severe malaria and mortality in substantial proportion but results in much lesser amalgamations of multi-organ involvements than P. falciparum. Pulmonary oedema/acute respiratory distress syndrome in P. vivax infection could lead to mortality and therefore should be diagnosed and treated promptly. Mounting complications and its broadening spectrum in ‘not so benign’ P. vivax warrants global vigilance for any probable impositions.The online version of this article (doi:10.1186/1475-2875-13-304) contains supplementary material, which is available to authorized users. Plasmodium falciparum is entrenched to be associated with life-threatening complications, leading to widespread global annual mortality. There has been a gradual increase in reports describing sporadic atypical manifestations  of patients with complicated malaria had received empiric antibiotics than uncomplicated malaria [7.2% (50/696)].Until preliminary febrile investigations and culture reports were obtained, empiric antibiotics were administered to 6.6% (32/486), 8.4% (17/202) and 12.5% (1/8) uncomplicated cases and to 9.1% (9/99), 17.4% (20/115) and 25% (3/12) complicated cases of P. falciparum than P. vivax. Hyperparasitaemia was significantly (P = 0.002) associated with P. falciparum only. In P. vivax mortality was significantly associated with PE/ARDS (P = 0.003) and respiratory distress (P = 0.001). In P. falciparum except shock and hyperparasitaemia, mortality was significantly associated with all the other severity determinants , haemoglobinuria (P = 0.04) and renal failure P <0.001) was significantly higher in  was signP. vivax patients succumbed due to ARDS and respiratory distress. Out of seven P. falciparum patients who died, only one succumbed with single complication, i e, acute kidney injury despite dialysis. More than one complication resulted in mortality in other fatalities with P. falciparum. In both mixed malaria patients who died, more than one complication was associated with mortality ]. In P. vivax, increasing respiratory rate [1.29 (1.15-1.46)], falling systolic blood pressure [0.96 (0.93-0.99)], leucocytosis [12.87 (1.43-115.93)] and haematuria [59.36 (13.51-260.81)] were the independent predictors of complicated malaria. In P. falciparum, rising parasite index [2.97 (1.11-7.98)] was found to be the only independent predictor of complicated malaria ] and [7.44 (3.60-15.60)] and packed red cells [6.06 (1.49-24.75)] and [13.40 (4.84-37.11)] were significantly higher in complicated P. vivax and P. falciparum, respectively. Figure P. vivax and P. falciparum cohorts. Mortality occurred in 2% (2/99), 6.1% (7/115) and 16.7% (2/12) of severe P. vivax, P. falciparum and mixed infection, respectively. There was no difference in mortality either across complicated groups of all species (P = 0.06) or between P. vivax and P. falciparum (P = 0.18).Compared to uncomplicated cohort, odds of blood transfusion [9.80 4.71-20.37)] and [10.70 (5.77-19.82)], more than seven days of hospitalization [6.54 (3.83-11.18)] and [2.72 (1.70-4.38)], and prolonged duration of intensive care [4.19 (1.79-9.78)] and [1.91(1.47-2.48)] was more in both complicated 7] and [1P. vivax and P. falciparum. This study encompasses the full spectrum (except ‘prostration’) of severity determinants in accordance with the WHO criteria [The spectrum of severity and its determinants in adult vivax malaria in comparison with falciparum malaria has not been studied adequately. Also, the popular notion of vivax malaria having only ‘benign’ course of infection has been belied , 3–7, 11criteria . Althougcriteria , 13, thecriteria .P. vivax cohort. Absence of fever in a significant proportion of complicated P. falciparum might have caused delayed presentation, diagnosis and onset of anti-malarial treatment thereby resulting in complicated malaria. Proportion of complicated malaria across all species in the current study varied substantially in comparison to other series  of complications in comparison with both P. falciparum (up to seven) and mixed infections (up to six). There is paucity of literature describing a comparable spectrum of complications similar to the current study. Nadkar et al.[P. vivax and P. falciparum. Sarkar et al.[P. vivax. Mohapatra et al.[P. falciparum and mixed infections whereas as Tjitra et al.[P. vivax, P. falciparum and mixed infection. Nonetheless, of the existing studies, it is apparent that the spectrum of complications in malaria is highly variable across the globe. Further multicentric prospective study comprising subjects across the globe, encompassing a uniform and full spectrum of severity determinants, might lead to true realization in this regard.Table ortality  in P. faP. vivax, 15. EviP. vivax–20 is mor et al. have desr et al. have repa et al. have desa et al. have expP. falciparum. Minimal malaria mortality at the study hospital is on par with the best worldwide [The present study is in concurrence with those , 21, 22 orldwide , 21, 23.et al.[P. falciparum was found to be more likely to cause complicated malaria than P. vivax. Rising respiratory rate, falling systolic blood pressure, leucocytosis, and haematuria were independent predictors of severe P. vivax malaria. These findings can be used by clinicians as an index to decide intensive care need for the better management and outcome of malaria patients. Occurrence of leucocytosis is inconsistent in all forms of malaria and depends on factors viz. severity of disease, parasitaemia, host immunity and concomitant infections. However, none of the study patients were having bacteraemia or concurrent infections, as this was an exclusion criterion of this study. Thus, either bacteraemia or concurrent infection as a cause of leucocytosis is overruled. Furthermore, low and unstable transmission intensity of malaria in the catchment area of the hospital might be responsible for non-immune status of patients having clinical malaria thus; vigorous inflammatory response might have resulted in baseline leucocytosis. This could be better understood by exploring parasite virulence/immunogenic differences as well as human host specific immune competence between severe and non-severe cohorts. Rising parasite index alone was found to be an independent predictor of severe P. falciparum. However, parasite biomass has been advocated to be the superior marker of severity than parasite index due to massive sequestration in P. falciparum[P. falciparum. These differing independent predictors of severity could indicate underlying pathophysiological differences between severe P. vivax and P. falciparum infections.In contrast with Tjitra et al., P. falclciparum. InteresP. vivax and P. falciparum malaria in adult cohort. The study cohort is substantially large comprising >60% P. vivax, thereby provides a robust comparison with P. falciparum to challenge the ‘benign’ tag with P. vivax. Determination of clinico-laboratory parameters as risk factors for severity by multivariate logistic regression analysis provides simple, inexpensive and independent predictors of severity  which could be applied in routine clinical practice. Retrospective design of the study possesses inherent limitations. This study lacks confirmation of the infecting species by polymerase chain reaction. Due to retrospective study design any supportive evidence could not be brought out to the involvement of immune response resulting leucocytosis, thus this must be confirmed and validated through future prospective study design. Furthermore, coagulation parameters and venous bicarbonate levels were not tested in all patients as deemed redundant by the treating physicians. Contribution of pre-existing co-morbidities on disease severity could not be described due to lack of sufficient data; however, none of those expired had any co-morbid etiology. Data on temporal onset of various complications and their progression during illness were not captured. Thus, the pathophysiology of progression of disease from infection to multi-organ involvement to final outcome could not be discerned. Future studies must try to overcome the limitations of this study.The current study has remarkable strengths. This is a unique study describing the full spectrum of severity determinants in accordance with WHO contemporary criteria  among P.P. vivax does result in ‘severe malaria’ and mortality in considerable proportion, the probability of such is significantly smaller than P. falciparum. Furthermore, P. vivax results in much lesser amalgamations of multi-organ involvement than P. falciparum and mixed infections. Occurrence of PE/ARDS in P. vivax infection could lead to mortality, therefore should be diagnosed and treated promptly. Rising respiratory rate, falling systolic blood pressure, leucocytosis and haematuria were the predictors of severity in P. vivax, whereas increasing parasite index alone was the predictor of severity in P. falciparum. By virtue of higher prevalence of P. vivax and substantial proportion of broad spectrum severity as manifested in the current study and other previous series, it is imperative to be vigilant for any probable impositions by P. vivax in addition to P. falciparum.In adult malaria, the spectrum of severity may be extensively different among various populations with diverse endemicity. Although Clinico-demographic and laboratory profile of malaria patients by malaria species.(XLSX 15 KB)Additional file 1:Spectrum of complications with outcome by malaria species.(XLSX 13 KB)Additional file 2:Comparison of present study with other reports describing complicated malaria in adults.(XLSX 17 KB)Additional file 3:


Whether using two languages enhances executive functions is a matter of debate. Here, we take a novel perspective to examine the bilingual advantage hypothesis by comparing bi-dialect with mono-dialect speakers’ performance on a non-linguistic task that requires executive control. Two groups of native Chinese speakers, one speaking only the standard Chinese Mandarin and the other also speaking the Southern-Min dialect, which differs from the standard Chinese Mandarin primarily in phonology, performed a classic Flanker task. Behavioural results showed no difference between the two groups, but event-related potentials recorded simultaneously revealed a number of differences, including an earlier P2 effect in the bi-dialect as compared to the mono-dialect group, suggesting that the two groups engage different underlying neural processes. Despite differences in the early ERP component, no between-group differences in the magnitude of the Flanker effects, which is an index of conflict resolution, were observed in the N2 component. Therefore, these findings suggest that speaking two dialects of one language does not enhance executive functions. Implications of the current findings for the bilingual advantage hypothesis are discussed. Bilingual individuals have the ability to speak two languages in everyday life. A major debate in bilingualism research is whether using two languages leads to specific enhancement in non-linguistic cognitive functions, such as executive control (for a review see ). On theTo maintain smooth verbal communications, bilinguals need to switch between two languages and inhibit interference from the unintended language to the target language –21. ThesHuman language is a complicated system containing a number of subsystems including phonology, orthography, grammar, syntax, morphology, and semantics. One novel perspective from which to study the effect of language on domain-general cognitive functions is to look at how the experience of controlling one, several, or all subsystems between two languages affects non-linguistic performance. This is an important question since the answer will further specify the nature of the interaction between language and cognition at the level of language subsystem. Previous studies comparing monolingual and bilingual speakers, who use two completely independent language systems, cannot tease apart the role of different language domains.To examine the relative contributions of language subsystems to cognitive functions, for the first time we compared two groups of Chinese native speakers, one of which uses only the standard Chinese Mandarin  and the other uses both the standard Chinese Mandarin and the Southern Min  dialect. Chinese has one of the most diversified and complicated dialect distributions. Some dialects in south China, for example the Southern Min dialect, the Hakka dialect, and the Wu dialect, have phonological variations  so distinct from the standard Chinese Mandarin and other Chinese dialects that speakers of different dialects cannot understand one another verbally. Interestingly, except for phonology, these dialects share all other subsystems including orthography with the standard Chinese Mandarin –31. TherThis study was approved by the Institutional Review Board of the Imaging Center for Brain Research of Beijing Normal University. All participants gave written informed consent prior to the experiment and were paid for their participation.t (18) = 2.43, p = 0.026). Between-group comparisons showed that the English proficiency between the two groups was not different (p > .01), but the Standard Mandarin proficiency of the bi-dialect group was rated lower than that of the mono-dialect group (t (37) = 2.83, p = 0.007). However, the dialect proficiency of the bi-dialect group was comparable with the Mandarin proficiency of the mono-dialect group (p > .1). The two groups were matched on factors that may influence executive functions. These factors include IQ . The other half speak the Standard Mandarin and another main Chinese dialect such as the Southern Min Nan dialect or the Hakka dialect . All the dialects have distinct phonological systems from that of the Standard Mandarin (see Matrices ), and deIn the classic Flanker task , the stiElectrophysiological data were recorded in reference to the right mastoid at a rate of 1 kHz from 32 Ag/AgCl electrodes (Neuroscan Inc.) placed according to the extended 10–20 convention. Impedances were kept below 5kΩ.Electroencephalogram (EEG) was bandpass filtered on-line between 0.05 and 100 Hz and refiltered off-line with a 30 Hz, low-pass, 24 dB/oct, zero-phase shift digital filter. Eye blinks were mathematically corrected , and remThe temporal windows of three critical components  were defined a priori , 41 and F  = 143.24, p < 0.001), no significant effect of group  = 0.23, p > 0.1), and no significant interaction between the two factors  = 0.19, p > 0.1). Post-hoc analysis  confirmed that the RT of incongruent trials was significantly longer than that of congruent trials (p < 0.001) and neutral trials (p < 0.001). In the analyses of error rate, the main effect of Flankers was significant  = 7.05, p < 0.005), but group difference  = 0.54, p > 0.1) and interaction  = 0.68, p > 0.1) were both non-significant. Post-hoc analysis showed that the error rate of incongruent trials was higher than that of congruent trials (p < 0.05) and neutral trials (p < 0.05).Only trials with correct responses made between 300 and 1400 ms and trials between mean reaction times ±2.5 standard deviations were included in the analysis. This resulted in the exclusion of 2.8% of total trials at the analysis. A series of repeated measures ANOVAs with language as the between-subject factor  and Flanker conditions as the with-subject factor  were performed on both RTs and error rate See . AnalyseF  = 4.38, p < 0.05), showing that the P2 was found earlier in the bi-dialect  as compared to the mono-dialect group . The analysis also showed a main effect of the Flanker task,  = 11.54, p < 0.001) and a significant interaction between group and Flanker task,  = 4.73, p < 0.05). Further analysis showed that there was a significant main effect of the Flanker task in the bi-dialect group  = 8.77, p < 0.05), but not in the mono-dialect group  = 3.18, p = 0.06). In the bi-dialect group, pairwise comparisons showed significant differences in the P2 latency between the neutral condition  and the congruent condition , and also between the neutral condition and the incongruent condition .Repeated measures ANOVAs conducted on the peak latencies of the P2 component revealed a significant main effect of group  = 8.03, p < 0.001), no main effect of group  = 1.83, p < 1), and a significant interaction between group and the Flanker task  = 3.84, p < 0.05). Post-hoc analysis revealed a significant group difference in the processing of the neutral stimuli,  = 2.20, p < 0.05), featuring a reduced P2 in the bi-dialect as compared to the mono-dialect group. Paired-sample t-tests showed that, for the bi-dialect participants, the amplitude in the neutral condition is significantly lower than that in the congruent and the incongruent condition (ps < 0.05).Repeated measures ANOVAs conducted on the mean amplitude of the P2 revealed a significant main effect of the Flanker task  = 10.13, p < 0.001). Pairwise comparison showed that N2 was significantly larger (i.e. more negative) in the incongruent as compared to the congruent and neutral conditions (ps < 0.005). Group difference  < 1) and interaction  < 1) were both not significant.A main effect of the Flanker task was found on mean amplitude analysis of the N2 component  = 4.04, p < 0.05). The main effect of the Flanker task was not significant  = 1.10, p = 0.33), and the interaction of the Flanker task and group was also not significant  = 2.48, p = 0.09). Post-hoc analysis showed that, in the congruent condition, a larger amplitude modulation was elicited in the bi-dialect as compared to the mono-dialect group (t (37) = 2.87, p < 0.05). In the neutral condition, between-group comparison showed a marginal significant difference toward the same direction as that of the congruent condition (t (37) = 1.98, p = 0.055). In the incongruent condition, the two groups were not significantly different (t (37) = 1.12, p = 0.27; See At the late positive component, a significant main effect of group on mean amplitude was observed  on a daily basis does not enhance behavioural performance in an executive function task , but it yields generic differences in underlying neural processes associated with the Flanker task between bi-dialect and mono-dialect speakers. Future studies will have to look at individuals with greater differences in language experiences beyond the monolingual-bilingual comparison. These individuals will include bilingual speakers of two distinct languages, bilingual speakers with two overlapping languages, bi-dialect monolingual speakers, and mono-dialect monolingual speakers, and they must be matched on non-linguistic variables that could potentially affect cognitive performance. Such studies will not only examine the validity of bilingual advantage effects that have been reported so far, but may also reveal the particular language experiences as the underlying causes of the effect.S1 Data(ZIP)Click here for additional data file.S1 Questionnaire(DOCX)Click here for additional data file.S2 Questionnaire(DOCX)Click here for additional data file.