The efficacy of RTS,S/AS01 as a vaccine for malaria is being tested in a phase 3 clinical trial. Early results show significant, albeit partial, protection against clinical malaria and severe malaria. To ascertain variations in vaccine efficacy according to covariates such as transmission intensity, choice of adjuvant, age at vaccination, and bednet use, we did an individual-participant pooled analysis of phase 2 clinical data.2–10), ranging from 5% to 80%. Choice of adjuvant was either AS01 or AS02.We analysed data from 11 different sites in Africa, including 4453 participants. We measured heterogeneity in vaccine efficacy by estimating the interactions between covariates and vaccination in pooled multivariable Cox regression and Poisson regression analyses. Endpoints for measurement of vaccine efficacy were infection, clinical malaria, severe malaria, and death. We defined transmission intensity levels according to the estimated local parasite prevalence in children aged 2–10 years (PrP2–10 10%) vaccine efficacy was 60% (95% CI 54 to 67), at moderate transmission (PrP2–10 20%) it was 41% (21 to 57), and at high transmission (PrP2–10 70%) the efficacy was 4% (−10 to 22). Vaccine efficacy also varied by adjuvant choice (p<0·0001)—eg, at low transmission (PrP2–10 10%), efficacy varied from 60% (95% CI 54 to 67) for AS01 to 47% (14 to 75) for AS02. Variations in efficacy by age at vaccination were of borderline significance (p=0·038), and bednet use and sex were not significant covariates. Vaccine efficacy (pooled across adjuvant choice and transmission intensity) varied significantly (p<0·0001) according to time since vaccination, from 36% efficacy (95% CI 24 to 45) at time of vaccination to 0% (−38 to 38) after 3 years.Vaccine efficacy against all episodes of clinical malaria varied by transmission intensity (p=0·001). At low transmission ; Bill & Melinda Gates Foundation Vaccine Modelling Initiative; Wellcome Trust. The increasing application of interventions for malaria control over the past 10 years has been linked to reductions in morbidity and mortality associated with malaria infection.RTS,S protects at pre-erythrocytic stages of the parasite's lifecycle. It is partly effective and has been described as a leaky vaccineVaccine efficacies are usually summarised with point estimates. However, if vaccine efficacy is heterogeneous by subgroups within the population, this efficacy figure will be a mean of the vaccine efficacy in the various subgroups, weighted according to the proportion of the population.Analysis of phase 2b data to date shows variations in measured efficacy between trials.We identified phase 2b trialsPlasmodium falciparum infection (ACDi),In the identified trials, healthy adults or children were recruited after clinical and laboratory screening to exclude participants with clinically significant disease. Five trials of children or adults used active case detection for P falciparum parasitaemia, with or without a measured fever. We defined clinical malaria as the presence of 2500 or more P falciparum parasites per μL of blood in association with reported or measured fever (≥37·5°C).We used four primary endpoints in our analysis: infection, clinical malaria, severe malaria, and death. In the trials we identified, infection was defined as any detectable We analysed episodes of infection identified by ACDi as one dataset. We combined clinical malaria episodes identified by ACDc and PCD and analysed these as a second dataset. ACDc and PCD were included in the initial study protocols, except for one trial,P falciparum parasitaemia, no alternative (or more probable) cause of illness, and either severe malaria anaemia (haemoglobin <50 g/L), cerebral malaria (Blantyre coma score <2), or another symptom .We identified episodes of severe malaria from safety data reporting. Criteria for severe malaria were derived from the WHO definitionOur primary analysis was vaccine efficacy, which was assessed per-protocol. Hence, cohorts monitored for infection or clinical malaria included all participants who had received three doses of vaccine, from 2 weeks after the third dose. We did not judge adults at risk of severe disease or death from malaria. Analysis of severe malaria or death was based on intention to treat and included all children who received at least one dose of vaccine, from the time of the first vaccination.In Mozambique,vs AS02), and clinical disease surveillance method at the site level . To ascertain transmission intensity, we used estimates from the Malaria Atlas Project (MAP) for prevalence of asymptomatic parasitaemia in children aged 2–10 years in 2007 (PrP2–10),We recorded the following covariates across the seven trials: sex, age at the time of vaccination, country, bednet use, adjuvant used , we pooled individual participant data and estimated the linear and non-linear effect of covariates in the data and the interactions of these covariates with vaccine efficacy. We used these empirically observed functions to estimate efficacy in subgroups by multiplying the fixed effect of vaccination  by the interaction term . We added variances and the covariance matrix to calculate SEs. All covariates were included in an initial model, and we excluded covariates or interaction terms with p greater than 0·05 to produce a final model. To examine the possibility that analyses of clinical malaria risk were biased by unequal durations of follow-up in some subgroups, we did an additional analysis restricted to 1 year of follow-up.We calculated vaccine efficacy as either 1 minus the hazard ratio or 1 minus the incidence rate ratio. We modelled the non-linear effects of age at vaccination and local parasite prevalence as multiple fractional polynomials, according to the method of Royston and colleagues.We examined parametric survival models to fit a γ distribution to the unmeasured heterogeneity in exposure. We used a Gompertz survival distribution for parametric models, since this method fitted the data better than the alternatives  and gave hazard ratios for vaccination that were nearly identical to those estimated using Cox proportional-hazards.We did this pooled analysis after a call for proposals initiated and facilitated by GlaxoSmithKline Biologicals. Employees of GSK Biologicals were investigators on the original phase 2 studies and were authors on the primary reports. Employees of GSK Biologicals reviewed the analysis plan and commented on early drafts of the pooled analysis, but were not required to give final approval of the manuscript. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.We analysed pooled data for 4453 participants in seven trials . 1376 paThe survival functions for the four endpoints are shown in 2–10 10%) efficacy varied from 60% (95% CI 54 to 67) for AS01 versus 47% (14 to 75) for AS02; however, efficacy did not differ by bednet use or by gender. Vaccine efficacy varied significantly by age for all episodes of clinical malaria (p=0·038), but not for first episodes of clinical malaria  with a local parasite prevalence of 20% ; data were from 39 children with severe malaria from a total of 2080 RTS,S vaccinated people, versus 58 children with severe malaria from a total of 1908 controls. Efficacy against death was 48% ; 11 deaths occurred in the 2080 people receiving vaccine and 19 deaths happened among the 1908 controls. We judged the frequency of severe malaria and death to be too low to justify further multivariable analysis.The survival plot of time to infection during ACDi by vaccination status shows convergence after the initial divergence , and theIn unadjusted analysis of ACDi, efficacy seems to wane rapidly , but aftvs AS01, hazard ratio 0·94, 95% CI 0·82–1·09, p=0·46; local parasite prevalence hazard ratio 1·35, 0·74–2·46, p=0·33; age in years hazard ratio 0·97, 0·93–1·01, p=0·15).We tested for heterogeneity in the rate of declining vaccine efficacy against clinical malaria by estimating the three-way interactions between time (in years), vaccination, and every covariate in turn. None of these three-way interaction terms were significant at the 5% level  had a non-linear effect on clinical malaria incidence.RTS,S can be regarded as a leaky barrier to infection, because it protects against some infectious bites but not against others.2–10) to gauge transmission intensity. These approximations were based on several thousand surveys in the countries where trial sites were located. We chose these standardised independent measures rather than within-trial factors, such as incidence of malaria among controls, because monitoring was not the same between trials. MAP estimates do not account for changes over time, but transmission intensity at our sites is likely to be stable enough over a few years for these data to be a reasonable approximation. Variations in seasonal transmission have a modest effect on the relation between entomological inoculation rate and asymptomatic parasitaemia, but in view of the limitations of using data from 11 sites, we did not feel that more complex characterisations of transmission intensity were warranted.We used MAP estimates of age-adjusted prevalence of asymptomatic malaria . Furthermore use of insecticide-treated bednets was not distributed evenly by site, varying from 4·5% to 100% of children. Tests for variation in vaccine efficacy by bednet use are, therefore, vulnerable to ecological confounding by site.We identified significant interactions between time and vaccine efficacy. We can confidently reject the null hypothesis that vaccine efficacy is constant over time (p<0·0001), but we cannot be confident about the shape of the plotted decline, which is reflected in the wide CIs surrounding estimates of efficacy at later timepoints . We chosA fall in vaccine efficacy over time might be attributable to systematic bias in estimates obtained using survival analysis because of heterogeneous exposure from a partly effective vaccine, as previously described.PanelSystematic reviewWe searched PubMed with the MeSH terms “RTS,S-AS02D vaccine [Substance Name]” OR “RTS,S-AS01E vaccine [Substance Name]” OR “RTS,S-AS01B vaccine [Substance Name]” OR “RTS,S-AS02A vaccine [Substance Name]” OR “PfCSP DNA vaccine [Substance Name]” AND “Malaria, falciparum/prevention and control”. All phase 2 trials identified are included in our analysis and no previous meta-analyses have been done. Two publications describe preliminary data from phase 3 trials, for which follow-up is ongoing. These preliminary reports have not reported subgroup analyses, and data were not available for inclusion in our analysis.InterpretationVaccine efficacy varied by transmission intensity, adjuvant, and time since vaccination. Age at first vaccination was of borderline significance, but bednet use or sex had no effect on efficacy. Vaccine efficacy against clinical disease was of limited duration and fell with increasing transmission intensity. Outcomes after vaccination cannot be predicted accurately on the basis of one pooled efficacy figure. When predicting public health outcomes of vaccination, variations in efficacy—by transmission intensity and by time since vaccination—will need to be accounted for.We report here all phase 2 data for RTS,S malaria vaccines , includiIn summary, we noted significant variation in estimated vaccine efficacy by population subgroups and a significant decline in protection against clinical malaria over time. One might argue that the unadjusted pooled estimates of efficacy nevertheless reflect what was actually seen in the population tested. However, the unadjusted pooled efficacy is merely a weighted mean of efficacies seen in the component subgroups of the population and, therefore, cannot be generalised to other populations. For instance, if the vaccine is more effective at lower transmission intensity, the pooled vaccine efficacy will depend on the proportion of children recruited in sites at low transmission intensity. Predictions of public health outcomes of vaccination will need to take account of these variations in efficacy by transmission intensity and by time since vaccination.


Plasmodium falciparum infection. It is unclear which antibody responses are responsible for, or even associated with protection, partly due to confounding by heterogeneous exposure. Assessment of response to partially effective antimalarial therapy, which requires the host to assist in clearing parasites, offers an opportunity to measure protection independent of exposure.Antibodies are important in the control of blood stage P. falciparum antigens using a quantitative indirect ELISA. Associations between antibody levels and risk of treatment failure were estimated using Cox proportional hazard regression.A cohort of children aged 1–10 years in Kampala, Uganda were treated with amodiaquine+sulfadoxine-pyrimethamine for uncomplicated malaria. Serum samples from the time of malaria diagnosis and 14 days later were analyzed for total IgG to 8 Higher levels of antibodies to apical membrane antigen 1 (AMA-1), but to none of the other 7 antigens were significantly associated with protection against treatment failure . Protection increased consistently across the entire range of antibody levels.P. falciparum, a tool which is currently lacking.Measurement of antibody levels to AMA-1 at the time of malaria may offer a quantitative biomarker of blood stage immunity to  Plasmodium falciparum prevents much of this morbidity in older children and adults, but it is slow to develop and requires repeated episodes of malaria. It has been shown that naturally acquired antibodies to P. falciparum can control malarial parasitemia P. falciparum proteins have been associated with a lower risk of malaria Malaria remains one of the leading causes of morbidity and mortality in children living in sub-Saharan Africa Assessing the response to partially effective antimalarial therapy offers an opportunity to estimate the level of blood stage antimalarial immunity independent of knowledge of prior exposure. In this context, acquired immunity enhances the efficacy of antimalarial therapy such that increasing immunity affords increasing ability of sub-optimal therapy to eliminate parasitemia P. falciparum antigens were associated with clearance of parasitemia, we measured IgG responses to 8 parasite antigens previously associated with clinical protection from malaria We have previously described an association between clinical surrogates of host immunity and protection from failure after treatment with amodiaquine plus sulfadoxine-pyrimethamine (AQ+SP) in a cohort of children in Kampala, Uganda ≥38.0°C or history of fever in previous 24 hours), parasitemia detected by microscopy, and absence of complicated malaria defined by evidence of severe disease The clinical study was conducted in Kampala, Uganda between November 2004 and December 2008 and has been previously described P. falciparum unexposed donors). Following a 2-hour incubation at 37°C, the plates were washed 3 times with PBST and 100 µl/well of alkaline-phosphatase-conjugated goat anti-human IgG  diluted to 1∶1000 in PBSB was added. Following a 1-hour incubation at 37°C, wells were extensively washed 6 times with PBST. BluePhos Microwell Phosphatase Substrate solution  was used to develop the plates, which were read at 650 nm on a Spectramax M2 ELISA plate reader using Softmax Pro Software v5.2 . Following the addition of substrate, optical densities (OD) were collected every 4 minutes for a total of 40 minutes. Serial 3-fold dilutions  of pooled serum from 50 African adults living in Kampala were analyzed on every plate as a positive control and to produce a standard curve for relative antibody quantification. The plate showing the greatest dynamic range for the standard curve was used. OD values were converted to antibody level expressed in arbitrary units (AU), where an AU of 100 was equivalent to the antibody level in the pooled serum. Samples with OD values above the linear range of the standard curve were further diluted up to two times to a maximum dilution of 1∶10,000 and re-analyzed to obtain a better quantitative estimate of antibody level.96-well microtiter plates  were coated overnight at 4°C with antigens of interest diluted in 0.01M phosphate buffered saline (PBS). All blocking and wash steps occurred at a volume of 200 µl/well. Plates were washed twice with a solution of PBS containing 0.05% Tween20 (PBST), followed by a 1-hour block with a solution containing 5% Blotto, non-fat dry milk  in PBS (PBSB) at 37°C. Plates were again washed twice with PBST, followed by addition of 100 µl/well patient serum samples diluted to 1∶200 in PBSB. Patient samples were run in duplicate, along with negative controls  from the 3D7 strain expressed in Escherichia coli (0.1 µg/ml) Saccharomyces cerevisiae (0.25 µg/ml) E. coli (0.25 µg/ml) E. coli (1 µg/ml) E. coli (0.5 µg/ml) E. coli (0.25 µg/ml) The following recombinant 5 repeat peptide for circumsporozoite protein (CSP); and the central amino acid repeat sequence LAKEKLQGQQSDLEQERLAKEKLQEQQSDLEQERLAKEKLQ for liver-stage antigen (LSA).In addition, the following synthetic peptides  were used in ELISA: the (NANP)Antibody prevalence and fold change between Day 0 and Day 14 were calculated for descriptive statistics. To determine antibody prevalence, the positive cutoff was set at 3 standard deviations above the mean response in samples from twenty healthy adults who have never been diagnosed with malaria or traveled within malaria endemic regions (negative controls). Fold change in antibody levels between Day 0 and 14 was determined by the difference in log-transformed values during the course of malaria follow up in an individual. Given that a number of subjects had multiple episodes of malaria, repeated measures within the same individual were accounted for by using generalized estimating equations (GEE) to estimate antibody prevalence, geometric mean antibody level, and the fold change of antibody level between Day 0 and 14. Pairwise correlation coefficients were obtained to determine correlation of antibody responses between each other. For all subsequent analyses, continuous antibody levels were used after log-transformation, truncating low values at the mean response in negative controls.P. falciparum exposure and antibody levels on Days 0 and 14, we evaluated the following predictor variables: age at study enrollment  as a continuous variable; date of the malaria episode as a continuous variable; whether or not the subject lived within 50 meters (m) of a swamp, previously shown to be associated with higher malaria incidence and higher antibody responses To determine associations between epidemiologic factors associated with To assess associations between antibody responses and the risk of treatment failure, hazard ratios were estimated for each response using Cox proportional hazard regression, adjusting for age at enrollment and parasite polymorphisms previously associated with treatment failure Written informed consent for their participation in the study was obtained from the children’s parents or guardians, in accordance with the Declaration of Helsinki. Approval was granted by the Uganda National Council of Science and Technology, the Makerere University Research and Ethics Committee, and the University of California, San Francisco Committee on Human Research.Over the course of the study, 601 total subjects were enrolled and 329 children were randomized in the clinical trial of combination antimalarial therapy. Children randomized to AQ+SP were followed up to 28 months on this therapy. The prevalence of asymptomatic parasitemia in this cohort was 18.6% at enrollment and fell to 2.3% during the study The prevalence of antibodies directed against LSA, AMA-1, MSP-1, and MSP-2 at the time of malaria diagnosis was greater than 50% . AntibodAntibody responses to AMA-1 at the time of diagnosis and treatment were most strongly associated with age at enrollment (44% increase per year of age) and living within 50 meters of a swamp (4-fold increase) . To a leTo determine whether antibody responses were associated with the ability to clear parasites after treatment with AQ+SP, we calculated the hazard ratio of treatment failure associated with each response, adjusting for parasite mutations previously shown to be associated with treatment failure. We additionally adjusted for subject age, a strong predictor of treatment failure in these subjects To further examine the dose-response relationship between anti-AMA-1 levels on Day 0 and treatment failure, antibody levels were divided into 5 equal intervals, each representing a 10-fold increase. There was a consistent, fairly linear relationship between anti-AMA-1 levels and protection against treatment failure over the entire range of measured responses . All malOur longitudinal study design allowed us to analyze the effect of changes in antibody responses to AMA-1 measured during different malaria episodes in the same subject, with 200 out of 244 episodes occurring in subjects with more than one episode. In these 200 malaria episodes, antibody responses to AMA-1 measured at the time of malaria diagnosis were highly correlated within individuals  and showed no consistent change within individuals over calendar time . Interestingly, anti-AMA-1 levels measured at the first episode of malaria in each subject were similarly predictive of treatment failure for all subsequent episodes in that subject  as anti-AMA-1 values measured at the time of the subsequent episodes themselves . Multivariate analysis of these 137 subsequent episodes indicated that the change in anti-AMA-1 level from the initial episode to the subsequent episode provided no additional information once the initial measurement was included (p = 0.9).P. falciparum antigens at the time of treatment of malaria with AQ+SP to determine whether any of these responses were associated with protection from treatment failure. We found that higher antibody responses to AMA-1, but to none of the other 7 antigens tested, were significantly associated with protection. Furthermore, we found a strong dose response relationship, with higher responses to AMA-1 consistently associated with increased protection against treatment failure.We measured IgG responses to 8 A, a recombinant protein based on AMA-1, may induce strain-specific protection P. falciparum exposure and anti-AMA-1 levels at the time of symptomatic malaria. Furthermore, antibody responses to all 8 antigens were significantly correlated with each other. In addition, despite our prior observation of decreasing efficacy of AQ+SP over time that was likely due to waning immunity Does the identified strong association between AMA-1 antibody level and clearance of parasites suggest a causal role in protection? Antibody levels against AMA-1 have been associated with protection from clinical malaria in this 19P. falciparum to observe a malaria episode and do not have complete immunity against symptomatic malaria. Of note, we obtained highly quantitative values for each response and demonstrated strong associations between anti-AMA-1 levels and protection, whether analyzing responses as reactive/non-reactive, in tertiles, or as a continuous variable.Associations between antibody responses to various antigens and antimalarial treatment outcome have been examined in prior studies 19 levels at the time of treatment to be protective against treatment failure 1919 processing and resultant invasion of the red blood cell. Indeed, a study in Nigerian children revealed that there was evidence for both blocking and neutral antibodies competing for MSP-119 binding sites as there was absent inhibitory activity observed despite high MSP-119 prevalence and levels by ELISA Interestingly, we found that, for anti-MSP-1 levels assessed 14 days following malaria treatment, lower levels were associated with clearance of parasites. This finding is in contrast to previous studies that found anti-MSP-1P. falciparum. A mechanistic role in protection is possible, but would need to be evaluated in the context of broader assessment of immunologic responses. The strong dose-response relationship we observed indicates that quantitative measurement of these antibodies may offer a robust biomarker of immunity and should be validated in other epidemiologic settings.In conclusion, we have shown that the levels of antibodies directed against AMA-1 at the time of presentation with malaria are strongly associated with concurrent and future blood stage immunity, as measured by the ability of the host to clear


Plasmodium vivax malaria remains a significant health burden in endemic areas like South East Asia, Central and South America. P. vivax infections are characterized by relapses of malaria caused by persistent liver stages of the parasite (hypnozoites) which are not present in P. falciparum infections. Currently, the only approved treatment option for the radical cure of P. vivax malaria is the 8-aminoquinoline, primaquine. However the long treatment course (two weeks) and severe side effects (hemolytic anemia in G6PD-deficeint patients) highlights the need for new chemical classes of drugs.P. cynomolgi rhesus monkey model. However recently developed assays that allow for the testing of new chemical entities on the parasite liver-stages (schizonts) and sexual stages (gametocytes) could be leveraged to identify dual acting compounds with potental anti-hypnozoite activity. In addition, compounds which target more than one stage in the Plasmodium lifecycle would be highly valued and compliment the current arsenal of antimalarial drugs.Currently the discovery of new anti-hypnozoite drugs is limited to testing in the


Plasmodium falciparum infection, in particular the central role of natural killer (NK) cell-derived interferon gamma (IFN-γ), is becoming increasingly recognised. Recently, it has been shown that IFN-γ production in response to P. falciparum antigens is in part regulated by killer-cell immunoglobulin-like receptor (KIR) genes, and a study from malaria-exposed Melanesians suggested an association between KIR genotypes and susceptibility to infection. This prompted us to determine and compare the frequencies of 15 KIR genes in Gambian children presenting with either severe malaria (n = 133) or uncomplicated malaria (n = 188) and in cord-blood population control samples (n = 314) collected from the same area. While no significant differences were observed between severe and uncomplicated cases, proportions of individuals with KIR2DS2+C1 and KIR2DL2+C1 were significantly higher among malaria cases overall than in population control samples. In an exploratory analysis, activating KIR genes KIR2DS2, KIR3DS1 and KIR2DS5 were slightly higher in children in disease subgroups associated with the highest mortality. In addition, our data suggest that homozygosity for KIR genotype A might be associated with different malaria outcomes including protection from infection and higher blood parasitaemia levels in those that do get infected. These findings are consistent with a probable role of KIR genes in determining susceptibility to malaria, and further studies are warranted in different populations.The relevance of innate immune responses to  Plasmodium falciparum can result in asymptomatic parasite carriage, an uncomplicated febrile disease or a potentially life-threatening illness. Apart from clinical immunity that gradually develops with repeated exposure Infection with the malaria parasite Natural killer (NK) cells are a key component of innate immunity. They kill their targets (diseased cells) by means of cytotoxic activity P. falciparum displays considerable heterogeneity  rather than cytotoxicity is the major contribution of NK cells to host defence ogeneity , 12 and ogeneity , 14. Thubright and CD56dim expressing cells bright cells produce more IFN-γ than CD56dim cells, CD56dim cells represent about 80% of the NK cell population dim population bright cells are KIR negative, whereas the majority of CD56dim cells express KIRs that are highly polymorphic  and 2 pseudogenes (KIR2DP1 and KIR3DP1). The expression and function of each of these genes influence the expression and function of other members of the gene family KIR molecules are glycoproteins encoded by a diverse and compact set of genes on chromosome 19. They are expressed on specialised lymphoid cells mainly NK cells and a subpopulation of γδ T cells and some memory αβ T cells KIRs and their interactions regulate NK cell activity in modulating disease outcomes  and KIR systems , 22 encooutcomes , 26–33.  alleles , 35, andP. falciparum infection, only a few studies have investigated the role of KIRs in malaria. A study that compared KIR genotypes in Melanesians with and without malaria parasitaemia found evidence for an increased number of activating KIR genes in parasitaemic individuals KIR genes among Gambian children with severe or uncomplicated forms of malaria, and cord-blood population control samples to assess whether individual KIR genes or genotypes are associated with the occurrence of disease or parasitaemia levels. Given that epidemiological findings have in the past implicated individual HLA class I alleles (e.g. HLA-B*53) Whilst there is accumulating evidence favouring an important role of NK cells in DNA samples were obtained from buccal swabs collected from Gambian children with uncomplicated malaria  and severe malaria .The typing technique used in this study (PCR-SSP) allowed us to detect the presence of inhibitory and activating KIR genes including those found in genotype A. The number of specific genes found at the centromeric end of B genotypes is variable including KIR2DS2, 2DL2, 2DL5, 2DS3 and 2DS5, in addition to the framework genes 3DL3 and 3DP1. However, telomeric B motifs are specifically identified with the presence of KIR3DS1 and 2DS1 genes in addition to 2DL4 and 3DL2 present in most motifs.In contrast to the conserved nature of genotype A in terms of gene content, KIR genotype B is highly polymorphic in gene content. Genotype B is a combination of activating and inhibitory KIR genes at the telomeric end all belong to KIR haplotype A . An opposite effect was observed for heterozygote (c-A/B) carriers . No such differences were seen with telomeric genotypes.We adopted the recent techniques and terminologies used by Cooley et al. lotype A . On the  KIR2DS4 . Analysi KIR2DS4  did not  KIR2DS4 . Howevercarriers , with a vs 12.5%, respectively, P = 0.004; adjusted for ethnicity and multiple comparisons P = 0.012), suggesting that having two copies of haplotype A may be protective against malaria infection. An interesting observation was that none of the 14 children with CM+SRD, considered to be the most critically ill patients, was homozygous for KIR genotype A, while a substantial proportion of children with less severe forms of malaria were homozygous for genotype A . Given the small number of individuals per group, this should be considered an exploratory analysis that encourages further investigations in other populations with larger sample sizes.The proportion of individuals homozygous for KIR genotype A varied significantly amongst the three groups . Whilst HLA-Cw*16:01 frequency  to be lowest amongst children with severe malaria . This observation, however, should be taken with caution given the small number of individuals used in this comparison. None of the HLA-B alleles was found to have any impact on malaria in this population. The frequencies of HLA-B or -C dimorphic groups and subgroups  were similar between groups ]. The frequencies of these two compound genotypes were significantly higher in cases compared with population control , suggesting that carriers of KIR2DL2+C1 and/or KIR2DS2+C1 were more at risk of being infected with malaria parasites than those without any of these genotypes. However, these two genes are in strong linkage disequilibrium (LD) even in African populations (It has been shown that KIR and HLA interact in an epistatic manner to modulate human immunodeficiency virus (HIV) disease outcome ulations , 48 makiKIR genes (I/A ratio) or the carriage of a particular KIR genotype is associated with parasite load measured on admission. When the ratios were grouped into four bins containing similar numbers of individuals, the level of parasitaemia was significantly different (PANOVA = 0.03) showing a significant increase with increasing I/A ratio , and parasitaemia levels declined significantly with increasing B content of the KIR genotype  and in a population control group. HLA class I (B and C) genotyping was performed on a proportion of samples with enough genomic DNA left after KIR typing. Possible KIR–HLA interactions were explored as compound genotypes in individuals expressing KIR molecules together with their putative ligand(s). Consistent with previous studies in other West African populations  compared with the population control group. Amongst the participants with available HLA type individuals carrying either 2DL2 or 2DS2 together with their corresponding ligand (HLA-C group 1) were significantly more frequent in the infected group. Taken together, these observations suggest that these genes could be involved in increased susceptibility to malaria infection caused by P. falciparum. Although a few of our study participants have one without the other, KIR2DL2 and 2DS2 genes are in strong LD in most populations worldwide including those of African origin (KIR gene (2DL2) and its corresponding activating counterpart (KIR2DS2) have been associated with other diseases. Absence of these genes has been associated with resistance to infection with herpes simplex virus type-1 (HSV-1) KIR2DS2 in the absence of its specific ligands has been associated with increased susceptibility to psoriatic arthritis In this study, we determined the presence or absence of 15 ulations , 48, we n origin , 48, 50.KIR genes did not differ significantly between disease subgroups, it is worth noting that prevalence of three activating KIR genes, KIR2DS2, KR2DS5 and KIR3DS1 were highest in the most critically ill children presenting with CM+SRD  contributes to exacerbated inflammatory responses that have been implicated repeatedly in the pathogenesis of severe malaria (Although the proportion of subjects with individual h CM+SRD . While t malaria , 56. ButKIR3DS1 was the least frequent KIR gene in the Gambian population, in line with previous data from other African populations (KIR gene evolution amongst different populations worldwide has suggested that KIR3DS1/L1 has been under positive selection in modern Sub-Saharan African populations, with KIR3DS1 being rare and KIR3DL1 allotypes predominating KIR3DS1, such as reduced susceptibility to malaria or protection from severe forms of malaria, for which our data provide some support. In a study that compared KIR genotypes in Melanesian individuals with and without malaria parasitaemia living in a malaria hyper-endemic area KIR3DS1 positivity was equally distributed in both groups, but KIR3DS1/L1 heterozygosity was significantly associated with being parasite positive.Present in 6.4% and 12.5% of our population control samples and malaria cases respectively, ulations , 50, 55.KIR genes influence susceptibility to malaria. The involvement of genes located at the centromeric part of the KIR locus in modulating the outcome of malaria and in particular the role of KIR2DL2 and KIR2DS2 in the susceptibility to P. falciparum malaria warrant further investigations, and we recommend that larger studies should be performed across different populations to test this hypothesis with additional power. Such studies are needed to complement genome-wide approaches KIR haplotypic diversity.Taken together, our data provide some support for the hypothesis that


Plasmodium falciparum with P. malariae and/or P. ovale sspp), despite use of AL for home-based management of malaria. As a first step to gather evidence on the effectiveness of AL in this setting, a community-based observational study was initiated in an area of intense malaria transmission.In Uganda artemether-lumefantrine (AL) is the first-line treatment for uncomplicated falciparum malaria. During a longitudinal study (SIMI project) investigating the dynamics of intestinal schistosomiasis and malaria in Ugandan lakeshore communities, a consistently high prevalence of malaria was found in young children  within the SIMI cohort was selected on the basis of a positive First Response rapid diagnostic test (RDT) result and microscopy-confirmed malaria. Children were actively followed up 7 and 17 days after baseline. Individuals who were RDT-positive on Day 17 were also followed up on Day 24. At each time-point, a blood smear archive was made and microscopy was performed two-three days after samples were taken. In addition, blood spots were collected onto Whatmann 3MM filter paper. Treatment decisions in the field were based on RDT results. All study participants were treated with AL at baseline. Children who were malaria positive by OptiMAL RDT on Day 7 were retreated with AL and those who were malaria positive on Day 17 were treated with oral quinine. All children were tested for Plasmodium species was performed. To distinguish between recrudescent and new infections, genotyping of merozoite surface proteins (msp1 and msp2) and glutamate-rich protein (glurp) was carried out.Genomic DNA was extracted from blood spots using chelex and real-time PCR diagnosis of Forty children (26.3%) were microscopy-positive for malaria on Day 7 and 33 (21.3%) on Day 17. After genotyping, 33 (21.9%) and 17 (11.7%) children were shown to have recrudescent infections on Days 7 and 17, respectively. Of the 28 children who had received two consecutive AL treatments, 11 were microscopy positive on Day 17.Plasmodium infections were common, with 41.1% of children positive for P. falciparum/P. malariae, 9.2% positive for P. falciparum/P. ovale sspp. and 8.0% for all three species at baseline. By real-time PCR 39.9% of those children infected with falciparum malaria at baseline were P. falciparum positive at Day 17 and 9.2% of those who were infected with P. malariae at baseline were P. malariae positive at Day 17. On Day 24, after two or three consecutive anti-malarial treatments, 10 children were infected with P. falciparum and two with P. malariae.Multi-species Our results suggest that AL may not be as effective as previously thought for treatment of malaria at a community-based level in Uganda and that further more formalised efficacy studies of this drug in high transmission settings are required, particularly in areas where mixed-species malaria infections are common and where mass administration of anthelminthic drugs is being carried out.


Plasmodium vivax and Plasmodium falciparum. In view of the severity of coinfection , overlapping symptoms, and a challenging obscurity of diagnosis, a multidimensional diagnostic approach is suggested.Dengue and malaria are both endemic in South Asia and represent a major public health burden in this region. Though there have been some case reports of concurrent infection with dengue and malaria, yet there are only few cases of such infections reported from South Asia. Here, we present a case of a young male returning from a dengue endemic area who tested positive for the virus along with  His renal and liver function tests, spot urine examination, chest X-ray, and USG abdomen were ordered and all parameters were within normal limits. Since the patient had visited Delhi during an outbreak of dengue and was found to have a low platelet count, a viral serology test using IgM ELISA for dengue virus was carried out that turned out to be positive. A diagnosis of dengue fever was made and supportive therapy was initiated. Patient continued to have fever spikes and chills despite the therapy. Since Delhi is also endemic for malaria, rapid card test for malaria along with thick and thin blood smears for malarial parasite was also carried out. The Malaria Ag (pLDH/HRP2) card test was positive for both Plasmodium vivax as well as Plasmodium falciparum and blood smears showed ring forms of Plasmodium falciparum. This suggested that the patient had concurrent dengue fever and malaria. The tests for Malaria were repeated to ensure conformity and the tests were again positive for both Plasmodium vivax and Plasmodium falciparum. The patient was put on 600 mg of oral Chloroquine Phosphate followed by 300 mg after six hours and then 300 mg daily for two days. Patient responded well to the treatment. Fever subsided and his general condition improved. He was also put on radical cure for malaria with 30 mg of Primaquine daily for 14 days . A 25-year-old male, resident of Srinagar, businessman by occupation returned to Srinagar after a weeklong trip to Delhi in October 2012. The patient started developing fever, chills, myalgias, and headache two days following his return and self-treated himself with Paracetamol. Prophylactic treatment for malaria was not given either before or during the trip. The patient subsequently developed severe headache and high grade fever of 102°F and was admitted to our hospital. Physical examination was unremarkable and haemogram revealed a normal study except for a low platelet count of 86,000/mmConcurrent infection with two different infective agents leads to an overlap of their clinical features that can pose a diagnostic challenge to the physician, especially in endemic areas. In addition, recent studies indicate that a coinfection may be more severe .Salmonella Typhi [Shingella Sonnei [Leptospira spp. [Plasmodium falciparum was published by Charrel et al. in 2005 [Plasmodium vivax in 2006 [Plasmodium falciparum and Plasmodium vivax). The first case was reported by Kaushik et al. in 2007 [Plasmodium vivax infection in two serum samples out of the total 111 samples investigated [P. Falciparum human blood positive samples, revealed that seventy-three percent were of P. falciparum single infections and 26.95% of mixed infections [Earlier there have been reports of concurrent infection of dengue virus with a flavivirus, Chikungunya  and withla Typhi , Shingela Sonnei , and Lepira spp. . The fir in 2005  where th in 2006  while Bh in 2006 . To the  in 2007 . In addistigated . StudiesPlasmodium falciparum and Plasmodium vivax was used twice to ensure confirmation. Earlier analysis by Bharti et al. revealed that overall the rapid diagnostic card test for Plasmodium falciparum and Plasmodium vivax was 93% sensitive and 85% specific with a positive predictive value (PPV) of 79% and a negative predictive value (NPV) of 95% [The accuracy of a serological test to diagnose dengue in patients experiencing malaria has been questioned earlier because reactivity is often nonspecific on certain rapid serological assays ; however) of 95% .According to Carme et al. in French Guiana the specific rate of concurrent malaria and dengue infection from overall febrile patients was equal to 0.99 , which i


Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases facing the global swine industry. Viremia profiles of PRRS virus challenged pigs reflect the severity and progression of infection within the host and provide crucial information for subsequent control measures. In this study we analyse the largest longitudinal PRRS viremia dataset from an in-vivo experiment. The primary objective was to provide a suitable mathematical description of all viremia profiles with biologically meaningful parameters for quantitative analysis of profile characteristics. The Wood's function, a gamma-type function, and a biphasic extended Wood's function were fit to the individual profiles using Bayesian inference with a likelihood framework. Using maximum likelihood inference and numerous fit criteria, we established that the broad spectrum of viremia trends could be adequately represented by either uni- or biphasic Wood's functions. Three viremic categories emerged: cleared ), persistent  and rebound (biphasic within 42 dpi). The convenient biological interpretation of the model parameters estimates, allowed us not only to quantify inter-host variation, but also to establish common viremia curve characteristics and their predictability. Statistical analysis of the profile characteristics revealed that persistent profiles were distinguishable already within the first 21 dpi, whereas it is not possible to predict the onset of viremia rebound. Analysis of the neutralizing antibody(nAb) data indicated that there was a ubiquitous strong response to the homologous PRRSV challenge, but high variability in the range of cross-protection of the nAbs. Persistent pigs were found to have a significantly higher nAb cross-protectivity than pigs that either cleared viremia or experienced rebound within 42 dpi. Our study provides novel insights into the nature and degree of variation of hosts' responses to infection as well as new informative traits for subsequent genomic and modelling studies. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious diseases threatening pig production worldwide in-vivo experimentally PRRSV challenged pigs are valuable indicators of the severity and progression of the infection in the host, and thus provide crucial information for the required subsequent disease control measures Viremia profiles of PRRSV challenge experiments with longitudinal viremia measures reveal substantial differences in the viremia profiles between hosts infected with the same PRRSV challenge dose, pointing to considerable variation in the host response to PRRSV infections. For example, numerous studies have shown breed differences in viremia levels and duration and also in antibody production 2 = 0.3), pointing to significant host genetic influence underlying disease severity and progression. Rebound was however not found to be heritable and thus thought to be controlled more by the virus than the host genotype Given the apparent diversity in viremia patterns, several important questions arise. For example, for vaccine development or consideration of genetic disease control strategies it is important to determine whether and to what extent the observed differences in the profiles are influenced by the host and the virus genotype. In the longitudinal study of Boddicker et al. One hallmark of PRRSV is its high genetic diversity due to its fast mutation rate, resulting in continuous emergence of new quasi-species that may evade the host's immune system From an epidemiological perspective, viremia rebound may constitute a problem as pigs diagnosed as cleared may have high levels of infectious virus a few days later. It would be useful to know whether viremia rebound reflects genuinely viremia reactivation rather than fluctuations in circulating virus load or measurement errors, and whether all or only a subset of pigs experience rebound. In particular, it would be useful to know whether virus rebound, or persistence, can be predicted based on early serum profile characteristics.Addressing the questions raised above would require frequent repeated measurements of viremia, as well as of nAb diversity, on a large number of pigs subjected to the same experimental PRRSV challenge conditions. Such data are now available from the PRRS Host Genetic Consortium The primary objective of this study was to find mathematical functions that adequately represent the full range of viremia profiles obtained from a large scale PRRSV infection experiment, and use these to determine quantitative characteristics of infection dynamics. The functions will be used to derive an objective method of classification of viremia profiles based on statistical inference and to assess the relationship between the breadth of nAb response and viremia profile.5 tissue culture dose50 (TCID50). The challenged pigs came from high health farms that were free of PRRSV, Mycoplasma hypopneumoniae and swine influenza virus. Pigs were placed randomly in pens of 10–15 pigs and were infected with PRRSV after a 7 day acclimation period, i.e. at 0 days post infection (0 dpi). Blood samples were collected immediately before infection (0 dpi) and at 4, 7, 11, 14, 19/21, 28, 35, 40/42 dpi and the level of PRRS viremia was measured using a semi-quantitative TaqMan PCR assay for PRRSV RNA. The viremia quantity data from RT-PCR was transformed on the logarithmic scale to the base 10 before the model fitting. Due to the sensitivity of RT-PCR the threshold of detection was set at 1 units on the log10 scale The data analysed in this study was obtained from the PRRS Host Genetic Consortium (PHGC) trials, the largest PRRSV in-vivo challenge study to date; a detailed description of the experimental protocol is outlined in For the purpose of this study, only individuals with a minimum of 6 serum viremia observations were retained. This resulted in a viremia dataset comprising 1371 pigs in total, with over 170 pigs per trial, with the exception of trial 6 for which data from 89 pigs with less than 6 viremia observations had to be discarded. The majority of missing observations in Trial 6 were from 14 dpi onwards due to the outbreak of a bacterial infection, thus reducing the potential to capture viremia rebound. All individuals from Trials 7 and 8 were missing the last observation at 40/42 dpi due to management issues in the experimental facility.in-vivo challenge experiment (NVSL-7985 denoted henceforth as NVSL) and three additional PRRSV isolates: KS06-72109 (KS06), P-129 and VR-2332 (VR). These type 2 PRRSV isolates were chosen for genetic differences based on viral ORF5 sequence. KS06 had been isolated in 2006 and as a more contemporary isolate is still found in the field. Excluding the relatedness between P129 and NVSL (95%), nucleotide comparisons within ORF5 show that the PRRSV isolates differed from each other by 10% or greater. Each serum sample was reacted with the panel of four type 2 viral isolates, where the NVSL isolate served as the homologous virus in the serum neutralisation assays. Serum samples were considered positive for PRSSV nAb at a titre of eight or higher. Individuals in PHGC Trials 1–3 were assigned to one of the following five nAb categories: 1) failed to produce a nAb response, 2) only produced antibodies against the challenge virus (homologous nAb response), 3) produced nAb against the original and a different isolate (mild heterologous response), 4) produced nAb against the original and two different isolates (moderate heterologous response), 5) produced nAb against all four isolates (broad response). Analysis was conducted on the combined nAb categories of no cross protection (categories 1–2) and cross-protection (categories 3–5) as this was the most biologically relevant grouping of the nAb classes.To explore hypotheses surrounding the association between viremia profiles and the variability of the nAb response to PRRSV, nAB data from serum collected at 42 dpi was obtained for 490 individuals from the first three trials using a virus neutralization assay as outlined in Visual inspection of the individual viremia profiles from the raw data indicated that the profiles can be empirically grouped into three categories, illustrated in 10 RT-PCR) at The Wood's function, a gamma-type function often used to empirically describe lactation curves in dairy cattle In order to represent the bi-phasic profiles, the Wood's function was extended to a biphasic function described by equation (0.2):10 scale the errors were assumed normally distributed around 0 with a standard deviation of 0.5 log units, and for observations less than or equal to the RT-PCR observation threshold the errors were assumed cumulative normal.Both the Wood's and Extended Wood's function were fitted to the individual data profiles using Bayesian inference with a likelihood framework. This was implemented by using an adaptive, population based Markov chain Monte Carlo method with power posteriors as described in Both the Wood's and Extended Wood's functions were fitted to all pigs. Thus the fitting procedure provided for each individual pig posterior distributions for every parameter of the Wood's and Extended Wood's function, respectively, from which parameter means, modes and credibility intervals were derived.The accuracy of the model fit and choice of best model was assessed based on the Akaike's Information Criterion (AIC), together with inspection of the predicted model profiles with 95% posterior credibility intervals for every individual, to gain insight into both the accuracy and potential bias of the model predictions over the time course of the experiment. This included plotting histograms of the parameter estimates, inspection of the residuals, as well as calculating the (product moment) correlation between observations and predictions at each sampling time point.Due to the high degree of fluctuation in the viremia measurements, classification of the raw data profiles into the three categories shown in 10 RT-PCR data and the Akaike's Information Criterion (AIC) was obtained. The AIC difference between the competing models for each individual pig is analogous to the likelihood ratio test statistic (D) when adjusted for twice the difference in the number of model parameters of the two models. The Wood's model has 3 parameters and the Extended Wood's model has 7 parameters, thus the AIC difference adjusted for the number of parameters becomes:An individual was classified as experiencing viremia rebound if the biphasic Extended Wood's model had a statistically superior fit to the data than the uni-modal Wood's model at the 95% significance level. For this purpose, both models were separately fitted to the individual's log10 scale then the profile was classified as ‘persistent’ within the 42 day observation period, otherwise it was referred to as ‘cleared’.Transient experimental persistence henceforth referred to as persistence was defined as a subset within the non-rebound profiles according to the Wood's model prediction at the end of the experiment, i.e. at 42 dpi. If the model prediction at that time point remained above the detection threshold of one RT-PCR unit on the logDescription of the individual viremia profiles by analytical functions provides the opportunity to explore whether viremia features associated with different phases of the infection are related, and to construct and test hypotheses. Pearson product-moment correlations were calculated between individual Wood's and Extended Wood's function parameter estimates and the derived parameters -Furthermore, to determine whether the phenomenon of transient persistence or viral rebound could be predicted by the shape of the profile during the earlier phase of infection, the Wood's model was fitted to the truncated dataset comprising observations from all pigs (rebounders and non-rebounders) from 0 to 21 dpi only. A linear mixed model analysis (using PROC MIXED of SAS 9.3) was then carried out to assess statistical differences in the individual Wood's curve parameter estimates associated with the different profile types , respectively. The dependent variables were the individual mean values of the estimated posterior distributions of the Wood's model and derived parameters obtained from the primary phase:To test the hypothesis whether viremia rebound, clearance or persistence was associated with greater within host viral diversity and thus more diverse neutralizing antibodies, logistic regression was carried out for the 439 pigs with nAB assays, using PROC GLIMMIX in SAS 9.3, assuming an exponential distribution of the data, conditional on random effects. In the full mixed model the dependent variable was the neutralising antibody class (i.e. binary as the nAB categories were pooled), the independent variables were the class of the viremia profiles . In addition, trial, profile class, parity, sex and corresponding interactions were fitted as fixed effects and dam within trial and pen within trial were fitted as random effects. Similar to the mixed model analysis above, the number of random and fixed effects in the full model was hierarchically reduced by examining their impact on the AIC model fit statistics, and according to statistical significance of individual fixed effects, respectively.Visual inspection of the predicted individual profiles , included fixed effects of profile class  determined on the complete dataset, as well as the trial and the trial by parity interaction, with pen within trial and dam within trial as random effects. Statistically significant differences for all the individual Wood's curve parameter estimates  model itself is constrained by the model parameters; the time of the peak viremia level is dependent on both parameters By parsimony, in the absence of more frequent measurements, the Extended Wood's model was chosen as it is a simple model representing the main features of the biphasic profiles. It encapsulates the assumption that the second phase of the profile has the same essential shape characteristics as the primary phase. Furthermore the Extended Wood's model parameters were also able to encapsulate the possible anamnestic nature of the immune response; the parameters allowed for variation in the size, timing, rate of increase and rate of decline in the secondary phase. The consistency of the model residuals and correlations indicate that the Extended Wood's model provides indeed a good fit during both phases of the profile; in fact the correlations between the data and model predictions were highest during the second phase. The second phase was generally shorter (28–42 dpi) and less severe than the first phase (0–28 dpi), as indicated by greater predicted rates of viremia increase and decline in the second phase of the profile  from predator-prey type interactions between the virus and the immune response Similar arguments may be used to interpret the observed high inter-host variation in viremia decline post the initial viremia peak. Variation in the rate of viremia reduction may be a function of the host's own immune response, virus mutation mechanisms or the interaction between both processes. The moderate negative correlation between parameters From the current study we cannot affirm that there would be infectious and epidemiological consequences in the secondary phase of rebound profiles. Inferences made on infectiousness and epidemiological consequences using the data obtained via quantitative RT-PCR may be limited due to the potential discrepancy between the measured viral genome load and the non-measured true viral load. Thus, the observed secondary phase of rebound may be the result of detecting circulating junk genomes rather than genomes of infectious particles and may thus have no significant epidemiological consequence. However, previous studies have explored the relationship between diverse viremia measurements an infectiousness of pigs infected with PRRSV Previous PRRS studies alluded to the phenomenon of viremia rebound in-vivo PRRSV infections in future studies.Lastly, there is accumulating evidence for substantial host genetic variation in response to PRRSV


Plasmodium falciparum malaria in hyperendemic regions. Premature elimination of non-parasitized red blood cells (nRBC) has been considered as one mechanism involved in the genesis of severe malaria anaemia. It has been reported that apoptosis can occur in RBC and, consequently, this cell death process could contribute to anaemia. This study was performed to evaluate the susceptibility of nRBC to apoptosis in a malaria anaemia murine model.Severe anaemia is a common complication of 6 P. yoelii 17XL parasitized RBC (pRBC) and, then, parasitaemia and anaemia were monitored. Apoptosis in both pRBC and nRBC was assessed during early and late phases of infection by flow cytometry using Syto 16 and annexin V-PE double staining and forward scatter measurement.Balb/c mice were intraperitonially inoculated with 1 × 10Plasmodium yoelii 17XL parasites was characterized by progressive increase of parasitaemia and acute anaemia, leading to death. Flow cytometry analysis showed that a number of pRBC was in the apoptotic process. It was noteworthy that the increase of nRBC apoptosis levels occurred in the late phase of infection, when anaemia degree was notably accentuated, while no significant alteration was observed in the early phase.As expected, experimental infection of Balb/c mice with The increased levels of nRBC apoptosis herein firstly reported, in malaria infection could represent a putative mechanism worsening the severity of malarial anaemia. Plasmodium falciparum is the most prevalent and the most frequent parasite species responsible for the severe and lethal forms of the disease. Complications associated to P. falciparum infection include severe anaemia, which affects mainly children and pregnant women living in malaria hyperendemic regions [Malaria remains the tropical disease of major prevalence in the world, representing great problem of public health with approximately 250 million cases and 900 thousand deaths annually . From al regions .The immunological processes involved in malaria anaemia cannot be implicated as the sole cause of erythrophagocytosis during malaria . It is w2+, which leads to cell shrinkage, membrane blebbing, phosphatidylserine exposure and protease activation [Apoptosis - a physiological process of programmed cell death related to nucleated cells - can also occur in RBC as a result of intracellular influx of Cativation . Althougtivation ,9. Enhantivation  and in mtivation -12. The Plasmodium yoelii 17XL parasites was used as a malaria anaemia model. Where indicated, non-infected, age-matched mice were used as control. All animal experimentation was approved by the Ethics Committee on the Use of Animals of the Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, RJ, Brazil.The lethal experimental infection of Balb/c mice with 6 P. yoelii 17XL-pRBC in 0.2 mL phosphate buffered saline (PBS). During infection, parasitaemia and anaemia degree were routinely monitored through mice tail blood samples. Parasitaemia was determined by counting the number of pRBC in a total count of 1000 RBC in thin blood smears stained by the Romanowski's method . Anaemia was evaluated by counting the number of RBC/mm3 of blood. Briefly, 2 μL of blood were suspended in 0.5 mL heparinized PBS, diluted 1:10 in the same buffer and, then, the number of RBC determined in a haemocytometer.For infection, female Balb/c mice aged 6-8 weeks, provided by the Centre for Laboratory Animals Breeding of the Fiocruz, were intraperitoneally inoculated with 1 × 10P. yoelii infection through the detection of phosphatidylserine exposure (PS) at the cell surface and cell shrinkage [g for 10 min at room temperature, washed twice with PBS and, then, incubated at 37°C for 40 min at a density of 1 × 105 cells/400 μL in PBS containing 100 nM Syto 16 (Invitrogen). After incubation, staining buffer was discarded and the RBC were stained with 5 μL annexin V (BD Pharmingen) for 15 min at room temperature in 100 μL annexin-binding buffer (BD Pharmingen) containing 100 nM Syto 16. Finally, cells were five times diluted in annexin-binding buffer containing Syto 16 and both forward scatter and fluorescence emission were assessed using a flow cytometer Apoptosis was identified in the early (day 4) and late days 6-7) phases of  phases oex-vivo analysis, apoptosis was also measured after incubation of RBC for 24 h at 37°C at a haematocrit of 0.5% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, and 1 CaCl2 (pH 7.4).In addition to Statistical analyses were performed using t student test in GraphPad Prism 5.0 software ; a p-value < 0.05 was considered significant.P. yoelii 17XL malaria murine model was used in the present report.Data from experimental rodent malaria infection, widely used as malaria models, have reinforced premature destruction of nRBC as a relevant process of malaria anaemia ,14. ThusP. yoelii 17XL infection was marked by an accentuated anaemia degree and it was lethal to Balb/c mice [As expected, b/c mice ,16. InfeP. yoelii 17XL infection, when anaemia degree was accentuated, was marked by RBC apoptosis. As expected, a percentage of P. yoelii-pRBC exposing PS and, therefore, undergoing apoptosis    Figure . Convers2+ and Na+ necessary for its intracellular growth [2+ influx leads to activation of phospholipid scramblase and Ca2+-sensitive K+ channels responsibles to PS exposure at outer membrane leaflet and cell shrinkage, respectively [The apoptotic process in pRBC has been described as a result of intraerythrocytic plasmodia development. During schizogony, malaria parasites activate non-selective cation channel in host RBC membrane, allowing entry of Car growth . Sequentectively . If one ectively , apoptosectively .P. falciparum [However, if the augmented elimination of nRBC  in both human and experimental malaria ,24,25 colciparum .P. falciparum-pRBC are also capable of inducing apoptosis in endothelial and neuroglia cells [P. falciparum in vitro culture undergo apoptotic process [Since nRBC apoptosis was increased during only the late phase of malaria infection, one may wonder which factors could be implicated to its induction? Notably, different types of RBC apoptosis-inducer, such as anti-RBC antibodies, oxidative stress and parasite antigens, are associated to malaria infection . Indeed,ia cells  and byst process . Moreove process  - has be process . Thus, bex vivo pRBC and nRBC are capable of exposuring PS and these cells were not significantly sequestrated at the present study, it is presumable that the simple presence of PS is not enough for in vivo endothelium adhesion, as previously suggested in in vitro-measured adhesiveness studies with both pRBC and nRBC [in vivo adherence capacity of apoptotic RBC on endothelium are necessary to clarify the role of PS, besides P. falciparum erythrocyte membrane protein 1 (PfEMP1), in cerebral and placental malaria pathogenesis. Intra-vital microscopy studies could help for clarifying these questions.Finally, since and nRBC ,33,34. FThe present report showed for the first time that malaria infection increase the levels of nRBC apoptosis, a process that could represent a putative mechanism worsening the severity of malaria anaemia. Attempts to decipher immune and parasitic factors related to RBC apoptosis during the course of malaria infection are currently underway on rodent and human malaria.The authors declare that they have no competing interests.PRRT participated in the design of the study, carried out the animal experimentation, flow cytometry and data analysis and drafted the manuscript. ADM and LAS helped in animal experimentation and carried out parasitaemia and anaemia measurements. DMB provided characterized parasite strains and revised the manuscript. CTDR helped in the design of the study and revised the manuscript. MFFC conceived the study, coordinated its design, and finalized the manuscript. All authors read and approved the final manuscript.


Prompt, accurate diagnosis and treatment with artemisinin combination therapy remains vital to current malaria control. Blood film microscopy the current standard test for diagnosis of malaria has several limitations that necessitate field evaluation of alternative diagnostic methods especially in low income countries of sub-Saharan Africa where malaria is endemic.The accuracy of axillary temperature, health centre (HC) microscopy, expert microscopy and a HRP2-based rapid diagnostic test (Paracheck) was compared in predicting malaria infection using polymerase chain reaction (PCR) as the gold standard. Three hundred patients with a clinical suspicion of malaria based on fever and or history of fever from a low and high transmission setting in Uganda were consecutively enrolled and provided blood samples for all tests. Accuracy of each test was calculated overall with 95% confidence interval and then adjusted for age-groups and level of transmission intensity using a stratified analysis. The endpoints were: sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). This study is registered with Clinicaltrials.gov, NCT00565071.Of the 300 patients, 88(29.3%) had fever, 56(18.7%) were positive by HC microscopy, 47(15.7%) by expert microscopy, 110(36.7%) by Paracheck and 89(29.7%) by PCR. The overall sensitivity >90% was only shown by Paracheck 91.0% [95%CI: 83.1-96.0]. The sensitivity of expert microscopy was 46%, similar to HC microscopy. The superior sensitivity of Paracheck compared to microscopy was maintained when data was stratified for transmission intensity and age. The overall specificity rates were: Paracheck 86.3% [95%CI: 80.9-90.6], HC microscopy 93.4% [95%CI: 89.1-96.3] and expert microscopy 97.2% [95%CI: 93.9-98.9]. The NPV >90% was shown by Paracheck 95.8% [95%CI: 91.9-98.2]. The overall PPV was <88% for all methods.The HRP2-based RDT has shown superior sensitivity compared to microscopy in diagnosis of malaria and may be more suitable for screening of malaria infection. The World Health Organization (WHO) recommends prompt parasitological confirmation by microscopy or rapid diagnostic test (RDTs) for all patients with suspected malaria before treatment is started ,2. TreatIn most malaria endemic countries of sub-Saharan Africa, the current standard for laboratory confirmation of a clinical malaria diagnosis is a peripheral blood film, examined microscopically. However, microscopic based diagnosis of malaria is labour-intensive requiring trained staff and quality equipment attributes that are scarce in resource-poor settings ,9. Thus,Uganda piloted the histidine-rich protein II (HRP2)-based RDT (Paracheck) and rolled it out as an instrument of choice for parasite-based malaria diagnosis and patient management in six districts in the first phase in 2008. Paracheck was distributed to parish-level and to selected sub-county health centres (HCs) without laboratory infrastructure. RDTs have been out of stock for twelve months. Currently, there are 955 sub-county and 2,008 parish-level HCs in 112 districts in the country. Depending on availability of RDTs, scaling up to additional 22 districts is planned for January 2011.Some data suggest that RDTs may not be very sensitive especially in varying transmission intensities ,14. HoweData was collected from June to July 2007 in three randomly selected sub-county level government HCs in Bushenyi and three in Iganga districts of Uganda. Bushenyi is categorized as low and Iganga as high malaria transmission intensity settings. The annual entomological inoculation rates are not known, but are reported to be <10 and >500 infective bites per person per year in the neighboring districts of Kanungu and Tororo respectively .A sub-county level HC laboratory is manned by one laboratory assistant who has two years of pre-service training. The laboratory personnel perform all investigations requested by the clinician. For malaria, at least one out of ten slides is stored daily for quality control. The district laboratory-focal person  performs quarterly technical support supervision to HC laboratories. During supervision the microscope, stains, staining of slides and reporting are checked. The supervisor performs fresh films with the laboratory assistant. Where necessary, an immediate feedback is given, but also takes a percentage of the slides for further examination. The external quality assurance is coordinated at the national level. As contribution to improvement in the delivery of services, the study provided HC laboratories with new binocular microscopes, reagents and supplies.p-value set at 0.05, with estimated sensitivity of Paracheck of 95% , the specificity of expert microscopy 98.6% [95%CI: 94.9-99.8] was significantly higher than that of Paracheck 90.6% [95%CI: 84.4-94.5], p < 0.001 for each comparison). Its specificity (78.1%) was significantly lower than that of HC microscopy 91.8% [95%CI: 83-96.9] (p < 0.001) or expert microscopy 94.5% [95%CI: 86.6-98.5] (p < 0.001), but similar to presumptive diagnosis based axillary temperature.In the high transmission setting, the sensitivity of Paracheck was 93.5% [95%CI: 85.5-97.9] and significantly higher than that of other diagnostic methods , HC microscopy (27.3%), expert microscopy (60%) and Paracheck (40.9%). In the high transmission areas, the PPV for presumptive diagnosis (66.7%) was significantly lower than for other diagnostic methods. In addition, the PPV for expert microscopy 90.5% [95%CI: 77.4-97.3], HC microscopy 86.7% [95%CI: 73.2-94.9] and Paracheck 81.8% [95%CI: 72.2-89.2] were statistically not different. The NPV for Paracheck 97.7% [95%CI: 93.3-99.5] in low transmission and 91.9% [95%CI: 82.3-97.3] in high transmission were significantly higher than that of other methods. Only Paracheck had a NPV >90% in both age-groups, being 94.7% [95%CI: 89.3-97.8] in patients ≥5 years and 98.3% [95%CI: 90.9-100] in those <5 years.The accuracy of clinical diagnosis routinely practiced at rural HCs, health centre microscopy, expert microscopy and Paracheck was compared in patients with uncomplicated malaria. The diagnostic accuracy of these methods was measured against PCR as gold standard. Some studies have however reported on the accuracy of RDTs using expert microscopy as gold standard -17. For If expert microscopy was the gold standard, the overall sensitivity was consistently high. The sensitivity (97.9%), specificity (74.7%) and NPV (99.5%) of Paracheck were similar to that reported elsewhere ,16,17,27This study reports that 37% and 47% patients who were negative by HC microscopy and expert microscopy respectively were confirmed to have malaria by PCR. These rates are slightly lower than that of another study , which rP. falciparum during treatment found that PCR remained positive for a median of 144 hours. In another study [P. falciparum DNA from circulating nonviable parasites after successful treatment. However, HRP2-based RDTs remain positive after treatment, and the HRP2 signal is of no value during the first week of treatment [P. falciparum with an HRP2 gene deletion or reduced HRP2 expression [Eight patients were negative with microscopy and Paracheck, but positive with PCR. Even a repeat of the analysis during quality control, the eight samples remained positive. An earlier investigation  into theer study  PCR detereatment . Therefopression , and sucpression .The overall specificity of Paracheck was lower than that of HC- and expert microscopy. This pattern was also shown when data was adjusted by transmission intensity and age-groups. The low specificity rates have been attributed to persistent antigenaemia even after successful treatment in some reports ,23,27,34falciparum infections [Uganda has adopted RDT as a method for parasitological diagnosis of malaria in addition to microscopy . RDTs arfections .The PCR assay was used as the gold standard because it has the ability to detect malaria parasites in patients with low levels of parasitaemia. Infections with ≤5 parasites per μl can be detected with 100% sensitivity and equal specificity . HoweverIn estimation of the sample size with the aid of nomogram , the preHigh sensitivity of malaria diagnosis is important in all settings, and essential for the most vulnerable population groups in which malaria infection produces an acute illness that can rapidly progress to death. The HRP2-based test demonstrated a superior sensitivity compared to microscopy and presumptive methods in the diagnosis of uncomplicated malaria in remote health facilities. Based on the current findings, the HRP2-baed RDT may be more suitable for screening of malaria infection in routine practice in primary health care centres.The authors declare that they have no competing interests.All authors conceived and designed the study; VB and FN collected, analysed, interpreted the data and drafted the manuscript; PM critically revised the manuscript. All authors read and approved the final manuscript.


This study aimed to evaluate the impact of home-based management of malaria (HBM) strategy on time to treatment and reported presumed malaria morbidity in children aged less than 5 years in Rwanda.The study was carried out in two malaria-endemic rural districts, one where HBM was applied and the other serving as control. In each district, a sample of mothers was surveyed by questionnaire before (2004) and after (2007) implementation of HBM.After implementation, we observed: i) an increase (P < 0.001) in the number of febrile children treated within 24 hours of symptom onset in the experimental district (53.7% in 2007 vs 5% in 2004) compared with the control district (28% vs 7.7%); ii) a decrease in the reported number of febrile children in the experimental district  compared with the control district .HBM contributed to decrease time to treatment and reported presumed malaria morbidity. In sub-Saharan Africa, children less than 5 years of age continue to pay the heaviest tribute to malaria , 2. In rIn Rwanda, the HBM strategy was adopted in May 2004. It aims to facilitate access to care and to reduce the time to effective treatment. In December 2004, the district of Nyanza was one of the first pilot districts where HBM was tested. In this country, HBM is based on voluntary community health workers known as drug distributors (DDs) who are elected by the population and trained for this activity. Currently, DDs use RDT to detect malaria prior to any treatment. However, by the time this study was conducted, DDs were trained to recognize malaria using a simple algorithm and to administer ACT according to an age-appropriate treatment protocol. Any child aged less than 5 years with febrile illness was considered to have malaria. The DDs examining the febrile children brought by their mothers had to decide to address them to the health center or to treat them at home if there was no contra-indication. In the latter case, the DDs sold to the mothers, at a subsidized price, pre-packaged artemether-lumefantrine (AL) tablets, and gave the first dose. A child was considered febrile if the DD personally noted, by touch, the presence of fever, or if the mother reported that the child has had fever.In addition to distribution of drugs, DDs were also in charge of sensitizing and providing information to the community and in particular to mothers on early consultation for fever and the correct administration of anti-malaria drugs to children aged less than 5 years.In Africa, studies on the health impact of HBM, including its effects on morbidity, are still sparse, particularly as many countries are changing their malaria treatment policy in favor of ACT . In RwanThe aim of this study was to evaluate, at a community level, the impact of HBM on time to treatment and on malaria morbidity (febrile episodes without RDT or laboratory confirmation), and on reported all-cause morbidity in children aged less than 5 years in the pilot district of Nyanza two and a half years after its introduction, independently of the possible impact of other community-based interventions.A comparative study was carried out in 2 districts, the HBM pilot district and a control district. The variables studied were measured in both districts 5 months before and 2 and a half years after implementation of HBM. Between these two measurements, HBM was implemented by the national malaria control program. During and after implementation of HBM, all other activities relating to malaria control continued as usual in both districts.Anopheles gambiae and Anopheles funestus. Plasmodium falciparum is the predominant species, responsible for over 90% of cases [The two districts in which the study was carried out are rural and malaria was endemic. The district of Nyanza was chosen by the Ministry of Health as a pilot district for HBM because of its malaria endemicity and the presence of a financial partner. The district of Bugesera was randomly chosen among the malaria-endemic districts where HBM had not yet been introduced. The district of Nyanza is located in the South Province of Rwanda, whereas the district of Bugesera is located in the East Province. In Rwanda, the major malaria vectors are of cases , 9. LikeThe pre-implementation study comprised 362 mothers (178 in the district of Nyanza and 184 in the district of Bugesera) with a participation rate of 98%. Its aim was to collect basic data to be used to evaluate the impact of HBM after implementation. For the post-implementation study, sample size was calculated to include at least 40% of febrile children aged less than 5 years treated within 24 hours of symptom onset in the experimental district (or two times more than in the control district). With a power of 90% and a 95% confidence interval, taking into account a cluster effect of 2 and a non-response rate of 2%, the minimum number of mothers required in the two districts was 376, or 188 in each district. All mothers took part in the study, a participation rate of 100%. During the two studies, before and after implementation, the mothers investigated were recruited in cells , which were selected by the cluster sampling method. In each cell selected, the number of mothers to be investigated was estimated in proportion to the size of the cell. The investigators visited the households following a circular centrifugal route (starting from the center towards the periphery of the cell) until the required number of persons had been included. In each household, the carers of the children aged less than 5 years were questioned.The questionnaires were designed to determine, before and after HBM implementation: i) time to treatment for febrile children aged less than 5 years reported by the mothers during the 2 weeks before our survey; ii) the occurrence of fever ; iii) the development of illness . The questions put were identical before and after implementation. After implementation, specific questions on HBM were added and were put only in the experimental district. The DDs and health facilities  were considered as formal sources of care.The first study was carried out in July 2004 and the second in July 2007. The questionnaires were administered in the households to the carer of the children aged less than 5 years . In this study, « mother » refers to the carer of the sick child in the household. The aims of the study were first explained to the mothers, who were then included in the study after giving their voluntary informed consent. Mothers who were not resident in the district were excluded from the study. The mothers were interviewed face to face and the questionnaires were directly filled in by the investigators.Experienced investigators were carefully selected and trained in administration of the questionnaire. The questionnaires were translated into kinyarwanda (the native language of Rwanda) and pre-tested before being applied. The completed questionnaires were checked the same day to ensure they were correctly and fully completed. Double data entry was performed using Epi Info 3.3.2 . Data were converted into SPSS 13.0  for statistical analysis. In each district, the variables studied were compared before and after implementation of HBM and the data of the experimental district were compared with those of the control district. Comparisons were made using the chi-squared test and Fisher's exact test for small series, and the conventional level of 5% was considered significant.The majority of mothers were aged less than 40 years, were married and had an agropastoral activity (94%). One in 5 was illiterate . In bothAfter implementation of HBM, the percentage of febrile children aged less than 5 years who were treated within 24 hours improved significantly in both districts P. In thisAfter implementation of HBM, the reported proportion of febrile children among the children aged less than 5 years  decreased in both districts, experimental  were taken to a formal health care source within 24 hours of fever onset, and in 8 of 10 cases they were taken to a DD. This suggests that HBM had a specific impact on the number of children treated within 24 hours. The results of our study confirmed those of the health facility reports  showing Our results suggested that implementation of HBM was followed by a significant decrease in the number of febrile children aged less than 5 years in the two districts. This decrease may be due to the combined effect of several interventions. In addition to introduction of the community-based health insurance scheme, insecticide-treated bed nets had been distributed on a nation-wide scale to children aged less than 5 years and pregnant women during the observation period. During the same period, in all health facilities in the country the Ministry of Health introduced AL as first-line anti-malaria treatment instead of amodiaquine and sulfadoxine-pyrimethamine.Studies have shown that cases of pediatric malaria seen in the health facilities decreased following the distribution of insecticide-treated bed nets  or introFew studies have found that programs for treatment of presumptive malaria in children aged less than 5 years by community health workers can reduce malaria morbidity , 18. A sNot all febrile children treated presumptively by DDs necessarily had malaria, for fever is a symptom of many other diseases, including respiratory tract infections. By the time this study was conducted, DDs did not have the possibility of confirming the presence or absence of the parasite in children reported to be « febrile » by their mothers. However, we assume that the situation in Rwanda probably did not differ from that of the other malaria-endemic African countries, where the majority of cases of fever were malarial .Our findings suggested that implementation of HBM led to a significantly higher proportion of febrile children receiving treatment within 24 hours of symptom onset and to a decrease in reported malaria morbidity. These effects were accompanied by a general trend towards improvement, probably attributable in particular to the development of community-based health insurance schemes, the distribution of insecticide-treated bed nets and the introduction of ACT in the health facilities.