The pre-erythrocytic malaria vaccine RTS,S/AS02A has shown to confer protection against clinical malaria for at least 21 months in a trial in Mozambican children. Efficacy varied between different endpoints, such as parasitaemia or clinical malaria; however the underlying mechanisms that determine efficacy and its duration remain unknown. We performed a new, exploratory analysis to explore differences in the duration of protection among participants to better understand the protection afforded by RTS,S.The study was a Phase IIb double-blind, randomized controlled trial in 2022 children aged 1 to 4 years. The trial was designed with two cohorts to estimate vaccine efficacy against two different endpoints: clinical malaria (cohort 1) and infection (cohort 2). Participants were randomly allocated to receive three doses of RTS,S/AS02A or control vaccines. We did a retrospective, unplanned sub-analysis of cohort 2 data using information collected for safety through the health facility-based passive case detection system. Vaccine efficacy against clinical malaria was estimated over the first six-month surveillance period (double-blind phase) and over the following 12 months (single-blind phase), and analysis was per-protocol. Adjusted vaccine efficacy against first clinical malaria episodes in cohort 2 was of 35.4%  over the double-blind phase and of 9.0%  during the single-blind phase.Contrary to observations in cohort 1, where efficacy against clinical malaria did not wane over time, in cohort 2 the efficacy decreases with time. We hypothesize that this reduced duration of protection is a result of the early diagnosis and treatment of infections in cohort 2 participants, preventing sufficient exposure to asexual-stage antigens. On the other hand, the long-term protection against clinical disease observed in cohort 1 may be a consequence of a prolonged exposure to low-dose blood-stage asexual parasitaemia.NCT00197041ClinicalTrials.gov  Plasmodium falciparum circumsporozoite surface protein (CSP) and the candidate malaria vaccine in the most advanced development phase, has been in development for more than two decades. After having demonstrated partial protection against infection in non-immune and semi-immune adults Developing a new vaccine is a long and complex process. For example, RTS,S/AS , a pre-erythrocytic vaccine based on P. falciparum asexual-stage infection, clinical malaria, severe malaria or death. Selection of the endpoint depends on several factors, including the type of vaccine, the phase of the trial and the evidence needed for advocacy and policy decision, and it will determine the sample size and have implications in terms of cost and time. Infection is the endpoint closest to the biological target of the vaccine and is influenced by fewer local cofactors, such as management of malaria cases and parasite and human genetics. As we go downstream , the clinical and public health relevance increases, providing stronger evidence for advocacy and policy decision, but the number of cofactors influencing the risk of malaria is larger, potentially decreasing the generalizability of results One of the most critical decisions when preparing a vaccine's clinical development plan is the proper selection of criteria by which the product will be advanced, re-engineered or terminated. Selection of appropriate study endpoints in the various trials that lead up to definitive Phase III efficacy studies is an important part of this process. Different endpoints can be used to estimate efficacy of a pre-erythrocytic vaccine: P. falciparum parasitaemia infection. By enrolling this second cohort it was possible to estimate efficacy for a more upstream endpoint and to evaluate how it correlated with efficacy against clinical malaria in Cohort 1. Participants in Cohort 2 were followed up through both active detection of infection (ADI) and PCD. During the double-blind phase the vaccine efficacy for time to first infection was 45.0%  In 2003, a randomized controlled Phase IIb proof-of-concept trial was conducted in Mozambique to provide a preliminary estimate of the efficacy, immunogenicity and safety of RTS,S/AS02A malaria vaccine in an age group (1 to 4 years) that would be close to the ultimate target population (infants) P. falciparum infection during the double-blind phase, therefore it was not possible to continue the ADI during the single-blind phase, in which children were only followed up for safety surveillance through health facility-based PCD.After unblinding data of the first six months of follow up, participants were followed up for an additional 12 months (single-blind phase), during which vaccine efficacy for the first or only clinical malaria episode in cohort 1 was 28.9% . Therefore the vaccine efficacy did not wane, showing sustained protection during at least 18 months The correlation between efficacy against clinical malaria in cohort 1 and efficacy against infection in cohort 2 showed that infection could be used as the primary endpoint for efficacy trials of pre-erythrocytic vaccines, which allows conducting smaller trials with high power, decreasing time and cost. Based on these results, a Phase I/IIb randomized controlled trial was recently conducted in infants in the same area to assess the safety, immunogenicity and efficacy of RTS,S/AS02D malaria vaccine, administered at 10, 14 and 18 weeks of age, staggered with the Expanded Program on Immunization vaccines In these two trials vaccine efficacy estimates vary for different endpoints, transmission intensities and age groups. To provide more evidence on the factors that may influence vaccine response and its duration, we performed a sub-analysis of cohort 2 data from the study in children, that was not included in the original protocol, to estimate vaccine efficacy for clinical malaria in this cohort, using information collected for safety through the health facility-based PCD system.417 children were recruited and randomized in cohort 2 . Details of the trial profile, the baseline characteristics, the safety, reactogenicity, part of the immunogenicity data and the efficacy against infection in cohort 2 have been previously reported In the analysis of the according-to-protocol (ATP) cohort over the double-blind phase (study months 2.5–8.5), 102 children had a first episode of clinical malaria (primary case definition) (46 per 72.8 person-years at risk (PYAR) in the RTS,S/AS02A group and 56 per 59.3 PYAR in the control group), giving a crude vaccine efficacy for the time to first or only clinical malaria episode of 34.3%  and an adjusted efficacy of 35.4%  and the adjusted vaccine efficacy for multiple malaria episodes was 30.0% .The primary case definition (fever and parasitaemia >2500/µL), that was chosen for cohort 1 based on baseline data from Manhiça, was estimated to be 97.4% sensitive (95% CI 88.8–100.0) and 72.3% specific (49.3–83.6) for cohort 2. Vaccine efficacies using other more specific case definitions are presented in During the single-blind phase (study months 8.5–21) 119 children of the ATP cohort had a first or only episode of clinical malaria, yielding a crude vaccine efficacy of 6.4%  and an adjusted efficacy of 9.0% . P. falciparum parasitaemia, giving an adjusted vaccine efficacy against first infection of 51.1% . Twenty-one children in the RTS,S/AS02A group and 40 children in the control group had a first episode of clinical malaria (primary case definition), yielding an adjusted efficacy for clinical malaria of 61.0% .To be able to compare the vaccine efficacy during the same follow up time period in cohort 2 to that in the infant trial conducted recently in the same area, an analysis was also done for a shorter follow up period of 3.5 months (study months 2.5–6). During this period 111 children in the RTS,S/AS02A group and 147 children in the control group had first episodes of asexual During the double-blind phase the geometric mean density of asexual-stage parasites at the first or only infection was significantly lower in asymptomatic children (1810 parasites/µL) than in children who presented fever .In the analysis of the intention-to-treat (ITT) cohort over the double-blind phase, 148 children had a first episode of clinical malaria , with a crude estimate of vaccine efficacy against clinical malaria of 9.3% . The corresponding estimate during the single-blind phase was of −4.2% .The relation between anti-CSP antibody titers and the risk of infection and clinical malaria during the double-blind phase was evaluated. The hazard ratio for first or only infection per ten-fold increase in the value of anti-CSP IgG was 0.41 . When comparing children in the higher tertile against those in the lower tertile value of anti-CSP IgG, the hazard ratio was 0.54 . The equivalent hazard ratios for first or only episode of clinical malaria were 0.99  and 0.90  respectively. Thus anti-CSP antibody levels correlated with protection against infection but not with protection against clinical malaria episodes.P. falciparum infections. Consequently the sample size for this cohort, based on this endpoint, was much smaller than for cohort 1. Nevertheless, a high incidence of clinical malaria episodes also allowed estimating vaccine efficacy against this endpoint, although with a lower precision.In cohort 2, adjusted efficacy of the RTS,S/AS02A candidate malaria vaccine against first or only clinical malaria episodes in Mozambican children aged 1 to 4 years was of 35.4% during the first six months of follow up (ATP cohort), decreasing to 9.0% in the subsequent 12 months of follow up. The follow up of cohort 2 participants, which included both ADI and PCD, was designed to estimate vaccine efficacy against new The primary case definition used (fever and a parasitaemia >2500 parasites/µL) was chosen for cohort 1 based on previous background data from the Manhiça area, where it had been estimated to be 91% specific and 95% sensitive Efficacy estimates for clinical malaria in cohort 2 are much lower in the ITT analysis, starting at dose 1 and including the vaccination period, than in the ATP analysis, starting post dose 3. In the ITT analysis time at risk is larger and there are smaller differences in the number of clinical episodes between the RTS,S/AS02A and control group during these first 2.5 months, possibly due to the administration of anti-malarial treatment before dose 3 and that children have not yet received the three vaccine doses, decreasing the differences between the total incidences of the double-blind phase. During the following months, maybe due to the children who have not completed the three doses vaccination course, there is a smaller difference in the number of malaria cases between the RTS,S/AS02A and control group compared to the ATP cohort, thus decreasing the vaccine efficacy.Data show that cohort 2 children that received RTS,S/AS02A were partially protected against infection and clinical disease in the first six months of follow up post dose 3 (35.4%), at levels similar to cohort 1 ) P. falciparum had, on average, longer periods of low density parasitaemia, as they were only treated when parasite density reached the fever threshold and the child was taken to the health facility for diagnosis and treatment. Geometric mean parasitaemias (GMPs) at the time of presentation with a clinical malaria episode in cohort 1 were 43522 for the RTS,S/AS02A and 41867 parasites/µL for the control group What are the main differences between cohort 1 and cohort 2 that might explain the discrepancy in vaccine duration of protection? Firstly, the different study design and follow up of participants. In cohort 1 no antimalarial treatment was given to clear parasitaemia before dose 3 and only health facility-based PCD was conducted to detect malaria cases. Therefore those children who became infected with Secondly, the malaria transmission intensity was higher in Ilha Josina, the area where cohort 2 was recruited, as reflected by the geometric mean of antibodies against the whole parasite measured by indirect fluorescent antibody test (IFAT) and the percentage of splenomegaly at baseline. Therefore participants in Cohort 2 may have had a higher level of naturally acquired immunity against infection and clinical malaria when the trial started.To understand the effect of vaccination and duration of protection, it is difficult to disentangle the effects of the study design from other potential factors such as the immunity level at the time of vaccination and the malaria exposure. RTS,S has shown to extend time to first infection, as seen in cohort 2, which results in reduced risk of clinical malaria, as seen in cohort 1 and 2 during the first 6 months of follow up. In cohort 1 sustained protection was shown for at least 21 months, and this must be a function of either sustained pre-erythrocytic immunity or induction of asexual-stage immunity. Moreover, waning efficacy in cohort 2 can be interpreted as waning pre-erythrocytic immunity. However, similar anti-CSP antibody levels between cohort 1 and 2 (data not shown) would argue against differential pre-erythrocytic immunity explaining the differences in the duration of protection. Based on this line of reasoning, we hypothesize that, as the vaccine-induced pre-erythrocytic immunity declines following the peak levels achieved after vaccination, it only partially inhibits hepatocyte invasion, liver-stage development and release of merozoites to the blood, decreasing the parasite load in the face of a new infection. A low-dose parasitaemia resulting from the partial pre-erythrocytic immunity might be critical to induce or boost the development of asexual blood-stage immune responses, which may confer long-lasting protection against clinical malaria. This low-dose parasitaemia has to be maintained for enough time to stimulate the asexual-stage immune response. Therefore the short-lived vaccine-induced pre-erythrocytic response facilitates the development of a long-lasting asexual-stage immunity in the presence of new infections that act as natural asexual-stage boosters. A vaccine inducing partial pre-erythrocytic protection, like RTS,S/AS, might allow the development of a better and more sustained asexual-stage protection than a more efficacious vaccine, by allowing this “leakage” of low-dose parasites.This is consistent with other recent hypotheses Children in cohort 1 were probably exposed to low-density parasitaemias for a longer time than children in cohort 2, in which the development of this enhanced asexual-stage immune response may have been impaired. We propose this may explain a waning of the vaccine-specific protective response in cohort 2.The short duration of protection in cohort 2 is not surprising and is similar to that observed in other RTS,S trials With regard to antibody responses to the vaccine in cohort 2 children, the level of anti-CSP IgG was correlated with a lower risk of infection but not with a lower risk of clinical malaria. In cohort 1 it had not been correlated with the risk of clinical malaria either P. falciparum infection as the primary endpoint and ADI can be used.When designing future pre-erythrocytic malaria vaccine trials it has to be taken into account that the study design might have a great impact on the duration of protection. ADI with rapid treatment of parasitaemias might impair the development of long-lasting protection, although initial efficacy seems to be independent of study design, age or malaria transmission intensity. If assessment of duration of protection is included in the trial objectives the design should consist only of a PCD follow up. Otherwise, if the main aim is to obtain quick efficacy results, a smaller trial with In conclusion, the preponderance of data discussed leads to the following hypothesis: that the long-term protection against clinical disease observed in RTS,S/AS02A recipients is a consequence of a partially protective vaccine-induced pre-erythrocytic response that lasts several months, and limits the number of viable sporozoites and merozoites emerging from the liver to initiate the blood stage cycle of the infection. This leads to prolonged exposure to low-dose asexual blood-stage parasites that allows the acquisition of long-lasting asexual blood-stage immunity. Study designs that include prompt ascertainment and treatment of infections in the absence of symptoms may modify long-term protection. Vaccination in infancy therefore has the potential to confer important levels of protection through a time of high susceptibility in early childhood. A fuller understanding of the mechanism of vaccine action including determination of the efficacy and duration under varying conditions of malaria transmission will be reached through the conduct of properly designed Phase III trials.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see P. falciparum, is perennial with marked seasonality. The trial was conducted in two different areas: Manhiça and Maragra, where cohort 1 (n = 1605) was recruited, and Ilha Josina, 55 km north of Manhiça, where cohort 2 (n = 417) was recruited. The estimated entomological inoculation rate for the Manhiça area in 2002 was 38 infective bites/person/year, being Anopheles funestus the main vector. In Ilha Josina the transmission is higher than in Manhiça, as reflected by a significantly higher geometric mean of antibodies against the whole parasite as assessed by IFAT and percentage of splenomegaly at baseline in study participants recruited in that area The trial was conducted at the Centro de Investigação em Saúde da Manhiça , in Manhiça District (Maputo Province), southern Mozambique. The area is under demographic surveillance system (DSS) and has been described in detail elsewhere The study was a Phase IIb double-blind, randomized controlled trial in children aged 1 to 4 years to assess the efficacy, immunogenicity and safety of RTS,S/AS02A candidate malaria vaccine according to a 0, 1, 2 month vaccination schedule The study design has been described in detail elsewhere P. falciparum infection, as detected by ADI or health facility-based PCD. According to protocol, information on clinical malaria episodes of cohort 2 participants was collected through PCD throughout the double and single-blind phases, with the objective of using these data for monitoring safety rather than the formal evaluation of vaccine efficacy. The rationale for that was that interpretation of efficacy results of clinical malaria disease collected at health centers would be compromised by the ADI visits and the sample size would be inadequate. However, despite the limitations of the study design and sample size, to gain further insights into the mechanism of long lasting protection, we have now performed an exploratory analysis, using these data to estimate incidence of clinical malaria and vaccine efficacy against clinical malaria in cohort 2.In the original protocol, the efficacy endpoint in cohort 2, assessed during the double-blind phase, was first The protocol was approved by the National Mozambican Ethics Review Committee, the Hospital Clínic of Barcelona Ethics Review Committee and the PATH Human Subjects Protection Committee. The trial was conducted according to the International Conference on Harmonisation good clinical practice guidelines, and was monitored by GSK Biologicals. A local safety monitor and a data and safety monitoring board closely reviewed the conduct, safety and data of the trial.The study was registered with the ClinicalTrials.gov identifier NCT00197041.P. falciparum CSP, that is fused to the S antigen of the hepatitis B virus, and is formulated with the AS02A Proprietary Adjuvant System. Details of the formulation and dosing of the vaccine have been reported elsewhere Prevenar™ Wyeth Lederle Vaccines) and one dose of Haemophilus influenzae type b vaccine  and for children aged ≥24 months the pediatric hepatitis B vaccine .Screening, informed consent, enrolment, randomization, immunization and safety assessment were done as previously described A round-the-clock health facility-based morbidity surveillance system was operating at the Manhiça District Hospital and the Maragra and Ilha Josina health posts throughout the study. A standardized questionnaire form, which includes personal and demographic data and clinical signs and symptoms, was completed for each child seen at the outpatient clinic. The axillary temperature was measured with an electronic thermometer and recorded and a finger-prick blood sample was collected from all children who present fever (axillary temperature ≥37.5°C) or report a history of fever in the preceding 24 hours. Blood was collected into heparinized capillaries to measure the packed cell volume (PCV) and thin and thick blood smears were prepared to determine parasitaemia. The first line malaria treatment for non complicated malaria at the time of the study was sulfadoxine-pyrimethamine and amodiaquine and the second-line treatment was Coartem® (artemether-lumefantrine) (Novartis).P. falciparum parasitaemia received antimalarial treatment, irrespective of symptoms, and were excluded from subsequent ADI visits.In cohort 2 participants were followed up by a combination of ADI and PCD during the double-blind phase. Antimalarials  were administered to all participants 14 days before dose 3 to presumptively clear parasites. Parasitaemia was checked two weeks later and, if positive, the child was treated with the second line antimalarial treatment and was not included in the assessment for ADI. Surveillance for malaria infection was started 14 days after dose 3 and was done throughout the double-blind phase (study months 2.5–8.5) through ADI visits performed every two weeks for 2.5 months and monthly for the following two months. During the ADI a field worker visited participants at home, completed a brief morbidity questionnaire and measured the axillary temperature. If the child was afebrile the field worker collected blood by fingerprick onto slides and filter paper. If the child had fever or a history of fever in the preceding 24 hours, the field worker accompanied the child to the Ilha Josina health post, where he or she was examined and blood slides and filter paper were collected. All children with Antibodies against CSP were measured before dose one and 30 days after dose three and IFAT and spleen size  were measured at screening.Blood slides were Giemsa-stained and read following standard quality-controlled procedures P. falciparum-infected red blood cells fixed onto a 12-well slide. Positive reactions were revealed with fluorescein isothiocyanate-labelled secondary antibody diluted in Evans blue. The highest dilution giving positive fluorescence under an ultraviolet light microscope was scored.IgG antibodies specific for the CSP tandem repeat epitope were measured by a standard ELISA with plates adsorbed with the recombinant antigen R32LR that contains the sequence [NVDP(NANP)15]2LR, using a standard serum as reference Trial results presented previously P. falciparum malaria. A clinical episode was defined as a child with an axillary temperature of ≥37.5°C and a P. falciparum asexual parasitaemia of >2500 parasites/µL on the blood slide (primary case definition), detected through the health-facility based PCD or the ADI visits. Analyses were performed on the ATP cohort, which was defined as children who met all eligibility criteria, received the complete vaccination course and contributed to the efficacy surveillance. Time at risk started 14 days after dose 3 and the analysis was conducted for the time periods 2.5 to 8.5 (double-blind phase) and 8.5 to 21 (single-blind phase) study months. For data pertaining to the period 2.5 to 8.5 vaccine efficacy for other definitions of clinical malaria using different cut-offs for parasitaemia and assessment of vaccine efficacy for multiple malaria episodes was also calculated.The results presented here are exploratory analyses of cohort 2 that were not described in the protocol, performed on data collected to 21 months post-study start. The endpoint of this sub-analysis was first or only clinical episode of Absences from the study area of two or more weeks and a time interval after antimalarial drug use  were not included in the time at risk. If the combination of drugs was given, the longest period was used. For the analysis of multiple episodes of clinical malaria, we did not judge a child to be susceptible for 28 days after the previous episode. Vaccine efficacy for the time to first or only clinical malaria episode was assessed using Cox regression models and defined as (1−Hazard Ratio). Vaccine efficacy was adjusted for the covariates: age at dose 1, bednet use at baseline and distance from the health centre . The interaction between age and vaccine efficacy was not significant for any of the follow up periods, so an interaction term was not included. The proportional hazards assumption was investigated graphically, using a test based on the Schoenfeld residuals For multiple episodes of clinical malaria the vaccine effect was assessed using Poisson regression models with normal random intercepts, including the time at risk as an offset variable. Vaccine efficacy was defined as (1−Rate Ratio). The difference in the geometric mean of the positive densities was assessed with the non-parametric Wilcoxon test.The sensitivity and specificity of different case definitions were estimated for cohort 2 participants using data from the study month 8.5 visit, following the methodology described by Smith and colleagues Results of the ITT cohort are also presented. All children who received at least one vaccine dose were included and efficacy estimates were not adjusted for covariates. Time at risk started from dose 1 and was not adjusted for absences from the study area or antimalarial drug use.The relation between anti-CSP antibody titers as measured 30 days post dose 3 and the risk of infection and clinical malaria was assessed in RTS,S/AS02A recipients. The hazard ratio of participants with anti-CSP antibodies in the higher tertile against those in the lower tertile was estimated, as well as the hazard ratio per ten-fold increase in the value of anti-CSP antibodies, using Cox regression models.The sample size of cohort 2 was calculated at the beginning of the study to estimate vaccine efficacy against first or only infection in cohort 2, which has been previously reported Analyses were done using STATA version 10.0 .Checklist S1CONSORT Checklist(0.19 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(1.19 MB DOC)Click here for additional data file.


In malaria endemic regions people are commonly infected with multiple species of malaria parasites but the clinical impact of these Plasmodium co-infections is unclear. Differences in transmission seasonality and transmission intensity between endemic regions have been suggested as important factors in determining the effect of multiple species co-infections.Plasmodium species  were randomly distributed amongst all children but not adults in the LIST and PT regions. Mean parasite density in children was lower in the HIST compared with the other two regions. Mixed species infections had lower mean parasite density compared with single species infections in the PT region. Fever rates were similar between transmission regions and were unaffected by mixed species infections. A history of fever was associated with single species infections but only in the HIST region. Reduced mean [Hb] and increased anaemia was associated with perennial transmission compared to seasonal transmission. Children with mixed species infections had higher [Hb] in the HIST region.In order to investigate the impact of multiple-species infections on clinical measures of malaria we carried out a cross-sectional community survey in Malawi, in 2002. We collected clinical and parasitological data from 2918 participants aged >6 months, and applied a questionnaire to measure malaria morbidity. We examined the effect of transmission seasonality and intensity on fever, history of fever, haemoglobin concentration ([Hb]) and parasite density, by comparing three regions: perennial transmission (PT), high intensity seasonal transmission (HIST) and low intensity seasonal transmission (LIST). These regions were defined using multi-level modelling of PCR prevalence data and spatial and geo-climatic measures. The three Our study suggests that the interaction of Plasmodium co-infecting species can have protective effects against some clinical outcomes of malaria but that this is dependent on the seasonality and intensity of malaria transmission. Plasmodium species is common in malaria endemic regions. It has been suggested that interactions between different species, in conjunction with differences in seasonality and intensity of malaria transmission may underlie variation in the epidemiology and clinical presentation of malaria Plasmodium falciparum causes the greatest morbidity and mortality but most malaria endemic regions are co-endemic for some or all of the other three human species: P. malariae, P. vivax and P. ovale. Individually, these species cause less severe morbidity and fewer deaths than P. falciparum but they are commonly found as co-infections with P. falciparumP. falciparum without reference to the potential effects of co-infecting species.Infection with multiple P. malariae has been implicated in protecting against fevers caused by P. falciparum in children in Cote d'Ivoire P. malariae are supported by experimental data from malaria-therapy infections in humans carried out in the 1940's, in which prior infection with P. malariae reduced fever caused by P. falciparumP. vivax infection reduced morbidity from subsequent P. falciparum infections in Vanuatu P. vivax or P. malariae protected against P. falciparum fevers Only a few studies have documented the effects of co-infection on uncomplicated clinical malaria Plasmodium co-infections and clinical measures of malaria under different epidemiological conditions. We compared two districts of Malawi with perennial (Mangochi) and seasonal (Dedza) malaria transmission and examined the effect of transmission intensity by comparing two distinct transmission regions within Dedza District. We used PCR detection of parasites to examine the prevalence and age-distribution of multiple Plasmodium species in these diverse regions. In Malawi, as in most of sub-Saharan Africa, the most common sympatric malaria combination is P. falciparum, P. malariae and P. ovale. Previous studies using PCR diagnosis have demonstrated that the prevalence of P. malariae and P. ovale are greatly underestimated by microscopy especially when these are present in mixed infections with P. falciparumPlasmodium densities, to determine how carriage of multiple Plasmodium species relates to clinical outcomes of malaria.In this study we examined the relationship of The study was carried out between March and April 2002 in two districts of Malawi: Dedza in Central Region (14°22′South 34°19′East) and Mangochi in Southern Region (14°28′South 35°15′East). Dedza district lies in a mountainous, semi-forested region at an altitude of >1000 m on the Rift Valley escarpment, bordering Mozambique. Mangochi lies 100 km to the east of Dedza, adjacent to the south western shore of Lake Nyasa, at a lower altitude of approximately 500 m. Annual rainfall is approximately 900 mm in both districts, with the rainy season November–March, during which >90% of rain falls. Dedza temperatures range from 10°C to 28°C, humidity 75% in the wet season and 3°C to 25°C, humidity 60% in the dry season. Annual mean temperature minima and maxima in Dedza are 7°C lower than for Mangochi and wet and dry season humidity values are 77% and 58% respectively The total population of the two districts at the time of the study was 544,334 for Dedza and 671,102 for Mangochi, within a total Malawi population of 11 million. Outside of the district towns, the populations of both districts reside in dispersed rural settlements where most dwellings are of traditional mud and wood construction with thatched roofs. The predominant occupation in both districts is subsistence farming and in villages adjacent to Lakes Malawi and Malombe, fishing provides additional income. The predominant ethnic group in Dedza is Chewa, who are part of the Nyanja group of Bantu, whilst the population in Mangochi is predominantly of the Yao tribe Plasmodium in the vector An. arabiensis and An. gambiae s.s. are the major vectors An. gambiae s.l. and An. funestusMalaria transmission in Dedza district is restricted to the wet season. During the dry season in Dedza, temperature falls below the minimum for development of Official statistics show that the number of clinical malaria cases and malaria deaths were more than three times greater in Mangochi than in Dedza  within a 25 km radius of the district hospitals of Dedza and Mangochi . SixteenMalaria community surveys were carried out between 8 March and 7 April 2002 in Dedza and Mangochi Districts of Malawi. Consenting individuals aged >6 months from each village were enrolled in the study. Oral consent for village participation was obtained from village elders and individual written consent was obtained from adults after explanation of the aims and methods of the study. Written consent for the participation of children was given by their parents or guardians.From a single finger prick, thick and thin blood smears and a filter paper blood sample for molecular analysis were prepared and haemoglobin concentration was measured using a portable haemoglobinometer  according to the manufacturer's instructions. Axillary temperature was measured with a digital thermometer and a questionnaire was administered to collect the following information from each participant: age (years), sex, occupation, religious denomination, history of fever in previous 2 weeks, medicine taken in the previous 2 weeks, overnight stay away from home in previous 4 weeks, use of bednets and other anti-malarial prevention measures.Following the analysis of blood smears by microscopy, parasite positive participants with axillary temperature ≥37.5°C, or [Hb]≤8.0 g/dl, or ≥5000 parasites/µl were treated within 24 hours of blood sampling with sulphadoxine-pyrimethamine (SP) and ferrous sulphate, according to government guidelines at the time of the study. Participants with overt signs of severe malaria or with haemoglobin≤5.0 g/dl or parasite density ≥100,000 parasites/µl of blood were taken to the district hospital. We did not follow up the clinical outcomes of treated or hospital referred participants. Ethical approval for the study was granted by the National Health Sciences Research Committee, Ministry of Population and Health, Government of Malawi and Glasgow University Ethics Committee for Non-clinical Research Involving Human Subjects.http://earth.google.com/; http://www.geographynetwork.com). As no climate data were available, vegetation cover was used as a proxy of greenness (leaf-top photosynthesis) and precipitation availability http://modis.gsfc.nasa.gov/). Differences between wet and dry seasons were examined and based on values from images 14–29 September 2001 (dry season), NDVI 30 September–15 October 2001 (dry season), NDVI 6–21 March 2002 (wet season), NDVI 22 March–6 April 2002 (wet season).Village location (latitude and longitude) was determined using ground GPS readings  and maps were drawn using ArcInfo 9.1 . Data on national boundaries, district hospitals, elevation, forested areas, water bodies, marshland and tributaries were obtained from scanned maps P. falciparum prevalence in relation to district hospitals and key environmental characteristics. The following geo-climatic variables were available for each village and extracted for statistical analysis: elevation, latitude, longitude, distance from district hospital, mean dry season NDVI, mean wet season NDVI, and difference between mean wet and dry season NDVI. Additional analysis in relation to elevation was undertaken by visualising topographical/contour differences between each village on a 3D wireframe map created in the surface mapping programme Surfer 7.0 .All epidemiological and environmental data were imported and geo-referenced in the geographical information system software ArcInfo 9.1. Maps were drawn showing each village location and Plasmodium and approximate parasite density, then parasite density counts per 200 leukocytes were made from positive smears. Parasite density per µl of blood was calculated assuming a standard leukocyte count of 8000 per µl of blood. In order to assess the reproducibility of microscopy, a random selection of 10% of smears from each district were re-read.Primary malaria diagnosis was carried out within 24 hours of blood sampling by microscopic examination of thick and thin blood smears. Blood smears were fixed with methanol (thin smear only) and stained with giemsa. Smears were initially graded for the presence/absence of Plasmodium species  whilst the remainder were tested only for P. falciparum, P. malariae and P. ovale. An estimated parasite density of 10 parasites per µl of blood was assigned per species detected by PCR in those samples that were microscopy negative. [The value of 10 parasites per µl is the approximate parasite detection level of PCR. In microscopy negative samples containing all 3 species detected by PCR in the study area  the use of this estimate results in an density value below the microscopy sensitivity level of 40 parasites per µl]. To assess the reproducibility of PCR detection a random selection of 5% of samples from each district were re-analysed (from the sample DNA extraction) for the presence of P. falciparum, P. malariae and P. ovale.To confirm microscopy results and detect parasites below the limit of microscopy (around 40 parasites/µl of blood) a retrospective, species-specific PCR 10 transformation was carried out to normalise parasite density data. Univariate and bivariate regression models (accounting for district and transmission region) were used to determine which variables were associated with the following clinical outcomes measured during community surveys: haemoglobin concentration ([Hb] g/dl), moderate anaemia ([Hb]≤8.0 g/dl) and fever (axillary temperature ≥37.5°C). Linear regression was used for [Hb] and logistic regression for the binary outcomes of anaemia and fever. In the linear regression analyses continuous variables were included as co-variates whilst categorical variables were included as fixed factors.Data were analysed using SPSS v10.0 . Data for each district were compared using Pearson's Chi squared test for categorical variables and Mann-Whitney U test for continuous variables. LogMixed effects logistic regression models were fitted for whether or not each individual had PCR-detectable infection, using the statistical software package MIwiN v2.01 . The log odds of being infected was allowed to vary at random between villages and between households within villages, according to a Normal distribution. A series of models was fitted: firstly, without covariates, to estimate the degree of variation between and within villages; secondly, allowing for individual-level fixed effects of sex and age (adult vs. child); thirdly, allowing for village-level fixed effects of elevation above sea level and the mean wet and dry season normalised difference vegetation index (NDVI) in each village; and lastly, allowing for both individual- and village-level fixed effects. Additionally, the final model was refitted allowing for separate random and fixed effects in each district (Dedza or Mangochi). For each model, the variation between villages and between households within villages are reported in terms of the variance in log odds of being infected, and in terms of the estimated odds ratio between units (villages or households) at the 75th and 25th percentiles of the assumed distribution of units. Also reported are the fixed effect estimates, in terms of the associated odds ratio estimates, with 95% confidence intervals. Finally, for the model allowing separate fixed and random effects in each region, p-values are reported for the comparisons of model parameters between villages.Plasmodium species  detected by PCR, differed from an independent random distribution P. malariae and P. falciparum were also analysed in the absence of the least prevalent species, P. ovale . We calculated ROE Mixed, ROE Negative or ROE Pm the ratio of observed/expected (and 95% CI) amongst values that deviated from expected mixed, negative or P. malariae samples, as a measure of the magnitude of the deviation from randomness.The multiple-kind lottery model was applied to determine if the distribution amongst individuals of Plasmodium infections in three transmission settings it has been necessary to use multiple testing. Unadjusted p values are reported throughout in order not to obscure possible associations and therefore individual p values should be interpreted with this in mind.In order to evaluate all possible associations between clinical measurements and the presence of multiple Clinical measurements and blood samples for parasitological analysis were collected between March and April 2002, from 2918 participants residing in 16 villages within Dedza District and 8 villages within Mangochi District, Malawi . DemograPlasmodium species) detected by microscopy in the two districts was remarkably similar during our wet season survey, given the variation in geography and difference in malaria transmission in the two areas. In Dedza, where transmission is seasonal, the mean microscopy prevalence was 23.6% whilst in Mangochi where perennial transmission occurs, prevalence was only slightly higher, 27.3%, a difference significant only at the 0.05 level  low intensity seasonal transmission (LIST) ; 2) high intensity seasonal transmission (HIST), 3) perennial transmission (PT)  were detected by PCR in both districts , whilst P. malariae  and P. ovale  were observed less frequently and usually only in combination with P. falciparum  were considered or when P. falciparum and P. malariae were considered in isolation from the least prevalent species, P. ovale  than in Dedza,  people with mixed P. falciparum & P. malariae infections and 8 out of 79 (10.1%) people with mixed P. falciparum and P. ovale infections. These data showed no deviation from expected fever rates in either district when data from all age groups or children (aged <1–14 years) only were analysed (data not shown). There were insufficient data from each of the two Dedza transmission regions for these to be analysed separately. No significant deviations from expected fever rates amongst P. falciparum only and mixed-species infections were seen when clinical malaria was defined more stringently as temperature ≥37.5°C or 38.0°C along with a positive microscopy reading or along with parasite density ≥1000 parasites/µl (data not shown). Only when clinical malaria was defined more stringently as temperature ≥37.5°C along with parasite density ≥5000 parasites/µl, was there a negative association of mixed infections with clinical malaria, and this was seen in children only (OR 0.43 (95% CI 0.19–0.96) p = 0.035).Fever was seen in 209 out of 1426 (14.6%) people with district and transmission region were not significantly associated but 14 out of 25 other variables were  but more people in Mangochi had taken painkillers  and had fewer mixed species infections than expected by chance alone . There was a near-significant association between bednet use and reduced current fever rate  but surprisingly, those using bednets were more likely to have a history of fever than those without . The low numbers of bednet users prevented similar analyses for Dedza district.Bednet usage varied between the two districts. Only 1.4% of individuals in Dedza slept under a bednet regularly, compared with 14.2% in Mangochi . In MangOverall, mean haemoglobin concentration ([Hb]) in Mangochi (10.6 g/dl) was significantly lower than Dedza . In children, mean [Hb] did not differ with bednet use . The impact of bednet usage on [Hb] could not be analysed within Dedza district because so few people in this district used bednets (n = 28).transmission region was strongly associated  therefore we carried out bi-variate regression analyses accounting for transmission region. All but 3 of the 24 measured variables showed significant association with haemoglobin level. There were significant interactions between transmission region and several variables  in univariate logistic regression (p<0.0001) and therefore we carried out bi-variate logistic regression analysis accounting for transmission region. When transmission region was included, 15 out of 24 measured variables were associated with anaemia  and perennial (Mangochi) malaria transmission. Within the seasonal transmission zone, we have explored the relationship of malaria prevalence with geophysical features and defined two regions in which prevalence varies markedly, almost certainly arising from elevation-linked variation in transmission intensity.In this study we have investigated the impact of infection with multiple species of Plasmodium species detected  between the three transmission regions. Prevalences of parasitaemia for each species were higher and peaked at a younger age in areas with high intensity seasonal or perennial transmission. In Dedza, high parasite prevalences were associated with low altitude and proximity to marshy ground. Higher P. falciparum prevalence was generally reflected in higher prevalence of minority species  raising the possibility of multiple Plasmodium species being co-transmitted. The relative abundance of these species showed micro-geographic variation. This could result from variation in the presence of mosquito species or sub-species, which may have Plasmodium species-specific transmission efficiencies. Alternatively, these differences could arise from stochastic variation in the seeding of infections by incoming human carriers within villages. The human populations in our two study areas come from different ethnic groups, the Chewa and Yao. Different ethnicity or religion can affect housing type, sleeping arrangements and bednet use – factors which may impact on mosquito exposure. However, we know of no evidence to suggest that these groups differ in their genetic predisposition or ability to raise immune responses to malaria infections.There were marked differences in the spatial and age-distribution of the three sympatric P. ovale in Malawi, although its presence has been noted previously P. vivax was not detected in our study populations, despite its presence in neighbouring Mozambique Plasmodium species amongst the human population showed randomness amongst children in all regions but was not random in adults from the PT and LIST regions. A non-random distribution is thought to result from the effects of acquired immunity. Our data contrast with those from Papua New Guinea, a holoendemic region where all four co-endemic human malaria species show random distribution across all age groups This study is the first to describe the prevalence of Plasmodium species. The lack of difference could not be explained by differences in anti-malarial drug use, which was similar between districts. People with a history of fever were more likely to have a single infection than those without but they were also more likely to have taken anti-malarial drugs. In 1998, SP  had a 2-week efficacy of 80% in symptomatic P. falciparum cases in Blantyre (170 km from our study sites) P. malariae and P. ovale co-infections, if not always against P. falciparum. In vivo drug resistance of P. malariae has been described only for chloroquine in vitro studies failed to find evidence of anti-folate resistance in this species in Africa Plasmodium species could explain these data.Fever rates were remarkably similar between the three transmission regions despite differences in the seasonality and intensity of transmission and the age-prevalence profiles of each P. falciparum morbidity P. falciparum-P. malariae infections than asymptomatic children Fever was equally likely in people with single or mixed infections. Only when clinical malaria was defined as fever ≥37.5°C with parasite density ≥5000 parasites/µl were there fewer mixed infections than expected, amongst children only. Parasite density was lower amongst children with mixed infections than those with single-species infections, but only in the PT region. This phenomenon may result from a greater level of acquired immunity under perennial transmission, resulting from the earlier age at infection and greater number of infections, acting to reduce density in more heavily infected children. Changes to the immunological control of parasite density and tolerance under different transmission settings can account for differences in P. falciparum densities, the detection of lower density species by PCR is masked. This phenomenon is known to occur when detecting multiple P. falciparum genotypes using common primers et al.,which we employed in this study, also uses common primers in the first round of amplification. This problem could be overcome by designing new PCR methods for malaria diagnosis in which each species was detected independently, without any amplification primers in common. This “common primer” explanation is given weight by the results of our analysis using different density thresholds and the less compelling results given in the Cote d'Ivoire study for children selected on less stringent clinical criteria, who may have had lower parasite densities. Differences in case-definitions of clinical malaria may underlie a similar discrepancy with data from Papua New Guinea A possible explanation for these differences is that in children with very high Anaemia has long been recognised as a complication of malaria, but only recently have studies have confirmed the causal link at the community level An association of anaemia with perennial malaria transmission P. malariae and P. ovale produce lower density parasitaemia than P. falciparum and therefore mixed infections with P. falciparum may be more likely to be detected at lower overall densities.The higher parasite density found in children compared to adults in all transmission regions may account for the lower mean [Hb] in children relative to adults. Children with mixed species infections had on average greater [Hb] than children with single infections, suggestive of a protective effect of multiple infections. However, this finding was only seen in the HIST region. The functional mechanism for this may be specific to immunity generated through seasonal, compared to perennial infection. The counter-intuitive, lower parasite density found in mixed species infections could result from the inherent bias in the PCR species diagnosis, in which mixed infections may be more likely to be detected in patients where the density of each species is equitable. The use of insecticide treated bednets is now a central focus of anti-malarial campaigns Plasmodium species decays during the dry season, and the corresponding effect on [Hb] and fever would give valuable insights into the dynamics of the relationships between these factors. Our study suggests that the interaction of multiple Plasmodium species can have protective effects against some clinical outcomes of malaria. This protection appears to be highly dependent on the level and seasonality of malaria transmission and most likely results from differences in the dynamics of acquired immunity. Our data will be informative in predicting the effects of anti-malarial intervention strategies on the clinical outcomes of malaria and on Plasmodium population dynamics across multiple transmission settings.This study took place during the peak of malaria transmission in the wet season. Dry season data would allow a greater understanding of the differences between the perennial and seasonal transmission regions. It would be useful to clarify the relationship between the HIST and LIST regions to establish the dynamics of carriage of each species during the dry season in these populations. An understanding of how the prevalence of each Table S1The number of samples in which each combination of Plasmodium species was detected by PCR for each of the 3 transmission regions.(0.05 MB DOC)Click here for additional data file.Table S2Univariate logistic regression analysis on fever (axillary temperature ≥37.5°C).(0.07 MB DOC)Click here for additional data file.Table S3Bi-variate linear regression analysis on haemoglobin concentration and bi-variate logistic regression on moderate anaemia ([Hb]≤8.0 g/dl), accounting for transmission region.(0.09 MB DOC)Click here for additional data file.


CRP gene have been strongly associated with the plasma concentration of CRP. The known lower susceptibility to malaria in the Fulani ethnic group, as compared to their sympatric neighbours in Africa, has been linked to different genetic backgrounds. The present study was performed to investigate if polymorphisms in the CRP gene could contribute to the lower susceptibility to malaria seen in the Fulani ethnic group.C-reactive protein (CRP) is an acute phase protein that can activate various immune cells and bind to certain Fcγ receptors. The latter may compete with the binding of IgG antibodies to these receptors and could thereby interfere with the antigen-specific immune response. Polymorphisms in the promoter region of the The CRP -717 T>C, -286 C>T>A, and +1444 C>T polymorphisms were analysed in asymptomatic Fulani and non-Fulani individuals from Mali and Sudan using Pyrosequencing T and TaqMan r MGB probes.The rare -286 A allele, previously shown to be associated with increased CRP expression and plasma levels, was shown to be more frequent in the non-Fulani ethnic groups as compared to the sympatric Fulani ethnic group both in Mali and Sudan. The common -717 T allele was more prevalent in the non-Fulani ethnic group compared to the sympatric Fulani ethnic group, but only in Mali. The parasite prevalence was increased for the -286 A allele, but not for the -717 T allele. No differences regarding genotype frequency or parasite prevalence were seen for +1444 C>T.This study indicate that CRP may play an important role in the immune responses to malaria, and that the -286 C/T/A CRP polymorphism may be a contributing factor to the lower susceptibility to malaria seen in the Fulani. Plasmodium genus, is a prevalent infectious disease worldwide. The Fulani ethnic group in Africa has shown a lower susceptibility to malaria as compared to their sympatric neighbours, despite being similarly exposed to malaria and having no obvious differences in socio-cultural circumstances. The Fulani have lower parasite rates and parasitaemia, higher anti-malarial immune responses and higher spleen rates than other sympatric groups [Malaria, caused by parasites from the c groups -3. It hac groups . A numbec groups ,6. Recenc groups ,8.Plasmodium falciparum infection, the plasma levels of CRP are low [P. falciparum symptomatic individuals are not merely an acute phase response. The role of CRP in P. falciparum infections is not clear. CRP has been associated with complement mediated haemolysis of infected erythrocytes and subsequent anaemia [CRP gene [C-reactive protein (CRP) is an acute phase protein, the levels of which increase rapidly in the circulation during infection and/or inflammation. CRP has the ability to activate various immune cells and has also shown a possible capacity to bind to certain FcγR . Fc rece are low ,17, sugg anaemia , but CRP anaemia . Importa anaemia , which c anaemia , and sin anaemia ,23 and tCRP gene  have beeCRP gene ,26, and CRP gene . PreviouCRP gene . This suIn order to investigate the possible involvement of CRP in innate immunity against malaria, and to further define potential immunological factors contributing to the ethnic differences in malaria susceptibility, the -717 T>C (rs2794521), -286 C>T>A  +1444 C>T (rs1130864) CRP genotypes and malariometric indices were analysed in individuals of sympatric ethnic groups from two countries, Mali and Sudan, with markedly different malaria endemicity.P. falciparum as the main parasite species. The entomological inoculation rate is similar in both ethnic groups [The study area in Mali is located in the Mopti area about 850 km Northeast of Bamako, the capital of Mali. Four villages, Mantéourou, Naye, Binédama, and Anakédié were identified for the study. Malaria transmission is mesoendemic in the area, with c groups . In thisP. falciparum is responsible for >96% of the malaria cases [The study area in Sudan is located in the Daraweesh village in the Gedaref State in eastern Sudan, 450 km from Khartoum and 16 km from the Gedaref town. Malaria transmission is markedly seasonal and unstable, and annual peak parasite prevalence ranges from 1 to 40% in different years, and ia cases .In Mali, blood was collected during the rainy season in September 2005 from 166 Fulani  and 608 non-Fulani represented by the Dogon (98%) and Rimaibé (2%) ethnic groups . All individuals were asymptomatic at the time of collection, and parasite prevalence was detected by microscopy. For PCR, finger prick blood was collected on filter papers. Spleen enlargement was assessed by palpation and graded according to the Hackett score (0–5). The proportion of individuals that had enlarged spleens was denoted spleen rate. Informed consent was obtained from all participants or their guardians and the Ethical committee of the Faculty of Medicine and Pharmacy, University of Bamako, in Mali and the National Ethics Committee in Sweden approved the study.In Sudan, blood was collected, as part of a longitudinal study, in May before the rainy seasons between 2004 and 2006, from 225 Fulani  and 100 non-Fulani represented by the Four (22%), Hausa (28%) and Masaleit (50%) ethnic groups . All the individuals were asymptomatic and parasite negative by microscopy at the time of collection. Three ml of peripheral blood were collected from all individuals into vacuum EDTA tubes. The study received ethical approval from the ethical Committee of University of Khartoum and national clearance from the Sudanese Ministry of Health.DNA was extracted from filter papers and venous blood with Chelex-100 as described previously , and the286 C>T>A polymorphism, using the forward primer 5'-TGT TTT CCT CAT TTC CCA GTC T-3' and the reverse primer 5'-biotinylated TGG CTA TCT ATC CTG CGA AAA T-3' . The PCR amplification was performed in a 40 μl reaction using 8 μl of genomic DNA template, 20 μl iProof T High-Fidelity Master Mix , and 1.5 μl of each primer (10 pmol/μl). The PCR was carried out in an Eppendorf Mastercyclerr  using a 30 sec denaturation at 98°C, followed by 35 cycles with 98°C for 10 sec, 59°C for 20 sec and 72°C for 15 sec. The final extension was at 72°C for 5 min. The sequencing primer was 5'-GTG CAC CCA GAT GGC-3' (MWG-Biotech AG), and the nucleotide dispensation order was GCA CGT ACA GT. Sequencing was carried using the PSQ™ 96MA pyrosequencing apparatus  according to the instructions from the manufacturer.The -286 C>T>A CRP polymorphism (rs3091244) was analysed using the pyrosequencing method. PCR primers were designed to amplify the -The -717 T>C (rs2794521) and +1444 C>T (rs1130864) CRP polymorphisms were analysed with Taqmanr MGB Probes from Applied Biosystems according to the manufacturers protocol.2 tests. Analyses of the estimated haplotype frequencies and associations of alleles were performed in Unphased (version 2.403) [All statistical analyses were performed in StatView version 5.0.1 unless stated otherwise. Differences in genotype frequencies and allele frequencies between the ethnic groups were tested for statistical significances using χn 2.403) . Linkage-8 and Sudan: OR = 2.51, P = 8.2 × 10-7, respectively). While the C allele was found to be at a lower frequency in the Malian non-Fulani as compared to the Fulani , the T allele frequency was lower in the Sudanese non-Fulani group .The genotype frequency of the -286 C>T>A polymorphism was similar in both countries Table  and was Further, the possible influence of the A allele on some malariometric data collected for the Mali study group were analysed, and the results revealed that individuals being parasite positive were more likely to be A-allele carriers than parasite negative individuals (p = 0.04) Table , but no The genotype frequency of the -717 T>C polymorphism was similar in both countries Table , and wasThe genotype frequency of the +1444 C>T polymorphism was similar in both countries, and was found to be in Hardy-Weinberg equilibrium. No differences in either genotype, allele frequencies  polymorphism showed a difference in allele frequency between the Fulani and non-Fulani groups in both Mali [Both the H rs180124 polymoroth Mali . These fThe present study demonstrates striking differences regarding CRP single nucleotide polymorphisms between sympatric ethnic groups, which may contribute to the differences in their susceptibility to malaria. In particular, the CRP SNP -286 C/T/A showed a marked difference in genotype frequencies between Fulani and non-Fulani individuals in two independent cohorts from Mali and Sudan. Lower frequencies of the A-allele of this SNP, previously associated with higher circulating CRP concentrations, were demonstrated in the two independent Fulani groups as compared to other sympatric ethnic groups. Moreover, a higher parasite prevalence was detected in individuals with the A allele, supporting the cause-relationship. The other two analysed polymorphisms did not show any consistent differences between Mali and Sudan, and in the haplotype analysis it was clearly the -286 A-allele that showed a consistent association with one ethnic group in both countries. The results suggest that the -286 CRP polymorphism may be a contributing factor in the lower susceptibility to malaria seen in the Fulani group.However, the hypothesis that the -286 A-allele is associated with protection against infectious diseases due to a selection in African populations was refuted. The reason for this is not clear, but the A-allele might be associated with a lower risk of other diseases, as discussed below.P. falciparum parasitaemia is dependent on a pro-inflammatory response, and an uncontrolled inflammation is suggested to cause severe symptoms [in vitro [So far, inconclusive results, suggesting both a beneficial and harmful role of CRP in malaria ,19, havesymptoms . Higher symptoms ,14, indisymptoms . The up-symptoms , and a rsymptoms . Moreovesymptoms , and neusymptoms . Since din vitro ,39, suchLow levels of CRP have been shown for a number of autoimmune diseases , and witCRP gene and the FcγRIIa gene are located in the same region on chromosome 1, the possibility of a susceptibility locus on this chromosome was investigated. Although the linkage analysis did not show any linkage between these two polymorphisms, associations were found between haplotypes containing the -286 A-allele, regardless of the FcγRIIa allotype or the -717 or +1444 CRP polymorphisms, and the non-Fulani groups. This finding suggests that the -286 CRP polymorphism could have a stronger impact on malaria susceptibility than the other investigated variants. The finding that there are more parasite positive individuals among the A-allele carriers than among the non-A allele carriers, further strengthens this suggestion. The sample size in this study was probably too small for making any firm conclusions, but the results strongly indicate a need for further studies on the impact of CRP in relation to susceptibility to malaria.The FcγRIIa 131 H/R polymorphism has been associated with malaria susceptibility in several studies ,42,43. Cin vitro, and associated with in vivo CRP levels in the circulation. This study further shows that the high producing -286 A-allele may be associated with parasitaemia, and it can, therefore, be speculated that CRP play a role in the early immune response to malaria infection. This may possibly occur by inhibiting dendritic cells and/or neutrophils, by competition with the binding of malaria specific IgG antibodies to Fcγ receptors, by enhance the clearance of infected RBCs, leading to more severe anaemia, or a deviation towards an anti-inflammatory cytokine response induced by CRP. Further research on the effect of CRP on malaria susceptibility is warranted, since recent studies on new therapeutic agents, that lower the circulating CRP levels [In conclusion, this study has demonstrated a marked difference in CRP genotype frequencies in two independent samples of Africans with low susceptibility to malaria as compared to sympatric ethnic groups. The -286 CRP polymorphism that was analysed has been shown to be functional P levels , might oThe authors declare that they have no competing interests.EI participated in the design and conception of the study, collection of samples, performed the CRP genotypings and the CRP measurements, carried out the statistical analyses and drafted the manuscript. ME participated in the design and conception of the study and helped to draft the manuscript. AN was responsible for design of sample collection in Sudan and helped to draft the manuscript. AD was responsible for design of sample collection in Mali and helped to draft the manuscript. SK helped in the design of TaqMan analysies and performed the CRP genotyping and helped to draft the manuscript. GA participated in the Pyrosequencing analysis and the CRP measurements and helped to draft the manuscript. MV helped in the collection of samples and helped to draft the manuscript. BM examined all participants from Mali and participated in design and collection of samples and helped to draft the manuscript. OKD was responsible for design of sample collection in Mali and helped to draft the manuscript. GE was responsible for design of sample collection in Sudan and helped to draft the manuscript. HAG was responsible for design of sample collection in Sudan and helped to draft the manuscript. MTB participated in the design and the conception of the study and helped to draft the manuscript. KB participated in the design and the conception of the study and drafted the manuscript. PT participated in the design and the conception of the study, performed the CRP genotyping and helped to draft the manuscript. All authors read and approved the final manuscript


Studies largely from the market research field suggest that the inclusion of a stamped addressed envelope, rather than a pre-paid business reply, increases the response rate to mail surveys. The evidence that this is also the case regarding patient mail surveys is limited.The aim of this study is to investigate whether stamped addressed envelopes increase response rates to patient mail surveys compared to pre-paid business reply envelopes and compare the relative costs. A sample of 477 initial non-responders to a mail survey of patients attending breast clinics in Greater Manchester between 1/10/2002 – 31/7/2003 were entered into the trial: 239 were randomly allocated to receive a stamped envelope and 238 to receive a pre-paid envelope in with their reminder surveys. Overall cost and per item returned were calculated.2 = 1.39; 2 tailed test, d.f. = 1; P = 0.239). The stamped envelopes were cheaper in terms of cost per returned item (£1.20) than the pre-paid envelopes (£1.67). However if the set up cost for the licence to use the pre-paid service is excluded, the cost of the stamped envelopes is more expensive than pre-paid returns (£1.20 versus £0.73).The response to the stamped envelope group was 31.8% (95% CI: 25.9% – 37.7%) compared to 26.9% (21.3% – 32.5%) for the pre-paid group. The difference (4.9% 95% CI: -3.3% – 13.1%) is not significant at α = 0.05  is similar to that demonstrated in a Cochrane review (5.3%) of strategies to increase response to general mail surveys. Further studies and meta analyses of patient responses to mail surveys via stamped versus pre-paid envelopes are needed with sufficient power to detect response gains of this magnitude in a patient population. Mail surveys are widely used both in the general population and specifically with patients to assess patient satisfaction and as part of clinical studies. This has advantages to surveying the patients at the hospital as patients are less likely to be influenced by the setting and may require less staff time . DisadvaA Cochrane review identified 21 trials comparing response rates from postal surveys enclosing stamped envelopes versus pre-paid envelopes ,6. The o8. Three of these studies concerned health topics, all of which surveyed health professionals or the general population rather than patients . SResponses to patient surveys may differ from other surveys due to adverse treatment outcome , interesBalancing the respective costs of stamps versus pre-paid returns against potential increases in response is of particular interest within the context of the resource limitations of contemporary health services. A meta-analysis undertaken in 1987 presented evidence that stamped envelopes produce a greater and more cost effective response than pre-paid . HoweverThe present study therefore investigates whether inclusion of a stamped addressed envelope (as opposed to a pre-paid reply envelope) increases responses to survey reminders in a UK-based patient population. Differences in costs per item returned are also examined.A randomised-controlled trial (RCT) was undertaken to determine whether postage stamps versus pre-paid envelopes increases patient response rates to a reminder mail survey. The RCT was nested within a wider study of health related quality of life amongst female patients, aged ≥ 65 years, attending breast clinics in 11 hospitals in Greater Manchester between 1/10/2002 – 31/7/2003. As the study reported here was nested within our main study, the sample size was constrained by the sample size needed to achieve the main studies objectives. Initially, 844 patients were sent a 6 page survey, measuring functional status (ELPHS ADL) , genericEach survey was assigned a unique identifier. Hospital staff responsible for mailing surveys to patients kept a log book linking patients to survey identifiers. Surveys were returned to the University of Manchester where random allocation of non-responders could be undertaken. The 477 patients who had not returned the survey within 3 weeks were entered into the trial. The unique identifiers of these non-responders were randomly assigned, using SPSS 11.5 for Windows, to have either a first class stamped addressed reply envelope or a pre-paid addressed envelope included in their reminder letters. The random allocation for each non-responder's unique identifier was returned to the hospitals from where, each non-responder was sent a reminder covering letter along with another copy of the questionnaire and either a stamped or pre-paid response envelope. Of the 477 women, 239 were randomised to the group receiving the stamped envelope and 238 to the group receiving the pre-paid envelope . There is, no evidence to reject the null hypothesis of no difference in return rates between those receiving the stamped and pre-paid envelopes. If the cost of setting up the  freepost licence is included, the pre-paid method is more expensive at a cost of £1.67 per item returned versus £1.20 per item returned using stamped addressed envelopes and total cost of returns is £106.66 versus £90.82 respectively . However, if the cost of the pre-paid licence is excluded, the overall cost of stamped envelopes is considerably more expensive than pre-paid (£44.16). This presents a cost of £3.68 for every extra return (£44.16/12).The results suggest that using stamped return envelopes rather than pre-paid replies does not significantly change the response rate to reminders for patient surveys. However, this study had 80% power to detect a difference of only 12.5% or more between the two groups; differences smaller than 12.5% would not be identified as significant in this study. Thus we need to treat our finding with some caution.Edwards et al's Cochrane review found that stamped envelopes increase the odds of response by 1.29 (95% CI: 1.18–1.42) compared to pre-paid . The resAlthough this suggests that, as with other types of surveys, stamped envelopes increase responses to patient surveys compared to pre-paid envelops, this assumption needs to be tested in further RCTs of patient surveys with sufficient participants to detect a response gain of approximately 5% or more at 80% power. However, this would require large sample sizes. For example, the present study would have needed 1305 participants in each group to detect a difference of 5% (27%–32%).An alternative approach would be undertake meta-analyses combining the results of smaller studies of patient returns to mail surveys, using similar methodology to the Cochrane review . HoweverOur study suggests that the stamped envelope strategy is cheaper per returned item compared to pre-paid returns if set-up costs are included. Previous evidence suggests that the use of pre-paid envelopes is usually more expensive per item returned because of the additional return yielded by first class post  under thOnly women aged ≥65 years where eligible to receive the survey in this study. As response rates to mail surveys differ by both age and gender  the geneThis study investigated responses to a second mailing, primarily due to methodological constraints of randomising at first mailing. Although this may limit the generalisability of our findings to first and final response rates, as the Cochrane review found both first and final response rates are increased with stamps compared to pre-paid envelopes, this seems unlikely . HoweverCompared with pre-paid business replies, including stamped envelopes did not produce a statistically significant increase in response rate to this patient survey. However, because our study was nested within a substantive study of older women's health and sample size was constrained by the demands of that main study we only had sufficient power to detected differences of 10 – 12.5%. The actual response gain of the stamped strategy (4.9%), is similar to that demonstrated in the Cochrane review (5.3%) suggesting that use of stamped envelopes versus pre-paid may provide similar gains as those demonstrated for general mail surveys . This neThe authors declare that they have no competing interests.All authors read and approved the final manuscript. KL devised study design, undertook data collection and analysis and produced the manuscript. CT devised study design, advised on analysis and approved and contributed to drafts and final manuscript. MC advised on sample size and analysis and approved drafts and final manuscript.The pre-publication history for this paper can be accessed here:


Characterization of severe malaria cases on arrival to hospital may lead to early recognition and improved management. Minimum community based-incidence rates (MCBIRs) complement hospital data, describing the malaria burden in the community.A retrospective analysis of all admitted malaria cases to a Mozambican rural hospital between June 2003 and May 2005 was conducted. Prevalence and case fatality rates (CFR) for each sign and symptom were calculated. Logistic regression was used to identify variables which were independent risk factors for death. MCBIRs for malaria and severe malaria were calculated using data from the Demographic Surveillance System.Almost half of the 8,311 patients admitted during the study period had malaria and 13,2% had severe malaria. Children under two years accounted for almost 60% of all malaria cases. CFR for malaria was 1.6% and for severe malaria 4.4%. Almost 19% of all paediatric hospital deaths were due to malaria. Prostration (55.0%), respiratory distress (41.1%) and severe anaemia (17.3%) were the most prevalent signs among severe malaria cases. Severe anaemia and inability to look for mother's breast were independent risk factors for death in infants younger than eight months. For children aged eight months to four years, the risk factors were malnutrition, hypoglycaemia, chest indrawing, inability to sit and a history of vomiting.MCBIRs for severe malaria cases were highest in children aged six months to two years of age. MCBIRs for severe malaria per 1,000 child years at risk for the whole study period were 27 in infants, 23 in children aged 1 to <5 years and two in children aged ≥5 years.Malaria remains the number one cause of admission in this area of rural Mozambique, predominantly affecting young children, which are also at higher risk of dying. Measures envisaged to protect children during their first two years of life are likely to have a greater impact than at any other age. Out of the 350–550 million malaria cases that are estimated to occur in the world every year ,2, only In Mozambique, as in other sub-Saharan African countries, malaria represents the main cause of paediatric outpatient consultations and admissions to hospital. However, no detailed characterization of the different malarial clinical syndromes on admission exists in the country. A comprehensive picture of the clinical and epidemiological characteristics of severe malaria is necessary to prioritize public health interventions and to guide national policies. This paper presents information on the clinical features, outcome and community incidences of malaria and severe malaria in children admitted to a rural hospital in Mozambique. Data on children with malaria who attend the outpatient clinic of the same hospital are presented in a companion article .The study area is located in Manhiça, Maputo Province, southern Mozambique. The Manhiça Health Research Center (CISM) runs a Demographic Surveillance System in the area  and a most of June 2003 to 31st of May 2005).Retrospective study of the data collected through the Manhiça morbidity surveillance system. This paper presents data from children younger than 15 years who were admitted to Manhiça District Hospital during a period of two years  to the hospital. A physician or experienced medical officer performed a physical exam of the children on admission and completed the questionnaire. An open clinical process was also filled, where the daily clinical evolution was recorded during admission.Plasmodium falciparum parasitaemia.Laboratory data was also recorded on the admission questionnaire. Upon arrival a finger prick blood sample was collected into heparinized capillaries to measure packed cell volume (PCV) and blood glucose concentration, and thick and thin blood films were prepared to quantify HIV status information was not routinely collected. Admission criteria for children with malaria included any sign of severe disease (see definitions below), inability to take oral medication, or moderate anaemia with a risk of cardio-respiratory decompensation. Hyperparasitaemia, although mentioned as an admission criterion in the national guidelines, was rarely a cause of admission at Manhiça hospital unless accompanied by moderate anaemia or other severity symptoms.Upon discharge or death up to four final diagnoses, based on the ICD classification of diseases, were recorded on the questionnaire by a project physician after review of clinical signs and symptoms and laboratory results.Children admitted with the diagnosis of malaria were managed according to Mozambican national guidelines, which, at the time of the study, included parenteral treatment with quinine  for a minimum of six doses if completed with treatment with sulphadoxine-pyrimethamine (SP), or 21 doses when used as monotherapy. Treatment was switched to oral as soon as the child was able to tolerate. Blood transfusions were restricted to children with a PCV < 12% or to children with higher PCVs but with clinical signs of decompensation (respiratory distress or signs of heart failure) or neurological impairment. Hypoglycaemia was handled with intra-venous 30% dextrose solution, repeated as necessary, and convulsions treated with up to two doses of rectal or intra-venous diazepam, followed by intramuscular or intravenous phenobarbitone if seizures could not be controlled. Lumbar punctures were normally performed in children with a witnessed convulsion or a history of convulsions in the previous 24 hours, with positive meningeal signs, focal neurological signs, suspected clinical sepsis or a Blantyre Coma Score <5. Facilities for intensive care do not exist. All clinical assistance and treatment of admitted children is free of charge. Children requiring specialized care were transferred to Maputo Central Hospital.®  at the bedside. All cerebrospinal fluids obtained from lumbar punctures were Gram-stained and cultured.PCV was measured using a microcentrifuge and a Hawksley haematocrit reader card . Thick and thin blood films were air-dried, Giemsa-stained, and examined using a light microscope fitted with a 100× oil immersion lens. Slides were declared negative only after 2000 leukocytes have been counted. Parasite numbers were converted to a count/μL by assuming a standard leukocyte count of 8000/μL. Glycaemia was determined using Accu-ChekP. falciparum asexual parasitaemia > 0 parasites/μL and not fulfilling the criteria for severe malaria.All case definitions were based on admission data from the admission questionnaires. An uncomplicated malaria case was defined as a child admitted with a clinical diagnosis of malaria with a Severe malaria was defined as a malaria case with at least one of the following criteria: PCV < 15%, deep coma (Blantyre coma score ≤ 2), prostration (inability to sit unaided or to look for mother's breast/feed in children who cannot yet sit), hypoglycaemia (< 2.2 mmol/L), convulsions (≥ 2 reported episodes in the 24 hours prior to admission) or respiratory distress (deep breathing or indrawing). Patients who met this definition but had a diagnosis of meningitis were excluded.Impaired consciousness was defined as a child having a Blantyre coma score of less than 5. Severe anaemia was defined as a PCV < 15% on admission and cerebral malaria was defined as children with malaria and deep coma.An increased respiratory rate (tachypnoea) was defined according to age: respiratory rate ≥ 60 breaths/min for children < 2 months; ≥ 50 for children 2 to < 12 months and ≥ 40 for children 1–5 years. Weight-for-age Z scores (WAZ) were calculated using the LMS method and the 2000 CDC Growth Reference.The clinical processes of all children who had been classified as a malaria death based on the admission questionnaire were reviewed by a paediatrician and reclassified according to the clinical evolution and other concomitant diagnoses. A malaria death was defined as a death in an admitted child in which malaria was considered the only or main cause of death. Children with malaria parasitaemia for whom the cause of death was not malaria, were not considered severe malaria cases.All admission questionnaires were double entered using a programme written in FoxPro version 5.0 . Statistical analyses were done with Stata 9.0 .Minimum community-based incidence rates (MCBIRs) for malaria and severe malaria were calculated referring cases to population denominators establishing time at risk (child years at risk (CYAR)) inferred from the DSS census information.Children did not contribute to the numerator or denominator for a period of 28 days after each episode of malaria or when they were outside the study area.Plasmodium falciparum parasitaemia results were missing in 10.7% of admitted children and PCV in 9.8%. Almost half of the parasitaemia and PCV missing results were in children younger than one month or older than five years. Children with missing PCV or parasitaemia results had a significantly higher case fatality rate compared to children with non-missing results. Outcome was missing in 0.1% of children. Children with missing age were dropped from the analysis.Case fatality rates (CFRs) were calculated for individual signs and symptoms and combinations of these as the number of patients who died having that clinical presentation divided by the total number of patients with known outcome admitted with that clinical presentation. These CFRs represent in-hospital mortality and do not include patients who absconded or were transferred.The seasonality of malaria and severe malaria admissions was assessed, using November to April as the defined rainy season.2 test or Fisher's exact test. Means of normally distributed variables were compared using the Student's t-test or ANOVA. Geometric means of parasitaemia were compared using the Wilcoxon Rank sum test.Qualitative variables were compared using a χA multivariate regression analysis was done to assess risk factors for malaria death in children aged one month to five years with malaria, the age group that concentrates most malaria deaths. The analysis was done separately for children aged one to seven months and for children aged eight months to five years, given that the ability to locate a painful stimulus, strongly associated with the risk of death , is unreclinical presentation and case fatality rates of children admitted with malaria includes all children who were admitted to the hospital during the study period (n = 8311). However, CFRs refer only to children with a known outcome. The multiple logistic regression analysis of the risk factors for death in children admitted with malaria was done for all children who were admitted with malaria during the study period and for which the final outcome  was known (n = 3859). Finally, the analysis of the minimum community-based incidence rates was restricted to study area children who were admitted with malaria (n = 1678).Thus, three different subgroups of data were used for the different analyses. The analysis of the During the two year study period, a total of 8,311 children younger than 15 years were admitted to Manhiça District Hospital. Almost half of them  had a diagnosis of malaria, and for 2.8% the diagnosis was missing. 27.0% of all admitted malaria cases fulfilled the definition of severe malaria, which corresponds to 1,100 (13.2%) of all admitted patients.Males accounted for 52.1% of the malaria  and 53.5% of the severe malaria admissions .The age distribution of uncomplicated malaria cases, severe malaria cases and malaria deaths among children admitted to the hospital is shown in Figure The CFR for all admitted children was 4.0% 332/8,311). During the study period, there were 62 deaths attributable to malaria, representing 18.7% of all paediatric in-hospital deaths. The age distribution of malaria deaths was similar to that of malaria cases  and were significantly lower for severe malaria cases  than for uncomplicated cases . Severe malaria cases had a significantly lower geometric mean parasitaemia  than non-severe malaria cases . The geometric mean parasitaemias among malaria deaths was also similarly lower when compared to non-lethal cases. Case fatality rates by parasitaemia level are shown in Table 62.8% of the malaria and 59.5% of the severe malaria cases (p < 0.0001), 59.4% of severe malarial anaemia episodes (p = 0.006) and 64.5% of deaths attributable to malaria occurred during the rainy season p = 0.03)  than for severely anaemic children  or children with respiratory distress . Figure 78.4% of children with severe malaria presented on admission with a single severe malaria criterion, 16.6% had two criteria and only 5.1% had three or more criteria. The case fatality rates significantly increased with increasing number of criteria fulfilled . None of these deaths occurred in the neonatal period and only seven in children above five years of age. The analysis of risk factors for death in malaria cases was restricted to children aged one month to five years (55 deaths), the age group where most deaths occur. Results are presented separately for children aged one to seven months and eight months to five years. Additional file .6%. NoneThe age-specific minimum community-based incidence rates (MCBIRs) for malaria and severe malaria in the study area are shown in Table MCBIRs for the total paediatric population admitted to the hospital were of 50 and 26 episodes per 1,000 CYAR for clinical malaria and 14 and seven cases per 1,000 CYAR for severe malaria in the first and second study year respectively. MCBIRs for clinical malaria per 1,000 child years at risk for the whole study period were 90 in infants, 86 in children aged 1 to <5 years and 6 in children aged ≥ 5 years, whereas for severe malaria, the numbers were 27 in infants, 23 in children aged 1 to <5 years and two in children aged ≥5 years.The overall MCBIRs for malaria and severe malaria, as detected through hospital passive case detection, were twice as high during the first year of study than during the second year and the age patterns were different. In the first year, incidences were highest in infants and decreased with age, whereas in the second year the burden was slightly shifted to the right and children aged one to four years carried the highest burden of malaria and severe malaria.This is a retrospective study of paediatric malaria cases admitted to a rural hospital in southern Mozambique. The sex and age-pattern of admitted children was similar to that reported in other countries in the region ,15. DataThe age distribution and prevalence of the three classical severe malaria clinical syndromes  is also concordant with a pattern of moderate transmission . RespiraThe case fatality rate of hospitalized malaria cases in this study (1.6%) was slightly lower than those recently published for six other African sites in the largest severe malaria study performed so far in Africa (ranging from 2.0 to 9.7%) . This maSixty-two malaria-attributable deaths occurred during the two year study period. Among children who died with malaria, a quarter died the same day of admission and more than half within the first 48 h of arriving to hospital. Most of those cases (73%) were already characterized as severe on admission, and the rest (27%) worsened during their stay in hospital before actually dying. Most of these early deaths occurred in children arriving to hospital in a much evolved and often irreversible clinical condition, raising the concern of how rapid access to hospital may determine the prognosis. In such cases, only aggressive resuscitation interventions, including artificial ventilation, may have had an impact on the outcome, but such measures are unavailable in most African settings. Early recognition of severe malaria cases at home or at the community level  and promLittle has been published concerning severe malaria in Mozambique ,24. The Alongside malaria, malnutrition is a recurrent underlying problem in all major child mortality causes ,26, and Neither deep coma nor impaired consciousness were associated in this model with a higher risk of dying, contrary to what most studies have shown ,27,30,31The identification of the real cause of the respiratory distress in severe malaria patients remains unclear. Neither peripheral lactate determinations, nor acid/base statuses were performed during the two years of the study, and acidosis was only diagnosed on clinical grounds. The real importance of metabolic acidosis in this setting, as well as additional assessment of the incidence of overlapping respiratory infections  need to be further investigated.Prostration (as defined above), present in more than half of the severe malaria cases, was also identified as a major risk factor for death in both age groups. Identifying prostrated children at the outpatient clinic may be challenging, as it may be difficult to assess and depends on age, but it is likely to have a great impact on their survival.A history of vomiting was also an independent risk factor for death, probably indicating an underlying dehydration, as vomiting often leads to clinical dehydration, implying an urgent need for parenteral treatment. In Tanzanian children  dehydratAnaemia, from mild to life-threatening, is highly prevalent among severe malaria patients. The real contribution of malaria to the burden of anaemia, the role of iron and other micronutrient deficiencies in anaemia, as well as the prevalence of haemoglobinopathies, still remains unclear in the Manhiça area. Preliminary data show that haemoglobinopathies, especially sickle cell disease, are relatively uncommon as compared to other African countries  and the effect on the risk of severe malaria of this low prevalence of a traditionally considered "protective" genetic trait, such as haemoglobin AS, is uncertain.Despite the existence of a life-saving intervention for severe anaemia, this highly prevalent presentation of severe malaria is the most important risk factor for death (adjusted OR 9.5) in children younger than eight months. Interventions focused on this age group, and targeted at preventing anaemia could be highly beneficial.These data show that the level of parasitaemia is not directly related to a higher risk of death. On the contrary, children with lower parasitaemias showed a tendency to have higher CFRs than children with higher parasite numbers, although differences were not significant. Parasitaemia level was not a significant risk factor in either the univariate or multivariate analysis. Poor outcome associated with low peripheral parasitaemias, has been previously associated to sequestration in the microvasculature ,34, but Fever on admission was absent in a third of the severe malaria patients, but a referred history of fever was almost omnipresent. Relying on the presence of fever or on the parasite count for identifying severe cases on admission may, therefore, be dangerous. Considering the high mortality associated to the first hours of admission, it would seem advisable that all ill children hospitalized with malaria parasitaemia receive prompt parenteral treatment, regardless of the parasitaemia level.This paper describes MCBIRs for severe malaria ranging from seven to 14 cases per 1,000 CYAR for the whole paediatric population, but rising up to 17–29 cases per 1,000 CYAR in children between one and five years of age. These findings are concordant with MCBIRs found in children of similar ages in other settings ,36. ThesLimitations arise when trying to estimate MCBIRs for both the neonates and the oldest children. Neonates are often admitted before their permanent identification number has been issued by the DSS, and the oldest children are more prone to present to hospital without reliable identification. Incidence rates can only be calculated among patients identified as belonging to the study area and may thus not include many malaria cases in those children. The substantial malaria burden among older children seen at the outpatient department , and theMCBIRs for hospitalized malaria were in general twice as high during the first year of study follow-up. The incidence of outpatient malaria was also higher for all paediatric age groups during the first year . Among hHospital-based results underestimate the real burden of disease and are greatly influenced by health-seeking behaviour. In a verbal autopsy study in the Manhiça DSS area, 53% of all paediatric deaths occurred at home , thus cases seen at the hospital may only be a fraction of the total paediatric community deaths . NeverthThis paper reviewed all malaria admissions during two years in a rural hospital of Mozambique, in which malaria still represents the principal cause of admission and an important cause of in-hospital deaths in the paediatric ward. Measures targeted at encouraging families to promptly take their sick children to hospital, at recognizing the risk factors for severity at presentation, and improving the initial care and management during admission, would be helpful to reduce malaria-attributable deaths. In populations with difficult access to hospitals, adequate pre-referral effective antimalarials should otherwise made available.The hospital surveillance system and the DSS in Manhiça were designed by JJA, CM and PLA. They were set up in 1996 and have received numerous contributions to its design and implementation since then. During the study period QB, EM, PA, BS, AB, JS and TN were involved in the diagnosis and management of malaria patients and collection of data. AN coordinates the DSS in Manhiça. QB and CG led the analysis, interpretation and write up of this data set, and received input from all authors. All authors read and approved the final manuscript.Prevalence and CFRs of signs and symptoms significantly associated with death in the univariate analysis in children admitted with malaria, according to age.Click here for file


Plasmodium falciparum parasites in longitudinal studies provided a new option to estimate force of infection (FOI). FOI, defined as the number of new P.falciparum clones acquired over time, is a molecular parameter equally suitable for describing basic malaria epidemiology and measuring outcomes of clinical trials of antimalarial interventions. We investigated the ability of molecular parameters to explain differences in the risk of P. falciparum infection and disease between wet and dry seasons, among age groups and with respect to insecticide-treated mosquito net use.Genotyping 264 children between one and three years of age from Papua New Guinea were followed over 16 months with active detection of infection at two-monthly intervals and during episodes of febrile illness. A polymerase chain reaction (PCR) for the highly polymorphic genotyping marker merozoite surface antigen 2 was performed in all blood samples. To track individual parasite clones in consecutive blood samples with maximal resolution PCR fragments were sized by capillary electrophoresis.P. falciparum clones than older ages. FOI was significantly correlated to incidence of episodes, irrespective of whether a parasite density cut off was applied or not. Seasonal variation was observed in FOI and thus risk of illness. FOI was significantly higher during the rainy season (7.47 children/year) than in the dry season . Following the high transmission season the risk of clinical illness was significantly lower than after a period of low transmission.FOI was moderately age-dependent with children under two acquiring less new P. falciparum malaria in our cohort. FOI almost completely explained spatial variation, age trends and effect of bed net use on incidence. Acquisition of new parasite clones seems to be a major factor for clinical illness in these children. This study highlights the suitability of a new parameter, molecular FOI, for understanding the epidemiology of clinical malaria in young children. We propose to apply the molecular determined parameter FOI for monitoring effects of malaria interventions.Our analyses suggest a central role of molecularly determined FOI for explaining differences in the burden of clinical