It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.P. falciparum blood stage antigens AMA1, MSP1-19, MSP2  and MSP3 . Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h2) estimates for 48% (20/42) of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested) among the adults, 48% (20/42) among the children in the dry season and 31% (13/42) among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20) of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.This study assessed the relative heritability of different plasma antibody isotypes and subclasses  naturally acquired to Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation. Genetic variation among individuals profoundly affects susceptibility to clinical malaria, although only a minority of the causal polymorphisms are known h2) An estimate of the heritability of a phenotypic trait can be derived by a Classical Twin Study (CTS) Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) were influenced by non-HLA genes P. falciparum antigens, with the apparent contribution of HLA to this being variable among assays and antigens tested P. falciparum antigens to be generally non-HLA-linked An early study of twins in Liberia indicated that antibody levels to the P. falciparum blood stage antigens which are considered to be vaccine candidates. The study employed samples from Gambian adult and child twins that were previously assayed for IgG and IgM with a small number of antigens. The aim was to estimate and compare the heritabilities of all the antibody isotype responses, to test whether these heritability estimates varied according to whether children were sampled during the peak or minimal malaria transmission periods, and to evaluate the influence of HLA class II and non-HLA genes.The basis of clinically relevant differences between individuals in antibody class and IgG subclass response polarisation needs to be investigated. For example, cytophilic IgG1 and IgG3 antibodies enable phagocytosis The study of genetics of immune responses to malaria in the adult and child twins was reviewed and approved by the MRC Gambia Scientific Co-ordinating Committee and the Gambia Government/MRC Joint Ethics Committee . At the time of approval in 1991, and during subsequent recruitment in 1991–1993, verbal informed consent was the standard practice for observational studies in The Gambia, reflecting the low literacy rate in the general population at that time, a practice guided by over 40 years of medical research experience which was found culturally appropriate and acceptable to observational study subjects. The purpose of the study was explained to potential participants in local languages and all subjects or both of their parents gave verbal informed consent. More recently, literacy rates have increased substantially and written informed consent has become standard practice for research, as incorporated in 2000 into the guidelines for the Gambia Government/MRC Joint Ethics Committee. The proposal for investigation of antibody isotypes in the samples presented here was further reviewed and approved by both the MRC Gambia Scientific Co-ordinating Committee and the Gambia Government/MRC Joint Ethics Committee in 2006.The plasma samples were prepared from heparinised blood samples (between 5 and 10 ml) collected from subjects living in malaria endemic areas of The Gambia between 1991 and 1993, and consist of three groups. The first group comprises 213 pairs of adult twins (58 monozygous and 155 dizygous) with a mean age of 27.0 years (range 14–92 years) sampled between January 1992 and May 1993. The second group comprises 199 pairs of child twins (32 monozygous and 167 dizygous) with a mean age of 5.0 years (range 1–10 years) sampled at the end of an annual dry season in April – May 1991 when there was minimal or no malaria transmission. The third group comprises 107 pairs of child twins (30 monozygous and 77 dizygous) with a mean age of 5.7 years (range 1–10 years) sampled at the end of the wet season (October - November 1991) when malaria incidence was highest. The children in the third group (wet season sample) were a subset of those in the second group (dry season sample). There were no significant differences between the mean ages of MZ and DZ twins for any of the three groups. The study sites and populations are described previously, when more limited analyses of antibody reactivities to malaria antigens were investigated Escherichia coli expressed recombinant proteins. The apical membrane antigen 1 (AMA1) recombinant protein represented the full length ectodomain containing amino acids 83–531 of the AMA1 sequence in the 3D7 clone The antigens used for assays were P. falciparum blood stage antigens AMA1, MSP1-19, MSP2-ch150/9 (MSP2A), MSP2-Dd2 (MSP2B), MSP3-3D7 (MSP3A) and MSP3-K1 (MSP3B), following protocols similar to those previously described 2CO3, 35 mM NaHCO3, pH 9.3), incubated at 4°C overnight and then washed 4 times with washing buffer (PBS with 0.05% Tween 20). Unoccupied binding sites were blocked with 200 µl per well of blocking buffer  for 5 hours at room temperature. Plates were washed 4 times prior to the addition of 100 µl plasma diluted with blocking buffer , these dilutions were chosen after optimisation of assays for each isotype. After incubation at 4°C overnight the plates were washed 4 times, and 100 µl of horseradish peroxidase-conjugated anti-human IgG subclasses , anti-IgM , anti-IgA  at 1/5000, or anti-IgE  at 1/2000 was added to each well and incubated at room temperature for 3 hours. Plates were washed 4 times and antibody reactivities detected with 100 µl of substrate  in developing buffer . The reaction was stopped with 50 µl of 2 M H2SO4 and optical density (OD) of plates measured at 490 nm. The OD reading was corrected for non-specific binding by subtracting the OD reactivity to the maltose-binding protein (MBP) fusion tag for MSP3, and glutathione s-transferase (GST) fusion tag for MSP1 and MSP2 recombinant proteins. All assays were performed with each plasma sample tested in duplicate wells, and the mean OD value determined. Each pair of twin plasma was run on the same plate to avoid plate-to-plate variations in measurements.Enzyme-linked immunosorbent assays (ELISAs) were used to detect plasma antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE to the panel of Antibody data generated by ELISA in the form of OD values were log transformed after adding a value of 1, and transferred from Excel spreadsheets to SPSS version 11  and GraphPad Prism version 4.0  for analysis. Pairwise correlations using Pearson's correlation coefficient were performed for MZ and DZ twins separately. The Mx Graphical User interface [GUI] version 1.3.65  The zygosities of the adult and child twins had been ascertained using minisatellite typing with 5 minisatellite probes, and HLA typing performed using Southern blot hybridisation with radiolabelled HLA-DRB and HLA-DQB probes, as described in previous studies on the samples P. falciparum blood stage antigens, was measured for all 1038 plasma samples from the adult and child twins. The analyses on heritability are shown separately from each of the three different studies, on the 213 adult twin pairs in The set of seven antibody isotypes and subclasses against the panel of six h2 values being above 0.5 for responses to all antigens by the child twins in both dry and wet seasons, indicating that IgG4 responses are most strongly genetically regulated . Significantly lower heritability in adults was also seen for IgG1 (P = 0.046 compared with children in the wet season) and IgG2 (P = 0.046 compared with children in the dry season). Heritability of IgE levels was significantly lower in the children sampled in the wet season than in the adults or the children sampled in the dry season .The significance of variation in HLA class II genes and non-HLA-associated genes to the antibody isotype responses was tested in the adult twin data. The correlations within HLA identical and non-identical DZ twin pairs were compared to test for the contribution of HLA class II genes, whilst the correlations within HLA identical MZ and DZ twin pairs were compared to show the contribution of non-HLA genes. Of the 20 antigen-specific isotype responses with significant overall heritabilities in the adults, 15 (75%) were significantly non-HLA restricted, whereas none showed significant HLA class II restriction .This study shows that there are differences in the heritabilities of different antibody isotype and subclass responses to malaria in children, with IgG4 having higher heritability than the other isotypes. There were no significant differences between heritabilities of different antibody isotype levels in the adults, and heritability of IgG4 levels was significantly lower in adults than in children recruited either in the dry season when malaria incidence is low or the wet season when incidence is at an annual peak.Reasons for higher heritability of IgG4 responses can be considered in the light of different pathways of antibody isotype production. The IgG4 subclass is in relatively low levels in sera, and its production is influenced by IL-4 and IL-13 in human B cells, as also occurs with IgE The genetic regulation of all antigen-specific isotype responses is shown to be mainly by non-HLA genes, as expected. An earlier analysis of IgG levels to parts of MSP1 and MSP2 had surprisingly not shown the contribution of non-HLA genes to be statistically significant IL-4 gene promoter region (-590C/T), considered to influence the transcriptional levels of IL-4IL-4 -590T allele is associated with higher levels of IgE among severe malaria patients P. falciparum specific IgE reactivity in the Fulani population, although not in another ethnic group A genome-wide approach is required for the discovery of the genetic determinants of the different antibody isotype responses, as our knowledge of candidate genes remains very limited In summary, this study has shown that genetic factors regulate the different antibody isotype and subclass responses to malaria antigens, mainly due to undefined non-HLA-linked genes. Identification of the genes responsible should be possible by conducting large genome-wide association studies of malaria-exposed individuals with different antibody isotype response profiles


Plasmodium falciparum malaria episodes may vary considerably in their severity and clinical manifestations. There is good evidence that host genetic factors contribute to this variability. To date, most genetic studies aiming at the identification of these genes have used a case/control study design for severe malaria, exploring specific candidate genes. Here, we performed a family-based genetic study of falciparum malaria related phenotypes in two independent longitudinal survey cohorts, as a first step towards the identification of genes and mechanisms involved in the outcome of infection. We studied two Senegalese villages, Dielmo and Ndiop that differ in ethnicity, malaria transmission and endemicity. We performed genome-scan linkage analysis of several malaria-related phenotypes both during clinical attacks and asymptomatic infection. We show evidence for a strong genetic contribution to both the number of clinical falciparum malaria attacks and the asymptomatic parasite density. The asymptomatic parasite density showed linkage to chromosome 5q31 , confirming previous findings in other studies. Suggestive linkage values were also obtained at three additional chromosome regions: the number of clinical malaria attacks on chromosome 5p15  and 13q13 , and the maximum parasite density during asymptomatic infection on chromosome 12q21 . While regions of linkage show little overlap with genes known to be involved in severe malaria, the four regions appear to overlap with regions linked to asthma or atopy related traits, suggesting that common immune related pathways may be involved. Plasmodium falciparum. Severe malaria episodes may vary considerably in the nature of clinical manifestations, and in the event of death, its precise cause. Indeed, death may result from severe anaemia, cerebral malaria, acidosis or multiple organ failure, or a combination of these P. falciparum infection may be an indirect cause of death, which results from e.g. concomitant septicaemia. The contribution of host genetic factors to the risk of severe outcome following infection has long been recognized, with Haldane reporting for the first time a major role of one genetic variant in the β-globin gene, the sickle cell mutation (HbS), in the protection against severe malaria Most malaria deaths occur as a consequence of complications following infection with vs. mild dichotomy is complex, because the mild malaria group may include patients who have not developed severe complications and yet are fully susceptible to severe malaria.Most of the genes that have been explored and reported to date encode red blood cell proteins, proteins involved in the immune response to infection, or in other pathophysiological mechanisms directly relevant to malaria infection. These genes have been identified through case/control association studies, comparing severe malaria to uncomplicated malaria cases. Such a study design is limited in four aspects: 1) it has been restricted to the examination of specific candidate genes, based on their presumed functional relevance to malaria; 2) it provides little understanding of the causal role of specific genes and variants; 3) there is considerable variability among studies, depending on the disease selection criteria, population background and environmental context, which may result in poor reproducibility; 4) the severe P. falciparum antigens) has been reported in four types of studies: concordance studies in monozygotic versus dizygotic twins Evidence for a contribution of host genetic factors to phenotypes related to mild clinical malaria , 11% Mandinka and 10% miscellaneous, whereas in Ndiop, there were 76% Wolof, 19% Fulani and 5% miscellaneous.P. falciparum (acronym PFA). This phenotype characterizes the individual tendency to become clinically ill following P. falciparum infection; 2) The maximum and mean P. falciparum trophozoite parasite densities (i.e. pathogenic parasite stage infecting red blood cells) during clinical episodes . Maximum density was chosen to assess whether there were individual human differences in tolerance of parasite burden prior to onset of clinical symptoms. Because the parasite density eliciting a clinical reaction may also vary according to the parasite genotype, we considered a second related phenotype, the mean density; 3) The prevalence of asymptomatic P. falciparum infection, which reflects the acquisition of clinical immunity and/or the tolerance of parasitic infection (acronym PtPF). The acquisition of non-sterilising clinical immunity occurs with repeated exposure to the parasite. However, the rate of acquisition may also be influenced by human genetic differences independently of exposure; 4) The mean and maximum P. falciparum parasite densities during asymptomatic infection (acronym me-tPFD and mx-tPFD). These phenotypes are equivalent to those of category 2, but emphasising the ability of the individual (and human-parasite interaction) to tolerate parasite density without clinical reactivity.For each person, the following malaria-related phenotypes were considered: 1) The number of clinical episodes of P. falciparum attacks (PFA) showed a significant positive correlation with the maximum parasite density during clinical attacks (mx-PFD) in both villages. It showed a moderate negative correlation with the mean and maximum parasite density during asymptomatic P. falciparum infection  in both villages, before correction for multiple testing.For each malaria phenotype, we first performed statistical analyses of the data using multivariate regression analysis to take into account non-genetic factors such as age, year of study and transmission intensity see . The uneP. falciparum clinical attacks (PFA), the mean of parasite density during clinical attacks (me-PFD), prevalence of asymptomatic infection (PtPF) in both villages, the mean and the maximum  asymptomatic parasite density. The mean asymptomatic parasite density (me-tPFD) was also studied in both villages despite the fact that it did not show a significant heritability in Ndiop, in order to enable comparison with previous studies We selected phenotypes that showed significant heritability for further linkage studies , namely:TNF, NM 000594; ICAM1, NM_000201; CD36, NM 001001547, NM 001001548 and NM 000072; CR1, NM 000651 and NM 000573), (ii) their high frequency of mutations known to be common in malaria endemic region and suspected to be protective  (iii) their role as a receptor for malaria antigens (e.g. the glycophorins) and (iv) their immunological response to malaria infection . An average information content of 0.68 (SD±0.12) was achieved at any point of the genome.Genome scan was carried out using 400 markers with average interval of 10 cM . In addition, we typed 66 markers localised near candidate genes selected from studies of severe malaria (marker list available on request). These candidate genes were selected according to (i) their previous proposed association with severe malaria  using SOLAR program P. falciparum attacks (PFA), linkage was detected in two chromosome regions in Dielmo: on chromosome 5p (near marker D5S419), with LOD scores of 2.57  and 2.81  using VCM and Regress respectively (p = 0.0014) and 2.65  using VCM and Regress respectively  with the mean transformed parasite density (me-tPFD) in Dielmo with LOD scores of 2.26  and 2.4   and 2.27   did not reach significance in Ndiop, genome scan linkage analysis revealed a LOD score of 2.08  by VCM and LOD = 1.06  by regression analysis in the region of the β-globin cluster on chromosome 11p (For asymptomatic Regress) . In NdioRegress) . It is isome 11p .p = 0.005 in Dielmo (near marker D2S168) and LOD = 1.75, empirical p = 0.0009 in Ndiop (near marker D2S319) by VCM in SOLAR and LOD = 2.16, empirical p = 0.0009 (near marker D2S319) when combining the 2 villages. LOD scores were similar with the maximum asymptomatic parasite density (mx-tPFD). This was not, however, reproduced when analysing with MERLIN that can not take into account large complex family structures.Interestingly, there was linkage to the same region of chromosome 2p with asymptomatic infection that was replicated in both villages with LOD scores >1. For mean asymptomatic parasite density (me-tPFD), we obtained LOD = 1.68, empirical P. falciparum malaria in both villages. With this study design, we were able to control for two major confounding factors of malaria infection rates, namely anti-malarial intake P. falciparum clinical attacks, P. falciparum density during clinical attacks and during asymptomatic infection. Based on genome-wide linkage analysis, suggestive evidence of linkage of these traits was detected in four chromosomal regions, including three novel ones.The duration of the survey, which is the longest active case follow-up period to date for a genetic study of malaria P. falciparum clinical attacks (PFA). The heritability observed confirms previous observations in cohorts of Kenyan children P. falciparum density in Kenya P. falciparum asymptomatic infection (PtPF) and maximal density of P. falciparum during asymptomatic infection (mx-tPFD). This suggests a genetic contribution to the control of peripheral blood parasitaemia. The equivalent measures for clinical episodes, PFA and mx-PFD, were positively associated with each other in both villages. This is consistent with the interpretation that the subjects experiencing many P. falciparum clinical attacks have a poor capacity to control parasite density, which frequently reaches the high threshold density necessary to elicit a clinical attack In both villages, we found evidence for a strong genetic contribution to the residual number of P. falciparum asymptomatic parasite density that has been previously reported in three independent studies Plasmodium chabaudi, where one quantitative trait locus for resistance to malaria has been mapped in homologous regions of human 5q31-q33 IL12B (NM_002187) seems to play a critical role since it has been associated with some immune-related diseases. An insertion/deletion polymorphism in the promoter region of IL12B has been reported to be associated with psoriasis We replicated the finding of linkage of the chromosome 5q31 region to ICAM-kilifi variant with PFA and other malaria-related traits in these villages P. falciparum infection in our study may reflect in part the constraints imposed by either approach, limited by the study of only selected candidate genes in previous studies, and limited in the power to detect small effects in our study. Moreover, variants responsible for susceptibility or protection against severe malaria, and that have a strong individual effect, may have a limited impact at the population level because of low frequency With the exception of the β-globin locus, where we found suggestive evidence for linkage to some of the selected traits, there was no overlap of the regions of linkage that we detected and the location of the genes that have been previously reported to be associated with severe/cerebral malaria. We also did not find any evidence for linkage and association of the Overall, genome scan linkage analyses showed peaks with higher LOD scores in Dielmo than in Ndiop. One reason for this is that the higher transmission intensity in Dielmo would reduce individual variation in exposure to infected bites, a major non-genetic factor. For asymptomatic phenotypes, prevalence (PtPF) gave lower LOD scores than parasite density (mean or maximum), although it showed highly significant heritability in Ndiop. Prevalence is strongly dependent on exposure and thus environmental factors, whereas parasite density will be determined by host-parasite biological interactions. Further analysis by the variance component method that included the effect of house supported this hypothesis: there was a strong effect of house on asymptomatic prevalence but not parasite density (data not shown).P. falciparum clinical malaria attacks (PFA) in Dielmo, and chromosome 12q21-q23 with the maximum parasite density during asymptomatic carriage (mx-tPFD) in Ndiop. Linkage results that we obtained in the two villages were notably different. This may be related to the important differences both in the ethnic backgrounds and in the prevailing transmission conditions in these villages. There was also no overlap between the regions that we detected in our study and those detected in an independent genome-wide linkage study of clinical P. falciparum malaria in Ghana We detected suggestive evidence of linkage to malaria-related traits in three new regions: chromosome 5p15-p13 and 13q13-q22 with the number of IL7R, NM_002185)], tumour necrosis factor synthesis [C1q and tumor necrosis factor related protein 3 ] and a gene involved in the complement system . Genes located within these regions might participate in development of “clinical” immunity. Among these genes, IL7R (5p13) is implicated in B and T cell differentiation and is also critical for the innate and adaptive inflammatory and immunological response IL7R gene with susceptibility to multiple sclerosis Plasmodium yoelii sporozoites develop into protective memory cells without undergoing changes in interleukin-7 receptor α expression IL7R polymorphisms and malaria, so further studies are needed to assess the implication of IL7R gene in malaria infection.The three novel regions of suggestive linkage that we identified harbour a number of genes that can be considered as candidates for a role in malaria response. These include several genes involved in innate immunity such as interleukin [interleukin 7 receptor (derm1) for atopic disease mapped close to the region controlling parasitaemia (char1 or pymr) on mouse chromosome 9 P. falciparum-specific IgE is elevated in malaria patients and has been proposed to play a pathogenic role in severe malaria It is intriguing to note that these three regions of suggestive linkage, as well as the 5q31 region, where we replicated previous suggestive evidence of linkage, have all been previously found to be linked to asthma/atopic disease or related phenotypes falciparum malaria related phenotypes investigated.Our study has limited power, as reflected by the relatively modest LOD scores. Indeed, similar limitations of linkage approaches have been encountered in most studies of multifactorial diseases, even when using much larger numbers of families An additional limitation and one shared by all studies and approaches to date, is the accurate characterisation of disease phenotypes when dealing with a human population. General health status of the population, malnutrition and co-infection can alter the clinical outcome of infection Our study provided several new and interesting findings, not only on the genetic basis to disease but also on how human genetics may influence the biology of the parasite within the host. We defined relevant malaria related traits and estimated their genetic contribution, and mapped genes involved in their control. In addition to replicating linkage to the 5q31, and to some extent to the β-globin locus, we identified linkage to three additional regions. While these regions are extensive and contain many putative candidate genes, it is remarkable that 5q31 and these three novel regions overlap with regions that have been previously identified to be involved in asthma/atopy, hence suggesting that common mechanisms may be involved between both pathogenic mechanisms. These results will provide highly valuable information in the context of integrated studies combining linkage and high density association studies for various malaria traits and conditions. Ultimately, these results will help understand the molecular mechanisms underlying pathogenesis, control of parasite density, and possibly immunotolerance to malaria antigens and the potential relationship with the asthma/atopic disease spectrum.The Dielmo and Ndiop longitudinal surveys have been described in detail elsewhere P. falciparum prevalence rates of infection in Dielmo (80+%) compared to the seasonal rates in Ndiop that change from 20% in the dry season to 70% in the rainy season P. falciparum occur in very young children (2–5 years old) in Dielmo and then decline rapidly with age compared to 5–10 years old in Ndiop with only a gradual decrease with age p <0.001).Two villages of differing malaria epidemiology were studied. Malaria transmission is perennial in Dielmo, where a river maintains larval breeding sites for the mosquitoes. By contrast, malaria transmission is strictly seasonal in Ndiop and dependent upon the rainy season that occurs from July-September. Such differing transmission has marked consequences on the epidemiology of malaria in the villages. This is most evident in the higher P. falciparum trophozoites to leukocytes was established. Measures of parasitaemia i) during pregnancy and ii) in the three days preceding or following any fever episode were excluded from the analysis of asymptomatic parasitaemia. Only individuals with at least six eligible estimations of parasitaemia were considered.During the first intensive survey period, malaria parasitaemia was determined by thick blood film for each inhabitant regardless of symptoms, twice a week from June to September 1990 in Dielmo and once a week from July 1993 to January 1994, then once a month from January to July 1994 in Ndiop. All thick blood films were examined by reading 200 oil-immersion fields on each slide (about 0.5 µL of blood) P. falciparum clinical malaria in any case of fever (axillary temperature > = 37.5°C) or fever-related symptoms  associated with i) a P. falciparum parasite/leukocyte ratio higher than an age-dependent pyrogenic threshold previously identified in the patients from Dielmo P. falciparum parasite/leukocyte ratio higher than 0.3 parasite/leukocyte in Ndiop.Following this initial period, only clinical malaria attacks were recorded. Case detection was both active and passive. A villager, who had volunteered to participate in the study, was visited daily at home by a physician, a technician, a nurse or medical field workers. Thick blood films were prepared and detailed medical examinations were made for villagers who had fever or any symptom that could be related to clinical malaria. A standardized questionnaire was completed for each episode of illness, recording clinical findings, treatment administered The survey periods were defined by trimester for each inhabitant. Only person-trimesters with more than 30 days under survey in the village were included in the analysis. Person-trimesters with pregnancy during the trimester were excluded.Data for each malaria-related phenotype were first analysed using multivariate regression analysis according to the distribution of the data. This method enabled us to take into account known confounding factors such as age, year of study and transmission intensity. The residual unexplained variation in each phenotype was then calculated from the difference between the observed and expected values and then used as the phenotype for further genetic analysis.P. falciparum malaria attack/person was 1 (0–69) in Dielmo and 2 (0–37) in Ndiop. The residual number of clinical P. falciparum attacks was transformed from the number of clinical malaria attacks during person-trimesters separately for each village by Poisson regression models including the effect of age, level of transmission and year of survey. To take into account the malaria exposure dependent acquired immunity in Dielmo, the models also controlled the effect of the proportion of time spent in the village for each individual from his/her birth to the beginning of person-trimester survey period. This confounding factor had no significant effect in Ndiop. Mean and maximum of decimal log transformed maximum parasite density during clinical attacks were analyzed as residuals after accounting for age using linear regression.For clinical malaria attacks, we considered the number of clinical malaria attacks recorded during June 1990 to May 1998 in Dielmo and during July 1993 to May 1999 in Ndiop. The median (range) number of clinical Asymptomatic parasitaemia was evaluated from the systematic thick blood films collected from each inhabitant during the first intensive survey period as described above. The median (range) number of measurements per individual was 33 (6–46) in Dielmo and 24 (6–34) in Ndiop. Asymptomatic parasitaemia was considered in two respects, namely prevalence and intensity. For the prevalence, the residual risk of having a positive blood smear for trophozoites was estimated. First, the probability of having a positive slide was separately estimated for trophozoites by logistic regression models, including variables for the effect of age, week of sampling and delay between the end of the preceding anti-malarial treatment and the date of sampling for each thick blood film. For each individual, the expected number of positive thick blood films was calculated by the sum of the probabilities estimated by the models divided by all observations of parasitaemia eligible for analysis from that individual. An Anscombe residual was then calculated for each individual as a function of the sum of the observed and the expected positive thick blood films. Densities were estimated as the number of trophozoites per 100 leukocytes. Addition of 1 to the density before decimal logarithm transformation allowed negative blood films to be taken into account. The expected parasite density of all thick blood films was estimated by multiple linear regression analysis controlling for the same confounding factors as for prevalence. A raw residual was calculated by subtracting the observed from the expected parasite density. The mean raw residual was calculated for each individual and was used as a phenotype for further genetic analyses. In order to test the maximum capacity of each individual to carry parasites without symptoms, we considered the maximum parasite density of all measurements during asymptomatic carriage. We performed decimal logarithm transformation and controlled for age and week of study.The transformed phenotypes were tested for normal distribution by estimation of skewness and kurtosis and Shapiro-Wilks test for normality. All phenotype analyses were performed using STATA version 7.DNA was extracted from 10 mL venous blood or 0.5 mL capillary samples and randomly amplified using a primer extension pre-amplification method Pedigree structures were checked by identity by state allele sharing of each relative pair from genotyping results of microsatellite markers used during genome screening using IBS_check program .2h), individual (2e) and quantitative trait locus (2q) effects. First, we tested for heritability of the phenotype by comparing likelihood between the reduced model, where total variation is due to environmental variation only, and the full model where total variation is composed of environmental and genetic effects estimated from the genetic relationship of each pair of individuals. When the null hypothesis was rejected, heritability (2h) was then estimated as the percentage of genetic variance of the total.Evaluation of the genetic contribution to the phenotypes was performed by estimation of the heritability using the variance-component model For phenotypes that showed significant heritability, we performed linkage analysis to estimate the genetic variance attributable to the region around a specific genetic marker p values were obtained by performing a simulation of 10,000 trials implemented in the SOLAR software Estimation of heritability and linkage analyses were run by use of the SOLAR program (version 2.1.3). SOLAR can analyse large complex families such as observed here. The complex family structure is accounted for in the genetic analysis via the polygenic variance component that uses the relationship coefficient between pairs of individuals as a parameter In addition to variance component analysis using SOLAR, we performed regression linkage and variance component analysis using the MERLIN program


Plasmodium falciparum infection in young African children is considered in the context of three current strategies for malaria prevention: insecticide-impregnated bed nets or curtains, anti-sporozoite vaccines and intermittent preventive therapy. Evidence is presented that each of these measures may permit attenuated P. falciparum blood-stage infections, which do not cause clinical malaria but can act as an effective blood-stage "vaccine". It is proposed that the extended serum half-life, and rarely considered liver-stage prophylaxis provided by the anti-folate combination sulphadoxine-pyrimethamine frequently lead to such attenuated infections in high transmission areas, and thus contribute to the sustained protection from malaria observed among children receiving the combination as intermittent preventative therapy or for parasite clearance in vaccine trials.The development of acquired protective immunity to  A significant reduction in the intolerable global burden of malaria morbidity and mortality is unlikely without the large-scale implementation of effective and sustainable prevention. However, it has been recognized for more than a decade that measures which reduce the level of exposure may interfere with the natural acquisition of immunity to malaria . Direct Plasmodium falciparum elicit effective and sustained immunity against clinical malaria [The suggestion that any short-term protection afforded by insecticide-impregnated materials (IIM) may be followed by longer-term increased susceptibility, or "rebound", applies equally to other interventions that reduce or prevent infection. For this reason, clinical trials of malaria vaccines and of intermittent preventive therapy (IPT) have used extended post-intervention follow-up to detect any rebound of excess malaria among recipients of the intervention. Published controlled trials of preventative interventions including IIM, IPT and vaccination are considered, and findings discussed in the light of evidence that low-dose blood stage infections of  malaria .It is hypothesized that the antimalarial sulphadoxine-pyrimethamine (SP), because of its long serum half-life and its activity against developing hepatic parasite stages, generates low dose blood stage inocula and attenuated infections, and can thus act as an ideal blood-stage vaccine when used for malaria prevention in highly endemic areas.An ambitious programme initiated in 1994 to provide a high-coverage of IIM in Oubritenga Province, Burkina Faso, was associated with a reduction in entomological inoculation rate of >90% . All-caupfcrt-76T chloroquine resistant genotype, comparing those under lifelong IIM protection, with those without IIMs in their village. IIM-protected children were significantly more likely to successfully clear resistant parasites after chloroquine treatment, compared to unprotected children . After stratification, children under three years of age remained significantly better at clearing resistant parasites than their unprotected peers, whereas children IIM-protected and unprotected at a later age were equally able to clear their infections. Thus, in the fourth and fifth years of life, unprotected children had levels of immunity similar to those enjoyed by children who have been protected by IIM.In the absence of immunological markers, the acquisition of immunity to malaria may be demonstrated by the ability to clear resistant parasites under treatment with a failing drug ,7. DiallP. falciparum infections. Older children are more likely to mount an effective immune response, preventing further progression of disease. The process of acquiring immunity to malaria in these children is likely to involve interplay between:These findings suggest that IIM-protected children in Burkina Faso commonly acquired protective immunity earlier. This may be because the protection afforded by IIM delays, on average, the age at which children are first challenged by P. falciparum leading to a disease episode• the risk of infection with • the age at which children first encounter infection.P. falciparum vaccine RTS, S is now under extensive testing in clinical trials in sub-Saharan Africa. Phase II controlled trials of the vaccine in Gambian men [P. falciparum parasitaemia, but differed in one remarkable respect. Vaccine-elicited parasitological benefit was transient in Gambian men, having diminished within 16 weeks of vaccination, whereas RTS, S-vaccinated children in Mozambique enjoyed a reduction in malaria episodes through 18 months of follow-up [The anti-sporozoite bian men  and Mozabian men ,11 demonollow-up . This maollow-up . Thus, bThe development of protective blood-stage immunity by this proposed mecanism would be dependent on:• the likelihood of natural parasite challenge during the transient period of anti-sporozoite protection• the level of immunity at the time of vaccination.et al [In a placebo-controlled trial in 700 infants in Tanzania, Schellenberg et al ,14 invesP. falciparum infections during a period of waning blood levels of the component drugs of SP. This could readily lead to attenuated blood-stage infections that do not flourish, and thus do not cause clinical malaria, but persist at low density, providing a challenge capable of eliciting protective immunity. This explanation is consistent with the results of a placebo-controlled clinical trial of the P. falciparum blood-stage vaccine Combination B, in which 120 participants (aged between five and nine years) were randomized into placebo or vaccine arms, with or without pre-intervention clearance of parasitaemia by SP [Under moderate to high transmission intensity, it can be imagined that a subset of IPT recipients are naturally challenged by ia by SP . A crudeia by SP . Key par• the probablility of infection during the period of effective drug levels• the level of immunity before IPT/chemoprophylaxis is givenP. falciparum infections enhance development of immunity, it is now proposed that presumptive administration of SP to young children in moderate to high transmission areas can accelerate their acquisition of protective immune responses. This occurs because, firstly, the elimination half-lives of sulphadoxine (11 days) and pyrimethamine (3 days) in serum provide extended pharmacological protection against the emergence of new blood-stage infections from the liver, but become permissive of such new infections as serum drug levels wane in due course. Secondly, SP provides a level of causal prophylaxis against liver-stage infection that is poorly understood [4 progeny merozoites into the blood. It is thus likely hepatic stages are exquisitely sensitive to anti-folate drugs such as SP, but no detailed studies have been published.Having presented evidence supporting the notion that low dose derstood -19. Livepfdhfr and pfdhps loci also protect hepatic parasites against SP, and it is a plausible possibility that SP remains effective against so called "resistant" parasites during rapid hepatic stage DNA replication, prior to further expansion of the total parasite burden during blood-stage infection. This offers an explanation for the continued utility of SP in preventative applications even where clinical cases commonly fail under SP treatment.In particular, it is unknown whether resistance-associated mutations in the Low-dose blood-stage infections enhance the acquisition of immunity against clinical malaria, perhaps partly by thwarting the immune suppression effects of clinical disease . Sustain


In clinical trials, the specificity of the disease endpoint is critical to an accurate estimate of vaccine efficacy. We used a logistic regression model to analyse parasite densities among children before and after treatment with antimalarials, in order to estimate the impact that clearing asymptomatic parasitaemia had on the specificity of the endpoint of febrile malaria. The malaria attributable fever fraction was higher after antimalarial treatment , implying that drug treatment prior to monitoring decreased the misclassification of febrile malaria. In intervention studies with febrile malaria as an endpoint, clearing asymptomatic parasitaemia increases the study's power more effectively than raising the threshold parasitaemia. It is an integral part of the strategy of intermittent preventative treatment 1.2The malaria attributable fever fraction (MAFF) is defined as the fraction of fevers that can be attributed to malaria infection . While it may not be possible to determine which individuals are “true” cases of febrile malaria, and so calculate this fraction by an arithmetically calculated numerator and denominator, using a logistic regression model allows the MAFF in a population to be estimated 1.3The specificity of the endpoint is critical to a trial's ability to correctly estimate vaccine efficacy. If a vaccine is efficacious against febrile malaria, but not against asymptomatic parasitaemia, then including cases of non-malarial fever that coincide with asymptomatic parasitaemia in the endpoint will reduce the apparent efficacy of the vaccine. Our objective in this analysis was to compare infections before and after parasite clearance was given, and so calculate the MAFF, sensitivity, and specificity at various parasite densities. This then allows the relative power of studies with and without drug clearance to be estimated.22.1Four hundred and five children, aged 1–6-years-old (inclusive), were randomised for either an experimental prime boost malaria vaccine or control vaccination (rabies). The children were healthy, and resident in Junju, within Kilifi District, a rural area of coastal Kenya. The process of randomization and vaccination is detailed elsewhere 2.2Cross-sectional surveys to examine peripheral blood parasitaemia were conducted at recruitment (January 2005) before any antimalarial treatment was given. After three vaccinations and antimalarial treatment, cross-sectional surveys were conducted 2 weeks after antimalarials were given (May 2005), then after 3 months in September 2005 (end of the rainy season) and after 8 months in January 2006 (during the dry season). Antimalarial treatment to clear asymptomatic parasitaemia was given 1 week after the final vaccination, using 7 days of directly observed dihydroartemisinin monotherapy . Four hundred and five children received at least one vaccination, and of these 387 finished the vaccination course and completed at least one further monitoring visit for malaria episodes. Three hundred and sixty children complied with the 7 days observed artesunate and were parasite negative 2 weeks later. Analysis here is restricted to these 360 children. Ninety-four fevers were assessed in the 3 months before clearance of asymptomatic parasitaemias treatment, and 353 in the 9 months after clearance. Further cross-sectional surveys were taken 3 months post-drug treatment (early August 2005) and 9 months post-drug treatment (January 2006).2.3Children were seen weekly by field workers, and blood films made when the temperature was >37.5°. Field workers lived in the study area, and parents brought their children for assessment between regular weekly visits if the child developed fever. Treatment for episodes of malaria was with the Government of Kenya recommended first line treatment, artemether–lumefantrine. When the mother reported the child was hot, but an objectively elevated temperature was not identified, blood films and rapid testing was not performed, but the field worker returned to the child a further three times in the next 24 h. Rapid testing and blood films were performed if the temperature was elevated on any of these visits. Parents brought their children for assessment in between regular weekly visits if they thought the child had developed fever, and the child was assessed as above. Field workers were recruited from the villages in which the study was conducted, and so were readily accessible to the parents.2.4Blood films were examined in duplicate by two microscopists, and examined a third time if there was a discrepancy. The results of blood films at cross-sectional bleeds were not immediately available, and asymptomatic parasitaemic children were not treated unless they developed fever.2.5The parasite densities from the cross-sectional survey and episodes of febrile malaria before drug treatment were used in a logistic model to relate parasite density to the likelihood of fever. The data are analysed by three time periods. The pre-treatment period included 6–7 weeks of dry weather and 6–7 weeks of rains. The post-treatment period was analysed separately for the first 12 weeks of rains, and for the subsequent 24 weeks of dry weather. The pre-treatment analysis used malaria episodes between January 2005 and May 2005, and the cross-sectional bleed from January 2005. Vaccinations were given between January and May 2005, and antimalarial drug treatment given in May 2005. The post-treatment rainy season analysis used malaria episodes between May 2005 and September 2005, and the cross-sectional bleed from September 2005. The post-treatment dry season analysis used malaria episodes between September 2005 and January 2006, and the cross-sectional bleed from January 2006.p) = a + bxτ, where p is the probability of fever, x the density of parasitaemia and τ is the power function of the parasite density which maximises the likelihood estimation for the different time points). The coefficient b can then be used to estimate the risk of febrile malaria in individual children, and the average risk of malaria among a group of children with fever gives the fraction of those children whose fever was a direct result of malaria . This calculation can be repeated after selecting children above a threshold parasitaemia, and used to calculate the sensitivity and specificity of threshold values for parasite density.The method for deriving malaria attributable fever fractions (MAFFs) has been described previously Separate analyses were conducted by vaccination status. MAFFs were not different by vaccination group, and vaccination had no effect on frequency of malaria episodes or parasitaemia 3In the pre-treatment cross-sectional survey (January 2005), 71% of children were parasitaemic. 2 weeks after drug treatment 2% were parasitaemic, but 3 months  and 9 months later , 27% and 33% of children were parasitaemic, respectively.3.1Before drug clearance of asymptomatic parasitaemia, 47 of 121 (38%) of children with fever had no parasitaemia, compared with 75 of 182 (41%) during the rainy season post-drug clearance , and 70 3.2A logistic model was fitted to the data as described previously −1).Pre-treatment, there were 74 febrile parasitaemic children over 12 weeks of monitoring. An MAFF of 37% (95% CI 26–48%) among all parasitaemic children suggests that there were only 27 true episodes of malaria among these children (i.e. 2.3 week−1).Malaria transmission was highest during the post-treatment rainy season. There were 107 febrile parasitaemic children during 12 weeks, and since the MAFF  was 74% (95% CI 63–83%) this suggests 80 true episodes of malaria (i.e. 6.7 week−1).During the 24-week post-treatment dry season, there were 49 febrile parasitaemic children, and an MAFF  of 51% (95% CI 37–63%) suggests that 24 had true episodes of malaria , was intermediate between the MAFF during the pre-treatment period, at 37% (95% CI 26–48%), and the MAFF seen during the post-treatment rainy season, at 74% (95% CI 63–83%).Using a threshold of 2500 paras/μl, the MAFFs were 52% (CI 35–69%), 87% (CI 73–87%) and 65% (CI 50–80%) pre-treatment, post-treatment rainy season and post-treatment dry season, respectively.4A phase 2b randomized controlled trial to measure the efficacy of a candidate malaria vaccine was conducted. In common with many phase 2b trials in children, the primary endpoint was episodes of febrile malaria. The specificity of the endpoint is critical to the accuracy of estimates of efficacy in clinical trials. Curative drug treatment before monitoring was used to clear asymptomatic parasitaemia before vaccination was complete. It is unclear whether this is necessary in vaccine studies, and there are recommendations to not do so in studies of blood stage vaccines 4.1The logistic regression model  suggesteThe sensitivities and specificity plots against parasitaemia were similar before and after parasite clearance , even th4.2A standard power calculation suggests that if vaccine efficacy was 50%, a trial with 90% power would require 85 subjects in each arm, given the rates of malaria seen here. How can one account for the situation where the MAFF is only 50%? If one assumes that the vaccine prevents fever causally related to parasitaemia, but not non-malaria fever that coincides by chance with asymptomatic parasitaemia, then the apparent vaccine efficacy would be 25%, and the required sample size would be 265 subjects in each arm.One could introduce a parasitaemia threshold to the case definition, in order to exclude cases of asymptomatic parasitaemia. However, this inevitably excludes some genuine cases of febrile malaria with low parasitaemia, and so the chosen threshold is a compromise between sensitivity and specificity. This then has the effect of increasing the sample size.In our data, a case definition of any parasitaemia occurred at an incidence of 0.55 per subject, but the MAFF was 36% . This would have required 512 subjects per arm to detect 50% protection with 90% power. At 50,000 paras/μl 485 subjects per arm would be required (because of increased specificity). However, higher parasitaemia thresholds do not result in further reductions in sample size, because of the competing effect of lowered sensitivity. At 100,000 paras/μl, 578 subjects per arm are required.However, after drug clearance, the MAFF rose to 0.64, without any loss in sensitivity. This would require 158 subjects per arm. Optimal power after drug clearance would have been seen using a threshold of only 100 paras/μl (143 subjects per arm) and the sample size required would rise to 217 per arm at 5000 paras/μl.Models have been used to demonstrate the theoretical underestimates and overestimates of blood stage vaccine efficacy against febrile malaria In recent studies of an efficacious vaccine


Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) and lactate dehydrogenase (pLDH). PfHRP-2 and pLDH are the malaria target antigens for the commonly used commercial RDTs.Rapid diagnostic tests (RDTs) for malaria are increasingly being used for management of patients. Different studies have shown significant failure rate of RDTs, especially in children and in areas of low malaria transmission. In this study, we sort molecular answers to RDT failure by re-screening RDT negative samples for malaria at genus and species level as well as for diversity of Patients (n=1054) with high fever but negative for malaria by RDT were recruited for the study from a network of clinical sites in Kenya. For RT-qPCR malaria diagnosis, total nucleic acids were isolated from whole blood and evaluated by a strategy that combines RNA and DNA using 18 S rRNA primers and probe for malaria genus and species. For diversity of PfHRP-2 and pLDH genes, PCR were subjected to gel electrophoresis to determine amplicons sizes.P. falciparum, either alone or in combination with P. malarie (7%), or P. ovale (2%). 31% of genus positive malaria could not be speciated. Of the P. falciparum, 56% had full size PfHRP2 gene (927 bp), 40% had truncation of up to 325 bp and 1 % appeared not to have PfHRP2 gene at all. All parasites had full size pLDH (922 bp) gene.Of the 1054 RDT negative febrile patients, malaria at genus level was detected in 160 patients (15%). Of the 160, half (52%) had a parasite density below the limit of detection of RDT in use at the hospital (100 parasites/ μL). On speciation, 60% of the patients had P.ovale and P.malarie is due to low parasite density commonly associated with these infections and not due to truncation of pLDH gene. Lastly, the unspeciated malaria infections are probably due to higher sensitivity of genus compared to species RT-qPCR.It is concluded that, 50% of the RDT failures are attributable to the detection threshold of the RDTs. It is unclear what impact the truncation of PfHRP2 gene has on sensitivity of RDT. That the failure to detect


IL12B gene. An IL12B promoter allele, CTCTAA, at rs17860508 has been reported to be associated with susceptibility to cerebral malaria in African populations. However, this association has not so far been replicated in non-African populations.Interleukin-12 (IL-12), a heterodimeric cytokine composed of p35 and p40 subunits, has been thought to play an important role in the pathogenesis of malaria. The IL-12p40 subunit is encoded by the Plasmodium falciparum malaria  were genotyped for rs17860508 by PCR-direct sequencing.To examine whether the CTCTAA allele is associated with susceptibility to cerebral malaria in Asian populations, 303 Thai patients with P-value = 0.030).The CTCTAA allele showed a significant association with susceptibility to cerebral malaria in the Thai population (allelic OR = 1.37; one sided The existence of a significant association between the CTCTAA allele and susceptibility to cerebral malaria was confirmed in Southeast Asian population, which was previously reported in African populations. Plasmodium falciparum malaria, causes the vast majority of deaths related to P. falciparum infection. Although the pathophysiology of severe malaria  is not fully understood, significant differences in serum or plasma concentrations of various cytokines have been observed between severe and mild malaria patients. The levels of interleukin-12 (IL-12) are significantly lower in patients with severe malaria in comparison to those with mild malaria [Cerebral malaria, a severe form of  malaria -5, althoIL12B gene. The IL12B has a functional polymorphism, CTCTAA/GC (rs17860508), with a 4 bp difference between the two alleles in the promoter region. The CTCTAA/GC polymorphism has been shown to affect the gene expression and the production of cytokines and nitric oxide [IL12B promoter allele, CTCTAA, is associated with susceptibility to cerebral malaria in Thais.Interleukin-12 (IL-12) is a heterodimeric cytokine formed by a 35-kDa light chain (p35 or IL-12a) and a 40-kDa heavy chain (p40 or IL-12b). The IL-12p40 heavy chain is encoded by the /GC rs1780508, witic oxide . The homic oxide . Recentlic oxide . HoweverP. falciparum malaria  living in northwest Thailand were analysed in this study. All patients underwent treatment at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University. Malarial infection by P. falciparum was confirmed in all patients by a positive blood smear for the asexual form of P. falciparum. Clinical manifestations of malaria were classified according to the definitions and associated criteria by the World Health Organization. Cerebral malaria was defined as unrousable coma, a positive result in tests for the presence of an asexual form of P. falciparum, and exclusion of other causes of coma. Mild malaria was defined as having a positive blood smear and fever without other causes of infection and no signs indicating severe malaria such as high parasitaemia , hypoglycemia (glucose level < 22 nmol/L), severe anaemia (haematocrit < 20% or hemoglobin level < 7.0 g/dl) or increased serum level of creatinine (> 3.0 mg/dl). All individuals were 13 years old or older, and the mean ages of patients with mild malaria and cerebral malaria patients were 25.5 and 28.6 respectively. This study was approved by the institutional review board of the Faculty of Tropical Medicine, Mahidol University, and the Research Ethics Committee of the Graduate School of Comprehensive Human Sciences, University of Tsukuba. Informed consent was obtained from all patients.A total of 303 adult patients with IL12B promoter polymorphism, rs17860508, was genotyped by PCR-direct sequencing with an ABI PRISMTM 3100 Genetic Analyzer (Perkin-Elmer Applied Biosystems). PCR was performed using a primer set: a 5' primer IL12B-proF: 5'-GGATCAGGGTCTGGATTGTG-3' and a 3' primer IL12B-proR: 5'-GGCTGATGCTTGGAGATTGT-3'. The amplification condition consisted of an initial denaturation at 96°C for 10 min, followed by 35 cycles of denaturation at 96°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min using a thermal cycler .Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp Blood Kit . The P-values for association were calculated by a one-sided test to avoid a false negative because the CTCTAA allele had been previously reported to be associated with both susceptibility to and mortality from cerebral malaria. The deviation from Hardy-Weinberg equilibrium (HWE) for each malaria group was assessed by a chi-square test. A P-value of less than 0.05 was regarded as statistically significant in this study.The differences in genotype and allele frequencies between the patients with cerebral and mild malaria were examined by the Cochran-Armitage test for trend and chi-square test, respectively. The P-value > 0.05). Consistent with observations in African populations, the CTCTAA allele showed a significant association with susceptibility to cerebral malaria in the Thai population . The Cochran-Armitage trend test for the genotype frequency also established the association between the CTCTAA allele and cerebral malaria . Although a previous study demonstrated that the CTCTAA/GC heterozygotes increased the risk of cerebral malaria [The genotype of rs17860508 was analysed in 303 Thai patients with malaria (Table  malaria , such a The significant association of the CTCTAA allele with susceptibility to cerebral malaria was found both in children living in Mali  and in TIL12B promoter polymorphism at rs17860508 plays an important role in determining the onset of cerebral malaria, although the role of IL-12 in malaria infection remains to be studied.The present results suggest that the IL12B promoter CTCTAA allele with cerebral malaria was replicated in a Thai population.A significant association of the The authors declare that they have no competing interests.NI performed the genotyping, helped conduct statistical analyses, and wrote the manuscript. KT helped genotype the samples. JP and HH collected blood samples and extracted DNA. JP and JO participated in the design and coordination of the study. NT was involved in the interpretation of the data and preparation of the manuscript. JO performed statistical analyses and helped write the manuscript. All authors read and approved the final manuscript.


Intermittent preventive treatment for malaria in Infants (IPTi) has been shown to give effective and safe protection against malaria. It has been suggested that IPTi might have long-lasting beneficial effects but, in most settings, the protection provided by IPTi appears to be short-lived. Knowledge of the duration of protection given by IPTi would help interpret the results of existing trials and suggest optimal delivery schedules for IPTi. This study investigated how the protective efficacy of IPTi against malaria and anaemia changes over time.A secondary analysis of data from a cluster-randomised, placebo-controlled trial of IPTi using sulfadoxine-pyrimethamine (SP) in Ghana was conducted. In this trial IPTi was given to 2485 infants at 3, 4, 9 and 12 months of age; children remained in follow-up until two years of age. Poisson regression with a random effect to adjust for the cluster-randomised design was used to determine protective efficacy of IPTi against clinical malaria and anaemia in defined time strata following administration of IPTi. Analysis of first-or-only clinical malaria episode following the individual IPTi doses showed that some protection against malaria lasted between 4 to 6 weeks. A similar pattern was seen when the incidence of all malaria episodes up to 2 years of age was analysed in relation to the most recent IPT, by pooling the incidence of malaria after the individual IPTi doses. Protective efficacy within four weeks of IPTi was 75.2% (95% CI: 66–82) against malaria, 78.9% (95% CI: 69–86) against high parasite density malaria, and 93.8% (95% CI: 73–99) against anaemia. Protection against these outcomes was short-lived, with evidence of any effect lasting for only 6, 6 and 4 weeks respectively. Protection in children who were parasitaemic when receiving IPTi appeared to be of shorter duration than in uninfected children. There was no evidence of any benefit of IPTi after the immediate period following the IPTi doses.Intermittent preventive treatment provides considerable protection against malaria and anaemia for short periods, even in an area of intense seasonal transmission. Due to the relatively short duration of protection provided by each dose of IPTi, this treatment will be of most benefit when delivered at the time of peak malaria incidence. Intermittent preventive treatment (IPT) is a promising strategy for preventing malaria morbidity in infants and children, particularly in sub-Saharan Africa where the disease burden is highest. Sulfadoxine-pyrimethamine (SP) given to Tanzanian infants alongside routine vaccinations gave high protective efficacy against malaria and anaemia; this protection appeared to be sustained long after the chemoprophylactic period of SP, suggesting that development of protective immunity might have been facilitated by the treatment Although there is now a large body of evidence that three courses of IPT given alongside Expanded Programme on Immunisation (EPI) vaccines can reduce the incidence of malaria in infants, the optimum timing and frequency of IPT doses is still debated This study used data from a cluster randomized trial of IPTi in Navrongo, Ghana described in detail elsewhere Covariates pre-specified for adjustment in analysis of the main trial were sex, urban residence and mosquito net usage. Reported bednet use among study children was low (13%) and insecticide treated net use very low (2%); there were no data for 26% of children. For this reason bednet use was not included in the models. The primary analysis was performed unadjusted for covariates; the effect of adjusting for age at time of dose, sex and place of residence  was then explored.Person time at risk of malaria was calculated from the date of a given dose of IPTi until a subsequent dose was administered, malaria was diagnosed, or the individual exited the study , whichever occurred first. Kaplan-Meier failure plots were used to examine incidence of malaria after each IPTi dose until the following IPTi dose or until the end of follow up. A poisson regression model was fitted with a gamma distributed random effect to account for potential correlation within clusters of children. Lexis expansion Person time at risk of malaria and anaemia was calculated from first dose of IPT until the child exited the study or reached two years of age. Lexis expansion was performed and a variable was created that showed the number of weeks since the most recent treatment throughout follow-up. This allowed incidence of any malaria or anaemia episode that occurred between the first treatment and two years of age to be related to time since most recent IPTi dose; pooling the analysis across doses enabled a more robust analysis of protective efficacy over time. For malaria and high parasite density malaria, follow-up was stratified by week since IPT; there were fewer episodes of anaemia so two-week strata were used. Incidence rate in a given time stratum was compared between trial arms using a poisson regression model with a cluster-level random effect.Participants who had received chloroquine or quinine, the drugs of choice during the study period, for the treatment of clinical episodes of malaria were not considered to be free of the risk of malaria during the post treatment period. This approach gives a more pragmatic estimate of the effect of IPTi, by making a direct comparison between IPTi and routine case-management of malaria. Consequently, our estimate of the protective efficacy of IPTi may be slightly more conservative than that reported by other studies that deducted 3 to 4 weeks post treatment for each episode of malaria to account for the prophylactic effect of the antimalarial treatment.To investigate if protection in Navrongo was compatible with that reported from studies that have used monthly IPT, an analysis that only included episodes that occurred within four weeks of an IPTi dose was performed.In addition to providing prophylaxis, IPT should in most cases clear parasitaemia present at the time of treatment. This could be beneficial since many children in this age group will not have developed sufficient immunity to prevent an existing infection increasing in density and causing disease The approach taken was the same as for the pooled analysis of all malaria episodes: a poisson regression model was fitted to the data and follow-up time stratified by week since most recent treatment. Blood smears were not taken at the dose given at four months of age (scheduled as IPT-2) but were available for 106 individuals who received IPT-2 at a later date due to vomiting or absence at the scheduled time. Follow-up time after doses at which parasitaemia was not determined was excluded from this analysis.All analyses were performed in Stata version 9 .Ethical approval for this study was granted by the Ethics Review Board of the London School of Hygiene and Tropical Medicine, number 05/176.Kaplan-Meier failure plots for clinical malaria following individual IPTi doses are shown in Poisson regression with a random effect to adjust for the clustered design was used to estimate protective efficacy against clinical malaria with increasing time since IPTi doses. Adjusting for age, sex and place of residence of children made negligible changes to the estimates of protective efficacy; data are presented from the unadjusted models. There was strong evidence of an interaction between IPT efficacy and time since IPT dose  suggesting that protective efficacy depends on how long ago the IPT dose was received. Protection is high initially but then appears to be lost rapidly. Consistent with other IPTi studies, protective efficacy is presented up to twelve weeks (3 months) post dose. No evidence of either protective or detrimental effects was observed after this period for any of the doses of IPTi. For all IPT doses there is evidence of significant protection for at least four weeks, protection appears to last for at least five weeks after IPT-3 and there is weak evidence of some protection up to 6 weeks for IPT-2 . This anThere was strong evidence of a high protective effect against all episodes of clinical malaria and high parasite density malaria in the first four to five weeks following IPT. Some protection was seen up to six weeks but from seven weeks onwards there was no evidence of any remaining benefit of having received IPTi. Protection against anaemia was high up to four weeks after IPT. No evidence of either a protective or detrimental effect was observed after the immediate period following IPTi for any of these outcomes. Our analysis included all of the follow-up period to two years of age, but since no protective effect was observed except for the immediate period following IPT, graphs are truncated at 20 weeks post-dose , table 2To compare the effect of our more pragmatic approach with those of previous papers, we repeated the analysis removing 28 days of person-time at risk after a clinical malaria episode. The results were remarkably similar to those presented, although there was evidence of a small amount of protection against clinical malaria lasting for an additional week after IPTi (i.e. into the seventh week after treatment rather than the sixth week). After this period there remained no evidence of any protection.Considering only incidence within the first four weeks following a dose of IPT, protective efficacy was high: 75.2% (95% CI: 66–82) against malaria, 78.9% (95% CI: 69–86) against high parasite density malaria, and 93.8% (95% CI: 73–99) against anaemia.Individuals who are parasitaemic when given IPT experience a higher incidence of malaria in the short term, but this risk wanes with time. Within four weeks of treatment the incidence rate for clinical malaria was 2.66 times greater in children who were initially parasitaemic (95% CI: 2.12 to 3.34). There was no evidence of interaction between parasitaemia and the overall protective efficacy of IPT (LRT p = 0.6521). However, there was strong evidence of interaction between parasitaemia at time of dose and the protective efficacy of IPT over time (LRT p<0.001). Adjusting for age, sex and place of residence of children made negligible changes to the estimates of protective efficacy; data are presented from the unadjusted model. In children parasitaemic at the time of IPT, protective efficacy is lower in the first week, but is then high in the period up to 5 weeks following the IPTi dose. There is a suggestion that protection may be lost faster in children initially parasitaemic than in children without parasites .The protection provided by IPTi in Navrongo was of limited duration. Protective efficacy against malaria and high parasite density malaria was high during the first four to five weeks after treatment, retaining some efficacy up to six weeks; there was no evidence of a protective effect from week seven onwards. Protection against anaemia lasted for four weeks following treatment. Since periods when children were being treated for malaria were not removed from time at risk in this analysis, our estimate of protective efficacy may be slightly conservative and the duration of protection slightly underestimated because more episodes of malaria occurred in the placebo group. However, removing 28 days person-time at risk after each malaria episode increased the duration of protection following IPTi by just one more week and there was no evidence of any protection after week seven post IPTi. The period of highest protection was still within the first four to five weeks. We do not therefore consider that our conservative approach has resulted in any important underestimation of the duration of protection given by IPTi.There is weak evidence of an elevated incidence of malaria in the SP group relative to those on placebo in weeks eight and nine following IPT-4. Increased malaria incidence eight to ten weeks after treatment with SP and artesunate was also observed in a mass-drug administration trial in the Gambia in vivo. What can be expected is that the direct parasiticidal effect of treatment cannot last indefinitely, and one would expect a gradual reduction in protective efficacy which accelerates over time, as seen here et al. have estimated that SP may be able to provide prophylaxis against fully sensitive Plasmodium falciparum for approximately 60 days The plasma half-lives of sulfadoxine and pyrimethamine are between 5–11 days and 3–5 days respectively in adults, but there are no data on pharmacokinetics in infants Protective efficacy against clinical malaria within four weeks of treatment was high: 75.2% (95% CI: 66–82). IPTi in this setting therefore attains an efficacy similar to that achieved in Niakhar, Senegal with monthly IPT There was no evidence of an elevated incidence of malaria in children who were parasitaemic when given IPT that would suggest that clearance of parasites results in loss of cross protective immunity against similar parasite genotypes and has a cost attached. In fact it appears that infections that are asymptomatic at time of detection are likely to lead to disease in the short term in this age group and should be treated, a finding that agrees with existing literature on this subject. A study in Uganda found that asymptomatic parasitaemia was a clear risk factor for clinical malaria within thirty days of detection Parasitaemic children experienced a markedly increased incidence in the first week after IPT, even those who were given SP. This effect was not age-related, so cannot be solely due to the possible under-dosing in older children. Presumably this reflects cases where the drug was given too late to prevent an incipient episode since antimalarial drugs do not reduce the parasite burden instantaneously, even if given intravenously For children free of parasites when they received IPT, the protective efficacy profile reflects the ‘true’ prophylactic effect of SP over time. Children who are parasitaemic are at the additional risk of recrudescence of the original infection if the parasite is resistant to SP, so protection may appear to last for less time than in uninfected children. Existing knowledge suggests that use of long acting antimalarials such as SP may result in very late recrudescence if not all parasites are killed but multiplication is suppressed until most of the drug is eliminated The protective efficacy curves including children with and without parasitaemia together , 4 & 5 r8 parasites) and a recently inoculated parasite on emergence from the exo-erythrocytic schizont (approximately 105 parasites) It is plausible that SP may remain effective for prophylaxis despite reduced therapeutic efficacy due to the difference in parasite burdens between a microscopically patent blood-stage infection (>10In all analyses there was strong evidence of a beneficial effect of IPT up to four weeks after treatment and in some cases this appeared to last up to five or six weeks. The largest drop in protective efficacy against both clinical malaria and high parasite density malaria was between five and six weeks after IPT ; before If regular delivery of IPT during the transmission season is to become a widespread approach to controlling malaria in areas where the majority of the burden is concentrated in a few months, several issues will need to be addressed. Some of these are not new: regular intermittent treatment is akin to seasonal chemoprophylaxis


In sub-Saharan Africa, knowledge of malaria transmission across rapidly proliferating urban centres and recommendations for its prevention or management remain poorly defined. This paper presents the results of an investigation into infection prevalence and treatment of recent febrile events among a slum population in Nairobi, Kenya.In July 2008, a community-based malaria parasite prevalence survey was conducted in Korogocho slum, which forms part of the Nairobi Urban Health and Demographic Surveillance system. Interviewers visited 1,069 participants at home and collected data on reported fevers experienced over the preceding 14 days and details on the treatment of these episodes. Each participant was tested for malaria parasite presence with Rapid Diagnostic Test (RDT) and microscopy. Descriptive analyses were performed to assess the period prevalence of reported fever episodes and treatment behaviour.Plasmodium falciparum using RDT; however, all were confirmed negative on microscopy. Microscopic examination of all 953 readable slides showed zero prevalence. Overall, from the 1,004 participants who have data on fever, 170 fever episodes were reported giving a relatively high period prevalence  and higher among children below five years . Of the fever episodes with treatment information 54.3% (95%CI:46.3%–62.2%) were treated as malaria using mainly sulphadoxine-pyrimethamine or amodiaquine, including those managed at a formal health facility. Only four episodes were managed using the nationally recommended first-line treatment, artemether-lumefantrine.Of the 1,069 participants visited, 983 (92%) consented to be tested. Three were positive for The study could not demonstrate any evidence of malaria in Korogocho, a slum in the centre of Nairobi. Fever was a common complaint and often treated as malaria with anti-malarial drugs. Strategies, including testing for malaria parasites to reduce the inappropriate exposure of poor communities to expensive anti-malarial drugs provided by clinical services and drug vendors, should be a priority for district planners. Most studies of urban malaria transmission in sub-Saharan Africa (SSA) suggest significant reductions in the risk of infection compared to neighbouring rural areas -7. TheseNairobi is a classic example of a rapidly growing human settlement. In 1900, Nairobi was a settlement restricted to a small area between the Ngong, Mathari and Nairobi rivers and expanded quickly with the building of a railway to become the colonial government headquarters in 1905. In 1910, the population of Nairobi was estimated to be only 1,700 people . By 1944Anopheles gambiae and Anopheles funestus were reported as present within the city [Plasmodium falciparum infection prevalence among children was reported to be 2.5% in 1929 [P. falciparum infection prevalence was reported to be between 1.8% and 13.5% at nine schools across Nairobi [Nairobi was historically an area of seasonal malaria transmission with as many as 939 clinical cases documented each year between 1930 and 1939 ,21,19. Ithe city  and confthe city . Plasmod in 1929 . Followi Nairobi . Despite Nairobi .The present study was undertaken at Korogocho, a slum settlement that forms part of an extended Nairobi Urban Health and Demographic Surveillance System (NUHDSS) established in 2002 to prospectively monitor vital, social and health events . Korogocde jure residents at 1st January 2008 as the sampling frame. All residents of the Korogocho NUHDSS on this date were eligible to be sampled and included in the study if they consented. Using simple random sampling 1,069 individuals were selected. Fieldworkers visited households of the sampled individuals to seek their consent to participate in a cross-sectional malaria parasite prevalence survey.The NUHDSS population census is routinely updated and the present study used the Four teams of trained interviewers, each comprising of a laboratory technician and a nurse collected the data on individuals who consented to participate in the study. Since the survey involved an oral interview and finger prick blood sampling, individuals could consent to be interviewed but refuse to provide a blood sample. Individuals who consented to participate were interviewed about the usual and previous night use of mosquito nets and whether these were treated with insecticide. Participants were also asked about fever symptoms experienced over the last 14 days preceding the visit and details on the treatment of these episodes. This information was asked for every reported fever episode and included how the fever was detected, the duration of the fever, the type (including formulation) of medicine taken and the origin of the medicine.®, Mannheim, Germany) performed. The RDTs were read 10 minutes after preparation and a confirmation reading was done 20 minutes later. The interviewers also prepared thick and thin slides and labelled them with the participant's identification number. Participants were provided with the results of the RDT and those positive were given treatment with Coartem® (artemeter-lumefantrine), the recommended first-line treatment for malaria in Kenya [After the interview, participants who consented to be tested were finger pricked and a rapid diagnostic test .Of the 1,069 participants 111 (10.4%) individuals reported to have travelled out of Nairobi in the eight weeks preceding the interview. Of the 1,069 residents interviewed, 87 (8.9%) refused to provide a finger prick blood sample. 52% of the 1,069 interviewees were female and 63% were aged 15 years or older.P. falciparum RDT result at the time of interview. After six separate examinations of 200 high power magnification fields of the thick smears of the three RDT positives, all were defined as microscopically negative. Among all the slides taken during the survey 12 (1.2%) were damaged before being read and an additional 18 (1.9%) were poorly stained and unable to be read. Of the 953 remaining readable slides all were reported as negative for the presence of sexual or asexual forms of P. falciparum, Plasmodium malariae and Plasmodium ovale.Three (0.3%) participants were reported to have a positive Of the 1,069 participants 110 (10.3%) reported owning a bed net and 103 (9.6%) sharing with another member of the household. The majority (55.8%) of the bed nets owned by study participants were treated with insecticide; of which 38% were pre-treated and 17.8% treated by the owner. Among participants who reported to have access to a bed net (owning and sharing), 88.7% slept under the net the night preceding the survey.Information on self-reported fever was missing for 31 (0.3%) individuals. Of the remainder, 34 (0.3%) said that they did not know whether they had had a fever or not and, therefore, analysis was restricted to 1,004 interviewees. Overall 170  fever episodes in the last 14 days were reported among 136 people. Thirty people reported more than one febrile event and four people reported more than two febrile events. The highest reported fever rate was reported for children less than five years by their parents or guardians . Self-reported fever prevalence was lower among age groups 5–9 years  and 10–14 year  however, period prevalence increased to 19.8% (95% CI: 16.7%–23.1%) among participants aged 15 years or older (Table Participants reported that fevers were diagnosed mostly by a feeling of the body being hot (71.8%) but some of the cases were diagnosed in health facilities (27.1%). 1.2% of the fever episodes were diagnosed at home using a thermometer.Among the 170 febrile episodes reported, 8 (4.7%) did not have information on treatment. Of the 162 febrile episodes with information of treatment, no action was taken to manage the event for 74 (45.7%). Among the 88 fevers for which an action was taken 85 reported using a modern medicine and three used herbs obtained from traditional healer . Of the identified anti-malarial drugs used to manage 53 fevers 16 were sourced from the informal retail sector as over-the-counter medicines, 12 were prescribed and purchased from a private clinic or pharmacy and 23 were prescribed at the formal, public sector. Within the public sector the tendency was to prescribe SP or AQ and in only four of consultations within this sector and for which the respondent could remember or show evidence of drug-type did the febrile patient take AL.Of the medicines prescribed or self-purchased 32 (36.4%) of interviewees could not adequately describe the medicines they used nor did they have any packaging remaining for interviewers to ascribe the formulation to either an anti-malarial preparation or other oral medications. Among the 56 febrile events for which a medicine type could be established 31 (55.4%) were identified as branded forms of sulphadoxine-pyrimethamine (SP) and 16 (28.6%) were classified as a formulation of amodiaquine (AQ). Interestingly one fever was treated with chloroquine, an anti-malarial drug discontinued as recommended therapy eight years earlier, and one fever was treated with oral quinine, a drug reserved for second-line therapy. Only four fevers, all among adults, were managed using the nationally recommended first-line treatment for malaria, CoartemAn. gambiae s.s, An. funestus or Anopheles arabiensis. The results do not preclude the possibilities of local malaria transmission in this area, but strongly suggest that should transmission occur it would be at exceptionally low levels, representing very rare events. Furthermore, the findings should not be interpreted as excluding the possibilities of malaria transmission in other parts of Nairobi with different settled population densities and surrounding environments, a wider series of investigations would be required to characterize malaria risks across Nairobi's complex and vast limits. There is also a need for an entomological study to characterize the mosquito population and assess the sporozoite rate to able to rule out any local transmission.There remains some controversy over the likelihood and extent of authochronus malaria transmission within Nairobi city ,34. NairImportantly, however, the observation that there were no individuals who were harbouring infection does provide a context to understand the appropriateness of fever case-management practices in this community. Fever was a relatively common complaint in two-week recall period among residents of Korogocho, 12.7% of the sampled population complained of having had a fever and 170 febrile events were recorded among 136 people. Consistent with other fever surveys across Africa ,6 many fOnly four of the anti-malarial treatments that could be identified were AL, the nationally recommended first-line treatment, and the majority were either mono-therapies drugs not recommended in the national treatment guidelines (AQ) or therapies abandoned because of wide-spread resistance (SP and chloroquine). Regardless of appropriate treatment regimens the combined findings of zero parasite prevalence and high anti-malarial use suggests that community and prescriber/dispenser education on the need to establish the diagnosis using parasitological detection is critical in this urban setting. These pleas have been made to support fever case management in other urban settings ,6,35) anShould diagnosis based on systematic testing for malaria parasites be promoted as a strategy in Nairobi to reduce inappropriate malaria treatment it is important to extend its implementation and support to sectors beyond the formal public sector. Over 52.8% of all fevers treated with a documented anti-malarial in the present study used medicines obtained from the private and retail sectors. These prolific and hard to regulate service providers must be engaged in strategies to reduce anti-malarial drug use in areas where malaria transmission is negligible or non-existent. Failure to do so will perpetuate the dogma that all fevers in all communities are due to malaria, which may be good for private sector market sales but poor for public health practice.The study could not demonstrate any evidence of malaria in Korogocho, an urban slum in the centre of Nairobi; however, fever was a common complaint and often treated as malaria with anti-malarial drugs. Strategies to reduce the population exposure to anti-malarial drugs should be a priority for all clinical services and drug vendors among the poor communities that make up Nairobi's informal settlements.The authors declare that they have no competing interests.YY designed and implemented the study. He performed the statistical analysis and drafted the manuscript; NM participated in the design and writing of the manuscript. RN took part in statistical analysis and writing of the manuscript; SO participated in writing the manuscript; RWS contributed to the study design, statistical analysis and drafting of the manuscript. All authors read and approved the final manuscript.Treatment of self-reported episode by participants.Click here for file