Plasmodium falciparum have been associated with protection from clinical malaria in independent studies. We determined whether this protection was allele-specific by testing whether children who developed clinical malaria lacked IgG/IgG3 antibodies specific to the dominant msp2 parasite genotypes detected during clinical episodes. We analysed pre-existing IgG and IgG1/IgG3 antibodies to antigens representing the major dimorphic types of MSP-2 by ELISA. We used quantitative real-time PCR to determine the dominant msp2 alleles in parasites detected in clinical episodes. Over half  of infections contained both allelic types. Single or dominant IC1- and FC27-like alleles were detected in 46% and 42% of infections respectively, and both types were equally dominant in 12%. High levels of IgG/IgG3 antibodies to the FC27-like antigen were not significantly associated with a lower likelihood of clinical episodes caused by parasites bearing FC27-like compared to IC1-like alleles, and vice versa for IgG/IgG3 antibodies to the IC1-like antigen. These findings were supported by competition ELISAs which demonstrated the presence of IgG antibodies to allele-specific epitopes within both antigens. Thus, even for this well-studied antigen, the importance of an allele-specific component of naturally acquired protective immunity to malaria remains to be confirmed.IgG and IgG3 antibodies to merozoite surface protein-2 (MSP-2) of  All antibodies were assayed in serum samples collected during a cross-sectional survey at the start of a malaria transmission season in May 1995. As the antibodies were collected before the clinical episodes, they are referred to as ‘pre-existing’ antibodies. Parasite DNA was collected from subsequent samples collected when children presented to hospital with acute clinical malaria. Ethical approval was granted by the Kenya National Research Ethics Committee.A case–control study of clinical malaria in which antibodies to a panel of P. falciparum contain a central domain comprised of repeats that vary in number, length and sequence, flanked in turn by nonrepetitive variable sequences, and by conserved N- and C-terminal domains. Dimorphic, nonrepetitive sequences internal to the N- and C-termini distinguish the two main allelic families (3D7/IC1-like or ‘type A’ and FC27-like or ‘type B’) (Merozoite surface protein-2 sequences of type B’) . For simm primers , 1 μL of template and the ROX™ dye for detection. Initial amplification steps were 50°C for 2 s then 95°C for 10 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, then a dissociation step of 95°C for 15 s and 60°C for 1 min, with a final post-dissociation cooling step of 95°C for 15 s. The dissociation step was used to exclude contamination and to determine the dissociation temperatures of the amplicons. The expected amplicon sizes were 65 and 76 base pairs for the msp2 type A and type B alleles, respectively. The default threshold setting of 0·2 was used to analyse all the data. The average amplification efficiency was determined from the slope of the standard curve for each reaction using the equation E = 10(−1/slope)−1.Reactions were performed using the 7500 Real-Time PCR System (Applied Biosystems). Twenty-five microlitre reactions were set-up in 96 well optical plates (Applied Biosytems) using Quantitect SYBR Green PCR Master Mix , 100 nP. falciparum clones ‘Malayan Camp’  and ‘Dd2’ (type B at the msp2 locus) were grown to 5–10% parasitaemia. A total of 100 μL of each of the culture media (20 μL pellet) was used to extract parasite genomic DNA using DNA Qiamp mini-kits . The amount of genomic DNA was quantified by UV spectrophotometry and adjusted to 10 ng/μL for each of the two standards. Tenfold serial dilutions were then prepared for each (MC and Dd2), in the range of 10–0·00001 ng and were run separately for each allelic type on each plate to enable comparable quantification of each major allelic type.Laboratory cultures of P. falciparum . Genomic DNA from each sample was amplified by RTQ-PCR in separate reactions on the same plate using primers for type A and type B alleles. Standards for each allelic family were run simultaneously on each plate for quantification. Samples were considered positive, if they contained >0·001 ng/μL of genomic DNA. This threshold was the lowest amount that could be reproducibly detected as determined from the standard curves run on each plate. Samples were considered to be dominant for one allelic type, if they contained at least double the amount of the alternative allelic type. The proportions of either allelic type of genomic DNA were calculated from the total amount of DNA detected in ng/μL from each allele-specific reaction. These experiments were designed to detect the dominant Escherichia coli fused to a Glutathione S-transferase (GST) sequence for purification, and each represents a major allelic type of MSP-2 (MSP-2_CH150/9 (type A) and MSP-2_Dd2 (type B)) ) , and then = 89) have been reported as part of a previous analysis . Diluted sera were pre-incubated with an excess (1000 ng) of competing heterologous antigens for 5 h, before addition to each ELISA plate well coated with 50 ng of homologous antigens and subsequent performance of the ELISA in the standard method. This ensured that only IgG antibodies to allele-specific epitopes of the homologous antigen were detected, while those to conserved epitopes would have been absorbed in the pre-incubation step. As a control, an aliquot of the same serum sample was also pre-incubated with an excess (1000 ng) of homologous antigen, before addition to ELISA plates containing 50 ng of the same antigen. The average ELISA OD in the homologous competition assays was 0·156 for the type A antigen and 0·221 for the type B antigens. The median difference in ELISA OD between heterologous and homologous competition assays was 0·510 range 0·014–1·920) for the type A antigen, and 0·386 (range −0·117 to 1·918) for the type B antigen. A difference of >0·3 ELISA OD units between the heterologous and homologous absorption assays was considered to indicate substantial allele-specific reactivity (–1·920 foLogistic regression was used to examine the effect of sero-positivity (or high levels) of total IgG to each MSP-2 antigen on the odds of getting a clinical episode caused by parasites bearing the corresponding homologous parasite alleles, compared with parasites bearing the alternative heterologous alleles. Analyses were conducted first for single allele infections and then repeated including children in whom a single dominant allelic type was detected. In separate analyses, the proportions of children who were sero-positive (or had high levels of IgG antibody) to each antigen were compared amongst children in whom a single or dominant corresponding allelic type was detected.P-values.The effects of high levels of IgG1 and IgG3 antibodies against each of the MSP-2 antigens on the risk of clinical disease were analysed using modified Poisson regression models as previously described . These amsp2 was confirmed by conducting cross-reaction tests, where type A genomic DNA (MC) was amplified with type B primers and vice versa [type B genomic DNA (Dd2) amplified with type A primers], with positive and negative controls. Type A genomic DNA was only amplified by type A primers and the positive control primers, but not by type B primers. Similarly, type B genomic DNA was only amplified by type B primers and the positive control primers, but not by type A primers. The dissociation curves for each allelic type showed single, allele-specific peaks free of contamination. Preliminary experiments in which laboratory cultured parasites of either msp2 allelic type were mixed in known proportions were highly reproducible on four independent runs  than type B alleles, this was not significant . A single allelic type was detected in 45% of the samples, while the remainder contained both type A and type B alleles. Neither allelic type was over-represented amongst children presenting with mild or severe malaria . There was no bias in the distribution of either allele amongst all age groups of children . Dominant (including single) type A alleles were detected in 46% (67/146) of samples, while dominant type B alleles were detected in 42% (61/146) of samples. Neither allelic type was dominant in children presenting with mild or severe malaria, or in any particular age category. A dominant allelic type  could not be confidently distinguished in a minority of samples, 18/146 (12%).Data on parasite genotypes was available for 146/165 study children who had a clinical episode of malaria. Parasite material was collected at the time of presentation to hospital with an acute clinical episode of malaria. One sample was negative by RTQ-PCR for both msp2 allelic types (above). The prevalence of IgG sero-positivity was 65% (95/146) for the type A antigen and 65% (95/146) for the type B antigen, while that of high level IgG antibodies was 20% (29/146) and 25% (37/146) for the type A and B antigens, respectively. Total IgG antibodies to the type A and B antigens were highly correlated, pairwise correlation coefficient P = 0·67, P < 0·001, and only a minority of children showed clear allele-specific IgG antibody reactivity when antibodies to full-length MSP-2 antigens were examined by indirect ELISA .Antibodies were analysed in samples collected during a cross-sectional survey at the start of a malaria transmission season. This was before the children presented with acute clinical episodes. Overall, the IgG antibody prevalence and levels to both MSP-2 antigens were similar and reflected the prevalence of the ct ELISA . Total Iz = −7·3, P < 0·001), while for the MSP-2 type B antigen the distributions of IgG1 and Ig3 antibodies were similar . We found that IgG3 antibodies to both the type A and B antigens were significantly associated with protection from clinical malaria, while IgG1 antibodies to both antigens were not. Children with high levels of IgG3 to the MSP-2 type A and B antigens were significantly less likely to develop clinical disease than those with low levels, RR 0·35 (95% CI 0·14–0·88) P = 0·026, and RR 0·22 (95% CI 0·08–0·60) P = 0·003, respectively. In contrast, children with high levels of IgG1 to the type A antigen were not protected from clinical malaria when compared to those with low levels (RR 1·04 (95% CI 0·56–1·93) P = 0·885). Although children with high levels of IgG1 to the type B antigen were less likely to develop clinical malaria than those with low levels, this was not significant [RR 0·57 (95% CI 0·30–1·12) P = 0·108].We had previously reported that total IgG antibodies to MSP-2 were strongly associated with protection from clinical malaria in this population . We furtmsp2 alleles. We therefore investigated whether children sero-positive for type A IgG antibodies were less likely to be infected with parasites containing type A compared to type B alleles, than sero-negative children and vice versa for type B IgG antibodies and alleles  type A infections were often sero-negative for the corresponding type A IgG antibodies, and per se to protection cannot be directly ascertained. To address this, we selected children whose clinical episodes were caused by parasites containing a dominant (or single) msp2 allelic type and who had high levels of pre-existing IgG antibodies to the homologous MSP-2 antigen. We performed competition ELISAs on these sera to verify that they contained IgG antibodies to allele-specific epitopes within the MSP-2 antigens. Sixteen children had high levels of pre-existing IgG antibodies to MSP-2 type A and subsequent clinical episodes caused by type A parasites. Competition with excess of the type B antigen revealed that 13/16 children had IgG antibodies to allele-specific epitopes within the type A antigen. Eleven children had high levels of pre-existing IgG antibodies to the type B antigen and subsequent clinical episodes caused by type B parasites. Competition with excess of the type A antigen revealed that 8/11 children had IgG antibodies to allele-specific epitopes within the type B antigen. Thus, the majority of these children developed clinical episodes despite having allele-specific IgG antibodies homologous to the major allelic types of the parasites that infected them.It may be argued that because full-length antigens utilized in this study contain allele-specific as well as conserved epitopes, the contribution of allele-specific epitopes P. falciparum were associated with protection from clinical episodes of malaria  protected against symptomatic episodes caused by type A parasites (In the population studied here, we previously found that high levels of IgG antibodies to MSP-2 were strongly associated with protection against clinical malaria . Furtherarasites .P. falciparum transmission. Although the sample size was limited, they found that pre-existing allele-specific IgG antibodies did not predict clinical outcome in infections with parasites bearing homologous rather than heterologous msp2 alleles. Clinical outcome was defined as the prevalence or severity of symptomatic infections, though none of their study participants developed severe malaria during follow-up (While many studies have examined the infecting MSP-2 parasite genotype and the corresponding allele-specific antibody response, these have been performed in concurrent samples –24 or inollow-up . A limitollow-up . Rather,Taken together, these data show that although pre-existing IgG/IgG3 antibodies to antigens representing the major dimorphic types of MSP-2 are strongly associated with a reduced incidence of clinical malaria in the majority of children , a minorThese data should be considered in the context of the evidence for immune selection on parasite diversity  and the


Plasmodium falciparum HRP-2-based rapid diagnostic test for malaria (RDT(Paracheck®). Given the higher cost of ACT compared to earlier anti-malarials, the objectives of the present study were i) to study the accuracy of Paracheck® compared to the thick blood smear (TBS) in two areas with different levels of malaria endemicity and ii) analyse the cost-effectiveness of the strategy of the parasitological confirmation of clinically suspected malaria cases management recommended by the NMCP.In 2006, the Senegalese National Malaria Control Programme (NMCP) has recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria and, in 2007, mandated testing for all suspected cases of malaria with a A cross-sectional study was undertaken in the villages of Dielmo and Ndiop  nested in a cohort study of about 800 inhabitants. For all the individuals consulting between October 2008 and January 2009 with a clinical diagnosis of malaria, a questionnaire was filled and finger-prick blood samples were taken both for microscopic examination and RDT. The estimated costs and cost-effectiveness analysis were made considering five scenarios, the recommendations of the NMCP being the reference scenario. In addition, a sensitivity analysis was performed assuming that all the RDT-positive patients and 50% of RDT-negative patients were treated with ACT.A total of 189 consultations for clinically suspected malaria occurred during the study period. The sensitivity, specificity, positive and negative predictive values were respectively 100%, 98.3%, 80.0% and 100%. The estimated cost of the reference scenario was close to 700€ per 1000 episodes of illness, approximately twice as expensive as most of the other scenarios. Nevertheless, it appeared to us cost-effective while ensuring the diagnosis and the treatment of 100% of malaria attacks and an adequate management of 98.4% of episodes of illness. The present study also demonstrated that full compliance of health care providers with RDT results was required in order to avoid severe incremental costs.A rational use of ACT requires laboratory testing of all patients presenting with presumed malaria. Use of RDTs inevitably has incremental costs, but the strategy associating RDT use for all clinically suspected malaria and prescribing ACT only to patients tested positive is cost-effective in areas where microscopy is unavailable. Malaria is a major cause of morbidity and mortality and the large, round numbers that delineate its burden have now become familiar. In 2006, WHO estimated that 3.3 billion persons were at risk of malaria infection of whom 1.2 billion were at high risk, mostly in Africa 49%). Of the estimated one million annual deaths due to malaria, approximately 91% of them were thought to occur in Africa and 85% in children under five years of age [9%. Of thPlasmodium spp. with a sensitivity of 100% and of giving rapid results (15 to 20 minutes) [Plasmodium falciparum secreted from erythrocytes infected with rings, trophozoites, schizonts and immature gametocytes. HRP-2-based tests have been shown accurate in detecting P. falciparum infections [Plasmodium lactate dehydrogenase (pLDH) is the other major targeted antigen. This enzyme is expressed at high levels in all blood-stages of the parasite, and all four human malarial parasites produce a unique pLDH activity. In addition, pLDH antibodies can also be used to detect Plasmodium knowlesi [Plasmodium infections and while following anti-malarial efficacy [Plasmodium glycolytic pathway [Plasmodium aldolase are pan-specific and have been used in combination with HRP-2, making tests capable of distinguishing an infection with P. falciparum from that due to non- P. falciparum.Therefore, as recommended by the World Health Organization (WHO), rapid diagnostic tests for malaria (RDT) offer this opportunity. They have the advantage of being simple to perform, easy to interpret, requiring minimal infrastructure and thus are particularly indicated where microscopy, still considered as the gold standard, is not available. In order to overcome the problems of availability of diagnostics the NMCP started in 2007 to provide health care facilities with a rapid diagnostic test for malaria (RDT). Different types of RDTs based on immunochromatographic antigen capture are marketed. According to WHO, they must be capable of reliably detecting 100 parasites/μl (0.002% parasitaemia) from all minutes) . The mosfections ,9. Plasmefficacy . Lastly, pathway . MonocloP falciparum is predominant and where skilled microscopy is not readily available [® , an HRP-2-based test with the objective to achieve the revised targets set by African Heads of State in Abuja in 2000, of 80% population coverage by 2010 [An earlier study in Senegal showed that HRP-2-based tests may be especially useful in areas where vailable . In 2007 by 2010 . The mai by 2010 -17. Sinc® compared to TBS in two areas with different levels of malaria endemicity and to conduct a cost-effectiveness analysis of the diagnostic and treatment strategy recommended by the Senegalese NMCP, taking into account the public health impact of the introduction of ACT and RDTs.The objectives of this study were to evaluate, under field conditions, the accuracy of ParacheckThe study was undertaken in the villages of Dielmo and Ndiop, located in a sahelo-soudanian region of Senegal approximately 280 km south-east of Dakar, and 10 km north of the Gambian border , and presenting with a clinically-suspected malaria  requiring a TBS. Excluded were individuals presenting with severe malaria or any other severe illness and those declining to give consent for this study.For all individuals consulting at the dispensary with a clinical diagnosis of malaria, finger-prick blood samples were taken both for microscopic examination, in accordance with the cohort protocol. For each patient included, a RDT was done and a standardized questionnaire was filled by the health personnel. All RDTs were carried out and interpreted before reading the blood smear, following manufacturers' instructions, by technicians present in each village. A second control reading of the slides was done by an experienced microscopist from the Institut de Recherche pour le Développement (IRD) in Dakar who was masked to the RTD and field slide results. All TBS readings followed a standard protocol,  with a tThe term "episode of illness" refers to any clinically-suspected cases of malaria according to the health worker's perception while "malaria" refers only to episodes of illness confirmed by positive control readings of the blood smears. The sensitivity, specificity, positive and negative predictive values of RDTs were calculated using the TBS as the reference test, as recommended by WHO . ConfideP. falciparum parasitaemia. For this reason, the primary measure of effectiveness was the proportion of patients that would have been correctly managed , the reference being the result of the blood smear. The secondary measure of effectiveness was the proportion of all malaria attacks that would have been treated with ACT. Full compliance of health care providers with tests results was assumed, what is consistent with some published studies [One objective of using RDTs is to limit as much as possible the prescription of ACT to people who actually have  studies -26.Five scenarios were considered:Presumptive treatment of all the febrile episodes of illness (body temperature ≥ 38°C): scenario frequently found in the literature and which supposes that all febrile episodes of illness are treated with ACT [1. with ACT ,28.Presumptive treatment of some episodes of illness according to the healthcare provider's feeling: here, was taken into account the attitude that the healthcare provider would have adopted in the absence of any malaria diagnostic tool. It is a more pragmatic approach, nearer to the field reality than the first scenario. It is also the attitude which was prevailing before the RDTs were deployed in Senegal.2. Treatment of all episodes of illness RDT positive: it corresponds to the current recommendations of the Senegalese NMCP, i.e. performing a RDT in clinically suspected malaria case and prescribing ACT if tested positive. This was the scenario of reference.3. Treatment of febrile episodes of illness with positive RDT: in this scenario, a RDT is performed in case of body temperature > 38°C only and ACT prescribed in the event of positive RDT. This scenario, which can constitute an alternative to the current recommendations of the NMCP, is also found in the literature [4. terature ,29.Treatment of all children under six and treatment of all episodes of illness RDT positive for patients over six: a scenario recommended by the WHO 2006 treatment guidelines.5. However, other studies have demonstrated that at least 50% of malaria-negative patients are treated with anti-malarial drugs ,31. For Epi-info Version 3.5.1  was used for data entry and Stata Version 10  for statistical analysis. Cost-effectiveness analysis results were displayed using the diagram proposed by Drummond .The Dielmo-Ndiop program, which was used as support for this work was approved by the Ministry of Health and the Prevention's National Ethics Committee in 2006 . A separate approval was also obtained from this committee for the present study. Written informed consents were obtained from all the participants in the study (or guardians of children under 15 years of age).A total of 189 consultations for clinically suspected malaria were recorded between October 2008 and January 2009, 122 in Dielmo and 67 in Ndiop. The proportion of females was statistically higher than males . The median age of the sample was 5.7 years . The distribution by age group was 51.8% under six years, 16.4% from six to 13 years and 31.8% older than 13 years. Age groups were the same as those used for estimating treatment doses. The distribution of the sample population by sex and age was not statistically different between the two villages.The most frequent symptom was a history of fever or a feeling that the body is hot (152 cases - 80.4%). A body temperature > 38°C was recorded in 94 cases (49.7%) at the time of the consultation. Headache and weakness were notified in 57.7% and 49.0% of cases, respectively.In half the cases (95/189) the health care provider stated that he would have given ACT on the basis of the clinical picture and in the absence of means of definitive diagnosis of malaria (blood smear or RDT). In the univariate analysis, body temperature > 38°C, diarrhoea, anorexia and an age < 6 years were associated with a presumptive treatment by ACT. The proportion of episodes of illness, which would have benefited from ACT decreased statistically with increased age (Chi² for linear trend: P < 0.001).P. falciparum gametocytes on the TBS. The six-month old child had no clear explanation for the positive RDT results.A total of 189 TBS and 189 RDT were performed at the initial consultation. Twelve (6.4%) malaria attacks were confirmed by positive blood smears (8/122 in Dielmo and 4/67 in Ndiop). No discrepancy (positive or negative) was found in the parasite density between the first reading at the time of the initial consultation and the control reading. Rapid testing was positive for 15 cases. False positive RDT results were found in three boys of six months, five years and 11 years of age living in Dielmo, the village with higher malaria endemicity. The five year-old had been treated for slide-positive malaria 14 days prior to inclusion in the study and the 11 year-old showed the presence of A body temperature > 38°C was recorded at the time of the initial consultation for six (50%) of the 12 cases of slide-positive malaria (range 38.2 - 39.8°C). In three (25%) cases of slide-positive malaria, no fever or history of fever was recorded. In these three cases, the main symptom was intense cephalgia. Cases from the two villages were not significantly different by gender, age, temperature, or parasite density.®, in comparison with TBS readings, by village and for the whole sample. Follow-up RDT and TBS were performed at D4 and D7 for 10 of the 12 confirmed malaria cases. Of these 10, while the follow-up TBS readings from D4 were all negative, six (60%) had a persistent HRP-2 antigenaemia at D4 and four (40%) at D7. Five (83%) of the six positive RDT at D4 and all four positive RDT at D7 were from Dielmo.As the test characteristics for RDTs done in Dielmo and Ndiop were not significantly different, the data from the two villages were pooled. Table ® tests cost 0.61€ each. Table The non-subsidized cost of a treatment course with artesunate-amodiaquine varies according to age: 0.47€ for children from 1 to 6 years, 0.87€ for children from seven to 13 years and 1.73€ for both children of more than 13 years and adults. ParacheckScenarios 1 and 2, i.e. those based on the presumptive treatment of suspected malaria, would have resulted in the correct management of only half of the episodes of illness while scenarios 3 and 4, based on the use of the RDT, would have resulted in correct management of nearly all of the episodes . The effectiveness of scenario 5 was estimated between these two values (58.2%). However, considering the secondary measure of effectiveness, scenario 4 would have resulted in ACT treatment for only half of the malaria attacks. Table Figure Costs of scenarios 3 to 5 were estimated assuming that all the RDT-positive patients and 50% of RDT-negative patients were treated with ACT. Costs of each scenario were estimated to be around 1.5 fold higher than when assuming full compliance to the test results Table . Under tP. falciparum [The accuracy of rapid testing was very good compared to TBS. At D0, sensitivity, specificity, positive and negative predictive values were of 100%, 98.3%, 80% and 100%, respectively. These results are consistent with other published studies showing that the sensitivity and specificity of HRP-2-based tests usually are > 90% for lciparum ,34 and tlciparum ,36. Howelciparum . In zonelciparum  and in alciparum  found 52lciparum . Howeverlciparum -43, whiclciparum . In the lciparum ,46 and tlciparum ,39.Another cause of false positive results with HRP-2-based tests found in Dielmo is the presence of early gametocytes ,48. The Through scenario 2, this study took into account the attitude of the healthcare personnel whether or not to prescribe ACT in clinically suspected cases of malaria when estimating costs. Although studies assume that only febrile patients are considered to have malaria, in reality almost 20% of anti-malarials are given to patients who do not have a history of fever . While t® but also shows the increased risk of false-positive results as transmission increases, probably related to persistent antigenemia after treatment, that could lead to misdiagnoses and thus of inappropriate prescriptions of ACT. However, in Senegal zones of high endemicity remain limited and the most representative epidemiologic setting is the strictly seasonal transmission of Ndiop. Under the scenarios based on rapid testing (3 and 4), ACT would have been prescribed in only 1.7% to 11.3% of the non-malaria episodes of illness, while under the scenarios based on clinical diagnosis (1 and 2) this proportion would reach nearly 50%, consistent with Luxemburger's findings [This study confirmed the high operational accuracy of Paracheckfindings . In addifindings  and can findings . It is, The cost of the scenario corresponding to the current recommendations of the NMCP in Senegal was estimated around 700€ per 1,000 episodes of illness, approximately twice as expensive as the others scenarios considered, except for scenario 5. Nevertheless, it still appeared to us cost-effective as it ensured the correct diagnosis and treatment of 100% of malaria attacks and an adequate management of 98.4% of episodes of illness. The other scenarios, while less costly, were also less effective. Scenario 4 was close to the reference scenario when considering the primary measure of effectiveness, but it would have resulted in the correct diagnosis and treatment of only 50% of malaria cases and thus cannot be recommended for ethical and public health reasons. Scenario 5 could be a possible alternative to the reference scenario when the primary measure of effectiveness is considered.However, the sensitivity analysis of the present study demonstrated that full compliance of health care providers with RDT results was required in order to avoid severe incremental costs.Studies undertaken in sub-Saharan Africa have shown that, when ACT is used, confirming cases with RDTs was cost-effective compared to presumptive treatment when the parasite prevalence was below 62% and may help to improve management of non-malaria febrile illness, particularly bacterial infections ,51. HoweA limitation of this study was the low malaria incidence during recruitment. This incidence was the lowest observed since the Dielmo-Ndiop cohort follow-up was begun in 1990. Such a drop in incidence, observed following the deployment of ACT and vector control measures in Senegal and described elsewhere , would oAccording to the results of the present study, strategies based on presumptive treatments of clinically suspected malaria, or febrile illnesses, or on performing RDT in case of fever only lead to a high level of misdiagnosis.In settings where microscopy is unavailable, using RDT can lead to a significant reduction in the overprescription of anti-malarials  Though tThe present study confirmed the benefits in promoting the use of RDTs in remote areas where microscopy is unavailable. However, it will be necessary to repeat these studies as malaria epidemiology changes and to test RDTs targeting both falciparum and non-falciparum parasites.The authors declare that they have no competing interests.RM, ABL ATall initiated the study and participated in the design of the study. LB was the head of the Unit of Infectious Diseases Epidemiology when the study was conducted. AB participated in the field study. JF performed data management. ABL and RM performed the statistical analysis and interpreted data, with the help of CR.The paper was essentially written by RM, with the input of ABL and RP, All authors read, critically revised and approved the manuscript.


Plasmodium vivax is the species responsible for most of the cases.Approximately 40% of the world's population is at risk for malaria. In highly endemic tropical areas, malaria is a major cause of morbidity and mortality during infancy. There is a complex interrelationship between malaria, malnutrition and intestinal helminths, and this may impair cognitive development in children. The aim of this study was to determine the relationship between malaria and school performance in children living in an endemic area where th percentile for the respective class and grade.The study was conducted in the Municipality of Careiro, Amazonas, Brazil, with five to14 year-old children, studying the first eight grades of public school, during the year 2008. After an initial active case detection, during nine months of follow-up, passive malaria cases detection was instituted, through a thick blood smear performed in every child with fever. School performance was evaluated by the final notes in Mathematics and Portuguese Language. Performance was considered poor when either of the final notes in these disciplines was below the 50Plasmodium vivax was detected in 69.2% of the attacks, Plasmodium falciparum in 25.5% and both species in 5.3%. In the multivariate analysis, adjusting for age, mother's education, time living in the study area and school absenteeism, presenting with at least one episode of malaria independently predicted a poor performance at school .The total number of students followed-up in the cohort was 198. Malarial attacks were reported in 70 (35.4%) of these students, with no cases of severe disease. P. vivax is responsible for the majority of the cases.Non-severe malaria compromises the school performance of children even during a nine-month follow-up, potentially contributing to the maintenance of underdevelopment in countries endemic for malaria. This is the first evidence of such impact in Latin America, where  Approximately 40% of world's population live in areas that have some risk of malaria infection. Every year more than 500 million people are infected, particularly children, and they are more susceptible to severe manifestations of the disease . The majPlasmodium vivax is frequent [Despite the implementation of malaria control strategies in Brazil, children are still affected by the disease on a large scale. In 2008, the annual parasite index (API) in older than 14 years-old was 11 cases/1,000, however the API for children from five to 14 years of age was 14.3/1,000 [frequent .et al [P. falciparum infection, but also appear to occur after less severe infections. The effects of cumulative or repetitive episodes on cognitive development of children need more clarification, as well as information about the effects of P. vivax infection on cognition, the major parasite found in the Americas.Studies in children with severe falciparum malaria  focusing on neurological sequelae show some impairment in developing cognitive abilities after the acute episode, both in the short-term -8 and thet al , deficitP. vivax (84.4% of the reported cases in 2008) than P. falciparum infections, this cohort study was conducted in an unstable transmission endemic area for malaria in the Western Brazilian Amazon, where severe malaria is rarely seen. The principal aim was to assess whether the occurrence of malarial episodes independently influences a child's performance in school.Considering that Brazil has more The Municipality of Careiro, in the Amazonas State (Western Brazilian Amazon), 112 km from the capital of the state, Manaus  followed by primaquine (0.5 mg/kg/day for 7 days). In the case of P. falciparum malaria, artemether/lumefantrine is prescribed for three days as the first-line regimen. During the follow-up period, children with symptoms of malaria submitted a thick blood smear for the diagnosis of malaria (passive case detection). All the teachers were informed about the objectives of the study and reported school absenteeism to the investigators.Students from two schools in grades 1-8, between 6-14 years old were followed from March to December 2008 in a cohort. In March, active case detection of malaria through thick blood smear and the haemoglobin (Hb) concentration were performed in all children from the schools . Children positive for idelines . In the Two thick blood smears were collected on the same slide, stained with Giemsa and examined under 1,000× magnification by the local microscopist in a laboratory of each community. The microscopists were formally and regularly trained according to the standard methods of slide reading. A minimum of 100 microscopic fields were examined for the final diagnosis of a negative smear.® photometer , in the cross-sectional evaluation.Haemoglobin concentration was measured in venous blood obtained from digital puncture, using a portable HemoCueth percentile for his classroom and grade. This was done to avoid the biases of evaluation by different teachers with distinct levels of qualification with the intent to compare children with their colleagues and year group.Two disciplines were selected to evaluate the school performance: Portuguese Language and Mathematics, because both are taught to children in all of the studied grades. To assess school performance the teacher's evaluation of each classroom was used, which is based on the evaluating system recommended by the Brazilian Ministry of Education . In each grade, a student was considered to have a poor performance if his final score in Portuguese Language and/or Mathematics was under the 50Weight and height were obtained by internationally recommended methods . Weight Odds Ratio (OR) with its respective 95% Confidence Interval (95% CI) was determined considering the school performance as the dependent variable, with a statistical significance at the level of 5%. Logistic regression was used for the multivariate analysis and the adjusted Odds Ratio with 95% CI was also calculated.A database was created in SPSS 16.0 software . Proportions were compared with chi-square test, using Fisher exact test when necessary. The crude A stepwise procedure for selecting variables in the multivariate analysis was used.The study protocol was approved by the Ethics Review Board of the Tropical Medicine Foundation of Amazonas . Both the Municipal Secretariat of Education and the school managers allowed the performance of the study. After educations regarding the objectives of the project, parents or guardians signed an informed consent.P. falciparum and was treated with oral artemether/lumefantrine. During the passive case detection, malarial attacks were registered in 70 (35.4%) students. 46 of these had only one malarial attack during the follow-up period, 21 children had two attacks and three children had three attacks. Considering all the malarial episodes, P. vivax was the involved species in 69.2%, P. falciparum in 25.5%, and both species in 5.3%. No severe case was clinically detected and no child needed hospitalization. At the end of the cohort follow-up, overall poor performance was detected in 94 (47.5%) school children.A total of 198 students were enrolled, 105 (53.0%) male and 93 (47.0%) female. In the preliminary cross-sectional evaluation, nutritional status was examined in 170 children, resulting in 143 (84.1%) with normal status, 20 (11.8%) in risk of malnutrition and 7 (4.1%) with malnutrition. Only one asymptomatic child was positive for Table P. vivax malaria.School performance in the native language (Portuguese) and/or Mathematics in a cohort of 198 children five to 14 years-old was associated with at least one episode of malaria during a nine-month follow-up interval, in an area highly endemic for P. vivax was also the major species, found the same association. Working with five to six year-old children, they also observed that the performance at school entry was poorer in children with history of five or more malarial attacks [Fernando et al , working attacks . These rChildren's cognitive development can be influenced by many factors, such as morbidity by infectious diseases, nutritional status (notably iron deficiency), anaemia, parental education, school absenteeism and other social, economical and environmental factors -19. In tLow mother's education has been considered as a risk factor for severe malaria in African children , but forSchool absenteeism is described as a relevant outcome of malarial infection, but it is still controversial. In Kenyan endemic areas, severe malaria was responsible for 3-8% of school absenteeism . On the P. falciparum. This is not unexpected in the Brazilian Amazon, with predominant P. vivax infections and easy and free access to diagnosis and treatment. In this area, therefore, the poor performance in school reflects essentially the effects of non-severe malaria, which is increasing in Latin America [Effects of malaria infection on cognition can be divided into direct effects of cerebral injury, as a consequence of severe malaria, and indirect potential impacts on performance mediated by chronic infection, repetitive infections and associated anaemia/malnutrition -27. No s America .In a similar endemic area in the Brazilian Amazon (Acre State), malaria morbidity was strongly associated with land clearing and farming . The impPlasmodium are treated, students improve their attention at school [There is scarce evidence that asymptomatic parasitaemia affects children's cognition in the long-term. When asymptomatic carriers of t school .Malaria is still considered a poverty related disease, affecting primarily people with the lowest socioeconomic status and difficult access to health services, leading to long-term impairment of health and education ,32. ManyStrictly urban areas would be affected to a lower extent, in part due to lower malaria incidence rates and a better quality of teaching . There hThe comparison of our data with others is limited by the distinct manner of evaluation of poor performance, since there is no standardized tool which could be applied worldwide due to cultural issues . HoweverThe previous episodes of malaria before the study were not analysed due to memory bias, and other factors associated with poor performance were not explored.P. vivax as the predominating species. Hence, social, economical and cultural development is potentially impaired in these tropical localities.Non-severe malaria causes a negative impact on school performance during an academic year in an unstable transmission endemic area in the Brazilian Amazon, with The authors declare that they have no competing interests.SVS designed the project, led field work, interpreted the results and drafted the manuscript. RCRL performed the statistical analyses, interpreted the results and drafted the manuscript. TRAP contributed with data collection in the field. MVGL designed the project, interpreted the results and critically read the manuscript. All authors read and approved the manuscript.


In a prospective cohort study of newborns residing in a malaria holoendemic area of Kenya, Christopher King and colleagues find a subset of children born to malaria-infected women who acquire a tolerant phenotype, which persists into childhood and is associated with increased susceptibility to malarial infection and anemia. Malaria in pregnancy can expose the fetus to malaria-infected erythrocytes or their soluble products, thereby stimulating T and B cell immune responses to malaria blood stage antigens. We hypothesized that fetal immune priming, or malaria exposure in the absence of priming (putative tolerance), affects the child's susceptibility to subsequent malaria infections.n = 246), (ii) exposed not sensitized , or (iii) not exposed . Exposed not sensitized children had evidence for prenatal immune experience demonstrated by increased IL-10 production and partial reversal of malaria antigen-specific hyporesponsiveness with IL-2+IL-15, indicative of immune tolerance. Relative risk data showed that the putatively tolerant children had a 1.61  and 1.34  greater risk for malaria infection based on light microscopy (LM) or PCR diagnosis, respectively, compared to the not-exposed group, and a 1.41  and 1.39  greater risk of infection based on LM or PCR diagnosis, respectively, compared to the sensitized group. Putatively tolerant children had an average of 0.5 g/dl lower hemoglobin levels (p = 0.01) compared to the other two groups. Exposed not sensitized children also had 2- to 3-fold lower frequency of malaria antigen-driven IFNγ and/or IL-2 production (p<0.001) and higher IL-10 release (p<0.001) at 6-month follow-ups, when compared to sensitized and not-exposed children. Malaria blood stage–specific IgG antibody levels were similar among the three groups.We conducted a prospective birth cohort study of 586 newborns residing in a malaria-holoendemic area of Kenya who were examined biannually to age 3 years for malaria infection, and whose malaria-specific cellular and humoral immune responses were assessed. Newborns were classified as (i) sensitized (and thus exposed), as demonstrated by IFNγ, IL-2, IL-13, and/or IL-5 production by cord blood mononuclear cells (CBMCs) to malaria blood stage antigens, indicative of in utero priming  and life-threatening organ damage and anemia (a lack of red blood cells).Each year, The age-dependent pattern of susceptibility to malaria suggests that young babies are protected by antibodies provided by their mothers, but that by 6 months old, when these antibodies have largely disappeared, babies have not yet fully developed their own anti-malaria immunity. However, little is known about the acquisition of anti-malaria immunity in infants, a process that needs to be understood in order to design effective vaccines for this age group. In particular, it is unclear how maternal malaria infection affects the acquisition of anti-malaria immunity. Malaria in pregnancy may expose the unborn child to malaria-infected red blood cells and to soluble malaria antigens (molecules that the immune system recognizes as foreign). This exposure could increase or decrease the child's immune responses to blood-stage malaria antigens and thus affect his/her ability to fight off malaria. In this study, the researchers investigated how prenatal malaria exposure affects anti-malaria immunity in young children and their susceptibility to subsequent malaria infections.P. falciparum before birth by looking for parasites in their mother's blood at delivery. They looked for malaria-specific immune responses in T cells (a type of immune system cell) in the newborn babies' cord blood by measuring the production of cytokines (molecules that either activate or inhibit the immune system) by these cells after exposure to malaria antigens. Finally, they examined the infants twice yearly for 3 years for malaria infection, malaria-specific immune responses, and anemia. The researchers classified the babies into three groups; cord blood cells of “sensitized” babies made activating cytokines in response to malaria antigens; cord blood cells of “exposed, not-sensitized” babies did not make activating cytokines but made an inhibitory cytokine (IL-10); and “not-exposed” babies were born to mothers with no P. falciparum infection at delivery. In their first 3 years of life, the exposed, not-sensitized group had a 60% greater risk of malaria infection (measured by counting parasites in their blood) than the unexposed group and a slightly higher risk of malaria infection than the sensitized group. They also had lower hemoglobulin levels (a sign of anemia) than the other babies. At age 6 months, the T cells of exposed, not-sensitized children were less likely to make activating cytokines in response to malaria antigens but made more IL-10 than the T cells of the other children; malaria-specific antibody levels were similar in the three groups.The researchers determined which of 586 newborn babies enrolled into their study in an area of Kenya where malaria is very common had been exposed to These findings suggest that some children who are exposed to malaria before birth become “tolerant” to blood-stage malaria antigens. Exposure to malaria antigens before birth “tricks” their T cells into recognizing these antigens as self antigens. This immune tolerance, which persists into childhood, reduces the ability of the immune system to attack and destroy parasites and increases the susceptibility of these tolerant children to malaria infection. Why some children who are exposed to malaria before birth become tolerant while exposure to malaria antigens “primes” the immune system of other children to respond efficiently to these antigens is not clear. However, these findings could have important implications for the design of malaria vaccines for use in areas where children are often exposed to malaria before birth and for the design of strategies for the prevention of malaria during pregnancy.http://dx.doi.org/10.1371/journal.pmed.1000116.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Lars HviidThis study is further discussed in a World Health Organization on malaria Information is available from the malaria (in English and Spanish)The US Centers for Disease Control and Prevention provides information on Roll Back Malaria Partnership on all aspects of global malaria control, including information on malaria in pregnancy and on children and malariaInformation is available from the malaria (in English and Spanish)MedlinePlus provides links to additional information on  Plasmodium falciparum (Pf) can develop due to repeated infections Falciparum malaria is one of the most important pediatric infectious diseases in sub-Saharan Africa, where it is estimated to kill at least 1 million children per year. In areas where malaria transmission is stable, infants and children typically experience clinical morbidity and high blood stage parasite densities. Infants up to 6 months old appear to be relatively resistant to clinical malaria and high-density parasitemia, while children between 6 and 36 months of age appear to have increased susceptibility to these clinical and parasitological outcomes The physiologic and immunologic effects of maternal malaria infection on the fetus complicate the acquisition of anti-malaria immunity in children. Pf-infected erythrocytes have the propensity to sequester in the intervillous blood of the placenta, a condition referred to as placental malaria. Placental malaria is most common in women with their first or second pregnancy, seemingly due to the lack of acquired immunity to Pf clones that preferentially bind to the placental vasculature 19, a protein required for entry of the parasite into red blood cells The significance of fetal malaria experience is underscored by epidemiologic studies suggesting that offspring of women with placental malaria are more susceptible to malaria during infancy than those of mothers without placental malaria We describe here the results of a prospective cohort study of infants born in a malaria-holoendemic area of Kenya. The study was designed to test the hypothesis that prenatal malaria experience, reflected by maternal malaria infection at parturition and neonatal cord blood T cell sensitization to several malaria blood stage invasion ligands, could influence susceptibility to malaria infection and risk of anemia in the first 3 years of life. To understand the mechanism associated with the altered risk for malaria, we measured whether this prenatal experience influences acquisition of cellular and humoral immune responses to these same invasion ligands, as measured biannually in children during the first 3 years of life. Merozoite surface protein 1 (MSP1) is the most abundant merozoite protein and a leading malaria vaccine candidate Approval for the study was obtained from the Kenya Medical Research Institute National Ethical Review Committee and the Institutional Review Board for Human Studies at Case Western Reserve University. Mothers provided witnessed written informed consent for participation and assent for their infants. All women included in the study were screened for HIV as part of a volunteer counseling and testing (VCT) program supported by the International Center for Reproductive Health and the Kenyan Ministry of Health starting in 2002. Counseling and perinatal treatment with nevaripine was provided through this program. All HIV results were kept strictly confidential. For women recruited before 2002 who had not participated in the VCT program, and following written informed consent, stored frozen plasma samples (at −80°C) from mothers were examined by HIV-ELISA as described Pregnant women were recruited from the antenatal clinic at Msambweni District Hospital, Kwale District in Coast Province, Kenya. They underwent a detailed questionnaire that queried their education level, spouse's occupation, and household income. Malaria transmission in this area is stable, with seasonal variation related to rainfall P. falciparum per microliter of blood, assuming a leukocyte count of 8,000/µl. In the PCR method, DNA was extracted from 200 µl of whole intervillous blood and the red cell pellet of anticoagulated maternal and cord blood (Qiagen). Real-time quantitative PCR (RTQPCR) was performed, with 2.5 µl of DNA used as a template for amplification of the multicopy Pf 18S small subunit ribosomal RNA gene Maternal venous blood, intervillous placental blood, and cord blood were examined for malaria infection status by two methods. In the LM method, thick and thin blood smears were prepared, fixed in methanol, stained with Giemsa, and examined by light microscopy (LM) for Pf-infected erythrocytes in 100 high-powered fields. The density of parasitemia was expressed as the number of asexual Wuchereria bancrofti microfilariae as described W. bancrofti was also assessed by a circulating antigen assay using stored frozen plasma samples collected at delivery, based on the Og4C3 antigen detection assay (TropBioMed). Samples were obtained during follow-up periods if participants were unable to provide samples at delivery. Women infected with intestinal helminths or schistosomes were treated with mebendazole (500 mg in a single dose) or praziquantel (40 mg/kg in a divided dose), respectively, according to the Kenyan Ministry of Health recommendations after delivery. No specific therapy was given for W. bancrofti infections, because a district-wide treatment program was started during the study (October 2002) as part of the WHO-sponsored global program to eliminate filariasis. Hemoglobin levels were assessed from fresh peripheral blood samples in children using the HemoCue system (HemoCue).Stool and urine were obtained at delivery and examined for the presence of intestinal helminths and schistosome ova, respectively, as described GYRKPLDNIKDNVGKMEDYIKK, codons 250–271; KLNSLNNPHNVLQNFSVFFNK, codons 101–121) DNVGSNQMASVRKSLR; codon 33–48), N2 , and C1  as previously described DRB1*0401, DRB1*0101, and DRB1*1501, which are common in Kenya Pichia42 was expressed in E. coli and properly refolded as described For the T cell studies in cord blood and peripheral venous blood, both peptides and recombinant antigens were used. For MSP1, two peptides were used that correspond to well-characterized T cell epitopes within the C-terminal 83 kDa fragment of MSP1 , in RPMI-1640 supplemented with heat-inactivated 10% autologous plasma, 4 mM L-glutamine, 25 mM HEPES, and 80 µg/ml gentamicin (Biowhittaker). For lymphocyte proliferation studies, CBMCs were cultured at 2×106/ml in a total volume of 200 µl in triplicate.Cord blood was obtained from the umbilical vein immediately after delivery and anticoagulated with heparin. Similarly, maternal peripheral venous blood was obtained by venipucture and anticoagulated with heparin. CBMCs and peripheral blood mononuclear cells (PBMCs) were isolated from the freshly obtained blood by density gradient centrifugation on Ficoll-Hypaque as described 6/ml in a total volume of 200 µl in triplicate for both cytokine production and lymphocyte proliferation assays. For lymphocyte proliferation studies 1 µCi of 3H-thymidine (New England Nuclear) was added to each culture and harvested 16 h later as described 3H-thymidine was measured with a beta-counter (Packard Matrix).In preliminary studies, heat-inactivated autologous plasma was not suppressive when compared to serum-free medium in activated lymphocyte cultures (unpublished data). By contrast, we occasionally observed high background and nonspecific lymphocyte reactivity using fetal calf serum or pooled human sera (unpublished data). For PBMC cultures 10% fetal calf serum was used instead of autologous plasma . Lymphocyte cultures were also performed at a density of 1×10For CBMCs the following culture conditions were used: medium alone (negative control); 10 µg/ml MSP1 P2 and P3 peptides; 10 µg/ml PfP0 N1, N2, or C1 peptide; 5 µg/ml EBA175; and PMA (50 pg/ml) plus ionomycin (1 µg/ml) in duplicate. CBMCs were cultured with individual peptides except when there were insufficient cells and MSP1 and PfP0 peptides were pooled. For PBMCs, MSP1 and PfP0 peptides were pooled for all participants, because of limited blood volume. In a subset of children with larger lymphocyte recoveries, peptides were cultured individually. Pooling peptides in CBMC or PBMC cultures did not affect the levels of antigen-induced cytokine production or lymphocyte proliferation (unpublished data). All culture supernatants were immediately frozen at −80°C following culture.In a subset of CBMCs , recombinant human IL-2 and IL-15  were added to parallel cultures, with medium alone or with malaria antigens, to assess the effect of these cytokines on reversing immune hyporesponsiveness. Lymphocyte proliferation responses, with addition of rIL-2+rIL-15 and malaria antigens, divided by lymphocytes cultured with rIL-2+rIL-15 alone , were calculated, and we designated a positive response when this ratio exceeded 2.Quantification of IFNγ, IL-5, IL-13, IL-2, and IL-10 was performed on culture supernatants collected at 72 h by ELISA 5 CBMCs or PBMCs in response to at least two malaria antigens when no IFNγ-secreting cells were present in negative control wells ; (2) in cases where IFNγ-secreting cells were observed in negative control wells, the number of spots generated by antigen-driven cord blood lymphocytes (CBLs) was at least 2-fold greater. Based on these criteria, no malaria antigen-driven IFNγ-secreting cells were detected in CBMC from 16 healthy North American newborns or PBMCs from ten malaria-naïve adults.IFNγ ELISPOT for both CBMCs and PBMCs was also performed at 48 h as described for all samples 42  and AMA1  and region II EBA175 were measured by ELISA as described previously 42 or AMA1, and 0.2 µg/ml of EBA175 in PBS. Plates were subsequently blocked with 3% bovine albumin in PBS (Sigma) to which 1∶200 final dilution of cord or peripheral blood plasma was added and incubated at 4°C overnight. Plates were subsequently washed, and a 1∶2,000 dilution of goat anti-human IgG conjugated alkaline phosphatase added and incubated for 2 h at 37°C . A pool of ten cord blood plasma samples with high antibody reactivity was used as a standard, with a 1∶200 dilution used as high standard and given the arbitrary value of 100. The pooled plasma was subsequently serially 2-fold diluted eight times to generate the standard curve. A positive control plasma sample with IgG antibodies that recognized all recombinant antigens tested was also added to each plate. Plasma from nine North American adults never exposed to malaria was used as the negative control. Positive values were greater than the mean+3 standard deviations (SDs) of the value of the individual negative control plasma samples.IgG antibodies to recombinant Pf MSP1t-test of log-transformed values, depending on data distribution and number of exposure categories analyzed. Categorical outcomes were analyzed using the Chi-square test. Repeated measures of categorical outcomes  were analyzed with generalized estimating equations All statistical analysis was performed using SAS version 9.1. Statistical comparisons of continuous variables according to prenatal malaria exposure were performed using the Mann-Whitney test, Kruskal Wallis test, or Student's An overview of the enrollment and follow-up of the participants is presented in Newborns were categorized as “exposed not sensitized,” “exposed sensitized,” and “not exposed” groups . Exposedp = 0.23, p = 0.11, and p = 0.37 respectively) and not-exposed group .The demographic characteristics of the three groups are also shown in n = 39) of 464 mothers tested were HIV positive. Mothers of the exposed not sensitized group had higher HIV positivity than offspring in the not exposed group. HIV was transmitted to 11 offspring (28%), with a similar proportion of HIV positive offspring in each group  was cultured in the presence of IL-2+IL-15 and malaria antigens and compared to cultures with IL-2+IL-15 in the absence of malaria antigens. In 21 of the exposed not sensitized children that previously lacked detectable lymphocyte proliferation responses or cytokine production, 11 (52%) had detectable antigen-specific proliferative responses in the presence of IL-2+IL-15. By contrast, malaria antigen with IL-2+IL-15 augmented four of 21 (19%) in the exposed sensitized group and two of 17 (11%) in the not-exposed children . At least one malaria infection assessed by PCR was observed in 67.2% (80/119), 64.2% (158/246), and 55.7% (121/217) of these children, respectively (p = 0.066). Since malaria transmission can vary over time and location, and to adjust for repeated measures on children, time, and location, we used a generalized estimating equation to model infection status  of exposed not sensitized (putatively tolerant) children, 35.0% (86/246) of exposed sensitized children, and 27.2% (58/217) of not-exposed children (n status . Exposedp = 0.043) or 1.78 parasites/µl , compared to sensitized children, assessed by LM or RTQPCR respectively every 6 mo, adjusted for season and location. Parasitemia was similar among infected individuals in the exposed sensitized, exposed not sensitized and not-exposed groups, respectively , indicating that differences in the frequency of malaria infection account for much of the difference in parasitemia between groups. Of note, when parity was entered in the analysis, it was not associated with child susceptibility to infection or parasitemia.When parasite density was analyzed using a repeated measures model, putatively tolerant children acquired an extra 1.30 parasites/µl  or the unexposed group .To determine whether prenatal experience influenced the risk of anemia, hemoglobin levels were measured over the period of follow-up for offspring that were sensitized, putatively tolerant or not exposed . HemogloIn order to understand the immunologic basis for the increased susceptibility of putatively tolerant offspring to malaria, we examined how prenatal exposure would affect the child's subsequent acquisition of cellular and humoral immune responses. A positive cellular response was defined as one or more cytokines and/or lymphocyte proliferation response to the malaria antigen or peptide, as indicated in the 42, or AMA1 with increasing age between the three study groups as determined from 12 mo of age  of children that acquired IgG antibody responses to MSP1o of age . Both aly of age . Antibody of age . General+ T cells in CBMCs from malaria-exposed newborns and in PBMCs from children, but not in cells from newborns in North America Results from the current study support the hypothesis that fetal malaria experience contributes to regulation of childhood malaria immunity in endemic areas where infection during pregnancy is common. We first described a subset of children whose mothers are infected with malaria in late pregnancy, but whose cord blood lymphocytes obtained at delivery failed to produce common T cell cytokines to several malaria blood stage antigens. We speculate that these children are likely to be exposed to malaria blood stage antigens in utero, but generate an immune phenotype that we refer to as putatively tolerant. This immune tolerant phenotype is based on the observation that approximately 90% of these children consistently failed to generate specific malaria blood stage antigen–induced Th1-type cytokine responses, compared with approximately 50% of other children, even though the risk of infection tended to be higher in the immune tolerant group. Further, these immune tolerant children showed increased malaria antigen–driven IL-10 production by cord blood lymphocytes that persisted through childhood, suggesting the presence of T regulatory cells specific for malaria blood stage antigens +CD25brightFoxP3+ cells in cord blood of malaria infected women that can suppress antigen-driven CD4+CD25dim cells . Brustoski and colleagues bright cells abrogates parasite-specific IL-10 production and increases IFNγ-specific responses, suggesting a population of IL-10–secreting T regulatory cells in newborns. Alternatively, there may be clonal deletion of malaria-specific lymphocytes, as previously demonstrated in offspring of malaria-infected mice Further evidence for prenatal immune experience in the putatively tolerant group is that antigen-specific proliferation responses can be detected in over half of a randomly selected subset of tolerant newborns following the addition of IL-2 and IL-15, indicative of lymphocyte anergy An important consequence of this antenatally acquired immune “tolerance” is increased risk for malaria infection during childhood. Prior studies have demonstrated that offspring of women with placental malaria and/or malaria infection at delivery are more likely to be infected with malaria A potential mechanism for this increased risk became apparent when offspring of infected women were divided into groups of children exposed to malaria in utero, in which immune priming to malaria antigens resulted, and children potentially exposed to malaria in utero but where evidence for immune priming by CBMCs was lacking (termed putatively tolerant). Tolerant children had a 61% and 34% higher incidence of malaria based on LM and PCR, respectively, using a generalized estimating equation that accommodates the individual correlation among repeated malaria measurements, as compared to not exposed children There was a stronger association with susceptibility to malaria infection among the putatively tolerant group as measured by LM compared to PCR diagnosis. This is not surprising since PCR can detect more infections with low-level parasitemia, ones less likely to result in overt clinical disease.42 and AMA1. It is possible that these measures do not provide a good correlate of protective immunity, and more restricted antibody responses may be involved. In this context, immune tolerant children failed to acquire antibodies to EBA175 compared to other children, although the overall antibody response to EBA175 was lower than to the other antigens. We have also previously observed that tolerant children in the same population had impaired functional antibody responses to MSP119There are several potential mechanisms that may increase susceptibility to malaria among tolerant children. The failure of 90% or more of tolerant children to acquire antigen-specific T cells that produce a Th1-type cytokine profile during the first 3 years of life may result in inadequate T cell support for antibody production and/or antibody-dependent cell mediated immunity. Humoral immunity among tolerant children, however, was similar to that observed in the other groups, as measured by the overall proportion (and levels) of malaria-specific IgG directed toward MSP1Putatively tolerant children were also at increased risk for developing anemia, showing mean hemoglobin of 0.5 g/dl lower during the follow-up period than children in the other groups. Previous studies have shown that babies born to anemic mothers  are more susceptible to anemia , relative to mothers with sensitized offspring (37%) and unexposed women and newborns (32%). This observation suggests that the amount or duration of malarial antigen exposure, or time of exposure during gestation, may determine whether a fetus becomes tolerant or sensitized. It is possible that some tolerant newborns may not have been exposed to malaria antigens during gestation, even though the mother was infected. This possibility is hard to exclude, although our preliminary data suggest that MSP1 is commonly found in cord blood from infected women, often as immune complexes , this coThere are several limitations in this study. First, children were only followed biannually for the presence of malaria infection and hemoglobin levels, so malaria infections occurring between sampling times would be missed. Second, mothers were only assessed for malaria at delivery and placental histopathology was not available. Uninfected women at delivery may have been infected earlier during pregnancy. This would have misclassified some putatively tolerant children as not exposed. The effect on the study would be to reduce the observed differences between the tolerant and not-exposed groups. Some children died during the first year after birth and were not included in the cohort. We were able to ascertain seven deaths, but could not determine whether these children died of malaria or not. This could introduce a bias into the results. Finally, the presence of clinical malaria in children was not examined. This omission reduces our ability to assess whether fetal immune tolerance protects against symptomatic malaria by modulating the host inflammatory response. This possibility is suggested by reports of asymptomatic malaria in children over 6 months of age that had been closely followed since birth Once the significance of fetal malaria experience is better understood, it should translate into more effective strategies for malaria chemoprophylaxis during pregnancy. Malaria chemoprophylaxis that completely prevents infection during pregnancy, for example, may reduce fetal exposure, thereby preventing the development of immune tolerance. If this reduces both the risk of infection and disease in offspring, then such treatment would be desirable. However, if fetal immune tolerance reduces the risk of clinical disease, then prenatal chemoprophylaxis should be targeted to limiting disease and not infection per se, and linked with intermittent presumptive antimalarial chemoprophylaxis during infancy.


An evaluation of the short-term impact on childhood malaria morbidity of mass distribution of free long-lasting insecticidal nets (LLINs) to households with children aged 9-59 months as part of the Togo National Integrated Child Health Campaign.The prevalence of anaemia and malaria in children aged zero to 59 months was measured during two cross-sectional household cluster-sample surveys conducted during the peak malaria transmission, three months before  and nine months after the campaign  in three districts representative of Togo's three epidemiological malaria transmission regions: southern tropical coastal plains (Yoto), central fertile highlands (Ogou) and northern semi-arid savannah (Tone).In households with children <5 years of age, insecticide-treated net (ITN) ownership increased from <1% to >65% in all 3 districts. Reported ITN use by children during the previous night was 35.9%, 43.8% and 80.6% in Yoto, Ogou and Tone, respectively. Rainfall patterns were comparable in both years. The overall prevalence of moderate to severe anaemia (Hb < 8.0 g/dL) was reduced by 28%  and mean haemoglobin was increased by 0.35 g/dL (95% CI 0.25-0.45).The effect was predominantly seen in children aged 18-59 months and in the two southern districts: PR (95% CI) for moderate to severe anaemia and clinical malaria: Yoto 0.62 (0.44-0.88) and 0.49 (0.35-0.75); Ogou 0.54 (0.37-0.79) and 0.85 (0.57-1.27), respectively. Similar reductions occurred in children <18 months in Ogou, but not in Yoto. No effect was seen in the semi-arid northern district despite a high malaria burden and ITN coverage.A marked reduction in childhood malaria associated morbidity was observed in the year following mass distribution of free LLINs in two of the three districts in Togo. Sub-national level impact evaluations will contribute to a better understanding of the impact of expanding national malaria control efforts. Insecticide-treated nets (ITNs) have been shown to cause marked reductions in all-cause child mortality, a 50%, 29% and 13% reduction in uncomplicated malaria, high density parasitaemia and any parasitaemia, respectively, and an increase in average haemoglobin level by 1.7% packed cell volume, in children under five years of age in areas of stable transmission . This caEvaluation of short-term programme impact is a core part of current efforts to rapidly scale-up malaria control efforts, but poses real challenges . The indAs the first example of rapid national scale up of ITNs using an integrated child health campaign, the Togo experience has been the subject of extensive evaluation. This impact evaluation was part of a multi-disciplinary monitoring and evaluation effort of the Togo campaign and included evaluation of ITN coverage, childhood morbidity, economic costing and the role of social mobilization. This paper assesses the impact of the campaign on malaria-associated morbidity, and consists of a set of two repeated prevalence surveys in children below five years of age. The results of the baseline morbidity survey have been presented previously , and indIn this paper, results are presented of the September 2005 morbidity survey, conducted nine months after the campaign, and compared with the baseline results from 2004 to assess impact of the campaign on malaria morbidity in children below five years of age.A detailed description of the study sites, population, survey design and methodology has been published . Brieflyet al [The methods, planning and implementation of the campaign have been previously described by Takpa et al . During Togo is 435 km long and 100 km across at its widest point and had a population of around 5.7 million in 2002. Approximately 17.2% or 980,000 children are under the age of five years ,12. MostTwo repeated community-based cross-sectional surveys were conducted involving children <5 years in Sept 2004 (three months pre-campaign) and Sept 2005 (nine months post-campaign). Both surveys were conducted during the estimated peak malaria transmission season  surveillance statistics) and used an identical design, with the difference in prevalence of moderate-to-severe anaemia (haemoglobin level (Hb) <8.0 g/dL) in children aged 0-59 months between surveys as the main endpoint. Haemoglobin levels were chosen as the primary outcome measure as they are sensitive to changes in malaria transmission, but have the valuable property of averaging out changing effects over time, and have been recommended by the RBM Malaria Monitoring and Evaluation Reference Group (MERG) -15. As tThe study was designed to allow evaluation of impact at district level and took into account the possible presence of effect modification by study region or age. A stratified two-stage cluster sample design was used. After selection of three geographically representative districts, a total of 90 enumeration areas were selected (30 per district) by random sampling with a probability proportional to size (PPS). Within each enumeration area all households were mapped using PDA-based GPS technology. A random sample of 25 (2004) or 27 (2005) households was selected regardless of the presence of children under the age of five years. Final clusters of enrolled households thus varied per EA. Selected households were revisited and invited to participate in the survey if there were children aged 0-59 months in the household, at which time the presence and hanging of an ITN was assessed. The morbidity survey took place two days later in a central location of each selected EA. The fieldwork for each of the two surveys was conducted during a three-week period. Six PDA teams of two staff members and six clinical survey teams consisting of five people each completed the survey in six enumeration areas per day. The use of PDA based GPS technology to determine a sampling frame in this study has been described in detail in a separate publication .The study had an 80% power to detect a 33% difference between pre- and post-intervention prevalence of moderate-to-severe anaemia in children 0-59 months in each of the three districts with 95% confidence, assuming a baseline prevalence of 20%, and taking into account a 10% refusal rate, and a design effect of 1.5. The pooled sample size of 2,700 children per survey had a power of >85% to detect a 20% difference between surveys.Refusal and non-response was recorded at household level in 2004. In 2005 this was changed to collection at child level for practical reasons. The 2004 household level data was converted into child level data, assuming an average of 1.45 children per household in 2004. For each household, a structured questionnaire was completed with the caretaker of the child to record background data on demographics, socio-economic status and the child's reported medical history. The used questionnaire was an adapted version of the MIS questionnaire. Axillary temperature was measured twice during the questionnaire process. A capillary blood sample was taken to assess haemoglobin level , the presence of malaria parasites were assessed by rapid diagnostic test , as well as a thin and thick malaria blood smear. Lastly, a brief medical exam was performed. Appropriate treatment was provided to children with anaemia (iron syrup 2-3 mg/kg/day of elementary iron for a minimum of four weeks) and parasitaemia as determined by RDT . Children with an acute illness that required further attention were referred to the nearest health facility. Malaria thick and thin smears were stained in the field with Giemsa  and read at a later date at the malaria reference laboratory of the Ministry of Health in the capital Lome. The total number of asexual parasites and gametocytes was determined per 500 white blood cells, assuming a total white blood cell count of 8,000/μl. A subset of slides were reread by a second microscopist for quality assurance purposes. Monthly rainfall data was provided by the National Meteorological Service in Lome.Data were entered directly into a database using PDAs in the field  and downloaded into a central database (Microsoft Access). Data were entered and cleaned using range and internal consistency checks. Analyses were performed using SAS . Analyses were weighed for the number of invited households as this varied over the first 3 days of the 2004 survey. The overall estimated effect of LLINs in Togo was based on pooled district estimates, weighted for the proportion of the population that the three districts represented, . Comparisons between pre- and post-campaign survey were made on the basis of intention-to-treat (i.e. independent of reported ITN use in 2005). Crude weighted frequencies, percentages, and Rao-Scott chi-square tests of association were calculated using PROC SURVEYFREQ. Dichotomous outcomes of interest were assessed while controlling for other covariates using log-binomial regression with the PROC GENMOD procedure. Adjusted prevalences were obtained from least squares mean estimates using the LSMEANS output group in this procedure. The village based cluster randomization was taken into account using an exchangeable correlation structure for observations obtained from residents within one village.A secondary analysis comparing current ITN users versus non-users in the 2005 survey used similar methods.Linear regression models (PROC MIXED procedure) were used to determine the mean difference in continuous endpoints (e.g. haemoglobin level) between the pre- and post campaign survey using a robust variance estimation correcting for village based cluster randomization, and assessing the same co-variates as was done in the models for dichotomous endpoints. The design effect associated with the primary endpoint (the ratio of the cluster-corrected standard errors and the standard error computed under the assumption of a simple random sample design) was assessed using an alternating logistic regression model (ALR) with the PROC GENMOD procedure. The actual observed design effect at village level for moderate to severe anaemia was 1.07 (95%CI 1.01-1.15).P-value < 0.05 was considered significant.Age was included in each model. Because of the rapid physiological changes in haemoglobin levels in childhood, narrow age categories were used . The presence of effect modification by district and age of the association between survey and various outcomes was assessed by adding a survey × district × age interaction term in the model, with age as a binary (0-17 and 18+ months) variable, and was determined by the P-value of the interaction term (using P < 0.05 as significant). As there was evidence of effect modification by district × age (as a binary variable 0-17 and 18+ months) of the impact of LLINs on moderately severe anaemia and clinical malaria, results are also presented separately for subgroups. Other variables considered for inclusion were: sex of the child, household socio-economic status, and level of education. If covariates were significant determinants of the outcome evaluated, but not confounders of the relationships of interest (i.e. pre- and post-intervention survey and outcome), and had no or minimal impact on the precision of the point estimates, they were excluded from the final models. For all statistical tests a two-sided A household was defined as a unit of one caretaker and her children. In polygynous households, one woman was randomly selected and invited to participate with her children <5 years. Socio-economic status was defined in quintiles according to a wealth index score based on criteria developed by the World Bank to classify each household's economic status . MalariaIn total, 2,304 and 2,430 households were randomly selected for participation in the 2004 and 2005 surveys, respectively. These households reported the presence of 2,721 and 3,123 children below the age of five years in 2004 and 2005, who were invited to participate in the survey. Of the invited children 70 and 42 were subsequently excluded because their dates of births revealed they were 60 months or older at the time of the survey. Dates of births were recorded from their under-5 clinic cards where available. Of the eligible children, 95.5%  and 96.3%  were enrolled. The main reason for non-enrolment was not being at home due to travel or work (4.4% and 3.4%). Only 3 (0.1%) and 9 (0.3%) children were not enrolled due to a refusal by their caretakers. A further 165 records were excluded from the final analyses because a) children were erroneously enrolled as part of a second or third mother-children pair within households of polygynous families in 2005 in Tone (147 records) or, b) because of data quality problems (18 records). In total, 5,334 children contributed to the results; 2,521  in the 2004 survey and 2,813 children  in the 2005 survey. Three hundred and sixty three households were involved in both surveys (19.9% of 2005 households).The median number of children per EA was 29 in 2004 range 11-51) and 31 in 2005 (range 17-54). District level characteristics of the enrolled population in the pre- and post intervention surveys are presented in Table 1-51 and Monthly district level rainfall patterns were compared for the pre- and post-campaign year Figure . While rBefore the campaign nets of any kind were reportedly present in 5% to 12.7% of the households in the three districts  and there was a 0.35 g/dL (95%CI 0.25-0.45) increase in mean haemoglobin between the two survey points. Importantly, these differences between surveys depended on a combination of the district and the age category involved, in particular whether children were targeted by the campaign and were ≥18 months old or not at the time of the 2005 survey. When adding a three-way interaction term, survey × district × age group, to the model, its In the central district of Ogou, with the long, stable rainy season, and 43% of the children reportedly sleeping under ITNs in 2005, substantial differences were observed across most of the anaemia and malaria indicators. Comparable reductions among both children below and over 18 months of age , but this was not accompanied by a change in mean haemoglobin, or a difference in the prevalence of the assessed malaria markers  had significantly less moderate to severe anaemia compared to other children who either did not sleep under a net or did not have an ITN hanging in their household . SES and sex were not found to be confounders. While ITN use or lack thereof during the previous night does not necessary relate well to persistent use, further analysis at district level suggested a trend where the difference between ITN users and non-users with respect to moderate to severe anaemia was biggest in the district with the lowest community coverage level, and smallest in the district with the highest community coverage level . Similarly, a difference between these same groups was also found in mean haemoglobin; ITNs users had higher mean Hb than nonusers: 0.17 g/dL, 95% CI 0.03-0.31, adjusted for age and district). There was no effect modification by district and/or age).The burden of malaria and anaemia in children <5 years was evaluated in three districts during two consecutive peak malaria transmission seasons before and after the 2004 Togo Integrated Child Health campaign. ITN ownership coverage reached 65% in 2005 in all three evaluated districts among households with children below the age of five years. This coincided with substantial reductions in moderate to severe anaemia and clinical malaria. Observed differences varied by region, and occurred mainly in the two districts from the southern coastal plain Maritime Region and hilly central Plateaux Region. The benefit was mainly observed in the age group of children ≥18 months who had been targeted by the campaign. The benefit was less clear in children <18 months. These children had not been targeted by the campaign themselves, but a considerable proportion shared a net with an older targeted sibling. No differences were observed in the northern district Tone in the semi-arid Savanes Region.Clearly, the cross-sectional design with a historical control group limits the interpretation of these finding and factors other than ITNs could have contributed to the observed effect. For example, the package of child health interventions included mebendazole, which is known to have a beneficial impact on anaemia. Furthermore, some of the children (approximately 20%) in 2005 had also participated in the 2004 survey when they would have received iron and anti-malarial treatment if they were found to be anaemic or to have malaria. This may have provided some residual benefits one year later. The effect however, if any, would likely have been small. Seasonal differences between surveys may also explain the observed effect, however, all three sites experienced average and comparable rainfall in 2004 and 2005 with respect to the duration and timing of the rains. Moreover, the prevalence of moderate to severe anaemia in 2005 was 19% lower in ITN-users when compared to non-users. It seems therefore reasonable to attribute a significant proportion of the observed differences to the rapid scale-up of ITN coverage that took place between surveys rather than to natural differences in malaria exposure and transmission. Even though the dynamics between the intensity of transmission and weather are complex, the largest differences were observed in the central district with the longest and most stable rainy season of the three sites. This setting was most suitable for a cross-sectional comparison as the malaria transmission pattern in this area was relatively stable, and less sensitive to the exact timing of the survey.It is not clear why the effect was observed in the southern two districts, but not in the northern district of Tone. ITN coverage was significantly higher in Tone than the two southern districts. In addition, if an ITN was present in the house, it was almost always hung, while in the southern areas nets were less likely to be observed hanging even though present in the house. This reflected that in Tone, a keep-up campaign was organized by the Red Cross which consisted of door-to-door home visits of Red Cross volunteers to explain the rationale and correct use of ITNs. Togo's most northern district has a much dryer semi-arid climate with a short rainy season and thus rapidly changing malaria transmission dynamics, making it more vulnerable to the potential confounding effects of season than the other sites. However, the rainfall patterns were almost identical in 2004 and 2005 and there was a lot of malaria in both years, and this is thus unlikely to explain the lack of effect of ITNs. Neither was there less malaria in Tone than in the other districts. Out of the three assessed districts, Tone was the only district with an active Lymphatic Filariasis Mass Drug Administration program. It is possible, of course, that other factors may explain the smaller effect of ITNs in the northern area of Togo than in the central and southern regions, such as a relative increase of nutritional deficiencies in Tone but not in the central and southern regions.Despite the remaining questions about findings in Tone, these findings broadly confirm the known strengths and weaknesses of the current malaria burden indicators. Prevalence of moderate to severe anaemia, expected to reduce within 12 month of achieving high intervention coverage in malaria endemic areas, showed substantial changes in the range of those seen in RCT settings . ParasitDespite their design limitations, national cross-sectional cluster-surveys play an important role in monitoring and evaluation of the impact of the increase in global malaria control efforts. The RBM MERG partnership has developed the Malaria Indicator Survey tool (MIS) , a natioThe Togo impact assessment, while a national cross-sectional survey using internationally recommended indicators and cluster sampling, was not a MIS survey. The main objective of the survey in Togo was to evaluate the short-term impact and effectiveness of the campaign as a delivery strategy, not to provide national or regional burden estimates in under 5 year olds. This survey had a limited budget , and was designed to capture some of the potential regional and age related variability in impact across the country. The observed complexity to burden changes at sub-national level is not captured by the current 'gold standard' MIS surveys and has not received much attention. As scaling-up of malaria control efforts is successful and reductions in the burden of malaria become apparent however, the focus of national malaria control programmes and their need for accurate estimates of progress soon requires more detail at sub-national level. Specifically, complementary M&E tools to the MIS surveys will be needed to capture the sub-populations and regions where control efforts need further strengthening, and guide national programmatic efforts, particularly now that countries are reconsidering their options for local malaria elimination. The assessment in Togo highlights both impressive progress, as well as the consequence of excluding young infants with this delivery strategy without additional keep-up or catch up distribution, even though a proportion of <18mo olds were reached by the campaign because they shared an ITN with a targeted sibling. The findings support subsequent adaptations of integrated campaigns, e.g. an integrated campaign in Tanzania in 2006 offered different intervention packages to different age groups .These findings add to the increasing number of reports that show the potential contribution of the use of integrated ITN distribution campaigns to reduce malaria morbidity in children. With this impressive scale-up of LLIN coverage and the switch of the first-line treatment policy to ACT, the Togo MoH is well underway to make significant progress in their efforts to control malaria in Togo.The authors declare that they have no competing interests.Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.DJT designed and coordinated the study, performed the statistical analyses and wrote the first draft of the paper. KM, AD1, AD2 and YS participated in the coordination of the study. AW, MJE and JvE supported the GPS component and participated in the coordination of the study. FtK and WAH participated in its design and participated in drafting the manuscript. All authors read and approved the final manuscript.We are grateful to the parents and guardians of the children who participated in the survey and the many field staff members and other people who assisted with and contributed to this project. This survey has been a joint international effort that has received support from various individuals and institutes. This study was financially supported by the Canadian Red Cross. We thank Poutougnima Tchamdja, Directeur Général de la Santé for the support from the Togo Ministry of Health. We are grateful to Mr Norbert Paniah, Antoinette Awaga, Blaise Edoh and Messan Nyonato from the Togolese Red Cross for their logistical support. At the International Federation of the Red Cross, we thank Jean Roy and for his assistance. We also express our gratitude to Sanofi-Synthelabo in France and DAFRA in Belgium for their kind donations of Arsucam, the anti-malarial drug used in this survey. Similarly we thank Part Peeters, Chris Weeks and their colleagues at DHL for their expert assistance with the transportation of all involved study shipments for the first survey. At LSTM we thank Philip Gichuru for his statistical support.