Plasmodium falciparum apical membrane antigen 1 (AMA1) and the adjuvant system AS02A, had strain-specific efficacy against clinical malaria caused by P. falciparum with the vaccine strain 3D7 AMA1 sequence. To evaluate a potential correlate of protection, we measured the ability of participant sera to inhibit growth of 3D7 and FVO strains in vitro using high-throughput growth inhibition assay (GIA) testing. Sera from 400 children randomized to receive either malaria vaccine or a control rabies vaccine were assessed at baseline and over two annual malaria transmission seasons after immunization. Baseline GIA against vaccine strain 3D7 and FVO strain was similar in both groups, but more children in the malaria vaccine group than in the control group had 3D7 and FVO GIA activity ≥15% 30 days after the last vaccination (day 90) . From baseline to day 90, 3D7 GIA in the vaccine group was 7.4 times the mean increase in the control group (p<0.0001). In AMA1 vaccinees, 3D7 GIA activity subsequently returned to baseline one year after vaccination (day 364) and did not correlate with efficacy in the extended efficacy time period to day 730. In Cox proportional hazards regression models with time-varying covariates, there was a slight suggestion of an association between 3D7 GIA activity and increased risk of clinical malaria between day 90 and day 240. We conclude that vaccination with this AMA1-based malaria vaccine increased inhibition of parasite growth, but this increase was not associated with allele-specific efficacy in the first malaria season. These results provide a framework for testing functional immune correlates of protection against clinical malaria in field trials, and will help to guide similar analyses for next-generation malaria vaccines.The blood-stage malaria vaccine FMP2.1/AS02A, comprised of recombinant Clinical trials registry: This clinical trial was registered on clinicaltrials.gov, registry number NCT00460525. Plasmodium falciparum apical membrane antigen 1 (AMA1) and the adjuvant system AS02A, was tested in a Phase 2 clinical trial in 400 malaria-exposed Malian children aged 1–6 years. Vaccination according to a 0, 1, 2-month schedule did not show efficacy against the primary endpoint, but showed approximately 20% efficacy against first and multiple clinical malaria episodes defined using different parasite density thresholds, and 64.3% efficacy (p = 0.03) against clinical malaria caused by parasites with AMA1 corresponding to the 3D7 vaccine strain at pre-defined polymorphic amino acid sites [in vitro correlates of protection. In this Phase 2 trial, three doses of the AMA1 (FMP2.1/AS02A) vaccine induced high levels of anti-AMA1 antibody comparable to those found in semi-immune adults living at the site. The difference in anti-AMA1 antibody titers from baseline to the time point just before the first malaria episode was associated with a lower risk of clinical malaria (hazard ratio (HR) 0.72, p<0.001) [The blood stage malaria vaccine FMP2.1/AS02A, comprised of recombinant 3D7 strain P. falciparum malaria antigens correlate with serum inhibition of parasite growth [in vivo have not been assessed for malaria vaccines based on blood-stage antigens like AMA1, because no previous blood-stage vaccine has demonstrated even partial protection against clinical malaria in field studies.Inhibition of merozoite invasion of erythrocytes as measured by a growth inhibition assay (GIA) represents a potential functional correlate of protection that has been studied in clinical trials of blood-stage malaria vaccines. GIA evaluates the functional activity of antibodies directed against blood stage antigens by measuring parasite growth in the presence of immune serum compared to non-immune serum. While previous studies have shown that antibody titers against blood stage e growth –5 and the growth , other pe growth –9, and ae growth . These sThe trial was conducted in compliance with the International Conference on Harmonization of Good Clinical Practices, the Declaration of Helsinki and regulatory requirements of Mali. The study protocol and informed consent process were approved by the institutional review boards of the University of Sciences, Techniques and Technology Faculty of Medicine, Pharmacy and Dentistry in Bamako, Mali; the University of Maryland Baltimore; the Walter Reed Army Institute of Research; and the United States Army Surgeon General. Written informed consent was obtained prior to screening and enrollment. Verbal consent of illiterate parents or guardians was administered and then documented using their thumbprints, a process verified by signatures of independent witnesses.P. falciparum infection with a parasite density of 2,500/microliter or greater. Clinical malaria episodes were classified as having AMA1 sequence matching or not matching that of the vaccine strain 3D7 based on complete concordance with eight polymorphic amino acid positions in the cluster 1 loop of AMA1 domain I that were identified before the analysis as important in the development of strain-specific immunity against AMA1 based on both in vitro [Details of the Phase 2 trial are published elsewhere . Brieflyin vitro  and molein vitro .P. falciparum, strain 3D7 and a genetically distant FVO strain, was assessed using a miniaturized, high-throughput method by laboratory staff who were blinded to treatment group. Samples were processed and evaluated for growth inhibitory activities by quantification of parasite lactate dehydrogenase (pLDH) in a 384-microtiter plate format [g to pellet RBCs. The required amount of serum needed was removed without disruption of the RBC pellet and transferred to and diluted to a 20% concentration . A 4% hematocrit parasite suspension (0.3–0.4% parasitemia) was added to the plates, and the plates were incubated for one cycle length  gassed  at 37°C. Plates were harvested, processed for pLDH detection  and optical density in each well was read at 650 nm. Children demonstrating ≥ 15% activity were considered responders, a reproducible cut-off value calculated based on the mean inhibition of U.S. non-immune pre-immune sera + 2 standard deviations.Strain-specific GIA was assessed for sera collected at baseline and at eight subsequent time points over the two year follow-up period, including study days 0, 30, 60, 90, 150 and 240 corresponding to the first malaria season following immunization, and days 364, 547, and 730 corresponding to the beginning, middle and end of the second post-immunization malaria season. Serum inhibition of the growth of e format ,13. In bSpecial considerations were made to quality control for plate variations by positioning negative and positive plate controls at various locations on the plate. Positioning the controls at opposite sides of each plate allowed monitoring of changing conditions during the enzymatic reaction (development of the plate). Every assay plate contained a complete set of assay controls; one of these assay controls is a historical reference sample . The growth inhibitory activity of this control against 3D7 and FVO parasites had to fall within a pre-determined range of inhibition and served as pass/fail criterion for each plate. The analysis of the samples was done using the pre-immune samples of each subject to determine the individual sample baseline activity.1/12, which is the level required for an overall type I error rate of 0.05 if the tests are statistically independent; since there are substantial positive correlations between GIA results at different times, these adjustments are likely to be conservative, making the overall type I error rate < 0.05 within each table.For both 3D7-specific and FVO-specific GIA results, means at various times since baseline and the mean difference in GIA results from baseline were compared between AMA1 and control vaccine recipients using Student’s t-test. Percentages of participants who met a minimum pre-specified threshold of 15% at each study time point, as well as percentages of participants who converted from a negative GIA at baseline (<15%) to a positive GIA (≥15%) at 30 days after the last vaccination, and at one and two years after the first vaccination, were compared using a chi-square test with no continuity correction. For each analysis of means or percentages, p-values are presented for 12 comparisons, along with an indication of whether the difference is significant at significance level 1–0.95The relationship between GIA results and risk of clinical malaria was examined by estimating the hazard ratio for the first or only episode of clinical malaria with onset between day 90 and day 240 after the first vaccination in Cox proportional hazards regression models for risk of clinical malaria. These models included both GIA at the study time point just before the first clinical episode or the difference in GIA from baseline to the study time point just before the first clinical episode included in the model as a time-varying covariate.Statistical analyses were done with SAS 9.2 and 9.3 statistical software  and NCSS software .http://dx.doi.org/10.7910/DVN/ZUQFHQ.GIA data used in the analysis are accessible at Before immunization, which began at the transition from the dry season to the malaria transmission season, mean 3D7 GIA activity was around 5% for sera from both AMA1 vaccinees and controls . At studBased on a predetermined threshold of 15% GIA, participants were classified as having significant GIA activity against the vaccine strain 3D7 or not. At baseline, both AMA1 vaccine and control groups were essentially equivalent with 15–16% in each group showing activity . This peFor Cox regression modeling of clinical malaria with onset from day 90 to day 240, with either 3D7 GIA or the change in 3D7 GIA since the day of vaccination treated as a time-varying covariate, there were 61 first or only malaria episodes among 163 AMA1 vaccinees and 59 such episodes among 149 recipients of rabies vaccine. There was a suggestion in AMA1 vaccinees of an association between 3D7 GIA and an increased risk of clinical malaria . A similar but slightly weaker association was seen with change in 3D7 GIA . There was no evidence of an association between 3D7 GIA and clinical malaria risk in recipients of rabies vaccine  or with change in 3D7 GIA .During the first malaria season, only three first or only episodes of malaria identical to the vaccine strain occurred between days 90 and 240 among AMA1 vaccinees, and 13 such episodes occurred among recipients of rabies vaccine. We did not fit Cox models to malaria identical to the vaccine strain.The mean baseline activity against FVO parasites was higher than the activity measured against 3D7 parasites. This activity reflects parasite inhibition induced by natural exposure and may target other blood stage antigens that are more cross-reactive than AMA1. At study start FVO GIA activity in AMA1 vaccinees averaged 35% and subsequently dropped. The activity recovered to 39% by day 150, corresponding to the peak of the first malaria season of follow-up. By day 240, FVO GIA values in AMA1 vaccinees had fallen to 30%, with subsequent measurements increasing up to the highest mean FVO GIA of 45% on day 730, the last day of follow-up , Table 3In the control group, mean GIA activity followed a similar pattern. Mean FVO growth inhibition was 33% at study start, with a subsequent fall to 19% on day 60, then an increase up to 35% on day 150 at the peak of the first malaria season. Average FVO GIA activity then dropped in control vaccinees to 27% at day 240, and subsequently increased to a peak value of 39% on the final study day, 730 , Table 3When comparing FVO GIA activity between the two groups, AMA1 vaccinee sera collected on day 60 showed slightly higher FVO GIA activity compared to the control group . By the next measurement at day 90, however, mean FVO GIA values were comparable in the two groups and were similar to baseline values . The difResults of Cox regression analyses of risk of clinical malaria with onset between day 90 and day 240 and FVO GIA in AMA1 recipients were similar to those for 3D7 GIA, but there was no suggestion of a significant association between risk of clinical malaria and FVO GIA or change from baseline in FVO GIA in either AMA1 or rabies vaccine recipients.Overall, these results demonstrate highly significant GIA activity against vaccine strain 3D7 parasites in the serum of children vaccinated with the FMP2.1/AS02A vaccine as compared to control rabies vaccine. In contrast, there were only modest and usually non-significant differences in GIA activity of the two serum sets against FVO parasites. These GIA activities did not correlate with the observed significant efficacy of the vaccine against 3D7-like parasites , althougThe current analysis reports results of serum growth inhibition, and differs from other published studies of growth inhibition using purified immunoglobulin G from plasma. The methodology used may influence results . A studyBaseline 3D7 GIA activity in this cohort of 400 children aged 1–6 years was similar to previously reported activities in semi-immune adults living at the same study site . Thirty The decreased 3D7 GIA activity at the start of the second year of follow up is also consistent with the lack of any measurable overall or allele-specific efficacy against clinical malaria in the AMA1 vaccinees during this time period. Subsequent to the start of the second year of follow-up (study day 364), mean 3D7 GIA activity increased in both groups by the end of the second malaria season (study day 547), likely due to natural boosting of antimalarial immunity with natural exposure. In contrast to waning GIA, anti-AMA1 antibody levels peaked in the AMA1 group 30 days after vaccination and remained at a high level for the entire 2-year follow up period, while levels in the control group remained low . These oUsing a threshold of 15% 3D7 GIA to define a specific response to blood stage parasites, more AMA1 vaccinees than controls had positive GIA at all post-vaccination study time points after day 30, and AMA1 vaccinees were more likely to “seroconvert” from negative to positive GIA. Post-immunization absolute GIA and increases in GIA were consistent with allele-specific efficacy against clinical malaria in the first year. Although both GIA and anti-AMA1 antibody levels remained higher in the AMA1 vaccine group for two years after immunization, no allele-specific vaccine efficacy was demonstrated in the second year of follow-up .Enhanced 3D7 GIA activity did not correlate with overall malaria risk due to both vaccine and non-vaccine strains in this study. It may be that GIA represents a surrogate measure of environmental exposure to and ongoing risk of malaria, similar to previous studies showing that anti-malarial immunity is associated with subsequent malaria risk because individuals with greater malaria exposure over time develop higher antibody levels .To date, other clinical efficacy trials of blood stage malaria vaccines have not shown efficacy against clinical malaria in children, precluding investigations of potential correlates of protection. While we were able to determine that children in the AMA1 group had a higher GIA measurement post-vaccination compared to controls, no statistically significant evidence of allele-specific protection was demonstrated. This functional assay did not correlate with the protection demonstrated in the vaccine trial, but it remains possible that GIA may correlate with protection against other candidate blood stage malaria vaccines.Correlation of 3D7 GIA activity with risks of clinical malaria in malaria vaccinees up to study day 240 showed a HR of 1.004 (p = 0.44), suggesting that the protection demonstrated in this group could be explained by other unknown factors. As it pertains to humoral immune responses, growth inhibition assays can only assess the biological function of antibodies. It is conceivable that AMA1 specific antibodies could display efficacy if acting in concert with phagocytic cells expressing Fc-receptors as has been shown for other blood stage antigens (e.g. MSP-3/GLURP) –29. AlteThe waning of allele-specific protection in the second year of follow-up occurred at the same time that 3D7 GIA activity in AMA1 vaccinees decreased compared to the initial follow-up period. This suggests that 3D7 GIA may be a surrogate of protection rather than a functional correlate. Perhaps a booster dose in AMA1 vaccinees just prior to the second malaria season would have increased immunity above a critical threshold necessary to provide the allele-specific protection observed during the first malaria season after vaccination.Baseline mean FVO GIA activity in the study population was greater than the 20% previously measured in adults at the study site ,21, and The observation that more AMA1 vaccinees had FVO GIA than did control vaccinees beginning with the time point after the first vaccination until 90 days after the last vaccination, suggests that immunization with one allele may broaden immune responses to other alleles.P. falciparum amino acid sequence increased strain-specific inhibition of parasite growth as measured by GIA against the 3D7 strain of P. falciparum. This increase was not associated with a reduced risk of clinical malaria caused by parasites with vaccine strain-type AMA1 in the two malaria seasons following vaccination. Additional immunologic analyses including affinity-purification of anti-AMA1 antibodies with subsequent characterization of these immunoglobulins regarding fine specificity, antibody isotype and avidity may increase our understanding of the functional basis of this allele-specific vaccine-induced immunity. Moreover, the antibody, GIA and efficacy data indicate that annual booster immunizations may be required to maintain protective immunity. The current study suggests that GIA is not an immune correlate of strain-specific protection against clinical malaria for this AMA1 vaccine, but assessment of GIA to other candidates may still provide information that can help guide the development of more broadly protective next-generation malaria vaccines.Vaccination with the AMA1 candidate malaria vaccine protein based on the 3D7


Plasmodium infection in primary school children often exceeds that seen in younger children and could affect haemoglobin concentration and school performance.School-aged children are rarely targeted by malaria control programmes, yet the prevalence of A cluster-randomised trial was carried out in 80 primary schools in southern Mali to evaluate the impact of a school-based malaria intervention package. Intervention schools received two interventions sequentially: (1) teacher-led participatory malaria prevention education, combined with distribution of long-lasting insecticidal nets (LLINs), followed 7 months later at the end of the transmission season by (2) mass delivery of artesunate and sulfadoxine-pyrimethamine administered by teachers, termed intermittent parasite clearance in schools (IPCs). Control schools received LLINs as part of the national universal net distribution programme. The impact of the interventions on malaria and anaemia was evaluated over 20 months using cross-sectional surveys in a random subset of 38 schools, with a range of cognitive measures  assessed in a longitudinal cohort of children aged 9–12 years in all 80 schools.Delivery of a single round of IPCs was associated with dramatic reductions in malaria parasitaemia  and gametocyte carriage  in intervention compared with control schools. This effect was sustained for 6 months until the beginning of the next transmission season. IPCs was also associated with a significant decrease in anaemia , and increase in sustained attention . There was no evidence of impact on other cognitive measures.Plasmodium transmission.The combination of malaria prevention education, LLINs and IPCs can reduce anaemia and improve sustained attention of school children in areas of highly seasonal transmission. These findings highlight the impact of asymptomatic malaria infection on cognitive performance in schoolchildren and the benefit of IPCs in reducing this burden. Additionally, malaria control in schools can help diminish the infectious reservoir that sustains  Plasmodium parasites is common in school-aged children in Africa.Asymptomatic infection with Asymptomatic infections increase the risk of anaemia and have been associated with reductions in cognitive function.While deleterious effects of malaria infection on cognition could translate into lower school performance, this has yet to be demonstrated.This trial found that malaria control delivered through schools can significantly reduce the risk of anaemia and improve sustained attention in primary school children.These data provide the first evidence from an area of highly seasonal transmission and corroborate findings from earlier trials of malaria parasite clearance in schools.Improvements in health outcomes lasted until the beginning of the next transmission season and indicate that for schoolchildren in the sub-Sahel region, a single annual intermittent parasite clearance treatment given in schools at the end of the transmission season may be sufficient to have a prolonged benefit spanning the entire schoolyear.Malaria control initiatives in schools have potential to generate multiple benefits—improving health and supporting educational progress—and should be an integral component of education and school health plans in malaria-endemic countries.Equally, national malaria control strategy should not overlook this age group.School-aged children comprise a sizeable proportion of the population, and by diminishing the infectious reservoir that sustains transmission, malaria control in schools could have an additional role in transmission-reduction strategies.Plasmodium infection in this age group often exceeds that seen in younger children.School-aged children are rarely targeted by malaria control, yet the prevalence of Plasmodium falciparum; 92% were anaemic (<110 g/L); and 35% had severe anaemia (<80 g/L).A two-arm stratified cluster-randomised trial was carried out in primary schools in Sikasso region, south-eastern Mali, to inform Save the Children’s school health and nutrition programme that aims to address key health problems that prevent children from participating and learning in school. The region is characterised by high humidity and rainfall, with an estimated entomological inoculation rate (EIR) in excess of 100 infective bites per annum.Sample size calculations for each of the primary biomedical and cognitive outcomes, taking clustering of students within schools into account, were carried out to determine the number of schools and students required.Rural government or community primary schools located within the Sikasso administrative region, with a total enrolment of 50 pupils or more, were eligible for inclusion in the study. Any school that lacked both classes 4 and 5 at baseline was not eligible to participate in the trial. A total of 114 schools met the eligibility criteria and were stratified into four groups according to the previous academic year’s pass rate , to account for differences in school quality and socioeconomic environment. Within each school performance stratum, 20 schools were randomly selected for inclusion in the trial. At this stage, one school refused to participate and was replaced with another school using the same selection method. Schools were randomly allocated to either the control or intervention arm; 40 schools per arm. Selection of schools and allocation to study arm were performed using a random number generator in Microsoft Excel. Cognitive outcomes were assessed at baseline and follow-up in all 80 schools randomised to the trial. Within each study arm, a subsample of 19 schools were randomly selected for inclusion in the evaluation of biomedical outcomes at baseline and follow-up .All children enrolled in the 40 intervention schools received two malaria interventions sequentially: (1) teacher-led participatory malaria prevention education in schools, combined with school-based distribution of LLINs, prior to the start of the malaria transmission season, followed by (2) intermittent parasite clearance in schools (IPCs) at the end of the transmission season. Control schools received LLINs as part of the national universal net distribution programme.All teachers were trained on malaria-specific knowledge, demonstrations of bednet use and participatory classroom activities based on the Child-to-Child approach to help incorporate malaria education into schooling during a 3-day workshop. Each participant received a teacher’s manual (available on request from the authors). Key messages in the education programme included the possible adverse effects of malaria in school age children, role of mosquitoes in transmitting malaria, advantages of insecticide treated nets and importance of continued net use throughout the year. In April 2011, teachers began the participatory malaria education activities in schools and organised a public event at which two LLINs were issued to every child in the 40 intervention schools, for use by the target child, as well as other household members.In December 2011, children in intervention schools also received intermittent parasite clearance, in which a treatment dose was given to all pupils irrespective of infection status, timed to coincide with the end of the malaria transmission season. IPCs comprised a single annual treatment of artesunate plus sulfadoxine-pyrimethamine (AS/SP), which was administered in school by teachers over three consecutive days. Dosage was given according to age: 5–6 years: 1 tablet AS (50 mg) daily for 3 days+1 tablet SP (500/25 mg) on day 1 only; 7–13 years: two tablets AS (50 mg) daily+2 tablets SP (500/25 mg) on day 1. Exclusion criteria included self-reported pregnancy, prior history of adverse reaction to sulfa-based drugs or current treatment for symptomatic malaria. One-day training on IPCs drug administration and monitoring was provided for all teachers. Children were observed and treatment was readministered if the child vomited or spat out the tablets within 30 min of drug administration. Pupils who were absent on the first day of treatment were excluded from treatment. Pupils who started treatment but were absent on subsequent days were traced and treated at home; if they could not be found at home, they were excluded from the remainder of the treatment. Adverse events were monitored by teachers for 7 days after treatment and referred to the local health facility.In May 2011, the National Malaria Control Program in Mali implemented a community-based LLIN distribution programme throughout Sikasso region, which aimed to achieve universal coverage by providing one net for every two people. The universal campaign was planned and implemented after the beginning of the school-based trial and covered all intervention and control villages enrolled in the trial.Plasmodium infection, mean haemoglobin concentration and performance in a range of cognitive tests. Cross-sectional surveys were carried out at baseline in November 2010 and repeated at two subsequent time points. Data gathered in November 2011 after the first phase of the intervention were used to assess the impact of the school bednet distribution and malaria education programme, while outcomes collected in February 2012 after the second phase of the intervention were used to evaluate the combined impact of the IPCs treatment, bednet distribution and malaria education programme. To preserve blinding, staff responsible for measuring study outcomes were unaware of group allocation, and data analysis was undertaken by a statistician with no prior involvement in the trial.As both study arms received long-lasting insecticidal nets via the government programme, study outcomes were of necessity compared between schoolchildren in schools receiving the comprehensive school-based malaria control programme  and schoolchildren in communities receiving LLINs through the universal coverage campaign alone. The primary biomedical endpoint was prevalence of anaemia. The primary cognitive endpoint was mean score in tests of sustained attention, chosen as less dependent on prior learning than other tasks in the cognitive battery. Secondary endpoints included prevalence and intensity of The effect of the intervention on cognitive abilities was investigated by comparing differences in test scores given to a cohort of students in classes 4 and 5 . This age group was selected for testing because they were old enough to understand the test instructions and could thus be assessed in groups. In each of the 80 schools, 15 students were randomly selected from class 4 at baseline. In schools without a class 4, 15 children were selected from class 5. The sample was drawn using a number counting system on the day of the survey: to ensure gender balance pupils were separated by sex, and the number of boys (or girls) divided by 15 to yield a ‘selection number’. Children of each sex were lined up in a random order and counted off with each child on whom the ‘selection number’ landed chosen for participation. The same children were followed longitudinally over the course of the study.Sustained attention was evaluated using two code transmission tasks, adapted from the TEA-Ch (Test of Everyday Attention for Children) batteryBiomedical outcomes were assessed, on the day following the cognitive assessments, in a randomly selected subset of 38 schools. The biomedical sample included the full age range of primary school pupils (age 5–18 years) and comprised the 15 pupils already enrolled in the cognitive cohort, plus an additional 35 pupils randomly selected from among the rest of the school . The sample was drawn on the day of the survey using a number counting system, as described above, with the total number of boys (or girls) divided by 35 to yield a ‘selection number’. This sampling procedure was undertaken at baseline and repeated afresh in November 2011 and February 2012. In this manner, data were collected at each time point on approximately 1900 students . As a result of the selection procedure, the biomedical dataset available for analysis included some data collected from the same 570 children evaluated at all three time points (15 per school in cognitive cohort) and a larger majority of data collected from mainly different individuals at each time point . The data available from each time point are summarised in Finger-prick blood samples were taken to assess haemoglobin concentration (Hb) with a portable photometer . Anaemia was defined according to WHO age-specific thresholds: Hb <110 g/L for children aged less than 5 years, Hb <115 g/L for children aged 5–11 years, Hb <120 g/L for children aged 12–14 years and female children >15 years and Hb <130 g/L for boys aged >15 years.Following the final evaluation in February 2012, intermittent parasite clearance was carried out in control schools. To investigate how long the effects of the intervention might persist, an additional cross-sectional biomedical survey was undertaken at the end of the school year in May 2012 in a randomly selected subsample of five schools in the intervention arm (250 schoolchildren). Malaria parasitaemia and anaemia were compared with 250 children in five randomly selected schools that had not previously participated in the study.Plasmodium infection), separate statistical models were fitted to assess (1) the impact of the school bed nets and malaria education programme (using data from the first follow-up survey in November 2011) and (2) the combined impact of the IPCs treatment, bed nets and malaria education programme (using data from the second follow-up survey in February 2012). For the cognitive outcomes , only the full intervention including the IPCs treatment was evaluated.For each biomedical outcome , time (follow-up vs baseline) and the interaction between study arm and time. The key effect of interest is the interaction, which estimates the change from baseline to follow-up in the intervention arm relative to change in the control arm. All analyses accounted for the nature of the distribution of the outcome and appropriate summary statistics and measures of effect are reported.For cognitive outcomes, test scores were standardised prior to analysis to create a z-score using the mean and SD of each measure at baseline. Potential practice effects were controlled for by including in analyses a covariate which indicated whether the student had already experienced the test on a prior occasion. For those outcomes where versions of the task differed between class 4 and 5, z-scores were calculated separately by class, and analysis controlled for variations in difficulty and scoring between different versions of the test used, and interactions between version and both practice and time. Where test scores were not normally distributed and demonstrated floor or ceiling effects, bias corrected bootstrap 95% CI estimated from 2000 bootstrap replications are reported, and statistical significance at p=0.05 is inferred from the 95% CI.For the biomedical outcomes, an adjusted analysis, which controlled additionally for age, sex, height for age z-score and village level prevalence of malaria parasitaemia at baseline , was carried out. Since this full set of covariates was not collected from every student in the larger cognitive sample (80 schools), adjusted analyses for cognitive outcomes controlled only for age, sex and school class.A supplementary analysis on the smaller subset of students for whom both biomedical and cognitive measures were available (38 schools) was also carried out. The adjusted analysis for this subset controlled for parental literacy, socioeconomic status (as measured by ownership of household assets), height for age z-score and village level prevalence of malaria parasitaemia at baseline, in addition to age, sex and school class.All analysis was by intention to treat and was carried out using Stata V.13.1.Ethical approval for the trial was granted by the Ethical Committee of the National Institute of Research in Public Health in Mali (00013/10/CE-INRSP). Community consent was obtained at the time of enrolment into the trial. Sensitisation meetings were held with village leaders, school staff and members of the school management committees (Comité de Gestion Scolaire) and read a standardised script detailing the justification, procedures, risks and benefits of the trial. Parents and students were informed of their right to withdraw from the study at any time. In communities agreeing to participate in the trial, written consent was provided by the president of the school management committee. Prior to each of the two follow-up surveys, community meetings were repeated to brief parents about the surveys, and verbal assent was sought from each student before participation.Students presenting with clinical malaria on the day of the surveys, defined as fever and a positive rapid diagnostic test, were started on artemisinin combination therapy with Coartem. Parents, older siblings or teachers were briefed to ensure the completion of the 3-day regimen. Students with severe anaemia, defined as a haemoglobin concentration of less than 70 g/L, were immediately given iron supplements and referred to the nearest health facility. Students with severe malaria or symptoms of other serious ailments were also referred.http://www.controlled-trials.com/ISRCTN26838440).The trial was registered with current controlled trials  and parasite density (9.0% and 5.2% missing). For all other measures, the quantity of missing data was low (<6%) and similar in both study arms. The prevalence of Plasmodium infection at baseline was similar in the two arms, although parasite density was marginally higher among infected children in the control schools. Reported household ownership of mopeds and telephones was slightly higher in the intervention arm. Nonetheless, biomedical outcomes at baseline were generally similar in the two study arms  is shown in Following the school-based and national universal community distribution of LLINs, reported net use among schoolchildren in August 2011 exceeded 80% in both intervention and control communities.Plasmodium infection was slightly lower in children in intervention schools compared with controls. Since Plasmodium infection increased from 79% in November 2010 to 86% in November 2011 in the control arm, while remaining stable at around 80% across both time points in the intervention arm, the differential change since baseline between the two groups corresponds to a 44% reduction in the odds of Plasmodium infection in intervention schools compared with control schools . After treatment, the prevalence of gametocytes was also lower in intervention schools compared with control schools that had not received treatment . Over the same period, the prevalence of anaemia fell in both arms of the study, but the reduction was greater in intervention schools: resulting in a 44% greater reduction in the odds of anaemia in intervention than control schools . The corresponding increase in haemoglobin levels was 2.8 g/L greater (95% CI 1.1 to 4.5) in intervention than control schools. Adjusted analyses that controlled for age, sex, height for age z-score and school-level prevalence of malaria parasitaemia at baseline  were broadly consistent with the unadjusted results reported above.In contrast, at follow-up in February 2012 (8 weeks after the IPCs treatment), we found strong evidence for an impact of the intervention on asymptomatic For cognitive outcomes, only the full intervention including the IPCs treatment was evaluated. Scores on both tests of sustained attention increased in all schools over the 15 months of the study, with the greatest improvements evident in the intervention arm . In analA supplementary analysis on the smaller subset of children for whom both biomedical and cognitive measures were available  in January 2012 found <1% children to be parasite positive, confirming helminth infection to be uncommon in this study population though undernutrition may remain a major cause of anaemia. Nevertheless, the similarities in nutritional status between the two study groups at baseline would indicate that the effect on anaemia can most likely be attributed to the marked reduction in malaria parasitaemia. Finally, the findings are remarkably consistent with previous trials in Kenya, Mali, Senegal and Uganda, in which the protective efficacy of intermittent preventive treatment on anaemia in school-aged children has usually exceeded 40%.Plasmodium infection and anaemia, where three rounds of parasite clearance were carried out per year.Plasmodium infection on other aspects of cognitive performance and education remain unknown. Whether these effects can be generalised to younger pupils at an earlier stage in their schooling is also unknown, and further research is needed.Performance in cognitive tests can be expected to increase over time in both groups, due to the increasing age of the test subjects and repeated testing, which increases familiarity with the testing methodology (practice effects); nonetheless, the size of the improvement in sustained attention was greater among children in intervention schools. We did not see any differences between groups in any of the other tasks included in the cognitive battery. However, unlike the sustained attention task that only requires knowledge of the numbers 1–9, these other tasks are more dependent on prior learning . The sustained attention task may therefore be less context dependent and more sensitive to changes in cognitive function. The ability to use the same test methodology with limited prior adaption also facilitates comparison with other recent studies.Plasmodium infection remained high, and no impact was observed for haematological outcomes at the end of the transmission season. Although more children in the intervention arm, where the LLIN distribution was supported by focused malaria education activities in schools, reported using nets in November 2011, it is possible that they did not sleep under a net every night during the preceding months and/or were exposed to infective mosquitoes prior to going to bed. Increased and consistent net use would be expected to reduce the incidence of new infections during the malaria transmission season between May and October 2011; however, as demonstrated by the follow-up data from May 2012, asymptomatic infections can persist for several months, and the limited impact on malaria parasitaemia and anaemia may also have been due to long-term parasite carriage of infections acquired prior to the distribution of insecticide-treated nets. Thus, although substantial impacts were not seen until after the administration of antimalarial drugs for parasite clearance, we cannot rule out the role that net use might have played in the overall effect, through reducing the risk of new infections. It is possible that the effect of net use could be more marked in the subsequent transmission season, following the first annual round of treatment, but was not examined in this trial.In this study, we cannot fully separate the effects of each component of the intervention. Furthermore, the government campaign of universal LLIN coverage in May 2011 meant that children in both the intervention and control arms received a LLIN. Despite increases in malaria knowledge and reported net use in intervention schools following the school-based malaria education and LLIN distribution,Qualitative studies undertaken after completion of the trial found the interventions to be well accepted by teachers, parents, school management committees and community leaders ; the antimalarial treatment was generally well tolerated, with most symptoms being mild and self-limiting. The malaria prevention education component of the intervention was estimated to cost $2.13 per child, and the intermittent parasite clearance to cost an additional $2.72 per child treated.Malaria transmission in the Sahel subregion, where this trial was carried out, is highly seasonal, with new infections concentrated in a few months each year. Schools are closed for most of the rainy season, which limits the role that schools can play in malaria control during the period of peak transmission. For example, as children are not in school in the months when the incidence of clinical attacks is highest, there is limited value in training teachers to diagnose and treat malaria. Our intervention package in schools instead focused on three complementary approaches that could be implemented in schools before and after the rainy season: (1) distribution of LLINs, accompanied by (2) malaria prevention education to promote net use and prompt treatment seeking for fever during the rainy season and school recess, followed by (3) parasite clearance at the end of the transmission season. A supplementary community-based approach to controlling malaria in school-aged children during the rainy season that might also be considered is the use of seasonal malaria chemoprevention, currently recommended for children under 5 years.32Plasmodium infection on cognitive performance in schoolchildren. This trial also provides further evidence that intermittent parasite clearance in schools is an efficacious control strategy to improve the health and cognitive performance of school-aged children. This approach is particularly suited to areas of seasonal transmission where a single annual treatment can be given at the end of the transmission season. Intermittent parasite clearance can be combined with the use of LLINs to reduce the risk of new infections between treatment rounds. In this area of seasonal transmission, malaria prevention education in schools encouraged continued use of nets for longer to ensure that children remain protected throughout the period of highest transmission risk. Malaria control initiatives in schools can also play a valuable role in supporting wider malaria control efforts. Malaria education activities in schools help to increase use of nets among schoolchildren and thereby contribute to higher levels of community coverage as well. The striking and sustained impact of a single round of intermittent parasite clearance on gametocyte carriage at the beginning of the next transmission season may have a further valuable effect in reducing the infectious reservoir that sustains transmission in areas of highly seasonal malaria.Our findings add to the growing body of evidence on the impact of asymptomatic


Complicated malaria remains an important public health problem, particularly in endemic settings where access to health services is limited and consequently malaria fatal outcomes occur. Few publications describing the clinical course and outcomes of complicated malaria in Latin America are found in the literature. This prospective study approached the clinical and laboratory characteristics of hospitalized patients with complicated malaria in different endemic areas of the Colombian Pacific Coast with the aim to provide epidemiological knowledge and guide to further reducing malaria severity and mortality.Plasmodium falciparum was the most common parasite species (70%), followed by P. vivax (28%), and mixed malaria . Overall, predominant laboratory complications were severe thrombocytopenia (43%), hepatic dysfunction (40%), and severe anaemia (34%). Severe thrombocytopenia was more common in adults (52%) regardless of parasite species. Severe anaemia was the most frequent complication in children ≤10 years (72%) and was most commonly related to P. vivax infection (p < 0.001); whereas liver dysfunction was more frequent in older patients (54%) with P. falciparum (p < 0.001). Two deaths due to P. vivax and P. falciparum each were registered. Treatment provision before recruitment hindered qPCR confirmation of parasite species in some cases.A prospective, descriptive hospital-based study was conducted in 323 complicated malaria patients (median age 20 years) enrolled in Quibdó, Tumaco and Cali between 2014 and 2016. Clinical evaluation was performed and laboratory parameters were assessed during hospitalization. P. vivax and hepatic dysfunction in adults with P. falciparum. Results indicated the need for earlier diagnosis and treatment to prevent complications development as well as more effective attention at hospital level, in order to rapidly identify and appropriately treat these severe clinical conditions. The study describes epidemiological profiles of the study region and identified the most common complications on which clinicians must focus on to prevent mortality.The study identified a high prevalence of complicated malaria in the Pacific Coast, together with more frequent severe anaemia in children infected by  Plasmodium falciparum (~90%). Meanwhile, Plasmodium vivax was responsible for ~4% of the global cases, 41% of which were reported in regions outside Africa . Prior to study initiation, laboratory tests such as automated complete blood cell count, biochemistry profile , and diagnostic tests (TBS and qPCR) were standardized to comply with similar SOPs, to ensure similar performance in the different laboratories. Laboratory technicians were trained to perform similar procedures.As quality control, a second analysis of the thick smears was performed by an independently trained malaria microscopist. In the case of discordant results, the slides were read by a third microscopist. The definitive result was one of which at least two reads matched. The quantitative PCR (qPCR)  was perfThe main objective of this study was to find associations between age groups (children and adults), the malaria parasite species  and the type and severity of the clinical complication(s). Therefore, outcomes measured were: parasitemia, haematological and blood chemistry parameters as well as clinical manifestation in patients infected with either P. vivax or P. falciparum. Categorical variables were summarized as frequency (proportion) of complicated malaria cases by P. vivax or P. falciparum species of each study sites. Chi-square or Fisher´s exact tests were used for comparison of categorical data: clinical presentation and complication pattern in adults/children and in P. vivax and P. falciparum infections. Patients enrolled (1–84 years old) were divided into two age groups: younger than 10 years old, and 10 and older. The severity of complication was determined by laboratory variables according to the numeric ranges established within the Colombian MOH and/or WHO guidelines for complicated malaria ,31. StudPlasmodium falciparum was the most frequent species , followed by P. vivax infections , and a low number of mixed Pf/Pv cases  which were all recruited in Quibdó .A total of 323 hospitalized patients were enrolled: 193 in Quibdó, 112 in Tumaco, and 18 in Cali ; with sin Quibdó . Ninety-P. vivax patients (48/91) were children between 0–10 years of age, while P. falciparum cases were more evenly distributed among all age groups , whereas most of the patients infected with P. vivax (41%) were indigenous (p < 0.01). The site of residency was almost equally distributed between rural (47%) and urban (52%) settings. Only 50 patients (15.5%) self-reported previous malaria episodes, and in 24 of them the last episode was in the previous year.Most of the e groups . NotablyP. falciparum in highly endemic areas of Africa, whereas malaria transmission in Colombia is of low to moderate intensity and is caused by both P. falciparum and P. vivax, predominantly the latter. Moreover, many complicated cases are referred from remote areas and would benefit from the use of more conservative criteria for hospital referral.The Colombian MoH adopted more conservative criteria than WHO since 2010 because the latter were highly focused on In general, malaria mortality in Colombia is considered low,  . We beliAnalyses were done to evaluate the adequacy of the severe malaria criteria for the specific Colombian settings. The group of patients with severe anaemia, severe thrombocytopenia and hepatic dysfunction was analysed by using both the MoH and the WHO criteria . Mean duAnalyses of patients with 1 and those with >1 malaria severity criteria including days of hospitalisation, ICU management, haemoglobin levels, platelets count, parasitemia, glucose levels, renal and hepatic function as surrogate markers indicated no significant differences in terms of days of hospitalization between the two groups, however, patients with 2 or more criteria required ICU management more frequently than those with only one (0.7% vs 7.0%). In terms of laboratory parameters, significantly worse values were found for platelet levels, hepatic and renal function in patients with >1 complication criteria .In addition, analyses to compare the surrogate markers of severity  between patients with 5 and > 5 days of disease (time between symptoms onset and recruitment day) were performed. However, no significant associations were found on any of the evaluated parameters between groups. Therefore, in this study a longer duration of disease was not associated with greater severity of the malaria episode.In this study, only 50 patients reported previous malaria episodes and this was not related with the severity degree (number of parameters) during the present illness.P. falciparum and 9,050 parasites/μL  in P. vivax  were negative, presumably due to the treatment initiated upon consultation to the POC, since every referred patient presented a positive TBS. Treatment of these patients consisted of chloroquine (25mg/kg divided in three doses) + primaquine (0.25mg/kg/day for 14 days) for most of P. vivax cases. Artemether + lumefantrine (20mg + 120mg in adults for six doses and according to weight and age in children < 34kg and/or <14 years of age) for most of P. falciparum cases. Artesunate and Quinine were also used in some cases. No specific characteristics distinguished them from other patients with positive qPCR.Most patients (69%) presented with low-to-moderate parasitaemia , with a median of 5,220 parasites/μL  in infections by P. vivax . HoweverPlasmodium species.Overall, patients attended late for diagnosis, most (78.2%) of them ≥72h after symptoms onset (IQR 3–7 days). No significant differences were found in terms of days of illness or hospitalization time between Plasmodium species. In P. falciparum cases, headache (p = 0.03), musculoskeletal pain (p = 0.0001), jaundice (p = 0.0008) and probable jaundice-related pruritus (p = 0.001) were the more frequent, whereas P. vivax patients presented significantly more asthenia/adynamia (p = 0.04)   . At phys <0.001) .vs. 482/612; 79%, respectively). The most frequent clinical complications were inability to oral intake (16%), prostration (7%) and abnormal bleeding (5%). Prostration was significantly more frequent in children ≤10 years of age than in older patients .A total of 612 complications were recorded among the 323 enrolled patients, with 170 presenting two or more complication criteria (range 2–8). Clinical complications were less common than laboratory complications , 40% hepatic dysfunction , and 34% severe anaemia (Hb <7.0 g/dL). The most common laboratory complication in children ≤10 years of age was severe anaemia , whereas in patients older than 10 years of age it was hepatic dysfunction , mainly due to nfection . The majP. vivax than in P. falciparum cases  were thrombocytopenic  and 81% (264/323) were anaemic (Hb <12 g/dL), with lower Hb levels in um cases . HoweverSalmonella spp, 112 blood cultures were either negative (81.7%) and 25 (18.2%) were positive for Staphylococcus epidemidis among others, probably due to accidental contamination.Of the 323 patients with complicated malaria, 121 (37.5%) required blood transfusion, 50 with Hb <5.0g/dL, 53 with Hb <7.0 g/dL and ≥5.0 g/dL and the remaining 18 with Hb <12.0 g/dL and ≥7.0 g/dL and blood cultures were performed in 137 patients. Only one patient presented a bacteraemia by Hepatic function was abnormal in most patients, as 147/186 (79%) presented elevated bilirubin levels (>1 mg/dL) and 169/226 (78%) elevated aminotransferases AST or ALT levels (>39 U/L). No associations between indirect bilirubin levels and haemoglobin levels were found among patients with hyperbilirubinemia. Renal function as per creatinine (>1.5 mg/dL) and BUN (>40 mg/dL) levels was altered in a low percentage of cases .P. falciparum . Their median age was 22 years (range 11–43 years) and the median gestational age was 30 weeks (range 10–40 weeks). Eight were primigravidae and the median parasitaemia was 4,800 parasites/μL for P. vivax  and 12,000 parasites/μL for P. falciparum . The main complication was severe anaemia , followed by severe thrombocytopenia , and hyperparasitaemia . Only one 20-year-old patient with mixed infection, first pregnancy, and unknown gestational age, presented an abortion. Most patients were discharged before delivery in good clinical conditions . Three gave birth during the hospitalization and one of them underwent a caesarean section.A total of 30 women were pregnant, mainly from Quibdó  and infected by P. falciparum, who died <72 hours after admission. The other patient was a 19-year-old male with P. vivax malaria who presented neurological impairment, hepatic dysfunction, severe thrombocytopenia and a HIV co-infection diagnosed during hospitalization. He presented progressive neurological decline and persistence of fever despite antimalarial treatment. The death occurred 13 days after hospital admission and was probably related to a central nervous system opportunistic infection. Although cerebrospinal fluid examination was not performed, the blood culture was negative.Two patients from Quibdó died during the study period. One was a 4-year-old female child who presented cerebral malaria, history of multiple seizures, severe anaemia, thrombocytopenia, and hyperparasitaemia due to Plasmodium species and the age range of complicated malaria patients. Most patients were adults presenting with P. falciparum malaria and moderate parasite counts. Severe thrombocytopenia was the most common complication overall and was significantly more common in patients older than 10 years of age. Severe anaemia was predominantly found in children, who were mostly infected by P. vivax, and hepatic dysfunction was the main complication in adults presenting P. falciparum infections. Two deaths due to P. vivax and P. falciparum each were registered, both with multiples complication including cerebral malaria.During a two year-long period a total of 305 complicated malaria cases were recorded in two malaria endemic areas of Colombia: Tumaco and Quibdó. Eighteen cases were enrolled in Cali, a non-endemic city that usually serves malaria patients who are referred from Buenaventura and Quibdó. Important associations were found between the complication parameters, the Because the recruitment of complicated malaria patients was performed at the best-equipped hospitals in each region, and strictly following the WHO and MoH guidelines, it appears this led to discordant results with the official SIVIGILA records. In 2015, SIVIGILA recorded total of 190 complicated malaria cases in the study region, 114 in Quibdó, 56 in Tumaco and 20 in Cali, whereas we recorded 162 complicated cases  . This ~1P. vivax, which is in contrast with previous Colombian reports in which the prevalence of this species ranged between 44–76% [P. falciparum is the most frequent parasite species. Despite P. vivax was for many years the predominant malaria species in Colombia, an increase in total malaria cases together with P. falciparum malaria prevalence have been reported over the last two years, from 18,642 of P. vivax and 18,343 of P. falciparum cases (45%) in 2014 [Although in the present study, only 28.2% of cases were caused by n 44–76% ,24, it i in 2014  to 33,05 in 2014 .P. vivax than in P. falciparum, similar to what has been reported in other sites of Colombia  [P. falciparum produces higher parasitaemias than P. vivax, as the latter only invades immature erythrocytes; higher mean parasite counts have also been reported in vivax than in falciparum complicated malaria patients in Sudan  [P. falciparum, as in this study where 87% of patients with hyperparasitaemia  were infected by this parasite species.In this series, most patients (75%) presented with moderate parasitaemia  and a higher median parasitaemia was found in saralda) ,24. Althctively) . HoweverPlasmodium species. Almost 90% of cases presented thrombocytopenia of any grade  with 43% of these corresponding to severe thrombocytopenia  cases. In contrasts to these study findings, there is evidence that thrombocytopenia is more frequent in P. vivax, than P. falciparum patients [Thrombocytopenia was the most common laboratory complication but no significant differences were found between patients , althougpatients . In somepatients . Howeverpatients .P. vivax complicated malaria was more common in children under 10 years of age. This is higher than reported in a systematic review of the Brazilian literature where P. vivax complicated malaria was recorded in all age groups, with 25% of P. vivax cases affecting children 0–14 years of age [P. vivax is considered to be a disease of children (< 9 years) because acquisition of immunity to this species occurs much faster than for P. falciparum in highly endemic areas [We found that s of age . In Soutic areas .P. vivax than in P. falciparum cases  and in 34% it was severe (Hb <7 g/dL); this is in agreement with previous studies in Colombia where anaemia prevalence in complicated malaria cases ranged between 75 and 92%. However, severe anaemia has been reported in up to 52% of cases ,16,18. Aum cases . Indeed,children ,47.et al reported that hepatic and pulmonary compromise were significantly more frequent in P. vivax, whereas cerebral malaria and renal dysfunction were present in P. falciparum infections regardless of age [P. falciparum cases  the patients, which is in agreement with recent reports from India , but contrast with the reports from Brazil  in the same study [P. vivax patients were treated with Chloroquine, including the 138 who had initiated treatment at the POC, before enrolment at the hospital. In our series, Chloroquine pre-treatment appear to associate with any particular malaria complication, as previously suggested [Interestingly, Chaparro-Narvaez s of age . On the um cases , in whomum cases . Conversme study . Whereasuggested .Two cases of death secondary to malaria infection were found in this study, both of them with multiple complications. Although they were recruited in Quibdó, their sites of origin were Sipi and Alto Baudó, two rural areas of Chocó that are very distant from the capital. This may represent a risk factor for the development of greater complications and death in those patients. However, the reported duration of illness was no significantly longer than in other cases.P. falciparum infection must be primarily assessed. Future prospective studies must evaluate all the required clinical and laboratory parameters in complicated and non-complicated malaria cases in order to find clear risk factors associated with disease severity.A limitation of the present study was that qPCR confirmation of malaria parasite species was not possible in ~40% of cases, as most of the patients received treatment before their recruitment at study hospitals, and therefore qPCR was negative. Thus, recruitment of complicated malaria patients should be performed at small hospitals or local point–of-care in rural areas where patients live. However, difficulties accessing these settings make this unfeasible. Additionally, as some of the laboratory results were taken from hospital, these were not complete in all patients, particularly the biochemistry tests. This study provides valuable information for the clinical approach and monitoring of complicated malaria patients in Colombia. Based on these results, physicians in endemic settings must strategically look with special attention for haematological parameters, mainly platelet counts as severe thrombocytopenia is the main abnormal feature among complicated malaria patients regardless of parasite species. Additionally, haemoglobin levels particularly in children and P. vivax patients, and hepatic function in adults with P. vivax and high frequency of hepatic and renal dysfunction in patients with P. falciparum, demand attention from health care professionals in order to rapidly identify and properly treat these malaria-related complications. Therefore, this study provides evidence that more conservative definitions for complicated malaria ensure a better patient management, although duration of hospitalization and ICU stay were similar in both groups criteria.The high prevalence of complicated malaria in the Pacific Coast of Colombia, along with more frequent severe anaemia in children infected by


Malariometric information is needed to decide how to introduce malaria vaccines and evaluate their impact in sub-Saharan African countries.Plasmodium falciparum and other Plasmodium species and gametocyte density by microscopy, and rapid diagnosis test in the event of fever within 24 h. Case report forms were used to evaluate malaria control measures and other factors.This cross-sectional study (NCT01954264) was conducted between October and November, 2013, corresponding to the high malaria transmission season, in four sites with Health and Demographic Surveillance Systems (DSS) [two sites with moderate-to-high malaria endemicity in Burkina Faso (Nouna and Saponé) and two sites with low malaria endemicity in Senegal (Keur Socé and Niakhar)]. Children (N = 2421) were randomly selected from the DSS lists of the study sites and were stratified into two age groups (6 months–4 years and 5–9 years). A blood sample was collected from each child to evaluate parasite prevalence of Plasmodium falciparum was identified in 707 (29.2%) children, with a higher prevalence in Burkina Faso than Senegal (57.5 vs 0.9% of children). In Burkina Faso, prevalence was 57.7% in Nouna and 41.9% in Saponé in the 6 months–4 years age group, and 75.4% in Nouna and 70.1% in Saponé in the 5–9 years age group. Infections with other Plasmodium species were rare and only detected in Burkina Faso. While mosquito nets were used by 88.6–97.0 and 64.7–80.2% of children in Burkina Faso and Senegal, other malaria control measures evaluated at individual level were uncommon. In Burkina Faso, exploratory analyses suggested that use of malaria treatment or any other medication within 14 days, and use of insecticide spray within 7 days decreased the prevalence of malaria infection; older age, rural residence, natural floor, grass/palm roof, and unavailability of electricity in the house were factors associated with increased malaria occurrence.Plasmodium falciparum infection prevalence in children younger than 10 years was 57.5% in Burkina Faso and 0.9% in Senegal, and variability was observed, among others, by age, study site and malaria control measures. In sub-Saharan Africa, malaria remains a major cause of morbidity and mortality, especially in young children . In 2013In many African countries, malaria control programmes have been implemented since 2000, including the use of insecticide-treated nets (ITNs), long-lasting insecticidal nets (LLINs), indoor residual spraying of insecticides (IRS), rapid diagnostic tests (RDTs), and effective anti-malaria medicines . AlthougAdding a malaria vaccine to the control programmes has been identified as a key component to complement current interventions , 9. RTS,Plasmodium falciparum and other Plasmodium species, the extent of use of malaria control measures, and other factors in four sites, where phase IV studies on RTS,S/AS01 were expected to be conducted. These results are needed to anticipate the impact of RTS,S/AS01 if this vaccine is implemented in focalized pilot programmes or national vaccination programmes.Malariometric information is needed to guide decisions on how to prioritize interventions and introduce malaria vaccines. Among others, the level of malaria transmission, which is very heterogeneous within geographical areas, may have an effect on the efficacy of malaria control measures, including RTS,S/AS01 , 14. TheThis epidemiological, cross-sectional study was carried out at four sites with Health and Demographic Surveillance Systems (HDSS) in Burkina Faso and Senegal NCT01954264). The first site was located in the area of Nouna at about 300 km from Ouagadougou (northwestern Burkina Faso), where malaria is hyper- to holo-endemic, the malaria transmission peak occurs at the end of the rainy season (June to October), and reduced transmission is observed during the dry season (December to May) . The sec54264. ThParticipants were children aged ≥6 months to <10 years, whose parents or legally acceptable representative had provided informed consent prior to any study-specific procedure. Children in care, or actively participating in any trial involving administration of an investigational malaria vaccine or drug were excluded. Children were randomly selected from the HDSS list at each site, and were stratified into two age groups for the analyses, as defined by the Joint Technical Expert Group (JTEG) criteria: children aged 6 months–4 years and children aged 5–9 years. Analyses were performed on all evaluable children for whom at least one laboratory result was available.This study was conducted during or just after the rainy season, at the peak of malaria transmission. Data were collected at one visit between 19 October and 25 November, 2013, and no follow-up was done , main house construction material , main source of drinking water, and availability of electricity.Each study site was requested to provide centre-specific information about control measures from the malaria control programme in the study area and, if available, meteorological data for the study site, such as rainfall and temperature. The information was collected in the form of a questionnaire recorded in a separate database.P. falciparum and pLDH (Plasmodium lactate dehydrogenase) specific to Plasmodium vivax . If the RDT was positive or the child was identified as being parasite-positive following microscopy, treatment was given according to national guidelines.Approximately 200 µL of whole blood was collected from all participants. A RDT was performed in the event of recent history of fever (axillary temperature ≥37.5 °C measured at the time of visit or reported in the previous 24 h). The RDT was used to detect HRP-II (Histidine-rich protein II) specific to , and counts for P. falciparum were made for both gametocytes and asexual parasites. Two slides, each containing a thick and thin blood smear, were prepared; positive parasitaemia was identified on the thick blood film, and species on the thin blood film, except in case of low parasitaemia. The parasite presence and density were determined independently by two readers for the same slide; if readings were judged to be discordant, a third independent read was organised. A child was defined as infected by a specific parasite if at least two readings were positive for the corresponding parasite. If the final decision was positive, the parasite density (parasites/μL) was calculated as the geometric mean of the two positive readings . The following classes were used to categorize the parasite densities: low (<2500 parasites/μL); medium (2500–9999 parasites/μL); high , and very high .The presence of parasites on capillary blood samples was assessed by 100-field microscopic examination, assuming 8000 leukocytes/µl of blood. The count was made by species A child was defined as having gametocytes when gametocytes were detected during at least one reading. In children with a positive status, the gametocyte density (units/μL) was defined as the geometric mean of the readings of the positive slides.The target sample size was 2400 children (600 at each participating site), according to the following stratification by age groups: 6 months-<1 year-olds (N = 60), 1 and 2 year-olds (N = 120 per group), 3 and 4 year-olds (N = 50 per group), and 5, 6, 7, 8, and 9 year-olds (N = 40 per group). To allow for 10% non-response, 660 children over all age categories were randomly selected from the demographic surveillance system databases of each site. This sample size ensured sufficiently narrow confidence intervals (CIs) around centre-wise PP estimates (with a maximum relative standard error of 0.25) in children from the 6 months–4 years (JTEG definition) and 29 years (WHO definition) age groups for sites with low (<10%), moderate (10–50%), or high (>50%) endemicity.Continuous variables were described with mean, standard deviation (SD), median, and range. Categorical variables were described in frequency tables with absolute numbers and percentages. For some categorical variables, 95% CIs were also calculated.P. falciparum infection status was evaluated as exploratory analysis by centre in univariate logistic regression analyses and summarized using unadjusted odds ratios (ORs) with 95% CIs. A potential impact was detected if the 95% CI of the unadjusted ORs did not include the value ‘1’. The statistical analyses were performed using the Statistical Analysis Systems, version 9.2.Potential differences between groups were detected in exploratory analyses based on non-overlapping 95% CIs. The impact of various malaria control measures and other factors on the In this study, 2421 children were enrolled, with a similar distribution among the four sites. A few minor protocol deviations were reported (inconsistencies in terms of RDT testing based on the temperature), but no child was excluded from the total cohort randomly selected to participate in the study. A total of 1210 children were enrolled in Burkina Faso (610 in Nouna and 600 in Saponé) and 1211 children in Senegal (603 in Keur Socé centre and 608 in Niakhar). Among all participants, 52.3% were boys, with an even gender distribution among the four centres. The proportion of children living in rural areas was higher in the two Senegalese centres (≥99.8%) and in Saponé (≥98.7%) than in Nouna (53.6% in the 6 months–4 years and 51.7% in the 5–9 years age groups)  and in terms of children with fever within the last 24 h (25.7% of infected children vs 11.6% of non-infected). In 5–9 years old children in Saponé, 0.7 and 15.0% of infected and non-infected children received any malaria treatment in the past 14 days, and 2.1 and 15.0% of infected and non-infected children received other medication in the past 14 days, respectively.There was a greater likelihood of fever in the last 24 h for children with very high malaria parasite density (48.4%) compared to low (15.8%) or negative (10.8%) parasite density. Moreover, the overall proportion of children hospitalized for malaria in the last 3 months tended to be higher in children with very high malaria parasite density (6.5%) compared to those with low (3.4%) or negative parasite results (1.8%).P. falciparum was identified in 80.6 and 86.0% of children with gametocytes in the 6 months–4 years and 5–9 years age groups, respectively.Gametocytes were more frequently detected in children from the centres with a higher PP . In the Senegalese centres, the number of gametocytes was extremely low, with a proportion of 0.3 and 0.0% in Keur Socé, and 1.0 and 0.5% in Niakhar in the same age groups. However, gametocytes were also frequently detected in children with low parasite density: 39.5% of 6 months–4 years old children and 64.9% of 5 to 9 years old children with gametocytes had a parasite density <2500 parasites/µL. Plasmodium species were identified in the Senegalese centres. In Burkina Faso, infection with P. malariae was observed in eight/407 (2.0%) and 12/203 (5.9%) children in Nouna, and 15/399 (3.8%) and 20/201 (10.0%) children in Saponé in the 6 months–4 years and 5–9 years age groups, respectively. Infection with P. ovale was observed in nine children. No infection with P. vivax was recorded.No other The actual implementation of recommended malaria control measures was evaluated at the site level. ITNs were distributed for free in Niakhar, but were not free of charge in Nouna. No policy regarding the distribution of ITNs was implemented in Saponé, and no information was reported for Keur Socé. LLINs were distributed for free to pregnant women in Nouna, to the entire population with a replacement period of ≥24 months in Saponé and Keur Socé, and to children younger than 5 years and pregnant women in Niakhar. Artemisinin-based combination therapy (ACT) was given for free to patients with malaria symptoms and positive RDT test at the four sites, except in Saponé where ACT was not free of charge.Among the malaria control measures evaluated at the individual level, mosquito nets were the most frequently used in both countries Table . In BurkIn Burkina Faso, the secondmost common malaria control measure was the use of mosquito coils, with a larger extent in Nouna (8.4% of children) compared to Saponé (0.8% of children). In Senegal, the secondmost common malaria control measure was the use of traditional repellents, which were not used in Burkina Faso, and the highest number of children using traditional repellents was registered in Niakhar (37.3% of children).P. falciparum parasitaemia  ) , and in 06–0.8]) . In Burkmeasures . Howevermeasures . This diP. falciparum was lower in infants than in older children, which is in line with previous observations [In this study, the prevalence of rvations . Potentirvations , 24, 25 Plasmodium species were scarce, with no infection with P. vivax and only a few cases with P. malariae and P. ovale, which is in line with other studies in Burkina Faso [Infections with other ina Faso , 26, andP. falciparum parasitaemia after the rainy season in the Nouna area [In Burkina Faso, malaria control policies include free distribution of ITNs, intermittent preventive treatment for pregnant women, treatment of malaria cases with ACT, and seasonal malaria chemoprevention . In Seneuna area . In Seneuna area ) were thP. falciparum infection status included the use of malaria treatment or other medications in the past 14 days and the use of insecticide sprays. Other factors influencing malaria occurrence included age, the main house construction material , lack of electricity in the house, and rural residence.In Burkina Faso, exploratory analyses suggested that malaria control measures having an impact on the The main strengths of this cross-sectional study are the indicators of the high quality of data collection, as no child was excluded from the total cohort, and the low number of protocol deviations, which were all considered as minor. The limitations included the low number of sites per country and the small sample size per site. Moreover, the results from a single cross-sectional survey should be interpreted cautiously because they do not take annual fluctuations of malaria transmission into account and they may not accurately represent the average MTI in endemic areas. The PP in Saponé could have been slightly underestimated since the peak in malaria transmission occurs in September in this area . AnotherP. falciparum infection status were based on exploratory analyses, which should be interpreted with caution because there was no adjustment for multiplicity and no analysis on multi-collinearity was done before the regression.The estimation of PP could be improved if microscopy results were confirmed by molecular detection techniques. Polymerase chain reactions (PCR) have a higher sensitivity than microscopy, but this technique is not frequently used in routine clinical practice due to its high cost. An estimate of PCR prevalence could be obtained by using a tool that has been developed to assess the relationship between microscopy and PCR measures . FinallyP. falciparum was much higher in the two sites in Burkina Faso with moderate-to-high malaria endemicity compared with the two sites in Senegal with low malaria endemicity. Mosquito nets were frequently used in both countries, but additional malaria control measures evaluated at individual level were uncommon. Exploratory analyses suggested that control measures having an impact on the P. falciparum infection status included the use of malaria treatment, other medications and insecticide sprays. In the centres with moderate-to-high malaria endemicity, age, main house construction material and lack of electricity in the house were also associated with an increased malaria occurrence. The high PP observed in the two sites in Burkina Faso suggests that new malaria control measures are needed in these areas.This study showed that the prevalence of


Plasmodium falciparum malaria. This has been supported by several studies in which clinical malaria cases of P. falciparum malaria were reported to be associated with low antibody avidities. This study was conducted to evaluate the role of age, malaria transmission intensity and incidence of clinical malaria in the induction of protective humoral immune response against P. falciparum malaria in children living in Burkina Faso.High parasite-specific antibody levels are generally associated with low susceptibility to P. falciparum antigens: Merozoite Surface Protein 3 (MSP3), Merozoite Surface Protein 2a (MSP2a), Merozoite Surface Protein 2b (MSP2b), Glutamate Rich Protein R0 (GLURP R0) and Glutamate Rich Protein R2 (GLURP R2) in plasma samples from 325 children under five (05) years with age, malaria transmission season and malaria incidence.We combined levels of IgG and IgG subclasses responses to P. falciparum infection in low transmission season compared to high malaria transmission season. While, parasite density was lower in low transmission than high transmission season. IgG against all antigens investigated increased with age. High levels of IgG and IgG subclasses to all tested antigens except for GLURP R2 were associated with the intensity of malaria transmission. IgG to MSP3, MSP2b, GLURP R2 and GLURP R0 were associated with low incidence of malaria. All IgG subclasses were associated with low incidence of P. falciparum malaria, but these associations were stronger for cytophilic IgGs.We notice higher prevalence of On the basis of the data presented in this study, we conclude that the induction of humoral immune response to tested malaria antigens is related to age, transmission season level and incidence of clinical malaria. P. falciparum malaria burden. It has been shown that there is a relationship between antibody production  and susceptibility to clinical malaria [Children and pregnant women in sub-Saharan African are carrying the global  malaria , 2. Bouh malaria . Similar malaria –7.There are several merozoite surface proteins with no specific function, including MSP2; MSP3 and GLURP . Many ofP. falciparum, endemicity of malaria and clinical incidence [MSP3 and GLURP have been tested in malaria vaccine antigens that have already undergone phase 1 trials. The efficacies of these malaria vaccines will be tested in phase II and III vaccine trials in Africa. For the purpose of the evaluation of the efficacy of these planned vaccine trials, it is important to investigate the induction of humoral immune response in protection against these vaccines antigens. However, the epidemiological evidence of the protective effect of naturally acquired anti-merozoite responses is not specifique. There could be many reasons for these inconsistencies. In malaria endemic areas the rate at which natural immunity develops is dependent on age, intensity and stability of exposure to ncidence , 16. ThiP. falciparum is the deadliest specie that causes more than 95% of infections [An. gambiae, and An. funestus. From February to May, the number of infectious bites per person per night ) was negligible. However, the EIR increased from June to September and decreased from September to November and remained low until the next rainy season [The study was conducted in four villages randomly selected out of the 74 villages of the Saponé Health District (SHD). The SHD is located at the center of Burkina Faso at 50 km south-west of Ouagadougou. In SHD, the climate is characteristic of the Sudanese savannah with two seasons: a rainy season from June to October and a dry season from November to May where the malaria transmission is nearly absent. Malaria is endemic and its transmission in these areas is very high during the rainy season. fections . The priy season , 17.Study population was children from 6 months to 5 years fulfilling the following inclusion and exclusion criteria.Inclusion criteria were (i) written/thumb printed informed consent obtained from the parent or legal guardian of each child enrolled to the study; (ii) permanent resident in the study area for at least 3 months prior to enrollment and expected to remain at least for the all the period of longitudinal follow up; (iii) aged between 6 months to 5 years.Exclusion criteria were (i) major congenital defect or any chronic disease  diagnosed by a physician/nurse based on medical history and physical examination; (ii) anemia defined as hemoglobin value less than 6 g/dl and (iii) any other circumstances and condition suspected by the physician to be the risk for the child health.Only children fulfilling study inclusion criteria and attending both cross-sectional surveys were enrolled to the study. Data obtained by cross-sectional survey was used to reach study objectives. Children were enrolled for the assessment of malaria infection and immunological endpoints in relation to protection against clinical malaria, age and malaria transmission level.Two cross-sectional surveys were carried out during low (January 2007) and high (September 2007) malaria transmissions seasons. During each cross-sectional survey, 5 ml of venous blood in a tube containing EDTA was collected from each child for a complete blood count. The remaining blood was centrifuged and aliquots of plasma were created and stored at −20 °C for immunological analysis. Thick and thin blood films were prepared from finger prick for microscopy diagnosis of malaria. Axillary temperature was measured at once. Children with fever, defined as axillary temperature ≥37.5 °C or history of fever reported within the last 24 h, had a malaria rapid diagnostic test (RDT) performed. A child with a positive test result was referred to the nearest health center for appropriate treatment of malaria which was given free of charge.A longitudinal survey with active case detection of malaria episodes was conducted starting from the beginning of the first cross-sectional survey. Twice weekly, each child was monitored clinically by a study nurse who resided in the village. During each visit, information regarding health status was recorded and axillary temperature was measured as described previously. If the child had fever or history of fever reported within the last 24 h, a RDT was performed. In addition, thick and thin blood films were prepared and sent to the Centre National de Récherche et Formation sur le Paludisme (CNRFP) laboratory for the determination of parasite density by using light microscopy. Patients with positive RDTs were referred at once to the nearest health care center for free treatment.Plasmodium falciparum parasite is identified by examining thick and thin blood films. Each slide was air-dried and stained with 5% Giemsa to give a parasites a distinctive appearance and was read by two different laboratory technicians. A slide was declared negative only after reading against 2000 white blood cells without observation of a malaria parasite. For blood smears collected during the cross-sectional survey, the number of parasites per microliter of blood was calculated according to the number of white blood cells obtained from a completed blood count; however, parasite densities for slides collected during the longitudinal survey were calculated assuming an average of 8000 white blood cells/µL of blood. In case of difference over the presence or absence of malaria parasites between different readers, or if parasite density estimates differed by more than 30%, the slide was read one again by a third laboratory technician. The arithmetic mean of the two final readings was used as the final parasite density. If there was no agreement after the third reading, the arithmetic mean of the two most closed results was considered.IgG and IgG subclass levels to merozoite surface antigens MSP3, MSP2a, MSP2b, GLURP R0 and GLURP R2 Table  were meaData was performed using EPI info version 6.0. Data generated from assays in the form of ELISA OD values were entered into Microsoft Excel worksheets. SAS software Version 9.2 was used to perform analyses.2 transformed values. The Wilcoxon signed-rank sum test was used to compare geometric mean parasite densities across transmission seasons. Age adjusted incidence rate ratios against multiple malaria episodes was determined by Poisson regression.Incidence rates of malaria which included varied thresholds of parasitemia  and axillary temperature ≥37.5 °C or history of fever within 24 h were calculated for the following time periods: date of 1st cross sectional survey to date of 2nd cross sectional survey. Malaria episodes occurring within 28 days of a previous episode were not treated as incident episodes. When calculating person time in analyses of multiple malaria episodes, 28 days following a new episode was subtracted from total time at risk. This was done to ensure that the infection causing the episode was recorded and subsequent episodes within the 28-day window were not counted. Overall incidence of malaria was calculated using the number of malaria events divided by time at risk. Geometric means for antigens were estimated using logWe performed immunological analysis of samples from 325 children under 5 years old (mean age 2.71 years and the sex ratio M/F 1.12). Participants were followed up from February 2007  to December 2007  to assess malaria clinical episodes and incidence.P. falciparum infection during low and high transmission seasons was 59.69 and 50.76%, respectively. By contrast, the mean parasite density was lower during low malaria transmission season compared to high transmission season; 2627 parasites/µL versus 6042 parasites/µL. Using increasingly stringent definitions of malaria by increasing the parasitemia threshold, during each of low and high transmission seasons, the incidence of malaria decreased. However, regardless of the clinical malaria definition, the incidence rates were always higher during high malaria transmission season.As shown in Table Children were categorized into five age groups to assess the age-dependent response of IgG to the tested antigens . In our study, older children had higher IgG responses than younger children for all tested antigens. Results are shown in Fig. Levels of specific IgG and IgG subclasses antibodies in 325 children were compared between low and high malaria transmission seasons , IgG4 to MSP2a, IgG2, IgG3 and IgG4 to MSP2b  and IgG1 to GLURP R0 (p = 0.2085).Clinical malaria incidence rates was performed as previously described , 14, 17 Among the four IgG isotypes, the two cytophilic classes (IgG1 and IgG3) were most associated with decreased malaria incidence. Association was strongest for IgG1 to MSP3 , GLURP R0  and GLURP R2  respectively for parasitemia threshold >0 parasites; parasitemia threshold >2500 parasites and parasitemia threshold >5000 parasites (Table p = 0.01302 and p = 0.03444) and IgG4 (p = 0.02417 and p = 0.02739); MSP2b (p = 0.03784 and p = 0.015) and GLURP R2 (p = 0.00056 and p = 0.00061) respectively for parasitemia more than 2500 parasite and 5000 parasites.When IgG2 and IgG4 were considered, there was no evidence of an association with malaria incidence for parasitemia >0, there was a moderate association, with borderline statistical significance of IgG4 to GLURP R2,  using standardized methods with age and malaria transmission season as done in previous studies [This study aimed was to continuously support malaria vaccine development based on specific  studies , 14, 17.P. falciparum infection prevalence was high in low transmission season compared to high transmission season. This finding is surprising because most malariometric parameters have been reported to be highest during malaria high transmission seasons in seasonal and stable malaria transmission areas. However in this study there was biweekly active follow-up, where children were treated with artemisinin combination therapy (ACT) when (a) axillary temperature was ≥37.5 °C and (b) there was reported history of fever within last 24 h, with a positive rapid malaria diagnosis test. Repeated treatment with ACT during these malaria illnesses might prevent malaria infection throughout the period of greatest malarial risk. As expected, the asexual parasite load and clinical malaria incidence regardless of parasite density thresholds peaked during the malaria intense transmission period. As shown in previous studies, these patterns are characteristic of our study area [Our data showed that udy area –20.As shown in this paper, recent studies with blood-stage antigens carried out in areas where malaria is endemic have reported increasing antibody (IgG and IgG isotypes) levels with age , 14. ThiP. falciparum. However we cannot exclude that this observation may also be due to a random variation. As shown in other studies, cytophilic antibody levels to the five antigens tested in this study were higher than non-cytophilic ones independent of the malaria transmission season, emphasizing their importance in anti-malaria immunity [Seasonal variation of IgG antibody responses to malaria parasite antigens has already been assessed in malaria endemic settings, where the responses were influenced by seasonal variation , 23. In immunity , 24–26. immunity , 27, 28.P. falciparum clinical malaria, but we find no evidence for associations with MSP3, MSP2a and MSP2 for malaria episodes defined by a parasitemia threshold >0. For malaria definitions with parasitemia thresholds >2500 and >5000, in addition to GLURP R0 and GLURP R2, we observed that IgG to MSP2b was correlated with reduced risk of P. falciparum clinical malaria, suggesting that by increasing the sensitivity of our clinical malaria case definition, IgG to these three antigens provide strong evidence of associations with protection against malaria. Previous studies carried out in the same area found that there is an association between the antibody responses to MSP3 and GLURP long synthetic peptides at the beginning of the malaria high transmission season and the reduced risk of clinical malaria [Previous studies using human sera from people living in malaria endemic areas have found evidence of an association between the levels of total IgG to MSP3 and GLURP and a reduced subsequent risk of clinical malaria –31. For  malaria , 32. The malaria .IgG1 responses to MSP3, GLURP R0 and GLURP R2 were associated with a reduced risk of malaria when the analysis is adjusted with age for all parasitemia thresholds defined. The same observation was seen for IgG3 to MSP3, and GLURP R2, with significant associations with IgG3 to MSP2b also observed for the >2500 and >5000 thresholds. These data suggest a role for cytophilic IgGs in protection against clinical malaria. IgG2 and IgG4 to MSP3 and GLURP R2 and IgG4 to MSP2b were also associated with a reduced risk of malaria. These data confirm those previously reported suggesting that IgG2, which preferentially binds the Fcγ receptor IIa 131H allele, is associated with protection against severe malaria –37. HoweOn the basis of the data presented in this study, we conclude that natural acquired immunity to malaria antigens is related to age and transmission season levels in children less than 5 years. All the tested antigens are inducing antibody responses, which are associated with clinical malaria protection. Notwithstanding the factors that may affect the pattern of the immunological responses induced by these malaria vaccine candidates, the data presented in this paper are strongly in support of the inclusion of these antigens in malaria vaccine formulations.