Plasmodium infection and anaemia among school children and to explore variations in effects across different malaria ecologies occurring in Kenya.To investigate risk factors, including reported net use, for Plasmodium infection and anaemia within different malaria transmission zones.This study analysed data for 49 975 school children in 480 schools surveyed during a national school malaria survey, 2008–2010. Mixed effects logistic regression was used to investigate factors associated with Plasmodium infection in coastal and western highlands epidemic zones and among boys in the lakeside high transmission zone. Other risk factors for Plasmodium infection and for anaemia also varied by zone. Plasmodium infection was negatively associated with increasing socio-economic status in all transmission settings, except in the semi-arid north-east zone. Plasmodium infection was a risk factor for anaemia in lakeside high transmission, western highlands epidemic and central low-risk zones, whereas ITN use was only associated with lower levels of anaemia in coastal and central zones and among boys in the lakeside high transmission zone.Insecticide-treated net (ITN) use was associated with reduction in the odds of Plasmodium infection and anaemia, including the protective associations with ITN use, vary according to malaria transmission settings in Kenya, and future efforts to control malaria and anaemia should take into account such heterogeneities among school children.The risk factors for  Plasmodium infection in school-aged children. However, the potential protective efficacy of ITNs in reducing Plasmodium infection and anaemia among school children may not be the same in all settings owing to differences in the underlying intensity of malaria transmission and the relative contribution of other factors that contribute to anaemia in this age group, including undernutrition (Insecticide-treated nets (ITNs), and more recently long lasting insecticide nets (LLINs), are a key tool in the control of malaria, with demonstrable health benefits of ITN use, especially among young children and pregnant women , which gave an on-the-spot diagnosis for malaria, and a thick and thin blood smear for subsequent microscopy. Different RDT types were used during the different survey phases. 72.8% of children were tested with either a ParaCheck-Pf device or a ParaCheck-Pf dipstick, while the rest were tested using OptiMAL-IT (17.3%) or CareStart Malaria Pf/Pv Combo (9.9%) RDTs. Blood slides were labelled and air-dried horizontally in a carrying case in the school and stained with 3% Giemsa for 45 min at the nearest health facility at the end of each day. All RDT-positive microscopy slides and a random sample of RDT-negative slides were examined by expert microscopists to ascertain Plasmodium infection. The RDT results were corrected using the microscopy results, and the slide-corrected RDT results were used as the definitive malaria diagnosis. Using the same finger-prick sample, haemoglobin concentration was assessed using a portable haemoglobinometer  and estimated to an accuracy of 1 g/l. A questionnaire was administered to children to obtain data on mosquito net ownership and use and whether the net was an ITN. Information was also collected on recent deworming, key socio-economic variables such as household construction and drinking water source. The children’s responses were entered electronically into ASUS Eee PC 1005P or Acer Aspire One d250 netbook computers using a customised Microsoft Access database and transmitted nightly to Nairobi through the mobile phone network. The geographical locations of schools were determined using a Garmin eTrex global positioning system .Selected children were asked to provide a finger-prick blood sample which was used to assess The study protocol received ethical approval from the Kenya Medical Research Institute and National Ethics Review Committee. Additional approval was provided by the appropriate national, provincial and district-level health and education authorities, who were briefed about the survey. At the school level, parental consent was based on passive, opt-out consent rather than written opt-in consent owing to the low risk and routine nature of the study procedures. Individual assent was obtained from each child before participation in the survey.tata version 11.0 . Plasmodium infection and anaemia were assessed for their association with reported net use for each of the five malaria transmission zones. Plasmodium infection was defined on the basis of RDT results corrected with expert microscopy results. Anaemia was defined as a haemoglobin concentration <130 g/l for boys aged >15 years, <120 g/l for children aged 12–14 years and girls >15 years, <115 g/l for children aged 5–11 years and <110 g/l for children younger than 5 years, with adjustments made for the elevation of the children’s school  models to account for the excess of zero prevalence, while the proportion of children using nets was estimated using a multilevel mixed effects model. The ZIP model was favoured over the standard Poisson model on the basis of the Vuong test  was pre-specified after a priori inclusion of age, sex and net use. Covariates included mosquito net use, household wealth indicators such as household construction , availability of electricity and latrine access. In addition, data on altitude and location  of the school were entered into the malaria models while data on Plasmodium infection status, deworming history and school-level estimated hookworm prevalence were also entered into the anaemia models. Backward-stepwise selection of covariates was used to generate minimum adequate models. Excluded covariates (P > 0.1) were retested in the final models using LR tests to confirm lack of association; however, reported net use, age group and sex were retained as fixed terms in all models regardless of statistical significance because of their known importance.Univariable analysis of risk factors for a priori interactions were investigated: (i) reported net use and sex, (ii) reported net use and age group in both models and (iii) age and sex in the anaemia model. The existence of heterogeneity in the odds ratios according to sex and age groups was assessed on the basis of LR tests in multivariable models, and interaction was included in the final model if P < 0.1. Stratum-specific odds ratios were derived from the final multivariable models.After identifying covariates for inclusion in multivariate regression models within each transmission zone, three Plasmodium infection. Data on anaemia were collected from 41 884 children in 399 schools and 98% of these had complete data and were therefore included in the anaemia analysis. A similar number of boys (50.7%) and girls were included  had complete data on all covariates of interest and therefore included in the analysis for included , and thePlasmodium infection was 4.4% , and the prevalence of anaemia was 24.0% . The prevalence of infection was highest in lakeside zone and lowest in central and semi-arid zones, whereas anaemia was highest in the coastal and semi-arid zones  reduction in the odds of infection (P = 0.006). Although there was evidence in the univariable analysis of a 15% reduction in the odds of infection in children who reported using nets in the lakeside zone , indicating no differences in the odds of anaemia in younger children (5–9 and 9–15 years) by sex while among children aged >15 years, girls had lower odds of infection.As expected, the risk factors for anaemia varied according to malaria transmission zone  and 5. IPlasmodium infection was associated with higher odds of anaemia in the lakeside, western highlands epidemic and central zones. Reported net use was associated with lower odds of anaemia in coastal and central zones and among male net users in the lakeside zone; no association was evident in the western highlands epidemic and semi-arid zones (P = 0.051); however, there was no evidence of variation in the other zones or by age group.id zones . Recent Plasmodium infection and anaemia in school children in a country with diverse malaria and nutritional ecologies. Results suggest that reported net use was associated with reduction in the odds of Plasmodium infection among all children in the coastal zone, and there was borderline evidence of a 35% reduction in the odds of infection among boys in the western highlands epidemic zone; no protective effect was observed in all other malaria transmission zones. Reported net use was associated with reduced odds of anaemia in the central and coastal zones and among the boys living in high lakeside zone.To effectively target malaria control interventions, an understanding of the potential efficacy of interventions against malaria and related co-morbidities, such as anaemia, is necessary. To our knowledge, this study presents the first nationwide analysis of the association between reported net use, Since the completion of the current work, the results of the 2010 Kenya Malaria Indicator Survey (MIS) have been published . Unlike The observed lack of an association between net use and infection in the lakeside zone contrasts other studies that have reported a reduced risk of malaria infection among school-aged children who use nets (Plasmodium and helminth infections and co-infection can be seen (The effect of reported net use on anaemia is likely to be mediated by malaria transmission intensity and the presence of other aetiological factors for anaemia . In the Our results also provide useful insight into the geographical variation in the relative importance of other risk factors for malaria and anaemia. Notably, there are differences in the magnitude and direction of the effect of risk factors such as sex, age and socio-economic indicators. First, the observed differences in infection risk by age  are consThis study is not without its limitations. First, net use and net treatment status were not directly ascertained. This may lead to misclassification of net users and non-users and ITNs and non-ITNs, thereby underestimating the effect size. However, a study in Uganda comparing school children’s reports on bed net ownership and community-based reports showed that school children can reliably report community-level bed net ownership . FurtherPlasmodium infection and anaemia vary according to malaria transmission zones. The results also identify differences by zones in other risk factors associated with infection and anaemia. The study highlights the need for scaling up ITN coverage in the lakeside high transmission and western highlands epidemic regions and that ITNs alone are unlikely to control malaria in such settings, and there is a need to implement a suite of malaria interventions, including IRS and IPT. A geographically targeted, integrated approach for tackling anaemia in the country is also warranted.In conclusion, our results demonstrate that the use of mosquito nets by school children varies markedly across Kenya, and importantly, their protective effects for


Plasmodium species and offer the potential to extend accurate malaria diagnosis in endemic areas. In this study, the performance of Plasmodium falciparum-specific histidine-rich protein-2 (PfHRP-2) RDT in the detection of asymptomatic carriers from a hyperendemic region of Burkina Faso was compared with microscopy to gain further insight on its relevance in community-based interventions.Rapid diagnostic tests (RDTs) are immune chromatographic tests targeting antigens of one or more P. falciparum asymptomatic carriers during three consecutive monthly community screening campaigns on the incidence of symptomatic malaria episodes over a 12-month period was compared with no treatment of asymptomatic carriers.The performance of HRP-2 test was evaluated in terms of sensitivity, specificity, positive and negative predictive values, discordant values, likelihood ratios, accuracy, and precision using microscopy as the 'gold standard’. This analysis was carried out in a controlled, parallel, cluster-randomized  study in children and adults. The effect of systematic treatment of Sensitivity of HRP-2 test in asymptomatic carriers was higher in campaign 1 (92.4%) when compared to campaign 2 (84.0%) and campaign 3 (77.8%). The sensitivity of HRP-2 test increased as parasite density increased across all the age groups. Highest sensitivity (≥97.0%) was recorded at parasite densities of 1,000-4,999/μl, except for children aged 10 to 14 years. The specificity of HRP-2 test was comparable across age groups and highest in campaign 3 (95.9%). The negative predictive values were high across the three campaigns (≥92.7%) while the positive predictive values ranged from 23.2 to 73.8%. False-positive and false-negative rates were high in campaign 1 and campaign 3, respectively.P. falciparum varied by age and parasite density. Although the use of HRP-2 test is beneficial for the diagnosis of acute malaria, its low sensitivity in screening asymptomatic carriers may limit its utility in pre-elimination interventional settings. The use of a practical and more sensitive test such as loop-mediated isothermal amplification in combination with a cost effective HRP-2 test may be worth exploring in such settings.The performance of HRP-2 test in detecting asymptomatic carriers of  Both microscopy and rapid diagnostic tests (RDTs) have been recommended by the World Health Organization (WHO) for diagnosis of malaria. The choice between the two methods varies according to available skills, patient-case load and local setting. Microscopy is the reference method for malaria diagnosis and can be used to identify species, parasite density and response to anti-malarial therapy. However, a major drawback of this tool is its requirement of well-trained, skilled staff and an energy source to power the microscope.Plasmodium falciparum, Plasmodium vivax or all human Plasmodium species). Positive likelihood ratio was defined as the probability of a positive test result in patients with the disease divided by the probability of a positive test result in patients without the disease , and negative likelihood ratio as the probability of a negative test result in patients with the disease divided by the probability of a negative test result in patients without the disease . Accuracy, the proportion of all tests that yielded a correct result, was defined as (TP + TN)/(TP + TN + FP + FN). Cohen’s Kappa value, the quantitative measure of the magnitude of agreement between two tests was calculated. The test performance was evaluated across the three screening campaigns and for categories of age and parasite density. Geometric mean parasite density by age group was also reported.The clinical trial protocol and informed consent forms were approved by the CNRFP Institutional Review Board and by the National Ethical Committee for Health Research of Burkina Faso. Prior to study initiation, a community meeting was held in each of the selected clusters to discuss the study objectives with the communities. The freedom of each individual household and household member to decide on study participation was discussed to minimize the potential influence of key opinion leaders in each cluster. Individual informed consent was obtained from each participant during a visit to the household prior to any study procedure.et al.[Of 14,075 subjects assessed for eligibility, a total of 6,817 subjects in the intervention arm and 7,258 subjects in the control arm were recruited and enrolled in the study  samples were detected to be positive for P. falciparum  samples were detected to be positive using microscopy  is an innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. There is evidence that LAMP may be a fast, simple, and cost-effective diagnostic approach in detecting malaria parasites. LAMP uses DNA polymerase and amplifies a target DNA sequence with high sensitivity and specificity under isothermal conditions. Several studies have evaluated the sensitivity and specificity of LAMP for the detection and differentiation of ent dyes. Studiesent dyes-30. CurrRDTs are still appealing since they are easy to use and can help overcome the challenges of microscopy in field-based trials. However, low sensitivity of RDTs in screening asymptomatic carriers limits their utility as single diagnostic tool in interventions targeting the parasite reservoirs . While LAMP may be an option, given its ease of use in the field and diagnostic accuracy, its role in MSAT trials needs to be explored especially in settings where prevalence of malaria is high. It might not be cost effective to use this tool as stand-alone in such settings. Rational combinations with RDTs need to be explored. One possible scenario would be to use both RDT and LAMP in the first screening and treatment campaign, where RDT is used in children and LAMP in individuals ≥15 years. This approach adds cost and complexity to the trial implementation, but could help to prevent spread of the infection (by those who were not detected by RDT) in the interval before the next campaign. In subsequent campaigns, using LAMP in newcomers to the area or in the whole population, based on malaria transmission dynamics, may be targeted.In areas with reports of high RDT sensitivity that is consistent across ages, exclusive use of RDT at the first campaign could be a better option to minimize cost. LAMP should be preferred for subsequent campaigns. Computer simulation analyses might be necessary to explore these scenarios and other possible ones to identify cost-effective solutions for better decision making.P. falciparum asymptomatic carriers varied by age and parasite density. This low sensitivity in screening asymptomatic carriers may limit its utility in pre-elimination interventional settings. LAMP is a practical and more sensitive test; but cost may be a barrier to its use as single diagnostic tool in large-scale MSAT trials. Cost effective combination of HRP-2 tests and LAMP may be worth exploring.The performance of HRP-2 test in ABT and AO have received honoraria from Novartis Pharma AG, Basel, Switzerland to attend Advisory Board meetings to discuss this study and manuscript. AM is an employee of Novartis Healthcare Private Limited. KH is an employee of Novartis Pharmaceuticals Corporation. AD, SC, IS, ATK, AB, and SBS declared no competing interests.ABT, AO, AD, SC, AM, SBS, and KH were involved in the design of the study, data interpretation, and defining the content for the manuscript. ABT, AO, AD, SC, IS, ATK, AB, and SBS were involved in data collection, while ABT, AO, AM, and KH conducted the data analysis. ABT, AM and KH were involved in writing the manuscript. All of the authors had full access to data in the study, discussed the results, critically reviewed the draft manuscript, and agreed on the final version. ABT, the corresponding author, had final responsibility for the decision to submit the manuscript for publication. Editorial assistance was provided by Ubhayabharathi Gurunath and Krishna Swetha Gummuluri, professional medical writers .


Plasmodium falciparum malaria as compared to other sympatric ethnic groups, such as the Dogon. However, the mechanisms behind this lower susceptibility to malaria are largely unknown, particularly those concerning innate immunity. Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive immune systems. Therefore, in this study we investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. Lower frequency and increased activation was observed in circulating plasmacytoid DCs and BDCA-3+ myeloid DCs of infected Fulani as compared to their uninfected counterparts. Conversely, a higher frequency and reduced activation was observed in the same DC subsets obtained from peripheral blood of P. falciparum-infected Dogon children as compared to their uninfected peers. Moreover, infected individuals of both ethnic groups exhibited higher percentages of both classical and inflammatory monocytes that were less activated as compared to their non-infected counterparts. In line with APC impairment during malaria infection, TLR4, TLR7 and TLR9 responses were strongly inhibited by P. falciparum infection in Dogon children, while no such TLR inhibition was observed in the Fulani children. Strikingly, the TLR-induced IFN-γ release was completely abolished in the Dogon undergoing infection while no difference was seen within infected and non-infected Fulani. Thus, P. falciparum infection is associated with altered activation status of important APC subsets and strongly inhibited TLR responses in peripheral blood of Dogon children. In contrast, P. falciparum induces DC activation and does not affect the innate response to specific TLR ligands in Fulani children. These findings suggest that DCs and TLR signalling may be of importance for the protective immunity against malaria observed in the Fulani.The Fulani ethnic group from West Africa is relatively better protected against  Plasmodium falciparum  infection between two ethnic groups; the Fulani and the Dogon. These populations live under similar social, cultural and geographic conditions and are exposed to identical malaria pressure P. falciparum-specific IgG subclasses Half of the world's population remains at risk of contracting malaria. During 2008 malaria was estimated to be responsible for 767.000 deaths in Africa alone, mostly in children below the age of 5 −, CD123+, blood dendritic cell antigen (BDCA)-2+ plasmacytoid DCs (pDCs) and the CD11c+, CD123− myeloid DCs (mDCs). The latter can be further divided into three subtypes; the BDCA-1+ DCs, the BDCA-3+ DCs and the CD16+ DCs Innate immunity has two important functions in the defence against the parasite. First, it acts to inhibit rapid parasite growth thereby limiting the onset of malaria pathology. Second, it is crucial for the development of parasite-specific adaptive immunity in vitro by the malaria parasite or by products derived from infection P. falciparum-infected children as compared to healthy controls + mDCs have been detected in the blood of severely infected children combined with reduced immunostimulatory ability. This indicates that malaria may impair mDC functionality and suggests that the BDCA-3+ DC subset may play a crucial role in the immune response against the parasite in humans It has been shown that monocyte/macrophage- and mDC functions are impaired P. falciparum also modulates pDC responses in vitro by inducing increased secretion of interferon (IFN)-α P. berghei in mice P. falciparum malaria episodes in humans P. falciparum stimulation in vitro. Nevertheless, the role of this cell subset during the infection in the natural host is only partially understood. Monocytes can trigger adaptive immune responses and contribute to parasite clearance through antibody-dependent cellular inhibition and phagocytosis +CD16−) and one inflammatory (CD14+CD16+) + monocytes were higher in children with severe P. falciparum anaemia, which could enhance erythrophagocytosis and TNF-α production in this cell subset Evidence demonstrates a fundamental role for monocytes in activation of NK cells Plasmodium DNA bound to either malaria pigment P. falciparum glycosylphosphatidylinositol (GPI) activates TLR2 APCs interact with pathogens through specialized receptors termed pattern-recognition receptors (PRRs) that have evolved to detect danger signals such as pathogen-associated molecular patterns (PAMPs) P. falciparum malaria infection in an endemic area of Mali. In addition, the impact of P. falciparum on the activation of APCs was examined by stimulating peripheral blood mononuclear cells (PBMCs) with specific TLR ligands. LPS was employed to activate TLR4 on monocytes and mDC subsets while imiquimod and CpG-A ODN were used to selectively stimulate TLR7 and TLR9, respectively, which are expressed in pDCs In this study, we evaluated the activation status of different DC and monocyte subpopulations in peripheral blood of Fulani and Dogon children with or without P. falciparum infection, the mean temperature was significantly higher in the infected Dogon (37.74°C) as compared to infected Fulani . The mean parasite density was 18758 [150-122000] asexual parasites/µl for Fulani and 13845 [575-48625] asexual parasites/µl for the Dogon.The mean age of the Dogon children was 6.36 years for uninfected and 6.70 years for the infected ones. In the Fulani children, the mean age was 6.44 years for uninfected and 4.50 years for the infected ones. The infected Fulani were younger than the infected Dogon . During Circulating DCs are defined as leukocyte lineage negative and HLA-DR positive mononuclear cells + (p = 0.034) and BDCA-3+ (p = 0.016) DCs than their uninfected peers (+ (p = 0.060) and BDCA-3+ (p = 0.038) DCs as compared to their uninfected peers. When comparing only the infected children from each ethnic group, the percentage of circulating BDCA-2+ and BDCA-3+ DCs was significantly lower in Fulani as compared to Dogon . No interethnic differences were observed between the uninfected children from each group. A similar trend was observed when evaluating absolute numbers of BDCA-2+ and BDCA-3+ DCs/ml of blood although differences were not significant (data not shown). The percentage and absolute numbers of circulating CD16+ and BDCA-1+ DCs did not show any inter- or intra-ethnic difference (The infected Dogon exhibited significantly higher frequency of circulating BDCA-2ed peers . Convers+ DCs (+ mDCs (p = 0.057) as compared to uninfected peers. In addition, expression levels of HLA-DR were reduced on CD16+ mDCs of both Dogon and Fulani children undergoing infection as compared to their uninfected peers.When examining DC-activation status, we observed that expression levels of the activation marker HLA-DR was significantly lower on all examined DC subsets in the infected Dogon as compared to uninfected peers . In addi+ DCs  as compa+, BDCA-2+ and BDCA-3+ DCs but not on CD16+ DCs as compared to the infected Dogon children. Thus, interethnic differences were observed in terms of blood DC activation status upon malaria and all circulating DC subpopulations excluding CD16+ DCs were significantly more mature in Fulani than in Dogon children during P. falciparum infection. In order to evaluate if these differences could be dependent on parasite burdens, we correlated levels of parasitaemia with HLA-DR expression levels on the affected DC subpopulations from each study participant. No significant correlation was observed in Dogon and Fulani children undergoing P. falciparum infection. For BDCA-1+ DCs the results of the correlation tests were Rho = −0.38 (p = 0.106) for Dogon children and Rho = 0.20 (p = 0.72) for Fulani children. For BDCA-2+ DCs the results were Rho = −0.16 (p = 0.49) for Dogon children and Rho = 0.20 (p = 0.72) for Fulani children. For BDCA-3+ DCs the results were Rho = −0.28 (p = 0.22) for infected Dogon children and Rho = −0.40 (p = 0.48) for infected Fulani children. This suggests that the differences observed in the DC subpopulations could not be correlated to the differences in parasite burden between the populations.When comparing children undergoing infection from each ethnic group, the Fulani exhibited higher levels of HLA-DR and CD86 on the BDCA-1+CD16−) and inflammatory (CD14+CD16+) monocytic subsets during P. falciparum infection in the children participating in the study.Given the importance of monocytes in inflammation and clearance of parasites, we investigated the classical  and inflammatory (p = 0.02) monocytes was observed in Dogon children undergoing infection as compared to their uninfected peers. A similar trend, although not significant, was found between infected and non-infected Fulani .When examining the activation status of circulating monocytes, a significantly decreased expression of HLA-DR was observed on classical monocytes from both Dogon and Fulani groups undergoing infection as compared to their uninfected peers  and TLR9 (p = 0.01) ligands, indicating an impaired pro-inflammatory activity in these specific TLR pathways upon P. falciparum infection in this ethnic group . This might indicate that the TLR4 signalling pathway may be less affected than the TLR7 and TLR9 signalling in Dogon undergoing infection. This ratio was not affected in infected Fulani as compared to uninfected peers upon TLR4, TLR7 and TLR9 stimulation. When comparing children undergoing infection from each ethnic group, the TNF-α/IL-10 ratio was higher in Fulani than Dogon children when cells were stimulated with TLR9 ligands (p = 0.04), suggesting a significantly higher proinflammatory activity induced by TLR9 ligation in Fulani than Dogon children undergoing P. falciparum infection. Similarly, the ratio of IFN-γ/IL-10 was decreased in the infected Dogon children as compared to their uninfected peers upon stimulation with TLR4 (p<0.0001) and TLR9  responses. These cells may be the counterpart of murine CD8-α+ DCs P. berghei in a murine model of malaria P. falciparum infection may contribute to more robust anti-malarial cellular IFN-γ responses Recent evidence suggests that BDCA-3P. falciparum infection. These results are in accordance with previous findings in Thai-Burmese patients where patients with acute malaria exhibited a higher frequency of inflammatory monocytes in peripheral blood P. falciparum infection impaired monocyte immunophenotypes, as reflected by the reduced expression of activation markers on classical and inflammatory monocytes in both Fulani and Dogon children, suggesting that P. falciparum alters the functionality of these cells in both ethnic groups. However, interethnic differences were observed during malaria infection as classical monocytes were significantly more activated in infected Fulani than in infected Dogon children.The frequencies of both classical and inflammatory monocytes were increased both in Dogon and Fulani children upon P. falciparum infection while such TLR inhibition was not observed in the Fulani children. This is in line with the impairment of DC phenotype that was observed in Dogon but not Fulani children upon malaria infection. In humans, TLR7 and TLR9 are mainly expressed in pDCs and it has been established that TLR9 is involved in recognition of malarial products P. falciparum infection in Dogon children, but not in the Fulani ethnic group.To further evaluate the innate response in the Fulani and Dogon children during malaria infection, we investigated cytokine release by PBMCs after stimulation with specific TLR ligands. Cytokines are crucial in the pathogenesis of malaria and the balance between pro- and anti- inflammatory cytokines plays a central role in the clinical outcome of this infection Plasmodium antigens by TLRs P. falciparum infection suppresses TLR responses in Dogon children while no effects were observed in the Fulani children. There may be several reasons for the discrepancies seen in these studies. Firstly, both the studies by McCall et al. and Franklin et al. were performed in adults while, in our study, only children were included. Secondly, these studies were performed in malaria naïve adults at the earliest stages of P. falciparum infection P. falciparum infection during the same season as estimated by previous observations in the same area (Arama C. unpublished results). Thus, malaria infection may have a biphasic influence on the innate immune system, initially inducing TLR priming and then, in case of frequent exposure to the parasite, tolerance will ensue. Interestingly, such a suppressive phase was observed in Dogon but not in the children belonging to the Fulani ethnic group.In general, the importance of TLRs in malaria is underlined by the fact that TLR4 polymorphisms can predispose to severe malaria P. falciparum in vitro release higher amount of IFN-γ than Dogon Moreover, we observed higher release of IFN-γ by PBMCs of infected Fulani children as compared to infected Dogon upon TLR stimulation. This is in line with recent findings indicating that PBMCs from Fulani specifically stimulated with P. falciparum infection merely depends on differences in parasitaemias in this ethnic group as compared to Fulani. However, we did not observe any significant correlation between parasitaemia and HLA-DR expression levels on distinct APC subsets in infected children. Therefore, differences in levels of parasitaemia do not appear to account for the APC dysfunction observed in Dogon children as compared to Fulani in our cohort.It is possible that APC dysfunction could be dependent on the levels of parasitaemia in an infected individual. This suggests that the APC alteration observed in Dogon individuals upon P. falciparum infection impairs the phenotype of blood DCs and alters TLR responses of PBMCs from Dogon but not Fulani children. In particular, malaria infection induces differential innate IFN-γ release in the two ethnic groups. These findings suggest that activation of certain DC subsets and additional mechanisms triggering cytokine release by APCs through TLR activation may represent immunological correlates of protection against P. falciparum malaria. However, the prospective evaluation of a larger cohort of patients is required to confirm our results and to analyze the potential occurrence of distinct TLR polymorphisms in these two ethnic groups. The understanding of innate immune responses to P. falciparum in naturally exposed children may yield important insights in the development of immunity to malaria with particular regard to rational vaccine development.In conclusion, in this study we found that Ethical approval was obtained from the institutional review boards of the University of Bamako, Mali (N°08_64/FMPOS) and by the Swedish ethical research committee (03-536). Before the inclusion of each child in the study, the community leaders' permission and individual consent form were obtained from the parents of each child.P. falciparum parasites. Presence of malaria infection was defined as a positive thick smear with or without any malaria symptom. Among the Dogon, 20 children were undergoing malaria infection and 20 children were healthy, while among Fulani 14 children were suffering from malaria and 23 were uninfected. Unfortunately not all slides could be recovered from the field study so we only have parasitaemias for eight of the Fulani and nineteen of the Dogon children.The study was conducted in a rural area of the Dogon valley of Mali where malaria is mesoendemic with intense transmission during the rainy season. The study area and the study population have been described in details elsewhere Venous whole blood samples were collected from healthy and infected children of both ethnicities. From each volunteer, 6 ml of peripheral blood was collected in heparin tubes  and were transported to the Malaria Research and Training Centre Laboratory in Bandiagara, north-east of Mali, for further experiments. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque  and used for APC immunophenotyping and TLR stimulation.Purified PBMCs were fixed and frozen immediately after purification as previously described PBMCs were resuspended in RPMI supplemented with 10% FCS, 200 mM L-Glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin to a final concentration of 1 million cells/ml. Two hundred µl of cell suspension were distributed into 5 different wells of 96-well flat bottom plate for cell cultures . Cells were stimulated with LPS  (100 ng/ml), CpG-A  (3 µg/ml), imiquimod  (1 µg/ml) and unstimulated control. The plates were incubated for 16 hrs at 37°C + 5% CO2 and then the cells and supernatants were collected and stored at −80°C for subsequent cytokine analysis.Levels of IL-1β, IL-6, IL-8, IL-10, IL-12 (p70) and TNF-α in supernatants obtained from stimulated PBMCs were determined by cytometric-bead array (CBA) technology  and the fluorescent signals were detected by Flow Cytometry according to the manufacturer's recommendation. This sensitive technique allows detection by flow cytometry of multiple cytokines in small quantities of samples. All samples were diluted 1∶10 in Assay Diluent . Data were acquired with fluorescent activated cell sorter (FACS) Calibur  and analyzed using CellQuest and FCAP Array software. The lower detection limit for the various cytokines was 4.8, 4.7, 3.2, 3.8, 3.7 and 4.9 pg/ml for IL-1β, IL-6, IL-8, IL-10, IL-12 (p70) and TNF-α, respectively.Levels of IFN-α and IFN-γ were measured by conventional sandwich enzyme-linked immunosorbent assay (ELISA) kits using pairs of capture and detection antibodies . All samples were tested in duplicates according to the manufacturer's recommendation. The optical densities were measured at 405 nm in an ELISA plate reader . The assay sensitivity was 2 pg/ml for IFN-α and 7 pg/ml for IFN-γ, respectively.Statistical significance was determined using non-parametric tests. A Kruskal-Wallis test was employed to analyze the results when more than two groups were involved. The Mann-Whitney U-test was employed to evaluate significance between two unpaired groups when the Kruskal-Wallis test showed a significant difference between the four groups. Linear regression analysis (Spearman rank correlation test) was employed when analysing possible correlations. For all tests p≤0.05 was considered significant and no correction was made for using multiple tests. The data were analyzed using the StatView software.


Policies concerning the prevention of transfusion transmitted malaria (TTM) are the responsibility of blood transfusion services and malaria control programmes. To prevent spreading drug resistance due to over-use of malaria drugs, recent malaria treatment guidelines recommend prompt parasitological confirmation before treatment is started. In contrast, blood safety policies from the World Health Organisation (WHO) recommend presumptive malaria treatment for recipients of blood in endemic countries but evidence supporting this approach is lacking. Our study documented how these conflicting policies relating to malaria transmission through blood transfusion impact on clinical practice in a teaching hospital in West Africa.We randomly selected and reviewed case notes of 151 patients within 24 hours of their receiving a blood transfusion. Transfusion practices including the confirmation of diagnosis and anti-malarial treatment given were compared across three departments; Obstetrics and Gynaecology (O&G), Paediatrics and Medicine. Overall, 66 (44%) of patients received malaria treatment within 24 hrs of their blood transfusion; of which only 2 (3%) received anti-malarials based on a laboratory confirmation of malaria. Paediatric patients (87%) received the most anti-malarials and only 7% and 24% of recipients in medicine and O&G respectively received anti malarials. In 51 patients (78%), the anti-malarials were prescribed at the same time as the blood transfusion and anti-malarials prescriptions exceeded the number of patients with a presumptive diagnosis of malaria.It is common practice in paediatrics to prescribe anti-malarials routinely with blood transfusions. This contravenes the malaria treatment guidelines of laboratory confirmation before treatment but is in accordance with the less-well evidenced blood safety guidelines. There is an urgent need for more evidence about the clinical impact of transfusion transmitted malaria to enable malaria and blood transfusion programmes to harmonize their policies and give clear guidance to clinicians who prescribe blood transfusions in malaria-endemic areas Malaria is one of the most important parasitic diseases in the world. In 2009, there were approximately 225 million malaria cases in the world with 781,000 deaths There are no evidence-based international guidelines for the prevention of TTM in sub-Saharan Africa and there is lack of harmonisation between policies produced by blood safety programmes and policies by malaria programmes. The World Health Organisation (WHO) recommends that donated blood should be tested for malaria “where appropriate and possible”, There are therapeutic dilemmas related to the use of anti-malarials and antibiotics in association with blood transfusions which are particularly apparent in paediatric practice. Anti-malarials are given presumptively on the basis that most febrile children have underlying malaria and that they are at high risk of transfusion-transmitted malaria. In the case of antibiotics the presumptions are that non-malaria infections are common in febrile children and that post-transfusion fever is frequently due to bacterial infections.There is very little information in the literature about how African countries have incorporated WHO's recommendations concerning transfusion-transmitted malaria into their national policies and how these policies have been translated into practice. The Democratic Republic of Congo, Comoros and Malawi are carrying out some screening of donated blood for malaria using microscopy Ghana's malaria policy recommends laboratory confirmation of malaria before starting treatment in all situations except for children under 5 years and for suspected severe malaria if laboratory confirmation is not immediately possible. The first line treatment for uncomplicated malaria which is the only type likely to be present in clinically well blood donors is oral artesunate-amodiaquine Approval was given for this study by 2 ethics committees: the Liverpool School of Tropical Medicine, Research Ethics Committee in Liverpool, UK and the Committee on Human Research Publication and Ethics (CHRPE) of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology in Kumasi, Ghana.This study was conducted at the Komfo Anokye Teaching Hospital (KATH) in the Ashanti region of Ghana between October and November 2009. KATH is the second largest hospital in Ghana and the referral centre for the middle and northern parts of the country. It has a 1000 bed capacity and carries out about 14,500 whole blood transfusions per year. There are no hospital based guidelines for the clinical use of blood or any written policies or protocols concerning transfusion transmitted malaria. The blood bank has transfusion monitoring forms which are given to ward staff when the blood is issued and are used for reporting any transfusion reactions.Transfusion recipients from the departments of Obstetrics and Gynaecology, Paediatrics and Medicine were included in the study. These departments were selected because they use the majority (66%) of blood in the hospital. All patients in these departments who had received a transfusion within the previous 24 hours were eligible for the study. Exclusion criteria were untraceable case notes, transfer to a department not included in this study or more than one transfusion within the 24 hour time period. Each day the blood bank staff provided AOO with the names of two blood transfusion recipients in each of the three departments. The recipients were selected randomly from the list of patients who had received blood in the preceding 24 hours. On six occasions during the two month study a department had less than two transfusion recipients in 24 hours and so the name of a replacement participant from another department was provided by the blood bank staff. On one occasion the case notes of a patient from obstetrics could not be traced and as no other obstetric patient was eligible, a replacement was recruited from paediatrics.At recruitment, a data collection form was used to extract information from the clinical notes of the transfusion recipients. The form was adapted from the KATH transfusion monitoring form, which is based on the national guidelines for the clinical use of blood in Ghana. The form was expanded to include information about the patient's diagnoses, laboratory tests  and treatment type . Information was also collected on monitoring of vital signs, duration of transfusion and documentation of adverse events of transfusion.Predictive Analytics Software (PASW®) statistics 18 package  was used to calculate frequency statistics including measures of central tendency and dispersion and the results were compared between the three departments. The practices relating to the prevention and treatment of malaria associated with transfusion were compared with known policies.151 transfusion recipients were recruited into the study from medicine , obstetrics  and paediatrics . The majority of patients (64.9%) were female, because of the inclusion of the obstetric department, and the median age of all patients was 22.0 years (IQR: 4.0–36.0) . 25 patiSixty six (44%) patients received malaria treatment within 24 hours of their blood transfusion. There was marked variation in the use of anti-malarial drugs between departments with 51 (87%) children receiving malaria treatment compared to 12 (24%) obstetric patients and 3 (7%) adult medical patients . In 51 oOverall 118 transfusion recipients (78%) received antibiotics, with the highest usage in obstetrics (96%) . 81% of In Ghana, as in many other malaria endemic countries in sub-Saharan Africa, blood for transfusion is not screened for malaria and there are no clear policies about whether or not anti-malarials should be prescribed presumptively with blood transfusions. 44% of all transfusion recipients in our study received malaria treatment with their blood transfusion (77%) or in the 24 hours following the transfusion (6%) but only two (3%) had parasitaemia confirmed on microscopy. This practice of routine prescription of anti-malarials with blood transfusions without laboratory confirmation of infection occurred predominantly in paediatrics. Many of the children had a clinical diagnosis of malaria so it is possible that the anti-malarials were prescribed presumptively for their suspected infection. However, in children, 98% of malaria drugs for which the timing was known, were prescribed simultaneously with the blood transfusion and the number of children prescribed anti-malarials exceeded the number with suspected malaria. This indicates that in paediatric practice anti-malarial drugs are prescribed routinely with blood transfusions.Prescribing malaria treatment routinely for transfusion recipients in endemic areas reflects WHO guidelines There have been no studies from endemic areas comparing presumptive versus targeted malaria treatment for transfusion recipients and there is very scanty evidence underpinning recommendations for presumptive malaria treatment of transfusion recipients. In contrast, current malaria treatment guidelines recommending that treatment should only be given to those with proven infections Our study found that the restrictive malaria treatment policy is not implemented in the context of blood transfusions in a malaria endemic area. International guidelines do allow for presumptive treatment of malaria if laboratory confirmation is not available The mean duration of transfusion in children was longer than in adults and diuretics were prescribed with the blood transfusion for 88% of children. These practices do not appear to be consistent with any policies or guidelines but are based on assumptions that the symptoms of severe malarial anaemia, particularly respiratory distress, result from biventricular failure The use of concomitant antibiotic in transfusion recipients ranged from 61–96% across the three departments and is likely to represent treatment for underlying conditions. In clinical practice in Africa it can be difficult to differentiate between severe malaria and sepsis, and both conditions may co-exist These results of this study have enabled us to make several recommendations concerning transfusion-transmitted malaria . We have


Malariometric parameters are often primary endpoints of efficacy trials of malaria vaccine candidates. This study aims to describe the epidemiology of malaria prior to the conduct of a series of drug and vaccine trials in a rural area of Burkina Faso.Malaria incidence was prospectively evaluated over one year follow-up among two cohorts of children aged 0–5 years living in the Saponé health district. The parents of 1089 children comprising a passive case detection cohort were encouraged to seek care from the local health clinic at any time their child felt sick. Among this cohort, 555 children were randomly selected for inclusion in an active surveillance sub-cohort evaluated for clinical malaria during twice weekly home visits. Malaria prevalence was evaluated by cross-sectional survey during the low and high transmission seasons.Number of episodes per child ranged from 0 to 6 per year. Cumulative incidence was 67.4% in the passive and 86.2% in the active cohort and was highest among children 0–1 years. Clinical malaria prevalence was 9.8% in the low and 13.0% in the high season (p>0.05). Median days to first malaria episode ranged from 187 (95% CI 180–193) among children 0–1 years to 228  among children 4–5 years. The alternative parasite thresholds for the malaria case definition that achieved optimal sensitivity and specificity (70–80%) were 3150 parasites/µl in the high and 1350 parasites/µl in the low season.Clinical malaria burden was highest among the youngest age group children, who may represent the most appropriate target population for malaria vaccine candidate development. The pyrogenic threshold of parasitaemia varied markedly by season, suggesting a value for alternative parasitaemia levels in the malaria case defintion. Regional epidemiology of malaria described, Sapone area field centers are positioned for future conduct of malaria vaccine trials. Despite suggestion of a general decline in malaria burden in sub-Saharan Africa, Burkina Faso has shown no evidence of decrease in high year-round endemicity; health facility reports in fact suggest an increase nationally from an estimated 1 million in the year 2006 to 4 million in 2009 Development and evaluation of malaria vaccine trials demands knowledge of the regional epidemiology of malaria; in fact malaria infection, clinical malaria, and mortality rates have represented primary endpoints of phase 2b or 3 of malaria vaccine trials Current malaria epidemiology data in endemic regions are needed to evaluate the public health significance of alternative interventions and to monitor effects of malaria control activities over time. We sought to describe the epidemiology of malaria prior to the conduct of a series of drug and vaccine trials by combing active and passive case detection of malaria episodes among children under five years living in a rural area of Burkina Faso.Enrollment was initiated in January 2007 in the Saponé health district in the Bazèga province where the Centre National de Recherche et de Formation sur le Paludisme (CNRFP) has maintained a continuous demographic surveillance system (DSS) since 2005. The Sapone DSS is located in the centre of Burkina Faso, 40 km at the south from the capital, Ouagadougou in the Sudan-Sahelian eco-climatic zone (isohyets 600–800). The DSS covers an area of 1700 Km2 within a plateau dissected by the Nazinon river (White Volta river). Mean temperature recorded during 2007 was 29°C and total rainfall for the same period was 713 mm. The climate is characterized by a rainy season from June to September and dry season from October to May. The total resident population is estimated as 100,000 inhabitants and infant and children mortality is 105.3 per 1000 live births. Children below five years represent 19% of the population and total fertility rate is an estimated 45‰. The predominating ethnic groups in the area are Mossi and Fulani. Most of the population depends on subsistence farming of millet and domestic animals . Houses are typically made of mud walls and grass or corrugated iron roofs Study randomization was performed at two levels to achieve target sample sizes of PCD and ACD cohorts: i) two of the 14 peripheral health centres catchment areas composed of 13 villages were randomly selected, ii) 9 villages of the 13 villages were randomized to conduct passive case detection and 4 to conduct active case detection of malaria over one year follow-up. All 13 villages were situated within ≈7.5 km of the peripheral health centres, facilitating access to each PCD surveillance system.Anopheles gambiae, An. Arabiensis and An funestus. P falciparum accounts for a 90% of malaria infections. Oral chloroquine was still in use in the communities during the study period despite parasite resistance of 57% . The first line treatment for uncomplicated malaria in Burkina is now Coartem® or Artesunate Amodiaquine. In the study region these medications are distributed primarily by peripheral health centres; there is no private clinic or pharmacy alternative although prohibited or sub-standard antimalarials are sold from the local market. In 2007, bed net coverage was less than 5% of the DSS population, reflecting the absence of large scale bed net distribution in the region.Malaria transmission in the region is markedly seasonal and intense during the rainy season. The entomological inoculation rate is estimated at 0.3 and 44.4 infective bites/person/month during the dry and rainy seasons, respectively Malaria surveillance over one year follow-up consisted of passive case detection among all patients attending the outpatient clinic; and active case detection by twice weekly home visits and two cross-sectional surveys among selected villages. The first cross sectional survey occurred during low malaria transmission season and the second during high transmission season. Overall and age-specific incidence of clinical malaria were compared among the passive and active cohorts.All children aged under five years and living for at least three months in the study area were eligible for inclusion in either cohort at study initiation. Exclusion criteria were presence of major congenital defect, chronic disease or severe anaemia ; these conditions were considered to increase participant risks associated with blood draws, and potentially require absence from the study area for receipt of specialized care. An age-stratified sample was generated from the updated census of Saponé demographic surveillance system area. Five age-specific age strata  included 245, 201, 225, 219 and 199 children respectively in the PCD, and 131, 103, 113, 115 and 93 children in the ACD cohort. Children under one year were recruited quarterly to maintain balance among cohorts. Signed or thumb-printed  written informed consent from child's parent/legal guardian was required prior to inclusion in the study. Each child was provided with an identity card and the caregiver was given instructions how to access health centre in the event of illness.In January 2007 the PCD cohort enrolled a total of 1090 children from 9 villages in the catchment areas of two health facilities. In the two community clinics providing routine care and in the Saponé District Hospital emergency unit, local health staff received training in study procedures and were provided with study case report forms to record clinical information of children enrolled in either cohort. At each attendance to a health facility , study children received a brief physical examination and parents reported previous health events. In the event of reported history of fever within 24 hours or measured axillary temperature ≥37.5°C, a finger-prick blood sample was collected for preparation of duplicate thick blood smears. One slide was read immediately (within two hours) in the Saponé District Hospital and each transferred to the CNRFP lab for quality assurance. Rapid diagnosis tests (RDT) were performed in the two community clinics of the study area where an operating lab was not present. The active surveillance cohort consisted of 555 children from four randomly selected villages who were visited in the home twice per week for detection of clinical malaria episodes over one year follow up. The target sample size was achieved after recruitment of children living in the first four of thirteen villages randomly selected for potential study inclusion.Trained fieldworkers visited the children in their homes twice a week on the same week day and before 11:00 AM in order to minimize the bias due to diurnal variation of the body temperature Diagnostic procedures were repeated during two seasonal cross sectional surveys, which contributed to analyses of prevalence of malariometric indicators and malaria attributable fraction of fevers. The first cross sectional survey occurred during the dry season and the second one during the wet season of the same year.Thick and thin blood films were prepared, air-dried, fixed, Giemsa-stained, and examined using a light microscope fitted with 100× oil immersion lens. Number of parasites and leucocytes were counted to reach 200 leucocytes for positive slides. Slides were declared negative only after 100 high power fields has been read. Parasite numbers were converted to a count/µL. All slides were read twice and slides with discrepant result were then read a third time. Haemoglobin was measured at each survey using a portable Hemocue.P. falciparum trophozoite count greater than 0). Analyses of clinical malaria incidence were repeated using the alternative parasitemia thresholds of >0, ≥2500, and ≥5000.Fever was defined as axillary temperature ≥37.5°C and or reported fever within the previous 24 hours. A clinical malaria episode was defined as an axillary temperature of ≥37.5°C or history of fever in last 24 hours, and positive asexual parasitemia . The low transmission season was defined as the date of first enrollment (January 23rd 2007) through May 31st 2007 and November 1st 2007 through date of last observation (February 29th 2008). The high transmission season was defined as June 1st 2007 through Oct 31st 2007.Overall and age-specific fever and malaria incidence rates over one year follow-up were calculated as the number of events malaria cases divided by the total person time at risk. Cumulative incidences were estimated by the Kaplan-Meier method. Children were censored for each day he or she recorded a fever or for 28 days after recording an episode, to ensure that the infection causing the episode was only recorded once. In analyses of both fever and clinical malaria incidence, children were censored after 365 days of follow-up, or at the date of death/out migration (N = 25). Those who did not experience death/migration but were followed for less than one year were censored at the last recorded passive case detection visit (Febuary 29i/(i−pi)) = βo+β1xiτLog or both objective and subjective fever Sample sizes expected to yield robust estimates of malaria incidence were calculated using Epi Info version 6 assuming weekly incidence densities estimated in previous years from regional DSS and targeted surveillance within one of the study site villages. Access was used for double data entry and query resolution and SAS 9.2 for data analysis.The spatial analysis map of community clinic accessibility was produced using Arc View GIS 3.2.Validity and completeness of data collection procedures were reviewed weekly by a team of physician researchers. In the ACD cohort, each week a sample of fifteen (average 50 children) randomly selected households were revisited by a field supervisor. The supervisor interviewed the parents/guardian to verify reporting of fever or collection of a blood sample.All slides were read twice independently; if the ratio of densities from the first two readings was >1.5 or <0.67 or if <30 parasites were counted with a difference in the number of parasites >10, the slide was evaluated a third time. The definitive result was the geometric mean of the parasite density. In the event of discrepancy the slide was evaluated a third time and the final result defined as the majority reading for positivity.Data queries were generated weekly to identify and resolve missing or erroneous values.The study protocol and the informed consent form were approved by the Burkina Faso ministry of health ethic committee. The study was conducted in compliance with principles set out by the International Conference on Harmonization Good Clinical Practices, the Declaration of Helsinki and the regulatory requirements of Burkina Faso. Individual written informed consent was obtained from all children's parents or legal representatives, in the presence of an independent witness for illiterate parents/legal representative. The conduct of the study was monitored by the sponsor.Of 1644 children enrolled, analyses include 1089 (66.3%) in PCD and 555 (33.7%) in ACD. Eight (8) children died and 17 (1%) withdrew during the follow up period. Enrolment within each cohort was approximately equally distributed among gender and age groups .ACD children where present during 55279 of 57720 (95.8%) home visits attempted. Losses to follow up were due to parents moving away from the DSS area. Median number of visits per child was 100 (range 1–104). ACD children presented to the community clinic without reference 3389 times; median number of visits per child was 6 (range 1–20).PCD children contributed 5742 visits; median number of visits per child was 5 (range 1–14).ACD scheduled & unscheduled visits identified 2668 cases of fever; 79% (n = 2129) of these were detected through unscheduled visits. 2382 cases of fever were detected by passive surveillance visits.In both ACD and PCD cohorts fever was more frequent among infants and markedly lower among children aged 4–5 years . Fever iP. falciparum was identified among 1071 (40%) of 2668 fevers in the ACD, and among 1208 (51%) of 2382 fevers in the PCD cohort. Clinical malaria incidence was higher in younger age groups among both the ACD and PCD cohorts  (95% CI) was 1.6 (1.5–1.7) in the ACD and 0.8 (CI 0.8–0.9) in the PCD cohort (Table4).Clinical malaria incidence was higher in younger children within each season and among alternative parasite thresholds for malaria case definition. Children aged ≥4 years experienced fewest malaria episodes with incidence rates ranging between 0.8 and 2.2 episodes per child-year-at-risk. This pattern was apparent by each method of incidence assessment .Among all age groups clinical malaria incidence was highest following the peak rainfall .Two cross-sectional surveys among children in the ACD cohort evaluated prevalence of malariometric indices within the low and high transmission seasons .P. falciparum infection was 57.5% in the low versus 49.3% in the high season (p = 0.01). Geometric mean parasite density (parasites/µL) (95% CI) was lower during the low relative to high season: 1554 (1307.5–1848.9) versus 2673.2 (2077.1–3440.4) (p<0.01) ; prevalence of asexual (p<0.01) .Median days to first malaria episode ranged from 187 (95% CI 180–193) among children 0–1 years to 228  among children 4–5 years in the ACD cohort. Cumulative incidence among these age groups ranged from 93%–71% .Malaria attributable fraction of fever was 41.9% in the high season and 34.5% in the low season, where fever was defined as reported fever in the past 24 hours and measured temperature ≥37.5°C. The alternative parasite thresholds for the malaria case definition that achieved optimal sensitivity and specificity (70–80%) were 3150 parasites/µl in the high and 1350 parasites/µl in the low season .The objective definition of fever (axillary temperature ≥37.5°C) yielded a malaria attributable fraction of fever of 59.6% in the high and 17.2% in the low transmission season. The thresholds that achieved sensitivity and specificity were 2930 parasites/µl in the high and 2780 parasites/µl in the low season .In this cohort of children under five years living in Burkina Faso, cumulative incidence by active case detection over one year follow-up exceeded 90% in the youngest children. Sixty-five percent of malaria episodes were detected during the rainy season. These data characterize the Saponé district as a stable malaria transmission area with marked seasonality Plasmodium falciparum is the most common vector of malaria in the Balonghin region. Clinical malaria incidence was highest among children under three years and showed graded decline through five years, a pattern consistent with previous reports Despite high all year-round endemicity, clinical malaria was markedly more common during the rainy months of June-November, a pattern previously reported in Burkina Faso Age and seasonal trends also persisted among passive (PCD) and active case detection (ACD) cohorts; however, fever and clinical malaria incidence detected by ACD were approximately twice that by PCD.These data suggest that as many as half of malaria episodes identified by active follow-up may be undetected by passive surveillance in this and comparable settings. This expected advantage of ACD highlights the common occurrence of unattended symptomatic malaria. Variation among cohorts may also in part reflect study design limitations. Passive surveillance outcomes may be underestimated due to alternative care seeking from non-study clinics or traditional healers or use of anti-malarials from the local market. Moreover, parents of ACD children may have been more likely to seek medical care for symptomatic illness as encouraged by field workers during regular home visits; by contrast, parents of PCD children received this instruction only at enrolment. It is also possible that time at risk for malaria, particularly among ACD children, is in part overestimated where chemoprophylaxis due to antimalarial treatment may have extended beyond the 28 day censoring window imposed after each observed episode. Refinement of censoring windows according to treatment duration information not available in this study may be expected to minimize associated inflation of time at risk, yielding higher than current incidence estimates.P.f. infection from 49–58% among transmission seasons. Age was not consistently associated with prevalence of clinical malaria, P.f. infection or splenomegaly. Prevalences of P.f. infection, gametocyte carriage, and splenomegaly were slightly higher in the low relative to high transmission season. These data may reflect protection provided by longitudinal active surveillance, in which frequent contacts initiated following the low season survey prompted early diagnosis and treatment and potentially chemoprophylaxis effect particularly through the subsequent high season. Prevalence of malariometric indices may be considered underestimated as a result of this potentially artificially healthy ACD cohort.Prevalence of clinical malaria in the ACD cohort ranged from 10–13%, and prevalence of Time to first malaria episode from the low season survey was lowest among ages 0–2 years, consistent with previously described vulnerability associated with the loss of protection by maternal immunoglobulin following the first six months of life. Cumulative incidences of malaria were also highest among this age group, potentially reflecting a critical period for induction of partial immunity associated with repeat malaria exposure in early life. Children 3–5 years were more likely to escape infection during the high transmission season, suggesting development of immune protection prior to our observation.Estimated proportion of fever attributable to malaria parasitaemia varied substantially by malaria case definition and were nearly two fold greater in the high relative to low transmission season. These data yielded optimal parastemia thresholds for malaria case definition ranging from approximately 3000 parasites/µl in the high and 1350 parasites/µl in the low season. Marked dependence of these characterizations on season and case definition have previously been described among children living in an endemic areas of Mali and Ghana Practicians in this region may be advised to investigate alternative etiology of fevers given parasitemia <1350 in the dry or <3150 µl in the rainy season, the optimal thresholds derived from the favored objective fever definition.In this high and seasonal transmission region of Burkina Faso, burden of malaria was highest among the youngest children, who may be considered the most appropriate target for malaria vaccine candidates. Active case detection identified nearly twice the incidence of malaria episodes relative to the passive system, highlighting the common occurrence of potentially clinically important unattended illnesses and resulting advantage of enhanced surveillance methods in estimation of disease burden. The pyrogenic threshold for malaria case definition differed markedly by season, suggesting that alternative etiologies of fever should be considered in the presence of low parasitemia levels particularly during the low transmission season. Characterization of malaria epidemiology in the Balonghin region reinforces the urgency of improved access to currently available medication and vector control tools, while setting the stage for conduct of early phase vaccine trials in local field sites.


Plasmodium falciparum infection, but immunoepidemiological data on longevity of immunity remain controversial. We quantified serum cytokines and chemokines by suspension array technology as potential biomarkers for durability of immunity in immigrants with clinical malaria after years without parasite exposure. These were compared to serum/plasma profiles in naïve adults (travelers) and semi-immune adults under continuous exposure, with malaria, along with immigrant and traveler patients without malaria. Immigrants had higher levels of IL-2, IL-5 and IL-8 compared to semi-immune adults with malaria (P≤0.0200). Time since immigration correlated with increased IL-2 (rho=0.2738P=0.0495) and IFN-γ (rho=0.3044P=0.0282). However, immigrants did not show as high IFN-γ concentrations as travelers during a first malaria episode (P<0.0001). Immigrants and travelers with malaria had higher levels of IFN-γ, IL-6, and IL-10 (P<0.0100) than patients with other diseases, and IL-8 and IL-1β were elevated in immigrants with malaria (P<0.0500). Therefore, malaria patients had a characteristic strong pro-inflammatory/Th1 signature. Upon loss of exposure, control of pro-inflammatory responses and tolerance to P. falciparum appeared to be reduced. Understanding the mechanisms to maintain non-pathogenic effector responses is important to develop new malaria control strategies.Immunity to malaria is believed to wane with time in the absence of exposure to  Plasmodium falciparum infection still causes millions of malaria cases and deaths worldwide, mainly in sub-Saharan Africa ) as we have observed before in previous studies ,23. CorrP>0.0001) and there were more males in the immigrant and traveler groups with malaria than in the other groups (P=0.0140). Immigrants were original of different African countries and most of the travelers were from Europe. Visiting countries were very heterogeneous among immigrants and travelers. Immigrants returned from visiting their countries of origin. Immigrants with malaria had lived for a median of 7 years in Spain and 9.6% had never returned to their original country before, 21.1% had returned 1 to 2 times, 50% 3 to 4 times and 19.2% had returned more than 5 times. Immigrants without malaria had a different time since immigration, but this difference was not statistically significant. No significant differences were detected in parasitemias between immigrants and travelers . However, only IL-2 differences remained statistically significant after applying a correction test for multiple comparisons.Immigrants showed a different cytokine profile than semi-immune adults during an acute malaria episode . ImmigraTo determine the effect of time since immigration on the cytokine responses in a malaria acute episode, Spearman correlation coefficients were calculated for cytokines and years since immigration . IFN-γ aP<0.0001; and 23.23  pg/mL, respectively, P=0.0334). Immigrants also had higher levels of IL-10 at day 28 of convalescence  compared to naïve adults . However, only differences in IFN-γ levels remained statistically significant after correcting for multiple comparisons.Cytokine and chemokine serum levels were measured in all immigrants and travelers during the acute malaria episode and in a subset of patients during convalescence . In immigrants, there was also a trend to have higher ratio of IL-12 (P=0.0513) and TNF (P=0.0684), and lower ratio of IFN-γ (P=0.0954). There were no differences in day 0 to day 28 ratios (data not shown).To evaluate the magnitude of the cytokine/chemokine responses as well as its kinetics from acute to convalescent phases, ratios of cytokine/chemokine concentrations between days 0, 7 and 28 were calculated. Day 0 to day 7 ratios of IL-4 and IL-1β were significantly higher in immigrants compared to travelers . Also, there were no correlations among cytokines/chemokines and antibody responses to antigens  in theseP=0.0003 and 5.64  pg/mL, P=0.0069, respectively). IL-6 levels were also higher in travelers  and immigrants  with malaria than in patients without malaria . Travelers and immigrants with malaria also had higher levels of IL-10  compared to individuals with other diseases . IL-8 and IL-1β were higher only in immigrants with malaria  compared to immigrants with other diseases .Patients presenting to the Tropical Medicine Units with an acute malaria episode showed a different cytokine/chemokine profile than patients presenting with other symptoms . TraveleEstimating duration of immune memory against clinical malaria in malaria-endemic populations is complicated due to re-exposure and boosting. Migrants moving from malaria endemic to non-endemic areas offer a good opportunity to study persistence of immunity and associated immune markers. This study was conducted in immigrants presenting with clinical malaria after returning from endemic areas, to assess how the loss of exposure affected their acute and convalescent peripheral blood cellular immune responses.First, when cytokine/chemokine blood levels in immigrants were compared to those in semi-immune individuals, both with clinical malaria, a different profile was observed. Loss of exposure was associated with increased levels of IL-2, IFN-γ, IL-8 and IL-5. In particular, immigrants showed higher serum concentrations of IL-2, IL-8 and IL-5 in acute malaria compared to semi-immune adults, although only IL-2 and IFN-γ showed to be dependent on time since migration. It is possible that recent visits to endemic countries or the total number of returns (69% of immigrants had returned at least 3 times) may have represented some malaria re-exposure that could have diluted the effect of time since immigration. The fact that cytokines/chemokine were measured in serum in immigrants and in plasma in semi-immune adults may have introduced certain error as it has been described that measurements may differ, depending on the cytokine, if the matrix plasma or serum ,28, but Second, when compared with naïve adults presenting a first malaria episode, immigrants with malaria had lower levels of IFN-γ, suggesting that even if there was a decline in cellular immunity this loss was not complete. In this line, we had previously observed that this group of travelers had higher levels of IFN- γ compared to malaria-exposed individuals (after few or continuous exposure) . Neverth+ T cells and TH1 CD4+T cells. IL-2 is mainly produced by T cells and reflects a strong cellular activation and proliferation. IL-2 stimulates NK differentiation and proliferation as well as B cell production of immunoglobulins. IL-8 is an important chemokine mediator of inflammation produced mainly by macrophages and endothelial cells, which recruits innate cells such macrophages, granulocytes and stimulates phagocytosis. IL-5 is produced by TH2 CD4+ T cells, but also eosinophils and mast cells and is involved in eosinophil activation, B cell growth and immunoglobulin secretion. Therefore, all these cytokines/chemokine are probably (mostly) reflecting a strong innate immune activation, although this should be demonstrated with data of cytokine producing cells. Not all patients were febrile at the time of day 0 serum sampling, although most of the patients had a history of fever during the previous days. This is of importance as some cytokines such IL-6, TNF, and IL-1β, considered pyrogens [The cytokines/chemokine measured come from peripheral blood cells as well as from endothelial cells  and sitepyrogens ,31, may pyrogens . Neverthpyrogens –34 and epyrogens  as well pyrogens ,33, althpyrogens ,33.H1 cytokines. Furthermore, IL-10 and IL-6 positively correlated with parasitemia in immigrants and travelers, whereas IL-1β only correlated with parasitemia in immigrants, and IL-8 only in travelers. Of note, semi-immune individuals did not show any correlation between cytokine/chemokine concentrations and parasite density. This may reflect a better capacity to regulate or mitigate the immune pro-inflammatory response induced in an acute infection, probably through mechanisms of tolerance that could result in milder malaria [We wanted to explore if parasitemia or parasite density was directly affecting the production of cytokines. There were no significant differences in parasitemia between immigrants and travelers, but parasite density data could not be compared with those of semi-immune people due to different slide reading methods. The assessment of correlations between each of the cytokines/chemokine and parasitemia showed that IL-2 and IFN-γ levels were independent from parasitemias, suggesting that other mechanisms could play a role in regulating those T malaria .H1 and pro-inflammatory cellular response, characteristic of non-immune patients with malaria compared with patients with other diseases. This is in line with what might be expected with increasing time since last malaria exposure: a shift in the cytokine/chemokine balance from an anti-inflammatory response towards a more pro-inflammatory response, reflecting a loss of malaria tolerance [+ T cells described recently in an area of low transmission in Thailand [+ T cell memory responses, whereas the IFN-γ concentration in plasma may come from other cell types, reflecting a predominantly innate response rather than an acquired one.Data about the persistence of protective immune responses is controversial and may depend on the antigen and the immune response assessed ,21,36–38olerance . Thus, iolerance ,40) thanolerance ,11. Howeolerance , and theA limitation of this study could be that immigrants and naïve adults were originally from very diverse countries (African or European), whereas semi-immune adults were from a unique African endemic area (Mozambique); thus, this could add genetic or environmental confounding factors. However, the immune response in clinical malaria is so pro-inflammatory compared to other diseases that it probably overcomes these limitations.H1 cytokines IL-2 and IFN-γ. However, immigrants did not show as high IFN- γ response as naïve adults in a first malaria episode, reflecting some persistence of responses associated with semi-immune individuals. Taken together, these observations may imply that immune mechanisms involved in malaria tolerance may be lost, explaining why previously immune patients appear to make strong inflammatory responses to clinical malaria. In addition data point out to immune responses that need further study to develop strategies to induce or potentiate immunity to clinical malaria.In summary, immigrants returning from endemic areas with malaria had higher serum concentrations for some cytokines/chemokines  compared to semi-immune adults with malaria, suggesting that this profile is associated with a partial loss of immunity. Time since immigration, and therefore, loss of exposure, correlated with increased T


Objectives. The aim of this study was to investigate the association of RCC and Ala16Val polymorphism in Turkish patients with RCC. Materials and Methods. A total of 41 patients with RCC who underwent radical or partial nephrectomy in our clinic and 50 healthy volunteers living in the same geographic area were included in this study. DNA samples from serum of RCC patients and controls were genotyped for MnSOD polymorphism analysis. Genotype ratios and allele frequencies were compared between two groups and odd ratios with 95% confidence intervals were calculated statistically. A P value of <0.05 was considered statistically significant. Results. There was a significant difference in the MnSOD genotype distributions between the RCC patients and the controls in terms of Ala/Ala+Ala/Val and Val/Val genotypes (P = 0.039). The Ala/Ala+Ala/Val genotypes were found significantly suspicious for RCC with an OR of 2.64 . In addition, Ala allele was found significantly suspicious for RCC with an OR of 2.26 . Conclusion. Our study indicated that MnSOD Ala16Val polymorphism may be one of the many genetic factors for renal cancer susceptibility in Turkish patients. Renal cell carcinoma (RCC) is the most common malignancy of the kidney and it constitutes approximately 3% of all adult malignancies and more than 90% of renal cancers . RCC oriβ Thalassemia, and cystic fibrosis may result due to SNPs [Single nucleotide polymorphism (SNP) is defined as a DNA sequence variation occurring when a single nucleotide, A, T, C, or G, in the genome differs between members of a biological species or paired chromosomes in a human . The ass to SNPs –7. As a  to SNPs . The SNP to SNPs .2) to hydrogen peroxide (H2O2) [The enzyme SOD is one of the most important members of the antioxidant defence system and it converts superoxide free radical (Oe (H2O2) . MnSOD Ae (H2O2) .In this study, we aimed to investigate the association of RCC and Ala16Val polymorphism in Tokat region and according to our knowledge this is the first study in the literature which investigated Ala16 Val polymorphism in RCC.After the approval of the local ethical committee permission, a total of 41  patients with RCC and who under went radical or partial nephrectomy operations in our urology clinic were included in this study. The control group included 50 healthy volunteers (37 men and 13 women), chosen at the same time, who were free of any chronic diseases, having no history of any cancer, and living in the same geographic area. They were matched with cases in age and gender. Smoking status and body mass index (BMI) of cases and controls were also evaluated. Tumor type, Fuhrman grade and tumor stage of the RCC patients were examined. Approximately 5 cc of venous blood was taken from patients and controls for the determination of genotype.Genomic DNA was extracted from the peripheral leukocytes of the collected EDTA-anti-coagulated blood by the High Pure PCR Template Preparation Kit  according to the manufacturer's instructions. To identify MnSOD Ala-9Val SNPs, genotyping was performed using PCR amplification and polymorphisms were detected with hybridization probes labeled with fluorescent dyes . Target fragments of the human MnSOD genes were amplified with specific primers. To detect the MnSOD Ala-9Val polymorphism, we applied 10 pmol of the forward primer 50-CAGCCTGCGTAGACGGTCCC-30 and the reverse primer 50 CGTGGTGCTTGCTGTGGTGC-30, and 3 pmol of the sensor probe 50 CTCCGGCTTTGGGGTATCTG-fluorescein-30 and the anchor probe 50-LC Red 640 GCTCCAGGCAGAAGCACAGCCTCCp-30. The LC FastStart Master Hybridization Probes buffer (Roche Diagnostics Inc.) was used as a reaction buffer. All primers and hybridization probes were designed and synthesized by TIB MOLBIOL . The genotypes were identified by running a melting curve with specific melting points (Tm). Wild type MnSOD Ala exhibits a Tm of 65 ± 0.5°C. The allele variant MnSOD Val exhibits a Tm of 56 ± 0.5°C. The PCR reaction was as follows: initial denaturation at 95°C for 10 min, followed by 20 cycles at 95°C for 10 s, and annealing at 60°C. And then a melting curve was recorded by an initial increase in temperature to 95°C, cooling the reaction mixture to 40°C at 20°C/s, holding for 30 s, and then slowly heating it to 85°C at 0.1°C/s with continuous acquisition. Finally, the fluorescence signal was plotted against temperature in real time to produce melting curves for each sample.t-test was used to compare the continuous data (age and BMI). Continuous data were presented as mean ± standard deviation. A P  value<0.05 was considered significant. Analyses were performed using commercial software .Pearson's, Yates corrected, and Fisher's Chi-Square test were used to compare the genotypes and other categorical data between control and case groups. All categorical data were presented as count and percentage. Two independent samples P = 0.039). In terms of allele frequencies, Ala allele frequency was found to be 55% and Val allele frequency was found to be 45% in RCC group. However Ala allele frequency was 35% and Val allele frequency was 65% in control group. Similarly, Ala allele was found significantly suspicious for RCC with an OR of 2.26 .The characteristics of the patients and control groups were presented in The etiology of renal cell carcinoma is still unclear. As in the other cancer types, oxidative stress may play a critical role in the development of RCC. The relationship between RCC and lipid peroxidation was shown in several reports , 13. AddThe inability of elimination of the ROS has been associated with the development of many diseases and various types of cancer as well. Three different isoforms of SOD have been identified. SOD1 contains copper (Cu) and zinc (Zn) and is located in cytoplasm. SOD2 contains manganese and is located in mitochondria. SOD2 is the only antioxidant enzyme known existing in the mitochondria. Mitochondrion is the most important accumulating area of the ROS that is produced during normal cellular metabolism. Because of this reason SOD2 (MnSOD) becomes more important in antioxidant defence mechanism. SOD3 or extracellular SOD also consists of Cu and Zn.2O2-O2 and carcinogenesis has been demonstrated in several studies [MnSOD is the most important enzyme that provide detoxification of the accumulated ROS in mitochondria and its gene is located at chromosome 6q25 . Numerou studies –26. Acco studies . Hence,  studies –31.2O2-O2 and SOD2 accelerated this reaction. Dasgupta et al. showed that increase in intracellular production of H2O2 by SOD2 was associated with decreased sensitivity to tumor necrosis factor-α mediated apoptosis. Therefore, as in the case of Val form, reduction of the SOD2 to enter mitochondrial matrix inhibits dismutation of superoxides to H2O2. Decreased level of H2O2 may lead to increased rate of programmed cell death ratio. As a result, the death of defective cells may supress the process of cancer development. This may be the possible cause of the association between Val form and lower risk of cancer [On the contrary, there were a lot of studies in the literature that advocated the presence of Ala allele as a risk factor for cancer. The reason of this opposition is still unclear. It was known that superoxide radicals dismutated to Hf cancer .The relationship between Ala/Ala genotype and cancer risk has been shown in many studies. Landi et al. found that the Ala/Ala genotype was associated with increased risk of malignant pleural mesothelioma . In a stThe association of Ala/Ala genotype and aggressive behavior of tumor has also been studied previously. Mikhak et al. reported that Ala/Ala allele was significantly associated with more aggressive form of prostate cancer in men with low lycopene status . SimilarIn conclusion, although it was studied with a very limited number of patients, this study indicated that Mn-SOD Ala16Val polymorphism may be one of the many genetic factors for renal cancer susceptibility in Turkish patients. It should be noted that Mn-SOD Ala16Val polymorphism in different populations showed variations due to ethnicity and various environmental factors should be considered in different geographic areas; therefore this result needs to be verified with further studies having larger sample size.