Yet host genetic factors which could influence placental malaria risk are little studied and there are no reports of the role of blood group polymorphisms on pregnancy outcomes in malaria endemic areas.Host susceptibility to P.This study analyses the association between ABO blood group phenotypes in relation to placental malaria pathology.A total of 198 mother/child pairs delivering in Banjul and the Kombo-St Mary District (The Gambia) were analysed. ABO blood group was measured by agglutination. Placental malaria parasites wee enumerated and the presence of malaria pigment noted. Birth anthropometry was recorded and placental weight. Maternal and infant haemoglobin was measured.89 (45%) subjects were primiparae and 110 (55%)multiparae. The ABO phenotype distribution was 38(A), 52(B), 6(AB) and 102(O). Placental histo-pathology showed active placental malaria in 74 (37%), past infection in 42 (21%) and no infection in 82 cases (41%). In primiparae blood group O was associated with a higher risk of active infection , and a lower risk of past infection . In multiparae the O phenotype was associated with reduced prevalence of active or past placental infection . The mean feto-placental weight ratio was significantly higher in multiparae with group O women compared to non-O phenotypes . Among primiparae with active placental infection, mean birth weight was higher in children of mothers with the O phenotype (p = 0.04).These results indicate that blood group O was significantly associated with increased placental malaria infection in primiparae and reduced risk of infection in multiparae. This parity related susceptibility was not present with other ABO phenotypes. Cell surface glycans, such as ABO and related antigens have special relevance in reproductive biology and could modulate specific cell interactions as those associated with the pathogenesis of placental malaria. P. falciparum infection during pregnancy, which is associated with placental parasitaemia, imposes a heavy burden on the health of mothers and newborns [P.falciparum infection is central for improving understanding of malaria in pregnancy, as a basis for improved control.In malaria endemic areas, the increased risk of newborns ,2, contrnewborns ,4. At prnewborns ,6. In thP. falciparum infection, and the risk of developing severe complications are determined by host genetic factors [There is increasing evidence that both the risk of acquiring  factors . The pro factors . These i factors .P.falciparum resetting [ABO blood groups are carbohydrate histo-blood antigens that are also expressed in many tissues and which have important roles in modulating protein activities both in infection and in some types of cancer . These aesetting , whereasesetting .No studies have been identified in the literature assessing associations of blood group types with placental malaria, despite the fact that placental parasites are unable to rosette , in contIn this analysis we present the first report to describe the association between ABO phenotypes, placental malaria and pregnancy outcomes.Data from a group of 198 mother/child pairs were analysed from a cross-sectional study of placental malaria undertaken in the Gambia. The original descriptive study assessed serum Ig levels in mothers and newborns in relation to placental parasitaemia. The primary outcomes and a detailed description of the methodology are outlined in the original publication .3 blood. Maternal and infant haemoglobin was measured.Mothers and their newborn babies were included in the study from September 1967 to May 1968 in Banjul and the Kombo-St Mary District (The Gambia). In this area malaria is endemic with peak transmission during and shortly after the rainy season which lasts from July to October . MothersData were entered on punch cards and for all births the following data was available: maternal age, parity, haemoglobin and peripherical parasitaemia; the babies gender, weight, length and haemoglobin. The ABO and Rhesus blood group phenotype of mother and baby were recorded and the placental weight (g), parasite count and presence of malaria pigment. All data was entered into a SPSS data file.Placental malaria definition was based on the pathological classification of Bulmer et al , which cDichotomous variables were assessed with chi-square or Fisher exact tests, with p values less than 0.05 considered statistically significant. Odds ratios and 95% confidence intervals were estimated. Differences between means were assessed by ANOVA where data was normally distributed, or the Mann-Whitney/Wilcoxon Test. Multiple linear regression was used to analyse factors associated with anthropometric outcomes. Factors included were those significant at p = 0.1 in the univariate analysis. For anthropometric variables the factors included where: maternal blood group (O versus non O), parity, placental infection, maternal haemoglobin, maternal peripheral parasitaemia and maternal height.Eighty-eight of the 198 women were primiparae (45%) who had a mean age of 17.8 years (SD ± 1.8). The mean age of multiparae was 29.7 years (± 6.1). Placental malaria was present in 116 cases (59%), with active placental malaria infection in 74 (37.4%), of which 26 (13.1%) were acute and 48 (24.2%) chronic infections. Past infection was detected in 42 women (21.2%). In the remaining 82 cases (41%) there was no evidence for current or previous placental malaria. Among primiparae, 56 (63%) had evidence of placental infection of which 41 (47%) were active, 12 (14%) acute and 29 (33%) chronic infections. Among multiparae 60 (55%) had evidence of placental infection with 33 (30%) active, 14 (13%) acute and 19 (17%) chronic , p = 0.04 and decreased in past infection OR = 0.45 (95%CI 0.22–0.90), p = 0.02.There was no significant association between O or non-O blood groups and placental infection when parity was not considered, except for past placental malaria which was less prevalent in women with blood group O . In primiparae blood group O was associated with more active placental malaria  and less past placental malaria  than in non-O women. In multiparae, blood group O was significantly associated with non-infected placentae  compared to non-O women  concentration was higher in O type mothers with active placental malaria  although this difference was not statistically significant (p = 0.48). There were no significant differences in mean infant haemoglobin, infant length, placental weight, ponderal index or placental parasitaemia prevalence by blood group category table .Mean feto-placental weight ratio was increased in blood group O compared to non-O (p = 0.06) and was significant in multiparae , Figure . Blood gThe Rhesus blood group type showed no significant associations in relation to placental malaria categories or birth outcomes.Blood group O was associated with an increased prevalence of active placental infection in primiparae and with a reduced risk of placental malaria in multiparae. Placental parasitaemia occurs at least twice as frequently in primiparae but only among blood group O women. This effect of parity, that is one of the cardinal features of placental malaria, was not observed in non-O phenotypes for any of the placental malaria histological types.In multiparae and specifically in mothers with past or non-infected placentae blood group O was also associated with higher mean feto-placental weight ratios compared to non-O individuals. These differences associated with multiparity can be interpreted to represent a parity-specific association of the blood group O phenotype with the protective malarial immunity which occurs in multigravidae . Mean biIn non-pregnant individuals blood group O has been associated with reduced risk of severe clinical malaria compared to patients with groups A or B ,16, but P.falciparum isolates which bind to trophoblast via chondroitin sulphate A (CSA) [This is the first time an association of birth outcomes with placental malaria and maternal ABO phenotype has been reported. This raises the question of the possible mechanisms underlying the association. A central mechanism in the pathogenesis of placental malaria relates to the cytoadherence of infected red cells to the syncytiotrophoblast which is in contact with maternal blood. The parity related susceptibility to placental malaria is partly dependent on the selection of  A (CSA)  and hyal A (CSA) . Infecte A (CSA) . Soluble A (CSA) .P. falciparum erythrocyte membrane protein 1 (PfEMP1) mediate binding to several independent host receptors [Duffy binding-like domains (DBL) of the eceptors  and in peceptors ,33. Howeeceptors .P.falciparum infection. For example the differential affinity of each phenotype for A.gambiae [P.falciparum leading to changes in immune response [P.falciparum entry into the red cell through GPA sialic acid, as ABH antigens have been described in the O-glycans of glycophorin A [The interaction between the ABO histo-blood group and placental malaria could relate to generic mechanisms affecting .gambiae , or antiresponse , or an aphorin A . In addiphorin A .It is well known that host genetic factors modulate the risk and severity of infection. In most cases these are genetic variants with subtle effects on the regulation or function of specific mediators which are often difficult to demonstrate in epidemiological studies . As ther


Previous trials of the RTS, S malaria candidate vaccine have shown that this vaccine is safe, tolerated and immunogenic. The development plan for this vaccine aims at administering it in the first year of life through the Expanded Program on Immunization (EPI). The objective was to evaluate the safety and reactogenicity of RTS, S/AS02D (0.5 ml dose), a pediatric formulation of GlaxoSmithKline Biologicals' current malaria candidate vaccine RTS, S/AS02A (0.25 ml dose). A 0.5 ml dose of AS02D is composed of the same active ingredients in the same quantities as in a 0.25 ml dose of AS02A and has been developed to be easily introduced into routine EPI practices.We performed a phase I/IIb randomized double-blind bridging study in a malaria-endemic region of Mozambique, to compare the safety and immunogenicity of both candidate vaccines with the aim of replacing RTS, S/AS02A with RTS, S/AS02D as the candidate pediatric vaccine. 200 Mozambican children aged 3 to 5 years were randomized 1:1 to receive one of the 2 vaccines according to a 0, 1, 2 month schedule.Both vaccines were safe and had similar reactogenicity profiles. All subjects with paired pre and post-vaccination samples showed a vaccine response with respect to anti-circumsporozoite (CS) antibodies irrespective of initial anti-CS serostatus. Geometric mean titers (GMTs) were 191 EU/ml (95% CI 150–242) in recipients of RTS, S/AS02D compared to 180 EU/ml (95% CI 146–221) in recipients of RTS, S/AS02A. For the anti-hepatitis B surface antigen (HBsAg), all subjects were seroprotected at day 90, and the GMTs were 23978 mIU/ml (95% CI 17896–32127) in RTS, S/AS02D recipients and 17410 mIU/ml (95% CI 13322–22752) in RTS, S/AS02A recipients. There was a decrease in anti-CS GMTs between months 3 and 14 in both groups .Our data show that the RTS, S/AS02D is safe, well tolerated, and demonstrates non-inferiority  of the antibody responses to circumsporozoite and HBsAg induced by the RTS, S/AS02D as compared to the RTS, S/AS02A. Plasmodium falciparum. Currently, malaria control strategies are chiefly based on the early diagnosis and treatment of infected individuals. While preventive measures such as vector control and insecticide treated bednets remain indispensable, a vaccine is viewed as an essential part of the long-term strategy to control malaria, especially in Africa, where ninety per cent of all deaths due to malaria occur.Malaria is still leading the list of killer diseases in Africa. Two million children die annually from this disease ,2 primarMore than 50 candidate vaccines are currently under development ,4. Glaxo® and QS21 immunostimulants). Adjuvanted vaccines based on RTS, S would potentially offer protection against malaria disease due to P. falciparum. In addition, studies with the RTS, S/AS02A candidate in African children have shown that the vaccine stimulates the production of high anti-HBsAg antibody titers 2LR. Briefly, R32LR protein was coated onto a 96-well polystyrene plate. Serum samples serial dilutions were added directly to the plate. The plates were washed and polyclonal rabbit anti-human IgG/HRP was added. After a final washing step and a color reaction with 3, 3',5,5' tetramethylbenzidine, the plates were read in an ELISA reader. The titers were calculated from a standard curve with the software SoftmaxPro (using a four parameters equation) and expressed as EU/ml. Anti-CS antibodies were tested at the CEVAC Laboratory, University of Ghent, Belgium. The cut-off for the anti-CS ELISA was 0.5 EU/ml.Anti-HBsAg antibodies were tested at GSK Biologicals, Rixensart, Belgium, using an AUSAB EIA, Abbott, which had a cut-off of 10 mIU/ml. Antibodies specific for the circumsporozoite protein tandem repeat epitope were assessed by a standard, validated ELISA with plates adsorbed with the recombinant antigen R32LR that contains the sequence [NVDP(NANP)Seventy-two evaluable subjects per group were required to demonstrate non-inferiority of RTS, S/AS02D compared to RTS, S/AS02A with 95% power, by comparing the upper limit of the 95% CI of the ratio of anti-CS GMTs  with a non-inferiority limit set at 3, assuming a log standard deviation of anti-CS GMTs of 0.72 . Allowing for a chronic carriage rate of Hepatitis B virus of 15% and a drop out rate of 15%, it was necessary to enroll approximately 100 children per arm in this study.Safety and reactogenicity were analyzed using the total cohort, defined as all enrolled subjects for whom safety and reactogenicity data were available. Immunogenicity was analyzed using the according to protocol (ATP) cohort, defined as all evaluable subjects  who tested negative for HBsAg at screening and for whom data concerning immunogenicity endpoint measures were available. These included subjects for whom assay results were available for antibodies against at least one study vaccine antigen component after Dose 3.For each group, the seropositivity rate for anti-CS antibodies (proportion of subjects with anti-CS antibody titers of ≥ 0.5 EU/ml), and the seroprotection rate for anti-HBsAg antibodies (proportion of subjects with anti-HBsAg antibody titers of ≥ 10 mIU/ml) and their 95% Confidence Intervals (CI) were tabulated by vaccine dose at each time point (day 0 and months 3 and 14). Geometric mean titers (GMTs) for anti-CS antibodies (EU/ml), and anti-HBsAg antibodies (mIU/ml) with 95% CI were calculated for each group at each time point when a serology sample was taken.The anti-CS vaccine response was calculated in two ways; the percentage of subjects with 3-fold increase in titers between day 0 and month 3 (day 90) and the geometric mean ratio , which was the fold increase in antibody GMTs at day 90 compared to day 0.In order to demonstrate non-inferiority for anti-CS and anti-HBsAg antibodies, the 95% CI for the ratio of GMTs  was computed using a one-way ANOVA model on the logarithm10 transformation of the titers. The 95% CI was also calculated using an analysis of covariance (ANCOVA) when adjusting for pre-vaccination titers. If the upper limit of the 95% confidence intervals of the ratio was below the clinical limits defining non-inferiority (three-fold difference), we can conclude that RTS, S/AS02D (0.5 ml dose) was non-inferior as compared to RTS, S/AS02A (0.25 ml dose).The percentage of subjects reporting solicited AEs within the seven-day follow-up period after vaccination was calculated with exact 95% CI by vaccine group, according to the type of AE (any and each specific solicited AE), their intensity (any and grade 3 only) and for general AE, according to their relationship to vaccination (any and those with suspected or probable relationship to vaccination). The percentage of subjects with any or related unsolicited symptom reported up to 30 days after vaccination, as tabulated per group. All serious adverse events occurring up to 30 days after dose three were listed for each treatment group.A total of 200 children (100 per group), aged between 3 and 5 years were enrolled in this 14-month study from March 2004 to April 2005. The two groups were comparable with respect to mean age, sex and race . We reported one SAE in the double-blind phase  and 10  during the single-blind phase. None of the reported SAEs were considered to be causally related to vaccination. One volunteer that developed AIDS died during the follow-up period.One month following the last dose of vaccine (day 90), all subjects included in the ATP analysis of immunogenicity were seropositive with respect to anti-CS antibodies Table . All subWith respect to anti-HBsAg, all subjects were seroprotected at day 90 Table , and theP. falciparum malaria concentrate in children less than 18 months of age [P. falciparum infection during the first 18 months.In most areas of stable malaria transmission in Africa, the prevalence of infection increases over the first few years of life and classically peaks around 5 years of age. This has led to the long term notion that immunity against the parasite tends to develop slowly, and that the main burden of malaria is in children around that age. However, emerging evidence shows that, a very significant proportion of deaths and severe episodes due to s of age ,12. Any Many African countries where malaria is endemic struggle with limited human and financial resources to run efficient National Health Services (NHS). The Ministry of Health of Mozambique estimates that approximately 50% of its population has no access to the services of its NHS. This rather common fact reminds us of the limitations of treatment of malaria cases and emphasizes the need to deploy preventive tools. One relatively well-functioning health service operating in developing countries is the WHO EPI, which manages to deliver a number of different vaccines, even to remote populations, during the first year of life. Renewed efforts to strengthen this system through GAVI/The Vaccine Fund, are yielding significant results with respect to vaccine delivery and the attainment of high coverage rates. Taking into consideration these elements, the Clinical Development Plan (CDP) of RTS, S malaria vaccine for public health use in Africa, aims to deliver vaccines as early as possible in life to prevent mortality due to malaria during the first months of life. Furthermore, the CDP ideally aims to deliver a malaria vaccine alongside other vaccines within the EPI schedule, thereby making use of already existing contacts between the health services and the target population.RTS, S/AS02D, a new formulation of the RTS, S candidate malaria vaccine, has been produced in order to match the volume of all other vaccines administered during the first year of life and therefore facilitate its administration and inclusion into the EPI schedule.This study shows that the safety and reactogenicity profile observed with RTS, S/AS02D is similar to that of RTS, S/AS02A, as well as that previously documented in other studies for RTS, S/AS02A . AlthougIn conclusion, our data show that the new formulation, RTS, S/AS02D (0.5ml) is as safe and immunogenic as the existing formulation, RTS, S/AS02A (0.25 ml), in this Mozambican pediatric population. These results allow us to proceed further in the clinical development of this candidate malaria vaccine and initiate Phase I/IIb trials in the infant population.MVI supports the development and testing of several malaria vaccines that can be seen as competitors. AL, ML, CPC, MK, M-CD, WRB, and JC are employees of GSK Biologicals. AO was on job training at GSK. AL, WRB, and JC own shares in GSK. JC and WRB are listed as the inventors of patented malaria vaccines; however, neither individual holds a patent for a malaria vaccine. None of the other authors in this paper have declared conflicts of interest.EVM, JS, MN, and PLA were involved in all phases of the study and were primarily responsible for the implementation. AL led the clinical team at GSK Biologicals. JA and ML, led the data analysis. EVM and JS were responsible for field, hospital activities and safety surveillance. JM, MN, MS and CG were the program managers. IM and YL coordinated all laboratory work at CISM. AO, was a clinical monitor at GSK Biologicals, MK was responsible for coordinating all laboratory work at GSK, M-CD, was the malaria vaccine project manager at GSK Biologicals. CPC was the scientific writer at GSK Biologicals, RT and FD, contributed to the design of the study, and interpretation. WRB, is the Vice President – Clinical Development Emerging Diseases Vaccines, at GSK Biologicals, JC, is Vice President Emerging Diseases and HIV Vaccines and heads the malaria vaccine Research and Development at GSK Biologicals. PA is the CISM scientific director. The manuscript was written by EVM and PA, with input from all other authors.


Plasmodium falciparum infection among children below five years in the North West of Burkina Faso.Identification and better understanding of potential risk factors for malaria are important for targeted and cost-effective health interventions. Housing conditions have been suggested as one of the potential risk factors. This study aims to further investigate this risk factor, and is focused on the effect of the type of roof on Plasmodium falciparum infection. In addition, data on bed net use and house characteristics was collected from the household were each child lived. Using adjusted odds ratios, children living in house roofed with iron-sheet were compared with those in house with mud or grass roof.In a cross-sectional study design, 661 children aged six to 60 months were randomly selected from three rural and one semi-urban site at the end of the rainy season (November 2003). The children were screened for fever and tested for P. falciparum infection prevalence was 22.8 % with a significant variation between . The prevalence in Cissé (33.3 %) and Goni (30.6 %) were twice times more than in Nouna (15.2 %) and Kodougou (13.2 %). After adjusting for age, sex, use of bed net and housing conditions, children living in houses with mud roofs had significantly higher risk of getting P. falciparum infection compared to those living in iron-sheet roofed houses .Overall P. falciparum infection and particular attention should be paid to children living in houses with mud roofs.These results suggest that house characteristics should be taken into consideration when designing health intervention against  Plasmodium falciparum infection.Malaria, a preventable disease, is still one of the most important causes of morbidity and mortality in developing countries, especially in sub Saharan Africa . For decP. falciparum infection [et al [et al [Anopheles mosquitoes and other vectors entering the room and may be an effective way to reduce malaria risk.Among the list of risk factors, ranging from biological, physical environment, health care systems and socio-economic conditions, the design of a house significantly affects the incidence nfection -8. Gamagn [et al  demonstrl [et al  found thBetter housing is one of the factors that reduced malaria infection risk in regions that used to be endemic and modification of houses was used to protect against malaria in Italy, Greece, Panama and the USA in the early 20th century . AlongsiP. falciparum among children below five years of age in the North West of Burkina Faso. This was done because, considering current knowledge, no study has specifically assessed and quantified the effect of different types of roofs upon P. falciparum infection in a sub-Saharan setting.Roof types determine indoor temperature, a parameter that is associated with malaria transmission. The objective of this study was to investigate the effect of the type of roof on The study area has dry savannah vegetation, with a hot and short rainy season. The total annual rainfall is about 700 mm concentrated in the June-September period. The average annual temperature is 29°C with a wide seasonal variation and diurnal variation in the cold period (December-January). In November, the mean temperature is around 29°C with a diurnal variation of 21°C to 38°C. Rainfall is almost inexistent, but some residual breeding sites from the rainy season remain and are sources of mosquitoes. Due to the climate conditions, malaria transmission, which is holoendemic in this area, has a strong seasonal variation with a high transmission period that starts one month after the onset of the rainy season until November . Four stAll the sites were part of a Demographic Surveillance System (DSS), which covers the Nouna health district located in the province of Kossi. In this area, since 1992 the population is followed longitudinally for demographical events  . These dHouses are mainly constructed out of local materials. About 80% of the houses were built of mud blocks and roofed with the same material Figure . Some muStudy participants (661) were children aged between six and 60 months from the four sites. They were selected randomly through cluster sampling of households (352), from the four sites, the sampling frame of which was obtained from the Nouna DSS database.Parents of selected children were visited prior to the survey to obtain informed consent. The objective and methods of the study were explained to them during these visits. They were also told that the finger prick procedure was very safe with the use of single-use lancets, although it may be slightly painful for the children; that the quantity of blood taken with this method was not harmful for the child; that the child would benefit from a medical check-up for malaria and other common pathology; that medical treatment would be offered free of charge if needed; and that referral was possible for severe cases.th to 18th November 2003. A mobile team of one physician, one nurse, two laboratory technicians and five interviewers visited each site. Mothers were asked to bring their children to a meeting point identified by the village chief. Children were screened for fever using digital axillary thermometers. Febrile (body temperature > = 37.5°C) children were physically examined, including weight measurement and spleen palpation and then treated with chloroquine  and paracetamol. A blood sample was then taken using the finger prick method. Severely sick children were referred to the nearest health unit. The slides were later stained with Giemsa and read for parasite count in the laboratory of the "Centre de Recherche en Santé de Nouna" (CRSN). Parasite density was estimated by counting 100 fields and equating this to 0.25 μl of blood [P. falciparum detected was classified as infected.Malaria infection data were collected by active case detection from each of the four sites from the 14of blood . Each chIn addition, mothers were asked if the child had been sleeping under a mosquito net, and whether the mosquito net was treated or not with insecticide. Use of anti-malarial drugs in the previous week was also noted.After the completion of the clinical phase, mothers were visited at home by the interviewers for detailed interviews about their housing conditions. Since a typical household compound in this area is comprised of several houses, the house where the participating child usually slept was selected. Using a structured questionnaire, the interviewer asked about the type of material used for the wall  and the roof . They also asked about the presence of animals, well and potential mosquito breeding site (open water body) within 50-meter radius of the house. All answers were cross-checked by observation.Proc logistic procedure was used to estimate the odds ratio (OR) of living in a house with a mud roof and one with a grass roof compared to living in a house with iron-sheets. The multivariate model included potential confounders such as age group , location of the house , presence of animal (binary: yes or no), presence of a well (binary: yes or no), presence of breeding sites and use of mosquito net (binary Yes or No). The logistic regression model was defined as follows:it(πi) = β0 + β1Roof_earthi + β2 Roof_grassi + β3 Xi + β4 Xi +...+ βx Xi logπi is the predicted probability for the ith child, of being positive Pf infection or having clinical malaria. The odds of the same child will be , β0 the intercept, and β1 ... βx the regression coefficients of the independent variables . The odds ratio associated with Roof_earth compared to iron-sheet roof (reference) is the exponential of β1 (ORRoof_earth = exp(β1 )). The significance of the OR was assessed using 95% confidence intervals (CI). All statistical analysis was done using SAS version 9.1 .Where The descriptive characteristics of the study population are given in Table A majority (75.3 %) of children lived in mud roofed houses. The same distribution was observed in all the four sites Table . In contReported mosquitoes net use among participants was relatively high (53.3 %) but varied significantly  between sites with the highest coverage in Goni (80.4 %) and Kodougou (68.3 %). Nouna had surprisingly the lowest coverage (15.6 %). Data on mosquito net use was missing for 3.8 % of the children and these were eliminated from subsequent analyses.Overall, 61.9 % of children lived in a house with an animal enclosure within a 50-meter radius but there was significant variation between the sites with a maximum in Cissé of 80.9 % . 18.5 % of households had wells and 13.5 % had a potential breeding site within a 50-meter radius of the house. In Nouna these figures were significantly higher than at the others sites 50.6 % and 40.9 % respectively Table .P. falciparum infection among participants is given in Table P. falciparum infection prevalence was 22.8 % with a significant difference  between the sites. The prevalence was more than two times higher in Cissé (33.3 %) and Goni (30.6 %) compared to Kodougou (13.4 %) and Nouna (15.1 %). There was no significant difference of P. falciparum infection between males (21.9 %) and females (23.8 %). Children aged between 12 and 23 months experienced the highest prevalence of Plasmodium falciparum (27.0 %) compared to those below 12 months (17.2 %) and above 23 months (14.3 %). The P. falciparum prevalence was high among children living in mud roofed houses, overall (25.9%) and consistently at all sites.The distribution of P. falciparum infection among participants who lived in iron-sheet roofed houses (12.7 %) was two times less than those who lived in mud roofed houses (25.6 %). A logistic regression model, controlling for site of residence, mosquito net use, presence of well, animal enclosure and potential mosquitoes breeding sites showed that inhabitants of houses with mud roof had significantly higher risk  of getting P. falciparum infection compared to those living in iron-sheet roofed houses  but not statistically significant.The prevalence of One of the principal issues in public health is the question of equity of treatment and variation in vulnerability of the relevant population. Of special relevance is the effect of socioeconomic class on the risk of morbidity and mortality. Malaria as a "poor man's disease", which affects either the children or parents in poverty stricken households of sub-Saharan Africa, epitomizes the problem. In this research one characteristic of financial status was tested, the quality of housing. Corrugated iron-sheets are relatively expensive and cannot be afforded by most villagers, in spite of their better protection from dampness. The question was whether this roofing material would also protect against malaria. There were two conflicting hypotheses as to the influence of the roof on the preponderance of house-dwelling mosquitoes, and hence on mosquito prevalence: (a) the iron-sheet would increase prevalence, due to the heating of the roof and resulting in increased indoor temperature and (b) the iron would decrease prevalence as the roof would no longer be a suitable resting place for blood-engorged mosquito, as opposed to cracks in the mud roof, and this would disturb the malaria-transmission cycle.The finding that there is a significant increase in malaria risk in mud roofed huts, after adjusting for other environmental conditions, suggests that the poorer sections of the population are at greater risk and thus require more intensive prevention. In this case it seems that internal spraying of roof areas would be useful. In addition, in areas where these types of dwellings are common, intensive surveillance would be required to reduce the high levels of child morbidity and mortality. From a biological point of view, it is expected that the microclimate conditions in grass roofed houses are more or less similar to the ones in mud roofed houses and similar precautions should be recommended. The non-significance of the results for the grass roofed houses in this case may be attributable to the small proportion of children living in this type of house, resulting in low statistical power.At a more theoretical level, weather-based models of malaria incidence, prevalence and mortality, require a clear evaluation of the ambient conditions of the mosquito environment. While it has been suggested that inside-outside temperature differentials are important, the results here suggest that the structural conditions are of greater importance.P. falciparum infection rather clinical malaria. The latter is likely to be influenced by better access to treatment. Further research will be required to test these explanations.Other possible explanations for the differences in prevalence between roof structures may also be raised. For instance, the more affluent houses may have better sealing around doors, hence preventing entry of mosquitoes. In addition, in the more affluent households children may be better nourished and hence less susceptible to infection. On the other hand, socioeconomic status would not have had much impact on the results since the main outcome was P. falciparum infection associated with the type of roof among under five children in North West of Burkina Faso, a holoendemic area. P. falciparum infection prevalence is consistently high in the four sites although there are differences between sites. After controlling for potential confounders, it was shown that children living in iron-sheet roofed houses have two times less risk of getting P. falciparum compared to those living in houses with mud roofs. This study adds evidence to the body of knowledge regarding the effect of house type on P. falciparum infection risk. In conclusion, taking house characteristics into consideration in any health intervention programme against malaria will be beneficial.The paper presents the risk of Yazoumé Yé has designed and implemented the study. He did the data analysis and wrote the manuscript. Moshe Hoshen and Valérie Louis have contributed to the data analysis and writing of the manuscript. Séraphin Simboro and Issouf Traoré have contributed to the data collection. Rainer Sauerborn Contributed to the design, implementation, analysis and writing of the manuscript.


Of these families, 392 siblings aged 0.5–11 y were characterized for malaria susceptibility by closely monitoring parasite counts, malaria fever episodes, and anemia over 8 mo. An autosome-wide linkage analysis based on 10,000 single-nucleotide polymorphisms was conducted in 68 selected families including 241 siblings forming 330 sib pairs. Several regions were identified which showed evidence for linkage to the parasitological and clinical phenotypes studied, among them a prominent signal on Chromosome 10p15 obtained with malaria fever episodes . The identification of genetic variants underlying the linkage signals may reveal as yet unrecognized pathways influencing human resistance to malaria.Although balancing selection with the sickle-cell trait and other red blood cell disorders has emphasized the interaction between malaria and human genetics, no systematic approach has so far been undertaken towards a comprehensive search for human genome variants influencing malaria. By screening 2,551 families in rural Ghana, West Africa, 108 nuclear families were identified who were exposed to hyperendemic malaria transmission and were homozygous wild-type for the established malaria resistance factors of hemoglobin (Hb)S, HbC, alpha In tropical Africa, virtually all children become infected with malaria parasites. Most of them experience several malaria attacks per year, and over a million die from disease complications. Sickle-cell anemia, thalassemias, and other inherited red blood cell disorders indicate that malaria has selected for human genetic variants, but no attempts have so far been reported to systematically screen the human genome for malaria-resistance factors. We describe a genome-wide linkage analysis performed in children living in rural Ghana, West Africa, including approaches to select an informative study cohort and to assess, over a period of 8 mo, individual disposition to malaria parasitemia, fever episodes, and anemia. Families carrying the known malaria-protective red blood cell disorders were excluded, infection intensities were adjusted to the use of mosquito-protection devices, and parasitological and clinical findings were corrected according to the state of partial malaria immunity, which, under constant exposure, gradually develops over the first 10 y of life. The study revealed several genomic regions showing evidence for linkage to the various malaria phenotypes recorded, among them a prominent signal on Chromosome 10 correlated to the frequency of fever episodes. Future identification of genes involved is expected to reveal previously unrecognized pathways that may protect children against malaria. Plasmodium falciparum is one of the leading causes of human morbidity and mortality worldwide, predominantly affecting populations of resource-poor countries in the south [Malaria caused by he south . DrawbacThe blood stages of the parasite comprise asexual forms, which maintain the infection and cause disease, and sexual forms, which transmit the infection . AsexualClinically, malaria presents as a mild form of acute febrile episodes and anemia, or as a severe form, which comprises a complex syndrome of life-threatening complications . While tA second clinical feature of mild malaria is anemia. It affects an enormous number of children in endemic areas  and may The effect of human genetics on malaria has long been recognized when the theory of balancing selection was substantiated for thalassemias, sickle-cell anemia, and other red blood cell disorders . Twin stP. falciparum infection intensity and mild clinical malaria among African children selected not to carry any of the classic malaria resistance genes. As markers, 10,000 single-nucleotide polymorphisms (SNPs) were used.Here we report on an autosome-wide linkage analysis for P. falciparum blood trophozoites, parasite densities, and interim or present malaria fever episodes were monitored weekly and anemia as indicated by the packed blood-cell volume (PCV) was determined biweekly. Compliance was as follows; 98.8% of 12,152 parasitemia assessments, 95.4% of 6,272 PCV assessments, and 98.5% of 12,152 assessments for malaria fever episodes were recorded with a maximum of data missing per planned visits of single participants of 13/31, 6/16, and 18/31 records, respectively. Results from regression models for analyzing the effect of age, bednet use, and intake of antimalarials on the various phenotypes are summarized in 392 siblings of 108 families resident in West Africa were followed over a period of 31 wk, which covered an entire rainy season. Prevalences of Based on a ranking that favored high levels of parasite densities in conjunction with high intrafamilial variability see , 377 indz score of 4.37  between SNP markers rs952153 and rs1964428 marking the interval of 5.9–12.0 cM and 2.5–3.5 Mb of the genetic and physical chromosomal maps, respectively  at marker rs1964428 corresponding to 12.0 cM/3.5 Mb , or the use of a wider definition of malaria fever episodes that included afebrile malaria episodes diagnosed by the study physicians (z score of 4.04). The 2.2 z score support interval (corresponding to a 1-LOD support) encompassed a 27.4 cM/11.0 Mb distance containing 71 annotated or hypothetical genes. Functional candidates include genes encoding a platelet-type phosphofructokinase (PFKP) also expressed in red blood cells, an inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2/PFKFB3), the alpha chain of the interleukin-15 receptor (IL15RA), the alpha chain of the interleukin 2 receptor (IL2RA), protein kinase C theta (PRKCQ), the GATA-binding protein 3 (GATA3), and a gene similar to that of the interleukin 9 receptor precursor (LOC439945).The nonparametric linkage analysis (NPL) and Haseman-Elston multipoint linkage analysis (HE) were applied A–2D. Parectively D. HE shoM/3.5 Mb D. The loz score of 3.73  between rs2147363 at chromosomal position 55.0 cM/51.4 Mb and rs726540 at 55.5 cM/52.3 Mb , S-formylglutathione hydrolase , the cysteinyl leukotriene receptor 2 (CYSLTR2), and the endothelin receptor, nonselective type, (EDNRB).A further region with evidence for linkage was found using parasite density as the phenotype. The NPL analysis yielded a signal on Chromosome 13q with a maximum asymptotic /52.3 Mb B. HE res/52.3 Mb B. The loreported –19. The p-value = 5.3 × 10−4), and PCV  at adjacent marker positions .Furthermore, a signal on Chromosome 1p36 at 18 cM/9 Mb provided evidence for linkage with both parasite prevalence   C.To our knowledge, this is the first genome-wide approach to identify human genetic variants influencing susceptibility and resistance to malaria. Since the seminal observations on balancing selection with inborn red blood cell disorders, malaria is a prominent element in human genetics. The importance of the classic malaria-protective red blood cell traits is in the present study highlighted by the large proportion of 86% of families found to be affected in the initial survey of our study population. These were excluded from the study in order to concentrate the search on as yet unrecognized human genetic variants ,20. As gAssessing the phenotype of malaria infection intensity remains a challenge because it is uncertain to which extent any limited number of parasite counts truly reflect the infection intensity . In addiAs expected, antimalarial treatments had an effect on all phenotypes studied. The influence on parasite prevalences and parasite densities was found to be limited to the two subsequent assessments, therefore it was addressed by correcting the respective values of prevalences and by excluding the corresponding densities see . In contOf all covariates tested, age had the strongest effect and was included in all phenotype adjustments. In children older than 6 mo as in the present cohort, the age effect on malaria in endemic areas is dominated by the gradual development of a certain degree of adaptive immunity, termed semi-immunity. This is reflected by a successive decrease over age of the number of fever episodes, the degree of anemia, parasite densities, and, at relatively high age, parasite prevalences –27. TherThe phenotypes studied showed significant correlations between each other. This is in agreement with the general understanding that all signs and symptoms of malaria result from parasitemia. The explained variances in most instances were low, however, leaving room for separate genetic influences. As expected, the correlation between parasite prevalences and parasite densities was exceptionally high. Despite this, both were included as separate phenotypes because there is evidence to suggest that they are under distinct genetic influences. First, epidemiological findings including those of the present study (unpublished data) indicate that semi-immunity suppresses high parasite densities significantly more efficiently than low parasite densities , which sEvaluation of the data using established linkage methods revealed several prominent linkage signals. Interestingly, locus-specific heritability calculations performed for two of these linkage regions indicated that, in both cases, approximately 35% of the total phenotype variability was attributable to these loci in families who did not carry any of the established malaria resistance factors. These estimates allow us to postulate the effect of a major locus in both instances, which would support a recent conclusion that susceptibility and resistance to infectious diseases may be governed by single major genes rather than by a large number of genes each exerting a small influence .(PFFE-1). Notably, the signal was found in both model-free approaches. Furthermore, it was robust to variations in phenotype definitions, which may be of particular importance because the non-specific symptoms of malaria fever episodes make the clinical diagnosis uncertain. That we found the strongest linkage signal with this particular phenotype may relate to the fact that fever regulation might be similar regardless of whether it is influenced by innate resistance or adaptive immunity, with respect to the age-dependent bias introduced into our study cohort by these two factors, as described above. The underlying genetic variant may be of more general interest because it may relate to the regulation of the systemic inflammatory response.The region showing strongest and significant linkage concerned the phenotype of malaria fever episodes A number of additional regions with evidence for linkage were identified which did not reach statistical significance. Therefore, they are not discussed in any detail, although experiences in other complex diseases have shown that weaker linkage signals may as well lead to the identification of relevant genetic variants . The linPlasmodium chabaudi infection, evidence has been obtained for linkage regions on Chromosomes 3, 5, 9, 11, and 17 [P. falciparum- parasite density . This is not unexpected because P. falciparum substantially differs from P. chabaudi in that P. falciparum-infected red blood cells adhere to the vascular endothelium [Plasmodium berghei infections [PFFE-1 on 10p.No support for our data can be derived from previous linkage studies in mouse malaria. Studying parasite density in murine , and 17  but not othelium , which mfections , which cTo our knowledge, this is the first time that the Affymetrix HMA10k chip was used for genotyping individuals of African descent. The raw genotypes yielded a call rate of 94.5%, which nearly reached 95% considered sufficient for optimal assay performance  and was P. falciparum.The study was conducted in the Asante Akim North District, Ashanti Region of Ghana, West Africa, a region classified as hyperendemic for malaria by a cross-sectional prevalence of 0.54 for + thalassemia deletion 3.7, and glucose-6-phosphate-dehydrogenase (G6PD) deficiency A-, which were considered to possibly influence susceptibility to P. falciparum parasitemia and mild malaria, were determined, and 346 (13.6%) families were identified not segregating any of the traits.Ethical approval was obtained from the Committee for Research, Publications and Ethics of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. All procedures were explained in the local language, and consent was obtained from both parents. Parents of 2,551 families were recruited who had three or more children below the age of 12 y and agreed to participate. Venous blood samples of 2 ml were obtained from both parents and preserved by addition of an equal volume of 8 M urea. The genetic variants of hemoglobin (Hb)S, HbC, alphap < 0.001) and other parameters of malaria infection intensity. All families belonged to the ethnic group of Akan.Of the 346 families, a study group of 392 siblings of 108 families was selected based on (i) the logistic criterion that their homes clustered in 16 of the 30 villages included in the initial survey and (ii) that they did not live in homes equipped with window screens, which a posteriori were found to significantly reduce parasite prevalences from 0.54 to 0.35 .A subset of 377 members of 68 families were selected from the study group by excluding siblings who were absent at more than two assessments and by a ranking that favored high levels of parasite densities of The children were phenotyped from May 20 to December 20, 2002. Weekly assessments by the visit of a trained physician included a medical history, measurement of body temperature by an infrared ear thermometer, a blood sample by finger prick or heel prick (approximately 100 μl), and in case of disease symptoms, a physical examination. The installation of window screens in homes and the use of bed-nets were recorded.Weekly malaria smears were prepared at the study site, and in the laboratory they were stained with Giemsa and examined . ParasitP. falciparum was the predominant species; Plasmodium ovale or Plasmodium malariae were found in 19% and in 90% of these in combination with P. falciparum. For the assessments of parasite prevalences and parasite densities, only P. falciparum and only the disease-causing asexual forms were included, which showed a median point prevalence of 53.1% and a period prevalence of 99%. Parasite densities of P. falciparum ranged from 0–317,360 parasites per μl, with an overall median of 32 and a 75th percentile of 680 in 12,011 assessments.Weekly point prevalences of malaria parasitemias showed a median of 54% (range 40%–61%) yielding a cumulative period prevalence of 99%. Contributing 98% of all parasitemias, 2+ over 6 wk. The median PCV value of 16 biweekly assessments was used as the phenotype in all cases because none of the children showed evidence for any other disease causing substantial anemia. As determined by 16 biweekly PCV measurements, mild anemia defined by a PCV of <33% in the age group of 0.5–6 y and of <36% in the age group of 6–14 y [Anemia was on the spot assessed as PCV by capillary hematocrit centrifugation using 70 μl EDTA anticoagulated capillary tubes  and mobile centrifuges. To reduce iron-deficiency as a possible confounder, prior to phenotyping, all children were treated against hookworm infection with 400 mg albendazole followed by oral iron supplementation of 2 mg/kg Fef 6–14 y  was founP. falciparum at any density. The number of fever episodes during the observation period of 31 wk was counted for each individual, whereby multiple episodes within 3 wk were considered recrudescences and counted as one episode [Mild malaria attacks were defined following WHO recommendations , first,  episode .Applying this definition resulted in the identification of 504 malaria fever episodes affecting 257 children, which corresponds to a period prevalence of 66%. In addition, 34 febrile states of other etiologies including upper respiratory tract infections, lower respiratory tract infections, pneumonia, measles, and urinary tract infections were diagnosed by the study physicians. Suspected malaria attacks were treated by a standard dose of chloroquine or amodiaquine following national guidelines, other illnesses as deemed appropriate.P. falciparum parasitemias . Fever attacks reported by parents or guardians to have occurred between two examinations were recorded at 870 visits, whereby blood smears at the time of examination were positive for P. falciparum at 522 of these instances, and 133 of the attacks had reportedly been treated with antimalarials. The study physicians at 587 instances suspected malaria attacks at the time of examination, 389 (66%) of which were retrospectively supported by the presence of P. falciparum parasitemia. Of 631 mild malaria attacks as defined by WHO recommendations, 57% were in agreement with malaria diagnoses made by the study physicians, whereas 3% were classified by the study physicians as a febrile illness other than malaria.In greater detail, a tympanic temperature of >37.7 °C was measured at 365 visits. Subsequent malaria smears showed that 248 (68%) of the fever attacks occurred in conjunction with p of covariate x is found, with the power p chosen from −2, −1, −0.5, 0, +0.5, +1, +2, +3, where x0 denotes ln x. For example, for p = 0, 0.5 the model is b0 + b1 ln(x) + b2 √x. The set includes the straight line, i.e., no transformation p = 1, and the reciprocal, logarithmic, square root, square, and cubic transformations. Even though the set is small, the powers offer considerable flexibility. The test is performed by comparing the difference in model deviances with a χ2 distribution on 1 degree of freedom. The resulting p-value is approximate and is justified by statistical arguments [Regression models were used for adjusting the raw phenotypes for influences of covariates . Continurguments . Pearsonrguments  were useP. falciparum parasitemias (1 or 0) minus the sum of predicted probabilities for parasitemias of an individual were calculated and used as phenotype. The predictions were derived from the study population by logistic regression models for parasitemia for each week's set of data with respect to the influences of age, time period since the last use of antimalarials, and the use of bed-nets (The difference between the sum of observed weekly bed-nets .p < 0.05) and were therefore excluded. Since the 31 weekly parasite-density values (or their log-transformations) of an individual deviated from a normal distribution, quantiles were used. The 75th percentiles were chosen because they were substantially more informative than the median values, which would have been zero in nearly half the assessments. The 75th percentiles were log-transformed, whereby half the detection threshold of 16 parasites per μl was added before taking the logarithm. The phenotype was adjusted to age by calculating residuals from a Tobit regression model, whereby 74 of 392 observations were left censored , and A-  [+ thalassemia, and G6PD A- were estimated to be 0.071, 0.058, 0.175, and 0.063, respectively. G6PD variant A, which was previously reported to show only a slightly decreased enzyme activity of 80% compared to the normal variant B (A376) [p = 0.13), parasite density (p = 0.55), anemia (p = 0.36), and fever episodes (p = 0.80); therefore children being homozygous, heterozygous, or hemizygous for G6PD B and/or A, were included in the study group. Paternities and maternities were assessed by typing the short tandem repeat markers D1S2782, D6S273 , D5S816, D7S2212, D11S1984, D17S1299, and D17S1294 , in(TG)n of CD36 [Venous blood samples of 2 ml were obtained at the end of the study period and preserved by the addition of urea. DNA was extracted by a standard procedure . HbS, HbC , and alplassemia  were det, G202A)  were typ, G202A) , wherebyB (A376) , was ret of CD36 , and IL1 of CD36 , all shohttp://www.affymetrix.com). Mapping order and genetic distances of markers were obtained from Affymetrix, the genetic position of 86 markers was unavailable, 295 were X-linked, and 10,279 were from autosomes. Allele frequencies were estimated from 134 founders. The physical positions of the markers were aligned to human DNA sequence information available from NCBI/NIH (http://www.ncbi.nlm.nih.gov/mapview/maps.cgi).A high density SNP genome scan was performed using a whole-genome sampling analysis (WGSA) approach  with thep < 0.001 test level, (ii) a genotype calling fraction of <80%, or (iii) a heterozygosity of <5%.In a first step, gender of participants was verified by investigating heterozygosity and hemizygosity, respectively, at X-linked markers. The relationships between individuals were confirmed by using Graphical Relationship Representation . Genotypz scores from NPL and LOD scores from HE are reported. Additionally, empirical p-values were determined using 100,000 replicates on the basis of the mean information content in the multipoint SNP analysis, which was 0.976 . Specifically, a single marker was simulated with 20 equally frequent alleles, thus yielding a heterozygosity of 0.95. For the simulation, family structures and phenotypes were left unchanged. Details on this Monte-Carlo approach can be found, e.g., in [p < 10−4 in either NPL or HE was considered significant [p < 5·10−4 and another signal of p < 10−3 obtained at the same genomic region with two largely independent phenotypes. We calculated locus-specific heritabilities of additive effects using the regression approach of Sham and colleagues [A quantitative trait locus autosomal linkage analysis was performed using the model-free nonparametric linkage method (NPL)  and the e.g., in . A linkanificant . In addilleagues . Applyin


Plasmodium falciparum, linked to the hepatitis B surface antigen and formulated in the proprietary adjuvant system AS02A. In a recent trial conducted in children younger than age five in southern Mozambique, the vaccine demonstrated significant and sustained efficacy against both infection and clinical disease. In a follow-up study to the main trial, breakthrough infections identified in the trial were examined to determine whether the distribution of csp sequences was affected by the vaccine and to measure the multiplicity of infecting parasite genotypes.The candidate malaria vaccine RTS,S/AS02A is a recombinant protein containing part of the circumsporozoite protein (CSP) sequence of P. falciparum DNA from isolates collected during the trial was used for genotype studies.The main trial was carried out in the Manhiça district, Maputo province, Mozambique, between April 2003 and May 2004.Children from the two cohorts of the main trial provided parasite isolates as follows: children from Cohort 1 who were admitted to hospital with clinical malaria; children from Cohort 1 who were parasite-positive in a cross-sectional survey at study month 8.5; children from Cohort 2 identified as parasite-positive during follow-up by active detection of infection.P. falciparum genotypes was also determined.Divergence of DNA sequence encoding the CSP T cell–epitope region sequence from that of the vaccine sequence was measured in 521 isolates. The number of distinct p = 0.035, p = 0.006), respectively, for the two cohorts. This was not the case for children in Cohort 1 who were admitted to hospital (p = 0.478).We found no evidence that parasite genotypes from children in the RTS,S/AS02A arm were more divergent than those receiving control vaccines. For Cohort 1 (survey at study month 8.5) and Cohort 2, infections in the vaccine group contained significantly fewer genotypes than those in the control group, (RTS,S/AS02A did not select for genotypes encoding divergent T cell epitopes in the C-terminal region of CSP in this trial. In both cohorts, there was a modest reduction in the mean number of parasite genotypes harboured by vaccinated children compared with controls, but only among those with asymptomatic infections. Background: A recent trial in young children in Mozambique found that an experimental malaria vaccine, RTS,S/AS02A, reduces the risk of clinical malaria, delays time to new infection, and reduces episodes of severe malaria over 18 months. RTS,S/AS02A is based on a particular protein, the circumsporozoite protein, which is expressed on the surface of the malaria parasite at a stage in its life cycle prior to the disease-causing blood-stage infection. Because there are different variants of the circumsporozoite protein in the parasite population, the researchers were concerned that the strain-specific protection offered by RTS,S/AS02A might select for parasites with particular variants of the circumsporozoite gene. They did a follow-up study to investigate this concern, looking to see whether vaccinated children who became infected with malaria had variant forms of the parasite.What this trial shows: The investigators found that there was no increase in how different the variants were from the vaccine-type allele between vaccinated and control individuals. There was a small decrease in the average number of parasite “strains” in vaccinated children compared with controls. However, in children within the trial who were admitted to hospital with clinical malaria, the number of different “strains” of the parasite was no different between vaccinated children and controls.Strengths and limitations: The study asks an important molecular question about whether vaccination with RTS,S/AS02A selects for different variants, specifically escape mutants of the malaria parasite. If the researchers had found such selection, this would suggest that the vaccine's success could be compromised once widely deployed. A limitation of this follow-up study is that it is difficult to interpret these findings because the mode of action of the vaccine is not yet fully understood. It is also difficult to understand how the results might apply to non-hospitalised patients with clinical malaria, because the malarial patients included in this study were exclusively identified through hospital admissions.Contribution to the evidence: This study adds molecular information to the previously reported results on the clinical effectiveness of the RTS,S/AS02A vaccine. The researchers found no evidence that the RTS,S/AS02A vaccine selected for different variants of the malaria parasite. Malaria continues to be a major cause of morbidity and mortality in many countries of Africa, and is estimated to cause more than one million deaths each year . WhereasP. falciparum fused to hepatitis B virus surface antigen formulated in the AS02A adjuvant system [P. falciparum infection in both experimentally infected volunteers and naturally exposed adults have been obtained with this vaccine [p = 0.004) for clinical malaria and 57.7%  for severe malaria over six months of follow-up. The vaccine efficacy for extending time to first P. falciparum infection was 45.0% . Extended follow-up has confirmed that protection is sustained over 18 months post-vaccination [Several malaria vaccine candidates have been tested in malaria endemic countries , includit system ,4. Effic vaccine –5, and t vaccine . VaccineP. falciparum have demonstrated a significant level of genetic polymorphism among isolates, the most variable domains being the T cell epitopes near the C-terminus of the protein, which are designated Th2R and Th3R [P. falciparum merozoite protein MSP-2 among vaccinated Papua New Guinean children [P. falciparum infection that was not strain-specific [P. falciparum clone 3D7/NF54. Of the csp alleles identified by dot-blotting, 10% encoded Th2R and 15% encoded Th3R epitopes that were indistinguishable from those in the vaccine. The prevalence of these alleles was similar among emergent infections in the vaccine and the control groups [p = 0.05). These studies indicate that integrated molecular typing will be a useful tool for the evaluation of malaria vaccines, especially where a strain-specific effect is possible.CSP is an abundant protein found on the surface of the sporozoite. The prevailing hypothesis in this field is that cell-mediated immune responses play an important role in protection against pre-erythrocytic parasites, and studies of the gene encoding CSP of and Th3R ,9. The ichildren . In adulspecific . The parl groups . InteresP. falciparum genotypes present in infected subjects.As part of the trial conducted in 2003–2004 by Alonso and colleagues to evaluate the efficacy of the RTS,S/AS02A vaccine in Mozambican children , DNA seqP. falciparum malaria over a six-month observation period starting 14 d after dose 3, corresponding to study month 2.5, until study month 8.5. In Cohort 2, 417 children were enrolled and the main endpoint was the efficacy of the vaccine for prevention of new infections, over the post-vaccination follow-up period spanning study months 2.5 to 8.5. In Cohort 2, subjects received a single dose of sulfadoxine-pyrimethamine and a daily dose of amodiaquine for 3 d, 4 wk prior to the commencement of surveillance and 2 wk prior to the last dose of vaccine, to clear any parasitaemia.Samples for molecular analysis were collected during the course of a vaccine trial conducted in the Manhiça district, southern Mozambique, between April 2003 and May 2004. Details of the study area, population, and study design have been previously reported . In brieSamples for parasite genotyping collected from children in Cohort 1 were obtained from two sources: 1) filter-paper bloodspots from a cross-sectional survey conducted at study month 8.5, and 2) frozen EDTA-blood from patients with malaria identified by passive case detection and admissions to the hospital as in-patients. In Cohort 2, filter-paper bloodspot samples were collected from patients with malaria infection identified by active detection of infection see . In bothThe protocol was approved by the National Mozambican ethics review committee, the Hospital Clínic of Barcelona ethics review committee, the PATH human subjects protection committee, and the London School of Hygiene & Tropical Medicine ethics review committee. Written informed consent was obtained from parents/guardians prior to study enrollment and blood sample collection.Filter-paper blood samples were air dried, individually wrapped in aluminum foil, placed in a box containing desiccant (silica gel), and stored at 4 °C until use. All samples were labelled with a unique sample identification number.DNA extraction from one spot  of filter paper and from about 200 μl of frozen blood samples  was performed using the QIAamp DNA blood minikit-extraction procedure as described by the manufacturer . DNA samples were eluted into 150 μl of buffer AE and stored at −20 °C until use.P. falciparum CSP was determined using a semi-nested PCR followed by sequencing of purified PCR products.The distribution of polymorphic sequence variants encoding the Th2R and Th3R regions of csp gene was performed using three primers binding to conserved sequences flanking the Th2R and Th3R regions of csp. For the first round PCR, the primers were: forward primer (CSo101), 5′-aatcaaggtaatggacaagg-3′; reverse primer (CSo102), 5′-ctaattaaggaacaagaagg-3′. For the second reaction, the primers used were: forward primer (CSo101), reverse primer (CSo104), 5′-ggaacaagaaggataatacc-3′. The primers CSo101 and CSo104 were used to prime sequencing reactions.Amplification by semi-nested PCR of a 321-bp fragment of the The first-round PCR mixture contained 1 μl of DNA in a total volume of 10 μl containing 200 nM concentrations of each primer constituted in 1× BioMix Red  PCR reaction buffer. The second PCR mixture contained 1 μl of DNA in a total volume of 20 μl containing 200 nM of each primer in 1× reaction buffer. PCR amplifications were performed in a MJ Research DYAD 96-well thermocycler with the following cycling conditions: 95 °C for 4 min, followed by 40 cycles of 94 °C (1 min), 50 °C (1 min) and 72 °C (1 min) and final extension at 72 °C for 4 min. Second-round PCR products were monitored on a 2.0% agarose gel in 1× TAE buffer to check the quality, size and yield of the PCR products before proceeding to product purification and sequencing.After purification of PCR products using the QIAquick multiwell PCR purification protocol (Qiagen), sequencing reactions were performed following the BigDye Terminator version 3.1 cycle sequencing kit protocol . In brief, the PCR-sequencing reaction mixture contained 2 μl purified product containing between 10 and 120 ng of PCR product together with 8.0 μl PCR master mix containing 2 μl of forward or reverse primer, 0.5 μl BigDye mix, 1.75 μl of sequencing buffer and purified water. The PCR amplification was performed in 96-well plates under the following conditions: 25 cycles of 96 °C for 0.30 min, ramp 1.0 °C/s to 50.0 °C, 50 °C for 0.15 min, ramp 1.0 °C/s to 60.0 °C, 60.0 °C for 4.00 min, ramp 1.0 °C/s to 96.0 °C.The sequencing products were then precipitated using the ethanol-sodium acetate precipitation method and placed in the ABI 3730 capillary sequencing machine for sample electrophoresis.The SeqMan software program  was used to analyse the electropherogram sequences. The sequence of each PCR product was manually checked by at least two investigators and a consensus reached or the assay repeated. The forward and reverse electropherogram sequences were assembled into a contiguous sequence and the consensus file examined to decide whether it represented a single allele or mixed alleles. Contiguous sequences showing two or more electropherogram peaks in the same position  indicated mixed alleles.The polymorphic variants at both regions of the CSP sequence (Th2R and Th3R) were scored using the 3D7/NF54 allele as reference. Eighty codons at positions 323–402 (numbered according to the 7G8 sequence ) were almsp-1 block 2 and msp-2 and the R11 repeat region of glurp as previously described [Nested PCR was used to discriminate alleles of escribed .Following electrophoresis, gels were double-scored independently by two investigators, and either consensus was reached or the assay repeated. The minimum number of genotypes at each locus was determined for each sample and the results entered in an Excel worksheet. The overall genotypic complexity of each isolate was taken as the highest of the three numbers calculated at each locus. The mean of this minimum number was then calculated for each group, and termed the “multiplicity of infection” (MOI). This must be regarded as a minimum estimate of the true mean MOI .All DNA extractions and laboratory analyses were performed by individuals blinded to vaccine assignment.An analysis plan was agreed upon prior to decoding the dataset into treatment groups. The endpoints for assessing the strain specificity of the vaccine were: 1) the relative proportion of the vaccine type allele, for each of the polymorphic amino acid sites at both T cell epitope regions (Th2R and Th3R), in the vaccine versus control group. The Fisher exact test (2-sided) was used to test the differences between RTS,S/AS02A and control group; and 2) the number of amino acids different from the vaccine type in each of the T cell epitope regions (Th2R and Th3R). The differences in the distribution of the number of amino acids different from 3D7/NF54 between the vaccine and the control groups were compared using a Mann–Whitney U test (2-sided Wilcoxon Rank Sum test). Statistical test results were not adjusted for multiple analyses.P. falciparum genotypes per infection, the frequency distribution of genotypes in vaccinated and control groups were compared using the Mann–Whitney U test as the primary outcome. Poisson regression was used to produce estimates of vaccine effect, adjusted for parasite density, age, and time to infection. However, the analysis did not produce an outcome that agreed with the non-parametric analysis, and therefore we concluded that the Poisson distribution did not adequately describe the data. An adjusted gamma regression was calculated and is presented as an exploratory (secondary) analysis, as this was able to fit the data more closely.To determine whether the vaccine affected the number of Overall participant flow is summarized in csp genes of 208 (98%) of these were successfully amplified by PCR and sequenced. The electropherograms of 109 isolates (52.4%) indicated the presence of a single csp allele, eight (3.8%) indicated a single clear majority allele , and 91 (43.8%) gave mixed alleles. A total of 95 samples were analysed from 104 Cohort 1 participants admitted to hospital with clinical malaria for the first or only time: DNA from 90 of these (94.7%) was successfully amplified and sequenced. Forty-one (45.5%) of the PCR products gave electropherograms consistent with a single allele, 11 (12.3%) a single clear majority allele in the presence of small background peaks, and 38 (42.2%) were mixed alleles.The RTS,S/AS02A trial conducted in Mozambique enrolled two cohorts of children, one for estimation of vaccine efficacy against episodes of clinical malaria (Cohort 1), and the other for estimation of efficacy against infection (Cohort 2) . In CohoIn Cohort 2, there were 323 children with first episodes of parasitaemia detected, but 82 of these children did not have a filter paper sample collected. The majority of these 82 samples corresponded to children who were febrile at the home visit and thus sent directly to the outpatient clinic where blood smear and microcapillary samples were taken, but filter paper was not. Thus DNA samples for genotyping analysis were available for 241 children with parasitaemia, and 223 (92.5%) of these samples were successfully amplified by PCR and the products sequenced. Of these, 157 (70.4%) contained a single allele, eight (3.6%) a single clear majority allele with small background peaks, and 58 (26%) gave mixed alleles. The observation of fewer mixed infections in Cohort 2 than in Cohort 1 may be at least partially explained by differences in both intervention and surveillance phases of the study. Children in Cohort 2 received anti-malarial treatment two weeks before the third dose of vaccine to clear parasitaemia prior to the start of the efficacy follow-up period, and underwent biweekly active detection and treatment of infection. Following up in Cohort 1 was by passive detection of malaria cases.In each cohort we determined 1) the proportion of isolates with the CSP vaccine type (3D7) at each polymorphic amino acid position in the Th2R and Th3R epitopes in the vaccine and control groups; and 2) the number of amino acids different from vaccine type (3D7) within the Th2R and Th3R regions in the vaccine versus control group. There were eight and six amino acid positions in the Th2R and Th3R regions of the CSP sequence, respectively, which displayed polymorphism in our population and were therefore informative. Primary comparative analyses were performed using a conservative dataset comprising both mono-allelic isolates, and those isolates with a single clear majority allele against a background of one or more minor alleles. Mixed-genotype isolates were included in secondary comparative analyses.Comparisons between groups were performed using Fisher's exact tests and by evaluating the 95% CI of the differences between groups. Post hoc, the study had the power to detect any between-group differences in the proportion of non-vaccine amino acid residues in the Th2R and Th3R epitope regions of at least 25%  and of at least 20% for the other datasets.p = 0.050), but there were no other significant differences between treatment groups in Cohort 2 at any position in either epitope region.The proportion of isolates that contained non-vaccine residues in the CSP Th2R and Th3R epitopes are shown in p = 0.066), and the vaccine type Lys present at residue 337 in the Th2R region was replaced by Thr, Arg, or IIe in 52% of vaccine group samples compared with 38% of control samples . In Cohort 2, the vaccine-type residue 374-Asp in the Th3R epitope was replaced by Asn in 3% of the vaccine group infections when mixed alleles were included, compared with 9% in the control group . Significant differences in substitution patterns between the control and vaccine groups were not seen at any other amino acid position in secondary analyses.In secondary analysis of Cohort 1 data at study month 8.5, including counts from mixed-allele infections, the vaccine-type Glu at residue 333 (Th2R) was replaced by Gln or Lys in 76% of vaccine group samples, compared with 64% of control samples , 95 from Cohort 1 children admitted to hospital, and 224 samples from Cohort 2 children, was typed successfully for s of MOI .p = 0.035, p = 0.006, respectively, for the two cohorts, Wilcoxon rank sum test) (p = 0.098). Mean MOI for Cohort 2 was 1.83 (sd 0.89) and 2.16 (sd 0.97), respectively, for vaccine and control groups . Among Cohort 1 participants who were admitted to hospital, there was no significant difference between the vaccine  and control groups  in the genotypic complexity of infection analysis as indicated by the Wilcoxon test (p = 0.478) and the gamma regression adjusted for parasite density and disease severity .For Cohort 1 (study month 8.5) and Cohort 2, the overall minimum MOI (combining the data from the three loci) was significantly lower among infections in the vaccine group than among those in the control group (um test) . The mea. [P. falciparum, includes regions that are highly polymorphic and may exist as dozens of variants within any given parasite population [P. falciparum infections in 222 vaccinated children as compared to 299 control individuals participating in the Mozambique trial of RTS,S/AS02A in 2003–2004 there was no evidence for selection of genotypes that differed in sequence from the vaccine type.Preventive measures against malaria disease and mortality are needed urgently as we enter the 21st century with a malaria burden among African children unchanged since the beginning of the previous century. The demonstration by Alonso et al.  that vac. , is timepulation ,9,11. If+ T cell responses to an invariant CSP epitope present in RTS,S have been associated with natural protection against infection [csp sequences encoding theTh2R and Th3R epitopes in 238 isolates from Thailand does not support the notion that diversity in these epitopes is generated in response to selection by sequence-specific human immunity in that population [+ T cells with other skin and genital HPV types [The absence of measurable sequence-dependent selection in the Th2R and Th3R regions does not imply that a contribution of vaccine-induced cellular immune responses to the observed efficacy of RTS,S/AS02A can be ruled out. Indeed, there is ample evidence that humans vaccinated with RTS,S/AS02A generate immune responses to both epitopes ,14, althnfection , and recpulation . Furtherpulation ,18 and cPV types  have beecsp, due to the presence of multiple genotypes with different csp alleles, to unequivocally assign a full sequence. Thus, 43.8% of isolates from Cohort 1 and 26% from Cohort 2 were of mixed csp genotype with no clear majority allele, and did not contribute to our primary sequence analysis. This may potentially bias our findings if sequence-dependent selection occurs more commonly among these complex infections. This limitation also applies to dot-blotting methods, but could be addressed in future studies by cloning out and sequencing individual csp alleles from such mixed infections, adding substantially to the analysis effort.Our analysis of sequence-dependent vaccine efficacy used direct-sequencing of DNA amplified directly from blood samples. All processes were performed in 96-well format from DNA extraction through to data collection. This innovative approach provides advantages over the dot-blot hybridisation method of Alloueche et al.  in beingp = 0.035) and in Cohort 2 (p = 0.006). No difference in MOI between RTS,S/AS02A-vaccinated children and control individuals was observed among Cohort 1 participants admitted to hospital with clinical malaria. The mechanism by which an efficacious vaccine might reduce MOI among emergent infections remains unclear, but in our analysis the reduction effect was shown to be independent of both parasite density and time to infection. A pre-erythrocytic vaccine such as RTS,S/AS02A may work in part by preventing or reducing emergence of parasites from the liver stage [An important parasitological parameter used in many clinical and epidemiological studies of malaria is the mean number of distinct parasite genotypes per infected individual, or MOI ,20. StudWe have found no evidence that the observed efficacy against malaria infection and morbidity of the RTS,S/AS02A vaccine among Mozambican children is sequence-dependent, and we have shown that fewer distinct parasite genotypes circulate in vaccinated hosts compared with controls. There has been no systematic review of genotypic multiplicity in the context of vaccination with RTS,S/AS02A. However, an analysis of a previous RTS,S vaccine trial conducted in Gambian adults  also revThese data were accrued in the context of a large Phase IIb trial. Nevertheless, should RTS,S/AS02A or similar pre-erythrocytic malaria vaccines be deployed on a large scale in the future, surveillance for evidence of sequence-dependent selection of breakthrough parasite genotypes would be a wise precaution. We have demonstrated that high-throughput direct sequencing of large numbers of field samples from parasite-positive individuals would be a feasible and robust approach to this end.CONSORT ChecklistThe trial protocol and CONSORT checklist list for the main trial have been previously published ,7.(49 KB DOC)Click here for additional data file.Trial Protocol(71 KB DOC)Click here for additional data file.


Plasmodium falciparum malaria because of protracted bone marrow suppression, possibly due to residual subpatent parasites.Severe anaemia can develop in the aftermath of Plasmodium antigens, lactate dehydrogenase , aldolase and histidine rich protein 2  were used to differentiate between patients with (1) no malaria, (2) recent cleared malaria, (3) persistent P. falciparum infection. Red cell distribution width (RDW), plasma levels of soluble transferrin receptor (sTfR) and erythropoietin (EPO) were measured as markers of erythropoiesis. Interleukin (IL) 10 and tumour necrosis factor (TNF)α were used as inflammation markers.Blood was collected from patients with recent malaria and negative malaria microscopy. Detection of the P. falciparum infection, but not recent malaria without residual parasites, was associated with bone marrow suppression i.e., low RDW (P < 0.001 vs. P = 0.56) and sTfR (P = 0.02 vs. P = 0.36). TNF-α and IL-10 levels were not associated with bone marrow suppression.EPO was correlated with haemoglobin, irrespective of malaria . Persistent In the treatment of malaria, complete eradication of parasites may prevent subsequent development of anaemia. Severely anaemic children may benefit from antimalarial treatment if antigen tests are positive, even when no parasites can be demonstrated by microscopy. Plasmodium falciparum malaria in African infants and young children. The anaemia develops when accelerated removal of erythrocytes is not compensated by the bone marrow. Bone marrow suppression is generally present in all malaria patients [P. falciparum infection [Anaemia is a common, life-threatening complication of patients  and has nfection . The facnfection . Data onnfection ,5, whilenfection ,6.In areas where malaria is endemic, a high number of children with severe anaemia but without detectable parasites are hospitalized each year coinciding with the peak of malaria transmission. The history of recent febrile illness and the presence of detectable circulating antigens strongly suggest that these children suffer from the late effects of a recent malaria attack . The quePatients with severe malarial anaemia have an increased ratio between the pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, and the anti-inflammatory cytokine, interleukin (IL)-10 , and it P. falciparum infection, two rapid tests for soluble malarial antigens, Now® ICT Malaria P.f/P.v  and Optimal®  were performed. Binax is based on detection of histidine rich protein 2 (HRP2) and aldolase and can remain positive for weeks following treatment of the infection [®, on the other hand, detects parasite derived lactate dehydrogenase (pLDH) and test results are positive only in the presence of live parasites [The purpose of the present study was to test the hypothesis that an impaired erythropoietic response to anaemia is associated with a persistent malarial infection in patients without microscopically detectable parasites. In order to differentiate between recent and persistent nfection . Optimalarasites .Red cell distribution width (RDW) and plasma levels of erythropoietin (EPO) and soluble transferrin receptor (sTfR) were measured as markers of erythropoiesis. RDW is a measure of size variation in red blood cells and is increased when erythropoiesis is stimulated . sTfR isThe study took place at the Department of Child Health, Korle Bu Teaching Hospital, Accra, Ghana during the malaria season, July and August, 2003. Two groups of children aged 0.5–12 years were consecutively recruited. Both groups consisted of children with a presumptive or confirmed diagnosis of recent malaria but without detectable parasites in Giemsa-stained blood films, examined by ordinary microscopy.Patients admitted to the emergency room with a presumptive diagnosis of acute malaria but with a negative microscopic test for malaria. The majority of these patients had received treatment for malaria either at home or in various health facilities prior to being referred to hospital. Those who had haemoglobin (Hb) ≤ 5 g/dl or Hb ≥ 8 g/dl were included, unless they had a history of a recent trauma or bleeding. Four hundred and five patients with the same age and sex distribution and with proven malaria by microscopy acted as a positive control group.Children who came for follow-up on day three after initiation of treatment for acute, uncomplicated falciparum malaria at the health clinic attached to the hospital and who had cleared their parasitaemia microscopically. Patients with febrile illness other than malaria, with severe malaria, concomitant infections or known chronic disease were excluded. The children had received amodiaquine, artesunate or a combination of the two as part of an ongoing drug trial.Children were enrolled after informed consent from parents or guardians. The ethics and protocol review committee at the University of Ghana Medical School had approved the study.group 1) and on day 0 (i.e. prior to inclusion), 3 and 7 (group 2). Blood films were considered to be negative if 200 leucocytes had been counted without finding parasites, corresponding to a detection level of approximately 50 parasites/μL. Antigen detection was performed on whole blood from day 0 (group 1) and day 3 (group 2). The rapid diagnostic tests, Now® ICT Malaria P.f/P.v  and Optimal® , were used according to the manufacturer's instruction.Blood was collected in EDTA-coated tubes and examined using an automated haematology analyser . Giemsa-stained blood films were prepared on day 0 .All data, except haemoglobin, were logarithmically transformed to achieve normal distribution. Differences between groups were analysed using two-tailed student's t-test, except differences in haemoglobin levels that were analysed by Mann Whitney two sample rank sum test. Associations between parameters were analysed by multiple regression analyses. P-values < 0.05 were considered statistically significant. All calculations were performed using Stata SE 8.0 group 1: 24 with severe anaemia and 24 with Hb ≥ 8 g/dl. Two (8.3 %) of the patients with severe anaemia and 15 (62.5 %) of those with Hb ≥ 8 g/dl had no detectable malaria antigens and were, thus, unlikely to suffer from malaria . On the day of inclusion (day 3) all had undetectable parasitaemia by microscopy but had a positive HRP-2 test. Only eight had signs of persistent parasitaemia as indicated by the pLDH test. In both groups, patients with persistent malaria tended to have lower Hb than those with recent malaria; although in group 1, the difference did not reach statistical significance  than in those with recent malaria or without signs of malaria . Persistent, microscopy negative malaria reduced the bone marrow response to the same extent as patent malaria. Thus, the slope of the regression line between RDW as the dependent and Hb as the independent variable was reduced to the same extent in microscopy negative patients with persistent malaria as in the 405 patients with a positive microscopy compared to patients without malaria  and those with negative pLDH,  in order to detect an effect of persistent P. falciparum infection did not affect the secretion of EPO  450.4 (291.5–609.3) pg/ml) and TNF-α (36.3 (25.7–51.3) pg/ml), but the levels were not correlated with Hb .The plasma levels of TNF-α and IL-10 were comparable to those in normal Ghanaian children without symptoms of malaria or detectable parasitaemia . There wChildren living in areas with endemic malaria are usually assumed to have malaria if they are hospitalized with severe anaemia that is preceded by an acute febrile illness. Such patients often have undetectable malaria parasites by microscopy of Giemsa-stained blood films, but their leukocytes commonly contain detectable malaria pigment . It has P. falciparum infection in vaccinated Aotus monkeys led to severe bone marrow suppression that was rapidly reversed in response to mefloquine induced parasite clearance [In the present study, patients with detectable pLDH, a sign of persistent parasitaemia, were distinguished from those in whom only HRP2 could be detected as a sign of a cleared, recent infection. Only patients with persistent parasitaemia had signs of bone marrow suppression. This corresponds with previous investigations in which release of immature erythrocytes from the bone marrow coincided with the time of parasite clearance ,6 and wilearance .P. falciparum infection and suppression of erythropoiesis has been debated. Menendez et al. found that levels of sTfR were increased in infants with malaria [P. falciparum infection have reduced Hb and signs of bone marrow suppression [P. falciparum infections are usually accepted as a necessary evil in order to maintain immunity in individuals, who are at great and constant risk of malarial infections [The association between  malaria . Similar malaria . However malaria . In the pression . Asymptofections , and thifections . The Hb fections , malariafections  and presfections . It is let al. unpublished data) compared with cerebral malaria and uncomplicated malaria. It is thus possible that the effect of cytokine perturbations in malarial anaemia is a lack of parasite control that leads to persistence of the infection and thus indirectly causes bone marrow suppression [Several studies have shown that the suppressed bone marrow response to anaemia, which is a general feature of malaria, is not caused by insufficient secretion of EPO ,25,26, bpression . It has pression .This study has shown that the majority of severe anaemia cases in children living in areas with malaria transmission is due to malaria. This is also the case when no malaria parasites can be detected by microscopy, in which case antigen detection may lead to the diagnosis. These patients have suppressed erythropoiesis, which persists until the parasites have been cleared completely.Marie Helleberg and Jorgen A. L. Kurtzhals designed the study in collaboration with all co-authors. Marie Helleberg, Bamenla Q. Goka, George Obeng-Adjei and Onike Rodriques performed the clinical work; Marie Helleberg and Bartholomew D. Akanmori did the laboratory analysis. Marie Helleberg did the data analysis and drafted the manuscript. All authors contributed significantly to the final version of the manuscript. The authors do not have any commercial or other association that might pose a conflict of interest.The study was supported by grants from the Danish Medical Research Council (SSVF grant no. 22-01-0343 and 22-03-0063) and the Danish International Development Assistance.


Mortality of falciparum malaria is related to the presence of severe complications. However, no scoring system is available to predict outcome of these patients. The aim of this paper was to devise a simple and reliable malaria prognosis score (MPS) to predict the outcome of adults with severe malaria.All slide-positive severe falciparum malaria patients admitted to Ispat General Hospital were studied. Eight clinical parameters that may potentially differentiate or influence the outcome were identified to predict recovery or deathOf 248 severe malaria cases, 35 died. There were 212 adults (34 deaths) and 36 children (one death). The malaria score for adults was (MSA) = 1(severe anaemia) + 2  + 3(Respiratory distress) +4 . The MSA ranges from 0 to 10. The mortality was 2% for MSA 0 – 2; 10% for MSA 3–4, 40% for MSA 5–6 and 90% for MSA 7 or more. The sensitivity is 89.9% and positive predictive value is 94.1% when 5 is taken as the cut off value.MSA is a simple and sensitive predictor. It can be administered rapidly and repeatedly to prognosticate the outcome of severe malaria in adults. It can help the treating doctor to assess the patient as well as to communicate to the relatives of the patients about prognosis. The score needs revalidation in other geographical areas. Falciparum malaria is a leading cause of mortality and morbidity in developing countries . Severe A simple scoring system is described to predict mortality in any severe and complicated malaria, based on available variables at the bedside with minimum laboratory parameters. The Malaria Score for adults (MSA) can be utilized at any time, and not as a "once only" predictor.The study was carried out at Ispat General Hospital, Rourkela, in the district of Sundergarh, in the northwestern part of State of Orissa, India. It is a 700 bed hospital, which caters to the medical needs of more than 500,000 populations. Being a large and well-equipped hospital, over the years it has established as a tertiary care center for seriously ill patients from the adjoining districts of Orissa, Jharkhand, Chhatisgarh, Madhya Pradesh and Bihar.This hospital-based study was conducted on patients over 12 years of age presenting with falciparum malaria who met the WHO criteria of severe and complicated falciparum malaria [Plasmodium falciparum in the peripheral blood smear, injection quinine dihydrochloride 10 mg/Kg body weight in 5% dextrose or in dextrose saline was infused over a period of 4 hours, every 8 hours, until the patient was able to take the drug orally. Oral quinine sulphate was given to complete the total duration of seven days therapy. Supportive measures like maintenance of hydration, antibiotics for any concurrent infections, blood transfusion, dialysis, ventilator support etc. were given according to individual needs. The patients who recovered and those who expired were grouped and the statistical analyses were performed [A detailed clinical evaluation was made with particular attention to neurological status, respiratory, renal condition and other complications. A sample of 5 ml heparinized venous blood was collected for determination of plasma glucose, urea, serum creatinine, bicarbonate, serum electrolytes, bilirubin, alanine aminotransferase, hemoglobin, and for a total and differential leukocyte count and a parasite count. Soon after confirmation of the presence of asexual forms of erformed .2) test with Fisher exact test where applicable; Odds ratio (OR) with 95% Confidence Interval. Those parameters which were significant were used for the multivariate analysis to get a linear discriminant equation showing the relationship between malaria score and the variables, i.e., a formula in the form of Malaria Prediction Score (MPS) = a + b1x1 + b2x2 + b3x3 + ... + bnxnThe clinical parameters associated with survival and deaths were compared by using) Chi-square  and xi = independent variables .where The study to develop the score included 248 severe malaria patients with 35 deaths. There were 36 children (one death) and 212 adults (34 deaths). All the variables were analysed  thus evolved was as follows:MPS = 2.13 + 0.02 × (Age) + 0.25 × (Creat) - 0.24 × (Hb)+ 3.05  + 0.8 (Pregnancy)+ 0.8(Ventilator)However, there were some practical problems in MPS : a) only one death from 36 children and b) the laboratory data for serum creatinine, bilirubin, haemoglobin and blood sugar must be available to get the MPS. This would delay in calculating the MPS, and thus it cannot be administered at the time of evaluation. Hence, the data from those patients aged above 12 years were analysed to develop the Malaria score to predict the outcome among adult patients with severe malaria. The clinical parameters of those who survived vs those who expired are given in Table So the score was accordingly modified (a) to include only the adults, and (b) the presence or absence of a clinical parameter was denoted as 1 or 0. Thus the malaria score for adults was (MSA) = 1(severe anaemia) + 2  + 3(Respiratory distress) + 4 . The MSA ranges from 0 to 10. The mortality was 2% for MSA 0 – 2; 10% for MSA 3–4, 40% for MSA 5–6 and 90% for MSA 7 or more. The sensitivity is 89.9%, specificity 70.6%, and positive predictive value is 94.1% when 5 is taken as the cut off value.6 ring forms, total leukocyte count > 15 × 109/l). This index is applicable only for the cerebral malaria cases, and there is no indicator for other forms of severe malaria. Moreover, each of the parameter was given a uniform weightage of 1. The positive predictive value of 4 or more of the above for an unsatisfactory outcome was 83% but sensitivity was only 66%.The clinical course of severe malaria is variable depending on the presence of one or several complications. The fatality rate of cerebral malaria, renal complications, acute lung injury is very high and they cannot be grouped together as similar to hypoglycaemia, hypotension, hyperpyrexia or seizure. It iscrucial to prognosticate the cases accordingly. As it was observed, the presence of haemolysis, hyperpyrexia, hypoglycaemia or hypotension alone was not a major factor; and not a single patient died due to any one of these. Moreover, combination if these also did not end with fatality. The prognostic indicators as suggested by Molyneux et al  for chilLewallen et al described the retinal findings to predict the outcome in cerebral malaria . PapilloP. falciparum parasitaemia. The definition of different degrees of severity of respiratory distress needs refinement. Many of the components of the clinical picture are subjective thus increasing the risk of inter observer variation [Marsh et al stressed the importance of respiratory complications as a simple bedside prognostic indicator in children with cerebral malaria . This coariation . In the Presence of multiple complications made the management difficult. Even in the tertiary care centers the management becomes challenging as a number of complications can appear at the time of admission or even during the course of hospitalization. To predict the survival of the index case is therefore very difficult and demanding from the relatives of the patient. A guarded opinion is invariably put forth, depending on the personal experience, intuition, clinical acumen of the physician; as no predictive scoring system yet available. The literature is scant about the relative risk of the complications in malaria In this situation, the severity classification APACHE-II  may be uIn some clinical situations, scoring methods are available for prediction of fatality (viz. burns , acute pAll the authors conceptualized the study, participated significantly in the analysis, drafting of the manuscript and writing the final version of the paper. SKM and RM contributed towards the mathematical modeling.