Plasmodium sp. infections. Some of the antibodies produced recognize self-components and are correlated with disease severity in P. falciparum malaria. However, it is not known whether some self-reactive antibodies produced during P. falciparum infection contribute to the events leading to cerebral malaria (CM). We show here a correlation between self-antibody responses to a human brain protein and high levels of circulating TNF alpha (TNFα), with the manifestation of CM in Gabonese children.Hypergammaglobulinemia and polyclonal B-cell activation commonly occur in P. falciparum cerebral malaria, we used a combination of quantitative immunoblotting and multivariate analysis to analyse correlation between the reactivity of circulating IgG with a human brain protein extract and TNFα concentrations in cohorts of uninfected controls (UI) and P. falciparum-infected Gabonese children developing uncomplicated malaria (UM), severe non-cerebral malaria (SNCM), or CM.To study the role of self-reactive antibodies associated to the development of P. falciparum infection may be associated with pathogenic mechanisms in patients developing CM.The repertoire of brain antigens recognized by plasma IgGs was more diverse in infected than in UI individuals. Anti-brain reactivity was significantly higher in the CM group than in the UM and SNCM groups. IgG self-reactivity to brain antigens was also correlated with plasma IgG levels and age. We found that 90% of CM patients displayed reactivity to a high-molecular mass band containing the spectrin non-erythroid alpha chain. Reactivity with this band was correlated with high TNFα concentrations in CM patients. These results strongly suggest that an antibody response to brain antigens induced by  Plasmodium falciparum who progress to severe malaria P. falciparum malaria, and CM displays the more acute clinical spectrum. In CM, the sequestration of mature P. falciparum-infected red blood cells to the cerebral endothelium is clearly a central event in the pathogenesis. However, the sequences of pathological events leading to fatal malaria in humans remain poorly characterized. Gaining an understanding of the mechanisms involved in CM will be crucial to the development of new preventive or curative therapies and/or vaccines, as well as to defining prognostic markers.Malaria remains one of the leading causes of death in most of sub-Saharan Africa, and mortality rates are particularly high for children under the age of five years, pregnant women and non-immune individuals P. falciparum infection in Kenyan children Plasmodium sp. Infections P. falciparum-infected patients P. falciparum-infected individuals suffering from severe anemia P. falciparum cerebral malaria.It has been recently shown that production of autoantibodies to brain voltage-gated calcium channels, but not other calcium channels, increased with severity of P. falciparum infection may depend on a fine balance between appropriate and inappropriate induction of theses immune regulatory factors.Another factor thought to influence clinical outcome of malaria is the fine balance between the pro- and anti-inflammatory cytokines, such as TNFα, IFNγ and IL-10 produced during the infection, that modulate parasite–induced immune responses Whether IFNγ, TNFα and IL-10 regulate the self-reactive antibody response during malaria, as is the case for primary biliary cirrhosis, remains to be shown P. falciparum malaria, and if so to identify the host molecules recognised, b) if there is a relationship between autoantibody levels and clinical symptoms, and c) whether self-reactive antibodies, in association with a defined pattern of circulating cytokines, are associated to the development of CM. We therefore recruited three groups of patients developing uncomplicated (UM), severe non-cerebral (SNCM) or cerebral malaria attending the Owendo Pediatric Hospital and Libreville Hospital Center in Gabon. We further investigated whether levels of circulating IFNγ, TNFα and IL-10 in these groups of patients were associated with the level of IgG self-reactivitive antibodies response.We wished to investigate a) whether autoantibodies directed against brain tissues occur in P. falciparum malaria: 1) uncomplicated malaria (UM), 2) severe non-cerebral malaria (SNCM) with severe anemia (hemoglobin level < 5 g/dl) or hypoglycemia (glycemia<2.2 mmol/ml), and 3) cerebral malaria (CM) with a Blantyre Coma Score inferior or equal to 2, or three convulsive episodes during the 24 hours before admission, with a post-critical comatose stated lasting>15 minutes. We also studied two control groups—the uninfected group (UI)—comprising healthy children with P. falciparum-negative thin blood smears, and European uninfected group (EUIC), who had never been exposed to malaria. All patients presenting diseases other than malaria were excluded from the study. All the UI subjects (18) were from the same area of Libreville City, and the patients in the UM (57), SNCM (24) and CM (14) groups were all recruited from the OPH and LHC hospitals. During the recruitment period, 7% (8/113) of the patients died in CM group. There was no significant difference in mean age and in sex ratio between the children in the various groups.Patients were included in the study only after informed consent had been obtained from their parents, after admission at the Owendo Pediatric Hospital (OPH) and Libreville Hospital Center (LHC) in Gabon, between 1996 and 1999. The ethics committee of the Gabon Health Office approved this study. Patients were assigned to the various groups on the basis of World Health Organization guidelines for the definition of uncomplicated and severe malaria. The children included in this study were aged between 2 months and 5 years, and fell into three groups for Patients were treated with amodiaquine or quinine, depending on disease severity. Children with uncomplicated malaria were given oral amodiaquine (25 mg/kg) for three days; those with severe malaria received intravenous quinine (25 mg/kg/day) for five days. Children with severe anemia underwent blood transfusion.P. falciparum on thin blood smears under a light microscope after staining with 10% Giemsa solution. Parasitemia is expressed as the percentage of infected red blood cells.Venous blood from each patient was collected into EDTA in sterile vacutainers on the day of hospitalization , at day-7 and day-30. Plasma was obtained by centrifuging blood samples at 5000 g for 15 min, and stored at −80°C until use. Parasitemia was assessed by counting asexual forms of Plasma total IgG of the patients were quantified by “Sandwich type ELISA” method as described in detail elsewhere Patterns of recognition of brain proteins by plasma IgG were detected by quantitative immunoblotting (PANAMA-blot method), using a protein extract from the brain of a healthy individual as the source of antigens as previously described Protein bands of interest were localised following migration on 10% acrylamide gels and staining with Coomassie blue. The proteins present in these bands were identified by peptide mass fingerprinting with a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS), Voyager DE-STR  2 test. The association between continuous quantitative parameters was assessed by linear regression, with the exception of correlations between two different types of parameters such as reactivity and cytokine profiles, which were tested by Spearman's rank correlation. P values<0.05 were considered significant.Immunoblot data were analyzed by multivariate statistical methods, using IGOR software , including specially written software packages. The standard migration scale was divided into sections around individual peaks of immunoreactivity. Section-wise absorbance values were subjected to principal component analysis (PCA), based on covariance calculation. For quantitative comparisons between groups, we used either Mann-Whitney (between two groups) or Kruskal-Wallis tests (>2 groups). Qualitative association was tested by Pearson'sχ2 = 11.2, p = 0.01). Thus, the number of brain antigens recognized by circulating IgG from P. falciparum-infected individuals increased with disease severity, with the sera of most UI subjects reacting with less than eight bands  before testing, so as to determine the proportion of total IgG accounted for autoantibodies. ht bands .We investigated the distribution of circulating IgG reactivities to brain proteins by carrying out a second quantitative immunoblot assay, using the same method as above but analyzing every plasma sample at a fixed dilution of 1/20 (non–adjusted assay). In this assay, we included 10 European children of similar ages who had never been exposed to malaria. We then analyzed the patterns of reactivity of the patients by principal component analysis (PCA), fitted to the Gabonese data. In PCA, the components are identified in decreasing order of importance. Thus, by definition, the first two components identified account for a large proportion of total reactivity. Factor 1 was characterized by overall positive factor loads . Factor 2All Groups = 10.4, p = 0.03)  appeared to be highest for CM patients, but no significant difference was found between the groups . The pro = 0.03) . RemarkaIn addition to the day 0 samples, we also analyzed samples taken from the same individuals  on days 7 and 30 after admission. The factor scores obtained for these samples were determined by projection, using factor loads calculated from day 0 data. Factor 1 scores increased significantly between days 0 and 7 in patients developing malaria whereas, on day 30, reactivity patterns were identical to those on day 7 for most of the children (data not shown). No such increase was observed in factor 2 scores.The reactivity to brain antigens represented by factor 1 scores was significantly correlated with age  in the non-adjusted assay, whereas no correlation was found between reactivity profiles, sex and parasitemia (data not shown). However, circulating IgG concentration was found to be significantly correlated with reactivity in all groups  except for uninfected controls (data not shown). These correlations with age and total IgG were less significant  for PCA calculated from the adjusted assay, previously used for the assessment of diversity, which may therefore better reflect intrinsic repertoire properties. Here, high factor 1 scores were observed only for patients with low total IgG concentrations . We assiP. falciparum infection.During the first twelve months of life, the plasma may contain maternal IgG. The differences between the three subgroups were indeed more pronounced when considering only children over the age of one year: subgroup α contained 89% of the UM and 79% of the SNCM patients but only 11% of the CM patients, whereas 78% of CM patients were found in subgroup β , and theProfiles of the reactivity of standard and patient plasma samples with brain proteins were separated into 33 sections on the standardized migration scale. PCA analysis revealed that reactivity with section 0 (band-0), corresponding to a set of high-molecular weight proteins, was the most informative . In the Proteins from human brain extract were separated on a 10% acrylamide gel. After Coomassie staining, three bands corresponding to reactive bands detected by Western blot were cut and their protein contents analyzed by peptide mass fingerprinting. In three independent experiments, band 1 corresponded to the beta chain of the non-erythroid spectrin isoform 2 while bands 2 and 3 to seven different isoforms of the alpha chain of the non-erythroid spectrin. In this latter case, it was not possible to distinguish if only one, several or all these isoforms were present in bands 2 and 3 .Plasma IL-10 concentrations were significantly lower in the UI group than in the UM (p = 0.002) and severe malaria groups (p<0.0001), but were similar in the UM, SNCM and CM groups (data not shown). IFNγ concentrations were highest in the UM and CM groups (data not shown). The differences between the CM and UI groups were statistically significant (p = 0.007), whereas no significant differences were observed between the UM, SNCM and CM groups. Plasma TNFα concentrations were significantly higher in the SNCM and CM groups than in the UM group . In addition, 75% of CM patients who died had a very high plasma level of TNFα  (data not shown).P. falciparum-infected patients. Plasma IFNγ and IL-10 concentrations were not associated with reactivity in the various groups. In the adjusted assay, reactivity to brain antigens, as measured by PCA factor 1 score, was positively correlated with TNFα concentration. This correlation was significant for children over the age of one year   (We assessed whether IgG autoantibody response and the type of disease were associated with cytokines concentrations in  = 0.02) . Factor = 0.002) . Reactiv= 0.002) .P. falciparum infection actually develops CM. Thus, it is difficult to recruit cohorts with sufficient numbers of patients meeting the CM case definition. Finally, the immuno-parasitological history of patients recruited in zones of moderate to high endemicity is generally difficult to establish, and thus the CM patients investigated might not be strictly homogeneous. Nonetheless, comparison of different cohorts with defined disease phenotypes has proven to be valuable in identifying factors specifically related to a particular pathology.In humans, detailed analyses of the events leading to CM and mortality from CM are restricted by ethical and practical considerations. First, sample collection is limited to peripheral blood, and post-mortem sampling is often unacceptable on cultural and religious grounds. Second, only a small proportion (ca. 1%) of the patients admitted with P. falciparum infection without apriori forming a hypothesis about the nature of the response. In this perspective, the PANAMA-blot method coupled to computer-assisted analysis was used to investigate the global repertoire of auto-antibodies reacting to brain proteins, the organ central to CM pathology. Using this strategy associating large scale antigen-antibody reactivities and multivariate statistical analyses, we assessed the relationship between self-reactive antibodies and CM pathogenesis.In contrast to previous studies analysing the immunoglobulin reactivity against one single antigen during malaria, in the current report we used a more global approach to analyze the response to P. falciparum-infected patients but not in UI or in EUIC controls. The size of the repertoire, in terms of the brain antigen diversity recognized by circulating IgG, increased significantly with disease severity. In addition, self-reactive serum IgG from 90% of CM patients displayed strong reactivity with the brain proteins found in band-0 of the Panama Blot. All the proteins in this region were demonstrated by MALDI-TOF analysis to be derived from the non-erythroid spectrin α and β chains, the α chain being more abundant. Non-erythroid spectrin is a major component of the cortical cytoskeleton of most eukaryotic cells. It forms heterodimers composed of α and β subunits P. berghei ANKA infected mice developing CM Our data showed high self-reactivity levels of IgG to brain antigens in the serum of We also observed that serum TNFα concentrations were correlated with brain antigen self-reactive antibodies (band-0 proteins). These concentrations were clearly higher in patients presenting severe malaria than in those admitted with uncomplicated malaria. Furthermore, the CM patients with the highest TNFα plasma concentrations (>100 pg/ml) also presented the highest levels of anti-brain reactivity. TNFα has been associated with pathogenic processes leading to CM The presence of self-reactive antibodies against non-cerebral antigens associated to malaria severity had been reported previously The function and origin of autoantibodies targeting brain protein observed in sera of CM patients is yet to be determined. In addition to the autoantibodies to α−ΙΙ spectrin, others reacting with CNS voltage-gated calcium channels were also found to be associated with an increased risk of developing severe disease in Kenyan children P. falciparum malaria may have distinct origins including polyclonal B-cell activation or the activation of specific B-cell clones. Parasite antigens and/or parasite-derived mitogens can lead to the non-specific polyclonal activation of B lymphocytes P. falciparum malaria patient cohorts. It is noteworthy that our results showed such differences, thus suggesting that the induction of a specific set of brain self-reactive IgG molecules was due to the activation of a selected repertoire. Several observations are consistent with this: 1) a single band was associated with strong anti-brain reactivity in CM patients, particularly those with low IgG concentrations, 2) high anti-brain reactivity was essentially observed in the adjusted assay, in which the effects of both age and IgG concentration were reduced, and 3) IgG concentrations were not correlated with anti-brain reactivity or parasitemia. Nonetheless, the two pathways of B-cell activation, mitogenic and antigen-specific, are not mutually exclusive in the malaria infection.Self-reactive antibodies to brain produced during 3) B-cell epitopes in the central nervous system triggers complement-mediated neuronal damage and contributes to the pathogenesis The hypothesis of a role played by autoantibodies to brain proteins in CM pathophysiology is in agreement with previous observations made for numerous autoimmune diseases involving the brain P. falciparum-induced defect or weakening of the natural regulation of homunculus autoreactivity An organized core autoimmune repertoire within the immune system has been termed the immunological homunculus Another mechanism which may explain the genesis of an autoimmune B cell repertoire to brain antigens linked to the neuropathology of malaria could be Fas-derived apoptosis during the course of the fatal disease, as it has been suggested for experimental cerebral malaria in macaques and mice P. falciparum malaria have antibodies to brain antigen in their plasma, may be the consequence of a cumulation number of sub-clinical brain damage resulting from previous episodes of severe non-cerebral malaria. Irreversible damage due to acute and chronic malaria episodes has been recorded in experimental infections of mice with PlasmodiumIn conclusion, the observation that children exposed to


Malaria is one of the most serious health problems in Kenya. In 2004, the Kenya Medical Research Institute and the US Army Medical Research Unit – Kenya surveyed adults in Samburu, Malindi, and Busia districts to determine socioeconomic risk factors for infection.Plasmodium falciparum parasites were observed in 100 high-powered fields. Univariate analysis was performed with Pearson's Chi-square test and univariate logistic regression. A multivariate logistic regression model was then created which included only variables found to be at least marginally significant in univariate analysis.Sociodemographic, health, and antimalarial data were collected along with blood for malaria testing. A smear was considered negative only if no P. falciparum were 1.7% in Samburu, 7.2% in Malindi and 22.3% in Busia. Interdistrict differences were statistically significant (p < 0.001) in univariate analysis and in a multivariate logistic regression model which included district, literacy, occupation, and recent illness as independent variables. In the model, literacy and recent diarrhoeal illness were positively and at least marginally significantly associated with parasitaemia . Neither age, sex, occupation, history of malaria in the previous three months, nor use of antimalarials in the previous four weeks were significantly associated with parasitaemia.A total of 1,141 subjects were recruited: 238 from Samburu, 442 from Malindi, and 461 from Busia. Smear positivities for While district of residence was the variable most highly predictive for parasitaemia among Kenyan adults surveyed, both a recent history of diarrhoeal illness and literacy were at least marginally statistically significant predictors. Malaria is one of the most serious health problems in Kenya. According to the World Health Organization (WHO), in 2002, there were over 124,000 reported cases of malaria nationwide in a population reported at just over 32 million  and by fBetween June and September 2004, researchers from the Kenya Medical Research Institute (KEMRI) and the United States Army Medical Research Unit – Kenya (USAMRU-K) carried out a community-based malaria parasitaemia and arboviral serologic survey of residents of three ecologically and culturally diverse Kenyan districts: Samburu, Malindi, and Busia, to assess potential socioeconomic risk factors for exposure to these pathogens. Extensive demographic, socioeconomic, health history, and recent antimalarial use data were collected along with blood for malaria smear and arboviral antibody testing. The malaria parasitaemia rates found in these three districts are reported here. Additionally, this study examines individual risk factors for parasitaemia, such as occupation, literacy, recent illness, health care utilization, and use of antimalarials.The study employed a population-based, cross-sectional design. Two villages were selected from each of the three districts under study. A two-stage cluster sampling technique was used to select villages for enrollment. The study population enrolled consisted of all consenting adults aged 18 years and above living in any of these six villages for at least five years. A standardized questionnaire was administered and a malaria smear taken at enrollment. This study was performed under a protocol approved by the KEMRI/National Ethical Review Committee.The districts of Samburu, Malindi and Busia were selected for their ecological and social diversity. Samburu is located in semi-arid northern Kenya, where malaria is mesoendemic, and is sparsely populated, mostly by semi-nomadic herdsmen. Malindi is an Indian Ocean coastal district, with endemic malaria and a population of mostly fishermen and agriculturalists. Busia is located in the Lake Victoria basin region on the Ugandan border. It is a center of cross-border commerce and trade, but also has a large agrarian population and is endemic for malaria.Kenya is divided into provinces, districts, subdistricts, divisions, locations, sublocations and villages. To obtain a representative sample of adults residing in the districts, a two-stage cluster sampling technique was used. A sampling frame was created for each district, from which two sublocations were selected using probability proportional to population size sampling without replacement . The secAt each of the selected sites, both the community leaders and the community were engaged prior to conducting the study. These leaders organized community meetings at which study staff explained the study and answered any questions posed in this open forum.Enrollment was conducted in community centers, such as schools or clinics, selected by local community leaders. These leaders verified that all potential subjects had been residents of the selected villages for at least five years. Before any potential subject was enrolled, a trained field worker explained the study in the local language and offered the opportunity for potential subjects to ask questions. Investigators were available to answer any questions posed. Witnessed informed consent was obtained if the potential subject demonstrated understanding of the study and was willing to enroll. In the case of an illiterate subject, a thumbprint was obtained on the consent forms and a separate Witness to Consent form was signed by a literate witness who had observed the consent process.The study questionnaire was developed to obtain demographic, socioeconomic, and health information. Local medical authorities participated in questionnaire design. The questionnaire was modeled partly on the Kenya Demographic and Health surveys: 1988–1989 , and 200P. falciparum parasites were observed in 100 high-powered fields. Subjects with positive smears were treated, free of charge, with an artemisinin-containing combination antimalarial, as recommended by the Kenyan Ministry of Health. This treatment constituted the study's direct benefit to the participants.Thick and thin smears were prepared and read on-site by the project's laboratory technicians. A smear was considered to be negative if no Questionnaire information included a categorical age variable , sex, literacy (defined as being able to read or write a simple line and being able to read a newspaper), and occupational group. Subjects reported whether or not they had been ill in the past three months and, if so, what illness they had . Additionally, subjects were asked to report whether they had used antimalarials in the past month and which antimalarials were taken. Malaria smear results were examined as a dichotomous variable (positive or negative).®  database and cross-checked for accuracy. Stata version 7 for Windows  was used for all statistical analysis. To obtain a valid estimate of associations between exposure variables and parasitaemia, univariate analysis was performed with Pearson's Chi-square test and univariate logistic regression. Mantel-Haenszel stratified relative odds (ROs) were calculated prior to further multivariate analysis to detect possible interaction between variables examined. As no interaction was detected, these results are not reported. A multivariate logistic regression model was then created which included only variables found to be at least marginally significant in univariate analysis , calculating maximum likelihood estimates of the adjusted ROs and 95% confidence intervals. If a categorical variable did not demonstrate a log-linear correlation with the outcome, dummy dichotomous variables were created and used in the logistic regression model. A p-value of <= 0.05 is considered statistically significant, while a p-value of > 0.05 and <= 0.10 is reported as marginally significant.Questionnaire and laboratory data were double-entered into a Microsoft AccessA total of 1,141 subjects were enrolled, representing 27.8% of the adult populations of the selected villages: 238 from Samburu, 442 from Malindi, and 461 from Busia. Of these, 731 (64.1%) were female and 410 (35.9%) were male. There was a marked difference in sex distribution in the different age ranges (p < 0.001), with younger males being less proportionally represented than younger females. However, there was no statistically significant difference in the age or sex distribution of subjects among the three districts . Of the 814 who answered questions about literacy, 322 (39.6%) were literate. Of the 1,110 subjects who responded to occupational questions, 585 (52.7%) were involved in agriculture, followed by home duties, which accounted for 167 (15.1%). No other occupational groups constituted more than 10% of the study population, so these other groups are not analysed separately.Malaria smear results are summarized in Table P falciparum. The rate of positives varied markedly by district (p < 0.001), with 1.7% positive in Samburu, 7.2% in Malindi, and 22.3% in Busia.Of the 1,141 subjects tested, 139 (12.2%) had a positive smear for Those who were literate had significantly higher parasitaemia rates than nonliterates with 13.7% of literates positive but only 6.9% of illiterates (p = 0.001). The rates of parasitaemia were lower in those engaged in home duties, at 2.4%, than those in agriculture, at 14.4%, or other employment areas, at 12.8% (p < 0.001). Neither age nor sex were associated with differences in parasitaemia rates .Those who reported either a malarial or a diarrhoeal illness in the past three months had significantly increased rates of parasitaemia . Of the 307 with a history of malaria, 15.3% were parasite positive on enrollment, while for the 45 reporting diarrhoea, the parasitaemia rate was 25.9%. The parasitaemia rate for those with other reported illness or no illness was 10.5%.Among those who had taken antimalarials in the past four weeks and those who had not, 13.3% and 11.9%, respectively, were smear-positive (p = 0.625). Analysis for each medication separately failed to demonstrate any statistically significant association with smear positivity (p = 0.603).Relative odds and 95% confidence intervals are presented in Table Overall, this study found an association between parasitaemia and district of residence, literacy, and a history of diarrhoeal illness in the previous three months.An interesting finding of this survey is that a history of diarrhoeal illness is a significant predictor for parasitaemia in Kenyan adults. The literature on the association of diarrhoea with malaria is somewhat conflicting and generally refers to the relationship between diarrhoea and acute malaria, while this survey correlates a history of diarrhoea in the past three months with a current parasitaemia. While diarrhoea is an often-cited symptom of malaria -24, therThis positive relationship between a history of diarrhoea in the past three months and a positive malaria smear reached only marginal statistical significance in the aggregate, but was consistent in the individual districts, making type I error an unlikely explanation for this association. There are certainly many possible explanations for this association. It is of considerable interest that self-reported diarrhoea was associated with smear-positivity, whereas self-reported malaria was not. In these semi-immune populations, it is quite possible for diarrhoea to be the predominant symptom of malaria. As a diarrhoeal illnesses may not be recognized as malaria, treatment for malaria may not be sought or provided. While this difference in obtaining antimalarial therapy could explain a higher risk of parasitaemia among those with self-reported diarrhoea than self-reported malaria, this study does not support this as the explanation. While 30% of those with a history of malaria report taking an antimalarial in the previous month, 29% of those with a history of diarrhoea also report doing so. This difference is not statistically significant (p = 1.000). However, both of these groups were more likely to have taken an antimalarial than those with other recent reported illnesses, of whom 14% took antimalarials  or those with no reported illness, of whom 6% took antimalarials . Of those with a history of either diarrhoea or malaria, those with a positive malaria smear were no more or less likely to have taken an antimalarial in the past month than those with a negative smear . There was no difference in the antimalarials taken by those with diarrhoea and those with malaria (p = 0.869). Further study of this association of diarrhea with parasitaemia would be useful in providing guidance for evaluation of diarrhoea in these populations and perhaps would prevent inappropriate antibiotic therapies from being administered.Literacy was another significant risk factor for parasitaemia in this study, even after controlling for potential confounding effects. This was a consistent finding in all three districts, although not reaching statistical significance for individual districts. This association cannot be attributed to occupation, recent antimalarial treatment, or other known factors. Literacy can directly impact health-seeking behaviors and also serves as a marker for other socioeconomic differences between individuals. It is plausible that differences in health-seeking behaviors or other socioeconomic factors would affect rates of malaria infection. However, literacy would have been anticipated to be protective against, not a risk factor for, infection as literacy and education are believed to be associated with more appropriate use of medical care -35. TherA clear finding of this study is that district of residence is the factor most strongly associated with differences in parasitaemia rates at the time of enrollment. Specifically, compared to those enrolled in Samburu, the subjects enrolled from Busia and, less so, Malindi, had higher odds of parasitaemia, even after adjustment for known potential confounders. The difference in ecologies of a semi-arid, a coastal, and a Lake Victoria basin district could well result in different disease transmission rates, particularly for vector-borne diseases -39. DistThere was no statistically significant association between history of antimalarial use in the past four weeks and the presence of parasitaemia on enrollment. This study did not have sufficient power to detect an association between specific antimalarial use and parasitaemia, even if such an association actually existed. Given an overall rate of 12.2% positivity, to find a 5% difference in malaria positivity with a power of 80% would require 826 subjects with a history of antimalarial use, a much higher number than the 187 enrolled.There are several further limitations of this study. First is the use of self-reported data, particularly in the area of recent health histories and antimalarial usage. As noted above, the low number of patients with a recent history of antimalarial use limits the study's ability to investigate antimalarial resistance. Also, as previously noted, the enumeration unit was the village, not the individual subject.While district of residence was the variable most highly predictive of parasitaemia among Kenyan adults surveyed, both a recent history of diarrhoeal illness and literacy were at least marginally statistically significant predictors. No other demographic or occupational variable, including age and sex, was associated with parasitaemia in these populations. Further study is required to determine the nature of these associations.RLC served as overall Principal Investigator on the study, involved in all aspects of protocol design and execution, data analysis, and manuscript preparation. TP and FO designed the sociodemographic questionnaire, supervised field workers, and assisted in manuscript preparation. VOO designed the laboratory system and supervised the reading of malaria slides. NA conceived the study, supervised field and data entry personnel, and provided significant contribution to the manuscript. All authors read and approved the final manuscript.


Zambia was the first African country to change national antimalarial treatment policy to artemisinin-based combination therapy – artemether-lumefantrine. An evaluation during the early implementation phase revealed low readiness of health facilities and health workers to deliver artemether-lumefantrine, and worryingly suboptimal treatment practices. Improvements in the case-management of uncomplicated malaria two years after the initial evaluation and three years after the change of policy in Zambia are reported.Data collected during the health facility surveys undertaken in 2004 and 2006 at all outpatient departments of government and mission facilities in four Zambian districts were analysed. The surveys were cross-sectional, using a range of quality of care assessment methods. The main outcome measures were changes in health facility and health worker readiness to deliver artemether-lumefantrine, and changes in case-management practices for children below five years of age presenting with uncomplicated malaria as defined by national guidelines.In 2004, 94 health facilities, 103 health workers and 944 consultations for children with uncomplicated malaria were evaluated. In 2006, 104 facilities, 135 health workers and 1125 consultations were evaluated using the same criteria of selection. Health facility and health worker readiness improved from 2004 to 2006: availability of artemether-lumefantrine from 51% (48/94) to 60% (62/104), presence of artemether-lumefantrine dosage wall charts from 20% (19/94) to 75% (78/104), possession of guidelines from 58% (60/103) to 92% (124/135), and provision of in-service training from 25% (26/103) to 41% (55/135). The proportions of children with uncomplicated malaria treated with artemether-lumefantrine also increased from 2004 to 2006: from 1% (6/527) to 27% (149/552) in children weighing 5 to 9 kg, and from 11% (42/394) to 42% (231/547) in children weighing 10 kg or more. In both weight groups and both years, 22% (441/2020) of children with uncomplicated malaria were not prescribed any antimalarial drug.Although significant improvements in malaria case-management have occurred over two years in Zambia, the quality of treatment provided at the point of care is not yet optimal. Strengthening weak health systems and improving the delivery of effective interventions should remain high priority in all countries implementing new treatment policies for malaria. By June 2006, 39 African countries had changed their policies to recommend artemisinin-based combination therapy (ACT) as the first line treatment for malaria  to reducOf these challenges, inadequate case-management practices are of particular concern. In health facilities across Africa, febrile children are often treated suboptimally -7, incorIn 2002, due to chloroquine resistance -15, ZambHealth workers' performance in treating febrile paediatric patients at government clinics in Zambia was reported when the new drug policy was in its nascent implementation phase . Here, aThe survey was undertaken in four districts purposively selected to represent major malaria ecologies in Zambia: Chingola, Kalomo, Chipata and Samfya . All govThe quality of antimalarial prescriptions, counseling and drug dispensing was studied using a cross sectional, cluster sample survey at all health facilities in four districts using similar methods in both the 2004 and 2006 surveys. A cluster was defined as all sick outpatients seen at a health facility during one working day. Each facility was randomly assigned to one survey day and all patients presenting to outpatient departments during the survey day were recruited. Here, case-management practices in children below five years of age were described, while data on patients five years and above will be presented elsewhere.Data were collected by four teams, each composed of two surveyors, using three methods: 1) exit interviews with caretakers, 2) health worker interviews, and 3) health facility assessments. Prior to the interviews, all caretakers and health workers were asked to provide written informed consent. For exit interviews, all caretakers of sick children were interviewed when they completed the health facility visit. Interviewers asked questions about the child's age, history of fever during the present illness, use of antimalarial drugs prior to the facility visit, and if the visit was an initial or follow-up consultation. Information was also collected from patient held records about routine diagnostic procedures requested and results reported, medications prescribed, and if the child was treated as an outpatient or referred for hospitalisation. For prescribed antimalarial drugs, interviewers assessed if the drug was dispensed, whether swallowing of the first dose was observed at the health facility, and if the caretaker received instructions on how to give the drugs at home. This information was obtained from caretaker interviews and referred not only to the consultation but also to any time during the facility visit. Finally, during the exit interview, each child was weighed and the axillary temperature was measured.Health worker interviews were conducted after working hours with all health workers who had attended sick children during the survey day. Health workers were asked about their demographics, pre-service training, working experience, supervisory visits, possession of guidelines, and exposure to in-service case-management trainings. Health workers' knowledge about recommended first-line treatment for various patient groups with uncomplicated malaria was assessed using open-ended questions.Finally, a health facility assessment was performed to record the availability of medical supplies and equipment related to malaria case-management. The presence of weighing scales, thermometers, malaria treatment wall charts and malaria diagnostics (microscopy and rapid diagnostic tests) was assessed. Particular emphasis was paid to the availability of antimalarial drugs on the day of the survey and stock-out periods in the past 12 months.Diagnosis definitions reflected recommendations from Zambian national guidelines ,19 and tAmbiguities exist regarding the role of malaria diagnostics in malaria case-management. The national malaria guideline states that the "presence of signs and symptoms of disease with negative blood smear does not preclude the diagnosis of malaria"  and simiRecommended treatment for uncomplicated malaria was defined according to national guidelines and training materials: artemether-lumefantrine for children weighing 10 kg or more and sulphadoxine-pyrimethamine for children less than 10 kg -23. FurtData were double-entered and verified in Microsoft Access and used STATA 8  for statistical analysis. Descriptive analysis was undertaken at health facility, health worker and child level. At health facility and health worker level chi-square test was used to test significant differences in proportions between 2004 and 2006 surveys. At child level the precision of proportions  was estimated accounting for the cluster sampling design using "health facility-day" as the primary sampling unit. Hypothesis testing and confidence interval estimation were done with an alpha level of 0.05.There were 94 facilities assessed in 2004 and 104 in 2006  programme in Zambia began to train health workers on the use of artemether-lumefantrine. The proportion of health workers receiving any form of training that included artemether-lumefantrine rose from 25% 26/103) in 2004 to 41% (55/135) two years later (P < 0.001) (Table 03 in 200During the 2004 survey 58% (60/103) of health workers had been provided with a draft version of new national guideline on malaria case-management. These guidelines were officially printed and distributed to health workers from August 2004 . FurtherDuring the 2006 survey, 1,498 children aged less than five years were evaluated as they left the clinic with their caretakers and were not referred for hospitalization. Four children were excluded from analysis because data were incomplete. Further 369 children were excluded because they presented for follow-up visit, had neither history of fever nor an axillary temperature of 37.5°C or more, or had a negative parasitological test. The influence of diagnostics on malaria diagnosis and treatment will be presented elsewhere. The remaining 1,125 children fulfilled our definition of uncomplicated malaria. Of these 1,125 children, 26 children weighed less than 5 kg, 552 were between 5 and 9 kg and 547 were 10 kg or more; comparable numbers of children were evaluated following the same selection criteria in 2004 .In 2004, during the early implementation of the treatment policy, artemether-lumefantrine was not recommended for children less than 10 kg; they were supposed to be treated with sulphadoxine-pyrimethamine. In October 2005, the Zambian Central Board of Health officially recommended the use of artemether-lumefantrine in patients weighing 5 to 9 kg ; and froThe treatment quality was first examined at all health facilities surveyed in 2004 and 2006, whether or not they had artemether-lumefantrine in stock  across both weight groups and both years (Table Of particular interest during the 2004 survey was the observation that only 22% (42/192) of children with uncomplicated malaria weighing 10 kg or more who presented to a facility where artemether-lumefantrine was in stock on the day of the survey received artemether-lumefantrine and 54% 103/192) of these children were prescribed sulphadoxine-pyrimethamine (table /192 of tThe correctness of prescribed artemether-lumefantrine dosages was assessed according to weight-specific criteria. The correctness was very high (81% to 96%) in all weight groups, except for children 15–24 kg because they tended to receive dosages designed for a lower weight category  had expired drugs. Ensuring adequate supplies of artemether-lumefantrine is a complex process in Zambia, critically dependent on the quantification of consumption for each of four different dose packages, processing of orders through districts to national stores, and subsequent prompt distribution of adequate quantities to districts and facilities. The effectiveness of the supply chain is further complicated with international procurement of artemether-lumefantrine, single source of the product and potential shortages of the drug on the global market. Further investigation of artemether-lumefantrine supply chain and strengthening of the drug distribution is an immediate priority in Zambia and probably in other countries implementing ACTs in Africa .Second, the implementation process of artemether-lumefantrine was launched in 2003 when there was little published data and no international guidance, on the use of this drug among patients weighing less than 10 kg. The national guidelines therefore recommended that artemether-lumefantrine be withheld from patient's weighing less than 10 kg and that sulphadoxine-pyrimethamine should be used in this weight group. This clearly posed problems in Zambia, where there was evidence that sulphadoxine-pyrimethamine failure rates were already high ,26. In JThe early introduction of new effective post-registration drugs into public health use is important when current therapies are failing to cure life threatening illnesses like malaria. However, the results of trials in special groups often emerge post-registration and national health officials need to respond to any changing recommendations. Changing and implementing revisions to a major drug policy, such as the treatment of malaria, is fraught with financing, coordination and human resource problems that can overwhelm the government agencies charged with this responsibility ,32. UpdaThe Zambian studies in 2004 and 2006 show that artemether-lumefantrine dosing was largely accurate among the two dominant weight group categories of 5 to 9 kg and 10 to 14 kg Table . Most ofThe studies reported here provide important information to measure the operational implementation of new drug policies. The Zambian experience as an early implementer of large-scale use of ACT offers valuable lessons for both further improvements in the national program and also for other countries in the region. Though improvements in health facility and health worker readiness and appropriate case-management occurred during the initial three year's experience, major areas of suboptimal performance still exist – especially in ensuring the effectiveness of the drug supply and management, improving effective translation of research into guidelines, and subsequently guidelines into clinical practice. Furthermore, repeated evaluation research needs to become a regular part of program implementation with consistent financial support so program and global policies can learn in a timely fashion from the efforts currently underway to mitigate the mortality and morbidity impacts of the malaria scourge in Africa.DZ, NS and RWS have received a fee for speaking at a meeting organized by Novartis Pharma AG, the manufacturers of artemether-lumefantrine. MN, PC, DHH and JLS declared no competing interest.DZ contributed to the conception and design of the study, analysis and interpretation of results, and drafting/finalisation of the manuscript.MN contributed to the conception and design of the study, analysis and interpretation of results, and drafting of the manuscript.NS contributed to data analysis and interpretation of results, and drafting of the manuscript.PC contributed to data analysis and interpretation of results, and drafting of the manuscript.DHH contributed to the study design, data analysis and interpretation of results, and drafting of the manuscript.JLS contributed to data analysis data analysis and interpretation of results, and drafting of the manuscript.RWS contributed to the study design, data analysis and interpretation of results, and drafting/finalisation of the manuscript.


There is a consensus among paediatricians that outcome of children presenting with life-threatening infections, irrespective of the infecting pathogen, can be improved by timely recognition and prompt intervention to correct disordered physiology using simple approaches to resuscitation –3. TheseMany children with severe malaria have signs of cardiovascular compromise or compensated shock on presentation to hospital and a smaller proportion are hypotensive . One of PLoS Clinical Trials [Recognising the importance of adequate fluid management to the outcome of the critically ill child, the group at Kilifi has conducted a staged series of studies over the last 15 years to address each of these questions. In a collaboration that included specialists in paediatric intensive care, neurology, and clinical trials, the group demonstrated the importance, prognostic implications , and cliGiven the continued controversy over the use of resuscitation fluids in the management of severe malaria, we suggest that this can only be resolved through a definitive clinical trial. Woodrow and Planche  have cho


Plasmodium falciparum. This grave complication involves malarial infection of the red blood cells that accumulate within the very small capillaries that flow through the tissues of the brain. Even when treated, cerebral malaria has a fatality rate of 15% or more.Malaria is one of the most serious of all tropical parasitic diseases: a severe and life-threatening form of which in humans is cerebral malaria, a complication that can occur in malaria caused by Numerous studies have pointed to a key role of tumor necrosis factor (TNF) and related proteins in the pathogenesis of cerebral malaria, and a clear relationship has been established between plasma concentrations of TNF and cerebral pathology. In experimental cerebral malaria, TNF-beta, now called lymphotoxin α (LT), has been shown to be a principal mediator of pathogenesis. LT and TNF belong to the same family, interact with a common receptor, and could act together during disease progression to effect the pathogenesis, according to recent evidence.Now Georges Grau and colleagues describe the anti-inflammatory activity of a transcriptional inhibitor of TNF, called LMP-420, which might offer a new way for treating cerebral malaria. The aim of their study was to assess the ability of LMP-420 to inhibit the in vitro TNF and/or LT effects on brain endothelium, with particular attention to endothelial cell activation, adhesiveness for malarial parasites, and vesiculation.P. falciparum–parasitized red blood cells, and endothelial MP release, three major features of cerebral malaria.Using an in vitro model of cerebral malaria based on human, brain-derived endothelial cells (HBEC-5i), they found that LMP-420 potently reduced endothelial activation, endothelial adhesiveness for P. falciparum–parasitized red blood cells on these endothelial cells. Identical but weaker effects were observed when LMP-420 was added with LT. LMP-420 also caused a dramatic reduction of HBEC-5i vesiculation induced by TNF or LT stimulation.The results provide evidence for a dual inhibitory effect of LMP-420 on both TNF and LT in an in vitro model of cerebral malaria pathogenesis, when added either before or simultaneously with both cytokines, they said. LMP-420 also abolished the cytoadherence of ICAM-1-specific Several molecules inhibiting TNF, such as monoclonal antibody to TNF or pentoxyfylline, have been tested in clinical trials of cerebral malaria but failed to improve disease outcome. These failures could be explained by the fact that LT was recently demonstrated to also have a crucial role in the pathogenesis of this cerebral syndrome, said the authors.They conclude that the anti-inflammatory activity of LMP-420 might be useful in targeting the wide variety of diseases in which TNF and its related family members play a role, and could represent a novel, stable, and efficient therapeutic way to improve the outcome of patients with cerebral malaria. However, the authors caution that the experimental in vitro results do not necessarily predict potential efficacy in either animal models or humans, especially since in their model the LMP-420 was administered before the disease process was established. Nevertheless, this avenue is a promising one to explore further.


The objectives were to evaluate the safety, reactogenicity, and allele-specific immunogenicity of the blood-stage malaria vaccine FMP1/AS02A in adults exposed to seasonal malaria and the impact of natural infection on vaccine-induced antibody levels.We conducted a randomized, double-blind, controlled phase I clinical trial.Plasmodium falciparum malaria.Bandiagara, Mali, West Africa, is a rural town with intense seasonal transmission of Forty healthy, malaria-experienced Malian adults aged 18–55 y were enrolled.42) from the 3D7 clone of P. falciparum, adjuvanted with AS02A. The control vaccine was a killed rabies virus vaccine (Imovax). Participants were randomized to receive either FMP1/AS02A or rabies vaccine at 0, 1, and 2 mo and were followed for 1 y.The FMP1/AS02A malaria vaccine is a 42-kDa recombinant protein based on the carboxy-terminal end of merozoite surface protein-1 . MSP-142 antibody levels followed a seasonal pattern that was significantly augmented and prolonged by the malaria vaccine. FMP1/AS02A was well tolerated and highly immunogenic in adults exposed to intense seasonal malaria transmission and elicited immune responses to genetically diverse parasite clones. Anti-MSP-1 Background: In sub-Saharan Africa the burden of death and disease from malaria is particularly severe. Most affected are young children under the age of five, in whom natural immunity against the malaria parasite has not yet developed. There are not yet any approved vaccines that would reduce this burden, although many research groups are currently developing potential vaccines. One such candidate vaccine is FMP1/AS02A. This vaccine is designed to trigger an immune response against a protein  found on the surface of the infectious, blood-stage form of the malaria parasite. Early-stage clinical trials have already been performed in healthy people in the United States, who were not exposed to clinical malaria, and in Kenyan adults who are exposed to malaria throughout the year. These studies did not identify any safety concerns regarding the candidate vaccine, which meant that it could progress further in clinical testing. As part of this next stage, a group of researchers wanted to examine the safety and ability of the vaccine to boost immune responses in an area of sub-Saharan Africa where people are not exposed to malaria throughout the year, but rather only in the wet season. The trial reported here was carried out in northeast Mali, in which 40 adults received either the FMP1/AS02A vaccine or a rabies vaccine for comparison, just at the start of the malaria transmission season. The researchers primarily looked at safety outcomes, collecting data on certain specific signs or symptoms up to 8 d after immunization, other reported symptoms up to 31 d after immunization, and any serious adverse events during a follow-up period of 364 d after immunization. The researchers also examined antibody levels in the participants' blood against the MSP-1 protein.What this trial shows: The researchers found that participants receiving the FMP1/AS02A vaccine had more immediate symptoms at the injection site  than the comparison group did. Other general symptoms, both solicited and unsolicited, such as headache, muscle aches, fever, and infections, were also more common in the malaria vaccine group than in the group receiving the rabies vaccine. There were two serious adverse events in the vaccine group, but these were not judged to be related to the vaccination. Antibody levels against the MSP-1 protein increased in both study groups through the course of the rainy season  and subsequently fell after the end of the malaria transmission season. However, participants receiving the vaccine had higher antibody responses at all timepoints measured; the differences were statistically significant at some timepoints, but not at others. Finally, the researchers looked at antibody reactions against three different variants of the MSP-1 protein in sera from participants receiving the candidate vaccine and found that the sera reacted similarly to all three variants.Strengths and limitations: The study protocol followed established procedures for phase I clinical trials of this type, which allows the data to be compared across studies. Randomization procedures were appropriate, and steps were taken to blind participants in the trial, as well as those assessing outcomes, to the intervention participants received. A limitation of this study, which can apply to other phase I studies in general, is that small numbers of participants were recruited. Therefore, the trial was not powered to detect statistically significant differences between participant groups. It is also not clear whether the higher antibody levels seen in the participants receiving the FMP1/AS02A vaccine would be biologically significant , a question that would need to be addressed in further trials.Contribution to the evidence: The safety results from this study are similar to those from other trials and confirm that no safety concerns have thus far been identified regarding the FMP1/AS02A vaccine, which has now progressed to efficacy testing. This study was also conducted in a population exposed to seasonal malaria, whereas previous trials had been done among people exposed to malaria year-round. Finally, results from the trial also suggest that this vaccine induces antibodies that recognize genetically diverse forms of the vaccine antigen. Plasmodium falciparum malaria would prevent severe disease and death in African children and other at-risk populations. Merozoite surface protein-1 (MSP-1) is a 195-kDa antigen found on the surface of P. falciparum merozoites. It is essential for merozoite invasion of erythrocytes, after first being processed to yield a carboxy-terminal 42 kDa fragment (MSP-142) that is further cleaved into the MSP-133 and MSP-119 fragments, as reviewed in [An effective vaccine directed against the blood stages of 42 from the 3D7 clone of P. falciparum produced in and purified from Escherichia coli [. Reconstituted with the AS02A adjuvant, an oil-in-water formulation containing the immunostimulants monophosphoryl lipid A and QS21, it constitutes the FMP1/AS02A malaria vaccine. At the time of this study, this vaccine had been evaluated in two clinical trials evaluating a total of 60 malaria-naïve North American adults  consists of recombinant MSP-1hia coli . Reconst42 (including subdomains) by enzyme-linked immunosorbent assay (ELISA), and measurably inhibited parasite growth. Kenyan adults exposed to intense year-round malaria transmission had high and variable background levels of anti-MSP-142 antibodies, and differences in antibody levels between malaria vaccine and control groups were not significant at any post-immunization timepoint; however, significant differences in antibody levels were measured when a longitudinal model was applied [The vaccine was highly immunogenic in malaria-naïve volunteers, inducing antibodies that recognized parasites by indirect fluorescent antibody (IFA), were reactive against recombinant fragments of MSP-1 applied .42 alleles and subunits were measured.The safety, immunogenicity, and efficacy of malaria vaccines may be affected by the intensity and pattern of local malaria transmission, which determines levels of pre-existing natural immunity and potential natural “boosting” of the immune response to vaccines, and may affect the prevalence of different allelic parasite types. Western Kenya and Bandiagara, Mali, represent two sites with very different transmission patterns, both representative of other malaria-endemic areas where a vaccine would eventually be offered. We assessed the FMP1/AS02A malaria vaccine compared to rabies vaccine in adults living in Bandiagara, Mali, where malaria is highly seasonal and naturally acquired immunity is lower than in western Kenya. To investigate the specificity of vaccine-induced antibody responses, antibodies to diverse MSP-1Anopheles gambiae is the principal malaria vector. Malaria transmission is strictly seasonal, with virtually undetectable transmission at the height of the dry season in March, less than one infected bite per person per month at the start and end of the transmission season in June and December, respectively, and peaks of up to 40–60 infected mosquito bites per person per month in August or September [P. falciparum represents 97% of malaria infections, with 3% due to P. malariae and rare infections with P. ovale. Despite the seasonal transmission pattern, the malaria burden is heavy: each year, children aged under 10 y old have an average of two clinical malaria episodes every transmission season [The Bandiagara Malaria Project research clinic is adjacent to the district hospital in Bandiagara, a rural town of 13,634 inhabitants in the Dogon Country in northeast Mali. It is relatively dry, with a mean annual rainfall of 600 mm in 2002. eptember . P. falcn season , and sevAfter obtaining community permission as described by Diallo et al. , the triP. falciparum and 17 non-MSP-1 amino acids encoding a 6-histidine tag plus linking sequence fused to its amino terminus. The antigen was expressed as a fusion protein in E. coli with the 6-histidine tag to facilitate purification. The clinical grade antigen was manufactured according to current Good Manufacturing Practices (cGMP) at the Walter Reed Army Institute of Research Pilot Bioproduction facility . The FMP1 protein met all purity, identity, and safety standards set for product release. After purification, less than 2 ng of host cell protein was detected per 50-μg dose. FMP1 protein was immunoreactive with several disulfide-dependent conformational monoclonal antibodies (mAbs), including growth inhibitory mAbs 12.10 and 12.8, implying correct structural conformation [The FMP1 antigen consists of the 42-kDa carboxy-terminal 392 amino acids of MSP-1 based on the sequence of the 3D7 clone of Quillaja saponaria [The AS02A adjuvant is composed of an oil-in-water emulsion and two immunostimulants, 3-deacylated monophosphoryl lipid A and QS21, a saponin agent derived from the soap bark tree, aponaria ,12. AS02The Imovax rabies vaccine  is a sterile preparation of killed rabies virus supplied in single-dose vials containing lyophilized antigen to which 1 mL of sterile water is added as a diluent before injection. Vaccines were administered by intramuscular injection in the left deltoid muscle.Forty adults were randomized in a 1:1 ratio to receive either FMP1/AS02A or the control rabies vaccine. Vaccines were given on a 0-, 1-, and 2-mo schedule. The first immunization was given in early July 2003, just as malaria transmission began; the second dose was given at the end of July, as transmission was increasing; and the third dose was given in late August, near the peak of transmission intensity. Study day 90 was in October, shortly after transmission crests and when severe and uncomplicated malaria episodes peak; study day 180 was at the end of the malaria season; and study day 272 was in the middle of the dry season. The final study follow-up on day 364 coincided with the beginning of the 2004 malaria season. Interim safety reports were reviewed by an independent safety monitoring committee before the second and third immunizations.42 and its subunits.The primary objective was to evaluate the safety and reactogenicity of the FMP1/AS02A malaria vaccine in malaria-experienced Malian adults. Secondary objectives were to evaluate the humoral immune response of the vaccine in malaria-experienced Malian adults in a setting of intense seasonal malaria transmission, to determine the impact of natural infection on malaria vaccine-induced antibody responses, and to measure antibody responses to genetically diverse forms of MSP-142 3D7 and its subunits and two genetically different parasite strains , at baseline and at specified times during and after immunization.The primary outcome was safety, measured as (1) occurrence of solicited symptoms during an 8-d follow-up period after immunization ; (2) occurrence of unsolicited symptoms during a 31-d follow-up period after immunization (day of immunization and 30 subsequent days); (3) occurrence of laboratory toxicities during the study period; and (4) occurrence of serious adverse events during the study period. Secondary outcomes were anti-MSP-1 antibody titers measured against recombinant MSP-1Following each immunization, participants were directly observed for 30 min, then evaluated at the study clinic 1, 2, 3, 7, and 14 d after immunization and on study days 90, 180, 272, and 364. Starting on day 180, monthly home visits were made to check the health status of participants and to encourage them to come to the research clinic if they felt ill. Study physicians were available at all times throughout the 12-mo study to assess and treat adverse events.Clinical evaluations consisted of measurement of vital signs and assessment for local injection site and general solicited signs or symptoms. Local signs and solicited symptoms included pain, swelling, erythema at the injection site, and limitation of arm abduction at the shoulder. General signs and solicited symptoms included fever , chills, nausea, headache, malaise, myalgia, and joint pain. Any other signs or symptoms were considered to be unsolicited, as were signs or symptoms that occurred more than 7 d after immunization. Solicited symptoms were considered to be related to the study vaccines. Unsolicited signs and symptoms were recorded during the 30 d after each immunization, whereas serious adverse events were monitored throughout the 12-mo study.Blood was collected at screening, on immunization days, 14 d after each immunization, and on study days 90, 180, 272, and 364 to determine complete blood count, alanine aminotransferase (ALT), and serum creatinine.Adverse events were graded by severity and judged for relatedness to study vaccines. Mild adverse events were easily tolerated, causing minimal discomfort and not interfering with daily activities. Moderate adverse events were sufficiently discomforting to interfere with normal activities. Severe adverse events prevented normal daily activities. Swelling, erythema, fever, and limitation of arm motion had specific definitions not based on interference with daily activities. Injection site swelling and erythema were graded based on their widest dimension: mild, >0–20 mm; moderate, >20–50 mm; and severe, >50 mm. Fever was classified as severe if the oral temperature was ≥39 °C , whereas severe limitation of arm motion was classified as abduction limited to 30°. For laboratory tests, toxicity grading was adapted to normal reference ranges determined for the local adult population.42 were measured by ELISA [42 [42, recombinant MSP-142 of the 3D7 and FVO alleles were prepared as described previously [42 of the Camp/FUP allele will be described elsewhere.Antibody responses to MSP-1by ELISA . BrieflyLISA [42  was usedeviously ,13. Prep42 corresponding to MSP-119  and the two epidermal growth factor (EGF)-like domains that comprise MSP-119, EGF1 . Codes were assigned in the order that participants arrived at the clinic on the day of first immunization.The only people at the study site with access to the randomization codes during the study were two study pharmacists, who had no contact with study participants and did not reveal vaccine assignments to anyone else. Study participants and investigators who assessed outcomes were blinded to vaccine assignment. Vaccines were prepared in a secure room communicating with the vaccine administration room through a small window with a sliding door. Reconstituted FMP1/AS02A is off-white and Imovax is pink. To reduce potential bias, syringes containing the vaccines were wrapped with opaque tape to conceal their contents from participants and vaccinators. Vaccines were administered by physicians who did not participate in assessing outcomes.The protocol was approved by institutional review boards of the University of Bamako Faculty of Medicine, the University of Maryland, the United States Army Surgeon General, and the National Institute of Allergy and Infectious Disease. Separate written informed consent was obtained for screening and for enrollment. Consent of illiterate participants was documented by their thumbprints and by signatures of independent witnesses. Permission to conduct the study was granted by the Republic of Mali Ministry of Health, and the trial was monitored by the United States Army Medical Materiel Development Activity and the World Health Organization.42 antibody titers were estimated by using log10-transformed values, calculating the 95% CI based on the normal distribution, and then converting the limits to the original scale for presentation. All tests were two-sided, and no correction of p-values was made for additional analyses. MSP-142 fragment-specific antibody responses were measured longitudinally and are reported as the median slope of the regression lines for responses for the malaria vaccine and control groups. Comparisons between groups were made using Mann–Whitney tests or Student's t test of log10-transformed antibody titers.Adverse event rates were analyzed using SAS version 8.2 . Fisher's exact test was used to compare rates between vaccine groups. Confidence intervals (CIs) for geometric mean MSP-1Of 108 screened adults, 40 were deemed eligible and enrolled in July 2003 . The maiAfter each immunization, the proportion of participants who had at least one local injection site reaction during the 8-d post-immunization periods was not significantly different between vaccine groups. Pain and swelling at the injection site were the most common local reactions for both groups and tended to be more common in the FMP1/AS02A group and after the first immunization .p = 0.001). No other severe reactions were observed. Severe local swelling was less common after the second and third immunizations and was similar in frequency between groups. Severe injection site swelling occurred in 13 FMP1/AS02A recipients across all immunizations  and in eight rabies vaccine recipients across all immunizations   . The swep = 0.02). Headache was the most common general adverse event in FMP1/AS02A recipients after each immunization, followed by myalgia and malaise . These were not associated with other clinical abnormalities or illness and resolved without intervention. Two participants in the control group had elevated ALT levels. One was a moderate elevation to 131 U/L detected during an acute malaria episode on day 272 that returned to normal by day 364. The second was a severe elevation to 359 U/L unaccompanied by symptoms or signs of hepatitis or other illness that was detected on day 74 and resolved by day 272. Serological tests for hepatitis A, B, and C were negative. The participant had been treated with erythromycin for a respiratory infection and was given a diagnosis of drug-induced hepatitis.Hemoglobin levels remained within or slightly above the normal range for all participants throughout the study . Mild abnormalities in white blood cell and platelet counts were infrequent and balanced by group.42 antibodies rose over the course of the malaria season, increasing more than 2-fold by day 60 and peaking at nearly 3-fold above baseline responses on day 180 as the malaria season ended (p < 0.001), and at day 180, they were 29,150 and 9,089 (p = 0.016). By the final follow-up timepoint 1 y after immunization, although titers had waned in both groups, they remained significantly higher in the FMP1/AS02A group .In the rabies vaccine group, anti-MSP-1on ended ; Table 342 titer was <3,000 in the six participants with an 8-fold or greater rise in titer, but >20,000 in those with a <2-fold rise.Individual responses to the vaccine varied. Of the 20 participants who received FMP1/AS02A, six developed very high antibody titers, with an 8-fold or greater rise in day-90 antibody titers compared to baseline; six had a 4- to 7-fold rise; four had a 2- to 3-fold rise; and four had a <2-fold rise. Much of this individual variation can be explained by the variation in background immunity at the start of the malaria transmission season: the baseline geometric mean MSP-142, i.e., days 74 and 90. Sera collected on study day 0 were analyzed to provide paired baseline data. Responses were measured by ELISA against three alleles of MSP-142  and subunit fragments of MSP-142 from both 3D7 and FVO . Antibody responses induced by FMP1/AS02A against the three alleles did not differ significantly . For all comparisons, slopes for anti-MSP-142 and subunit antibodies were significantly higher in the FMP1/AS02A group than in the rabies control group  in which protective efficacy is demonstrated. For this small phase I trial in malaria-experienced adults, we chose to use MSP-142 antibody titers as the immunogenicity endpoint to inform clinical development decisions.While cellular immune responses, antibody avidity assays, growth inhibition assays, and other potential alternative surrogate markers may eventually prove more predictive of clinical protection than MSP-142 antibody titers in the control group, which rose nearly 3-fold over the malaria season. The difference in MSP-142 antibody levels between the FMP1/AS02A and rabies vaccine groups was much greater in this trial than in a virtually identical trial conducted in an area of intense year-round malaria transmission in Kenya [42 and MSP-119, as measured by the slopes of the regression lines fitted using data from day 0, 74, and 90, were not different between the two study sites (data not shown).Immunizing at the start of the of the malaria transmission season permitted observation of the effect of natural boosting on MSP-1in Kenya . The samin Kenya . This co19, and the most divergent in MSP-133). Longitudinal antibody responses showed a tendency toward stronger reactivity against the 3D7 and Camp/FUP alleles than against the FVO allele, which is consistent with the greater degree of homology between 3D7 and Camp/FUP than between 3D7 and FVO. Whether these in vitro allele-specific analyses predict in vivo efficacy against diverse parasites is not yet known.The vaccine induced responses against all alleles tested: 3D7 (homologous to the vaccine antigen), Camp/FUP (different in three amino acids in the second EFG-like domain), and FVO Trial ProtocolClick here for additional data file.(376 KB PDF)


Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/−). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions.Erythrocyte Duffy blood group negativity reaches fixation in African populations where p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/− vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype.We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/−) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, in vivo observations provide evidence that Pv-targeted control measures can be developed safely.Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our  P. vivax (Pv), P. falciparum (Pf), P. malariae (Pm) and P. ovale (Po), and endemic in regions below 1600 meters elevation Plasmodium species (mixed infections) are common, and there is growing interest in the potential that cross-species regulatory mechanisms control parasitemia and malarial disease In PNG, malaria is caused by Plasmodium species interactions observed previously in Vanuatu led Williams et al. +-thalassemia predisposes young children to Pv infection and that early exposure to this more “benign” species reduced the risk of severe falciparum malaria. Under this hypothesis host genetic adaptations that reduce the risk of Pv would not be favored to arise in regions that were co-endemic for Pf.Ministry of Health statistics show that malaria represents the most significant public health burden in PNG nullFY*B (B−) allele on the FY*A allele nullFY*A allele (A−) and no homozygous negative (A−/A−) individuals in vitro assays, erythrocytes from A+/A− donors (compared to A+/A+ donors) formed significantly fewer rosettes when mixed with COS-7 cell transfectants expressing the Pv Duffy binding protein in vivo while not affecting the risk of Pf infections.It was surprising then that our earlier studies in PNG discovered the same single nucleotide polymorphism (SNP) in the Duffy gene promoter observed on the African Duffy negative We conducted two sets of serial cross-sectional surveys to estimate malaria prevalence in the Wosera, East Sepik Province, Papua New Guinea. The first set of seven prevalence surveys was conducted monthly from July 1998 to January 1999 in six villages in the Wosera (cumulative population 1845), where the Papua New Guinea Institute of Medical Research (PNGIMR) has performed numerous malaria epidemiologic surveys Plasmodium species blood-stage infections to Duffy genotype.A second set of four malaria prevalence surveys was conducted at approximately 6-month intervals from August 2001 to June 2003 in 29 villages of the Wosera Ethical approval for these studies and procedures for oral/written informed consent were obtained from the Medical Research and Advisory Council of Papua New Guinea, the Institutional Review Board for Human Investigation of Case Western Reserve University and University Hospitals of Cleveland, OH, and the National Institute of Allergy and Infectious Diseases, International Clinical Studies Review Committee (ICSRC).+-EDTA Vacutainer™ tubes, and stored at −20°C until DNA extraction could be performed. DNA was extracted from whole blood (200 µL) using QIAamp 96 spin blood kits .Samples were collected in KPlasmodium parasite species was performed by a post-PCR ligase detection reaction/fluroescent microsphere assay (LDR-FMA) Thick/thin smears were prepared by standard procedures, stained with 4% Giemsa and examined by PNGIMR-trained microscopists under oil-immersion (100×). Parasite species and species-specific densities were identified and recorded while counting the number of microscope fields containing 200 leukocytes as described previously FY) corrupts the GATA-1 transcription factor binding site regulating erythroid Duffy antigen expression specifically (−33 T→C a and Fyb antigenic variation. Combinations of these two SNP lead to FY*A (A+), nullFY*A (A−), FY*B (B+) and nullFY*B (B−) alleles A single nucleotide polymorphism (SNP) in the Duffy gene ( −33 T→C . A seconFY gene was PCR amplified as previously described FY+ (−33T) and 65 bp FY− (−33C) allelic fragments using the restriction endonuclease StyI following recommended protocol . Restriction fragments were separated by electrophoresis on 4% 1xTBE agarose gels , stained with a 1∶10,000 dilution of SYBR® Gold , and visualized on a Storm® 860 Scanner . Genotyping assays for the FYA vs FYB SNP SLC4A1Δ27) GYPCΔex3) α−thalassemia were performed using methods previously described.A 329 bp fragment inclusive of the GATA-1 SNP in the Plasmodium species by Duffy genotype. To address this aim, we compared the incidence rates of blood-stage infection (estimated as infections per 100 person-months of observation) among children who were A+/A− with children who were A+/A+ using the rate ratio and 95% confidence intervals The primary aim of the analysis was to compare the susceptibility to Pv blood-stage infection according to Duffy genotype. Further analyses compared susceptibility to all other We performed all statistical analyses using SAS® software .Plasmodium species parasites in recent surveys was similar to that reported previously Plasmodium species infections were observed in 2.4% (LM) or 16.8% (LDR-FMA) of individuals From surveys conducted since July 1998, our primary objective was to investigate the relationship between the Duffy A− allele and susceptibility to Pv blood-stage infection. For these studies we performed genotyping on DNA samples from 9,092 of the 13,000 Wosera residents (69.9%) to characterize the distribution and frequency of the A− allele and investigate whether this new allele alters susceptibility to Pv and thereby confers some fitness advantage. We identified 223 Duffy A+/A− individuals and no A−/A− (Duffy-negative) individuals . The distribution of A+/A− individuals was not random within the Wosera villages and the proportion of A− carriers within the villages ranged from 0 to 13.0%. Within the community, overall infection prevalence by the four human in vivo susceptibility to blood-stage Pv infection in vivax-hyperendemic PNG. To test this hypothesis, we analyzed data from two different epidemiologic studies in the Wosera.Our study setting in PNG is therefore distinct from African populations. In comparison to vivax-hypoendemic Africa where Duffy negative (B−/B−) homozygosity predominates and confers complete resistance to Pv erythrocyte infection, reduced Duffy erythrocyte antigen expression in PNG heterozygotes (A+/A−) may significantly reduce natural p = 0.026; hazard ratio 0.336, 95% CI: 0.198, 0.901; p = 0.875; hazard ratio 1.051, 95% CI: 0.471, 2.420; p = 0.16).First, in an unmatched longitudinal cohort analysis (July 1998 to January 1999), we compared the rate of Pv blood-stage infection in 11 A+/A−  and 456 A+/A+  children under 15 years of age who attended at least four of the monthly malaria prevalence surveys. Overall we found that the rate of Pv infection determined by conventional LM analysis was 52.0% lower in A+/A− compared with A+/A+ children : 0.24, 0.96; 12.1 vs. 25.2 infections per 100 person months of observation). When we estimated the proportion of children who escaped Pv blood-stage infection during the survey period, we found that a greater proportion of A+/A− heterozygotes escaped Pv infection (72.7%) during the 7-month survey compared with A+/A+ homozygotes (30.6%)  who attended at least four of the 1998–1999 cross-sectional malaria prevalence surveys. In a longitudinal analysis, we observed that A+/A− compared to A+/A+ was associated with a risk reduction of 46.0%  was greater than the proportion of A+/A+ children (23.6%)  compared to A+/A+ individuals (µ = 2.96) (Mann-Whitney U: p = 0.023) (p<0.0001). These results suggest that the A+/A− genotype confers a level of protection against Pv blood stage infection that would otherwise be attributable to acquired Pv-specific immunity. Interestingly, this difference in mean Pv parasitemia conferred by A+/A− was no longer observed in individuals over 15 years of age . Finally, when these studies were performed following a real-time quantitative PCR strategy to estimate Pv parasitemia, results between molecular diagnostic strategies (LDR-FMA to RTQ-PCR) were highly concordant , and comparisons between Pv susceptibility, Duffy genotype and age were identical to those reported above. In an analysis of repeated measures using generalized estimating equations to estimate the impact of the Duffy genotype on the mean Pv fluorescent signal intensity and adjust for Pf co-infection and correlations over time, we found that children with the A+/A− genotype had a 1.25-fold lower mean Pv parasite density based on LDR-FMA than children with the A+/A+ genotype (p = 0.017). These findings are consistent with our evaluations on infection prevalence and infection intensity showing that a reduced risk of Pv blood-stage infection was associated with the A+/A− genotype.Recognizing that our observations in the initial set of surveys were based on a limited number of heterozygous children, we compared the intensity of Pv infections by age  in a sub= 0.023)  This difp = 0.742; 15 years and older–2.51 vs. 2.69, Mann-Whitney-U: p = 0.648). Similar to results for Pv parasitemia, we did observe that older individuals had lower Pf parasitemia than younger individuals. Our results suggest, therefore, that while we observed a reduction in Pv parasitemia in Duffy A+/A− compared to A+/A+ individuals, we did not observe that a reciprocal increase in Pf parasitemia was associated with Duffy A+/A−.Because some studies have suggested that Pv infection reduces the severity of Pf infection, we were interested in determining whether Duffy genotype influenced susceptibility to Pf blood stage infection. In additional comparisons between children under and 15 years and older individuals , we obseP. falciparum infection and therefore reduced fitness. In contrast to this expectation the Duffy A− allele has been consistently inherited over at least four generations within the Wosera community, and the frequency of this new PNG allele has risen above 6.5% within some villages. These observations imply that Duffy A− confers some selective advantage within the population. As it is clear that vivax malaria can cause significant morbidity Whether these results offer insight into events of human evolution responsible for fixation of the Duffy B− allele in African populations would be difficult to substantiate. What is intriguing about our observations in a region of PNG endemic for both Pv and Pf is the juxtaposition between a significant reduction in Pv risk in A+/A− children and proposals that Pv infection reduces severity of falciparum malaria It is important to acknowledge that we have yet to identify a homozygous Duffy negative (A−/A−) individual in PNG and, based on a review of the Wosera Demographic Surveillance database, we have not yet identified parental partners who are both A+/A− heterozygous. Thus, it is difficult to predict, based on allele frequency, when we should expect to identify Duffy negative individuals in the Wosera. This data alone, however, cannot be used to rule out the possibility that the A−/A− homozygous genotype might be associated with significant negative fitness and constrain the Duffy A− allele to the role of a balanced polymorphism When assessing the potential of Pv as a cause of human genetic adaptation, it is important to remember that previous characterization of Pv as a “benign” human malaria parasite are based upon data collected from adults receiving malaria therapy


Data suggest that antibody responses to malaria parasites merozoite antigens are generally short-lived and this has implications for serological studies and malaria vaccine designs. However, precise data on the kinetics of these responses is lacking.Plasmodium falciparum merozoite antigens  among Kenyan children were monitored using ELISA for 12 weeks after an acute episode of malaria and their half-lives estimated using an exponential decay model.IgG1 and IgG3 responses to five recombinant The responses peaked mainly at week 1 and then decayed rapidly to very low levels within 6 weeks. Estimation of the half-lives of 40 IgG1 responses yielded a mean half-life of 9.8 days (95% CI: 7.6 – 12.0) while for 16 IgG3 responses it was 6.1 days (95% CI: 3.7 – 8.4), periods that are shorter than those normally described for the catabolic half-life of these antibody subclasses.This study indicates antibodies against merozoite antigens have very short half-lives and this has to be taken into account when designing serological studies and vaccines based on the antigens. An effective malaria vaccine is urgently needed, but to date it remains elusive. A common way of trying to establish if a given malaria parasite antigen is a good candidate for a malaria vaccine is by determining if an with protection against subsequent infections of malaria. However, a number of studies suggest that naturally acquired responses to malaria merozoite antigens are short-lived. Among the majority of people living in endemic areas, levels of antibodies to merozoite antigens appear to vary with the levels of malaria transmission i.e. they are highest during periods of intense transmission and lowest or undetectable at the end of periods of low transmission -3. FurthPlasmodium falciparum merozoite antigens among Kenyan children recovering from a clinical infection of malaria and the data used to estimate the half-life of the responses. The results of the study indicated that both IgG1 and IgG3 antibodies to merozoite antigens have very short half-lives.The implication of these observations is important as they suggests that during serological surveys, individuals who can nonetheless mount a rapid secondary antibody response to malaria antigens upon re-infection are likely to be classified as antibody negative depending on how recent their last malaria infection was. Conversely, individuals who are positive at the survey may be negative by the time they encounter the next infection. If indeed the antibodies responses are very brief, then data from longitudinal studies with long intervals between sampling days will not reflect well the dynamics of the responses. Unfortunately, estimates of the half-lives of antibody responses to malaria that can help guide the design of such studies are lacking. In this study, a closely spaced sampling schedule was used to monitor the kinetics of antibody responses to five recombinant g for 5 min to obtain plasma, which was stored at 20°C. The children were examined by a clinician and a thick malaria film prepared during the follow-up visits or any other time during the study when they were unwell. Malaria treatment (SP) was given for parasitaemia in the presence of fever (axillary temperature ≥ 37.5°C). Seven children from whom weeks 1 and 2 samples could not be obtained were considered lost to follow up, so the cohort for analysis comprised 41 children.This study was carried out at Kilifi District Hospital (KDH) on the Kenyan coast. Ethical clearance for the study was given by the Kenya Medical Research Institute ethics review board. Forty eight children admitted to the pediatric ward of KDH with a primary diagnosis of malaria, but who did not fulfill the World Health Organization criteria for severe malaria , were reP. falciparum apical merozoite antigen 1(AMA-1), the 11 kDa carboxyl portion of merozoite surface antigen 1 (MSP-119), region II of the 175 kDa erythrocytes binding antigen (EBA-175 RII), and two recombinant proteins representing the two major allelic types of MSP-2 was assessed in plasma samples from 41 children . Levels of IgM reactivity against the two allelic types of MSP-2 were also assessed. The AMA-1 antigen and EBA-175 region II were kind gifts from Sheetij Dutta and Arnoldo Barbosa  and have been previously described t are concentrations at time 0 and t respectively, k is a rate constant and plateau is the concentration at the end of the decay and half-life of the decay (t1/2) is calculated as follows:Where [C]Association between kinetics half-life of the responses and age of responder or peak level of antibodies at the beginning of the decay was assessed by linear regression using Stata ver. 8 , while differences between half-lives of response to the various test antigens was assessed using one-way ANOVA. In addition, ANOVA was used to test if the mean level of responses to the test antigens differed between individuals. The profiles were then grouped together depending on whether or not they had peaked before the child presented to hospital, and the mean half-lives of the groups compared using a student t-test.19 in subjects 23, 36, 38, 40, 41,47, 48 and 56, Figure There was considerable variation in the children's overall pattern of responses to the five test antigens but for each antigen, the children generally responded in one of three ways. Either, (1) they already had moderate to high levels of antibodies to the antigen by the time they presented to hospital , but not always . Only in 10 of the 43 individual antigen-specific boosting instances were IgG1 responses boosted to similar or higher levels than the initial peak . There was no association between half-life and age , peak level of antibody , or antigen specificity . The half-lives of the responses did not differ significantly between different individuals . The mean half-life of the IgG3 responses analysed was lower  than that of IgG1 but not significantly . As with IgG1, there was no association between half-life and age , peak levels of antibody , or antigen specificities   (8.9 days [95% CI: 5.5–10.8]) but the difference was not significant (P = 0.17). The mean half-life of IgG3 responses that peaked before presentation to hospital (n = 5) was 7.3 days (95% CI: 5.0 9.5), while that of the responses that peak post presentation (n = 10) was 8.1 days (95% CI: 4.8 – 11.5), a small difference that was not significant (P = 0.685)The profiles were grouped depending on whether or not they had peaked by the time the child was presented to hospital and compared their mean half-lives. IgG1 profiles which peaked before presentation to hospital (n = 8) had a longer mean half-life (13.1 days [95% CI: 8.2 – 21.4]) than those that peaked after presentation .Data from 11 children where responses to two or more antigens were analysed for half-life was used to determine the correlation between the half-life of responses to different antigens within the same child. A high level of correlation between responses to different antigens by the same child was observed  was similar to that of antibodies injected into healthy adults (8 days) [Estimating the half-life of antibody responses to malaria antigens among residents of malaria endemic areas is difficult as the kinetics of the antibodies are continually altered by reinfection. In this study, eventual boosting of the initial responses was evident in many of the children even in the absence of patent re-parasitization. As such, half-lives could only be estimated for those responses that showed no obvious boosting over at least four time points. However, it is still possible that the half-life estimates obtained may still have been subtly affected by sub-patent of re-parasitization. In addition the half-life of the some of the profiles that were not analysed because they did not meet the inclusion criteria may have been different from those estimates obtained but it was not possible to clarify this. As such, a future study is being planned that will include a group of children who are kept parasite-free over study period by chemoprophylaxis. Nevertheless, this study shows that among Kenyan children recovering from clinical malaria, antibody responses to (8 days) , that ofP. vivax merozoite surface protein 1 (MSP-119) during a clinical episode of malaria became sero-negative within two months of treatment [P. falciparum MSP1 and rhoptry associated protein 1(RAP-1) in Sudanese individuals often last for less than three months after treatment for clinical malaria [Such short half-lives could explain observations from previous longitudinal studies that reported a rapid decline of antibodies to malaria antigens within a few months of malaria treatment. For instance, 44% of Brazilian individuals who made antibodies to the C-terminal fragment of reatment . Similar malaria ,14.Data on the half-lives of natural responses to other infectious agents such as bacteria are generally lacking and it is not possible to say if the short-lives observed here are unique to malaria or common to acute infection by different pathogens. Future studies will have to incorporate patients with other conditions for comparison. On the other hand, antibodies responses to viral infections and vaccines such as measles, appear to last for many years ,16 althoAlthough the half-life of IgG1 is usually cited as 21 days , early sSince in natural responses, the kinetics of antibodies is a balance between production and decay the profiles observed in this study suggest that production of antibody against merozoite antigens is not sustained following an acute episode of malaria. Antibody production can be sustained through re-stimulation of memory B cells by persistent antigens ,26 or byInterestingly, in many instances, re-parasitization after the episode did not boost the initial responses or only did so mildly. While polymorphism in the new infection might explain some of the failure, the fact that boosting occurred in many instances but only to a lower level than the initial responses suggests that there may be a period of suppression of antibody responses following a clinical episode of malaria. Reduced reactivity to viral vaccines during malaria episodes and recovery of reactivity following malaria treatment has been reported among Nigerian children .This study shows that following an acute episode of malaria in children living in an endemic area, antibody responses to malaria merozoite often have very short half-lives; less than those reported for normal antibody clearance. This has implication for studies monitoring responses to these antigens and indicates the need for closely spaced sampling within the first few weeks of an episode. Further studies on the fate of antibody producing cells following a malaria episode are required in order to elucidate the mechanism underlying this brevity. In addition, it is important to establish whether the brevity of the responses is due to malaria disease processes or whether it is inherent to the antigens. Would vaccines based on the antigens also induce short-lived responses in healthy children, and would this compromise their use, or would memory B cell populations be adequate for a sufficient antibody response upon infection?The author(s) declare that they have no competing interests.SMK carried out longitudinal sampling, the ELISA and prepared the manuscript DJC developed the ELISA and helped in the manuscript preparation DEL participated in the preparation of the antigens used in the study KM helped in designing the study and setting up of the longitudinal framework