Plasmodium falciparum parasites, implications in the aetiology of common conditions that prevail in individuals of African origin, and a wealth of pharmacological knowledge, the stimulatory G protein (Gs) signal transduction pathway presents an exciting target for anti-malarial drug intervention. Having previously demonstrated a role for the G-alpha-s gene, GNAS, in severe malaria disease, we sought to identify other important components of the Gs pathway. Using meta-analysis across case-control and family trio  studies of severe malaria from The Gambia and Malawi, we sought evidence of association in six Gs pathway candidate genes: adenosine receptor 2A (ADORA2A) and 2B (ADORA2B), beta-adrenergic receptor kinase 1 (ADRBK1), adenylyl cyclase 9 (ADCY9), G protein beta subunit 3 (GNB3), and regulator of G protein signalling 2 (RGS2). Our study amassed a total of 2278 cases and 2364 controls. Allele-based models of association were investigated in all genes, and genotype and haplotype-based models were investigated where significant allelic associations were identified. Although no significant associations were observed in the other genes, several were identified in ADORA2A. The most significant association was observed at the rs9624472 locus, where the G allele (∼20% frequency) appeared to confer enhanced risk to severe malaria . Further investigation of the ADORA2A gene region is required to validate the associations identified here, and to identify and functionally characterize the responsible causal variant(s). Our results provide further evidence supporting a role of the Gs signal transduction pathway in the regulation of severe malaria, and request further exploration of this pathway in future studies.With the functional demonstration of a role in erythrocyte invasion by  Plasmodium falciparum, the most virulent species of human malaria parasite, and their impact at the disease level  and severe malaria disease using a case-control association approach GNAS locus warranted further investigation of the Gs pathway to enhance our understanding of the disease mechanism(s) involved and to identify other components which might present suitable targets for anti-malarial drug intervention. Indeed, owing to implications in common disorders such as hypertension and diabetes, the pharmacology of the Gs pathway is well defined www.sequenom.com) and the opportunity for lower cost and higher throughput genotyping (∼10-fold increase in SNP number) relative to the previous Sequenom (hME) platform, we were able to undertake economic genotyping in a larger selection of genes, allowing further interrogation of the Gs pathway to identify other disease-regulatory candidates that might present suitable targets for anti-malarial drug intervention.Harrison and colleagues demonstrated a role for the host erythrocyte stimulatory G protein (Gs) pathway in erythrocyte invasion G protein pathways provide a means for intracellular components to respond appropriately to extracellular stimuli Negative feedback processes regulate the Gs pathway in order to prevent excessive cell signalling. Following dissociation from the G-alpha-s subunit, the beta-gamma dimer binds and translocates a G protein coupled receptor kinase such as the beta-adrenergic receptor kinase 1 (ADRBK1) to the membrane The inhibitory G (Gi) protein pathway also opposes the effects of the Gs pathway, having an inhibitory effect on adenylyl cyclase and cAMP production. A well studied component of this pathway is the Gi beta subunit 3 (GNB3), which has been implicated in complex disorders such as hypertension ADORA2A, ADORA2B), beta-adrenergic receptor kinase 1 (ADRBK1), regulator of G protein signalling 2 (RGS2), adenylyl cyclase 9 (ADCY9), and G-protein-beta 3 (GNB3). A meta-analysis approach was chosen to enhance sample size whilst allowing for heterogeneity between studies. Owing to the challenges of collecting severe malaria cases in African study sites, large sample sizes are difficult to obtain Using meta-analysis pooled across four association studies , Malawi family trio , Gambian case-control and Gambian trio), we tested SNP associations with severe malaria in six genes related to the Gs pathway; adenosine receptor alpha 2A and 2B (P<0.001) and failure rates below 10% (results not presented).Allele frequencies for the assays  in the Gambian and Malawian studies are presented in ADORA2A SNPs, rs9624472 and rs5751876, demonstrated significant association with severe malaria, as illustrated in ADORA2A loci, rs2267076 and rs3761422, demonstrated a trend of association (0.05≤P≤0.1). A summary of the single-locus associations identified in ADORA2A are presented in P = 0.001), with increased susceptibility to severe malaria conferred by the G allele [OR = 1.22 (1.09–1.37)] in an additive manner . Upon moving up- or down-stream of the rs9624472 locus, the strength of associations weakened. Approximately 2.5 kb downstream of rs9624472, the rs5751876 C allele conferred significant susceptibility to severe malaria  with an apparent recessive genotype effect . Four kilobases upstream of rs9624472, the rs2267076 T allele demonstrated a trend of susceptibility to severe malaria  and, further upstream (8 kb), the rs3761422 T demonstrated a weaker trend of susceptibility to severe malaria . No significant associations were demonstrated in the other candidate genes  and rs5751876 (C) alleles, accordingly demonstrated significant risk to severe malaria . Furthermore, the A2A-1 haplotype which comprises the protective alleles at rs9624472 (A) and rs5751876 (T) demonstrated significant protective from severe malaria . With a less significant P-value, the A2A-2 haplotype, which comprises the rs9624472 A and rs5751876 T alleles demonstrated risk to severe malaria .P. falciparumGNAS, in severe malaria disease ADORA2A in severe malaria, we demonstrate significant association with this gene using meta-analysis across case-control and TDT studies from The Gambia and Malawi.Owing to implications in the aetiology of erythrocyte invasion by ADORA2A gene whereby single-locus associations strengthened in significance with increasing proximity to rs9624472, at which the most significant association was observed (P = 0.001). At this locus, the G allele demonstrated enhanced susceptibility to severe malaria. With respect to the rs9624472 locus, 2.5 kb downstream, the rs5751876 C allele demonstrated susceptibility to severe malaria (P = 0.024); 4 kb upstream, the rs2267076 T allele demonstrated a trend of susceptibility to severe malaria (P = 0.051); and 8 kb upstream, the rs3761422 T allele demonstrated a weak trend of susceptibility to severe malaria (P = 0.096). Using haplotype-based analysis, the two most significant ADORA2A haplotype associations concurred with the single-locus associations at rs9624472 and rs5751876; A2A-3 (CCGC) risk to severe malaria (P = 0.002), and A2A-1 (CCAT) protection from severe malaria (P = 0.003). Association at A2A-2 (TTAT) demonstrated discordance with the single-locus effects of rs9624472 A and rs5751876 T but at lower significance (P = 0.019). Further investigation of this region is required to identify the true functional variant responsible for the observed associations.We identified a pattern of association in the ADCY9, ADRBK1, ADORA2B, GNB3 or RGS2. Further investigation of these genes in other populations and with greater sample size should provide more conclusive evidence on their role in severe malaria pathogenesis.We used meta-analysis across the Gambian and Malawian TDT and case-control studies in order to enhance our sample size. Across the four studies, we amassed a total of 2278 cases and 2364 controls. A rough estimation of the power of this study to detect the rs9624472 association was 97% at the 5% significance threshold P≈0.008. At this threshold, the rs9624472 allelic association (P = 0.002), and the A2A-1 (P = 0.003) and A2A-3 (P = 0.002) haplotype associations remain significant.The results presented here have not been corrected for multiple testing. The application of the Bonferroni correction in this study is debatable as the candidate genes are members of the same biological pathway and, thus, partially interdependent. Due to LD, the SNPs within each gene also exert a measure of interdependence. If we correct the associations observed here by the number of genes tested (6), the threshold for significance is ADORA2A associations presented here may represent a complex network of interactions with other human genetic variants in the Gs pathway such as the GNAS locus and, perhaps, other pathways. Indeed, it is feasible that inter-gene interactions may have masked our ability to detect possible associations at the other loci investigated here. However, with regard to multiple hypothesis testing, our study was not sufficiently powered to investigate the numerous possible epistatic interactions between the genes investigated here. In addition to other host genetic factors, it should also be considered that a number of parasite genetic factors and environmental factors may interact with variants in the Gs pathway and, thus, influence the outcome of the disease trait. Indeed, in the context of erythrocyte invasion, which presents an intimate interaction between host and parasite proteins, it is most likely that the composite of numerous parasite and host genetic variants involved in the process ultimately determines the overall rate of invasion. Unfortunately, owing again to limited sample size, this study was underpowered to investigate such variables.As a component of a signal pathway, it should be considered that the ADORA2A associations presented here might elicit a pathogenic effect in malaria disease remain unclear. ADORA2A and ADORA2B are both adenosine-activated GPCRs, the latter exhibiting lower affinity for this agonist The mechanism by which the ADORA2A association reported in this study, indicates that the Gs signal transduction pathway is involved in the regulation of malaria, and more specifically, in the severe, life threatening manifestations of this disease ADORA2A gene region is necessary to validate the associations identified here, and to identify and functionally characterize the causal variant(s) responsible for the signals of association. This should enhance our understanding of how ADORA2A is involved in the regulation of severe malaria pathology.Accumulating evidence, including the The Gambia Government/Medical Research Council Ethical Committee approved the Gambian samples in this study. The Malawian samples in this study were approved by the College of Medicine Research and Ethics Committee. All samples were obtained with informed written consent from a parent or guardian.P. falciparum asexual parasitaemia. CM was defined by a Blantyre coma score ≤2, indicating unrousable coma not attributable to convulsions, hypoglycaemia or meningitis in a patient with P. falciparum parasitaemia Nuclear software package http://www.hapmap.org) was used to test the accuracy of the genotyping assays and was extracted from lymphoblastoid cell lines provided by the Coriell repository .Individuals were recruited in a clinical setting. Children under the age of 12 years presenting with severe malaria at the Royal Victoria Hospital in Banjul, The Gambia, and the Queen Elizabeth Central Hospital in Blantyre, Malawi, were enrolled as cases. Severe malaria was defined as severe malarial anaemia (SA), cerebral malaria (CM), other severe complications such as respiratory acidosis, and fatalities due to malaria infection. The threshold for SA was a haemoglobin concentration <5 g/dl in the presence of ADRB1 and ADRB2), which have previously been implicated in the regulation of P. falciparum erythrocyte invasion Genes were selected on account of one or more of the following properties; implication in common disorders which prevail in individuals of African origin possibly as a result of malaria selection http://www.hapmap.org).SNP selection was balanced between literature-based evidence of functionality and apparent signatures of selection. This was narrowed down to an economic subset of SNPs for which genotyping assays could be designed on the mass spectrometry iPLEX platform. Three measures were used to identify signatures of positive selection; Wright's Fst http://www.sequenom.com). Prior to genotyping on the Malawian and Gambian samples, the SNP assays were tested on the Yoruba HapMap parental DNA samples. Only assays with high concordance (>95%) to the official HapMap data (http://www.hapmap.org), genotype distributions conforming to Hardy-Weinberg equilibrium at the 0.1% significance threshold, and high call rates (≥90%) were genotyped on the malaria cases and controls.Genotyping was undertaken using Sequenom's mass spectrometry iPLEX gold platform (P<0.05). Individual SNPs were investigated using allele- and genotype-based models. Multi-allele tests were undertaken using haplotypes. Genotype associations were tested using pseudo case-control analysis with correction for HbS using the GenAssoc package (http://www-gene.cimr.cam.ac.uk/clayton/software/). Associations were corrected for the HbS locus, which exerts a strong influence on severe malaria outcome Meta-analysis was undertaken across all the case-control and family studies using the methods outlined in Kazeem and Farrall http://www-gene.cimr.cam.ac.uk/clayton/software/). Only haplotypes with SNPHAP probabilities ≥80% were included in association analysis. In the family trios, haplotypes were reconstructed using an Expectation Maximisation algorithm in R.In the case-control studies, haplotypes were reconstructed using SNPHAP . Z scores and corresponding P-values are presented for the pooled associations. Q scores and corresponding P-values are presented for Cochran's test of heterogeneity between the studies. Odds ratios refer to the minor allele at each locus. Cochran's Q-test for heterogeneity on 4 degrees of freedom. CC: Case-control.(0.30 MB RTF)Click here for additional data file.Table S2Severe malaria subphenotype frequencies. CM: Cerebral malaria. SA: Severe anaemia.(0.04 MB RTF)Click here for additional data file.Table S31Region investigated using haplosimilarity (HS) and extended haplotype homozygosity (EHH) tools for detecting signatures of selection.Properties of Gs pathway candidates. (0.03 MB RTF)Click here for additional data file.


Plasmodium falciparum incidence rate was estimated at 84/1,000 person-months, and its prevalence was estimated at over 40% in the two first surveys and 68.9% in the third survey. Multivariate analysis showed that girls and people living in closed houses had a lower risk of clinical malaria. Bed net use was associated with a lower risk of malaria infection. Conversely, children of families owing a pirogue were at higher risk of clinical malaria. Considering the high pyrethroids resistance, indoor residual spraying with either a carbamate or an organophospate insecticide may have a major impact on the malaria burden.Clinical malaria incidence was determined over 18 months in a cohort of 553 children living in a peri-urban area near Cotonou. Three cross-sectional surveys were also carried out. Malaria incidence showed a marked seasonal distribution with two peaks: the first corresponding to the long rainy season, and the second corresponding to the overflowing of Lake Nokoue. The overall  Malaria transmission and consequently, the disease burden may vary widely, even within a small geographical area.In Benin, malaria remains the first cause of attendance to the health centers, despite the control activities carried out by the National Malaria Control Program.The study was part of a longitudinal randomized trial carried out in southern Benin, West Africa, in three adjacent sites: Ladji, Awansori (Toweta 1), and Toweta 2, all located in a large suburban depression north of Cotonou bordering Lake Nokoue . The stuThe climate is subequatorial, with two rainy seasons (from April to July and from October to November) and two dry seasons (from December to March and from August to September). In 2003, monthly mean temperature varied between 23.6°C and 33.1°C, and the average relative humidity index was between 75.7% and 83.7%.27During the long rainy season (from April to July), the environment does not change radically, with some fresh water pools mostly disappearing a few weeks after the end of the rains. In contrast, during the second season, this area is subject to recurrent flooding because of the overflowing of Lake Nokoue as a result of the water influx coming through the Oueme River, the main affluent of the lake, after the rains in Northern Benin.26Anopheles gambiae s.s. although An. melas (4%) can also be found, is seasonal and lasts for about 8 months.Malaria transmission, mainly by In 2001, the local authorities and the population were informed about the study and its objectives. A census was carried out in 2002–2003. Houses were numbered, and information on the household's residents  were collected. A list of children 6–59 months old was produced according to the following criteria: absence of chronic illness with parents willing to participate in the study and unlikely to emigrate. Before inclusion, parents or legal guardians were asked to sign a written informed consent. Refusal to participate did not affect access to basic health services. Parents/guardians were instructed to attend the nearby health facility, where a physician was always available, whenever their child was sick. They were also asked to administer only drugs given or prescribed by the research physician to the child.From February to March 2003, all participants' households were mapped using a handheld Global Position System  with an accuracy goal of < 15 m. If children lived in the same house or in an immediately adjacent one, only one point was recorded. A structured questionnaire combined to direct observation was used by a team of research assistants trained for this purpose to collect information on demographic variables, malaria recognition, treatment, and prevention as well as housing and environmental condition.During the follow-up period, three cross-sectional surveys were carried out: the first from February to March 2003, the second from February to March 2004, and the third at the end of the flooding period between October and November 2004. During surveys, each child was examined by a physician, and a blood sample for hematology, the detection of malaria infection (microscopy), and later, genotyping was collected.Plasmodium falciparum asexual stages, regardless of parasite density. Children with a P. falciparum monoinfection, a parasite density between 1,000–200,000/μL, and without the following exclusion criteria .49The apparent difference in the last survey (October–November 2004) between the relatively low number of children with clinical malaria and the high prevalence of infection may be explained by the active detection of clinical cases (two times per week) and their prompt treatment.Use of bed nets was relatively common, although most of them were probably not treated with insecticide. The protective effect of both treated and untreated mosquito nets against malaria infection is well-documented.56An. gambiae, the principal African malaria vector, with ceiling and closed eaves has been recently reported.In the early 20th century, improved housing and screening were considered as valid methods for controlling malaria.Few families owned a pirogue, but this was a strong risk factor for clinical malaria. In this impoverished community, mostly composed by immigrants, fishing has ceased to be a major economic activity, and pirogues are not used anymore but left outside the houses. Nevertheless, unused pirogues probably play a role as a breeding site after being filled with water during the rainy season. This should be an easily solvable problem, although unlikely to have a major impact on transmission.In conclusion, malaria transmission in this peri-urban area is substantial and perennial, with two peaks: the first corresponding to the long rainy season, and the second corresponding to the overflowing of Lake Nokoue caused by the increased water flow after the rains in Northern Benin. Considering the high resistance to pyrethroids and the high population density, indoor residual spraying with either a carbamate or an organophospate insecticide may have a major impact on the malaria burden. Improvement of the population's socio-economic status and housing conditions could reduce it further.


Results are reported on the sensitivity of various gynaecological tumour cell lines to 5-aminolaevulinic acid-induced protoporphyrin IX-sensitised photodynamic therapy (ALA-PDT) in vitro. All cell lines tested accumulated ALA-induced protoporphyrin IX (PpIX) and demonstrated good sensitivity to ALA-PDT. Localisation of PpIX in the mitochondria was demonstrated by fluorescence microscopy. Subcellular damage following ALA-PDT was observed using transmission electron microscopy. This damage was localised initially to the mitochondria, with damage to membranes and the nucleus and complete loss of intracytoplasmic organisation being observed subsequently. There was no apparent difference in ALA-PDT response between a multidrug-resistant ovarian carcinoma cell line and its parent line. These results indicate that ALA-PDT has potential for application to therapy of gynaecological malignancies.


A polymorphic malaria parasite antigen, merozoite surface protein 3 (MSP3), is among the blood stage malaria vaccine candidates. It is believed to induce immunity through cytophilic antibodies that disrupt the process of erythrocytes invasion by merozoites. This study aimed at assessing natural acquisition of antibodies to MSP3 in individuals living in an area with different malaria transmission intensity in preparation for malaria vaccine trials.The study was conducted in individuals aged 0-19 years from villages located in lowland, intermediate and highland strata in Korogwe district, northeastern Tanzania. Blood samples from 492 study participants were collected between May and June 2006 for malaria diagnosis and immunological investigations. Reactivity of MSP3 to different types of antibodies  were analysed by Enzyme Linked ImmunoSorbent Assay (ELISA).Plasmodium falciparum infection was associated with higher IgG3 levels (p = 0.008). Adjusting by strata and age in individuals who had positive blood smears, both IgG and IgM were associated with parasite density, whereby IgG levels decreased by 0.227  while IgM levels decreased by 0.165 .Malaria parasite prevalence was higher in the lowland (50%) compared to the intermediate (23.1%) and highland (9.8%) strata. Immunogloblin G subclasses 1 and 3 (IgG1 & IgG3), total IgG and IgM were found to increase with increasing age. IgG3 levels were significantly higher than IgG1 (p < 0.001). Furthermore, Individuals with higher levels of IgG3 might be partially protected from malaria infection. Higher levels of total IgG and IgM in highlands might be due to low exposure to malaria infection, recent infection or presence of cross-reactive antigens. Further studies of longitudinal nature are recommended. Data obtained from this study were used in selection of one village (Kwashemshi) for conducting MSP3 phase 1b malaria vaccine trial in Korogwe. Malaria is one of the most serious public health problems in the world, affecting tropical developing countries and killing young children mainly . RelatioPlasmodium falciparum offers good prospects for a potent vaccine whereby epidemiological and laboratory data suggest that, immune responses targeting this antigen is associated with protection [in vitro [Currently, a number of malaria vaccines candidates are at different stages of clinical development. Promising results have been obtained with some of the vaccine candidate -8. Amongotection . It is botection . Antibodotection , by actiotection  or by inotection . Immunogin vitro . Individin vitro ,14. Clinin vitro ,16.The aim of this study was to employ a standardized ELISA assay to assess natural acquisition of antibodies to MSP3 in individuals living in an area with different malaria transmission intensity in preparation for malaria vaccine trials.This study was conducted in Korogwe district, north-eastern Tanzania. The district is about 100 kilometers inland from Tanga. The population of Korogwe district in year 2002  was estimated to be 261,004, with a growth rate of 1.4% per annum. The area is topographically stratified into three strata namely; lowland, intermediate and highland. The strata are characterized by marked differences in malaria transmission profiles. The altitude of Korogwe district ranges from 300 - 1,200 meters above sea level (mASL). In these areas, malaria transmission decreases with increasing altitude ,18 and iP. falciparum is the predominant malaria species accounting for a little over 90% of all infections, the rest being P. malariae and P. ovale [In Korogwe district, the estimated average entomological inoculation rate (EIR) ranges in the past from 30-100 infective bites per person per year . In lowlP. ovale . In KoroP. ovale .Study villages were grouped into three strata namely; lowland, intermediate and highland based on the altitude and level of transmission. Mng'aza village was categorized as lowland stratum, Kwashemshi village as intermediate while Magundi, Kwamhanya and Vugiri villages were grouped into highland stratum. The study involved individuals aged 0-19 years. The details of the study villages are as explained elsewhere [This study was part of a longitudinal project being conducted in Korogwe. Samples used in this study were obtained from a cross sectional malaria survey conducted in May and June 2006 . Plasma Ethical clearance was granted by the Medical Research Co-ordinating Committee of the National Institute for Medical Research and the Ministry of Health and Social Welfare, Tanzania. Prior to commencement of the study, sensitization meetings were held in each village to explain the objectives and methodology of the study as well as seeking for community consent. Informed consent was obtained in writing from all individual participants or parents/guardians in case of children during the survey as described elsewhere .® machine. The remaining blood was centrifuged at 2,000 revolutions per minute (rpm) to obtain plasma, packed red blood cells (RBCs) and buffy coat. Plasma was stored at -80°C and later analyzed for immunological assays.Clinical examination was done in all 492 individuals at a central point in each village. Venous blood (about 3 ml) from each individual was drawn into vacutainer containing EDTA anticoagulant. Out of the 3 ml of blood collected, 8 μL was used for preparation of thick and thin blood smears for malaria parasite diagnosis, species identification and quantification. Also, 10 μL of blood of each individual was used for estimation of haemoglobin using Haemocuehttp://www.amanet-trust.org. Briefly, wells of the microtiter plates  were coated with 100 μL at 0.5 μg/ml of a recombinant protein covering the nonpolymorphic carboxy-terminal region of the MSP3 protein (aa 212 - 380) [2O2 with tetramethylbenzidine (TMB) substrate . Absorbance was read at 450 nm with reference at 650 nm. For each antibody isotype, a calibration (standard) curve was obtained from two-fold serially diluted purified myeloma proteins coated directly to duplicate wells of the first two columns of each plate. First well concentrations in ng/ml were: 100 (IgG & IgG3), 500 (IgG1) and 250 (IgM). Four pooled plasma from clinically immune adult Liberians and two Danish donors never exposed to malaria were used as positive and negative controls respectively. Antibody units were calculated using the Afro Immuno Assay (AIA) Network protocol of 2005 volume 1.01 excel based curve fitting program as described elsewhere [Plasma samples were used to determine levels of IgM, total IgG and IgG subclasses (IgG1 & IgG3) responses to MSP3 malaria vaccine candidate by Enzyme Linked ImmunoSorbent Assay (ELISA) technique as described elsewhere All data were entered and verified in Microsoft Access while statistical analysis was done using R version 2.8.0 and STATA software version 8 . All antibody levels were transformed to normality using the log transformation. Malaria parasite densities were converted from parasites per 200 white blood cells (WBCs) to parasite per microliter (μL) by multiplying by 40. A multiple regression analysis was used to assess variables associated with antibody levels. A p-value < 0.05 was considered significant.P. falciparum parasite prevalence in the three strata were 50.0% (95%CI: 37.4 - 62.6) in the lowland stratum, 23.1% (95%CI: 16.0 - 31.7) in the intermediate stratum and 9.8% (95%CI: 6.7 - 13.7) in the highland stratum. Intermediate stratum had low malaria parasite prevalence but its geometric mean parasite density (GMD) among the positive cases was the highest among the three strata . Bednet use was highest (91.4%) in the intermediate stratum. Spleen rate was higher in the lowland (30.8%) compared to the other two strata.Table P. falciparum parasites .The increase in IgG3 levels with age was 0.190  and was slightly higher than in IgG1. Individuals who had P. falciparum infection had significantly high levels of total IgG by log 2.552 . Adjusting by strata and age among individuals who had positive blood smears, IgG levels were associated with parasite density, whereby, the log mean levels of IgG decrease was 0.227 .Individuals living in highlands had higher total IgG antibody levels compared to negative controls while individuals aged 10-19 years had higher mean levels than positive controls  for conducting MSP3 phase 1b malaria vaccine trial in Korogwe.2O2: hydrogen peroxide; TMB: tetramethlylbenzidine; nm: nanometer; ng/ml: nanogram per millilitre; μg/ml: microgram per millilitre; aa: amino acid; AIA: Afro Immuno Assay; AIC: akaike information criteria; n: number; mASL: metres above sea level; Hb: haemoglobin; g/dL: grammes per decilitre; AU: arbitrary unit; WBCs: white blood cells; Hb: haemoglobin; temp: temperature; <: Less than; =: equal to; NC: negative controls; PC: positive controls.ml: millilitre; RBCs: red blood cells; IgG0: total immunoglobulin G; IgG1: immunoglobulin G subclass 1; IgG3: immunoglobulin G subclass 3; IgM: immunoglobulin M; EIR: entomological inoculation rate; ELISA: enzyme linked immunoSorbent assay; EDTA: ethylenediaminetetraacetic acid; rpm: revolutions per minute; μL: microlitre; °C: degrees Celsius; PBS: phosphate buffered saline; CA: Canada; UK: United Kingdom; DK: Denmark; HThe authors declare that they have no competing interests.MDS assisted with proposal writing, study design, blood sample collection, laboratory analysis of blood samples, data interpretation and manuscript writing. BPM assisted with study design, data analysis, data interpretation and manuscript writing. MDS assisted with blood sample collection, laboratory analysis of samples and manuscript writing. JPL and MML assisted with proposal writing, study design, supervision of sample analysis, data interpretation and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/55/prepub


IL4-590 gene polymorphism has been shown to be associated with elevated levels of anti-Plasmodium falciparum IgG antibodies and parasite intensity in the malaria protected Fulani of West Africa. This study aimed to investigate the possible impact of IL4-590C/T polymorphism on anti-P. falciparum IgG subclasses and IgE antibodies levels and the alteration of malaria severity in complicated and uncomplicated malaria patients with or without previous malaria experiences.The P.falciparum IgG subclasses and IgE antibodies in plasma of complicated and uncomplicated malaria patients with or without previous malaria experiences were analysed using ELISA. IL4-590 polymorphisms were genotyped using RFLP-PCR. Statistical analyses of the IgG subclass levels were done by Oneway ANOVA. Genotype differences were tested by Chi-squared test.Anti-IL4-590T allele was significantly associated with anti-P. falciparum IgG3 antibody levels in patients with complicated (P = 0.031), but not with uncomplicated malaria (P = 0.622). Complicated malaria patients with previous malaria experiences carrying IL4-590TT genotype had significantly lower levels of anti-P. falciparum IgG3 (P = 0.0156), while uncomplicated malaria patients with previous malaria experiences carrying the same genotype had significantly higher levels (P = 0.0206) compared to their IL4-590 counterparts. The different anti-P. falciparum IgG1 and IgG3 levels among IL4 genotypes were observed. Complicated malaria patients with previous malaria experiences tended to have lower IgG3 levels in individuals carrying TT when compared to CT genotypes (P = 0.075). In contrast, complicated malaria patients without previous malaria experiences carrying CC genotype had significantly higher anti-P. falciparum IgG1 than those carrying either CT or TT genotypes .The IL4-590C or T alleles participated differently in the regulation of anti-malarial antibody isotype profiles in primary and secondary malaria infection and, therefore, could play an important role in alteration of malaria severity.The results suggest that  Plasmodium falciparum specific antibodies play a critical role in immune protection against asexual blood stages of the parasite, in which anti-P. falciparum IgG antibodies involved in reducing severity of the disease [P. falciparum, whereas IgG2 and IgM are not, and even suggested to block protective effects of the former Ig subclasses [P. falciparum IgE, as well as total IgE antibodies, which are elevated in individuals exposed to malaria in Thailand, have been implicated to play a pathogenic role during malaria infection [P. falciparum IgE levels in asymptomatic individuals in Tanzania were associated with a reduced risk for subsequent malaria disease [Anti- disease . In partnfection . In cont disease .IL4-590 C/T transition in IL4 promoter was shown to influence the IL4 production as well as the elevated levels of total IgE [IL4-590T allele corresponded to IL4-524T and IL4-589T alleles in an alternative numbering scheme [IL4-524T allele was found to be associated with the elevated levels of anti-P. falciparum IgG antibodies and protection against malaria [IL4-589T allele was associated with the elevated levels of total IgE in children with severe malaria living in Burkina Faso [IL4-590T allele and have been living in Ghana [IL4-590T allele in regulating antibody profiles and malaria severity is controversial. In mice infected with Plasmodium chabaudi chabaudi, the specific IgG2a and IgG3 antibodies are predominant in primary polyclonal B cell activation. At that point, IFN-γ is markedly stimulated, while IL4 is moderately enhanced. In secondary IgG1-restricted response, only IL4 is produced [IL4-590 C/T polymorphism influences the balance between IL4 and IFN-γ and thus, could alter the severity of malaria [P. falciparum IgG subclasses and IgE antibodies, the results showed different regulation in patients with complicated and uncomplicated malaria [The regulation of antibody profiles in patients with complicated and uncomplicated malaria is still largely unknown. In both human and mice, different cytokines are thought to induce particular Ig isotypes. In humans, IL4 regulates B cells to express the γ1, γ2, γ3, γ4, α1, α2 and ε, germline gene transcripts (GLT) and to secrete the corresponding proteins . IL10 protal IgE ,13. The g scheme -16. In t malaria , while Iina Faso . Conversproduced . Accordi malaria . When th malaria .IL4-590 C/T single nucleotide polymorphism (SNP) on the production of anti-P. falciparum -IgG1, -IgG2, -IgG3, IgE antibodies and to determine whether such polymorphism and specific antibodies levels were associated with previous malaria experiences and clinical outcome of the disease in people living in a malaria endemic areas in Thailand.Therefore, it is of interest to evaluate the impact of P = 0.849), gender (P = 0.137) and nationality, i.e Thai, Burmese, Mon and Karen (P = 0.614). The median age was 25 years (ranged 14-67) for complicated and 24 years (ranged 13-65) for uncomplicated malaria. Based on the interview and clinical records, 27.3% of patients with complicated and 55% of those with uncomplicated malaria had previous malaria experiences. The number of previous malaria episodes in these patients varied widely, describing mostly 1-2 episodes of malaria (84/125), 3-8 episodes (27/125) and many episodes (12/125). However, the duration of malaria infection upon admission and the previous malaria episodes among patients were far different, varying from one month to more than 10 years (mostly between one month and two years). However, both groups of patients had acute malaria infection upon admission. Therefore, complicated malaria patients with or without previous malaria experiences  and uncomplicated malaria patients with or without previous malaria experiences  were identified. Clinical manifestations of complicated and uncomplicated malaria were defined according to World Health Organization criteria [P. falciparum in blood smears, while other causes of coma were excluded. Severe malaria but not cerebral  was defined as patients exhibiting one or more of the following signs, hyperparasitaemia , hypoglycaemia (glucose <22 nmol/l), severe anaemia (haematocrit <20% or haemoglobin <7.0 g/d) or increased serum level of creatinine more than 3.0 mg/dl. The patients with complicated malaria comprised of all severe forms including cerebral malaria. Uncomplicated malaria was characterized by a positive blood smear and fever without other causes of infections and no manifestations of severe malaria as described. The patients were transported for admission to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok. The study has been ethically approved by the Institute Review Board of the Faculty of Tropical Medicine, Mahidol University, Bangkok and the fully informed consent was obtained from all patients. Other details about the patients are delineated in Table The same sets of patients previously reported were studied ,19. Briecriteria . CerebraBlood samples from patients with complicated and uncomplicated malaria were collected in EDTA sterile tubes before treatment on admission. The plasma and packed cells were separated and frozen at -20°C until use.P. falciparum parasites per ml of blood obtained from the number of parasites observed per 1,000 erythrocytes in thin film or per 200 leucocytes in thick film.Thick and thin films were made for microscopic examination after standard Giemsa staining. Parasite densities were determined by calculating P. falciparum strain F32 was used to prepare the antigens for malaria antibodies detection. The late stages of P. falciparum infected erythrocytes were enriched by 60% Percoll and sonicated as described [The laboratory escribed .P. falciparum -IgG1, -IgG2, -IgG3 and IgE antibodies levels were determined by enzyme-linked immunosorbent assay (ELISA) as described previously [Anti-eviously  with someviously . NegativIL4-590 C/T polymorphism was detected by restriction fragment length polymorphism analysis as previously described [DNA was extracted from buffy coats by phenol-chloroform extraction . Genomicescribed .t-test or Mann Whitney U test. The highest fever, haemoglobin, creatinine, and glucose levels between the two patient groups were tested by unpaired t-test, while the other characteristics were tested by Mann Whitney U test. The Chi-squared test was used to compare allele and genotype frequencies between the two patients groups. One-way ANOVA and unpaired t-test after log transformation were used to evaluate the significant associations between anti-P. falciparum IgG subclasses or IgE antibodies and the IL4-590 genotypes. The IgG subclasses and IgE were taken in the extreme 3 tiles. The genetic distribution of the IL4-590 genotypes between the low and high quartile was compared using Chi-squared test. A P-value of less than 0.05 was considered to be significant.The data were analysed using SPSS or StatView computer software. The differences in characteristics of the patients with complicated and uncomplicated malaria were analyzed by unpaired IL4-590 T allele in patients with complicated malaria did not differ from those with uncomplicated malaria (0.72 in both groups). When the allele and genotype frequencies in patients with or without previous malaria experiences were explored, the allele and genotype frequencies between complicated and uncomplicated malaria groups either with or without previous malaria experiences did not differed   and uncomplicated malaria (UCM) patients are illustrated in Table P. falciparum IgG3 were significantly different among individuals carrying IL4-590 TT, CT and CC genotypes (P = 0.031), while the levels of anti-P. falciparum IgG1 tended to be significant among IL4-590 genotype (P = 0.095). To study the impact between IL4-590 genotypes on anti-P. falciparum IgG1 and IgG3 levels, the antibody levels in individuals carrying different genotypes were compared by TT to CT, TT to CC and CT to CC genotypes. As shown in Table P. falciparum IgG1 levels were significantly different between individuals carrying TT genotype compared to those carrying CC genotype (median 18.04 vs 223.80: P = 0.025) and between individuals carrying CT genotype compared to those carrying CC genotype . In contrast, the anti-P.falciparum IgG3 levels were significantly different only between individuals carrying TT compared to those carrying CT genotype . When complicated malaria patients with or without previous malaria experiences  were analysed separately, similar patterns were seen by the anti-P. falciparum IgG1 levels were significantly different between individuals carrying TT and CC genotypes (median 8.54 vs 223.80: P = 0.002) as well as between CT and CC genotypes (median 13.94 vs 223.80: P = 0.004). Interestingly, these observations were seen only in complicated malaria patients without previous malaria experiences. While the difference in anti-P. falciparum IgG3 levels between patients carrying TT and CT genotype tended to be significant in complicated malaria patients with previous malaria experiences (median 1.32 vs 5.62: P = 0.075). No such tendency could be observed in the levels of anti-P. falciparum IgG2 and IgE in complicated malaria patients either with or without previous malaria experiences. Among uncomplicated malaria patients carrying TT, CT or CC genotypes, the levels of anti-P. falciparum-IgG1, -IgG2, -IgG3 and IgE did not differ.The anti -P. falciparum IgG subclasses and IgE were associated with certain IL4-590 genotypes and whether the distribution of the C and/or T allele differed between patients with complicated and uncomplicated malaria either with low or high specific antibody levels, the antibody profiles were divided into three tiles, i.e. those with low, medium and high antibody levels. This division was based on the findings that there was a great individual variation in antibody responses to the crude plasmodium antigens. Because of the low numbers of homozygotes for the IL4-590 C allele frequency, the individuals homozygous and heterozygous for C alleles (CC+CT) were pooled and compared with those homozygous for T alleles. In complicated malaria, individuals homozygous for T alleles had significantly lower anti-P. falciparum IgG3 antibody (P = 0.0054), while no significant difference was found in uncomplicated malaria (P = 0.079). Conversely, the IL4-590 genotypes were not significantly associated either with anti-P. falciparum -IgG1, -IgG2 or with anti-P. falciparum IgE antibodies when low and high tiles in complicated and uncomplicated malaria were compared  (P = 0.0156) while higher anti-P. falciparum IgG3 antibody levels were seen in uncomplicated malaria patients with previous malaria experiences (UCME) (P = 0.0206). In contrast, no such associations could be found when the IL4-590 genotypes were compared between complicated malaria patients without previous malaria experiences (CMN) and uncomplicated malaria patients. Conversely, high anti-P. falciparum IgG1 antibody levels seem to be associated with the IL4-590TT genotype in uncomplicated malaria patients with previous malaria experiences (UCME) (P = 0.1219) To investigate whether the anti-P = 0.015 while hiIL4-590 genotypes could differently regulate anti-P. falciparum -IgG1 and -IgG3 responses. The association between individuals homozygous for IL4-590 T alleles and the levels of anti-P. falciparum IgG3 was found only in patients who had previous malaria experiences with low levels in complicated malaria and high levels in uncomplicated malaria. When the differences in antibody levels between genotypes were explored, similar results were seen for anti-P. falciparum IgG3 in that complicated malaria patients with previous malaria experiences tended to have lower IgG3 levels in those carrying TT compared to CT genotypes. In contrast, the complicated malaria patients without previous malaria experiences who carried CC genotype had significantly higher IgG1 than those carrying either CT or TT genotypes. Thus, repeated exposure and host genetics, i.e the polymorphism of IL4-590 or other related genes has an impact on the modulation of antibody isotype profiles and altered clinical outcome.This study demonstrated that IL4 polymorphism and anti-P. falciparum IgG subclasses antibody levels has not been studied thoroughly. This is the first finding to show that a single nucleotide polymorphism at position IL4-590 transition from C to T is associated with the levels of anti-P. falciparum IgG3 antibody in malaria patients. In complicated malaria especially those with previous malaria experiences, the levels of anti-P. falciparum IgG3 were significantly different among patients carrying IL4-590 CC, CT or TT genotypes. In particular, complicated malaria patients homozygous for IL4-590 T alleles and with previous malaria experiences exhibited significantly lower levels of anti-P. falciparum IgG3 antibody than their IL4-590 counterparts, whereas significantly higher levels were found in uncomplicated malaria patients homozygous for IL4-590 T alleles and with previous malaria experiences. Similar trends were observed for the anti-P. falciparum IgG1 in uncomplicated malaria patients with previous malaria experiences although this did not reach statistically significant differences. These results are consistent with the previous findings that low levels of IgG3 may lead to the onset of complicated malaria while high IgG3 antibody levels may protect against malaria [P. falciparum surface proteins and/or inhibiting cytoadherence of infected red blood cells, thus, interfering the parasite sequestration [IL4-590 polymorphism may differ in patients with complicated and uncomplicated malaria and thus, alter the IgG1 and IgG3 production. This may also explain why different anti-P. falciparum IgG3 levels were observed only among patients with complicated malaria carrying IL4-590 TT genotype compared to their counterparts.The relationship between  malaria ,26 and w malaria . IgG3 ca malaria  and co-o malaria , thus, I malaria  which arstration ,32. In hstration ,8,33 whistration . FurtherIL4-590 C/T transition and the severity of malaria in either with or without previous malaria experiences, although there was an association between IL4-590 TT genotype and the levels of anti-P. falciparum IgG3 antibody in both complicated and uncomplicated malaria patients with previous malaria experiences. The reason for this discrepancy is probably due to: firstly, the polymorphism itself may not be functional but act as genetic markers of other functional polymorphisms; secondly, the polymorphisms may directly regulate IL4 production only in the presence of other genetic determinants. In contrast to the results recently demonstrated in Mali of West Africa. The IL4-590 T alleles were associated with the increased levels of both total and anti-malarial IgE in the Fulani, but not the Dogon. However, IL4-590 polymorphism did not influence on the levels of anti-malarial IgG1-3 subclasses in both the Fulani and the Dogon [The present study did not reveal any association between he Dogon . The reaIL4-590 C or T alleles in association with primary and secondary anti-P. falciparum -IgG1 and -IgG3 responses were different. The IL4-590 T allele was associated with anti-P. falciparum IgG3 levels in which the low levels were observed in complicated and the high levels were observed in uncomplicated malaria with previous malaria experiences. In complicated malaria with previous malaria experiences, the anti-P. falciparum IgG3 levels tended to be lower in patients carrying TT than those carrying CT genotypes. Conversely, the IL4-590 T allele seems to be associated with high anti-P. falciparum IgG1 in only uncomplicated malaria patients with previous malaria experiences, whereas, complicated malaria patients without previous malaria experiences who carried CC genotype had significantly higher anti-P. falciparum IgG1 than those either with CT or TT genotypes. Therefore, it is likely that T allele associated with secondary malaria infection, while C allele associated with primary infection. The shift of Ig-isotypes from primary to secondary responses has been demonstrated in mouse models to be correlated with the levels of either Th1 or Th2 cytokines [IL4-590 T creates a hair trigger for IL4 transcription, it is advantageous when a Th2 response is required, while IL4-590 C is favored when a Th1 response is required [IL4-590°C or T in combination with previous exposure to malaria antigens probably partly modify the antibody isotype profiles and thus represent a wide spectrum of clinical symptoms. However, some of these observations need to be confirmed in a larger sample size in order to correct the confounding factors, such as age, number of previous episodes and parasite-virulence.The ytokines . Th1 subrequired . TherefoIL4-590 C or T is likely to play a modulating role in the regulation of antibody profiles in primary and secondary immune responses. The IL4-590 T allele participated in the regulation of anti-P. falciparum -IgG1 and -IgG3 in secondary infection, whereas IL4-590 C allele might participated in the regulation of anti-P. falciparum IgG1 in primary infection. In individuals carrying the IL4-590 TT genotype with previous malaria experiences, an increased risk of complicated malaria was associated with low anti-P. falciparum IgG3 antibody, whereas a lower risk was associated with high antibody levels. However, a panel of genetic variations in humoral immune responses and their related genes in larger sample size should be further investigated which may provide clue for better understanding in the alteration of disease severity.This study showed that CM: complicated malaria patients; CME: complicated malaria patients with previous malaria experiences; CMN: complicated malaria patients without previous malaria experiences; UCM: uncomplicated malaria patients; UCME: uncomplicated malaria patients with previous malaria experiences; UCMN: uncomplicated malaria patients without previous malaria experiences.The authors declare that they have no competing interests.IL4-590 genotyping and ELISA, collected clinical data, analysed statistics and drafted the manuscript; HP contributed to ELISA; MH assisted in IL4-590 genotyping; SK and MTB supervised the work, revised the manuscript and financed the research. All authors read and approved the final manuscript.SK and PT designed the study; PT prepared the blood samples, performed the


Severe anaemia requiring emergency blood transfusion is a common complication of malaria in children. To ensure access for urgent blood transfusion, the World Health Organization has developed clear guidelines with haemoglobin thresholds prevent unwarranted transfusion,. Few studies have reported outcome and haematological recovery of children with severe malaria where transfusion practice complies with WHO recommendations.A prospective observational study of survivors of severe and complicated malaria transfused in accordance with WHO guidelines. Children were invited for review at one month post-discharge. Non-attendees were traced in the community to ascertain survival.Outcome was assessed in 213 survivors. Those transfused were younger, had a higher base deficit, mean lactate levels and a higher prevalence of respiratory distress. As expected mean admission haemoglobin (Hb) was significantly lower amongst transfused [5.0 g/dL SD: 1.9] compared to non-transfused children [8.3 g/dL SD: 1.7] (p < 0.001). At discharge mean Hb was similar 6.4 g/dL [SD: 1.5] and 6.8 g/dL [SD: 1.6] respectively (p = 0.08), most children remained moderately to severely anaemic. At one month follow up 166 children (78%) returned, in whom we found no differences in mean Hb between the transfused (10.2 g/dL [SD: 1.7]) and non-transfused (10.0 g/dL [SD: 1.3]) survivors (p = 0.25). The major factors affecting haematological recovery were young age (<24 months) and concomitant malaria parasitaemia; Hb being 8.8 g/dL [SD: 1.5] in parasitaemic individuals compared with 10.5 g/dL [SD: 1.3] in those without (p < 0.001).This data supports the policy of rational use of blood transfusion, as proposed in the WHO guidelines, for children with anaemia in areas where access to emergency transfusion is not guaranteed. We have provided empirical data indicating that transfusion does not influence superior recovery in haemoglobin concentrations and therefore cannot be justified on this basis alone. This may help resolve the disparity between international policy and current clinical practice. Effective anti-malarial treatment at discharge may prevent reoccurrence of anaemia. Plasmodium falciparum resulting in over 500 million clinical cases and about one million deaths predominantly in children less than five years living in the sub-Saharan Africa (SSA) [Annually approximately two billion people are exposed to ca (SSA) . Malariaca (SSA) -4. Anaemca (SSA) . Previouca (SSA) .For children with severe, symptomatic anaemia, it is now recognized that an urgent blood transfusion is life saving. HoweverThe World Health Organization (WHO) guidelines, which are largely based on expert opinion -17 and aWhilst there is compelling evidence to support use of blood in children with profound anaemia and severe anaemia with respiratory distress,3 evidenWe have previously reported in-hospital outcome in children with severe malaria including those with and without symptomatic anaemia -27. In t+ thalassemia deletion genotypes[The study was conducted between May 2002 and January 2005 at the Kenya Medical Research Institute, Kilifi District Hospital, Kenya. Patients attend this hospital from a well mapped Demographic Surveillance Site (DSS) in which a census is conducted three times a year. Malaria is endemic in Kilifi District, where falciparum parasitaemia at the time of the study was present in over 30% of children in the community and 46% of hospital admissions. Althouggenotypes.P. falciparum parasitaemia (at any density), a clinical feature of severe malaria  and metabolic acidosis (base deficit of >8). Such children are at the greatest risk of death, including the subgroup of children with symptomatic severe malaria anaemia (Hb<5 g/dl in association with signs of cardiorespiratory compromise).Demographic and clinical data were recorded on admission using a standard proforma. Children were eligible for inclusion in this study if they manifested all of the following features: In accordance with WHO guidelines whole blood transfusion (20 mls/kg of whole blood) was administered (without diuretics) for children admitted with or subsequently developing a Hb <4 g/dL or 4–5 g/dL if associated with respiratory distress. Those with these features at admission were transfused immediately (early transfusion group) while those without these features at admission were transfused later if they developed these features (delayed transfusion group). Children with features of decompensated shock meeting above transfusion criteria (early transfusion group) were administered pre-transfusion management with 10 mls/kg fluid while awaiting transfusion. ChildreDischarge from hospital was consequent upon establishing both clinical and parasitological recovery . Children were discharged with any level of haemoglobin ≥ 4 g/dL as long as they did have respiratory distress. At discharge all survivors received a course of iron sulphate (20 mg/kg/day in three divided doses for 28 days which was continued to complete three months), folate (2.5/5 mg once day for body weight <10/>10 kg respectively for 28 days and continued to complete three months), and mebendazole (100 mg twice daily for three days). Children reviewed before 21 days or after 35 days were excluded from the primary analysis of haematological recovery due to temporal effects on haemoglobin recovery, but included in survival analysis. Follow-up review included a full clinical assessment, anthropometric measurement, full blood count, and a malaria slide. Investigation of parasite genotyping was not performed (to distinguish re-infection from recrudescence). Survival of study children was also obtained by linking the clinical data to the census data routinely done in the DSS reference to find out the long term progress of the study participants. In this study, information from census done in May 2005 was used.2 or Fisher's exact test for proportions. The haemoglobin at follow-up was further categorized into <9.3 g/dL (WHO criteria for anaemia) and >9.3 g/dL with those who failed to attain a Hb of 9.3 g/dL classified as anaemic according to WHO criteria[a priori decision was made to include age in each model due possible confounding effect of age on incidence of anaemia in children with malaria. Formal survival analysis was not performed, instead survival was described as the proportion of cases that were ascertained at discharge, follow-up and long-term follow-up by DSS.Data was analysed using Stata version 8.2. Patients were retrospectively classified as transfused or not transfused and by compliance with follow-up. In children attending follow-up within one month of post discharge admission, discharge and follow-up characteristics of children who were transfused according to WHO guidelines were compared to those of their relatively less sick counterparts who did not received blood transfusion, because they did not fulfil the WHO criteria, by comparing means and standard deviations for continuous variables and Chi criteria. Mantel-Of the 241 participants in three intervention studies ,27) admiOut of 241 children studied 65 (27%) had SSMA requiring urgent transfusion according to WHO guidelines: 64 (98%) received a blood transfusion while one child died before transfusion could be obtained. Mortality was low, only five (7.7%) children in this group died: four of these children were in coma at admission and died within eight hours of admission. All cases had a cardiorespiratory arrest.In addition 45 (18.6%) children received a whole blood transfusion later during the course of their admission after fulfilling the criteria outlined by WHO. Among the fourteen children initially with severe anaemia (Hb of 4–5 g/dL), but without respiratory distress, ten (71%) were eventually transfused because they developed impaired consciousness and/or respiratory distress. For children with Hb> 5 g/dl with moderate or severe metabolic acidosis (base deficit >8 and >15 respectively) who were initially treated for shock with volume expansion, 5/74 (7%) and 30/88 (34%) children respectively also went on to receive a whole transfusion due to a fall in haemoglobin below 5 g/dL. No differences in the proportion requiring delayed transfusion were found between the different resuscitation fluids. Four children (8.8%) in the delayed transfusion group died; all had been admitted in deep coma. No cases developed pulmonary oedema or a transfusion reaction.Of the 213 survivors, 166 (78%) patients returned for follow-up assessment: 158 (74%) children were reviewed between 21–35 days post discharge and eight (4%) were reviewed outside this interval. Out of these eight children, three attended earlier due to malarial fever (2) or malnutrition (1) and five were well, but attended on the wrong date. Haematological recovery was assessed in 158 survivors who subsequently attended follow-up within 22 to 35 days post-admission5 (95% Reference Range (RR) 1.7, 3.5) and non-transfused group 2.3 × 105  (p = 0.62) as were sex, prevalence of hypoglycaemia or mean corpuscular volume (MCV) (Table Baseline data are summarized in Table Significant changes were observed in mean Hb between admission and discharge which increased on average by 1.4 g/dl (SE 0.2) in those receiving a blood transfusion and fell by a mean of 1.5 g/dl (SE 0.1) of Hb in the non-transfused group. Although there were clear differences in Hb at admission there were no differences in discharge Hb in the two groups   Table . OverallThe presence of malaria parasitaemia at follow-up and young age were the only factors independently associated with follow-up haemoglobin. Nutritional status (determined by mid-upper arm circumference (MUAC) and weight), mean corpuscular volume (MCV) and review interval did not have a significant influence on the follow-up haemoglobin.Presence of malaria parasitaemia was found to be the most significant determinant of haemoglobin recovery in patients following admission with severe malaria Figure . For thoAge <24 months was found to be a significant determinant of haemoglobin at admission (p < 0.001), discharge (p = 0.008), and at follow-up (p = 0.01). Younger children (age <24 months) had a lower Hb at admission , discharge  and at review . Children under 24 months old accounted for 31/61 (51%) of all transfusions; 14 given for SSMA and 17 delayed transfusions.Medium term survival was ascertained in the 166/213 (78%) patients who returned to follow-up, and the demographic surveillance was used to trace survival in those who failed to attend follow-up review. Of the 47 non-attendees 32 (68%) came from within the study area, of whom we could confirm longer term survival on 30. Twenty one were reported to be still alive and living in the census area, 9/32 were still alive when they migrated out of the study area and two were not traceable. This increases the overall medium term survival to 196 (92%) children with severe and life threatening malaria who were managed conservatively with respect to blood transfusion in accordance with WHO transfusion guidelines.This study demonstrates a number of important points. First, if provides evidence that the WHO policy for rationale use of blood transfusion in children with severe malaria is justified. In these children both immediate and longer-term child survival were optimized by strictly limiting blood transfusions to a high-risk group with urgent need of transfusion, and preventing unnecessary exposure of those not warranting transfusion to the hazards of blood transfusion.Secondly, the study demonstrates that despite fall in haemoglobin during admission for those who are less sick and do not fulfill the WHO criteria for transfusion, they have satisfactory haematological recovery even if they are discharged with moderate anaemia. They also do not have increased risk of mortality either during admission or at discharge. The absence of deaths among those children who did turn up for one month follow-up but resident in the study area at least six months after the last patient was recruited allays fears of bias due to loss of follow-up.Thirdly, this study shows that complete parasitological recovery is very important for a satisfactory hematological recovery. Once malaria parasitaemia is cleared the Hb recovers rapidly with haematinic supplementation in this study. We found that haematological recovery at one month follow-up of these children was remarkable: 74% attaining an effective haematological recovery Hb >9.3 g/dL) and 89/158 (56%) achieving values greater than 10 g/dl. Neither blood transfusion nor nutritional status affected mean Hb at follow-up. Although not directly addressed by this study we suspect that anti-malarial drug resistance was a major factor threatening hematological recovery and is supported by similar studies in Kenya and Tanzania  g/dL and. TogetheThe relative severity of anaemia in those less than 24 months is consistent with findings in other studies and is likely to be a reflection of poor immunity to malaria ,4,15. A In severe malaria this study provides some support that blood transfusion can be avoided, and that while the majority of children were moderately to severely anaemic on discharge, most demonstrated a good haematological recovery. This latter finding provides encouraging justification for investigation for the use of the low volume umbilical cord blood as an alternative source of blood for transfusion. StudiesA major limitation of this study was that these children were in another clinical trial that was investigating the safety and efficacy of volume expansion in children with severe malaria and features of impaired perfusion. It is thus not very clear whether fluid administration altered the outcome in terms of the number who would have required a blood transfusion. It is also unclear how the WHO transfusion guidelines would work where frequent re-assessment is not possible as this study found that 70% of children with severe anaemia who were stable at admission were eventually transfused.In conclusion, this study provides empirical data to support the rational use of blood transfusion in hospitalized children with severe malaria, which may help resolve the disparity between recommended policy and current clinical practise. When the guidelines are followed, there is neither an increase in mortality nor an increase in anaemia in children with severe malaria after discharge. Effective anti-malarial treatment at discharge prevents the development of rebound anaemia.CI: confidence interval; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDU: High Dependency Unit; HIV: Human immunodeficiency virus; DSS: Demographic surveillance site; KDH: Kilifi district hospital; MUAC: mid upper arm circumference; SP: sulfadoxine-pyrimethamine; SSMA: severe symptomatic malaria anaemia; WHO: World Health OrganizationSOA enrolled patients to the studies, reviewed patients at follow-up, entered and analysed the data, and wrote the manuscript; OH helped analyse the data and reviewed the manuscript; AP enrolled patients to the studies, reviewed patients at follow-up, and reviewed the manuscript; RI enrolled patients to the studies, reviewed patients at follow-up, analysed the data and reviewed the manuscript; TW provided the DSS data for follow-up and reviewed the manuscript; CRJCN enrolled patients to the studies, analysed the data, and reviewed the manuscript; KM designed the study, analysed the data, reviewed all drafts of the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum-malaria, intestinal schistosomiasis, soil-transmitted helminth infections, and the respective co-infections, among schoolchildren in northwest Tanzania.Malaria, schistosomiasis and intestinal helminth infections are causes of high morbidity in most tropical parts of the world. Even though these infections often co-exist, most studies focus on individual diseases. In the present study, we investigated the prevalence of S. mansoni and soil-transmitted helminth eggs. Giemsa stained thick and thin blood smears were analysed for the presence of malaria parasites.A cross sectional study was conducted among schoolchildren living in villages located close to the shores of Lake Victoria. The Kato Katz technique was employed to screen faecal samples for P. falciparum and S. mansoni were 13.5% , and 64.3%  respectively. Prevalence of hookworm infection was 38% . A. lumbricoides and T. trichiura were not detected. Of the children 26.5%  that harbored two parasite species, combination of S. mansoni and hookworm co-infections was the most common (69%). Prevalence of S. mansoni - P. falciparum co-infections was 22.6%  and that of hookworm - P. falciparum co-infections 5.7% . Prevalence of co-infection of P. falciparum, S. mansoni and hookworm was 2.8% .Of the 400 children included in the study, 218 (54.5%) were infected with a single parasite species, 116 (29%) with two or more species, and 66 (16.5%) had no infection. The prevalences of Multiple parasitic infections are common among schoolchildren in rural northwest Tanzania. These findings can be used for the design and implementation of sound intervention strategies to mitigate morbidity and co-morbidity. Plasmodium falciparum inflicts the largest burden [Parasitic infections present a major cause of disease and morbidity in Africa . Among tt burden ,3. It ist burden . In Tanzt burden .Schistosomiasis is endemic in 76 countries worldwide and besides malaria is the second important parasitic disease for public health . In TanzThe World Health Organization estimates that more than one billion people are chronically infected with soil-transmitted helminths . HookworP. falciparum malaria and helminth infections are endemic throughout Tanzania, the affected populations often endure infections with a number of different species [Due to the fact that  species , and ind species . Such in species .P. falciparum malaria depends on the conditions that favour multiple parasitic species survival and transmission [P. falciparum malaria, schistosomiasis and soil-transmitted helminth have been reported from various epidemiological settings in Africa [Overlap of schistosomes, soil-transmitted helminth and smission . These csmission . Polyparn Africa ,16,17, yP. falciparum malaria, S. mansoni and soil-transmitted helminth, and co-infections, among schoolchildren living in villages surrounding the Lake Victoria shore. Understanding the extent of polyparasitism in high-risk groups will serve as a guide in developing sound interventions strategies to reduce the burden of these diseases and co-morbidity.This study was therefore conducted to determine the prevalence of Nyamatongo ward is located in Sengerema district in the Mwanza region, northwest Tanzania. The ward is located on the eastern side of the district and boarders Lake Victoria to the east. It has a population of about 21,000. The study area is located at 1140 m altitude above sea level, and the inland area is covered by seasonal rivers and stream flowing down to the lake. The area is characterized by a tropical climate, with an average annual temperature of 26.5°C, and experiences long rainy seasons between January and May. Annual average rainfall is 1065 mm. The majority of inhabitants are involved in rural subsistence farming, fishing and livestock keeping.The study was carried out between March - May 2009 among 400 schoolchildren from four primary schools, located 500 m-1500 m from the shore of Lake Victoria in Sengerema district, northwest Tanzania. The inclusion criteria for the study were: (1) children aged 8-16 years; (2) parents or guardians gave written informed consent; (3) in addition to the written consent from caretakers, children also were supposed to agree and provide informed assent; (4) children lived in the study area.P. falciparum malaria, S. mansoni and soil-transmitted helminth and any co-infections of P. falciparum, and intestinal helminths among schoolchildren. A two-step random sampling method was used to select children participating in the study. First, four schools of Nyamatongo ward were selected randomly. Second, using the class attendance registers, each child was given a number. Selection of the study participants was achieved by using tables of random numbers. Demographic data including age, gender and school/village where participants studied/lived were recorded by a structured questionnaire.A cross-sectional study was undertaken to determine the prevalence of The day before parasitological screening commenced, schoolchildren were issued with plastic containers and instructed by a research team (HDM and NJSL) on how to collect a portion of their morning stool samples the next day. Stool containers were then collected at the school and labeled with identification numbers. These samples were brought to the field laboratory on the same day, and duplicate Kato-Katz cellophane thick smears was prepared from each specimen ,19. As aP. falciparum. The counts of P. falciparum were converted to the number of parasite per μl of blood, assuming as standard a WBC count of 8000/μl [A finger prick blood sample was collected after cleaning the finger surface using a sterile cotton wool soaked in methylated spirit. Two thick and one thin smears were prepared and stained with 10% Giemsa  in phosphate buffer . Species 8000/μl .S. mansoni infections were treated with mebendazole 500 mg and praziquantel 40 mg/kg [Children found positive for hookworm and 40 mg/kg . Sweet pThe study was approved by the Institutional Review Board  of the Weill-Bugando University College Health Sciences and Bugando Medical Centre, Mwanza, Tanzania.Prior to conducting the study, meetings were held with parents or guardians, teachers and community leaders to explain the aims and procedures to be used to collect data. Informed written consents were obtained from children's parents or guardians and in addition assent was subsequently obtained from children.S. mansoni was classified into four groups according to WHO criteria: (1) light infection (1-100 eggs/g faeces (epg), (2) moderate infections (101-400 epg), (3) heavy infections (401-1000 epg) and (4) very heavy infections (> 1000 epg). For hookworms, infections were classified as follows: (1) light < 1000 epg; (2) moderate infection (1000-3999 epg) and (3) heavy infection (> 4000 epg) [Data were entered into Microsoft Excel 2003 spreadsheets , checked for entry errors and analysed using SPSS for Windows version 11.5 . Intensity of helminth infection was expressed as arithmetic mean from two Kato Katz thick smears . InfectiP. falciparum was specified on the basis of Giemsa stained blood smear examination, and infection intensities were classified into four groups: 1-50, 51-500, 501-5000 and > 5000 parasites/μl of blood [of blood .2) test was used to assess whether single and double or triple parasitic infections were associated with sex and age. Logistic regression analysis was applied to investigate whether gender and age were significantly associated with S. mansoni, P. falciparum and hookworms. A model of with S. mansoni infected children defined as cases incorporated age and sex. The adjusted odds ratios, including 95% confidence interval and P-values were calculated. Similar procedures were repeated for hookworm and P. falciparum infections.The prevalence of single and multiple species of parasites were assessed and classified by gender and three age groups . Chi-squared  harboured at least one parasitic infection and 66/400  had none of the investigated parasitic infections.S. mansoni was 64.3% (95% CI: 59.6-68.9) with no significant difference among males and females  had light infection (1-100 epg).The prevalence of es Table . The majT. trichiura or A. lumbricoides were not detected. Table The overall hookworm prevalence was 38.5% (95% CI: 33.2-42.8) with no significant differences between males and females (Table S. mansoni and hookworm infections with the age group (14-16 years), S. mansoni  and hookworm infection , as compared to the ≥ 10 year age group. Infection with S. mansoni  as well as with hookworms  was independent of gender.Logistic regression analysis revealed significant associations between P. falciparum of 13.5%  and females had higher prevalence of asymptomatic malaria (16.6%) . Co-infection with more than one parasite species was common. The overall prevalence of at least two parasite species in study participants was 26.5% (n = 106), 95% CI, 22.2-30.8) , hookworm + P. falciparum . The prevalence of dual infections did not differ significantly by gender (P = 0.068). The age group 14-16 years exhibited high prevalence of dual infection . Female children had the highest prevalence of triple infections .Out of the 400 children tested for malaria parasites, 57  were positive, with a prevalence of 5% Table . Three cS. mansoni was the most prevalent parasitic disease identified with almost 2/3 of the schoolchildren infected. Prevalence of malaria was relatively low. Parasitic co-infections were common. Ascariasis and trichuriasis were absent.The findings from the current study confirm that Nyamatongo ward in northwest Tanzania is highly endemic for intestinal schistosomiasis and hookworm infections. S. mansoni) than in the present study probably due to focal exposure to S. mansoni by the population living in endemic areas as reported previously [S. mansoni in children were light to moderate, and only 7.5% of the children had heavy infections. These findings support previous observations that only few individuals in an endemic community excrete large numbers of eggs and that S. mansoni infections starts at early ages [S. mansoni. Similar observations have been made for other areas [S. mansoni was observed to decrease with increase in age of the children, which has also been reported in previous reports from Cote d'Ivoire [S. mansoni appears to coincide with the onset of puberty [Earlier studies conducted on other localities within the lake basin showed that both schistosomiasis and hookworm infections are common in the area ,23. Howeeviously ,25. Inferly ages . In the er areas . On the d'Ivoire . The dec puberty  but slig puberty .A. lumbricoides and T. trichiura, which occur in various ecological settings of sub-Saharan African [et al. [A. lumbricoides and T. trichuris. Variability in endemicity or prevalence of these infections, low sensitivity of the diagnostic method, the use of single stool sample, environmental contamination and inability of the helminth eggs to withstand high temperature could partly explain the observed difference. The prevalence of P. falciparum malaria in Nyamatongo ward was relatively low (13.5%) in spite of the locality being within a malaria holoendemic area [Our data confirm that among soil-transmitted helminths, hookworm infection is the major public health problem within the Lake basin ,25. A po African . Interes African  and a remic area . Our finmic area ,32, but mic area .S. mansoni and hookworms being the most common. Co-infections of helminth and P. falciparum infections also have clinical importance [P. falciparum with hookworms or schistosomes tend to exacerbate hepato-splenic, anaemia and malnutrition morbidities among school children [This difference could be attributed to low malaria prevalence in the study area or high mosquito net coverage which was not assessed in the present study. A particular concern is the high occurrence of parasitic co-infections, with concomitant infections of portance . Previouportance ,36. Hookportance . Childreportance . Studieschildren .S. mansoni and hookworm could partly be attributed to the co-endemicity of the two species in the study area and poor sanitations [S. mansoni and P. falciparum observed in the present study have also been observed previously from Senegal [P. falciparum among schoolchildren were high in those infected with S. mansoni. One reason for this observation could be the availability of breeding sites for the intermediate host (fresh water snail) and malaria vectors (Anopheles mosquitoes) in the study area. Furthermore, heavy intensities of S. mansoni infection did not correlate with having heavy intensities of P. falciparum. The findings of our study were similar to a study from Uganda [Co-infections of itations ,39,40. Titations  Côte d'Iitations  and Chin Senegal  and Zimb Senegal  where prm Uganda .P. falciparum co-infections were more common in our study than reports from Cameroon (0.2%) [P. falciparum infection showed no significant association with hookworms.Hookworm and n (0.2%)  but lowen (0.2%) ,43. DiffP. falciparum, hookworm and S. mansoni have been reported in studies from Côte d'Ivore, Cameroon and Zimbabwe [S. mansoni, hookworm and P. falciparum are prevalent among schoolchildren in the study area. The findings of the present study, supports the need for initiatives to implement a new framework for an integrated approach in disease management. We also recommend longitudinal studies to identify the associations between parasites and associated morbidity.Similar to our results, triple infections of Zimbabwe ,33,42. TZimbabwe ,29,39,40Zimbabwe . In concThe authors declare that they have no competing interests.HDM, GMM, RW and NJSL designed the study and participated in data collection. JS and AK assisted in data collection. JH, HDM, SEM, LML, AMM and EJK analysed the data. HDM and SEM wrote the first draft of the manuscript. All authors contributed to the manuscript and approved its final version.


The reason for this cerebral malaria mortality is still unknown although host proinflammatory factors have been shown to be evidently important. The current study investigated the role of circulating MIF in the pathogenesis and outcomes of cerebral malaria.Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine implicated in the pathogenesis of a number of human diseases including inflammatory neurological diseases. Its role in the pathogenesis of cerebral malaria is unknown. Cerebral malaria is a life-threatening complication of Three categories of subjects contributed to this study: healthy controls subjects, mild malaria patients, and cerebral malaria patients. The cerebral malaria patients were further grouped into cerebral malaria survivors and cerebral malaria non-survivors. MIF levels in the peripheral blood plasma, obtained at the time of enrollment, were measured using standard ELISA methods. In logistic regression on cerebral malaria patients, log MIF levels were found to be significantly associated with fatal outcome . In multinomial logistic regression log MIF levels were found to be significantly associated with patient category (p = 0.004).This study suggests that elevated levels of MIF in the peripheral blood of cerebral malaria patients may be associated with fatal outcomes. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine whose role as an important regulator of immune and inflammatory responses in a number of human diseases, such as sepsis, rheumatoid arthritis, cancer and inflammatory neurological diseases, has been confirmed (reviewed in ). Its roThe role of circulating MIF in the pathogenesis of cerebral malaria (CM) and its outcome has not been investigated. CM is a life-threatening complication of falciparum malaria; approximately 20% to 30% of CM patients die despite anti-malarial treatment and some survivors develop neurological complications . It is nP. falciparum parasitemia of <25,000 parasites/μl of blood  and no evidence of impaired consciousness, seizures, and no past history of mental illness, meningitis, or accidental head injury, and cerebral malaria (CM) patients who met the World Health Organization's criteria for CM [P. falciparum and no other clinically evident cause of impaired consciousness. Peripheral blood samples were collected from the subjects after informed consent was obtained and plasma samples were stored at -80°C until used for experiments. A total of 138 samples were available for this investigation of which 23 were from HC, 47 were from MM patients, 54 were from CM survivors (CMS) and 14 were from CM non-survivors (CMNS). Plasma MIF levels were determined using standard ELISA methods as described previously [For this investigation, plasma samples obtained from a well characterized prospective cohort study of malaria conducted in Jabalpur, India were utilized. This study area is a low endemic area with seasonal transmission of malaria. The study details were recently reported elsewhere . This coa for CM  with a Geviously . PairwisThe highest median MIF level was in the CMNS group, followed by HC, MM, and CMS, respectively Figure . None ofFigure Several studies have demonstrated that elevated MIF levels were associated with inflammatory neurological disorders such as neuron-Behcet's disease and different forms of multiple sclerosis (reviewed in ). In a rThe observation from this study that MM and CMS patients had low MIF levels compared to HC may suggest a protective role of MIF against malaria and is consistent with previous studies that demonstrated significantly reduced circulating MIF levels in children with acute malaria infection compared to matched healthy controls . AlthougResults from this study suggest a dual role for MIF during malaria infection: a protective and pathological role. This duality in MIF functions has been suggested previously using in vitro studies . HowOverall, these studies suggest that MIF may play a role in the fatal outcomes associated with CM. However, further studies are needed to better understand the role of MIF in malaria pathogenesisThe authors declare that they have no competing interests.VJ: Participated in the cohort study design, enrolled patients, collected clinical and epidemiologic data and biological samples and editing of the manuscript. SM: Participated in statistical analysis of the data and editing of the manuscript. ACN: Participated in designing the cohort, enrollment of subjects, clinical evaluation and editing of the manuscript. APD: Participated in planning of the study, supervision of the study in India and editing of the manuscript. JKS: Participated in the cohort study design, planning of experiments and assisted in the editing of the manuscript. VU: Participated in the study design, data analysis, and contributed to manuscript writing and editing. NS: Participated in the cohort study design, patient recruitment, planning of the experiments and editing of the manuscript. NWL: Participated in the study design, performed the experiments and data analysis and wrote the manuscript. All authors read and approved the manuscript