The hypothesis is that hemoglobin-based metrics are useful tools for estimating malaria endemicity and for monitoring malaria control strategies. The aim of this study is to compare population hemoglobin mean and anemia prevalence to established indicators of malaria endemicity, including parasite rates, rates of enlarged spleens in children, and records of (presumptive) malaria diagnosis among populations living with different levels of malaria transmission.2) between population Hb mean and anemia prevalence and altitude, parasite rate, and spleen rate were investigated in children ages 2 to 10 years, and in the general population; 21,664 individuals from 156 different communities were surveyed. Altitude ranged from 5 to 2120 meters. In young children, correlations between altitude and parasite rate, population Hb mean, anemia prevalence, and spleen rate were high . In the general population, correlations between altitude and population Hb mean and anemia prevalence were 0.83 and 0.85, respectively. Among young children, parasite rate correlated highly with anemia prevalence, population Hb mean, and spleen rate . Population Hb mean  increased with altitude, from 10.5 g/dl at <500 m to 12.8 g/dl at >1500 m (p<0.001).Convenience sample, multisite cross-sectional household surveys conducted in Papua New Guinea. Correlations (rIn PNG, where Plasmodium vivax accounts for an important part of all malaria infections, population hemoglobin mean and anemia prevalence correlate well with altitude, parasite, and spleen rates. Hb measurement is simple and affordable, and may be a useful new tool, alone or in association with other metrics, for estimating malaria endemicity and monitoring effectiveness of malaria control programs. Further prospective studies in areas with different malaria epidemiology and different factors contributing to the burden of anemia are warranted to investigate the usefulness of Hb metrics in monitoring malaria transmission intensity. Malaria, with about 500 million cases reported per year worldwide, remains one of the predominant diseases in tropical countries. The intensity of malaria transmission depends on a variety of factors such as climate and geographical context, human population, socio-economical level and vector ecology, as well as the type of control strategies in place. Nowadays, several efficient strategies are being used to decrease the malaria burden. Most prominent among them are insecticide treated bed nets (ITN), indoors residual spraying (IRS), artemisinin combination treatments (ACT)et al. that the spleen rate may vary between sub-populations of non-pregnant women with similar levels of exposure to malariaDifferent tools and metrics have been used for more than 150 years to investigate the level of malaria endemicity in a given population. Traditionally, the spleen rate (SR), i.e. the prevalence of splenomegaly in a population, has been used to classify malarial endemicityet alIn the 1950's, the prevalence of blood stage malaria parasites  was introduced as the principal metric for monitoring malarial endemicity and remains the method of choicewww.map.ox.ac.uk), used the Plasmodium falciparum (Pf) parasite Rate (PfPR), usually in children 2–10 yearsPf Annual Parasite Incidence (PfAPI) to map Pf endemicity. As part of this work, Hay and colleagues published an extensive and detailed review on the tools and strategies available to monitor malaria endemicity All these different metrics have been integrated into control strategies such as the Global Malaria Eradication Programme coordinated by the World Health Organization (WHO) since the mid 1950's. More recently the Malaria Atlas Project , often resulting in anemia Plasmodium falciparum (Pf) responsible for more than 50% of all clinical malaria episodes, and Plasmodium vivax (Pv) being responsible for most of the remaining cases. In past surveys, entomological inoculation rate (EIR) has varied between <1 and 400PNG is situated in the South-West Pacific and presents a wide range of malaria endemicity due to its mountainous geography, ranging from highly prevalent at sea level to absent in the highlands (up to 4000 meters)Individual informed consent was obtained from all participants or in case of children their guardians before enrolment into the study. Malaria surveys conducted in the Highlands of PNG et al. (2003–2009) The study was performed in PNG alongside a series of household malaria surveys performed in three different regions of the country: the Highlands , the Middle Sepik  and Madang . The different villages were chosen according to their location and representativeness in terms of presumptive malaria endemicity and population diversity. Further details of the selection of Highlands and Sepik survey locations and of the survey methodology are given by Mueller All blood smears were stained with 2.5% buffered Giemsa (pH 7.2) for 35 minutes and examined by light microscopy. Slides were declared negative if no parasites were seen in 100 thick film fields by two different microscopists. The parasite species in positive films were identified and densities were recorded as the number of parasites/200 WBC. Densities were calculated assuming 8,000 WBC/µl 2Hb [g/L] = −0.32 x +0.22 x .There is no standardized cut-off value for defining anemia for individuals All metrics  were calculated for both the general population and children 2–10 years. Indeed, spleen rate and parasite rate are usually calculated in this age group for defining malaria endemicity categories2); significance was assessed by calculating p-values. The same software was used to draw a graph of the correlations between the different metrics weighted for the number of individuals per village. We performed also an ANOVA analysis to investigate the variation of the different metrics across altitude strata. As both Hb metrics and prevalence of infection are subject to measurement and sampling errors, major axis regression All data were analyzed using STATA software, version 10.0. Correlations between the different metrics and altitude were investigated by calculating Pearson's correlation coefficients . Overall, the median for age was 14.9 years (range [0–99]) with 51.4% of participants female. Among the villages, 27 were at elevations below 500 m, 17 were between 500 and 999 m, 55 were between 1000 and 1499 m and 57 were above 1500 m altitude. The ratio of Pf/Pv prevalence rates among the 145 villages is 2.04 (range [0–17]).3, 148 = 75.6, p<0.001) and SR 2–10  were found to decrease significantly from 58.1% and 37.8% in villages <500 m to 4.3% and 1.6% in those >1500 m. AP also decreased significantly with altitude  while PopHb increased . Complete altitude stratification data are shown in The PR in 2–10  relationship between Hb metrics and the prevalence of infections in children 2 to 10 years and Hb metrics: PUsing a very large data set from Papua New Guinea, this study has investigated the correlation between altitude, commonly used metrics (spleen rate and parasite rate in children 2–10 years and records of febrile illnesses) and hemoglobin levels expressed as population mean hemoglobin (PopHb) and prevalence of anemia (AP) per community. As shown in earlier studies in Tanzania and Kenya In an altitude-stratified analysis, a progressive increase of PopHb is observed, from 10.5 g/dl on average below 500 m, to 12.8 g/dl above 1500 m, after correction for altitude. Of factors other than altitude itself which may explain these variations, malaria is likely to be the most important one. Other “prevalence” metrics  all show progressive decreases with increasing altitude. Correlation of Hb metrics and PR 2–10 are stronger at altitude between 500 and 1500 m, where endemicity is higher see .By nature, single cross-sectional surveys are not well suited to describe malaria endemicity in areas of significant seasonality. Although Hb level also show some seasonal variations P. falciparum, P. vivax, P. malariae and P. ovale are co-endemic. In most areas P. falciparum is the most common infection followed by P. vivax and in particular in highly endemic areas mixed species infection are very common P. falciparum infection tend to be associated with a stronger reduction in Hb levels P. vivax. However, as prevalence of infection with all species increases in parallel with increasing endemicity, it is difficult to attribute parts of the observed effect of malaria on Hb levels to individual species. Consequently, overall prevalence rates show stronger association with Hb metrics than either P. falciparum or P. vivax specific prevalence rates (data not shown). Finally, it is likely that increasing drug resistance to malaria infections will not have the same effect on the different metrics. A recent study in the Gambia showed higher rates of severe anemia in children infected with chloroquine resistant P. falciparumP. falciparum and P. vivax patients in Indonesian Papua was linked to the very high rate of drug resistance Throughout PNG Besides malarial infections there are a number of different factors that also impact on Hb levels. Of these, it is straightforward to control for altitude, sex and age. As shown in this study, such adjustment may however not necessarily improve the strength of the association between Hb and malarial endemicity.Other important contributors to the burden of anemia include infections, nutrition status and host genetic traits such as common red blood cell polymorphisms. Among infections, hookworm infestation is probably the most important in PNG. It is highly prevalent throughout the country Secondly, dietary factors such as malnutrition or micronutrient deficiencies impact on Hb levels. A recent review by Metz has shown that folate and vitamin B12 deficiency, even though very frequent in developing countries, do not significantly affect the prevalence of anemiaFinally, there are a number of genetic traits that are known to impact on hemoglobin levels. By far the most important one in PNG is alpha-thalassemia 2 (-α) which is very common in lowlands PNG but virtually absent in highlands populationsAfter consideration of these important potential confounders, the correlations between Hb metrics and altitude, parasite prevalence and spleen rates appear to remain robust. This study therefore provides good evidence that popHb and AP are valuable metrics to estimate the burden of malaria in PNG even without adjusting for potential confounders. Before applying them to other settings, in particular those where schistosomiasis is highly prevalent, it will be important to cautiously consider the impact of such confounders when interpreting Hb levels as malaria metics.The major advantages of using Hb metrics as measures for malaria endemicity is that they require limited resources to be recorded, they are immediately available in the field and are more reproducible than estimating spleen rate. In addition, popHb is very easy to calculate and AP easy to interpret. Last but not least they also provide important additional information on the health status of a population.Hb metrics thus appear to be promising alternative tools for measuring malaria burden, on their own or in combination with other traditional or more recent serologicalSupplementary Material S1How to define the best Hb cutoff for anemia prevalence?(0.03 MB DOC)Click here for additional data file.


To the Editor: Studies indicate that Plasmodium falciparum infection increases HIV replication in adults . When 15 malaria episodes with malaria-free visits 1 month before and after the episode were compared, mean difference (signed-rank test) in viral load “before” and “after” malaria was not significant.After adjusting for age and baseline viral load, we assessed logOur findings suggest that low-density malaria infection may not dramatically affect plasma HIV-1 levels in infants. This finding is similar to results of studies of perinatally HIV-infected children in which, although viral loads were unavailable, number of malaria episodes did not significantly affect development of AIDS-related symptoms  for malaria . Most ofOur assessment was limited in size and duration. Furthermore, in attempting to provide optimal patient care through conducting monthly surveillance and encouraging mothers to bring children in during febrile episodes, ability to assess the effect of high-density malaria was diminished because parasitemia levels never reached clinically significant levels. Finally, because malaria was diagnosed by microscopy, rather than PCR, some subclinical malaria infections may have been misclassified as malaria-negative.Although we found no evidence of an association between subclinical, low-density malaria and infant HIV-1 viral load, the consequences of high-density or clinical malaria need to be explored. If clinical malaria in infants increases HIV-1 viral load as it does in adults (


Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies.In endemic regions naturally acquired immunity against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were compared amongst groups of individuals with different levels of exposure to P. falciparum infections.Naturally occurring IgG antibodies against P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens.IgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to In the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria. Plasmodium falciparum is associated with the acquisition of anti-parasite antibodies of the cytophilic subclasses [P. falciparum malaria [Immunoepidemiological studies have demonstrated that immunity against blood stage bclasses , and in bclasses -9. No subclasses ,3. For Ibclasses ,2,10, or malaria ,12. It i malaria ,13-16. T malaria . It has  malaria .The subclass profile of naturally occurring IgG responses has therefore been extensively studied for several major blood-stage malaria vaccine candidate antigens. These analyses have mainly been carried out by ELISA using recombinant proteins or synthetic peptides usually representing subdomains of malarial proteins as test antigens. Such antigen preparations do not always accurately mimic native parasite protein conformations, including post-translational modifications. A more global approach was therefore adopted to study the targets of the naturally occurring anti-parasite IgG subclass responses through IgG subclass specific Western blot analysis of total parasite proteins expressed in mature blood stage schizonts. Purified Parasitophorous Vacuole Membrane-Enclosed Merozoite Structures (PEMS)  were useP. falciparum infection is reported. Plasma samples were collected from four distinct sub-groups including: Group A: non-immune Danish travellers with a single episode of P. falciparum malaria; Group B: young (0-5 years) and Group C: older (6-10 years) Ghanaian children with frequent episodes of clinical malaria; and Group D: clinically immune Liberian adults. A group of non-immune Danish healthy adults (Group E) never exposed to malaria was included as control group.Profiling of different naturally acquired IgG responses, in terms of their subclass specific recognition of parasite PEMS proteins, in individuals with different levels of exposure to Plasmodium falciparum (F32 strain) was cultured in vitro in human RBCs as previously described [3, 2 mg/liter hypoxanthine, 0.5% (w/v) AlbuMAX I, and 1% (v/v) penicillin-streptomycin at 5% v/v hematocrit. Cultures were synchronized by repeated treatment of parasites with D-sorbitol [et al [. Briefly, highly synchronized cultures at approx. 20% parasitemia were grown until the parasites had reached the schizont stage (> 8 nuclei stage), at which time they were treated with 10 μM E64  for 8-10 h until the PEMS emerged. These PEMS were brought into suspension, incubated for another 2 h to allow for the vast majority of RBCs to sediment and then purified from the supernatant by centrifugation through 60% v/v Percoll. PEMS were collected from the top of the cushion, centrifuged for 10 min at 2000 × g, washed three times in PBS containing protease inhibitor cocktail (Roche), and stored as packed cells at -80°C until use. The quality and homogeneity of the purified PEMS were routinely evaluated by light microscopy of Giemsa-stained smears and by SDS-PAGE. When adjusted for protein concentrations and under identical sample processing conditions, samples from different batches showed very similar protein patterns on Coomassie stained 1D-SDS-PAGE and silver stained 2-D gels (data not shown).escribed  using RPsorbitol . PEMS prl [et al . BrieflP. falciparum malaria, and ii) had their malaria episode confirmed by a positive IFA test. Enzyme-linked immunosorbent assay (ELISA) was performed essentially as described [Plasma samples from Liberian adults clinically immune to malaria (n = 12), Danish donors never exposed to malaria (n = 35), and Ghanian children (n = 14) were selected randomly from previously published studies ,22. Plasescribed  using anOne dimensional SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting was carried out as per standard procedures described earlier . MembranThe differences between the distributions of parasite antigen bands recognized by plasma IgG from different individuals were compared by calculating two-sides P-values based on Wilcoxons rank sum test with correction for ties. The differences between ELISA values were compared by the u-test.P. falciparum asexual blood forms [Levels of parasite-specific IgG antibodies were determined in plasma from four groups of individuals who differ in their level of exposure to malaria Figure . Levels od forms . In ordePurified PEMS preparation was used as a source of proteins expressed in mature schizonts. Microscopic examination of the PEMS preparation showed merozoite clusters surrounded by the PVM (data not shown). Proteins extracted from the PEMS preparation were separated by SDS-PAGE and subjected to immunoblot analysis.P. falciparum malaria resulted in increased antibody reactivity against wider range of low-molecular weight parasite antigens.Representative immunoblots displaying profiles of different antibody isotypes reactivity are shown in Figure P. falciparum asexual blood stage antigens at different exposure levels with a particular focus on parasite antigens expressed in mature schizonts. Using parasite PEMS preparation as the source of antigens, antigenic profiling of the naturally acquired IgG has been carried out using ELISA and Western Blot analysis of parasite antigens separated by SDS PAGE. Conventionally, the approaches widely used to study host immune response against malaria antigens have relied on the use of recombinant proteins or synthetic peptides usually representing subdomains of parasite proteins which could differ from their corresponding native parasite counter-parts in terms of their overall structures and post-translational modifications both of which could potentially alter the antigenic property of the target protein. Since the selection of antigenic targets of interest has traditionally been the first step for studying naturally acquired immunity against malaria, very few studies have attempted an unbiased pan-profiling of antibody reactivity against a broad range of parasite antigens [Schistosoma mansoni showed that the antigenic profiling was the same whether antigens had been denatured and reduced or not [Naturally acquired immunity against malaria is highly prevalent in adults residing in malaria endemic regions. It is the strongest known resistance against severe clinical malaria, and it develops over a long period with repeated infections. Passive transfer of purified IgG from hyperimmune sera to malaria patients has demonstrated that IgG mediates anti-parasite activity and protection against malaria. However, the mechanisms involved in the acquisition of naturally acquired immunity and the targets of the protective IgG have not been completely elucidated. This report demonstrates a differential parasite antigen reactivity of IgG responses acquired against antigens . Alternaantigens ,26, an aantigens -29. The d or not . Other ad or not .P. falciparum reactive IgGs with the highest number of antigens being recognized by plasma from clinically immune Liberian adults. A degree of antigenic influence on subclass specificity is particularly evident for the IgG1 and IgG3 subclasses, with some bands being recognized predominantly by IgG1 or IgG3 whilst others are recognized by both classes. In contrast, the IgG2 antibody response seems relatively independent of host age and exposure to P. falciparum. It appears that the antigens recognized by IgG2 are few and in most cases of high molecular weight (> 70 kDa). One antigen of approximately 70 kDa was found to be particularly strongly recognized by majority of the plasma samples from all exposure groups. The apparently low prevalence of IgG2 antibodies to some malaria antigens is in agreement with many previous reports [P. falciparum is dominated by non-cytophilic antibodies (IgG2/IgG4 and IgM), and that this response gradually develops into a more protective response dominated by cytophilic IgG1 and IgG3 antibodies [P. falciparum IgG2 response is directed against a few high-molecular weight antigens and that the age-dependent IgG1 and/or IgG3 responses observed against several low-molecular weight proteins are not preceded by corresponding IgG2 responses.The analysis of total anti-parasite IgG in individuals subject to different levels of exposure revealed unexpectedly high levels of anti-parasite IgG in the Danish travellers' plasma after a single episode of clinical malaria. Though polyclonal activation of host B-cell response has been suggested to be mediated by certain parasite antigens , this do reports ,33-35. Htibodies . The datThis observation suggests that clonal B-cell activation is age and exposure dependent. It seems that upon subsequent malaria attacks, additional B-cell clones are activated, which primarily generate IgG1/IgG3 responses and which may contribute to the gradual development of immunity. The immune response to a wide range of antigens is weak upon the first few exposures to malaria infections. It is likely that the parasite may produce immune-suppressive factors -38 resulP. falciparum malaria is not only related to isotype switching towards ADCI-effective antigens but also to a gradual development of IgG1 and IgG3 antibodies against some previously non-targeted antigens like the distinct low-molecular weight P. falciparum proteins detected in this study.Results from this study suggest that the progressive development of anti-parasite immunity in older children is not mediated by a general switch of malaria-specific antibodies from the non-cytophilic to the cytophilic subclasses. Taken together, the data suggests that the long time required to acquire clinical protection against P. falciparum IgG subclass responses increase with age and exposure, however, subclass switching from pre-existing non-cytophilic antibody responses to cytophilic subclass may not be a general phenomenon.The present study confirms and extends previous observations suggesting that the cytophilic anti-The authors declare that they have no competing interestsBV, SS and MT conceived the study. CHO, KB, SLD, PSJ, LSV, DD, PH, MC, SOL and SS performed the laboratory work and the statistical analysis. SS and MT wrote the manuscript. All authors have read the manuscript and agree with its contents.We thank M. Paulli Andersen for technical assistance.


The T-cell mediated immune response plays a central role in the control of malaria after natural infection or vaccination. There is increasing evidence that T-cell responses are heterogeneous and that both the quality of the immune response and the balance between pro-inflammatory and regulatory T-cells determines the outcome of an infection. As Malaria parasites have been shown to induce immunosuppressive responses to the parasite and non-related antigens this study examined T-cell mediated pro-inflammatory and regulatory immune responses induced by malaria vaccination in children in an endemic area to determine if these responses were associated with vaccine immunogenicity.Using real–time RT- PCR we profiled the expression of a panel of key markers of immunogenecity at different time points after vaccination with two viral vector vaccines expressing the malaria TRAP antigen (FP9-TRAP and MVA-TRAP) or following rabies vaccination as a control.The vaccine induced modest levels of IFN-γ mRNA one week after vaccination. There was also an increase in FoxP3 mRNA expression in both TRAP stimulated and media stimulated cells in the FFM ME-TRAP vaccine group; however, this may have been driven by natural exposure to parasite rather than by vaccination.Quantitative PCR is a useful method for evaluating vaccine induced cell mediated immune responses in frozen PBMC from children in a malaria endemic country. Future studies should seek to use vaccine vectors that increase the magnitude and quality of the IFN-γ immune response in naturally exposed populations and should monitor the induction of a regulatory T cell response. Plasmodium falciparum malaria is high and has risen over the last 20 years, with an estimated minimum of one million deaths per year P. falciparum malaria is short lived Global mortality and morbidity from Plasmodium specific CD8 Both pro-inflammatory and regulatory responses to malaria infection are important in determining the outcome of the disease. Chronic infection with malaria parasites has been associated with immunosuppressive responses to the parasite and to unrelated antigens In a phase 2b malaria vaccine trial we evaluated the efficacy of a viral vector vaccine based prime-boost regime (FFM ME-TRAP), comprising of two vaccinations with an attenuated fowlpox virus (FP9) then one vaccination with modified vaccinia virus Ankara (MVA), both recombinant for a pre-erythrocytic antigen construct containing a multiple-epitope string and thombospondin-related adhesion protein (ME-TRAP). The trial recruited 1–6 year old children in the Kilifi district, Kenya and the end-point of the efficacy trial was clinical malaria. A rabies vaccine was used as the control in this trial. Over 18 months follow-up, there was no evidence that FFM ME-TRAP provided protection against malaria This study aimed to look at T-cell mediated pro-inflammatory and regulatory immune responses induced by vaccination in children in a malaria endemic area to examine whether regulatory responses could be influencing vaccine immunogenicity in this setting.6 had been preferentially selected for analysis, the IFN-γ ELISPOT immunogenicity was significantly higher in selected versus excluded subjects. Due to the small size of the rabies vaccine group (n = 10) not all features of excluded subjects were represented by the included subjects  when compared with baseline levels. At 9 months IFN-γ expression had returned to baseline levels. FOXP3 showed a trend to increase at 3 months but these responses were not significant (P = 0.2471) when compared with the baseline levels. IL-10 and TGF-β1 showed minor differences in expression across the different time points. In the Rabies group there were no significant changes in the expression of any gene tested at any time point. When the responses were compared between the two groups, only IFN-γ expression at 1 week showed significant response to the vaccine  from the circumsporozoite protein of CR assay . Using h6 PBMC. These 4 children were excluded from the correlation analysis as we had determined that the sensitivity of our qPCR assay was between 25 and 250 SFC/106 PBMC. Using the week 1 TRAP stimulated samples we then correlated IFN-γ mRNA and protein expression in the remaining 26 children. No significant correlation was observed using data from the 26 children . Children with an IFN-γ ELISPOT response >100 SFC/106 PBMC were preferentially selected for inclusion in the study as these children were most likely to have a detectable change in gene expression as measured by qPCR.Frozen samples were obtained from children recruited in a phase 2b malaria vaccine trial that evaluated the efficacy of the regimen of FFM ME-TRAP in preventing episodes of clinical malaria among 1–6 year old children in Kilifi, Kenya. Rabies vaccine was used as a control. Peripheral blood mononuclear cells (PBMCs) were isolated and frozen as previously described 6 cells in 100 µl of media were transferred in duplicate wells to a 96-well plate. Cells were incubated in a 37°C/5% CO2 incubator for 4 hours, then stimulated by adding 100 µl of TRAP peptide pools . 1.0×106 PBMC using Rneasy® Mini kit  according to the manufacturer's instructions.RNA was extracted from 1×10Reverse TranscriptionReverse transcription of mRNA was performed using oligo-dt  and the OmniscriptRT® Kit . Briefly, 5 µl of RNA extracted was added to 15 µl of the reaction mix in a 1.5 ml eppendorf tube. This was then incubated for 1–2 hours at 37°C followed by 5 mins at 94°C.Real-Time PCR1 µl of the template cDNA was added into the master mix with the relevant primer make a final volume of 20 µl. Each of the samples was run in duplicate with 2 negative controls included in each PCR run. Hypoxanthine phosphoribosyl transferase (HPRT) was used as the house-keeping gene and PCR data was normalised against HPRT. Real -Time PCR was performed using LightCycler® (Roche) with QuantiTect®SYGR® Green kit . Primers were targeted towards the following sequences;5′-CACTTACAGGCACTCCTCCAGG-3′ and R 5′-CACCGTTGAGAGCTGGTGCAT-3′), TGF-β1 (F5′-GGACATCAACGGGTTCAC T-3′ and R 5′-CCGGTTCATGCCATGAATGG-3′), IFN-γ (F 5′-ATTCGGTAACTGACTTGAATGTCC-3′ and R 5′-CTCTTCGACCTCGAAACAGC-3′), IL-10 (F 5′-GGCCGTGGAGCAGGT-3′ and R 5′-CACTCATGGCTTTGTAGATGCC-3′), HPRT (F 5′-TATGGACAGGACTGAACGTC-3′ and R 5′-CTACAATGTGATGGCCTCCC-3′) mouse IFN-γ(F 5′-GGGTTGTTGACCTCA AACTTGGCA-3′ and R 5′-CAGGCCATCAGCAACAACAT-3′).FOXP3  supplemented with 10% heat-inactivated FCS, 4 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, 100 µM β-mercaptoethanol and 10 U/ml IL-2  expanded T cell clones were washed and counted. A 10 fold dilution series of pb9 T cell clones was prepared to obtain 250,000 to 2.5 T cell clones. T cell clones were mixed with naive splenocytes to obtain a total cell number of 1×106. Cells were then stimulated overnight with 10 µg/ml pb9 peptide, pelleted and then RNA extracted for qPCR analysis as described above.To determine the minimum number of IFN-γ secreting T cells that could be detected by the qPCR assay a mouse T cell clone specific for the H-2Kd-restricted pb9 CTL epitope (SYIPSAEKI) from the circumsporozoite protein of t test. P<0.05 was considered significant. The results in the graphs are expressed as log values . Paired analysis of data points over time were performed using Wilcoxon signed rank and correlations were performed using Spearman's rho (SPSS Statistics 17.0).The mean copy number for each of genes was normalised against the HPRT copy number for both TRAP stimulated and media stimulated cells. The TRAP stimulated value was then divided by media stimulated value to give a fold change ratio. Geometric means (95% CI) were calculated from fold change ratio at each time point. Statistical analysis was performed on log transformed values using Student's


In the rural areas of sub-Saharan Africa, the majority of young children affected by malaria have no access to formal health services. Home treatment through mothers of febrile children supported by mother groups and local health workers has the potential to reduce malaria morbidity and mortality.in vivo CQ efficacy study was nested into the trial. The study is registered under  (ISRCTN 34104704).A cluster-randomized controlled effectiveness trial was implemented from 2002–2004 in a malaria endemic area of rural Burkina Faso. Six and seven villages were randomly assigned to the intervention and control arms respectively. Febrile children from intervention villages were treated with chloroquine (CQ) by their mothers, supported by local women group leaders. CQ was regularly supplied through a revolving fund from local health centres. The trial was evaluated through two cross-sectional surveys at baseline and after two years of intervention. The primary endpoint of the study was the proportion of moderate to severe anaemia in children aged 6–59 months. For assessment of the development of drug efficacy over time, an P. falciparum parasitaemia, fever and palpable spleens was lower at follow-up but there were no differences between the intervention and control group. CQ efficacy decreased over the study period but this was not associated with the intervention.The intervention was shown to be feasible under program conditions and a total of 1.076 children and 999 children were evaluated at baseline and follow-up time points respectively. Self-reported CQ treatment of fever episodes at home as well as referrals to health centres increased over the study period. At follow-up, CQ was detected in the blood of high proportions of intervention and control children. Compared to baseline findings, the prevalence of anaemia  and malaria parameters such as prevalence of The decreasing prevalence of malaria morbidity including anaemia over the study period can be explained by an overall increase of malaria prevention and treatment activities in the study area. The lack of effectiveness of the intervention was likely caused by contamination, pre-existing differences in the coverage of malaria treatment in both study groups and an unexpectedly rapid increase of resistance against CQ, the first-line treatment drug at the time of the study. At least one million annual malaria deaths occur among young children in rural sub-Saharan Africa (SSA). Most of these deaths are in populations with little access to health services -4 In sucAs malaria treatment provided through formal health services is currently not a sufficiently effective strategy for malaria control in rural SSA, interventions aiming at improving malaria home treatment by main caretakers, usually the mothers, may be considered as a complementary strategy. There is some evidence that improved home management of malaria in young children of SSA will result in earlier and more effective treatment with consequently reduced morbidity and mortality ,15.MAlaria: an experiment to bridge the gap between MOthers and health care Providers). It is a controlled malaria community intervention with a pre-post design conducted in rural Burkina Faso and Tanzania in 2002 – 2004. The overall objective of the MAMOP study was to evaluate the feasibility and effectiveness of an intervention aimed at improving case management of malaria in underfive children through primary caretakers in collaboration with local women groups and existing health centres.This project is an EU INCO-DEV funded collaboration between the Heidelberg University (Germany), Karolinska Institute (Sweden), Muhimbili University College of Health Sciences (Tanzania) and Centre de Recherche en Santé de Nouna (Burkina Faso) called MAMOP project  in Nouna Health District, north-western Burkina Faso Figure . The NouThe study was designed as a cluster-randomized controlled effectiveness trial. Using the data base of the Nouna Demographic Surveillance System (DSS) , villagein vivo CQ efficacy was measured in all study children with uncomplicated falciparum malaria at baseline and at follow-up using a modified version of the standard WHO protocol [The primary outcome of the study was the proportion of moderate to severe anaemia (haematocrit ≤ 24%) in children aged 6 to 59 months. Secondary outcomes were prevalences of fever, malaria, of palpable spleens (Hackett score ≥ 2), of other illnesses, mean species-specific number of blood films positive for malaria parasites, mean species-specific malaria parasite densities, mean haematocrit values and mean weight. Finally, protocol .To be able to detect a 10% difference in anaemia prevalence between the intervention and control group with 80% power and at a significance level of 5%, and assuming a prevalence in the control group of 20%, and a conservative design factor of 2.5, 992 under-five children were required . As the The intervention was targeted at three groups: health workers (nurses) from five peripheral health centres , women group leaders, and the main care takers  of preschool children. The community members of the six intervention villages selected a total of 70 women group leaders, two by sub-village (sub-village numbers ranged from 3–9 per village). Inclusion criteria for group leaders used by the communities were permanent residency in the sub-village, age 30–50 years, honesty, and respect by the community. The main components of the intervention were:• Training of health staff, women group leaders and mothers• Sensitisation of the communities• Drug supply to women group leaders, revolving fund• Supervision of health workers and women group leaders• Intervention information system.A five days training course for the health workers of participating health centres was conducted by one of the investigators (FS) together with the District Medical Officer and included an update on malaria case management and an introduction to adult non formal education methods. Afterwards, all women group leaders were trained in respective peripheral health centres by the health workers under the supervision of the investigators for two days on all relevant aspects of malaria knowledge and management including referral criteria. The training included discussions as well as practical sessions using locally produced pictorial sensitisation material and role play, with one refresher course over the study period Figures , 4, 5. AAll women group leaders were regularly provided with CQ and paracetamol from the essential drug stock of Nouna Health District. Pre-packed tablets have been shown to be better for ensuring compliance compared to loose tablets in African countries including Burkina Faso ,27,28. TMothers with febrile children were advised to go to the house of women group leaders for early treatment. Children were treated with a total dose of 25 mg/kg CQ over a period of three days . In addition, all children received standard doses of paracetamol (10 mg/kg every 12 hours) over the first two days. Women group leaders were advised to directly supervise the first dose of CQ and paracetamol, and to visit the sick child again on the second and third day. In case of danger signs at any time point or ongoing fever at the end of the treatment time, the women group leaders had to refer the child to the health centre.At the beginning of the intervention, a six months stock of drugs was provided free of charge to the women group leaders, who had to afterwards renew their stock by buying new drugs from the health workers. Drugs were sold by the women group leaders to the mothers/caretakers at prices which allowed them to make a small profit as an incentive table . The heaThe intervention was monitored through a specific intervention information system which used forms based on pictorial charts, which were filled in every month by the women group leaders figure . The firInformation about the process of the intervention in health facilities was gathered through specific data collection tools  as well as through the routine health information system .The intervention had been pre-tested in three villages outside the study area. For the main study, training of health staff and women group leaders took place from February until June 2003. The intervention itself was fully implemented from July 2003 until October 2004, thus covering two rainy seasons.A baseline survey took place in September/October 2002, and a follow-up survey in September/October 2004.Plasmodium falciparum parasites per μl) were followed up for in vivo drug efficacy testing over a two weeks period. The WHO definitions for early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and adequate clinical and parasitological response (ACPR) were applied [All households with children below 5 years of age were included into the two surveys. In households with more than one eligible child, the survey child was chosen by lottery. All survey children were examined by the study physician (FS). Children found ill during the surveys were treated according to national guidelines. Those fulfilling the criteria for uncomplicated falciparum malaria  according to national guidelines. Children with danger signs were referred to the health centre.All children included into the Treatment procedures were followed through documentation of self-reported behaviour during surveys as well as through CQ determination in blood samples.From all children seen at the baseline and follow-up survey, a finger-prick blood sample was taken. From this, thin and thick blood smears were prepared for malaria diagnosis while anaemia determination was done through measurement of haematocrit values with a portable microhaematocrit centrifuge .For those children included into the CQ efficacy study, additional finger-prick blood samples were taken on day 2, day 3, day 7, day 14, and on any other day the child presented with symptoms during follow up according to standard procedures . MoreoveBlood films were kept in closed slide boxes until they were transported to the CRSN laboratory in Nouna town. After Giemsa-staining, all films were examined by experienced laboratory technicians. Thick and thin blood films were analysed for the species-specific parasite density per μl by counting against 200 white blood cells and multiplying by 50. Slides were declared negative if no parasites were seen in 400 fields on the thick film. For quality control a 10% random sample of blood films is regularly cross checked at the laboratory of the Heidelberg School of Tropical Medicine .® Institute Inc, Cary, NC, USA).To assess the effect of the intervention, logistic regression was used. In separate models, anaemia (haematocrit ≤ 24%), fever (≥ 37.5°C), spleen enlargement (Hackett score ≥ 2), clinical malaria (fever + ≥ 5000 parasites/μl), and malaria parasitaemia (>0 parasites/μl) were considered as binary outcomes. Age (continuous), weight (continuous) and ethnic group  were considered as co-variables for adjustment in the model. The distribution of the outcome variables in intervention and control group was compared at baseline to check adequacy of randomization. Fisher exact test was used to compare rates. The analysis was performed with SAS release 8.02  and a total of 1006 children (496 from intervention and 510 from control villages) were included into the study at baseline and follow-up time points respectively  and a total of 181 children (88 from intervention and 93 from control villages) were included into the CQ efficacy study at baseline and follow-up time points respectively Figure . The difSome 12.500 pre-packaged malaria treatments were sold in the intervention villages over the period July 2003 until October 2004. Table Due to technical problems, no measurements of CQ blood levels were possible during the baseline survey. During the follow-up survey, CQ was detected in the blood of 33/43 (77%) and 37/43 (86%) while the median CQ blood level (range) was 212 nmol/L (23–26.878) and 407 (23–13.734) nmol/L in intervention and control group children respectively.P. falciparum parasitaemia but not of falciparum malaria was significantly lower during the follow-up compared to the baseline survey, but there were no significant differences between intervention and control group. A multivariate analysis showed no effect of the intervention on any of the outcome variables considered. However, there was a highly significant improvement regarding the prevalence of anaemia, fever, malaria, P. falciparum parasitaemia and splenomegly at the follow-up survey independent of the intervention (p < 0.001).Table in vivo CQ efficacy study are given in table The results of the This study was designed as a cluster-randomized controlled trial with a sufficient sample size to show intervention effects of public health importance on malaria parameters. The intervention was shown to be feasible under programme conditions and the uptake was documented by an increase of malaria treatment with CQ in the intervention households and by an increase of referrals to the local health centres. Unfortunately, at baseline the use of CQ was higher in the control compared to the intervention villages which points to the fact that intervention and control area differed with regard to treatment behaviour. However, although CQ treatment increased in the intervention villages it was observed to a similar degree in the control villages. This is to a large degree explained by the high degree of pre-existing CQ treatment in control villages. Moreover, contamination could have occurred by communication between the villagers of both study arms but also by local health workers who partly were responsible for both intervention and control villages. Such an interpretation is also supported by the fact that some 10% of women in control villages also received support from women group leaders. A further explanation for the high degree of CQ treatment in the control villages is a possible contamination by a parallel community-based CQ-distribution project in the area. In this GTZ (Gesellschaft für Technische Zusammenarbeit) – supported reproductive health project, women of 18 villages neighbouring the MAMOP study area received contraceptives but also CQ through trained volunteers. This unforeseen additional contamination has likely contributed to making the control zone not a real control zone.P. falciparum parasitaemia and spleen enlargement over time, but without differences between study groups. The differences in the prevalence of anaemia and malaria parameters between the baseline survey in 2002 and the follow-up survey in 2004 are likely caused by a higher use of CQ in the study area. This hypothesis is supported by the finding of high CQ blood levels both in intervention and control children during follow-up. However, the effects of another community based malaria study which was implemented in all villages of the rural CRSN study and during which all newborn children received insecticide-treated mosquito nets (ITN) free of charge may also play a role in this overall dynamic [The main findings from the study were significant reductions in the prevalence of anaemia,  dynamic . As the in vivo CQ resistance lends support to the hypothesis that better compliance with the full course of antimalarial treatment will likely delay resistance development [Another important observation from this study is the significant increase in clinically relevant CQ resistance in the study area over the two years observation period. This finding supports similar findings from a number of other studies which looked at the efficacy of CQ in Burkina Faso in recent years ,19. Howeelopment ,29.Obviously, all-cause mortality and cause-specific mortality would have been the best outcome measure for this kind of intervention study. Due to sample size considerations and costs moderate to severe anaemia was chosen as the primary endpoint. The MAMOP community intervention had no effect on the prevalence of anaemia. This could at least partly be explained by the finding from another cohort study in the rural CRSN study area which has demonstrated that malnutrition but not malaria is the main determinant of anaemia development in young children of the area . HoweverOnly a few studies have tried to measure the effects of complex malaria treatment interventions at the community level in endemic areas. One study in Ethiopia has measured the effectiveness of treating malaria episodes of young children through their mothers on mortality and was able to show a major reduction in all-cause mortality and malaria-specific mortality attributed to the intervention . In a coIn conclusion, this study has shown the feasibility of a complex malaria community intervention which has bridged the gap between the health workers at the peripheral health centres and the mothers of young children in individual households through women groups. The coverage of malaria treatment has substantially improved during the trial. Although such an approach is promising given the continuous lack of access to formal health services in much of rural SSA, there was no major difference in effectiveness between intervention and control areas. A possible intervention effect may have been masked by contamination, pre-existing differences in the coverage of malaria treatment in both groups, and the rapid rise in CQ resistance in the study area. Future studies should try to avoid confounding influences of other malaria interventions, employ more effective malaria first-line drugs such as artemisinin-based combination therapies (ACT), and preferably use mortality as the primary endpoint.BK, FS, AJ, OM, RS and GT designed the study. FS, BC and BK were responsible for the conduct of the study in Burkina Faso. FS, OM and HB analysed the data. All authors contributed to the interpretation of the data, helped write the paper, and read and approved the final manuscript.


It may be assumed that patterns of clinical malaria in children of similar age under the same level of exposure would follow a Poisson distribution with no over-dispersion. Longitudinal studies that have been conducted over many years suggest that some children may experience more episodes of clinical malaria than would be expected. The aim of this study was to identify this group of children and investigate possible causes for this increased susceptibility.Using Poisson regression, we chose a group of children whom we designated as ‘more susceptible’ to malaria from 373 children under 10 years of age who were followed up for between 3 to 5 years from 1998–2003. About 21% of the children were categorized as ‘more susceptible’ and although they contributed only 23% of the person-time of follow-up, they experienced 55% of total clinical malaria episodes. Children that were parasite negative at all cross-sectional survey were less likely to belong to this group .The pattern of clinical malaria episodes follows a negative binomial distribution. Use of lack of a clinical malaria episode in a certain time period as endpoints for intervention or immunological studies may not adequately distinguish groups who are more or less immune. It may be useful in such studies, in addition to the usual endpoint of the time to first episode, to include end points which take into account the total number of clinical episodes experienced per child. The standard immune-epidemiological approach to studying immunity in malaria uses the lack of an episode(s) of clinical malaria over a certain period of time as a marker of ‘being protected’ However, in Senegal, children followed up for five years were found to experience between 0–40 episodes of clinical malaria Over distribution is common for many infectious organisms, for instance Woolhouse and colleagues We examined a group of children that have been under malaria surveillance for between 3 to 5 years to investigate the pattern of susceptibility to clinical malaria over time and to identify factors associated with this increased susceptibility in an area of low-moderate malaria transmission in Kilifi District, Kenya.Using total malaria episodes experienced per child while controlling for mean age and time at risk, it was found that the negative binomial regression fitted better than the Poisson regression . The dispersion parameter ‘k’ was 1.9 (95% CI: 2.4–1.5). When k→∞, then the data follows the Poisson distribution. The values obtained for ‘k’ suggest moderate over-dispersion of clinical malaria episodes suggesting that there are some children who are at increased risk of clinical malaria compared to others.Children experiencing more episodes of clinical malaria appeared to be at greater risk of developing disease severe enough to require admission to hospital. Poisson modelling was used to estimate the predicted number of clinical malaria episodes per child after controlling for mean age and time at risk. Children who experienced >2 episodes of clinical malaria above the total predicted were arbitrarily considered ‘more susceptible’. A total of 78 (21%) children comprised this ‘more susceptible’ group. Out of a total of 1,173 malaria episodes experienced during the period of the study, 55.3% was experienced by this ‘more susceptible’ group, representing only 23% of the cohort person years of follow-up. The children in the ‘more susceptible’ group were also 4.9 times more likely to be admitted to hospital for malaria than others . The incidence of clinical malaria (episodes/child/year) among those slide negative at all cross-sectional surveys [0.69 (95% CI: 0.6–0.79)] was lower than that of those who had a GMPD at the cross-sectional surveys of <5,000 parasites/µl of blood [1.19 (95% CI: 1.1–1.3)] which was lower than that of those with a GMPD ≥5,000 parasites/µl of blood [1.78 (95% CI: 1.52–2.03)].Sickle cell trait was associated with decreased risk of being in the ‘more susceptible’ group but this was not statistically significant  while children from households with average parasite rate ≥50%  were more likely to be in the ‘more susceptible’ group .Using Poisson regression and controlling for sickle trait, high geometric mean parasite density, household level of transmission as well as the clustering effect of household, there was evidence that children who were parasite negative in all six cross-sectional surveys were less likely to belong in the ‘more susceptible’ group . None of the other factors (including household level of transmission) showed statistically significant associations in this multiple regression.Using the random effects Poisson regression likelihood ratio test, there was evidence, of clustering of episodes of clinical malaria within households (p = 0.03). However, the number of households was small (n = 60) with large differences in the numbers of children per house (6.2±4.1). Half of the households did not have a single ‘more susceptible’ child but none of the households were made up purely of ‘more susceptible’ children .Data collected in one malaria season (4 months from May to August of 1999) was analysed to find out whether it was possible to identify this high risk group over a short period of time. Within this 4-month period, 78% of the children in the ‘more susceptible’ group experienced at least one episode of clinical malaria while only 29% of the others experienced an attack. Five children (4 from the ‘more susceptible’ group) had 3 episodes of malaria within the four months of surveillance.A total of 77 (21%) children did not experience a single episode of clinical malaria over several years of follow-up. In this group, 23% had sickle cell trait, 49% slept under an intact bednet, 59% were over five years of age and 36% lived in houses with transmission above average . Figure one illustrates that there were more children observed to have no clinical malaria episodes than would be expected if the data were to fit a Poisson distribution especially among those under 5 years of age.We found that within a small geographical area and over several years, the risk of experiencing clinical episodes of malaria was markedly heterogenous, some children being ‘more susceptible’ than others of the same age group. This increased susceptibility appears to be malaria specific as it was not observed for non-malaria fevers.The pattern of clinical malaria attacks followed a negative binomial distribution. For the purposes of further analysis, we arbitrarily chose children who over the study period experienced a total of more than two episodes of clinical malaria above what was expected from the Poisson distribution. This group designated as ‘more susceptible’ were also at higher risk of clinical malaria severe enough to warrant admission to hospital and some were admitted more than once. This is consistent with earlier work by Snow and colleagues We also noted that there were a large number of children who do not get any malaria episodes in the whole period of follow-up. This is a phenomenon that was also observed from 11 years of malaria surveillance in an area in the Sudan with low malaria transmission Children who were parasite negative at all cross-sectional surveys were less likely to belong to the ‘more susceptible’ group of children. Whilst this may seem obvious, it is important to recognise that being repeatedly negative may stem from different circumstances. Some children may be less exposed (some homes did not have a single ‘more susceptible’ child), in which case they may in fact have increased susceptibility that is not manifest due to lack of exposure to infected bites. However, the effect of local variations in exposure (measured by household parasite rates) was weak in the multiple regression model though there was evidence of spatial clustering of clinical malaria attacks. Alternatively, some children could be parasite negative in the face of continuous exposure, in which case they have genuine reduced susceptibility. Such protection could stem from a range of factors including behavioural differences between households and genetic factors which either act directly to control the risk of clinical disease or by influencing immune responses. Sickle cell trait probably provides the greatest degree of protection of any single genetic polymorphism. In this study, the odds ratio for association with the less susceptible group was consistent with this, though not statistically significant. However, it should be noted that we have previously estimated in the same group of children that sickle cell trait contributes only 2.1% of the variance in incidence of non-severe clinical malaria Children with low parasite densities at cross sectional survey (i.e. lower geometric mean densities over time) had a lower incidence of clinical malaria compared to those with higher densities, suggesting that ‘resting’ levels of asymptomatic parasitaemia may reflect the immune status of the individual. Although the “strain specific” aspects of immunity to malaria are often emphasised, such an observation would argue for a significant degree of cross protective immunity. For instance, in a study in Senegal on ten children who experienced more episodes than expected over a short time period, it was found that each new clinical episode was caused by a distinct genotype Whatever the underlying mechanisms, the phenomenon of increased susceptibility was evident over even short periods of time: within a 4-month period in a single malaria season, 78% of the ‘more susceptible’ group (as defined by their experience over the entire period of study) of children experienced at least one episode of clinical malaria compared with 29% in the rest of the population. However, depending on the relative size of the two groups, it may be difficult to tell these groups of children apart over a short time period. The significance of this for longitudinal studies may vary depending on the reasons for the heterogeneity observed and the questions being asked. Whatever the case, it is important to be aware that simply detecting a clinical episode of malaria in a time limited longitudinal study may not identify groups with homogenous levels of ‘protection’ and ‘susceptibility’.It has previously been suggested that concentrating control resources on the ‘more susceptible’ children may lead to more successful control interventions In conclusion, we have confirmed over-dispersion in the distribution of clinical malaria episodes in a relatively small area. Although this may have possible implications for targeting interventions, we suggest that such heterogeneity is especially relevant to studies of both naturally acquired immunity and vaccine trials. It may be useful in such studies, in addition to the usual endpoint of the time to first episode, to include end points which take into account the total number of clinical episodes experienced per child.The study was conducted as part of a longitudinal study defining and describing non-severe malaria among people living in Ngerenya, an area of low-moderate malaria transmission in Kilifi District on the coast of Kenya. Consent was sought from parents of 373 children <10 years of age in August-September 1998. Verbal consent was sought from village leaders and heads of households. The study was then explained in the local language to the mother or child's direct care giver. Information sheets were left with the family for discussion and the study explained again the following day and any questions answered. The mother or child's direct care giver was then asked to sign or thumb-print on a consent form to show willingness for her child/children to participate in the study after fully understanding it. They were also made aware that they could withdraw their child/children from the study at any point.et al. Details of the weekly surveillance, and parasitological surveys are described in Mwangi The primary measure for analysis was episodes of clinical malaria among study participants during follow-up. Clinical malaria was defined as fever and any level of parasitaemia for children under one year old and fever accompanied by parasitaemia ≥2,500 parasites/µl of blood for children 1–10 years old Six parasitological surveys were conducted, half in the high transmission, wet season  and half in the dry season when transmission was low . Blood samples were taken for genetic and immunology studies. A study to investigate bednet condition and use was conducted in June 2000, details of which are described elsewhere Haemoglobin types  were characterized by electrophoresis using cellulose acetate gels , while participants were typed for the common African 3.7-kb α-globin deletion by polymerise chain reaction P. falciparum. A primary diagnosis of malaria was made if the child had a positive blood film for P. falciparum at admission with no other detectable cause for the clinical presentation.Admission data were collected at the paediatric wards at the Kilifi District Hospital (KDH) which is the main referral hospital for residence of the study area and would capture most admissions. All paediatric admissions to the KDH were assessed by a clinician and a standard set of laboratory parameters collected routinely All records were double entered into a database (FoxPro® version 2.5) and both entries cross-checked for errors before cleaning. All data analyses were done using STATA® software, version 9.0. . We generated a map using ArcGIS® version 9.0 plotting all households within the study and indicated the total number of “more susceptible” children within each study household.http://www.R-project.org). A better fit using the negative binomial regression model would indicate the presence of over-dispersion. We calculated a parameter that inversely measures the extent of over-dispersion, the ‘k’ factor, which is calculated using 1/α Kruskal-wallis test was used to investigate the difference in the median total number of malaria episodes experienced per child among those admitted and those not admitted with malaria. Poisson regression models, negative binomial regression models and Pareto distribution were compared for fit using total episodes of clinical malaria per child for the period of follow-up as the outcome measure. These distributions and their plots were generated using the R statistical package, version 2.62 . Due to the small number of children per house in some of the homes, houses were merged into 13 zones (<2 km apart) and the parasite rate calculated. Households were then classified as above average (parasite rate ≥50%) and those average or below (parasite rate <50%).


PLDH-based tests become negative more quickly, but sensitivity has been reported below the recommended standard of 90%. A new pLDH test, CareStart™ three-line P.f/PAN-pLDH, claims better sensitivity with continued rapid conversion to negative. The study aims were to 1) compare sensitivity and specificity of CareStart™ to Paracheck-Pf® to diagnose falciparum malaria in children under five years of age, 2) assess how quickly false-positive CareStart™ tests become negative and 3) evaluate ease of use and inter-reader agreement of both tests.Most malaria rapid diagnostic tests (RDTs) use HRP2 detection, including Paracheck-PfParticipants were included if they were aged between two and 59 months, presenting to a Médecins Sans Frontières community health centre in eastern Sierra Leone with suspected malaria defined as fever (axillary temperature > 37.5°C) and/or history of fever in the previous 72 hours and no signs of severe disease. The same capillary blood was used for the RDTs and the blood slide, the latter used as the gold standard reference. All positive participants were treated with supervised artesunate and amodiaquine treatment for three days. Participants with a persistent false-positive CareStart™, but a negative blood slide on Day 2, were followed with repeated CareStart™ and blood slide tests every seven days until CareStart™ became negative or a maximum of 28 days.®, 98.8% . Specificity of CareStart™ was 96.0%  and of Paracheck-Pf®, 74.7%  (p < 0.001). Neither test showed any change in sensitivity with decreasing parasitaemia. Of the 155 eligible follow-up CareStart™ participants, 63.9% (99/155) had a false-positive test on day 2, 21.3% (33/155) on day 7, 5.8% (9/155) on day 14, 1.9% (3/155) on day 21 and 0.6% (1/155) on day 28. The median time for test negativity was seven days. CareStart™ was as easy to use and interpret as Paracheck-Pf® with excellent inter-reader agreement.Sensitivity of CareStart™ was 99.4%  and of Paracheck-PfBoth RDTs were highly sensitive, met WHO standards for the detection of falciparum malaria monoinfections where parasitaemia was >100 parasites/μl and were easy to use. CareStart™ persistent false positivity decreased quickly after successful anti-malarial treatment, making it a good choice for a RDT for a hyperendemic falciparum malaria area. Plasmodium falciparum malaria . Howe. Howe®, gs [i.e. . This fags i.e. -13]..®, whichPlasmodium species that infect humans and is produced by sexual and asexual stages of the parasites. PLDH is cleared more quickly from the bloodstream than HRP2 after starting an effective treatment, as it is an enzyme only produced by viable parasites . -16. Plass .There is an ongoing debate whether or not anti-malarial treatment should be given to children under five years of age only on the basis of clinical suspicion for malaria ,22-24. T® to diagnose Plasmodium falciparum malaria in children under five years of age using Giemsa-stained blood smears as gold standard reference and 2) to assess the time required for false-positive CareStart™ three-line P.f/PAN-pLDH tests to become negative after successful treatment with ACT.The two main aims of the study were 1) to compare the sensitivity and specificity of CareStart™ three-line P.f/PAN-pLDH test and Paracheck-PfSecondary aims included assessing ease of use and inter-reader agreement of both malaria RDTs.A prospective, single-blind evaluation of two RDTs compared to slide microscopy.The study was carried out in the Gerihun community health centre (CHC) in Bo district, one of five CHCs in which MSF is working. Bo district, situated in south-east Sierra Leone, is a hyperendemic malaria region where transmission is perennial. In 2008, of the 417,576 consultations in all MSF health facilities, 181,711 (43.5%) had uncomplicated malaria confirmed by RDT. Severe malaria is the principal cause of morbidity in the area, accounting in 2008 for 54.3% (3733/6875) of all admissions of children under five years of age in the paediatric department of the MSF-run referral hospital . For its catchment population of 152,000 people MSF offers free malaria diagnosis, treatment and prevention in a setting of free primary heath care.All children under five years of age consulting Gerihun CHC for fever and/or history of fever in the previous 72 hours were screened for clinical suspicion of malaria according to routine CHC protocols. Children were included in the study if they satisfied all of the following screening criteria: age between two and 59 months, suspected malaria defined as fever (axillary temperature > 37.5°C), and/or history of fever in the 72 hours prior to attending Gerihun CHC, no signs of severe disease and/or clinical danger, no treatment for malaria administered within the previous four weeks, and signed, informed consent by responsible caregivers.Patients were included for follow-up of the CareStart™ three-line P.f/PAN-pLDH test if they satisfied all of the following criteria in addition to the initial inclusion criteria: positive CareStart™ three-line P.f/PAN-pLDH test for at least one malaria species and positive malaria blood smear at Day 0 (day of inclusion), place of residence less than one hour walking distance from Gerihun CHC, and able to attend follow-up for up to 28 days.Plasmodium falciparum with a sensitivity of 90%, 138 blood smear positive patients were required . Similar calculations applied to the specificity, so an additional 138 blood smear negative patients needed to be tested. To allow for an estimated 5% of patients with incomplete information, patients were continuously recruited until a minimum of 145 positive and 145 negative blood smears had been included. The study was not powered to evaluate detection of malaria species other than Plasmodium falciparum, or mixed infections that included Plasmodium falciparum.To detect A convenient sample size of 145 patients with a positive CareStart™ three-line P.f/PAN-pLDH test result and positive malaria blood smear at Day 0 (day of inclusion) was chosen to describe the time taken for the false-positive CareStart™ three-line P.f/PAN-pLDH tests to become negative.®) performed. Treatment was based on the results of the blood smear, taken as the gold standard reference diagnosis. All positive patients were treated for free according to the following treatment scheme recommended by World Health Organization (WHO), the national and MSF protocol: artesunate 4 mg/kg body weight and amodiaquine 10 mg/kg body weight once a day on Days 0-2. Patients eligible for the follow-up part of the study had all been treated with three doses of artesunate and amodiaquine, administered at the study site, and were observed over the first 30 minutes for immediate vomiting. Failure to complete the full course of treatment at the study site resulted in exclusion from the follow-up study part.On the day of inclusion, demographic and clinical information were recorded, thick and thin blood smears prepared, and the two malaria RDTs  and had a guaranteed history of proper storage and transport conditions. All RDTs were performed and interpreted according to the manufacturers' instructions.Plasmodium infections by pLDH detection, distinguishing between Plasmodium falciparum and the other malaria species Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale. It requires 5 μl of whole blood to be collected with a pipette provided by the test kit. Test results need to be read after 20 minutes.1) CareStart™ three-line P.f/PAN-pLDH  is an individually packaged test cassette, diagnosing ®  is also an individually packaged test cassette, diagnosing Plasmodium falciparum infections by HRP2 detection. It requires 5 μl of whole blood to be collected with a sample applicator provided by the test kit. Test results need to be read after 15 minutes.2) Paracheck-PfPlasmodium falciparum or other Plasmodium species) or invalid. All tests without a control line were considered invalid by the first reader and were repeated.Test results were read by two independent readers, who received training before the beginning of the study. The readers were taught the procedure and interpretation of the RDT results. The study did not start until the study supervisor was satisfied that the readers were familiar with handling and reading of both tests. Preparation and reading of the RDTs were continually reviewed under the observation of the study supervisor. The first reading was performed at the time specified by the manufacturer. The person doing the first reading was a trained laboratory technician and also prepared the two RDTs beforehand. As the two manufacturers provided no maximum time before reading the results, the second reading was performed less than 10 minutes after the first. The second reading was done by a trained layperson. The readers were blinded to each other's results and to that of the blood smear. For each RDT the result was classified as negative, positive , or 500 WBC for low-density infections. Parasite density was calculated assuming 8,000 WBC per microlitre. The thin smears were examined to confirm the parasite species for positive samples. Gametocyte presence or absence on all slides was also recorded, and species identification and gametocyte-density counts  performed.Thick and thin blood smears and RDTs were performed from the same finger-prick of blood. Thick and thin smears were prepared on the same slide and were air-dried. The thin blood smears were fixed with methanol and the thick smears left unfixed. Each slide was then stained with 10% Giemsa solution for ten minutes. All blood smears were examined microscopically under oil immersion . The thick smears were used for diagnosis of All inclusion slides were double-read, blinded, by experienced technicians . A third reading was performed in case of discordance, such as positive/negative discordance for asexual stages, species discordance for asexual stages, asexual density discordance (difference in parasitaemia ≥50%), and/or positive/negative gametocyte discordance. Third readings were performed by the Department of Infectious and Tropical Diseases at the London School of Hygiene and Tropical Medicine and were taken as the definitive results for these slides being disconcordant between the first and the second reader.At the end of the field part of the study, the ease of use of both RDTs was compared using quantitative (storing conditions) and qualitative criteria. The study laboratory technicians who performed the RDTs were asked to rank the tests independently in order of preference where "2" corresponded to their most preferred test and "0" to their least preferred in each of the following categories: Ease and safety of taking blood, ease of adding reagents, ease of interpretation and quality of instructions. The trained laypersons who were performing second readings of tests were asked to rank the tests independently based on the ease of interpretation, and for any additional comments where "2" corresponded to their most preferred test and "0" to their least preferred.All data were recorded directly on an individually numbered case report form. Data were entered into EpiData 3.0 software . Data cleaning was done to check for inconsistencies in data entry and responses. Data analysis was conducted using Stata 8.0  and SPSS 11.0 .Baseline characteristics  were analysed using descriptive statistics . Sensitivity was defined as the proportion of true malaria cases (positive blood smears) that were correctly identified by positive RDTs. Specificity was the proportion of true negative malaria cases (negative blood smears) that were correctly identified by negative RDTs. Positive predictive value was the proportion of true malaria cases (positive blood smears) among the total number of positive RDTs. Negative predictive value was the proportion of true negative malaria cases (negative blood smears) among the total number of negative tests.Sensitivity, specificity, positive and negative predictive values of each test were calculated for RDTs performed at the day of inclusion and estimated using microscopy as the reference standard. Sensitivity, specificity, positive and negative predictive values were also stratified by category of parasitaemia . The first reading of the RDTs was used for these calculations. A 95% confidence interval (95% CI) was given for each parameter. Proportions (paired data) were compared using the McNemar test. Agreement between readers of each RDT was assessed using the kappa coefficient. A Kappa coefficient greater than 0.8 was considered as a measure of good agreement.Approval was received from the Ethics Review Board of MSF and the Research and Ethics Committee of the Ministry of Health and Sanitation of Sierra Leone. Informed, written consent was sought from responsible caregivers of all children participating in the study. The caregivers were provided with an information sheet and had the study purpose explained in their own language by the study personnel . Participation was entirely voluntary.Plasmodium falciparum. Parasitological characteristics of smear-positive study participants are given in Table Between 17 March and 15 July, 2008, 343 participants under five years of age were included in the study. Baseline characteristics of the study population are shown in Table ®  (Table ® with 96.0% (167/174) and 74.7% (130/174), respectively (p < 0.001). Stratification by level of parasitaemia  did not show any differences in terms of sensitivity and specificity results of the two RDTs. The only false negative result for CareStart™ three-line P.f/PAN-pLDH test had a parasitaemia of 1-2 parasites/μl. The two study persons with false negative results for Paracheck-Pf® test had a parasitaemia of 288,000 and 580,000 parasites/μl.There was no significant difference between the sensitivity of CareStart™ three-line P.f/PAN-pLDH  and Paracheck-Pf%) Table . CareStaOut of the 167 study patients eligible for follow-up, 12 were lost to analysis. Five defaulted at Day 1, one had an incorrect artesunate and amodiaquine intake on Day 2, one needed referral to the hospital due to deteriorating health status, and five blood slides from Day 2 were broken during transport and consequently were unreadable. Altogether, 155 study patients were included in the follow-up analysis. There were no positive blood smears for any of the 155 study patients on any of the follow-up days. A positive CareStart™ 3 line P.f/PAN-pLDH test during that period was considered as a false-positive result.On Day 2 of follow-up, 63.9%  of the study participants had a CareStart™ three-line P.f/PAN-pLDH false-positive test. Similarly, on day seven, 21.3% , on day 14, 5.8% , on day 21, 1.9%  and on day 28, 0.6%  had false-positive tests  for CareStart™ three-line P.f/PAN-pLDH and 0.3% (1/343) for Paracheck-Pf®.Remarks made were that the two RDTs were equally safe for personnel to handle and easy to use in terms of adding blood and reagents. Both the control and the positive test lines were equally readable in both RDTs. The instruction sheet provided with Paracheck-PfThe control and the positive test lines were equally readable for both RDTs. Kappa coefficient for the inter-reader reliability for both RDTs tests was above 0.9 Table .®, were highly sensitive for the detection of falciparum malaria monoinfections in children under five years of age. When a parasitaemia of more than 100 parasites per microlitre was considered, both tests were >95% sensitive for the detection of Plasmodium falciparum, meeting the criteria set out by WHO [Plasmodium falciparum malaria in Africa. Both studies used the two-line test and did not exclusively focus on children under five years of age. They included a field study in Uganda, which found sensitivity of the CareStart™ two-line PAN-pLDH test to be 95.6% [Both malaria RDTs, CareStart™ three-line P.f/PAN-pLDH and Paracheck-Pft by WHO ,25. A libe 95.6% , and a sbe 95.6% . In a sube 95.6% . The resBoth malaria RDTs were very reliable, having less than one percent invalid tests. Inter-reader reliability was equally good, indicating that wherever the tests are used, readers will be able to give trustworthy results. Both tests were perceived by the study team as similar in terms of ease of use. Therefore, neither RDT was preferable based on these characteristics.Heat-stability has been a major concern for pLDH tests, especially under field conditions . Due to ® that remains false-positive for up to four weeks after successful treatment. In the Democratic Republic of Congo, 73.5% of effectively treated children were still Paracheck-Pf® false-positive at Day 35 [® false-positive at Day 14 [® false positivity rate was around 30% at Day 28 of follow-up [th day of follow-up less than 6% of the study patients still tested false-positive, approximately half the percentage found false-positive during the Uganda study at the same follow-up day. At Day 28, less than one percent of the study patients were still false-positive. Especially in a hyperendemic setting, it is extremely important to have a short parasite antigen clearance time after parasites are removed. This characteristic allows health personnel to confidently interpret positive RDT results without concerns over possible false-positive results from previously treated infections.The major advantage of pLDH-based tests over HRP2-based tests is their ability to rapidly become negative after effective treatment with an anti-malarial. This is in contrast to Paracheck-Pft Day 35 . In Ugant Day 14 . In soutollow-up . The firollow-up . This ra®. All tests were evaluated using prepared blood panels of cultured Plasmodium falciparum parasites and patient-derived Plasmodium falciparum and Plasmodium vivax parasites, and a parasite-negative panel.At the time this study was planned (early 2008), WHO did not endorse any particular RDT, but listed products submitted to the WHO malaria RDT product testing programme . By Apri® in terms of detection rate of parasites, inter-reader agreement, and heat stability. However, they both showed an equivalent high level of detection at a high parasite density . It has to be noted that the WHO testing was an evaluation of the performance under laboratory conditions. In contrast, this study was carried out in the field and was the first to do so focusing on children under five years of age under these conditions. Future procurement decisions should be based on WHO test results, but need to take into consideration local conditions of malaria transmission and illness where the test will be used. Nevertheless, to centralise RDT product testing in vitro is a very welcome initiative from WHO to facilitate the evaluation of reliable RDTs in the field.In the WHO evaluation, CareStart™ two-line PAN-pLDH performed better than Paracheck-PfPlasmodium falciparum malaria species to detect. CareStart™ three-line P.f/PAN-pLDH tests might perform less satisfactorily in settings where other malaria species predominate, which was the case in a study in Myanmar [Plasmodium falciparum no conclusions can be drawn in terms of sensitivity of CareStart™ three-line P.f/PAN-pLDH test for the other Plasmodium infections. This confirms the need to carry out RDT assessments in different geographical areas and with different types and levels of transmission in order to find the most suitable test for each region.There are some potential limitations in generalizing this promising CareStart™ three-line P.f/PAN-pLDH test as a suitable candidate for malaria diagnosis. The study team was well-trained and constantly supervised in test preparation and interpretation. This might not be the case in all health facilities using this technology. Also, the study cohort only had  Myanmar . As the Several studies demonstrate inconsistencies in blood smear reading and, therefore, possibly doubtful results for sensitivity and specificity of malaria RDTs ,31. SomeBefore choosing a malaria RDT for use in the field it must fulfil a number of requirements. In this study CareStart™ three-line P.f/PAN-pLDH fulfilled all the requested criteria: (1) Sensitivity for the diagnosis of falciparum malaria was more than 95% with a parasitaemia greater than 100/μl. (2) All further accuracy parameters  were above 90%. (3) The test had an excellent inter-reader agreement and was reliable, easy to use and interpret. (4) False positivity decreased rapidly after successful anti-malarial treatment and became, on average, negative after seven days.® in hyperendemic settings where Plasmodium falciparum malaria is the predominant species.This study confirms the value of RDTs for use in children under five years of age by refuting the idea that RDTs may miss a significant number of diagnoses in this age group. Widespread use of RDTs should improve diagnostic accuracy of malaria in the field and should be adopted by malaria control programmes. These results confirm that, in terms of accuracy, CareStart™ three-line P.f/PAN-pLDH test can be used as an alternative to Paracheck-PfOn the basis of this study, policy makers and governmental and non-governmental health providers can consider CareStart™ three-line P.f/PAN-pLDH test as a valuable tool for diagnosing falciparum malaria in children under five years of age, especially in more remote areas with hyperendemic conditions. This supports the wider use of RDTs to accurately diagnose malaria and avoid over or under treatment.The authors declare that they have no competing interests.SG was the principal investigator of the study. She designed the study and collected data in the field. She did the study analysis, interpretation of data and wrote the paper. SD, AM and SB supported the principal investigator considerably in carrying out the field work. MDS and JM did a critical revision of the paper. All authors read and approved the final manuscript.


Salmonella (NTS) infection in children. In the last 10 years, indices of malaria infection in The Gambia have fallen substantially.Malaria is a risk factor for invasive non-typhoidal We compared temporal trends of childhood malaria and NTS infection in two Gambian locations. In Fajara, on the coast, the incidence of NTS infection at three time points between 1979 and 2005 was compared to the percentage of malaria positive outpatient thick blood films and the percentage of admissions associated with malaria over time. In Basse, in the eastern part of the country, the incidence of NTS infection at three time points between 1989 and 2008 was compared to the prevalence of malaria parasitaemia at four time points between 1992 and 2008.The estimated incidence of NTS infection in Fajara fell from 60 (1979–1984) to 10 (2003–05) cases per 100,000 person years. The proportion of outpatients in Fajara with suspected malaria who were parasitaemic fell from 33% (1999) to 6% (2007) while the proportion of admissions associated with malaria fell from 14.5% (1999) to 5% (2007). In Basse, the estimated incidence of NTS infection fell from 105 (1989–1991) to 29 (2008) cases per 100,000 person years while the prevalence of malaria parasitaemia fell from 45% (1992) to 10% (2008). The incidence of pneumococcal bacteraemia in Fajara and Basse did not fall over the study period.These data support an association between malaria and NTS infection. Reductions in malaria infection may be associated with reduced rates of invasive childhood NTS infection. Salmonella species (NTS) are a leading cause of invasive bacterial infection in African children and are responsible for many deaths Non-typhoidal Evidence suggests that malaria is a risk factor for invasive NTS infection in children. In regions of seasonal malaria transmission, annual peaks of NTS and malaria infection coincide Over the last 10 years, indices of malaria infection in The Gambia have fallen substantially Plasmodium falciparum. The Medical Research Council (MRC) Laboratories in Fajara, on the Atlantic coast, have provided clinical services for the surrounding population since the late 1950s. Basse is situated in the rural eastern part of the country about 400 km from the coast, where MRC has conducted studies since 1982.We reviewed data from studies conducted in two locations in The Gambia, Fajara and Basse. The Gambian climate is sub-tropical with one wet season (June to November) during which most malaria transmission occurs. Almost all cases of malaria are due to Haemophilus influenzae type b (Hib) conjugate vaccine conducted in Western Region of The Gambia from 1991 till 1995 To relate indices of invasive NTS and malaria infection in Fajara, four data sets were used  and 2. TSalmonella typhi and non-typhi isolates were differentiated in the two hospital-based studies but not during the vaccine trial. However, S. typhi infections are very uncommon in young children in The Gambia Manual blood culture methods were used in the first two studies, after which an automated system was used. Salmonella isolates is known. The incidence of NTS infection in the trial was extrapolated by multiplying the incidence of IPD by the ratio of NTS to pneumococcal isolates detected in blood.To determine the approximate incidence of blood culture positive NTS and invasive pneumococcal disease (IPD), an estimate was made of the catchment population for the MRC Hospital during the 1979-84 study (100000 with 20% aged less than 5 years). Population growth of 3% per year was used to give an estimated population for the 2003–2005 study. Because children in the Hib vaccine trial were aged 2 to 35 months, the overall incidence of IPD in children aged less than 5 years during the vaccine trial was estimated based on reported age-specific rates of IPD. Age-specific rates of IPD and NTS infection were extrapolated using data from two Kenyan studies which documented that the incidence of invasive IPD and NTS among children aged 36-59 months was approximately half the incidence among those aged 24–35 months Three studies in Basse have documented the incidence of invasive NTS and IPD  and 3: . AutomatDuring the 1989–91 study, the under-5 population of Upper River Region (URR) was estimated to be 24793 and approximately half the population lived in areas covered by the disease surveillance Three studies in Basse documented the prevalence of malaria parasitaemia in children aged less than 5 years during the study period  and 3:  isolates detected in blood cultures in all the studies (NTS∶P). We also compared trends in the incidence of blood culture positive IPD with the incidence of NTS infection in children less than 5 years of age in all the studies.At the end of 2004, the government guideline for first-line therapy of mild malaria was changed from chloroquine to a combination of chloroquine, sulfadoxine and pyrimethamine. Artemether plus lumefantrine was introduced through government health facilities in 2008. Quinine was the treatment of choice for severe malaria throughout the observation period. Multiple indicator surveys in 2000 and 2006 documented the proportion of children under 5 years of age sleeping under insecticide treated nets as 14.5% and 49% respectively Data were extracted directly from publications and derived from current studies. Incidence rates were calculated assuming Poisson distributions.All the clinical trials from which data presented in this paper have been derived were approved by the Gambia Government/MRC Joint Ethics Committee and all research study participants provided written informed consent.Salmonella spp. and 76 pneumococci were isolated from blood (NTS∶P = 0.51) Over a 69 month period from 1979 until 1984, pathogenic bacteria were isolated from 259 patients less than 5 years of age (approximately 20000 population and 115000 person-years at risk) who presented to the MRC Hospital, Fajara; 69 were NTS and 38 were pneumococci (NTS∶P = 1.8) The fall in invasive NTS infection followed a similar pattern to the fall in malaria infection . The proTwenty-four months of community-based surveillance for invasive bacterial infection under 5 years of age was conducted between 1989 and 1991 (24793 person-years at risk) The end of transmission season prevalence of malaria parasites in cross-sectional surveys of young children resident in URR fell from 45% (1992), 39% (1994) and 35% (1995) to 10% in 2008  [17]–[19As far as we are aware, this is the first report to document reduced rates of invasive NTS infection in children associated with reductions in malaria.The time point at which the incidence of NTS infection in Fajara began to fall is uncertain, but it is somewhere between 1994 and 2004 . MalariaThis study has several weaknesses inherent in a retrospective analysis of data collected for other purposes. Temporal changes in disease would have been best described by continuous data collection. At Basse, blood cultures were collected from children identified in populations under surveillance whilst at Fajara samples were obtained from children who came from a wide area and chose the MRC hospital as their centre for care. Thus, the Basse data are more representative of the population than those of Fajara, but the similar patterns of falling malaria and NTS infection in both locations strengthen the case for an association between malaria and invasive NTS infection. We made intuitive assumptions on the catchment populations for the MRC Hospital at Fajara, and so these incidence estimates are only approximate. Calculation of disease incidence assumed similar under-5 age structure in the Hib and pneumococcal vaccine trials as in the other studies providing data. Likewise, the association of NTS and IPD with age strata less than 5 years was assumed to be the same for both organisms. If these assumptions are false, the interpretation of the data would not change but be strengthened as the true difference between the incidence of IPD and NTS in the latter studies would be greater than that which was observed.There were differences in selection criteria for blood culture in the Fajara studies, where blood cultures may have been collected more assiduously during the vaccine trial than during routine hospital admissions. However, the comparability between NTS studies conducted in Basse should be good as similar criteria for blood cultures were used in the three studies. To reduce confounding by variable patient selection or factors reducing the risk of any bacteraemia, we calculated rates of pneumococcal blood stream infections during the same surveillance periods. Greatly reduced NTS∶P ratios of blood culture isolates suggests that there has been a marked decline in the prevalence of NTS infection which is not due to changes in selection criteria for blood culture nor due to non-species specific reductions in rates of childhood bacteraemia. Although automated blood culture systems were introduced during the study period this is unlikely to have changed the sensitivity of NTS detection. Methods for calculation of malaria admissions and slide positivity in Fajara were consistent throughout as were the population-based malaria prevalence survey techniques used in Basse. Unfortunately, we do not have detailed information on the age distribution of children under the age of 5 years admitted to the hospital in Fajara and changes in this pattern could have influenced the apparent decline in the prevalence of malaria. However, experience of those involved in patient care at the MRC hospital indicates that if there has been a change in age pattern this has been small. As malaria prevalence estimates from Basse were derived from population-based surveys less than 5 years of age, these data are not subject to bias due to changes in age. The similar pattern and magnitude of NTS reduction in parallel with reduced malaria prevalence in Fajara and Basse suggests that the age-related bias in the Fajara data is not substantial. Therefore, our interpretation of a potential association between reduced prevalence of childhood malaria and reduced incidence of NTS infection remains valid.A decline in the incidence of NTS infection in parallel with a decline in malaria does not prove cause and effect and we have considered other possible explanations for our findings. Improved socio-economic and environmental conditions may be associated with reduced risk of childhood malaria and enteric infections associated with fecal-oral transmission, and hence reduced rates of NTS infection. One study has documented reduced rates of diarrhoea in The Gambia between 1979 and 1993 Salmonella antigens, increased circulating immune complexes which inhibit Fc receptor-mediated phagocytosis, reduced complement levels, alteration of phagocytic and bactericidal function by ingested malaria pigment, altered splenic function, free haemoglobin resulting in production of methaemalbumin and increased availability of iron stimulating bacterial growth through the many iron uptake pathways possessed by NTS.Previous epidemiological, clinical and pathophysiological studies support our hypothesis that the reduction in NTS infection which has been observed in The Gambia is likely to be associated with the substantial fall in malaria which has been recently observed in the country. Compared to other forms of bacteraemia, invasive NTS has previously been associated with anaemia An intervention study in The Gambia has shown that the use of insecticide treated bed nets was associated with reductions in overall child mortality of a magnitude greater than what was expected due to reductions in malaria-attributable child mortality alone If similar findings to ours are found in other locations which have experienced reductions in the incidence of malaria, the implications are substantial. Investments in research and public health to control malaria may have added value, reducing rates of NTS infection and the associated child mortality, in addition to reducing mortality related directly to malaria.