Plasmodium infections and an inability to control malaria-induced immunopathology. This Review discusses humoral, cell-mediated and innate immune responses to malaria and how each contributes to protection – focusing on how deficiencies in infant and paediatric immune responses might influence malaria vaccine efficacy in this population. In addition, burgeoning evidence suggests a role for inhibitory receptors that limit immunopathology and guide the development of long-lived immunity. Precisely how age or malaria infections influence the function of these regulators is unknown. Therefore the possibility that infants may not have the immune-dexterity to balance effective parasite clearance with timely immune-regulation leading to protective immunologic memory is considered. And thus, malaria vaccines tested in adults and older children may not be predictive for trials conducted in infants.Naturally acquired immunity to malaria requires repeat infections yet does not engender sterile immunity or long-lasting protective immunologic memory. This renders infants and young children the most susceptible to malaria-induced morbidity and mortality, and the ultimate target for a malaria vaccine. The prevailing paradigm is that infants initially garner protection due to transplacentally transferred anti-malarial antibodies and other intrinsic factors such as foetal haemoglobin. As these wane infants have an insufficient immune repertoire to prevent genetically diverse  We often reduce, into a single sentence, the accumulation of decade’s worth of clinical and epidemiologic studies on malaria with an estimated number of cases and of children each year who die. We all agree that events leading to the manifestations of severe malaria in a paediatric population are multi-factorial and are influenced by intrinsic as well as extrinsic factors. These include, but are not limited to, malaria transmission intensity, parasite genetic diversity and complexity of infection, the degree of maternal antibody protection and prenatal malaria experience, foetal haemoglobin, haemoglobin S heterozygosity, and nutritional status. These factors aside, children remain more susceptible to malaria compared to adults. This age-associated susceptibility has in part been explained by children being immunologically naïve whereby repeat infections are required to develop a repertoire of immune cells capable of recognizing the various and antigenically variant malaria-derived proteins. Once infected, however, children are more likely to succumb to the immunopathology associated with malaria [reviewed in ] which iTo better understand age-associated differences in anti-malarial immunity, immuno-epidemiologic studies of naturally exposed populations have been conducted with the express purpose of discovering which immune mediators may be responsible for semi-protective immunity that could be enhanced or sustained with a malaria vaccine. What immune responses do asymptomatic malaria-infected people possess compared to those who have symptomatic infections? And by extension, which immune responses confer the semi-immune status benefiting adults that are lacking in infants and young children? This review highlights humoral, cell-mediated and innate immune responses to malaria under natural exposure conditions that may contribute to the development of protection – focusing on how ‘deficiencies’ in infant and paediatric immune responses might influence malaria vaccine efficacy in this population. Within this context, malaria vaccines that have shown promise in children will be discussed. As this review concludes, a better understanding of infant immunity is needed in order to design an efficacious malaria vaccine that accomplishes what natural infections do not.Plasmodium falciparum (Pf) malaria  activity decreased with age (children screened were 2–14 years old), even though GIA activity was significantly associated with less risk of malaria infection after adjusting for age  region encoding the B-cell epitopes that are associated with protection against infection and disease (in vitro MSP-119 invasion inhibitory antibody (MSP-119 IIA) assay utilizes transgenic parasites differing only in their MSP-119 domain and by comparison of activity in parallel cultures can attribute inhibition to antibodies solely against MSP-119 ; was associated with protection from infection in a highland area where malaria infections are more likely to be symptomatic  that appear to survive in the absence of antigen and play a role in protection from re-infection. The observation that overall functional anti-malarial antibody activity decreases with age may be countered by the development of more efficient cell-mediated adaptive immunity, in keeping with the concept that different components of ‘protective immunity’ may vary with age.Taken together, a possible interpretation is that MSP-119 specific memory B cells were not producing antibodies in asymptomatic individuals  compared to individuals from nonendemic areas. A general definition of an exhausted lymphocyte is one that is antigen-experienced, has undergone some differentiation but is no longer functional. This subset of tissue-homing dysfunctional memory B cells  found in malaria-experienced children expressed an inhibitory receptor Fc-receptor-like-4 similar to those described in HIV-infected individuals who undergo persistent antigen stimulation  B-cell antibody responses and T-cell dependent (TD) antibody responses [reviewed in ]. DuringCell-mediated adaptive immunity is arguably as important in protection against malaria as humoral immunity; the discovery and details of which have been extensively reviewed by others ,49,50. SMeasuring the network of cell-mediated immune responses has additional challenges over assays designed to measure humoral immunity, including ethically justified limitations in blood sample volume, especially from children under 5 years of age. Therefore, such studies are sorely lacking in human malaria vaccine trials, especially those conducted in children. However, recent advances in multiplex technologies have begun to shed light on the full functional potential of antigen-specific T-cell responses against many infections, including malaria. Luminescent microsphere assays and multiparameter flow cytometry have significantly expanded our ability to examine human lymphocyte populations and their functions. Resources availed for capacity building and technology transfer in malaria endemic countries have also facilitated more sophisticated investigations of human cell-mediated immunity.et al. reported considerable variation between individuals during the course of their malaria infection and thus categorized them into three groups: those with moderate levels of IFN-γ and IL-10, but no IL-12p70; those with IL-12p70 and high levels of IFN-γ and IL-10; and those that failed to up-regulate pro-inflammatory cytokines but had the highest TGF-β levels in response to Pf-infected erythrocytes in vitro. Pro-inflammatory in vitro recall responses  were associated with the ability to control parasite growth (length of time to first detection of 1000 parasites/mL) after challenge infection. In contrast, higher ratios of IL-12 or TNF-α to IL-10 were correlated with a more rapid onset of clinical symptoms . This study clearly demonstrates individual host differences in the magnitude and quality of cell-mediated immunity despite the same parasite exposure, and hints at the far-reaching consequences of innate immune signals in generating protective adaptive immunity. Simultaneously measuring the sequence and magnitude of pro-inflammatory events  in conjunction with antigen presentation signals (i.e. IL-12 and IL-18), balanced by down-regulatory cytokines  within the context of a malaria infection or vaccine trials in children would significantly improve our understanding of immunologic harmonization (or lack thereof) to specific malaria antigens. Especially if these children continue to be exposed to natural infections that may subvert the desired response. Longitudinal repeat measurements of relative cytokine contributions during the course of a malaria infection also gives us the ability to determine which down-regulatory mediators are missing, suboptimal or delayed, and may, by their absence, contribute to the development of immunopathology, especially in paediatric populations.A plethora of cytokine profiles have subsequently been revealed, similar to sero-profiles, that give us a broader picture of cell-to-cell signalling in response to a malaria infection. This technology has been used to investigate the kinetics of cytokine responses in a sporozoite challenge study in malaria-naïve adults . WaltherTaking the lead in utilizing and optimizing polychromatic flow technology are studies of acute and persistent viral infections which describe a mosaic of T-cell subsets associated with immune memory and protection [reviewed in ]. Multipin utero against a strong pro-inflammatory response as a residual effect of having recently been in the intra-uterine environment [reviewed in (H1 mediated pro-inflammatory cytokines (i.e. IL-12 and IFN-γ), neonatal immunity is thought to be ‘suppressed’. However, more recent studies have shown that neonatal monocytes and antigen-presenting cells do express pro-inflammatory and regulatory cytokines  and at higher levels than adults . Mouse models have been used to examine these four T cell subsets during a P. yoelii malaria infection and found that IL-10 producing Tregs impede parasite clearance  produce more IL-23 than IL-12 (2 has been shown to inhibit INF-γ production from naïve human cord blood mononuclear cells (CBMCs)   and TD I (CBMCs) . PGE2 annterface . The rolThe few studies directly investigating the impact of malaria on human DC function have yielded conflicting results. A recent balanced review attempts to resolves these apparent contradictions concluding that disparate findings could be explained by low-dose immune induction vs. high-dose immune suppression . The aut+ monocytes and myeloid DCs and decreased expression of TLR9 on plasmacytoid DCs compared to adults with no history of malaria exposure  pre-incubation experiments with Pf followed by TLR ligand stimulation demonstrated a temporal-synergistic activation of TLR-4 and TLR-2 significantly increasing TNF-α and IFN-γ but reducing IL-10 production. This is a distinct contrast to the effect observed when PMBCs are primed with LPS, which induces subsequent TLR tolerance (+ monocytes isolated from PBMCs compared to uninfected children (aged 5–16 years old) and enhanced TLR2 (Pam3Cys) activation but in this case resulted in higher production of both IL-10 and TNF-α (in utero exposure) CBMCs produced more IFN-γ in response to both TLR3 (poly I : C) and TLR4 (LPS) ligand stimulation if the mother was infected with malaria during the last month of pregnancy, whereas CBMCs without prenatal malaria exposure had significantly higher TNF-α in response to the same ligands  or the control rabies vaccine (n = 407). The cumulative 8-month incidence of all clinical malaria episodes was 9% in the group that received RTS,S compared to 21% in the control group, with an adjusted efficacy of 56%  (P = 0.01) . The vaccine did not induce IL-10 or TNF-α however IL-4 levels were lower in response to MSP-1 though not to MSP-2 or RESA. Clearing circulating malaria parasites prior to immunization appeared deleterious for inducing MSP1-specific IFN-γ responses; perhaps by preventing ‘boosting’ afforded by natural infections. This vaccine reduced parasite density  but did not reduce prevalence of infection and was strain-specific for MSP-2. Similar to RTS,S, immunizing children with combination B failed to achieve anti-malarial antibody levels observed in adults and titres significantly waned over time.The goal of RTS,S is to generate neutralizing humoral immunity directed against the sporozoite stage of the malaria parasite before they invade the liver, in addition to inducing CSP-specific CD8 and CD4 Th1+ T cell immunity. An RTS,S/AS01E efficacy trial was conducted in Kenya and Tanzania from March through August 2007 in which children 5–17 months of age were randomly assigned to receive either the malaria vaccine (< 0.001) . Vaccine = 0.01) . Of note = 0.01) ,107. The = 0.01) . Choice  = 0.01)  that may = 0.01) . This stAs malaria vaccine trials in infants provide optimism, progress has been made in decreasing the incidence of clinical and severe malaria in young children as the result of exposure-reduction programmes such as insecticide-treated bednets (ITBN) and intermittent preventative therapy in infants (IPTi). Both of these interventions decrease the cumulative parasite burden by preventing some  malaria infections, with the latter also decreasing the duration of asymptomatic infections experienced by a child . It is iMonitoring the impact of these strategies on decreasing the incidence of clinical and severe malaria has yielded some interesting findings that are relevant to understanding the development of anti-malarial immunity in infants and young children. Several studies have addressed the possibility that exposure-reduction programmes could result in a delay in acquisition of immunity thereby shifting clinical sequelae to an older age group – referred to as a rebound effect. Evidence from ITBN studies has alleviated this concern as bednets appear to mediate reductions in malaria-associated morbidity by decreasing parasite densities and delaying median age of first parasitaemia from 4 to 10 months of age . Even thet al. (19 (3D7) and AMA-1 (3D7) compared to placebos starting at 9 months of age, the point at which maternal antibodies are waning and infant immunity develops (A parallel assessment of humoral immune responses within the context of a three-dose IPTi schedule was conducted in the study by Quelhas et al. . Antiboddevelops . This scThe most evident impediment against basic immunologic investigations of infants and young children are limited blood volumes, an acceptable frequency of repeat sample collections, and gaining ethical and parental approval for enrolling often healthy children in research studies. Unfortunately, these understandable obstacles have left children, who are the most vulnerable to the adverse consequences of malaria, understudied. In the midst of technological advancements and ethical deliberations, some of these long-standing issues are being overcome. Yet, if a malaria vaccine is deemed efficacious in adults the question remains if that vaccine will also protect infants and young children. Current opinion in the field of immunology is that the next generation of vaccines will need to be designed to do what nature does not in order to confer long-lasting protective immunity. With the ultimate goal of designing a malaria vaccine that will be delivered within the first year of life, when there appear to be inherent down-regulatory mechanisms preventing the induction and maintenance of protective immunologic memory, the composition of such a vaccine may need to differ from one that would protect an adult. It may not be enough to teach an infant immune system to behave like an adult’s by priming and boosting a pro-inflammatory response against an array of malaria antigens that in turn induce potent humoral immunity. A malaria vaccine for infants may also need to be designed to subsequently mute pro-inflammatory responses and further guide the selection of the appropriate T and B cells to be maintained as memory – a ‘prime-boost-mute-select’ vaccine design, if you will. Therefore, increasing our understanding of basic immunologic differences between adults and young children will provide the mechanistic insights necessary to design a malaria vaccine that will be safe and successful in infants.


Fever has traditionally served as the entry point for presumptive treatment of malaria in African children. However, recent changes in the epidemiology of malaria across many places in Africa would suggest that the predictive accuracy of a fever history as a marker of disease has changed prompting calls for the change to diagnosis-based treatment strategies.Plasmodium falciparum infection recorded using a rapid diagnostic test was evaluated across a range of endemicities characteristic of Africa today.Using data from six national malaria indicator surveys undertaken between 2007 and 2009, the relationship between childhood (6-59 months) reported fever on the day of survey and the likelihood of coincidental P. falciparum compared to children without a history of fever on the day of the survey after adjusting for age and location . There was a strong linear relationship between the percentage of febrile children with infection and infection prevalence (R2 = 0.9147). The prevalence of infection in reported fevers was consistently greater than would be expected solely by chance and this increased with increasing transmission intensity. The data suggest that in areas where community-based infection prevalence in childhood is above 34-37%, 50% or more of fevers are likely to be associated with infection.Of 16,903 children surveyed, 3% were febrile and infected, 9% were febrile without infection, 12% were infected but were not febrile and 76% were uninfected and not febrile. Children with fever on the day of the survey had a 1.98 times greater chance of being infected with The potential benefits of diagnosis will depend on the prevalence of infection among children who report fever. The study has demonstrated a predictable relationship between parasite prevalence in the community and risks of infection among febrile children suggesting that current maps of parasite prevalence could be used to guide diagnostic strategies in Africa. Plasmodium falciparum is not the only cause of fever and only one of many pathogens that cause identical pyrogenic responses in young African children [P. falciparum transmission is declining across many parts of Africa [The aetiology of fevers in malaria endemic areas has been the subject of considerable basic and applied public health research for many years. children -6. Althochildren -12. Therf Africa  and thisf Africa ,14. The f Africa There is an increasing body of data derived from national household sample surveys where the presence of reported fever among children below the age of five and malaria infection are recorded using interviews with caretakers and rapid diagnostic tests (RDTs). An evaluation of these data provides a new opportunity to assess the proportion of childhood fevers likely to be associated with malaria infection across a range of contemporary malaria transmission settings in Africa.Malaria indicator surveys (MIS) are promoted as tools to monitor national level progress toward milestones set by national malaria control programmes and the Roll Back Malaria movement on the coverage of key malaria prevention and disease management strategies . These sTo provide an analysis across malaria risk classifications we have reconstructed individual level data to represent the administrative units used principally as part of the multi-stage sampling, hereafter referred to as ADMIN1 level. These ADMIN1 units represented provinces or regions in Kenya and Senegal. In Angola, sampling was designed to represent three recognized malaria epidemiologic regions: hyper-endemic, meso-endemic stable and meso-endemic unstable and these divisions were used within the present analysis. In Liberia, survey data were reported for Monrovia and each of five regional groupings that do not correspond directly to national administrative boundaries. Due to very low parasite prevalence in Djibouti, data have been summarized at larger spatial resolutions as the Capital and Rural Djibouti. For similar reasons, the data to four regional groupings in Namibia were aggregated as shown in Table P. falciaprum infection (PfPR) among all surveyed children and those that were afebrile. First, individual child level data were used to investigate the risks of infection if the child was reported as having a fever using the Mantel-Haenszel estimate of the odds ratio that adjusted for the age of the child and the overall prevalence of infection within each of the ADMIN1 units. Second, the relationships between the percentage of febrile children with infection was examined against the overall prevalence of infection among all children sampled in each ADMIN1 unit. In addition the prevalence of infection among those who did not report a fever on the day of the survey was examined against the percentage of febrile children with infection thus specifically excluding the children in the dependent variable from the predictor (x-axis). In both cases it is assumed that true zero infection prevalence would equate to zero prevalence in febrile children to anchor the association through the X-Y intercept (i.e. using a zero intercept). Confidence intervals (CIs) around the best-fit association between infection prevalence and prevalence of infection in febrile children were computed providing the boundaries of all possible linear fits to the data. Linear prediction plots were defined and included CIs around the predicted line using the twoway lfitci command in Stata. All data were analysed using STATA version 11.0 Data were assembled across each of the 33 ADMIN1 units assembled from the six national sample surveys. Each of the 33 sites was classified according to the overall prevalence of P. falciparum. 535 (3%) of the children were both febrile and infected, 1,536 (9%) were febrile without evidence of infection, 1,936 (12%) were infected but were not reported as febrile, and 12,896 (76%) were neither infected nor febrile. The 33 sampling units surveyed represented a wide range of transmission intensities ranging from P. falciparum parasite prevalence among all sampled children 0.5 to 4 years (PfPR0.5-4) values of 0% in rural Djibouti to as high as 44.4% in North Central, Liberia. 13 sites were identified as having a PfPR0.5-4 <5%, 18 sites were characterised as having a PfPR0.5-4 within 5-39% and only two sites were located in the highest transmission class with PfPR0.5-4 ≥ 40%. The median parasite prevalence across the entire range was 11.74% (IQR: 2.78-22.94%). The median fever prevalence across the entire data series range was 9.25% (IQR: 6.50-16.43%).Data were assembled on fever and infection prevalence among 16,903 children surveyed at 33 different locations across six countries. The surveys recorded a total of 2,071 (range 10-192 per site) children with a fever on the day of survey and 2,471 (range 0-313 per site) children with a positive RDT result for P. falciparum compared to children without a history of fever on the day of the survey after controlling for age and ADMIN1 residence . The continuous relationship between the percentage of febrile children with infection and infection prevalence  among all children is shown in Figure 2 = 0.9147, p < 0.0001). Comparing this correlation to the 50%-50% equivalent chance of fevers and all children being infected  and a prevalence of infection among asymptomatics of approximately 34% corresponding to 50% of fevers infected with P. falciparum.Children with a reported fever on the day of the survey had a 1.98 times greater chance of being infected with P. falciparum infection as judged by an RDT and 74% were aparasitaemic. Clearly, among these communities the use of fever alone as a presumptive prompt for anti-malarial treatment would result in a huge over-treatment burden. Conversely, among the 15% of children with evidence of infection they were almost twice as likely to be febrile on the day of investigation when compared to children without infection suggesting that infection in these areas is a reasonable predictor of symptoms. These results are specific to the range of transmission conditions represented by the six national surveys and did not include many sites at the highest transmission intensity. Nevertheless there is increasing evidence that low parasite prevalence is a common feature of most of Southern Eastern and Horn of Africa countries [P. falciparum is very likely to result in symptoms that include fever, presumably because of poorly developed functional immunity through the first five years of life, but fever alone remains a poor discriminator of malaria infection suggesting that all fevers should be tested to confirm or refute the role of malaria in the febrile presentation.Recognizing the combined needs to mitigate the threats of drug pressure on the emergence of drug resistance, the changing malaria epidemiology in Africa and the need to improve the way febrile children are managed clinically has provided support for calls to move away from presumptive treatment of African children . Childreountries  and thosountries -26. In tP. falciparum transmission maps for Africa which are able to provide a prediction with definable uncertainty of malaria prevalence in the community [P. falciparum malaria endemic countries in Africa.These findings are not new. However, previous studies have largely been undertaken among clinic attending populations at a time when asymptomatic parasite prevalence was higher across more countries in Africa. The results presented here are of randomly selected children resident within a wide range of communities experiencing very different malaria transmission conditions. Figures ommunity  and coulThe analysis comes with several caveats. First, the application of community-survey data on reported fever prevalence assumes that fever reporting on the day of the survey is accurate and similar to what might be reported by mothers of sick children attending clinic. The recognition of fever as a morbid event to interviewers or health workers varies within and between communities who have developed an elaborate cultural vernacular to describe fever -33. Not Africa has made significant progress in the control of malaria and this has led to a call for diagnosis-based treatment strategies. The cost-effectiveness of imposing diagnosis for febrile children will depend on the prevalence of infection among those who report fever. The data presented here indicate a predictable relationship between parasite prevalence in the community and risks of infection among febrile children implying that mapped estimates of parasite prevalence can be used to guide diagnostic strategies in Africa.The authors declare that they have no competing interests.EAO assembled all the data, restructured and analysed the data and wrote the manuscript. RWS was responsible for the project and its overall scientific management, interpretation and preparation of the final manuscript. Both authors read and approved the final manuscript.


Information on the burden of malaria in early infancy is scarce. Young infants are relatively protected against clinical malaria during the first six months of life due to the presence of maternal antibodies and foetal haemoglobin, and have received relatively little attention with respect to research and treatment guidelines. The World Health Organization provides treatment guidelines for children from six months onwards, without specific treatment guidelines for the younger infants. A number of recent reports however suggest that the burden in this young age group may be underestimated.A retrospective review of paediatric hospital records at the Queen Elizabeth Central Hospital in Blantyre from 1998 to 2008 from three data sources was carried out. The number of admitted infants <6 months and ≤ 15 years was obtained from the registry books of the Paediatric-Nursery-Department and the Malaria Research Laboratory. For the period 2001 - 2004, more detailed malaria related admission information was available as part of an ongoing study on severe malaria, allowing a calculation of the proportion of infants < 6 months of age among admissions in children < 5 years.Retrospective analysis of hospital records showed that over the course of these years, the average annual proportion of paediatric admissions in children ≤ 15 years with confirmed malaria aged <6 months was 4.8% and ranged between 2.8%-6.7%. This proportion was stable throughout the seasons. Between 2001-2004, 9.9% of admissions with confirmed malaria in children <5 years occurred in infants <6 months, with numbers increasing steadily during the first six months of life.These findings are consistent with recent reports suggesting that the burden of malaria during the six first months of life may be substantial, and highlight that more research is needed on dose-optimization, safety and efficacy of anti-malarials that are currently used off-label in this vulnerable patient group. Infants below the age of six months are thought to be relatively protected against clinical malaria due to the transfer of maternal antibodies and the presence of foetal haemoglobin, and consequently receive relatively little attention with respect to research and policy guidelines on treatment of uncomplicated malaria,2.However, several recent reports of facility- and community-based settings across sub-Saharan Africa include scattered information indicating a higher burden of disease in this age group than what is generally assumed and the period of protection might be shorter than the widely quoted six months ,3. Succeet al conducted a community survey in under 5s in Togo in 2004 and showed that the prevalence of parasitaemia increased rapidly from two months of age and the prevalence of parasitaemia with document fever remained stable from three months onwards . Registry books did not record the clinical course or outcome of patients.Information on the total number of children ≤ 15 years old admitted to the hospital with malaria  was obtained from record books kept by a dedicated "Malaria Research Laboratory". This Laboratory conducts all the malaria tests for admissions to the general and research paediatric wards in children ≤ 15 years of age. The records books contain limited information, including patient identifiers, gender and lab results  but not age.For the period 2001 - 2004, more detailed information was available for individual admissions from the malaria research laboratory database and clinical records. This additional data was captured as part of an ongoing study on Severe Malaria in African Children (SMAC) as described in more detail previously. The metAll the information was copied from the registry and laboratory records into an Excel database. The descriptive statistical analyses were done with Excel 2007  and SPSS version 15 . In all analyses a malaria case was defined as any child admitted to the hospital with a positive malaria slide  regardless of the parasite density or disease severity.A total of 26,413 children ≤ 15 years of age were admitted with malaria in the eight-year period . Of these, 1,572 were admitted in the Paediatric-Nursery-Department, including 1,087 children <6 months and 439 infants aged ≤ 6 months  , with a peak of admissions in 1998 (8947 patients), with 25,326 children <15 yr and 1,087 infants < 6 months  during the 2001-2004 when body weight data was collected, Among this group, 47 infants (15.4%) had a weight <5 kg and 63.3% weighted between 5-7.5 Kg. The minimum weight registered during the study period was 2.3 Kg  admitted to the Queen Elizabeth Central Hospital in Blantyre with confirmed malaria parasitaemia per year were aged <6 months. This proportion was broadly similar across the seasons and years.et al in Togo also observed a rapidly increasing parasite prevalence and clinical malaria in a random sample of children at community-level from the age of two months onwards; with parasitaemia prevalence increasing from 18.2% in children aged 0-2 months to 43.0% in children aged 3-5 months, and the prevalence of parasitaemia with document fever remained stable from three months onwards [et al reported similar findings in a study conducted in Nigeria, where 27.1% of all the admitted infants ≤ 6 months had positive malaria parasitaemia, and malaria rates increased after the second month of life[et al estimated that 59.0% of febrile episodes during the first five months of life in western Kenya were attributable to malaria[This finding supports a growing number of reports suggesting that clinical malaria in infants <6 months old is not a rare event. Although the risk is less than in older infants, it appears to increase steadily from early infancy onwards. Eliades onwards . Afolabio malaria. Alonso o malaria.El Niňo had on the level of rainfall and the subsequent increase in malaria transmission during this particular year. Despite this considerable increase in malaria transmission data form 1998 was poled with the rest of the years included in the study because the proportion of infants under six months admitted with malaria during 1998 (3.9%) was similar to the one reported among the rest of the study period. A similar season distribution in malaria cases among infants was observed in 1998 compared with the rest of the study period.While our findings contribute to the available data, this retrospective review has several clear limitations. Because of its retrospective nature and reliance on mostly routine hospital records the quality and completeness of the available data varied over the years. The main finding of this study is the annual proportion of young infants admitted (4.8%), but this may underestimate the absolute number of malaria cases in all age groups that were admitted. Not all cases were recorded in the Registration Book at the time of admission, and malaria smears may not have been done for all children at the time of admission due to time and staff constraints in a poor-resource setting. Neonates with congenital malaria were not included either, as they were admitted to the neonatal ward. A second limitation is that multi-source data may generate some (biased) level of error when all the data are pooled together. Furthermore, these in-patient data are not necessarily representative of the age distribution of clinical malaria in children attending the out-patient clinics, or in the population. The greater amount of malaria cases detected in 1998 can be explained due to the effect that the meteorological phenomenon of Despite these limitations, the results of this retrospective review are consistent with a growing number of observations from both facility and community-level assessments suggesting a higher burden in early infancy than generally assumed. Children below five kg are not included in the WHO treatment guidelines for the new ACT that are currently being implemented across the globe. Infants under six months of age have also been systematically excluded from the efficacy and safety trials that have been conducted as part of the standard drug development process for ACT. DespiteThe burden of malaria during the six first months of life is probably underestimated, and may represent up to 5% of all admitted paediatric malaria cases in children below 15 years. With the wide-scale implementation of artemisinin-based combination therapy (ACT) for the case-management of uncomplicated malaria throughout sub-Saharan Africa, accurate estimates of burden, pharmacokinetic profiles, safety and efficacy of ACT in early infancy are urgently needed to guide the development of evidence-based treatment guidelines for the different artemisinin-based combinations that are currently used off-label in this vulnerable patient group.The authors declare that they have no competing interests.BL, EM and DJT developed the surveillance methods. EM, MM and TT contributed data and supported the compilation of data conducted by BL. BL conducted the analyses, with support from DJT, and prepared the initial draft. All authors contributed to the development of the final manuscript.


A recent school-based study in Benin showed that applying a policy of anti-malarial prescriptions restricted to parasitologically-confirmed cases on the management of fever is safe and feasible. Additional PCR data were analysed in order to touch patho-physiological issues, such as the usefulness of PCR in the management of malaria in an endemic area or the triggering of a malaria attack in children with submicroscopic malaria.PCR data were prospectively collected in the setting of an exposed (with fever)/non exposed (without fever) study design. All children had a negative malaria rapid diagnostic test (RDT) at baseline, were followed up to day 14 and did not receive drugs with anti-malarial activity. The index group was defined by children with fever at baseline and the control group by children without fever at baseline. Children with submicroscopic malaria in these two groups were defined by a positive PCR at baseline.PCR was positive in 66 (27%) children of the index group and in 104 (44%) children of the control group respectively. The only significant factor positively related to PCR positivity at baseline was the clinical status (control group). When definition of malaria attacks included PCR results, no difference of malaria incidence was observed between the index and control groups, neither in the whole cohort, nor in children with submicroscopic malaria. The rate of undiagnosed malaria at baseline was estimated to 3.7% at baseline in the index group.Treating all children with fever and a positive PCR would have led to a significant increase of anti-malarial consumption, with few benefits in terms of clinical events. Non malarial fevers do not or do not frequently trigger malaria attacks in children with submicroscopic malaria. Restricting anti-malarials to parasitologically-confirmed cases has been recommended in many countries for children of five years of age or above. Discrepancies between laboratory results and anti-malarial prescriptions show that healthcare providers may not trust blood smear results in routine practice -3. ProviHowever, submicroscopic malaria is frequent in endemic areas, and no study yet has dissected the consequences of applying the algorithm of management of fever by asking the following questions: what would be the consequences in many endemic areas of treating with anti-malarials all febrile children with a malarial infection, as detected by PCR? and are children with submicroscopic malarial infections prone to develop malaria attacks soon after the occurrence of a non malarial fever?Thus, the results of our recent study were re-analysed in the light of the PCR results collected during this trial.The data were prospectively collected through an exposed (with fever)/non exposed (without fever) study design. This study was performed from February through June 2008 in four schools, located at Allada, Southern-Benin, where malaria transmission is intense and perennial . AltogetThe primary objective of the study was to assess whether applying the algorithm of management of fevers in a school setting is consistent with an adequate management of fevers, i.e. it does not lead to a high number of undiagnosed (and thus untreated) malaria attacks, and by comparing the incidence of malaria attacks between the two groups (with and without fever) of children.Plasmodium falciparum infection at enrolment, using the most sensitive diagnostic tool currently available (PCR). Based on one study from the literature, it was estimated that about 15% the proportion of children attending for fever with a negative RDT and a positive PCR for malaria [The secondary objective was to compare malaria incidence between children with submicroscopic malaria during the follow-up in the index group and the control group, respectively. Children with submicroscopic malaria in these two groups were defined by the detection of  malaria , and it P. falciparum malaria at enrolment was estimated, using data collected both at enrolment and during the follow-up. At enrolment, all cases with P. falciparum parasitaemia > 1,000/μL were considered. During the follow-up an undiagnosed P. falciparum malaria at enrolment was defined as 1) at least one test (including PCR) positive at enrolment, 2) fever occurring during follow-up 3) at least one test (including PCR) positive for a P. falciparum infection at the time of fever.The number of undiagnosed DNA was prepared from blood collected at day 0 and on the last day of follow-up, as well as at occurrence of fever during follow-up. Blood collected on Whatman 3 MM filter paper was dried and conserved at room temperature until extraction. DNA was prepared by Chelex extraction, as described and subjected to a single PCR amplification protocol using primers specific for the small subunit ribosomal DNA (ssrDNA-PCR) as follows: rfal1 5'-TTAAACTggTTTgggAAAACCAAATATATT, rfal2 5'-ACACAATgAACTCAATCATgACTACCCgTC ,8.Parametric tests (Pearson's chi-square and chi-square for the comparison of crude rates ) and nonBaseline data are presented in Table P. falciparum. In the control group, blood smears were positive in three children ; PCR was positive in 104 (44%) children. Results of multivariate analysis on PCR positivity are shown in Table In the index group, PCR was positive in 66 27%) children, including the 11 children with a blood smear positive for 7% childrChildren of the index group were followed-up a total of 465 person-weeks Table . When taAmong the 66 children who had a positive PCR at baseline, eight presented with a malaria attack  during the follow-up. In 58 cases, no fever occurred in the follow-up .P. falciparum malaria at enrolment, based on baseline criteria  and eight cases based on follow-up data. The total number of undiagnosed P. falciparum malaria at enrolment was estimated to nine (one overlap) cases (3.7%).There were two cases of undiagnosed Children of the control group were followed-up a total of 477 person-weeks Table . Eleven Among the 104 children who had a positive PCR at baseline, nine presented with a malaria attack  during the follow-up. In 95 cases, no fever occurred in the follow-up. Incidence of malaria in children at risk for malaria  had a very low parasitaemia, in both groups. The concomitance of a negative RDT and a positive PCR is a proxy for submicroscopic malaria.P. falciparum, treating them would be of poor benefit in terms of clinical events. These findings should not be extrapolated to the management of fever in younger children, who are less immune than children of this study population. Similarly, these findings may not be representative of school children from low transmission intensity areas who have developed very few functional clinical immunity. Similar follow-up type study should be repeated in different population and epidemiological settings in order to explore more extensively the implications of RDT treatment strategies.A recent study performed in Italy in African migrants found a high discrepancy between malaria blood smears and PCR data . SubmicrMost children in the index group had another infection than malaria at baseline. The transient immunosuppression related to many infectious diseases might trigger malaria attacks in children with submicroscopic malaria at enrolment. The proportion of children who had malaria during the follow-up was 8% in children with a positive PCR at enrolment in the index group, while this proportion was 2% in the entire index group, and 4% in children with a positive PCR at enrolment in the control group. This suggests that non-malarial infections do not or do not frequently trigger malaria attacks in children with submicroscopic malaria.The baseline status of the two groups was the negativity of RDT. Interestingly, microscopically detectable parasitaemia at baseline, although rare in both groups, was more frequent in the index group, while PCR detection at baseline was markedly more frequent in the control group. Differences in terms of self-medication can hardly explain these parasitological differences between groups, and the only factor related to PCR positivity was the clinical status at baseline. Because chloroquine and quinine were the most commonly drugs used for self-medication in the study area, they were chosen as markers of self-medication, though other anti-malarials like SP, were widely available at the time of the study. The higher rate of asymptomatic parasitaemia (as detected with PCR) in the control group may indicate that these children are less prone to develop symptomatic malaria and why not symptomatic infections in general.The rate of estimated undiagnosed malaria attacks at enrolment was low. Baseline parasitological data may be used to estimate undiagnosed malaria attacks, but asymptomatic parasitaemia is a common status in the study population. Therefore, in an attempt to define the rate of undiagnosed malaria attacks at enrolment a combination of enrolment and follow-up data were used. It was postulated that undiagnosed malaria cases at baseline would lead in most cases to recurrence/persistence of fever concomitant to a positive malaria diagnostic test in the index group during follow-up. This definition has at least two main limitations: 1) it does not take into account that malaria may spontaneously heal in this population, and thus may lead to underestimate the "true rate"; 2) the use of a very sensitive diagnostic tool like PCR may lead to overestimate the "true rate" of undiagnosed malaria attacks.P. falciparum in this population would have led to a significant increase of anti-malarial consumption, with few benefits in terms of clinical events. Non-malarial fevers do not, or do not frequently, trigger malaria attacks in children with a submicroscopic infection.Treating all children infected with The authors declare that they have no competing interests.JFF designed the study, led field work interpreted the results and drafted the manuscript. PM, GA and TB participated in data collection. GA contributed with data collection in the field. AA, JD performed molecular assays. EO designed the study. PH performed pharmacology assays. PD designed the study and critically read the manuscript, MC designed the study, performed the statistical analyses and critically read the manuscript. All authors read and approved the manuscript.


Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes.The protection afforded by human erythrocyte polymorphisms against the malaria parasite, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of α+-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA.To test this hypothesis, No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA.P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms.Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage  Case-control studies have demonstrated that these polymorphisms reduce the risk of severe falciparum malaria rather than mild malaria [In vitro experiments have suggested reduced ability of Plasmodium falciparum to invade or sustain growth in abnormal erythrocytes [P. falciparum-infected abnormal erythrocytes to oxidative stress, have also been proposed [+-thalassaemia, CR1 deficiency and blood group O may protect via reduced rosetting [Human erythrocyte polymorphisms have long been thought to play a role in resistance to malaria. High frequencies of host erythrocyte polymorphisms such as α malaria . Experimhrocytes . Enhanceproposed -28. It hosetting ,21,29-32P. falciparum is lower in HbAS individuals after drug treatment [+-thalassaemia [It is well documented that individuals with sickle cell trait (HbAS) have lower parasite densities compared to HbAA individuals in asymptomatic, mild and severe malaria -20,33-44reatment , provideassaemia ,46, SAO assaemia ,48, G6PDassaemia -53 and Aassaemia ,54-65. DP. falciparum-infected erythrocyte variant surface antigens (VSA), which include P. falciparum erythrocyte membrane protein-1 (PfEMP-1) and rifins, has been shown to be dependent on the intensity of transmission [P. falciparum-infected erythrocyte VSA [P. falciparum children receive in malaria-endemic areas, the younger children will seroconvert to VSA.There are two possible ways to measure exposure to malaria blood stage infection: 1) by comparing parasite prevalence/density and, 2) by comparing age-specific antibody seroconversion to major blood stage antigens among host genotypes. Seroconversion to smission . Antibodcyte VSA . Furthercyte VSA . Seroconcyte VSA . The mor+-thalassaemia, CR1, ABO blood group, SAO and G6PD deficiency were determined in a group of children from Papua New Guinea (PNG). Parasitological criteria and levels of anti-VSA antibodies were measured in these children in two consecutive surveys, one year apart. The relationship between these genetic polymorphisms, parasite prevalence/density and age-specific antibody seroconversion to VSA was investigated in a malaria endemic region of PNG.If human erythrocyte polymorphisms were to hamper invasion and/or growth of the parasite or enhance the antibody-dependent control of malarial infection, this should manifest as differences in both mean parasite densities and patterns of age-specific seroconversion to VSA. The frequency of the host polymorphisms α® tubes  and EDTA as anti-coagulant. A small aliquot of whole blood was blotted onto Isocode® Stix  following the manufacturer's instructions, for further human genetic analyses. The remaining blood was centrifuged and separated into plasma (stored at -80°C), buffy coat and erythrocyte pellet, both stored in guanidine hydrochloride at 4°C. 555 paired samples were available for antibody determination.Details of study design and data collection have been reported previously . Briefly+-Thalassaemia genotype was determined by polymerase chain reaction [® Stix. G6PD deficiency was evaluated qualitatively  and ABO blood groups were determined using anti-A and anti-B antibodies from a kit . CR1 genotype was determined by polymerase chain reaction and restriction fragment length polymorphism digest with HindIII as previously described with modifications [αreaction . SAO genreaction  using huications ,72.P. falciparum isolate PNG/M 12, was collected from a child presenting with acute symptomatic malaria at an aid post in Madang, during a longitudinal cohort study from 1990/91 [in vitro using the method of Trager and Jensen [2, 2% O2, and 93% N2, sub-cultured into O positive erythrocytes  and stabilates frozen down. Adaptation and maintenance of PfEMP-1 expressing lines were made following a previously published method [ 1990/91 . Erythrod Jensen  with modd Jensen . Cells wd method .®  flotation[Detection of antibodies bound to the surface of infected erythrocytes (IE) have previously been described . Brieflyflotation. After wflotation. The perflotation.P. falciparum presence in no surveys, one survey and two surveys was assessed by ordinal regression with adjustments for age. Differences in frequency of positive slides were assessed using χ2 tests for each study year. Linear mixed models were used to examine the variation of parasite density among host genotypes with adjustments for age. Linear mixed models were also used to investigate the effect of age, parasite density and genotype on antibody levels: the two values were treated as a repeated measure with a heterogeneous compound symmetry covariance structure. Since the antibody levels showed considerable heteroscedasticity, they were transformed before analysis, using the transformation log(antibody + 2.5). The P. falciparum counts were transformed by log to reduce skewness while leaving the zero values unchanged. The models examined the effect of P. falciparum (logarithm of density), age (continuous), and either α+-thalassaemia genotype, CR1 genotype, ABO blood group , SAO genotype or G6PD phenotype . Interactions between host erythrocyte polymorphisms were also examined but some subgroups obtained were too small for reliable analysis. An interaction between sex and G6PD deficiency was also examined as G6PD deficiency is an X-linked disorder [+-thalassaemia genotype [Due to the repeated measures study design, appropriate statistical tests were chosen to analyze paired data. The association of host polymorphisms and disorder . CR1 expgenotype  and SPSSInformed consent was obtained from subjects and their parents or guardians. The project received ethical approval from the PNG Medical Research Advisory Committee and Institutional Review Board of the New York University School of Medicine.P. falciparum in both years of the study and 24.7% were positive for Plasmodium vivax. Only 57 samples (5.2%) were positive for both P. falciparum and P. vivax. Geometric mean parasite density of P. falciparum was 680 parasites/μl (95% CI 567–814) and parasite density was negatively correlated with age .1,100 blood samples and thick smears were collected from 555 children aged 1–17 years (mean age 9.68 ± 0.18) in the Amele region, Madang Province, PNG at two surveys in November-December of 1999 and 2000. Thirty-eight percent of samples were slide-positive for +-thalassaemia were found in this population (92.9%) with a predominance of the 4.2 deletion allele (frequency = 0.71). The CR1 L allele was also predominant with a prevalence of 94.6%, whereas the host erythrocyte polymorphisms SAO and G6PD were relatively uncommon in this population (< 10%).Table P. falciparum positive for one survey, two surveys or no surveys, according to host polymorphism. There was no association of α+-thalassaemia (P > .7), SAO (P = .29) or G6PD deficiency (P > .3) with P. falciparum prevalence either at both surveys, after adjustments for age. There was also no significant association of α+-thalassaemia (P = .78), SAO (P = .66) or G6PD deficiency (P = .64) with mean parasite density, after adjustments for age. Children who were blood group B were less likely to be positive at both surveys compared to those who were blood group O (P = .005). When P. falciparum prevalence was analysed separately for each year, blood group B had a significantly lower prevalence of P. falciparum (27.8%) compared to O (43.3%) in 2000 only (P = .02). There was no significant difference in mean parasite densities between ABO blood groups (P ≤ .4) as assessed by a mixed linear model, with adjustments for age. Children with the CR1 HL genotype were more likely to be P. falciparum positive for both surveys compared to HH individuals but this was of borderline significance (P = .07). There was also weak evidence of CR1 HH genotype having lower geometric mean parasite densities  compared to those who were HL  or LL genotype .Table β = 0.16, SE 0.009, P < .001) and parasitaemia  were found to positively influence antibody levels. This model was then applied to each of the five host erythrocyte polymorphisms investigated in this study. There was no significant difference in the age-dependent acquisition of VSA-specific antibodies with respect to ABO blood groups (P ≥ .39), SAO (P = .39), or CR1 genotype (P = .99). There was also no significant difference in age-dependent seroconversion in children with G6PD deficiency (P > .2), nor was there evidence of an interaction between G6PD deficiency and sex (P = .16). There was an effect of α+-thalassaemia on age-dependent anti-VSA antibody acquisition . There was no significant difference in rate of antibody acquisition between children of normal genotype and those that were homozygous for α+-thalassaemia (P = .14).A multivariate linear mixed model was used to examine the effect of age and parasitaemia on antibody levels in samples from the cohort. Both age . This result could have occurred by chance given that only 15 children with HH were P. falciparum positive during the study. There was also evidence of blood group B having lower P. falciparum prevalence in the 2000 survey, but there was no significant difference in parasite densities among ABO blood groups. Neither ABO blood group and CR1 genotype had no effect on age-dependent seroconversion to anti-VSA antibodies, indicating that differences in parasitological data in these groups must have been due to chance. This study would support the hypothesis that ABO blood group and CR1 deficiency associated with the L allele protect against malaria by reduced rosetting and associated pathology rather than parasite density.CR1 deficiency and blood group O are thought to protect against malaria by decreased rosetting. A positive correlation has been shown between rosetting and  evasion . PolymorP. falciparum VSA with respect to G6PD and SAO genotypes. These data conflict with several studies that have proposed that innate resistance to malaria involved the inability for the malaria parasite to grow in and/or invade G6PD deficient and SAO erythrocytes [There was no significant difference in geometric mean parasite densities or rates of seroconversion to hrocytes . There whrocytes , but theP. falciparum infection by a mechanism that does not influence parasite density or immunity to VSA.Data presented here highlight the importance of measuring malaria exposure at two time points to investigate the interactions of host polymorphisms with the malaria parasite. Results can occur by chance, and previous studies have reported data from a single time point. No polymorphism showed both a reduction in parasite prevalence/density and age-specific seroconversion to anti-VSA antibodies. We interpret these findings to indicate that the mechanisms of protection by these polymorphisms are not fully explained by either structural aspects of the erythrocyte preventing infection or by enhanced immune responses to VSA. Our observations from PNG differ significantly from those we and others have seen for African children with HbAS and/or Hb C carriers. HbAS individuals have lower parasite prevalence/densities and delayed time to reinfection, as well as increased prevalence of anti-VSA antibodies, compared to those of normal genotype ,44,82,83FJIF performed data analysis, interpreted data and prepared the final manuscript with KPD. PM helped to draft the manuscript and undertook laboratory work (FACS assay and PCR-based host genotyping except CR1). LP and CMW helped undertake laboratory work and interpret data. HI and LT undertook laboratory and field work. RR participated in the design of analysis. CSM, AJFL and KPD conceived the study, participated in its design, and obtained funding for the project. All authors discussed the results and approved the final manuscript.


Current recommendations to prevent malaria in African pregnant women rely on insecticide treated nets (ITNs) and intermittent preventive treatment (IPTp). However, there is no information on the safety and efficacy of their combined use.1030 pregnant Mozambican women of all gravidities received a long-lasting ITN during antenatal clinic (ANC) visits and, irrespective of HIV status, were enrolled in a randomised, double blind, placebo-controlled trial, to assess the safety and efficacy of 2-dose sulphadoxine-pyrimethamine (SP). The main outcome was the reduction in low birth weight.Two-dose SP was safe and well tolerated, but was not associated with reductions in anaemia prevalence at delivery , low birth weight , or overall placental infection (p = 0.964). However, the SP group showed a 40% reduction  in the incidence of clinical malaria during pregnancy, and reductions in the prevalence of peripheral parasitaemia (7.10% vs 15.15%) (p<0.001), and of actively infected placentas (7.04% vs 13.60%) (p = 0.002). There was a reduction in severe anaemia at delivery of borderline statistical significance (p = 0.055). These effects were not modified by gravidity or HIV status. Reported ITN's use was more than 90% in both groups.Two-dose SP was associated with a reduction in some indicators, but these were not translated to significant improvement in other maternal or birth outcomes. The use of ITNs during pregnancy may reduce the need to administer IPTp. ITNs should be part of the ANC package in sub-Saharan Africa.NCT00209781ClinicalTrials.gov  Every year 50 million women become pregnant in areas where malaria is endemic, and at least half of them live in Africa nd trimester onwards at least one month apart Currently, prevention of malaria in pregnancy in Africa relies on intermittent preventive treatment (IPTp) and insecticide treated nets (ITNs) Although IPTp and ITNs have shown separately to be efficacious in reducing the harmful effects of malaria during pregnancy, limited information exists on the safety and efficacy of both interventions together IPTp, is now being implemented throughout most malaria endemic areas of Africa, and has become somewhat controversial. This is fuelled by two elements; on the one hand, the spread of SP resistance, and the other, the overlap in some areas of malaria transmission and high HIV prevalence. IPTp with SP is not recommended to HIV-positive women receiving cotrimoxazole prophylaxis or antiretroviral drugs In order to evaluate the safety and efficacy of two intermittent doses of SP in women of all parities and regardless of HIV status, who had been given a long-lasting ITN (LLITN) through the antenatal clinic (ANC), we carried out a randomised, double blind, placebo-controlled trial of IPTp in Mozambican pregnant women. This information should help guide policy towards the rational use of control tools for malaria prevention in pregnancy.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see P falciparum. Anopheles funestus is the main vector, and the estimated entomological inoculation rate for 2002 was 38 infective bites per person per year. Data on the efficacy of SP in children in this area showed a therapeutic efficacy rate of 83%, with an in vivo parasitological sensitivity of 78.6% at day 14 The study was undertaken at the Centro de Investigação em Saúde da Manhiça (CISM) in Manhiça District, southern Mozambique. A demographic surveillance system covering 36 000 inhabitants is carried out by the CISM and constitutes the study area. Adjacent to the CISM is the Manhiça District Hospital (MDH), a 110 bed health facility. The characteristics of the area have been described in detail elsewhere This double blind, individually randomised, placebo controlled trial had the primary objective of estimating the additive protective effect of two-dose IPTp with SP to that of ITNs on LBW prevalence. Based on previous estimates in the area, a LBW prevalence of 20% and an estimated 25% reduction to 15% in the presence of ITNs From August 2003 to April 2005 pregnant women were enrolled at the MDH ANC if their gestational age was ≤ 28 weeks, they did not report allergies to sulpha drugs, and they were permanent residents of the CISM study area. After written informed consent was obtained, the lowest available study number was assigned. A computer-generated sequential list contained the study numbers linked to treatment identification letters, randomly ordered in blocks of 10. Tablets of SP or placebo, identical in shape and colour, were stored in 10 bottles labelled only with a single treatment identification letter. Women were randomised to receive 3 tablets of SP (1500 mg sulphadoxine/75 mg pyrimetamine) or placebo. The project health nurse administered study drugs to women with at least 12 weeks of gestational age. Doses were given twice from the second trimester, at least one month apart. Regardless of their gestational age, all women at recruitment received a LLITN. Assessment of gestational age was made by bimanual palpation of the fundal height. Haemoglobin and the rapid plasma reagin test  were assessed as part of the routine antenatal care. In accordance to the National Programme for HIV control, women were offered voluntary counselling and testing; HIV positive women and their babies were given Nevirapine prophylaxis and referred for further clinical follow-up.Plastic photo cards of mother and child facilitated identification at every contact throughout the study (from recruitment until 8 weeks postpartum). A health facility-based, passive surveillance system was established at the MDH. At each consultation, a standardized questionnaire was completed documenting signs and symptoms. Blood films were prepared for malaria parasite examination and the packed cell volume (PCV) measured if there was a history of fever in the preceding 24 hrs, or the axillary temperature was ≥37.5°C. Clinical malaria episodes were treated with chloroquine or SP in the first and subsequent trimesters, respectively, for uncomplicated malaria, and parenteral quinine for severe malaria.At delivery, venous blood was collected by venipuncture from the mother and the umbilical cord, and two thick blood smears and a filter paper prepared for haematological and parasitological determinations. Newborns were weighed on a digital scale, accurate to the nearest gram. The gestational age was assessed by the Dubowitz's method Eight weeks after delivery a capillary blood sample was collected from the mother and the infant for parasite and haematological determinations. The axillary temperature was measured and the weight of the baby recorded.ITN use was assessed by asking the women if they had slept under the LLITN the night before.Thick and thin blood films were stained and read according to standard, quality-controlled procedures HIV serostatus was assessed using a rapid test , and positive results confirmed using Unigold rapid test .Tissue samples were collected from the maternal side of the placentas and placed into 10% neutral buffered formalin. Biopsies were processed and stained following standard procedures Data were analysed by intention-to-treat (ITT) analysis whereby all randomised women were included regardless of whether or not they had received the intervention and the number of doses.2 test and proportions with the Fisher's exact test. Continuous values were evaluated with the non-parametric Wilcoxon test. Data analysis was performed using Stata 8.2 .The risk of the first or only episode of clinical malaria between recruitment and 8 weeks postpartum, or censoring due to withdrawal or death, was estimated using Cox regression models. The protective effect (PE) of SP was estimated from the hazard ratio (HR) as PE = 100(1-HR) %.The effect of time on efficacy since dose 1 was estimated using time versus treatment interaction in a Cox time-dependent model. In the secondary analyses, crude and adjusted  prevalences were calculated using the Risk Ratio. Likelihood Ratio tests were calculated to evaluate the interaction between the intervention group and parity, or HIV status. Differences in prevalence were estimated with the χMalaria infection was defined as the presence of asexual P. falciparum parasites of any density in a blood smear. A clinical malaria episode was defined as the latter plus an axillary temperature ≥37.5°C. The duration of a malaria episode was estimated as 28 days. Overall and severe anaemia were defined as a PCV lower than 33% and 21%, respectively.Placental infection was classified according to a previous definition Women with a multiple delivery (twins or triplets) were also included in the analysis.Only one woman from the SP group vomited after IPTp dose 1. There were no differences in the incidence of hospital admissions and outpatient visits during pregnancy or postpartum period, or in infant visits until age 8 weeks. Nine women, similarly distributed between the groups, had mild skin reactions within 27 days after any IPTp dose . No seveThere were no significant differences between the intervention groups in either the prevalence of LBW   or in meThe risk of foetal anaemia was halved among women in the SP group compared to those who received placebo  . A non-sOverall, 7% of the women receiving two-dose SP and 10% receiving placebo had a first or only episode of uncomplicated malaria (protective efficacy (PE), 40% ; p = 0.020) . The anaThere was no difference in the prevalence of anaemia at delivery between the intervention and control groups . However, although not significantly, there were fewer women in the IPTp/SP than in the placebo group with severe anaemia at delivery . This difference was not modified by adjustments for gravidity and HIV status .Prevalence of any placental infection was comparable between the groups , and so it was the proportion of placentas with only pigment deposition on histology (past infection). However, there were significantly less infected placentas with parasites in the SP group as assessed by impression smears  or histology (acute and chronic infection) . This diThere were less women in the SP group than in the placebo group with malaria parasitaemia at 8 weeks after delivery . There were no differences in the prevalence of women or infants with clinical malaria or anaemia . SimilarThis randomised placebo-controlled trial of IPTp in Mozambican pregnant women has shown that administering two IPTp-SP doses to women concurrently using LLITNs was safe and well tolerated, and there were no significant drug reactions despite the high prevalence of HIV among the study women. The intervention was associated with a moderate reduction in the incidence of clinical malaria during pregnancy, and with a statistically significant reduction in the prevalence of parasitaemia at delivery and at 8 weeks postpartum. However, these positive effects did not appear to translate in an improvement on relevant foetal birth outcomes such as birth weight or prematurity, nor on maternal anaemia.in vivo drug efficacy data in children in this area showed a parasitological sensitivity of 78,6% at day 14 in vivo drug resistance in sick children and the use of the same drug for prevention in asymptomatic individuals It could be argued that parasite resistance to SP may explain the modest impact on some malariometric parameters and the lack of effect on the more significant birth outcomes. The available The risk of malaria during pregnancy seems to concentrate in primi and secundigravidae women As previously reported, SP reduced the prevalence of malaria parasitaemia at delivery and post-partum The prevalence of active placental infection  in the SP group  was similar to the one reported in Kenyan and Malawian HIV-positive women who received monthly SP  Despite the fact that bednets are not traditionally used in this area, the delivery of ITNs through the ANC was well accepted and the compliance with their use was very high.In recent years, IPTp with SP has been the cornerstone of malaria control in pregnant women living in stable transmission areas. However, the recent controversy has raised questions as to whether this was the most appropriate approach for malaria prevention in African pregnant women given the reported increased in parasite resistance to SP in some areas In conclusion, this study provides information of public health relevance in deciding the adequacy of adding IPTp when ITNs are used, and suggests that IPTp may not be required if an ITN is used. To confirm these findings, more studies of similar design are needed in areas of different malaria endemicity in Africa. In the meantime, efforts should be focused to promote the delivery of long-lasting ITNs as part of other routine ANC health interventions.Protocol S1Study Protocol(0.10 MB DOC)Click here for additional data file.Checklist S1CONSORT Checklist(0.05 MB DOC)Click here for additional data file.


Many malarious countries plan to introduce artemisinin combination therapy (ACT) at community level using community health workers (CHWs) for treatment of uncomplicated malaria. Use of ACT with reliance on presumptive diagnosis may lead to excessive use, increased costs and rise of drug resistance. Use of rapid diagnostic tests (RDTs) could address these challenges but only if the communities will accept their use by CHWs. This study assessed community acceptability of the use of RDTs by Ugandan CHWs, locally referred to as community medicine distributors (CMDs).The study was conducted in Iganga district using 10 focus group discussions (FGDs) with CMDs and caregivers of children under five years, and 10 key informant interviews (KIIs) with health workers and community leaders. Pre-designed FGD and KII guides were used to collect data. Manifest content analysis was used to explore issues of trust and confidence in CMDs, stigma associated with drawing blood from children, community willingness for CMDs to use RDTs, and challenges anticipated to be faced by the CMDs.CMDs are trusted by their communities because of their commitment to voluntary service, access, and the perceived effectiveness of anti-malarial drugs they provide. Some community members expressed fear that the blood collected could be used for HIV testing, the procedure could infect children with HIV, and the blood samples could be used for witchcraft. Education level of CMDs is important in their acceptability by the community, who welcome the use of RDTs given that the CMDs are trained and supported. Anticipated challenges for CMDs included transport for patient follow-up and picking supplies, adults demanding to be tested, and caregivers insisting their children be treated instead of being referred.Use of RDTs by CMDs is likely to be acceptable by community members given that CMDs are properly trained, and receive regular technical supervision and logistical support. A well-designed behaviour change communication strategy is needed to address the anticipated programmatic challenges as well as community fears and stigma about drawing blood. Level of formal education may have to be a criterion for CMD selection into programmes deploying RDTs. Globally 3.2 billion people remain at risk of malaria and nearly one million malaria deaths occur each year, mostly in children under five years of age in sub-Saharan Africa . Besideset al  even asks us to take our children when they are sick to give us drugs. She has been very good because even if I go to her place at night she opens and gives me drugs. Even if I find her in the garden digging she comes back home to give me medicine. I trust her." The spirit of volunteerism as demonstrated by CMDs not demanding to be paid for their services was greatly appreciated by community members, and their trust and confidence in the CMD services was often the main motivating factor for the CMDs to do voluntary work. The community members who had used the CMDs perceived the medicines provided to them as effective in treating children with malaria, and the recovery of sick children after treatment by CMDs was said to enhance the reputation of the CMDs amongst their neighbours and community."According to me, they [CMDs] have done commendable work. They have a good relationship with the people and they have helped them a lot. They have been effective in distributing Homapak® and Coartem®. I heard that the tablets are to be distributed freely and they are doing it - they fulfilled the intention. My twins are making 3 years and I've never taken them to hospital. They are treated at home and given Coartem®. They [CMDs] have never asked for money for treatment." Health workers reported to have observed a reduced workload at the health facilities as a result of CMD management of children with fever in the communities."We have trained CMDs in many workshops. We taught them about the danger signs, so in case of such they refer them to the health centre immediately. The CMDs also sensitize the communities where they stay on malaria prevention. They have really helped us health workers. The work load has reduced. We appreciate [their contribution]". "There is a good relationship with us because they know where they stop. They know that they can't manage [everything] according to how they were trained. [For complicated cases] they immediately refer them to the health facilities. These people have really helped very much in the areas where they operate. We have not heard cases that a child has died at the CMDs level." However, there were also some reservations about CMDs performance in the management of malaria at community level. Some informants reported that CMDs are sometimes difficult to find and in some instances they are looked down upon by caregivers because they are not trained professionals. Others reported that medicine distribution is a difficult task and that CMDs can easily give medicines without understanding what the underlying problem is. Some KIs indicated that CMDs are not trusted because of their low level of education."In some cases [instances] they are minimized because they are not trained professionals. Medicine distribution [prescription] is very hard and they can easily give you medicine without understanding what you are suffering from. If you explain to him that you have malaria or that you are not feeling well, of cause; for him he has the medicine he just gives you". Although it emerged that community members had some doubts about CMD skills, there was consistency across the FGDs and KIIs a high degree of confidence that CMDs can perform the test once they are provided with adequate training. This trust and confidence was based on the education level of the CMDs, their experience, as well as perceptions about their commitment to work."If a person is trustworthy in a small thing they can work equally well even in a bigger one. These people have worked well and we never got problems with them. If my child falls sick, I just cross the road to him instead of going to Namungalwe where a person from Marina [name of place outside Iganga] knows nothing about me." Community members also echoed the need for refresher training for CMDs in addition to training on RDTs."That will be very good [introducing RDTs] only that they will need to be trained like how they were trained before starting to give out coartem.""I would second it [introducing RDTs] provided they are trained to do it safely. It is very important." CMDs expressed willingness to use RDTs as part of the services they provide, and are confident they can perform it if they receive adequate training. CMDs contended that they have been voluntarily distributing drugs and would now volunteer also for this new task. CMDs were optimistic that the community would welcome their use of RDTs."If they know that we have been trained, they will accept [the use of RDTs]. Even for the giving out drugs they first resisted but later accepted." Also the majority of health workers supported the idea of CMDs conducting RDTs as long as they are given practical training to acquire additional skills, accurate information, and guidelines to ensure safety. They asserted that this is one way of taking services nearer to people, encourage early treatment seeking behaviour, and is one way of providing CMDs with additional skills crucial for managing malaria. They also appreciated the fact that the CMD treatment of malaria could be performed more effectively if an RDT was introduced, so as to distinguish between malaria and other types of fever."That is a good thing [the use of RDTs], and it will work because people have been coming here a lot because of malaria. So if it [RDT and malaria treatment] is taken near them it will help them so that a person knows what is required and will help them get early treatment. But these CMDs need to be given additional skills because they need to have enough information to avoid questions like why they are taking blood." Even though most community members were positive towards the use of RDTs by CMDs, many had mixed feelings about the taking of blood from children. The fear included concerns that their children could get infected with HIV in the process of undertaking an RDT and that the blood could be used to test children for HIV rather than malaria."People have fears about blood and that it can lead to contracting HIV. It would also imply to some people that when they prick to test the blood, they think you are testing for HIV but not malaria. They believe you can prove malaria by mere looking at signs like vomiting, shivering, diarrhea and other basic signs." Another frequently mentioned concern was that the blood could end up in the wrong hands and be used for witchcraft."Since people use it [blood] for bewitching, someone can draw it from you without you being aware that it will be used for other things. If you have a co-wife she can use it to bewitch you to death or stop producing children. Some say that they use it as sacrifice." Given the tasks that the CMDs are given, views regarding the challenges that CMDs are likely to face were solicited. The reported challenges that were foreseen included that CMDs will lack transport for follow up of patients and for replenishment of supplies from the health facilities, that adults will try to force CMDs to also test them for malaria, that caregivers will insist that CMDs treat their children even when the child needs referral, and that some leaders will try to seek for favours for themselves and their relatives."I have seen some of them [CMDs] walking all the way to the health center to get drugs, and when following up children. They need to be assisted with transport for drugs". "These tests [RDTs] will create a lot of interest. I am concerned that adults will also demand to be tested. I think also some of our people will insist that their children are treated when they should be referred as now CMDs will be seen as "little doctors" who can take blood and examine it". et al [et al [et al [et al [et al [® (chloroquine and sulphadoxine/pyrimethamine) - a combination drug known to have low clinical efficacy due to parasite resistance.The study found that community medicine distributors (CMDs) were trusted by their communities because of their voluntary services, ease of access, and perceived effectiveness of the anti-malarial drugs they use. This is in line with the findings from a multi-country study (including Uganda) which showed high community acceptability of CMD management of sick children, with most children receiving treatment on the same day or within one day of onset of symptoms . Kilian et al  found sil [et al  also conl [et al  demonstrl [et al  evaluatel [et al  evaluateCMDs were trusted by health workers who recognized that the CMDs have contributed to a reduced workload in the health facilities. This concurs with what was found in a rural district in Burkina Faso, where a reduction in workload at peripheral health facilities was observed after HMM was implemented . SimilarIn this study, the formal education level of CMDs appeared to be a key factor in their acceptability by the community, with CMDs with low education being viewed less positively. This finding needs to be explored further, as it could have major implications on what criteria should be used to select CMDs under home management of malaria (HMM) programmes. Current selection criteria for the community to consider when they choose their CMDs include: age, literacy levels, and gender but different districts and sub-districts may however, choose different criteria for nominating CMDs and ability to read and write may not be prioritized in certain areas . If show® and RDTs in HMM to be high, with a marked increase in treatment-seeking behaviour following the implementation [A majority of community members, health workers and CMDs welcomed the use of RDTs by CMDs, provided that they are properly trained in their use. A similar study in Sudan also found community acceptability of Coartementation . In the While RDTs have the potential to greatly enhance the management of febrile illness  the succFears and stigma of taking blood of children in the community were common, relating both to risk of exposure to HIV and witchcraft. Myths about blood could have serious consequences to the HMM programme, especially if there is a bad outcome for a child. In Tanzania, though there was a high degree of provider and caregiver acceptance and satisfaction with RDTs, some caregivers had reservations about RDTs, with some thinking they were HIV test kits . To counIn this study, community members anticipated that CMDs would face difficulties with adults demanding to be tested and caregivers insisting that their children should be treated instead of being referred. Compliance to guidelines is often a major challenge and a study in Burkina Faso found very low health worker compliance to negative RDT results, as they still went on to prescribe anti-malarial drugs for patients who tested negative . This waOne of the limitations of qualitative methods is that the magnitude of a variable of interest across different categories of respondents cannot be measured. However, it was not the intent of this study to quantify the issues under study as a household survey is planned for this purpose. Triangulation of information from caregivers, health providers, CMDs and community and opinion leaders using both FGDs and KIIs, was a useful strategy for checking consistency and contradictions across and within groups ,57. The Use of rapid diagnostic tests (RDTs) by community medicine distributors (CMDs) is likely to be acceptable by community members given that CMDs are properly trained, supported and provided logistical support. A well-designed behaviour change communication strategy is needed to address the anticipated programmatic challenges as well as community fears and stigma about drawing blood. Level of formal education may have to be a criterion for CHW selection into programmes deploying RDTs.The authors declare that they have no competing interests.DM, JT, JK, GP, PW, FB, BM, HC, GO and KK took part in designing the study, in tools development, in data analysis and in manuscript writing. DM, JK, FB, BM, GO and KK did field work. All authors approved the final manuscript.