However, their performance must be established taking into the account the particularities of each endemic area. In the Democratic Republic of Congo, the validity of Optimal-ITThis was a two-stage cluster randomized survey, conducted in one health centre in 12 health zones in Kinshasa city. All patients with malaria presumptive diagnosis were eligible. Gold standard was microscopy performed by experts from the parasitology unit, Kinshasa University.® and microscopy performed at PHC. Specificity was 97.0% (95% CI: 95.5-98.5), 91.6% (95% CI: 89.1-94.0) and 49.1% (95% CI: 44.7-53.4). The proportion of confirmed cases seemed similar in under-fives compared to others. Any treatment prior to the current visit was a predictor for malaria , but not malaria treatment . Bed net ownership tended to protect against malaria .624 patients were enrolled. 53.4% (95% CI: 49.4-57.3) owed a bed net, obtained in 74.5% of cases (95% CI: 69.4-79.1) through community-based distribution by the National Malaria Control Programme. Microscopy expert reading confirmed 123 malaria cases . Overall sensitivity were 79.7% (95% CI: 72.4-86.8), 87.8% (95% CI: 81.9-93.6) and 86.2% (95% CI: 79.9-92.3), respectively, for Optimal-IT®, Paracheck-PfAlthough microscopy is considered as the "gold standard" for malaria diagnosis at point of care level, this study showed that its accuracy may not always be satisfactory when performed in health centres. Despite the efforts engaged in control, malaria remains a major concern for the public health, especially in sub-Saharan Africa. The hope to control malaria was hampered by spread of resistance to anti-malarial drugs. Irrational anti-malarial drug use contributed to the selection of drug resistant strains . The sprPlasmodium falciparum, lactate dehydrogenase (LDH) or aldolase, two enzymes produced by all human Plasmodium species.Microscopy is considered the gold standard for malaria diagnosis. However, this requires a well-maintained microscope, staining performed according to standard procedures and a good expertise in microscopy. These conditions are not always easy to meet in limited-resource HC . Many auMany studies have shown that HRP2 remains longer in circulation as LDH or aldolase following a successful malaria treatment -21. This®) and a HRP-2-based RDT (Paracheck-Pf®) at primary health care level in DRC.The aim of this study was to assess, the validity of a LDH-based RDT .Considering a 98% sensitivity of Optimal-IT®, a sample size of at least 264 patients was needed to find, with 90% power and 95% confidence, a sensitivity of at least 95%. Taking into the account a possible cluster effect, 624 patients were included in this survey.Optimal-IT®  was chosen for its good performance in areas of stable transmission ,26 and tData collection in HCs was carried out by laboratory technicians who were members of the study team. They obtained informed consent, filled out a questionnaire, prepared blood slides and performed RDTs, under the supervision of researchers from the Tropical Medicine Department, Kinshasa University. The aim of the supervision was to ensure the compliance to the study procedures as well as to recheck RDT interpretation. A one-day training workshop was conducted on the study standards operating procedures (SOPs). The expert microscopists were senior laboratory technicians with over fifteen years of work/research experience at the Kinshasa UniversityAll male and female outpatients attending study centres with clinical suspicion of malaria and to whom a blood smear was asked for confirmation, were eligible for inclusion in the survey. Lack of informed consent constituted the exclusion criteria. Those who gave consent were consecutively enrolled. Medical history, ownership and use of bed nets, socio-demographic data and attitudes before and during the illness course were recorded on CRFs.Blood for thick/ thin smears and RDTs were collected from the same finger prick and prepared on the same slide bearing the patient’s identification code. About 5 μl of blood was drawn by the study team using a loop provided with the RDT device. The test preparation and interpretation was done following manufacturer’s instructions. The tests were considered positive when the antigen and control lines were visible in their respective windows, negative when only the control band was visible and invalid when the control band was not visible. In case of invalid result, the RDT was done once again.Blood smears were stained with 10% Giemsa for 10 minutes. Thin smears were fixed with methanol prior to staining. Slides prepared by the study team were first examined by the HC laboratory technicians, blinded to RDTs results and using the WHO semi-quantitative method . Their rData were double-entered and validated in Epi info version 3.5.1 software and analysed using Stata version 11 . The Sensitivity, Specificity, PPV and NPV of the history of fever (presumptive treatment), HC microscopy, Optimal-IT® and Paracheck-Pf® were determined with expert microscopy as gold standard.Ethical approval for this study was provided by Ethical committees of the University Hospital, Antwerp, Belgium and the School of Public Health, Kinshasa University, DRC. Before inclusion, written informed consent was obtained from participants/ legal guardians for minors.From 9 May to 7 June 2011, 632 patients were screened. Eight were excluded: three for not meeting the inclusion criteria and five who refused to consent , 86.2% (95% CI: 79.9-92.3), 79.7% (95% CI: 72.4-86.8) and 87.8% (95% CI: 81.9-93.6). Overall, no technique reached the positive predictive value (PPV) of 90%, whereas the negative predictive value (NPV) was over 90% for all cases  there is constant training, supervision of staff and quality control of their tasks;(ii) the structure of the programme is practical and sustainable with adequate staff and resources;(iii) slide collection, staining and reading are accurate, timely and linked to clinical diagnosis;(iv) results are quickly provided to clinicians;(v) clinicians can trust the results; and(vi) there is logistic support to provide quality supplies and equipment.et al[The training may be the backbone of the improvement. In Uganda, refresher training in malaria microscopy has shown a substantially impact . Such exet al, to prefet al, taking The parasite load in HC was not assessed as all HC laboratory technicians used the semi-quantitative scale expressed from + to ++++ according to numbers of asexual parasites per high-power field , whereasPresumptive treatment based on history of fever appears to be the worst diagnostic method of all assessed, especially in children under five years old. Indeed, the low specificity of 11.7% in area of low malaria endemicity created a situation where three-quarters of the presumed malaria cases had no malaria (PPV = 27.3%). Therefore, this practice has to be discouraged in order to reduce unneeded prescription of ACT, reduce anti-malarial drug pressure and decrease delay of specific non-malaria treatment.et al[Malaria in patients presenting at health centre level was low as only 19.7% of presumptive malaria cases were confirmed by expert microscopy. This may be the result of several interventions, including ACT availability and community-based distribution of mosquito nets. However, the impact of these interventions on malaria transmission is not documented in DRC. In fact, in Kinshasa, more recent data published 19 years ago reported an entomological inoculation rate of up to 1,200 infecting bites/man/year . It is let al in the hAlthough microscopy is considered as the "gold standard" for malaria diagnosis at point of care level, this study showed that its accuracy may not always be satisfactory when performed in health centres. This suggests an urgent need for capacity building of microscopists and/or the implementation of accurate RDT on a large scale. Once this has been achieved, the next step and also the cornerstone to maintain the accuracy of malaria diagnosis will be the implementation/ strengthening of the quality control system.ACT: Artemisinin-based Combination Therapy; DRC: Democratic Republic of Congo; HC: Health Centre; NGO: Non Govermental Organization; NMCP: National Malaria Control Programme; PHC: Primary Health Care; RDT: Rapid Diagnostic Test; WHO: World Health Organization.Authors declare that they have no conflict of interest.HMM, PL and JPV designed the study protocol. HMM, RM, PM and GI supervised activities on the field. HMM performed the statistical analysis. HMM, PL and JPV wrote the first draft of the manuscript. All authors read and approved the final version.


The clinical manifestations of cerebral malaria (CM) are well correlated with underlying major pathophysiological events occurring during an acute malaria infection, the most important of which, is the adherence of parasitized erythrocytes to endothelial cells ultimately leading to sequestration and obstruction of brain capillaries. The consequent reduction in blood flow, leads to cerebral hypoxia, localized inflammation and release of neurotoxic molecules and inflammatory cytokines by the endothelium. The pharmacological regulation of these immunopathological processes by immunomodulatory molecules may potentially benefit the management of this severe complication. Adjunctive therapy of CM patients with an appropriate immunomodulatory compound possessing even moderate anti-malarial activity with the capacity to down regulate excess production of proinflammatory cytokines and expression of adhesion molecules, could potentially reverse cytoadherence, improve survival and prevent neurological sequelae. Current major drug discovery programmes are mainly focused on novel parasite targets and mechanisms of action. However, the discovery of compounds targeting the host remains a largely unexplored but attractive area of drug discovery research for the treatment of CM. This review discusses the properties of the plant immune-modifier curcumin and its potential as an adjunctive therapy for the management of this complication. Plasmodium, remains a serious public health threat in tropical and sub-tropical countries. It is the most important parasitic disease in humans with 300 to 500 million infected individuals every year. A proportion of these patients, mostly children under five, is at risk of developing severe anaemia or cerebral malaria (CM), which claims at least 1 million lives annually [Malaria, a disease caused by the Apicomplexan parasite of the genus annually . The effannually , and is Plasmodium falciparum infections, is associated with various pathophysiological processes [in vitro[Plasmodium chabaudi-infected mice, significantly decreased parasitaemia levels in wild type compared to CD36 knock-out mice and improved survival in Plasmodium berghei-ANKA infected groups [P. falciparum malaria. That trial showed a reduction in parasite clearance time and inflammatory markers in patients with uncomplicated malaria under a treatment regimen consisting of atovaquone + rosiglitazone compared to patients treated with atovaquone + placebo [PPARγ with improved survival in a rodent malaria model [Cerebral malaria, one of the most severe complications of rocesses . CM is mrocesses . Understrocesses . This aprocesses -13 as werocesses ,15. One rocesses . Pharmac[in vitro. In a mud groups . These f placebo . This evia model , led us P. berghei-ANKA murine model of ECM, injection of high doses of Epo at the beginning of symptoms, significantly reduced the expression of pro-inflammatory cytokines (TNF and Interferon-γ) and improved the survival of mice with ECM compared to untreated mice [Erythropoietin (Epo), a hormone produced by the kidneys which modulates the survival of developing erythroid precursors and production of new erythrocytes in the bone marrow, has been explored for the management of CM in animals and humans . In the ted mice . Furtherted mice . Clinicated mice . These fted mice .in vitro, the damage to the endothelial barrier resulting from PE adhesion [P. berghei-ANKA infected mice [in vitro[P. berghei-infected mice with the antioxidant N-acetylcysteine (NAC) or the iron chelator, deferoxamine, in combination with chloroquine at the first signs of CM, prevented the development of persistent cognitive dysfunction in infected mice [P. falciparum malaria [P. berghei infected mice [Activated charcoal is also being explored as a potential adjunctive therapy for the treatment of CM. Oral activated charcoal (oAC) is highly effective at adsorbing a range of endotoxin-induced cytokines from the bloodstream including TNF, IL-1 and IL6 ,24. It wadhesion ,26. It iadhesion . Furtherted mice . PE-induted mice . However malaria . Althougted mice .Artemisia annua, respectively. Although artemisinin and its derivatives have had a major impact on treatment when given in combination with other anti-malarials, they are limited by their short half-life, cost and safety concerns in pregnant women [For thousands of years, some of the most effective anti-malarial drugs have been derived from plants. Quinine and artemisinin, the only two molecules of choice for the treatment of severe malaria were isolated from the bark of the cinchona tree and the Chinese plant nt women . Poor dint women .Curcuma longa L. (family Zingiberaceae) , curcumin has been considered an effective drug for the treatment of various disorders, and recent studies have substantiated and provided scientific evidence regarding its prophylactic and therapeutic potential, unravelling the anti-inflammatory, anti-carcinogenic, and anti-infectious activities of this natural product -1,6-heptadiene-3,5-dione) is a polyphenolic compound extracted from the rhizome of iewed in -35).in vitro and in vivo indicated that curcumin possesses a moderate anti-malarial activity with an IC50 ranging from 5-10µM in vitro[in vitro with an IC50 of ~400nM [2+ ion pump involved in metabolic arrest which was first thought to be the biological target of artemisinin [P. berghei-infected mice, prevented recrudescence usually associated with α-β-arteether monotherapy and ensured almost 100% survival of animals [P. chabaudi artemisinin-resistant phenotype [P berghei -ANKA, P chabaudi causes a non lethal infection and does not lead to CM in CD1 mice [Studies carried out both  in vitro. In ordef ~400nM . It was f ~400nM . Despitef ~400nM . Curcumif ~400nM . Moleculf ~400nM ,43 an ATemisinin -46. Inte animals . By conthenotype . The modCD1 mice .P. falciparum malaria [scid/bg mouse model of malaria, a pro-oxidant vitamin E-deficient diet enriched with fish oil suppressed a lethal P. yoelii infection [The severity of malaria infections is often associated with a large parasite biomass which triggers defence mechanisms that hinder parasite multiplication and are, therefore, important contributing factors in host survival . A key f malaria . Furthernfection . Althougnfection  and CD36nfection ,55.in vivo to mice, a significant increase in macrophage phagocytic activity was observed [in vitro[In addition to its specific anti-malarial activity, the immunomodulatory properties of curcumin affect various cell types of the immune system [Reviewed in ). When aobserved . Further[in vitro. From th[in vitro. Thus, t[in vitro. CD36 ex[in vitro,59. Inte[in vitro,61, thou[in vitro. To summPlasmodium-infected host [P. falciparum to endothelial cells [P. falciparum to human brain endothelial cells in vitro creates an inflammatory environment via stimulation of NF-κB which in turn upregulates expression of ICAM1 [P. berghei model as discussed by Riley at al [Cerebral malaria is the result of deleterious patholophysiological processes that take place in a ted host . In humated host . This seted host ). The seted host  or by thal cells . In factof ICAM1 ,67. Mostof ICAM1 . Howeverey at al , making P. berghei-ANKA prevented CM and delayed death by 10 days [in vitro that curcumin reduces the production of proinflammatory cytokines TNF, IL12p40 and IL6 in PBMC primed with trophozoites/schizonts stages of P. falciparum and downregulated the expression of ICAM1, VCAM1 and E-selectin in TNF-activated human endothelial cells [in vitro, is perhaps the result of Nrf2 or PPARγ activation which exert anti-inflammatory actions by blocking NF-κB activation (Figure in vitro[PPARγ might have in malarial survival [in vitro as well as in vivo in order to validate the Nrf2 activation pathway as an additional therapeutic target for CM.Curcumin exhibits profound anti-inflammatory activities due to inhibition of NF-κB activation . This ca 10 days . In addial cells . The dow in vitro which ar in vitro. Recent survival , supportP. falciparum malaria in African children, intravenous artesunate has now been declared to be the most effective treatment for severe malaria [In malaria endemic areas, both severe malaria and sepsis may often occur together . In an a malaria . This giAlthough the information discussed above suggests a plausible and beneficial use of curcumin in the management of CM, an important limitation hindering the clinical advancement of this promising molecule is its poor ADME properties. Curcumin shows low oral bioavailability, high tissue distribution, rapid metabolism and elimination ,82. The Curcumin has a long history of therapeutic use in the Ayurvedic and Chinese traditional medicine and multiple clinical trials are ongoing to evaluate its efficacy in the management of various human disorders including cancer, neurodegenerative diseases and diabetes  . CurcumThe authors declare that they have no competing interests related to this work.PNM drafted the manuscript. DT and LV participated in the writing and made corrections to the manuscript. All the authors read and approved the final version.Financial support: This work was supported by a grant from the European Commission Sixth Framework Programme FP6- IP-18834, AntiMal to DT from the University of Milan and LV from the London School of Hygiene and Tropical Medicine. PNM is recipient of a studentship from the AntiMal International PhD programme, an EMBL collaborative training programme. The European Commission had no role in study design, data collection, analysis or interpretation, the writing of the manuscript, or the decision to submit the work for publication.


This study aimed to determine the epidemiological impact of rice cultivation in inland valleys on malaria in the forest region of western Côte d’Ivoire. The importance of malaria was compared in terms of prevalence and parasite density of infections and also in terms of clinical malaria incidence between three agro-ecosystems: (i) uncultivated inland valleys, (R0), (ii) inland valleys with one annual rice cultivation in the rainy season, (R1) and (iii) developed inland valleys with two annual rice cultivation cycles, (R2).Between May 1998 and March 1999, seven villages of each agro-ecosystem  were randomly selected among villages pooled by farming system. In these 21 villages, a total of 1,900 people of all age groups were randomly selected and clinically monitored during one year. Clinical and parasitological information was obtained by active case detection of malaria episodes carried out during eight periods of five consecutive days scheduled at six weekly intervals and by cross-sectional surveys.Plasmodium falciparum was the principal parasite observed in the three agro-ecosystems. A level of holoendemicity of malaria was observed in the three agro-ecosystems with more than 75% of children less than 12 months old infected. Geometric mean parasite density in asymptomatic persons varied between 180 and 206 P. falciparum asexual forms per μL of blood and was associated with season and with age, but not with farming system. The mean annual malaria incidence rate reached 0.7 (95% IC 0.5-0.9) malaria episodes per person in R0, 0.7 (95% IC 0.6-0.9) in R1 and 0.6 (95% IC 0.5-0.7) in R2. The burden of malaria was the highest among children under two years of age, with at least four attacks by person-year. Then malaria incidence decreased by half in the two to four-year age group. From the age of five years, the incidence was lower than one attack by person-year. Malaria incidence varied with season with more cases in the rainy season than in the dry season but not with farming system.In the forest area of western Côte d’Ivoire, inland valley rice cultivation was not significantly associated with malaria burden. Anopheles gambiae s.l. populations. Furthermore, the epidemiological impact of rice cultivation varies according to the local malaria situation [Africa has the highest population growth rate of any continent . This exituation . A revieituation ,5. Conveituation -9. Most ituation .Thus, an interdisciplinary study of relationships between rice cultivation systems and malaria was conducted between 1997 and 1999 at regional level in three West African important settings: Sahel, savannah and forest. The Sahel study carried out in Mali showed that rice cultivation altered the transmission pattern from seasonal to perennial and reduced the incidence of malaria fevers in children aged less than 14 years . In savaThe present study was carried out thirteen years ago in the forest region of western Côte d’Ivoire. Its objective was to compare malaria burden in three inland valley rice production systems in terms of prevalence and parasite density of infection, and also in terms of clinical malaria incidence. Furthermore the results of the present study may be put in perspective with the situation in Côte d’Ivoire after implementation of malaria control strategies.A longitudinal study was carried out in the forest zone in the west of Côte d’Ivoire Figure . The metThe same methodology of sampling as the one used in the savannah area was applied here . The popActive case detection (ACD) for malaria episodes was done during eight periods of five consecutive days at six-weekly intervals in the year. During these periods the monitoring was made as described previously . BrieflyPlasmodium species were identified and asexual forms of each species counted on 200 leucocytes. The parasite density was calculated by assuming an average of 8,000 leucocytes/μL of blood. A randomly selected 10% sample of the thick smears was double-read for quality control. The readings of the six technicians were compared using a set of blood smears.Thick blood smears were stained with Giemsa in the field and examined using a microscope  at Institut Pierre Richet (IPR) in Bouaké by six experienced technicians under the supervision of a parasitologist. Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale asexual blood forms, (2) density of P. falciparum asexual blood forms in parasite-positive thick smears, and (3) prevalence of P. falciparum gametocytes. A generalized estimating equation (GEE) approach was used for statistical analysis of repeated measures [Demographic, clinical, parasitological and attendance data were double-entered independently in an Access database (ver.7 1995). Data were analysed using Stata V11 software. Only one blood sample per person per monitoring period was considered for the analysis. When a pathological condition was detected, it was the blood sample taken during the clinical episode that was considered for the analysis. When several blood samples from an asymptomatic period were available, one of them was randomly selected for the analysis. Parasitological data were analysed separately in terms of (1) prevalence of measures . The GEEP. falciparum gametocyte presence were analyzed as a binomial response in logistic regression model. The parasite density was log transformed and analysed with a link function for a linear regression model. These two dependent variables were analysed according to demographic , seasonal and agro-ecosystem variables.The prevalence of asymptomatic malaria infection and the prevalence of versus asymptomatic state) as dependent variable, and parasite density, age and season as independent variables. In this type of model, a random intercept variable is allowed to vary with subjects and this random subject-specific intercept allows taking into account the interdependency of observations made on the same person. For each clinical episode, the probability that it was caused by malaria was estimated by the Attributable Fraction (AF) calculated from the odds ratios associated with the estimated parasite density in each logistic model [The association between the parasite density and the occurrence of clinical episodes was tested using a random effect logistic regression model for each agro-ecosystem, taking clinical status  years in R0 and 22 (21–23) years in R1 and R2. The female/male ratio was 1.20 in R0, 1.02 in R1 and 1.03 in R2  in R0, 44.9% (95% CI: 41.7-48.2) in R1 and 47.1% (95% CI: 45.6-48.7) in R2 ) asexual forms per μL of blood and significantly decreased with age.The geometric mean parasite density in positive asymptomatic persons varied in the three agro-ecosystems between 180 and 206 em Table . ParasitP. falciparum gametocytes was higher in the rainy season than in the dry season .The mean annual gametocyte rates were similar between the 3 farming systems, 2.1% (95% CI: 1.8-2.4) in R0, 2.5% (95% CI: 2.2-2.7) in R1 and 2.2% (95% CI: 1.9-2.5) in R2, respectively. The highest rates were observed in the 0–4 old children (P < 0.0001). Prevalence of Plasmodium-positive thick blood films of asymptomatic subjects was 0.994, 0.042 and 0.007 for respectively P. falciparum, P. malariae and P. ovale. According to multivariate GEE linear regression model, the prevalence of P. malariae infections was similar in R0 (2.3%) and R1 (2.0%) (OR: 0.76 (95% CI: 0.52-1.11); P = 0,151), but slightly lower in R2 (1.7%) than in R0 (OR: 0.67 (95% CI: 0.48-0.93); P = 0.019). Children 0–4 years old were the most frequently infected (P < 0.0001). According to season prevalence of P. malariae species was higher in dry season (2.5%) than in wet season (1.7%) (OR: 0.66 (95% CI: 0.49-0.87); P = 0.006). The P. ovale asymptomatic infection rates were similar in the three agro-ecosystems (P = 0.564) and seasons (P = 0.654). Children 0–4 years old were the most frequently infected (P < 0.0001).The plasmodial formula on the 13,746 P. falciparum alone, three with P. malariae alone and 58 individuals with mixed infection . The P. malariae infections showed densities of 100 and 40 parasites/μL. Among 47 out of the 58 mixed infections P. falciparum was more frequent than the other plasmodial species. Whatever the age group sick patients had higher geometric mean parasite densities than healthy people . To quantify malaria morbidity, the method of estimation of the fever fraction attributable to malaria was used . In the P. falciparum because the transmission is intense throughout the year, with a peak during the rainy season [In the three farming systems, clinical malaria decreased strongly and quickly with age. Infants were more affected than children and from the age of five years, people experienced less than one episode a year. All the parasitological and clinical findings indicate that the naturally acquired immunity to falciparum malaria starts at a very early age and the majority of infected adults and older children rarely experience overt disease . Indeed y season . The parIn the forest area the inland valley rice cultivation did not influence the rates of malaria infection and disease although it appeared as a risk for malaria transmission. An economic study carried out on 750 households (21 villages) in the three agro-ecosystems also showed that malaria had no effect on coffee and cocoa productions and that lowland irrigated rice cultivation was not a risk factor for malaria infection .People living in the R0 agro ecosystem where no inland valley was cultivated spent probably more time in the camp for working in the coffee and cocoa plantations than in the other agro systems. This situation could explain the high rate of absence of inhabitants during the surveys of the medical team (18% in R1 and R2 and 30% in R0). Despite this potential behavioural difference and the differences in ethnic group of the populations, there was no significant difference in malaria burden between the three agro-ecosystems.et al.[Two other studies carried out within the framework of the Health Research Consortium hosted by the West African Rice Development Association (WARDA) (currently known as the Africa Rice Center), following a North–south transect in West Africa showed quite different results. In Côte d’Ivoire in the savannah zone where the same methodology as in the forest zone was used, malaria was hyper-endemic. The mean annual incidence rate reached 0.9 (95% IC: 0.8-1.0) clinical malaria episodes per person in R0, 0.6 (95% CI: 0.5-0.7) in R1 and 0.8 (95% CI: 0.7-1.0) in R2 . The devet al.,32 showeThe study carried out in Mali in the semi-arid sub-Saharan environment showed that irrigated rice cultivation altered the malaria transmission pattern from seasonal to perennial and reduced annual morbidity incidence more than two-fold, compared to cultivation without irrigation .This study, together with the other studies conducted in the framework of the Health Research Consortium in the forest, the savannah and the Sahel were performed on a large scale with a similar methodology in the context of farming systems rather than in single villages. The findings in these ecologically different zones  clearly confirm the conclusion that Carnevale and colleagues  and IjumThe study also provides an important basic data set before Côte d’Ivoire implemented the Roll Back Malaria strategies. It should permit to put the results of the current malaria control programme in Côte d’Ivoire into perspective.The authors declare that they have no competing interests.


Parasite transmission is intense in the highlands, and these areas are vulnerable to epidemics. Plasmodium falciparum prevalence, asexual and sexual parasite density, and spatial distribution to establish rates of infection at a site in western Kenya. Three cross-sectional surveys were carried out in western Kenya highlands. Blood samples were taken from 1,388 persons from 6 months to 75 years of age. Parasite prevalence and densities in the population decreased with age and distance from valley bottoms. Children from 1 to 4 years of age had the highest parasite prevalence (38.8%–62.8%); in adults, prevalence declined to 2.9%–24.1%. Malaria prevalence declined by an average of 19% from July to December 2002 across age groups. These observations suggest that parasite transmission is intense at this altitude. Asexual parasite density indicated clustering near major vector breeding habitats. Variability in seasonal prevalence indicates transmission instability and susceptibility to epidemics.We carried out a population-based study to determine the unbiased, age-specific  The reemergence of epidemic malaria in the East African highlands  is a public health problem , Kakamega district, western Kenya, with population ≈11,000. This area experiences 2 rainy seasons and averages 2,000 mm rainfall per year. The long rainy season usually occurs between April and May, with an average monthly rainfall 150–260 mm, while the short rainy season usually occurs between September and October, with an average monthly rainfall 165 mm. Malaria prevalence peaks usually lag 1–2 months after the rain. The mean annual daily temperature is 20.8°C. The area has experienced extensive deforestation and swamp reclamation in recent years as a result of rapid human population growth and the demand for settlement and agricultural land; therefore, only patches of forest remain. Malaria vectors in the area are unestus ,; most moThe working hypothesis was that no difference would be seen in the prevalence among various age groups and that infections would be randomly distributed. The binomial model was used to estimate the confidence interval (CI). Because the prevalence of malaria was not well known in the area, a 50% estimate, which gives the best sample size, was used for the peak-transmission season, and a 25% estimate was used for the low-transmission season. We calculated the 2 sample sizes with a 95% CI and precision level of 5%:n is the sample size, z is the critical value of the standard normal distribution at the 5% level (1.96), p is the malaria prevalence estimate, q = 1 – p, and d is the precision level. The population size was estimated to be 3,000. The sample size obtained was 341 persons for the peak-transmission season and 263 for the low-transmission season.In this equation, Plasmodium infections in July 2002. The sample size for each age group during each sampling period is shown in the The study, which involved 1,388 participants, was carried out in 3 cross-sectional surveys undertaken in July 2002 (n = 396), December 2002 (n = 283), and June 2003 (n = 709). Peak malaria transmission occurs 1–2 months after the rainy season; thus, surveys were conducted 1 month after the rains. The short rains before December are usually not adequate to increase transmission, and this period is considered entomologically dry. We examined the monthly malaria infection dynamics in primary school children, age 6–14, in the study area from June 2002 to June 2003, with the microscopic test. Daily rainfall data were collected by using automated weather stations . These data were offloaded monthly from the weather stations.P. falciparum prevalence and parasite density was determined across age, season, and distance to the nearest stream/valley. To examine the effects of age on parasite prevalence and infection density, ages were stratified into 6 age groups: <1, 1–4, 5–9, 10–14, 15–19, and >19 years  with logarithm-transformed parasite density was used to examine the difference among age groups and among surveys in parasite density. Nonspatial statistical analyses were conducted by using the JMP statistical package .Most of the homes of the participants in the prevalence survey were georeferenced by using a global positioning system (GPS) unit. A database was created with participants' names, ages, GPS location, and parasite density. Participants' names were coded for confidentiality. The relationship between P. falciparum infections were tested for clustering by household with the global spatial statistic, the K function, weighted by parasite density (–L(d), examines the spatial dependence of malaria infection by household over a wide range of scales of pattern (L(d) function values were tested against the null hypothesis that the spatial distribution of all infected residents in the study area was random, by using 1,000 Monte Carlo iterations. Because children and adults showed significantly different prevalence and parasite density, the global spatial cluster analysis was conducted separately for the 0- to 9-year age group, the 10- to 19-year age group, and adults (>19 years old). The local spatial statistic Gi*(d) (Gi*(d), significance levels were determined by using Table 3 in reference  was used to create spatial-distribution maps of infected and uninfected participants for the 3 surveys. P. falciparum, we found P. malariae and P. ovale in our study populations. P. falciparum constituted 97.1%, 94.9%, and 94.6% of malaria cases in July and December 2002, and June 2003 surveys, respectively. P. malariae constituted 2.9%, 5.1%, and 4.6% of malaria cases, while P. ovale was observed only in June 2003 (0.8%). P. malariae was often seen in mixed infection with P. falciparum. For example, 83.3% of P. malariae infections in July 2002 occurred with P. falciparum, while 50% of P. malariae infections in December 2002, and 81.8% of P. malariae infections in June 2003, were accompanied by P. falciparum.In addition to P. falciparum prevalence varied significantly among age groups  and sampling months  or age groups , malaria prevalence showed a general decreasing trend . HoweverL(d), was used to examine the spatial dependence of malaria infection by household over an interpoint distance of 100–1,400 m for each of the 3 age groups . L(d) and the 95% CI plotted for various values of interpoint distance for the July 2002 survey. The spatial distribution of infections is considered evenly dispersed if the observed K function values are below the lower limit of the 95% CI, clustered if above the upper limit, or random if within the 95% CI. The weighted K function indicated that the parasite density distribution pattern was significantly different than expected under complete spatial randomness for age groups 0.6–9  adjacent to the highlands reaches >80%  had a prevalence of 86% in the lowlands, while in the highlands prevalence in this group was 2.9%. Children 5–9 years of age had the highest rate of infection with gametocytes (4.9%) at Iguhu, which is comparatively lower than that reported in the lowland (40%) for the same age group (,Spatial analysis of asexual parasite densities indicated clustering in relation to distance from major larval breeding habitats and in relation to age. This phenomenon was not observed in persons living >1,000 m from major breeding sites, which suggests that the rate of parasite transmission may be higher closer to major breeding habitats. This finding is consistent with the spatial distribution of indoor resting vector densities in the area. Our results are consistent with the fact that the risk of malaria is strongly associated with distance from breeding sites (This cross-sectional study in a highland site where malaria epidemics have been reported in the past shows that transmission is intense, particularly after the rainy season, but variable with regard to season and distance from major mosquito breeding habitats. The risk of infection is highly variable within the site, and subsequently, the stability of transmission may reflect this variability. A large host population is available for infection before periods of hypertransmission. Although the population has a functional age-dependent immunity to malaria, as indicated by parasite densities, its development is slower than that found in the holoendemic lowlands, which suggests that this population may be more susceptible to malaria infections and more prone to epidemics compared to the lowland populations.


Levels of complement regulatory proteins (CrP) on the surface of red blood cells (RBC) decrease during severe malarial anaemia and as part of cell ageing process. It remains unclear whether CrP changes seen during malaria contribute to the development of anaemia, or result from an altered RBC age distribution due to suppressive effects of malaria on erythropoiesis.Plasmodium falciparum parasitaemia and illness. Levels of CD55 and CD59 were measured by flow cytometer and compared between anaemic (8.08 g/dl) and non- anaemic children (11.42 g/dl).A cross sectional study was conducted in the north-east coast of Tanzania to investigate whether the changes in glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins (CD55 and CD59) contributes to malaria anaemia. Blood samples were collected from a cohort of children under intensive surveillance for Levels of CD55 and CD59 decreased with increased RBC age. CD55 levels were lower in anaemic children and the difference was seen in RBC of all ages. Levels of CD59 were lower in anaemic children, but these differences were not significant. CD55, but not CD59, levels correlated positively with the level of haemoglobin in anaemic children.The extent of CD55 loss from RBC of all ages early in the course of malarial anaemia and the correlation of CD55 with haemoglobin levels support the hypothesis that CD55 may play a causal role in this disorder. Plasmodium falciparum. The pathogenesis of malarial anaemia is complex, multifactorial and incompletely understood. Direct destruction of infected red blood cells (RBC) during malaria seems to be a relatively minor contributing mechanism because parasite densities do not correspond to the severity of anaemia ; old  and very old  but not to the non-anaemic malaria cases i.e. median (IQR = 25-75%) for young ; mature ; old  and very old . CD59 levels were low during malaria compared to normal controls but the differences were not significant for all RBC subsets.Overall levels of CD55 but not CD59 were higher in the healthy controls than in other groups but this difference was only significant for the anaemic malaria cases. CD55 MFI levels were significantly higher in the healthy control group in all RBC subsets when compared to anaemic malaria cases i.e. median (IQR = 25-75%) for young [2.52 (0.36) versus 2.15(0.8), Levels of CD55 and CD59 decreased progressively as RBC aged, in all children. CD55 levels were lower in anaemic compared to non-anaemic children for all RBC age subsets, and these differences were significant for all except the very old RBC subset Figure . CD59 dir = 0.506, P = 0.001; mature RBC, r = 0.421, P = 0.0063; old RBC, r = 0.526, P = 0.0008; very old RBC, r = 0.375, P = 0.0246) but not in non-anaemic children with malaria. CD59 level did not correlate with haemoglobin in either group of children.CD55 levels correlated significantly with the level of haemoglobin in anaemic  and non-anaemic populations . Multivariate linear regression analyses for parasite density, CD55 levels and child's age as independent variables, and haemoglobin level as the dependent variable indicated that decreases in CD55 was associated with the odds (OR 95% CI) for low haemoglobin levels of 0.19 (0.05-0.83).CrP levels did not vary with child's age. CD55 but not CD59 levels were negatively associated with parasite density. The relationship of CD55 to parasite density was significant for both anaemic  levels, but specifically do not account for the decrease in CrP during early malarial anaemia. The correlation between CD55 and haemoglobin levels in anaemic children suggests that CD55 loss may at least partially mediate the disease.The results confirm previous findings that CD55 levels on the surface of RBC are lower in children with malarial anaemia , and in This study focused on acute episodes of mild to moderate anaemia. Children in the cohort were monitored intensively with routine blood smears, clinical examinations and prompt treatment in case of illness, and therefore chronic malaria cases were unlikely. Significant increase in number of young RBC in anaemic children suggests body's attempt to correct the reduction in cell mass resulting from malaria infection, and also suggests that these cases were acute and not chronic episodes . EarlierRed blood cells were separated into fractions of different ages using Percoll density gradients method. Density gradient separation is a well validated technique which yields distinct cell populations with progressive shift in the cell age with density, and differ by physical and biochemical properties -17. DecrApparently, this is the first study to show variations in CD55 and CD59 levels in relation to RBC age during malaria. Earlier study reported a progressive decrease in CD55 and CD59 levels with RBC age in healthy donors ,19. ReduAlternatively the same mechanism responsible for physiological loss may be taking place, but at an accelerated rate for CD55. A process resembling transfer reaction of CR1 has been proposed to explain the loss of CD55 on RBC during malaria . During P. falciparum malaria [The correlation between CD55 and haemoglobin as observed in this study suggests that CD55 depletion contribute to RBC loss during malarial anaemia. Erythrophagocytosis resulting from C3b deposition on RBC could explain the concomitant decrease of CD55 and haemoglobin. Severe malarial anaemia is associated with elevated levels of circulating immune complexes ,23, whic malaria .et al [Alternatively, direct lysis of RBC by membrane attack complex (MAC) could explain the concomitant decrease of CD55 and haemoglobin, but seems less likely. Children with severe malarial anaemia have been reported to have low levels of CR1 and CD55 . Such deet al  found thThe differential loss of CD55 and CD59 may be related to their different roles in protection from complement attack. Activation of complement occurs in a step-wise fashion, and each regulatory protein acts at a different step in the cascade. CD55 acts at the initial enzymatic step to prevent the activation of C3 to C3b by accelerating the dissociation of the C3 convertase C4-2a and C3bBb -30. CD59Demonstration of higher amounts of C3 activation and degradation fragments bound to the RBCs of the anaemic children and an inverse correlation with CD55 would be another approach to support the hypothesis that CD55 loss supports the causal role in malarial anaemia. Moreover, demonstration of C3b on the CD55 low cells, would argue that sublytic C5b-9 induced the ectocytosis of infected and bystander cells, which resulted in the preferential loss of CD55 over CD59. This phenomenon was not explored in the current study, but earlier studies by other researchers did demonstrate an increase in C3b deposition on low red cell CR1 and CD55 levels in children with severe malarial anaemia ,25.Reductions in CD55 during malarial anaemia are evident in RBC of all ages, correlate with haemoglobin level, and develop early in the course of disease. Although the RBC age distribution changes during malarial anaemia, and CD55 levels decrease as RBC age, these changes do not account for the CD55 loss seen in children with new onset disease. Taken together, the results support the hypothesis that CD55 loss may play a causal role in malarial anaemia. It is suggested that the loss of CD55 during malaria compromises the complement regulatory function thereby allows the deposition of opsonin C3b on RBC leading to increased erythrophagocytosis and anaemia.The authors declare that they have no competing interests.MF and PED designed and managed the Mother Offspring Malaria Studies Project. MG designed and performed the study, analysed the results, and together with PED prepared the manuscript. GD supervised the laboratory work. All authors reviewed and approved the manuscript.


Plasmodium vivax was associated with a substantial share of the burden of morbidity and mortality caused by malaria in this hypo- to meso-endemic community.Records of 3,449 patients admitted to Karitas Hospital at Waitabula in eastern Indonesia with microscopy-confirmed malaria through 2008 and 2009 were systematically reviewed. Falciparum, vivax, and mixed species malaria occurred among 1,541, 1,837, and 71 admissions, respectively. Among these, 400 (26%), 199 (11%), and 15 (21%) had serious illness. Fatalities occurred in 46 (12%), 18 (9%), and 2 (13%) of these patients, respectively. Although patients with a diagnosis of falciparum malaria were more likely to have serious illness compared with those with vivax malaria , this diagnosis nonetheless was associated with 32% of serious illness and 27% of fatalities. Among the seriously ill with a diagnosis of falciparum or vivax malaria, no significant difference in risk of death occurred . Serious and fatal illness was predominantly anemia or altered mental state syndromes among patients with either of the species diagnoses.  Plasmodium vivax malaria has long been considered only rarely fatal.P. vivax may often become pernicious and directly threaten life.P. vivax, perhaps few would credit even a minor share of mortality attributed to malaria to this parasite. Nonetheless, recent retrospective and prospective hospital-based studiesPlasmodium falciparum or P. vivax, with 10% and 7% of those ending in death, respectively. Such evaluations, comparing relative burdens associated with the two species, may mitigate some of the many confounding factors  inherent to endemic zones like Indonesia.Recent work from India emphasizes the uncertainty in estimates of mortality caused by malaria. One study used verbal autopsy methods and the findings suggested underestimation of the burden of malaria mortality in that nation by at least 9- and as much as 19-foldP. falciparum alone was over 12 million, and the number of Indonesians at risk of P. falciparum in 2007 was 151 million,P. vivax in 2009 numbered 175 million.In 2006 Indonesia reported 347,597 confirmed and suspected malaria cases to the World Health Organization (WHO), most of those having a laboratory-confirmed diagnosis.Most endemic malaria in Indonesia is relatively low transmission intensityKaritas Hospital is located at Waitabula near the northern coast airport of Tambolaka in West Sumba, Indonesia. Karitas serves as the primary hospital in that community, and referral site for health centers throughout much of West Sumba. The catchment population was ∼280,000 people. Karitas had 115 beds for inpatient care, surgical capacities, and a clinical laboratory service. People coming to the hospital seeking treatment of febrile illness first had blood smears examined for malaria by microscopy. Technicians trained and certified as competent both by the Eijkman Institute for Molecular Biology and U.S. Naval Medical Research Unit 2P. falciparum of 4.88% and 2.88%, respectively, and 2.16% and 2.14% for P. vivax.P. falciparum and P. vivax.A systematic sampling of villages in West Sumba in 2007 for cross-sectional blood surveys of 8,870 residents of 45 villages showed wet and dry season prevalence for The ethical review boards of the Eijkman Institute for Molecular Biology and of the London School of Hygiene and Tropical Medicine reviewed the protocol describing this study and approved its conduct. This retrospective, cross-sectional study retrieved the records of patients who sought treatment at Karitas Hospital and who had a laboratory-confirmed malaria diagnosis.Outpatient and inpatient records for all of 2008 and 2009 were examined. Relevant data from the hospital patient file, with exclusion of personal identifiers, was directly transferred into two duplicate case report forms (CRF) for each patient by two different nurse technicians. Each CRF was then manually entered into an electronic database (EpiData) twice. The four electronic versions per patient were examined for discrepancies that were resolved by manual cross-check of the CRF copies and, if necessary, the hospital patient record. Concordant electronic CRFs were uploaded to a permanent storage device in Stata version 11  for analysis.Infants were < 12 months of age, children were < 10 years of age, adolescents were between 10 and 15 years of age, adults were > 15 years of age, and people > 60 years of age were classified as elderly. Women were classified as pregnant with affirmative evidence in the record; absent of such evidence they were classified as not pregnant. The malaria microscopists did not count and record parasite densities from blood films. Instead they used a scoring system based on the number of parasites seen among microscopic fields of the thick film under oil immersion magnification  as follows: 1+ = 1–10 parasites/100 fields, 2+ = 11–100 parasites/100 fields, 3+ = 1–10 parasites/field, and 4+ ≥ 10 parasites/field. The 4+ score may be estimated at > 6,000/μL by assuming that each high power field represents ∼1/600th of a microliter.Classification of disease severity did not comply with standard definitions such as those provided by WHO because most measurements required for that definition were not available at Karitas Hospital. Strict application of it would have caused exclusion of most records as incomplete. Therefore, before examining the records, a system for classification of illness associated with a diagnosis of malaria was developed using what was understood to be broadly available information at Karitas.1.Not admitted to intensive care unit; and2.No record of seizure or altered consciousness; and3.No record of severe anemia (< 5 or < 7 g/dL for children or adults) or transfusion; and4.No record indicating pulmonary distress; and5.No record indicating renal failure; and6.No record indicating hepatic failure.Patients meeting the following criteria were classified as having NOT SERIOUS illness:1.Treated in any ward; and2.Intravenous (IV) antimalarial therapy; or3.Record of seizure or altered consciousness; or4.Record of severe anemia (< 5 and < 7 g/dL for children or adults) or transfusion; or5.Record indicating pulmonary distress; or6.Record indicating renal failure; or7.Record indicating hepatic failure.Patients were classified as having SERIOUS illness in accordance with the following:1.Severe anemia: Patient record contained notes documenting blood transfusion or a laboratory measurement of hemoglobin < 5 g/dL for children and < 7 g/dL for adults.2.Altered mental state: Patient record contained notes documenting patient was disoriented, stuporous, delirious, unconscious, comatose, suffering at least two seizures, and receiving intravenous or intramuscular antimalarial therapy.3.Respiratory distress: Patient record contained notes describing labored or rapid breathing (> 50 breaths/minute).4.Hepatic illness: Patient record contained notes describing physical signs of jaundice or containing record of laboratory values aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) elevated 2.5-fold above normal values .5.Bleeding: Patient record contained notes describing patient had spontaneous bleeding from any site.6.Prostration: Patient record contained notes describing patient was unable to sit up or stand without assistance.7.Double syndrome: Any two of the above.8.Multiple syndromes: More than two of the above.Patients classified as having SERIOUS malaria were further classified according to the apparent organ of primary illness. All of these classifications were made on the basis of notes or data in the patient record. The following guided these syndromic classifications:Patients were classified as FATAL only with evidence that death occurred in the hospital and the attending physician recorded malaria as the cause of death.P values comparing rates and proportions were calculated using χ2 contingency tables and Fisher's exact test. The CI that included 1 and/or the P values that were > 0.05 were considered insignificant.The statistical analysis used Stata version 11. Descriptive analyses were performed to determine the proportions for categorical data. Odds ratios (OR), 95% confidence interval (CI), and P. falciparum, P. vivax, or mixed infections of these two species among demographic groups. Among the 3,449 patients admitted with a diagnosis of malaria, 614 (18%) were classified as having serious disease, and 66 (11%) of those patients died. Falciparum malaria occurred among 45% of admissions, 65% of serious disease, and 70% of deaths, whereas vivax malaria occurred in 53%, 32%, and 27% of these, respectively. No significant differences appeared between rates of serious illness with a diagnosis of P. falciparum versus mixed species infection , and the same was true of case fatality rates . Mixed infections were excluded from further analyses comparing trends in morbidity and mortality between species.P. falciparum and 110 P. vivax), 85% and 72% received no antibiotics, respectively. Antibiotic therapy was with amoxicillin, ampicillin, or ciprofloxacin.Presumptive antibiotic therapy was not routinely administered with a diagnosis of malaria. Among the 220 patient files randomly selected for the survey of antibiotic therapy  more likely to be classified as having serious illness compared with admission with a diagnosis of vivax malaria. This trend was strongest among infants, children, and adolescents (OR 3.0 to 8.3), and weakest among adults and the elderly (OR 1.3 and 0.4). Pregnant women were no more likely to have serious illness with a diagnosis of falciparum compared with vivax malaria , and this was also true among women classified as not pregnant . Compared with not pregnant women, pregnant women with a diagnosis of falciparum malaria or of vivax malaria were similarly likely to be classified as having serious disease .P. falciparum and 54% of that for P. vivax. Overall, these young age groups accounted for 73% of serious illness and 77% of fatalities, and represented only 55% of the general population. They were 4.5 times (95% CI: 3.3–6.1) more likely to have serious outcomes with a diagnosis of P. falciparum compared with adults and the elderly with the same diagnosis. The same was not true for a diagnosis of P. vivax, where risk of serious illness appeared more evenly distributed between infants/children/adolescents versus adults/elderly . Infants, children, and adolescents contributed 82% of serious illness with a diagnosis of P = 0.02). Most patients with serious illness and a diagnosis of falciparum (90%) or vivax (96%) malaria presented with only a single syndrome, and only one patient presented with more than two syndromes  accorded with similar frequencies in other hospital-based studies .10Among fatalities, however, death obviously occurred despite such therapy and the likelihood of other endemic infections playing a role in that outcome cannot be ruled out. Studies elsewhere,P. vivax for therapy of tertiary syphilis.34Clinical studies support a relationship between serious illness and risk of death with vivax malaria. Rates of reported death under vivax malaria therapy for neurosyphilis in patients during the 1920s and 1930s show consistency with the data from hospitals in endemic zones today. The therapeutic efficacy of induced malaria for tertiary syphilis, an otherwise fatal disease before antibiotics, was only about 35% and hinged upon the severity and number of malaria paroxysms.P. vivax as the sole agent of serious and fatal illness in any patient. Indeed, this may be unlikely with either species of parasite, and the observed relative weights of association with serious and fatal illness may encourage and guide more detailed prospective analyses of this question.As with death caused by many infectious agents, such extremes of virulence by the parasites that cause malaria may hinge upon complex host genetic, nutritional, immunological, and many other factors, as well as co-infections or other underlying disease states. The study at Karitas Hospital does not confirm A diagnosis of falciparum malaria carried a higher risk of serious illness among hospitalized patients , but vivax malaria was nonetheless associated with 32% of that burden. Risk of death among patients with falciparum malaria and classified as having serious illness was indistinguishable from patients with a diagnosis of vivax malaria . These findings accorded with a similar retrospective study from Jayapura, Papua; OR = 2.8; 95% CI: 2.0–3.0 for severe disease with falciparum versus vivax malaria, and OR = 0.9; 95% CI: 0.4–2.1 for risk of death with those diagnoses.The spectrum of syndromes among seriously ill patients with vivax or falciparum malaria resembled those observed in the prospective hospital-based study from Timika, Papua in eastern Indonesia, and in the Sepik region of Papua New GuineaP. vivax, along with myriad possible co-determinants, contributes substantially to the burdens of hospitalizations, serious illness, and death associated with the malaria endemic to this community. Although risk of serious illness with a diagnosis of vivax malaria was significantly less than with that of falciparum malaria, risk of death among patients with serious disease was approximately equal between those diagnoses. Infants, children, and adolescents bore most of the burdens of serious and fatal illness associated with falciparum malaria, whereas all age groups appeared vulnerable to serious and fatal illness associated with vivax malaria. Despite the perception of vivax malaria as a benign infection, this parasite may be contributing to or causing serious and fatal illness in eastern Indonesia.In summary, this retrospective study of the 24-month malaria experience of a single hospital in eastern Indonesia suggests


Plasmodium falciparum malaria  and Epstein Barr Virus (EBV) infections coexist in children at risk for endemic Burkitt's lymphoma (eBL); yet studies have only glimpsed the cumulative effect of Pf-malaria on EBV-specific immunity. Using pooled EBV lytic and latent CD8+ T-cell epitope-peptides, IFN-γ ELISPOT responses were surveyed three times among children (10 months to 15 years) in Kenya from 2002–2004. Prevalence ratios (PR) and 95% confidence intervals (CI) were estimated in association with Pf-malaria exposure, defined at the district-level  and the individual-level. We observed a 46% decrease in positive EBV lytic antigen IFN-γ responses among 5–9 year olds residing in Kisumu compared to Nandi . Individual-level analysis in Kisumu revealed further impairment of EBV lytic antigen responses among 5–9 year olds consistently infected with Pf-malaria compared to those never infected. There were no observed district- or individual-level differences between Pf-malaria exposure and EBV latent antigen IFN-γ response. The gradual decrease of EBV lytic antigen but not latent antigen IFN-γ responses after primary infection suggests a specific loss in immunological control over the lytic cycle in children residing in malaria holoendemic areas, further refining our understanding of eBL etiology. Plasmodium falciparum (Pf) malaria and Epstein Barr Virus (EBV) have been identified as co-factors in the pathogenesis of endemic Burkitt's lymphoma (eBL) Pf-malaria infections promote eBL in two mutually-compatible ways. In developing countries, most children experience primary EBV infection by 3 years of age, followed by life-long infection in memory B-lymphocytes P. falciparum induces polyclonal B-cell expansion and lytic EBV reactivation Pf-malaria infections could hence lead to exhaustion or hypo-responsiveness of EBV latent or lytic antigen CD8+ T-cells, thus increasing the chance for this EBV-associated malignancy to arise.It has been hypothesized that Pf-malaria. Using an in vitro regression assay as a measure of cytotoxicity, children with acute Pf-malaria demonstrated a transient loss of control over B-cell outgrowth Pf-malaria infected individuals with healthy adults came to the same conclusion Pf-malaria infections on EBV persistence has not been thoroughly studied Limited evidence supports an impaired EBV-specific T-cell response in association with Pf-malaria on EBV latent and lytic antigen CD8+ T-cell IFN-γ ELISPOT responses in children over a two-year period.The objective of this study was to examine the influence of cumulative P = .11 and P = .30, respectively). Children in Kisumu experienced more Pf-malaria infections than children in Nandi (P<.001); only 3% of Kisumu children were never infected compared to 78% in Nandi. This was despite a classically defined malaria outbreak in Nandi during the survey periods  were seropositive for EBV  periods .Pf-malaria exposure does not appear to influence the magnitude of EBV-specific IFN-γ responses.The proportion of positive IFN-γ responses to PHA (positive control) demonstrates that children from both districts were equally able to elicit an IFN-γ response indicating no global signs of immune dysfunction . There wWe observed a few intriguing patterns in the prevalence of positive EBV lytic antigen CD8+ T-cell IFN-γ response when children were stratified into age groups by their baseline age (age group cohorts) . In KisuPf-malaria and the weighted model, we estimated the prevalence of positive responses in Kisumu was 0.70 (95% CI: 0.45–1.08) times the prevalence in Nandi although this 30% difference was not significant. In Kisumu, there were no significant differences in positive responses in children 0–4 years  and 5–9 years  when compared to children ≥10 years  times the prevalence in Nandi, although not significant. In Kisumu, the prevalence of positive responses in children 0–4 years  and 5–9 years  was not significantly different from children ≥10 years, although there was a decrease in prevalence with increasing age group . SimilarPf-malaria in our weighted model, we found the association between recurrent Pf-malaria infections and EBV lytic antigen CD8+ T-cell IFN-γ response varied by age group and survey period. We therefore used two weighted models. In the first model, we stratified results by age group, while adjusting for sex and survey period. Similarly in the second model, we stratified by survey period while adjusting for sex and age group.Using the individual-level definition of Pf-malaria infections and positive IFN-γ responses among Kisumu children were consistently lower than Nandi children for all age groups and survey periods (P = .32). Secondly, the association between recurrent Pf-malaria infections and IFN-γ responses varied by age group. In both Kisumu and Nandi, the prevalence of positive responses among children 0–4 years with recurrent Pf-malaria infections was higher than that of similarly aged children never infected. In Nandi, the difference was statistically significant. Finally, the PR of recurrent Pf-malaria infections and IFN-γ responses to EBV lytic antigens varied by survey period in both districts. At baseline, for both districts, the PR of positive responses among children with recurrent Pf-malaria infections was greater compared to children never infected; this result was statistically significant in Nandi, but not Kisumu. However, the PR decreased at subsequent study periods; the prevalence of positive responses among children with recurrent Pf-malaria infection diminished over time compared to children never infected. This could reflect functional diminishment of responsive EBV lytic antigen T-cells under continuous pressure from Pf-malaria.We noted three observations from our analysis. First, the PR of recurrent  periods . In genePf-malaria infections compared to those never infected  district-level according to malaria transmission intensity, and 2) individual-level based on measured Pf-malaria infection. Although our findings of EBV lytic antigen CD8+ T-cell IFN-γ responses were consistent with both definitions, our findings of EBV latent antigen CD8+ T-cell IFN-γ responses were inconsistent. This may have been due to the limited power or an underestimation of the influence of Pf-malaria infections in hypoendemic areas. However, it also highlights the potential pitfall in attributing district-level results to the individual, also known as the ecological fallacy. The inconsistency may have been due to other factors that differed between the districts and unrelated to malaria transmission intensities. Therefore, we conclude that the use of malaria transmission intensity as a surrogate for malaria infection has been informative yet future studies should endeavor to prospectively collect Pf-malaria and EBV co-infections information from individuals to more accurately describe this complex relationship.A strength of our study was the use of two definitions for Pf-malaria and EBV response relationship indicated adjusting for schistosomiasis infection would have biased our analysis. Finally, is socioeconomic status had been measures, participants and their families would likely have been classified as low socioeconomic status because the main occupation was fishing (Kisumu) and farming (Nandi) in both rural study areas with homes constructed of locally available materials.There were several potential confounders that were not captured in our study, specifically HIV status, nutritional status, schistosomiasis infection, and socioeconomic status. However, we do not believe the absence of these confounders materially affected our findings. When data were collected in western Kenya from 2002–2004, HIV testing in infants was conducted only when medically warranted. All children enrolled in this study were examined by a clinician and had no obvious signs of illness or malnourishment, and no deaths were reported as of 2009. Therefore, even though it was possible that an underlying HIV infection might influence the rate of malaria parasitaemia Pf-malaria infections on EBV-specific T-cell response. We observed fewer positive EBV lytic antigen CD8+ T-cell IFN-γ responses among 5–9 year old than older children Pf-malaria infection.Our findings on EBV lytic antigen CD8+ T-cell IFN-γ responses were consistent with the studies that have used residence area  to explore the cumulative effect of Pf-malaria.However, we did not detect the same statistically significant district-level difference in positive EBV latent antigen CD8+ T-cell IFN-γ responses among 5–9 year olds as a previous study Pf-malaria infections and EBV-specific T-cell IFN-γ responses and identifies a potential difference between children recurrently infected with Pf-malaria compared to children never infected. To adequately quantify this effect, a longitudinal study should be considered which could accurately measure Pf-malaria infection and changes in Pf-malaria and EBV-specific T-cell immunity over time. The temporal aspects of future studies will be vital to elucidating the precise mechanism by which repeated Pf-malaria infections affect EBV persistence and immunity.This study design marks a step toward examining the individual-level association of Pf-malaria transmission intensities: Kisumu is characterized as holoendemic and Nandi as hypoendemic. Due to the age-related incidence of eBL, an equal distribution of children by age and sex were enrolled from each area: children 0–4 years have an elevated risk of eBL whereas 5–9 year olds are at highest risk and ≥10 years old have the lowest risk. Data were collected from 2002–2004 using a standardized survey. Three face-to-face interviews were conducted at baseline (July–August 2002), six month follow-up (February–March 2003), and two-year follow-up (July–August 2004). Blood was also collected for malaria and EBV testing.The Kisumu/Nandi cohort has been previously described Pf-malaria infection was confirmed on thick and thin blood smears by microscopy. Testing of EBV-specific T-cell response by IFN-γ ELISPOT has been previously described P<.05), EBV lytic and latent epitope-peptide CD8+ T-cell IFN-γ responses were categorized as positive or negative. A positive response was recorded if the proportion of SFUs in the stimulated well was significantly different from the proportion of SFU in the unstimulated well. The magnitude of response was calculated by subtracting the SFU in PBS wells (negative control) from the SFU in the stimulated wells. The median value for the negative control wells was 4 SFU per million PBMCs (range 0 to 772 SFU/million PBMC). Median values were calculated among positive responders only.Pf-malaria. First, Pf-malaria exposure was defined according to the malaria transmission intensity of the district (district-level definition): Kisumu (holoendemic) or Nandi (hypoendemic). Next, Pf-malaria infection was defined as the cumulative average of P. falciparum infection (parasitemia) in a participant over the three survey periods . The value ranged from 0 (never infected) to 1 ; results and discussion focus on children who were always infected (referred to as recurrent) and never infected. With the individual-level definition, we also included the covariates age group, district, sex, and when the survey was conducted (referred to as survey period) in the analysis. We first examined covariates as potential effect measure modifiers using an a priori cutoff of P = .20. In the absence of evidence of effect measure modification, we included covariates in the model as potential confounders.Analyses were restricted to EBV seropositive children at baseline U)/Kruskal Wallis test for continuous outcomes. For multivariable analyses, we used weighted log-binomial regression with robust variances to estimate the prevalence ratios (PR) and corresponding 95% confidence intervals (CI). We used generalized estimating equations (GEE) with robust variance estimators to account for correlation due to repeated measurements. A weighted model with inverse probability weights (IPWs) was used to address missing observations due to children not participating in all surveys. When using IPW, observations are assumed to be missing at random; missing data are dependent on observed data but independent of unobserved data For descriptive analyses, we used the Chi-square statistic to measure associations between categorical exposures and outcomes. We used the two-sided Wilcoxon rank sum  participating in all surveys. Taking the inverse of the predicted probabilities, the mean IPW was 1.44 and ranged from 1.14–1.92. Children who participated in all three surveys were assigned the mean IPW value whereas children with missing observations were assigned an IPW of 0. We also conducted complete case analyses and found no differences in the PR or 95% CI; therefore we report results from the weighted analyses. Data were analyzed in SAS 9.1.3 .To calculate the predicted probabilities for our study, we used logistic regression with first order interaction terms using the following equation:Written informed consent was obtained from a parent or guardian of the participant. This study was approved by the Institutional Review Boards at the University Hospitals of Cleveland, Case Western Reserve University where Dr. Moormann was affiliated at the time this study was done and also obtained from the Ethical Review Committee for the Kenya Medical Research Institute. It was deemed exempt by the Institutional Review Board at the University of North Carolina at Chapel Hill.


A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.Plasmodium infection by using microscopic, serological and nested-PCR techniques.Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate 19-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-119, using ELISA.Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region. Plasmodium vivax, P. falciparum mono-infection and a mixed-infection of both species, respectively .Malaria is the most important vector-born and parasitic disease and half of the world's population is under the risk of acquiring malaria . This inThe Iranian CDMC with the technical support from WHO started the malaria elimination program in Iran since 2009. It should be noted that to achieve successful malaria elimination in any given endemic region, one of the main requirements is active case detection  and, P. falciparum (1.5%) is less prevalent . The most important anopheles vectors in Bashagard are Anopheles stephensi, An. culicifacies and An. fluviatilis in Bashagard District. Pregnant women and children below four years old were excluded from the study. After signing an informed consent form, finger-prick blood, thick and thin blood smears, were prepared from all cases for microscopy. But, for nested-PCR, and serological analysis, blood samples were collected in tubes containing EDTA. Plasma was obtained after the centrifugation of the collected blood samples and stored at -20 °C until use. If the positive result would be detected by microscopic examination, in this case the patient will be received standard anti-malarial drugs in accordance with CDMC of Iran. This study was approved by the Ethical Committee of Pasteur Institute of Iran.This study was conducted in 500 volunteers , aged from 4 to 60 years old, living in Bashagard District. All randomly selected participants were found actively without having any malaria symptoms including chill, fever and sweating in order to evaluate asymptomatic × 1000 with oil immersion for detecting malaria parasites by a trained microscopist at the Hormozgan Provincial Health Center laboratory.Thick and thin blood smears were prepared according to a standard method. Briefly, after cleaning the finger with 70% ethanol it was punctured. Thick and thin blood smears were air-dried, and then only the thin film was fixed using methanol. All slides were stained with 10% Giemsa and examined at 19) of P. vivax and P. falciparum as recombinant antigens (19 (32 ng) or rPfMSP-119 (200 ng) antigens per well overnight. Then the plate was blocked with the blocking buffer (1% BSA in PBS 1X) and washed three times with the washing buffer (1% PBS- 0.05% Tween-20). Afterward, 100 µL of sample sera was added to the wells  and incubated at room temperature for 1.5 h. Then the plate was washed and 100 µl of horse radish peroxidase-labeled anti-human IgG  was added to each well. After 1 h of incubation the plate was washed and 100 µL orthophenylene diamine hydrochloride  substrate was added to each well. The reaction was stopped by adding 50 µl of 2 N H2SO4 and the absorbance (OD) was measured at 492 nm. It should be mentioned that all samples were analyzed in duplicate.IgG antibodies in serum samples were detected by ELISA using the 19 kDa C- terminal region of merozoite surface protein 1  as secondary antibodies at dilutions of 1:2000 and incubated at room temperature for 1 h. After washing, the procedure was followed by adding streptavidin–peroxidase conjugate  at a dilution of 1:3000 and incubated at room temperature for 1 h. The enzyme reaction was developed with OPD-H2O2  and stopped with 2 N H2SO4. The samples with an optical density greater than the cut-off point were considered positive. The cut-off was calculated as the mean OD of the 30 samples collected from healthy individuals who are living in non malaria endemic region plus three standard deviations.All IgG positive samples were also analyzed to determine the IgG1 and IgG3 subclasses. To measure the anti-PvMSP-1P. vivax and P. falciparum as described previously . DNA samples were used to amplify the small sub-unit ribosomal ribonucleic acid (18ssRNA) gene with species specific primers for eviously  with somrd to 10th August 2009). All study samples were categorized by sex and age group  had anti PvMSP-119-specific IgG antibody to PvMSP-119. Among them, 0.2 was under and 0.8% was ≥ 15 years old. Serological results using rPfMSP-119 as an antigen showed anti PfMSP-119-specific IgG antibody in only one of the samples (0.2%) who was a 35 years old woman  in this survey.As the malaria elimination program started in 2009 in Iran, active case detection has become a crucial element during achievement. In this situation one of the main challenges is the detection of asymptomatic infection which is not detectable by routine laboratory tests due to the lack of malaria symptoms and having low levels of parasitaemia. So these patients become gametocyte carriers and have a key role in continuing disease transmission that might combat the malaria elimination program , 4, 11. 19 have been shown to correlate with recent infection (Microscopic technique was used as a gold standard and nested-PCR as a sensitive and specific method for asymptomatic malaria diagnosis which can detect low parasite densities . Despite19 and -PfMSP-119 IgG antibodies, respectively. The present serologic data is parallel with the previous study (The results of this study did not indicate any asymptomatic infection neither by microscopic nor nested-PCR examinations in the studied population. However, in the case of serological examinations, five and one cases were positive and had anti-PvMSP-1The emerging of asymptomatic malaria infection could be a result of the repeated exposure of individuals to the parasite population which leads to the development of effective humoral immune response. Thus despite the presence of parasite, malaria symptoms might not be observed . InteresAlthough it is expected that asymptomatic infection would mainly be detectable in high endemic areas, there has been other contrary reports from hypoendemic areas of the world that indicated high prevalence of asymptomatic malaria such as Peru and Brazil , 4, 12. Taken together, the results of the present survey showed that there was no asymptomatic malaria infection in the studied population of Bashagard District. Therefore, it could be postulated that, as no asymptomatic malaria cases were found, the malaria elimination program is feasible and can be successfully carried out in this area. As reported previously from Iranshahr District of Sistan and Baluchistan Province, asymptomatic infection was detected by microscopy among both Iranian individuals and Afghani immigrants . Therefo


Malaria is a major public health problem, especially for children. However, recent reports suggest a decline in the malaria burden. The aim of this study was to assess the change in the prevalence of malaria infection among children below five years of age between 2004 and 2010 in a mesoendemic area of Uganda and to analyse the risk factors of malaria infection.P. falciparum infection among children less than five years of age. Rapid diagnostic tests and blood smears were used to diagnose malaria infection. In 2010, sampling was stratified by urban and rural areas. In each selected household, knowledge of malaria and bed nets, and bed net ownership and use, were assessed.Two cross-sectional surveys were conducted in 2004 and in 2010 at the end of the rainy and dry seasons to measure the prevalence of P = 0.001) and remained stable from 3% to 4% (95% CI: 1-7) in the urban area (P = 0.9). The proportion of households reporting ownership and use of at least one bed net increased from 22.9% in 2004 to 64.7% in the urban area and 44.5% in rural areas in 2010 (P < 0.001). In 2010, the risk of malaria infection was consistently associated with child age and household wealth. In rural areas, malaria infection was also associated with geographic factors.In 2004 and 2010, respectively, a total of 527 and 2,320  children less than five years old were enrolled. Prevalence of malaria infection declined from 43% (95% CI: 34-52) in 2004, to 23% (95% CI: 17-30) in rural areas in 2010 and 3% (95% CI: 2-5) in the urban area in 2010. From the rainy to dry season in 2010, prevalence decreased from 23% to 10% (95% CI: 6-14) in rural areas (This study reports a significant drop in the prevalence of malaria infection among children below five years of age, paralleled by an uptake in bed-net use. However, prevalence remains unacceptably high in rural areas and is strongly associated with poverty. Malaria is still a major public health problem in sub-Saharan Africa, where 85% of cases and almost 90% of malaria-related deaths occur ,2. HowevThese epidemiological changes were observed after several interventions to combat malaria , includiA common challenge in analysing the effect of these interventions is considering a combination of interlinked factors instead of separate ones. A limitation of the available data is that they often come from routine surveillance systems, which do not necessarily reflect the situation in the community and usually rely on clinical presumptive diagnoses. Therefore, additional population-based data are needed to confirm observed surveillance trends and analyse the observed heterogeneity in malaria prevalence changes, as well as to document the actual impact of the various interventions. For malaria control and eradication, a better understanding of the factors associated with malaria prevention is important to enhance the quality of interventions and inform public health decisions.The situation is even more complex for children aged less than five years. Young children pay a heavy price for malaria infection, accounting for nearly 88% of deaths related to malaria in sub-Saharan Africa. However, the heterogeneity of the risk of malaria infection and fatal outcome are the highest for this population and not fully understood .Plasmodium falciparum in the general population. The second survey was conducted in 2010 as part of a wider global study aiming to collect epidemiological data on P. falciparum infection among children aged less than five years; gather information on knowledge, perception, and behaviours related to malaria; and assess the performance of various diagnostic methods. This report focuses on the changes in the epidemiology of P. falciparum infection in children aged less than five years over a six-year period and on the analysis of associated risk factors.This study presents the results of two cross-sectional surveys done at a six-year interval in a rural area of south-western Uganda. The first cross-sectional survey was conducted in 2004 to measure the prevalence of confirmed infection with This study was conducted in four districts located in south-western Uganda formally referred to as the Greater Mbarara district. The total population was estimated at 1,088,356 inhabitants in 2002, and the main town is Mbarara municipality with a population of 69,363 . This arTwo cross-sectional surveys were conducted. The first survey was performed between mid-January and mid-February in 2004. The second survey was conducted in two parts: first round in mid-January to mid-February 2010 at the end of the rainy season; and second round in August 2010 at the end of the dry season.In 2004, three-stage cluster sampling was used to select individuals to be included in the survey. A total of 30 parishes were sampled with probability proportional to the size. Within each cluster, one village was randomly selected, and within each village 30 households randomly selected using the EPI method ,24. All In the first round of the 2010 survey (January), two-stage stratified cluster sampling was used to select households and the children within the households. Stratification was performed by classifying Greater Mbarara district into an urban area  and rural area (remaining clusters). A total of 33 villages were randomly selected in each stratum with a probability proportional to their population size. Households were then randomly selected in each village using the EPI method, and all children less than five years of age from the households were included in the survey until a total number of 20 children per village was reached. For all children, RDT and blood smear were performed.In the second round of the 2010 survey (August), the same 33 villages were selected for the rural stratum, whereas only 17 clusters were sampled from the urban stratum using the same method.Household locations and altitudes were recorded using a global positioning system (GPS) receiver . Standardized questionnaires were administered to the child's parent or legal representative by qualified medical personnel in their preferred language . Demographic and socioeconomic data of each household were collected.An indirect measure of the socio-economic status (SES) was derived from characteristics of the house  and whether or not the household possessed the following assets: mobile phone, radio, television, motorbike, bicycle and electricity. From the factor analysis of the eight items, two distinct factors emerged. The first factor was made of the housing characteristics whereas the second factor of five out of the six asset indices. As loading factors were similar and close to one for each factor, two simple scores were eventually computed by summing the indicator variables and later referred as "housing score"  and "economic score" (sum of the responses whether the household possessed the assets) .Knowledge of malaria and prevention strategies was assessed. Bed net ownership and actual use of bed nets were directly controlled by the investigator. Each child in the study received a medical examination, including height, weight, and temperature.Plasmodium species and a thick blood film for microscopic detection and parasite density count were prepared and stained with 10% Giemsa. Asexual parasites were counted until 200-500 leucocytes were counted and converted to the number of parasites per unit volume, assuming 8,000 leucocytes/μL of blood. Gametocytes were reported after examining 100 microscopic fields. Slides were considered negative if no parasites were detected after viewing 100 microscope fields.Blood smear and RDT were carried out on the same finger-prick blood sample. A thin blood film for confirmation of In the 2010 survey, the slides were stained in the laboratory by a technician different from the one in the field and double-read following blinding. In case of discordant results between the two microscopists , a third reading was performed by the most experienced microscopist and taken as the definitive result. In the 2004 survey, the slides were read by only one microscopist.® . RDTs were stored in their original packaging in a room with monitored temperature. RDTs were performed by trained laboratory technicians. A test result was considered positive if both the internal control and the test band were stained; negative if only the internal control was stained; and invalid if the internal control was not stained.The RDT used for both studies was Paracheckhttp://www.r-project.org/) [Data were double entered using EpiData . Statistical analysis was performed using the open software R  .P. falciparum was computed using positive RDT results corrected by blood smear for the 2004 survey, and both positive RDT results corrected by blood smear and blood smear only for the 2010 survey. Comparison between the prevalence estimates of 2004 and 2010 was done using RDT results corrected by blood smear for both surveys. Prevalence was computed separately for 2004 and each stratum  of 2010 using the Horvitz-Thompson estimator [Prevalence of infection with stimator . Variancstimator . CategorProportions of respondents with relatively high knowledge of malaria, bed-net ownership, and bed-net use were separately estimated for urban and rural strata in the 2010 survey. The relationship between malaria knowledge, bed net ownership and use, and household characteristics was assessed through logistic models with robust variance.Associations between risk of malaria infection (assessed using blood smear-corrected RDT for 2004 and blood smear only for 2010) and the variables collected were analysed using generalized, multilevel, mixed-effects models with a logit link. Village size was included in all models to account for unequal selection probabilities proportional to village size. The variables assessed fell under three classifications: (1) individual-level variables: child's age, gender, weight (Z score), weight-for-height (Z score), and use of a bed net by the child during the night preceding the survey; (2) household-level variables: household size, wealth measured by the housing and economic scores, education level of the head of the household, bed net ownership, number of bed nets owned, bed-net use by the head of the household the night preceding the survey; and (3) village (cluster)-level variables: village size, bed net coverage, mean economic score, and for rural areas, mean altitude and geospatial coordinates . In the 2004 survey, only child's age and gender, household size, housing score, education level of the household, village size, mean altitude and geospatial coordinates were collected and available for the analysis. In all models, unexplained non-spatial variation was accounted at the village (cluster) levels. As preliminary analysis indicated that the variance estimates in the rural and urban clusters were highly different, separate variance components were estimated for each surveyIn all models, goodness of fit was checked using graphical assessment of the residuals and general diagnostics methods ,29.Written informed consent for study participation was obtained from the children's parents or guardians. Children with a positive RDT result were immediately treated according to the Ugandan national malaria policy. The study was approved by the institutional review boards of Mbarara University of Science and Technology (MUST), Uganda National Council for Science and Technology, and France's Comité de protection des personnes - Ile-de-France XI.A total of 264 households were included in the 2004 survey, and 1,598 in the 2010 survey (Table P < 0.001). However education level and housing scores were similar between households surveyed in 2004 and those in rural areas surveyed in 2010 (P = 0.4). As expected, occupations of the head of household differed between rural and urban areas, with rural heads of households more likely to be farmers compared with the urban area .Characteristics of the households were different between rural and urban areas Table . On averP < 0.001).In the 2004 survey, 527 subjects were aged less than 5 years and therefore included in this analysis Table . In 2010P. falciparum infection among children less than five years old was 43%, 95% CI: 34-52, based on RDT diagnosis corrected for false positives by blood smear. Compared with 2004, in 2010 at the end of the rainy season prevalence decreased significantly to 23%, 95% CI: 17-30 in rural areas and 3%, 95% CI: 2-5 in the urban area (P < 0.001).In 2004, the prevalence of P < 0.001).The proportion of clusters with estimated prevalence >50% decreased from 43% in 2004 to 9% in rural areas and 0% in the urban area in 2010, whereas the proportion of clusters with estimated prevalence <1% increased from 20% in 2004 to 24% in rural areas and 70% in the urban area in 2010 (P = 0.9). However, prevalence decreased significantly to 10%, 95% CI: 6-14 in rural areas, compared with 23%, 95%CI: 17-30 during the rainy season (P = 0.001).In the dry season in 2010, estimated malaria prevalence remained relatively low and stable in the urban area at 4%, 95% CI: 1-7, compared with 3%, 95%CI: 2-5 during the rainy season . In adjusted analysis, other factors associated with knowledge of mosquitoes as the malaria vector were higher education level  and higher socioeconomic level .In terms of knowledge about malaria, most respondents mentioned mosquitoes as the vector of malaria, though less often in rural areas  compared with the urban area . Knowledge of bed nets as a prevention method was also positively associated with knowledge of mosquitoes as the vector of malaria  and socioeconomic level .When knowledge of malaria prevention methods was assessed, 96% (95% CI: 93-98) of those in the urban area and 76% (95% CI: 71-84) in rural areas mentioned the use of a bed net as a prevention strategy (P < 0.001). However, after adjusting for education, socioeconomic level, and knowledge of prevention methods, this association weakened .Significantly more households in the urban area  reported owning at least one bed net compared with the rural areas . A majority of the households owning at least one bed net reported its use by an adult in the night prior to the interview , as well as by children . Bed net use was more frequent amongst adults with higher education and socioeconomic levels  in the urban area and 45%, 95% CI: 36-53 in rural areas  of the 2010 survey.P falciparum infection amongst children less than five years old and various individual, household, and village-level factors was assessed in univariate analyses in the 2004 survey and for each round and stratum  in the 2010 survey. In univariate analysis, lower socioeconomic score of the household and higher age of the child were consistently associated with increased risk of malaria infection across the various settings . In sensitivity analysis, exclusion of clusters with an observed prevalence of zero did not alter the estimations, except for the effect of altitude in rural areas. This was further explored using logistic regression to assess the association of factors measured at cluster level with probability of a cluster having a prevalence equal to zero in rural areas. In adjusted analysis, altitude was the only significantly associated factor, with an increased probability of cluster prevalence equal to zero as altitude increased .No systematic spatial pattern was found in the model residuals as indicated by variogram (data not shown) and Moran index  was not collected. In 2004, the information collected was less comprehensive and the diagnosis method was less accurate, compared with 2010. Although the same area was investigated, the cross sectional design of this study leads to limitations that include the inability to account for short term parasitemia fluctuations. Also, it should be noticed that malaria risk is known to vary on very small scale, thus only a rough estimation of the spatial variation of malaria could be done with the sampling design used. This study focused on children less than five years of age, since they constitute the most vulnerable population for malaria morbidity and mortality. However, as a consequence of changes in malaria transmission, the peak in malaria morbidity and mortality might shift toward older age groups . Future This study suggests a large decline in malaria prevalence over a 6-year period in a mesoendemic region. However, high disease prevalence remains present in rural villages. This analysis shows also the important role poverty may play as a risk factor for malaria infection, and highlights unexplained heterogeneity in spatial structure as related to malaria prevalence that should be investigated further.The authors declare that they have no competing interests.CN and PDB participated in study design, data collection, statistical analysis, and manuscript drafting. ET participated in data collection, statistical analysis, and manuscript drafting. FG participated in study design, data collection, and manuscript review. YB participated in data collection, data analysis, and manuscript review. BS provided scientific advice for the study design, participated in data collection, and reviewed the manuscript. JFE participated in the study design, provided scientific advice for data analysis and interpretation, and reviewed the manuscript. All authors read and approved the final manuscript.Factors associated with bed-net ownership and use, 2010, n = 941 households.Click here for file