Plasmodium vivax accounts for 85% disease incidence. The role of genetic factors in host immune system conferring resistance/susceptibility against P. vivax infections is still poorly understood.Malaria is among the most prevalent parasitic diseases worldwide. In Brazil, malaria is concentrated in the northern region, where P. vivax. A total of 263 healthy individuals (control group) and 216 individuals infected by P. vivax  were genotyped for 33 single nucleotide polymorphisms (SNPs) in IL1B, IL2, IL4, IL4R, IL6, IL8, IL10, IL12A, IL12B, IL12RB1, SP110, TNF, TNFRSF1A, IFNG, IFNGR1, VDR, PTPN22 and P2X7 genes. All subjects were genotyped with 48 ancestry informative insertion-deletion polymorphisms to determine the proportion of African, European and Amerindian ancestry. Only 13 SNPs in 10 genes with differences lower than 20% between cases and controls in a Poisson Regression model with age as covariate were further investigated with a structured population association test.The present study investigates the influence of polymorphisms in 18 genes related to the immune system in patients with malaria caused by IL1B gene -5839C > T and IL4R 1902A > G polymorphisms and IL12RB1 -1094A/-641C and TNF -1031 T/-863A/-857 T/-308 G/-238 G haplotypes were associated with malaria susceptibility after population structure correction .The Plasmodium vivax malaria pathophysiology is still poorly understood. The present findings reinforce and increase our understanding about the role of the immune system in malaria susceptibility. Plasmodium parasites. Plasmodium vivax is the second most common cause of malaria in the world after Plasmodium falciparum, with high incidence in Asia, Central and South America causing high morbidity to these populations [P. vivax) were reported in this country [Malaria remains one of the most important parasitic infections in the world with almost 250 million new cases diagnosed annually . It is c country ,6.Plasmodium vivax has unique biological features that distinguish it as a species. The most obvious features that distinguish P. vivax from P. falciparum include the development of dormant forms (hypnozoites) in the liver that cause subsequent infections in the blood called relapses, which add a substantial number of cases to the general burden of the disease and present one of the most challenging bottlenecks for vivax malaria eradication [dication .P. falciparum malaria in Africa and Asia have been reported [P. vivax infection is still poorly investigated. In patients with P. vivax malaria from India, two single nucleotide polymorphisms (SNP) in the TNF promoter (−308 G > A and -1031C > T) were associated with tumour necrosis factor (TNF) levels and clinical symptoms but not with susceptibility [IFNG gene with Interferon gamma (IFN-γ) plasma levels but not with -1082 T > C in IL10 gene with interleukin (IL) 10 plasma levels in patients infected by P. vivax from Brazil [A sensitive balance between pro- and anti-inflammatory immune response, primarily mediated by cytokines released by T helper (Th) cells and macrophages, is necessary for an adequate response to malaria. An early pro-inflammatory response mediated by a set of Th1 cells and macrophages helps to reduce parasitaemia; however, a second anti-inflammatory response mediated by a set of Th2 cells and monocytes is required to control malaria by preventing organ damage and more severe symptoms. Timing and intensity between Th1 and Th2 response and auto-regulation can influence both pathology of infection and its progress -10. Inflreported , howevertibility . A recenm Brazil .P. vivax predominance [P. vivax infection allied to several particularities of the Brazilian Amazonian region, including the diverse genetic background of its population, indicate that the generalization of findings from Southeast Asia may not be appropriate for this region [P. vivax malaria in an Amazonian population.Brazil has a peculiar epidemiological situation, as one of the few countries around the world with ominance . Data ons region . The preP. vivax malaria at the Evandro Chagas Institute, Belém, Pará between 2002–2009. All patients were diagnosed by thick blood smear as recommended by the Brazilian Ministry of Health [A total of 216 patients were diagnosed with f Health . The patGenomic DNA was extracted from peripheral blood leukocyte subjects using proteinase K digestion and standard phenol-chloroform procedures . All subThe 33 immune system gene SNPs were determined by allelic discrimination with Taqman 5’-nuclease assays. A total of 30 polymorphisms were genotyped with validated genotyping assays . Three variants -174C > G (rs1800795), -863A > C (rs180030) and 874A > T (rs2430561) were genotyped by custom genotyping assay by design . All assays were genotyped according to the manufacturer’s recommended protocol.Allele and genotype frequencies were estimated by gene counting. Deviation from Hardy-Weinberg equilibrium was assessed by Qui-square tests with Bonferroni correction. Haplotype frequencies and linkage disequilibrium were estimated with PHASE 2.1.1 . DiffereThe 33 SNPs investigated, their location in the gene and the allele frequencies observed in malaria cases and controls are shown in Additional file Age and mean ancestry proportions of the subjects enrolled in the study are shown in Table IL1B -5839C > T and IL4R 1902A > G polymorphisms were associated with malaria susceptibility. The IL1B -5839C and IL4R 1902A alleles are 8.2% and 6.2% respectively more frequent in malaria patients than in controls. Haplotype association tests adjusted for population stratification by the STRAT software are shown in Table IL12RB1 and TNF haplotypes were associated with malaria susceptibility. The IL12RB1AC (−1094/-641) haplotype is only present in the malaria sample whereas the TNF TATGG (−1031/-863/-857/-308/-238) haplotype is 2.5% more frequent in individuals with malaria.Because ancestry proportions differ between malaria patients and controls Table , the assIL1B, IL4R, IL12RB1 and TNF genes were associated with susceptibility to P. vivax malaria in a population of Pará state, Brazil.In a gene-based association study with 18 candidate genes for malaria susceptibility using 33 SNPs as genetic markers, this study demonstrated that IL1B, IL4R, IL12RB1 and TNF genes in P. falciparum malaria pathology, the influence of these variants in P. vivax infections is unknown.Cytokines are immunomodulatory proteins produced by a wide variety of cells, and with very complex activities. A functional cytokine network is a central element in the homeostasis of the immune response and its alteration may lead to an abnormal immune response. Hence, recent interest has focused upon genes regulating the cytokine expression; in particular on gene polymorphisms that may influence the levels of expression and therefore the overall immune response. Despite evidence demonstrating the importance of Independent studies reported differences in immune system gene polymorphisms frequencies in distinct malaria-endemic regions . The relThe IL-1β, IL-12 and the TNF together with IFN-γ are the major cytokines in pro-inflammatory Th1 immune response. IL-1β is predominantly secreted by monocytes and macrophages in initial immune response against infections -24 and hIL1B gene was associated with P. falciparum malaria in African populations only [P. vivax malaria susceptibility. The -5839C allele presented a higher frequency in malaria patients then in controls. Despite that, the function of this intronic SNP is not completely elucidated, variability of this important pro-inflammatory gene could represent an important factor in immune regulation. The association of haplotype -31C/-511A in IL1B gene promoter with severe malarial anaemia and circulating IL-1β low levels in children with P. falciparum malaria from Kenya have been shown recently [IL1B -31C > T polymorphism was not associated with cerebral malaria in Thailand [IL1B gene and IL-1β levels are important to help understand how this gene influences malaria susceptibility and severity.ons only ,31. The recently , howeverThailand . In the P. falciparum demonstrated that IL12RB1 rs4229774 and rs383483 polymorphisms were associated with protection against severe malarial anaemia and high parasitaemia levels, but not susceptibility [IL12RB1 -1094A/-641C haplotype with P. vivax malaria susceptibility. Despite the lower frequency in the study subjects, this haplotype is present only in the individuals with malaria, suggesting a possible influence in malaria response. The -641C allele leads to a missense variant (i.e. encodes a different amino acid) and can modify the receptor properties and interfere with IL-12 ligation and function. These results suggest that IL12RB1 variants are important in malaria susceptibility and severity. Further studies will be necessary to better understand the IL12RB1 influence on susceptibility and severity in P. vivax malaria.Recent work in Kenyan patients infected with TNF gene promoter have been reported to be associated with symptoms and severity of P. falciparum malaria in different African and Asian populations [TNF haplotype is associated with P. vivax malaria in a Brazilian population. TNF is an important pro-inflammatory cytokine and the TATGG haplotype diverge in two alleles (−1031C and -308A) associated with TNF levels in vivax malaria infection in India [Polymorphisms in ulations -39. The in India . In thatIL4 and IL4R genes have high resistance to malaria liver stage caused by sporozoites. The absence of modulation mediated by IL-4 to Th2 immune response, the Th1 response mediated by IFN-γ is maintained and promotes a rapid cellular response against sporozoites. The present study shows that 1902A allele is more frequent in malaria patients, although 1902 G presents a higher overall frequency in the study population, 1902A allele could influence the co-regulation between Th1 and Th2 immune response against malaria infections. New complementary studies will be necessary to elucidate the IL4R gene importance in vivax malaria infection.Interleukin 4 receptor (IL-4R), together with IL-4 and IL-13 are important Th2 anti-inflammatory immune response modulators. IL-4R is the principal receptor of these interleukins, and when it is blocked IL-4 and IL-13 function is aborted preventing Th2 immune response modulation . Only a IL4, IL10, IL12B, IFNG and IFNGR1 genes polymorphisms were associated with symptoms and severity of P. falciparum malaria in studies from African and Asian populations [P. vivax. Differences in malaria pathophysiology caused by these two species of parasites could be a possible explanation for the divergences reported. Plasmodium falciparum malaria presents a more acute form of infection reaching more than 50% of erythrocytes leading to cyto-adherence, organ damaged, severe malarial anaemia and cerebral malaria. Malaria caused by P. vivax is characterized by a long incubation period and milder initial symptoms. The parasite infects approximately 2 to 5% of erythrocytes, and can remain dormant in the liver as hypnozoites leading to subsequently relapses [P. falciparum infections [P. vivax infections [P. falciparum and P. vivax malaria response, pathology and outcome.ulations . In the relapses . Studiesfections -47 and Tfections ,48-50. TP. vivax infection influence susceptibility to malaria. The present findings reinforce and increase our understanding about the role of the immune system in malaria susceptibility. However, to expand knowledge about the actions of the immune system in the pathophysiology of vivax malaria it is necessary to conduct population-based longitudinal studies.Knowledge of the relationships between genetics, susceptibility to and severity of malaria infections is essential to identify subjects at higher risk and to develop specific preventive measures. This study showed that genetic polymorphisms of some interleukins involved in the immune response to Th: T helper; SNP: Single nucleotide polymorphism; TNF: Tumour necrosis factor; IFN-γ: Interferon gamma; IL: Interleukin; Indels: Insertion/deletion; NK: Natural killer; IL-12R: Interleukin 12 receptor; IL-4R: Interleukin 4 receptor.The authors declare that they have no competing interests.VAS designed the research project, carried out the laboratory assays, and wrote the manuscript; MGC participated in the study design, planning and data collection, revised the manuscript for important intellectual content and approved the version to be published; MDOO participated in the study design, planning and data collection, performed the malaria diagnosis and revised the manuscript for important intellectual content and approved the version to be published; JMS participated in the study design, planning and data collection, performed the malaria diagnosis and revised the manuscript for important intellectual content and approved the version to be published; NPCS participated in the study design, planning and data collection, performed the malaria diagnosis and revised the manuscript for important intellectual content and approved the version to be published; AKCR participated in the study design, planning and data collection, revised the manuscript for important intellectual content and approved the version to be published; SMC-J participated in the study design, planning and performed the statistical analysis and approved the version to be published; SEBS genotyped the ancestry informative markers, revised the manuscript for important intellectual content, and approved the version to be published; MHH conceived the study, and participated in its design and coordination, analysed the data, and wrote the final version of the article. All authors read and approved the final manuscript.Table S1. List of SNPs investigated in the present study. List of SNPs investigated, their location in the gene, pubmed database SNP identification, manufacturer’s assay identification and allele frequencies in case and control groups.Click here for fileTable S2. Control and malaria group genotypic frequencies. Genotypic frequencies of all SNPs in case and control groups.Click here for fileTable S3. Poisson Regression for association between malaria and control samples controlling for age. Poisson Regression results for association between malaria and control samples controlling for age.Click here for file


Malaria transmission in most of Latin America can be considered as controlled. In such a scenario, parameters of baseline immunity to malaria antigens are of specific interest with respect to future malaria eradication efforts.Plasmodium parasites and naturally acquired antibodies to Plasmodium falciparum antigens in serum. The GMZ2 vaccine candidate proteins MSP3 and GLURP were chosen as serological markers.A cross-sectional study was carried out in two indigenous population groups in Amazonas/Venezuela. Data from the regional malaria documentation system were extracted and participants from the ethnic groups of the Guahibo (n = 180) and Piaroa (n = 295) were investigated for the presence of P. falciparum in both communities was found to be less than 2%, and none of the participants harboured P. falciparum at the time of the cross-sectional. Nearly a quarter of the participants  had positive antibody titres to at least one of the antigens. 53/475 participants (11.2%) were positive for MSP3, and 93/475 participants (19.6%) were positive for GLURP. High positive responses were detected in 36/475 participants (7.6%) and 61/475 participants (12.8%) for MSP3 and GLURP, respectively. Guahibo participants had significantly higher antibody titres than Piaroa participants.The incidence of P. falciparum, submicroscopical infections may explain the comparatively high anti-P. falciparum antibody concentrations.Considering the low incidence of  Plasmodium vivax (ca. 80%) followed by Plasmodium falciparum . Plasmodium malariae is only occasionally detected [To date, malaria is among the top ten causes of death in low-income countries . In Venedetected ,4.P. falciparum would be a powerful tool in the control of malaria [P. falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP), which has been evaluated during phase I trials as a promising vaccine candidate in Germany and Africa [Saimiri sciureus monkeys [P. falciparum is low.As drug resistance continues to be a major problem, an effective malaria vaccine against  malaria ,6. The vd Africa ,8. Antib monkeys  and were monkeys . However monkeys . This isP. falciparum antigens.In 2008, a pilot programme for the evaluation of blister-packed treatment in distinct ethnic groups was initiated. An initial cross-sectional study was carried out in two indigenous population groups in order to gain knowledge about the micro-epidemiology of malaria in the project area. A second cross-sectional study is planned to close the project. As serological markers are useful indicators to measure transmission variations especially in low endemicity areas , immune Anopheles breeding sites. The majority of residences in the communities are non-traditional governmental housing projects. The communities of Platanillal and Paria Grande have a health post, each with a malaria microscopist.The study took place in June 2009 in the municipality of Atures, Amazonas, Venezuela. Three indigenous communities were visited. They were inhabited by two ethnic indigenous population groups, the Guahibo  and the Piaroa: 1) Platanillal ; 2) Cerro de Oro ; 3) Paria Grande . The distance between the communities is small (less than 20 km linear distance) and they are comparable with respect to ecological, geographical and malariological conditions such as distance to P. falciparum: artesunate, mefloquine, and primaquine. P. vivax: chloroquine, primaquine) [Ethical clearance was obtained from the institutional ethical committee of the Amazon Center for the Investigation and Control of Tropical Diseases 'Simón Bolívar', Autonomous Service, Puerto Ayacucho, Amazon State, Venezuela (SACAICET). Residents were informed about the ideas and procedures of the study, when necessary with the help of translators. All residents were invited to take part. Those consenting orally were interviewed and examined. Individuals with a blood smear positive for malaria were treated according to the national guidelines (maquine) .Data from the regional malaria documentation system were extracted. Malaria cases detected in the health posts of Platanillal and Paria Grande before the study took place, from January 2003 to May 2009, were analysed.Every participant, or the legal guardian, was interviewed using semi-structured interviews. Translators assisted if it was necessary, and standardized questions were phrased with the support of anthropologists. The structured part of the interviews concerned malaria antecedents, possession and use of mosquito nets, general condition, unspecific symptoms during the last seven days, and pregnancy. Every participant was examined physically including a check for splenomegaly and body temperature.Thick blood smears and thin films were taken from every participant, air-dried and stained for 45 min with 3% Giemsa using standard procedures1 . Slides Escherichia coli as described elsewhere [212-318 and GLURP27-500 were used.Recombinant proteins were produced in lsewhere . The 48 lsewhere . The 220lsewhere . For allSerum antibodies to MSP3 and GLURP were measured applying a modified version of a quantitative ELISA . In brie®. Optical densities (ODs) were averaged and normalized. The cut-off value for the difference between duplicates was set at 25%. If the difference was higher, the measurement was repeated. All results were converted to values in mg/dl using the positive standard serum. Positivity and high positivity of samples was defined as any OD higher than the mean of negative controls plus three and five standard deviations (SD), respectively. Statistical analysis was performed using SPSS 19® software. T-Tests were run to screen for significant differences between the variables subgroups concerning antibody titres, Chi-Square-Tests to learn about disparities in respect to antibody positivity. Age in years, sex, self-reported incidence of malaria episodes during the last 12 months and lifetime, possession and frequent utilization of mosquito nets and ethnic affiliation were included in a binary logistic model to predict anti-MSP3 and anti-GLURP antibody positivity. This quite conservative model has the advantage to show an Odds ratio (OR) for each expression of a variable. Furthermore, a linear regression model was performed to expose the influence of the aforementioned independent variables on the direct IgG-titre against the GMZ2-subunits.To do so, antibody levels had to be transformed to their decadic logarithm to gain Gaussian distribution. The resulting model was then de-logarithmized to show comparable and comprehensible results.All data were subjected to plausibility, double-entered and checked for transcription errors in Epidata v3.0Four hundred and seventy-five persons participated in the study. Demographic data of participants and questionnaire results are summarized in Table Within the six years preceding the cross-sectional study (2003 - 2008), the health post of Platanillal reported 573 cases of malaria in Guahibo patients .P. falciparum in Platanillal and Paria Grande, respectively. All other infections were caused by P. vivax. P. malariae was not detected.The health post of Paria Grande reported 270 cases of malaria in Piaroa patients within the six years preceding the cross-sectional study . Of these, 45/573 cases (7.8%) and 36/270 cases (13.3%) were caused by P. vivax, and 5/154 slides (3.2%) were positive for P. falciparum.Within the five months preceding the cross-sectional study (January 2009-May 2009), 154 slides were taken in the health post of Platanillal; 43/154 slides (27.9%) were positive for P. vivax, and none (0%) was positive for P. falciparum.Within the five months preceding the cross-sectional study, 148 slides were taken in the health post of Paria Grande; 15/148 slides (10.1%) were positive for P. falciparum at the day of the cross-sectional study. Two participants  were found positive for P. vivax (both <100 parasites/μl). Both patients were from the Guahibo ethnic group and reported malaria antecedents. One patient was a 12-year-old anaemic boy (Hb 12.5 mg/dl) in reduced general condition and with normal body temperature (36.5°C) reporting chills during the last seven days. The other patient was a 26-year-old anaemic pregnant woman (Hb 9.7 mg/dl) with normal body temperature (37.1°C) complaining about a reduced general state of health.Participants (n = 475) complained about the following symptoms within the preceding seven days: headache , body ache , flu-like symptoms , cough , chills , fever , diarrhoea , abdominal pain , nausea , fatigue . The mean temperature of all participants was 37,02°C . 9/475 participants (1.9%) had a body temperature over 38°C. None of the participants had palpable splenomegaly and none of the 475 participants was found positive for The cut-off value for a positive sample was set at 0.193 mg/dl and at 0.224 mg/dl for MSP3 and GLURP, respectively. 111/475 participants (23.4%) were positive for at least one of the antibodies. 53/475 participants (11.2%) were positive for MSP3, and 93/475 participants (19.6%) were positive for GLURP. 35/475 participants (7.4%) had positive antibody levels against both antigens. The average value (geometric mean) of positive samples was 0.36 mg/dl (95% CI 0.32 mg/dl - 0.41 mg/dl) and 0.47 mg/dl (95% CI 0.41 mg/dl - 0.55 mg/dl) for MSP3 and GLURP, respectively. The cut-off value for a high-positive sample was set at 0.27 mg/dl and at 0.31 mg/dl for MSP3 and GLURP, respectively. 78/475 participants (16.4%) were high-positive for at least one of the antibodies. 36/475 participants (7.6%) were high-positive for MSP3, and 61/475 participants (12.8%) were high-positive for GLURP. 19/475 participants (4%) had high-positive antibody levels for both antigens. The highest titres measured were 2.8 mg/dl and 3.36 mg/dl for MSP3 and GLURP, respectively.SpGLURP = 0.130, p = 0.005. RSpMSP3 = 0.259, p < 0.001). When the study population was grouped by age - each group with a span of 10 years - a significantly higher number of positives and high-positives was seen in the older age groups . No significant differences in antibody titres or positivity rates were found regarding the sex of the persons .Antibody titres were significantly increasing with age , but there was no significant difference respective to MSP3 . Participants who reported daily use of mosquito nets during the previous 30 nights were defined frequent users. 392/475 participants (82.5%) were found to be frequent users. No significant difference in antibody titres nor positivity rates were found between frequent users and the rest of the study population .Comparing possessors  and non-possessors  of mosquito nets, holders were found to have lower levels of GLURP , but not for GLURP . Those who stated a malaria episode within the previous 12 months had slightly more positive responses against both antigens. This difference was significant for GLURP and for MSP3 .282/475 participants (59.4%) stated they had at least one malaria episode in their life, and 85/475 (17.9%) reported a malaria episode during the previous 12 months. Those who reported to have suffered malaria at least once in their life showed significantly higher IgG-levels than the group reporting no previous malaria episode. This difference was significant for MSP3  and GLURP . Non-possession of a mosquito net increased the chance to have GLURP by 4.05-fold . Guahibo individuals had a 8.04-fold  chance for MSP3 antibody positivity compared to Piaroa individuals. With respect to GLURP, the chance for positivity was 2.81-fold higher for Guahibo in comparison to Piaroa individuals . Results of the linear regression model are shown in Table Results show that the two GMZ2 antigens are recognized by antibodies acquired through natural infections in the Venezuelan Amazon. Overall, the results correspond - except high responders - to rather low responses which are roughly two-fold higher than the cut-off value for positives. As antibody subclasses are of great importance  further P. falciparum malaria, and 2) the significantly higher prevalence of MSP3 and GLURP antibodies in the Guahibo group when compared to the Piaroa group.The two main findings of this study were 1) the high prevalence of antibodies against conserved parts of MSP3 and GLURP antigens in the two population groups with low incidence of The total population of the Piaroa is about 12,000 persons. Anthropologists describe them as peaceful and egalitarian. Traditionally, the Piaroa's agroforestal subsistence strategy implicated small, dispersed and mobile ways of settlement in groups of about 50 individuals. Nowadays they are living in bigger communities mostly built by the Venezuelan government in the periphery of their ancestral territories, which they still use as cropland and hunting grounds .The Guahibo consist of about 30,000 persons settling mainly around Puerto Ayacucho and neighbouring Colombian areas. As a patriarchal society, great importance is placed on the nuclear family. Traditionally, they inhabited vast flatlands in Colombia and Venezuela as nomadic foragers. Nowadays, they inhabit communities constructed by the Venezuelan government. They are mainly assimilated according to language and clothing style, but still are threatened by pauperisation .The serological results of this study confirm previous investigations hinting to an elevated risk for malaria in the Guahibo population ,23. LiviThe positive correlation of antibody titeres and prevalences with age has been known for a long time . When peP. falciparum parasites is expected to be minimal. Indeed, only two persons in nearly half a thousand participants were found harbouring parasites . Both patients were positive for P. vivax, none was positive for P. falciparum. Nevertheless, nearly a quarter of participants had antibodies to at least one of both antigens. As adding up immune responses to individual antigens might not be an appropriate procedure for results, which show - not surprisingly for outbred human populations - an extensive variation between individuals, it is important to note that 7.6% and 12,8% of participants had high antibody titres against MSP3 and GLURP, respectively. Possibly only high-positive antibody responses are relevant and/or specific results in this endemic area. Moreover, it should be taken into account that higher antibody responses might persist longer than lower ones.This cross-sectional study evaluated immune responses against two blood stage antigens considered as promising vaccine candidates on the basis of studies carried out in completely different settings. Samples were taken in the period of low transmission when the direct contact with P. falciparum in particular. The average pan-malarial incidence was higher in the Guahibo population of Platanillal (ca 18%) than in the Piaroa population of Paria Grande (ca 8%). However, this was due to P. vivax infections, whereas the transmission of P. falciparum was surprisingly low in both population groups. Within a time period of 6.5 years, only 50 and 36 cases of P. falciparum were detected in Guahibo and Piaroa people of the health posts of Platanillal and Paria Grande, respectively, and the average incidence of P. falciparum was estimated to be less than two percent in both population groups, without a significant difference.Using the records of the health information system of Amazonas the level of malaria transmission in the communities during the years and months before the study was approximated. As the health posts of Platanillal and Paria Grande were comparable in the volume of slides taken per year  and data of ethnic origin, origin of infection, and population census were available, the prevalence in both communities could be estimated for malaria in general, and for P. falciparum antigens by P. vivax [MSP3 and GLURP antibodies last six-12 months if no boost infection takes place . In ordeP. vivax .Controlled malaria is the result of an interplay of host defense systems, public health measures, ambiental conditions, behavioural features, and epidemiology and infection biology of the parasite. Interventions should be weighed carefully in order not to destabilize an established equilibrium. The epidemiology and immunology of controlled malaria might be subject of growing interest for malariologists and decision makers, as lessons learnt in low endemic areas could be instructive after eradication and control efforts have been successful in high endemic areas.The authors declare that they have no competing interests.AB coordinated and conducted field and laboratory work, analysed and interpreted the data, and drafted and revised the manuscript. MM conceived the study, supervized field and laboratory work and interpreted the data. SVM conceived the study, facilitated the overall collaboration and revised the manuscript. MLU and TH coordinated and conducted field and laboratory work. RD collaborated as anthropologist. ME and BGM supervized the laboratory work, gave statistical input, interpreted the data and revised the manuscript. MT and LA were responsible for the conception of the work. WGM conceived the study, interpreted the data, and drafted and revised the manuscript. All authors revised and approved the final manuscript. WGM and MM are the guarantors of the paper.


Plasmodium knowlesi is a common cause of human malaria in Malaysian Borneo, with a particularly high incidence in Kudat, Sabah. Little is known however about the epidemiology in this substantially deforested region.The simian parasite Malaria microscopy records at Kudat District Hospital were retrospectively reviewed from January 2009-November 2011. Demographics, and PCR results if available, were recorded for each positive result. Medical records were reviewed for patients suspected of representing family clusters, and families contacted for further information. Rainfall data were obtained from the Malaysian Meteorological Department.“Plasmodium malariae” mixed or mono-infection was diagnosed by microscopy in 517/653 (79%) patients. Of these, PCR was performed in 445 (86%) and was positive for P. knowlesi mono-infection in 339 (76%). Patients with knowlesi malaria demonstrated a wide age distribution  with P. knowlesi predominating in all age groups except those <5 years old, where numbers approximated those of Plasmodium falciparum and Plasmodium vivax. Two contemporaneous family clusters were identified: a father with two children (aged 10–11 years); and three brothers (aged one-11 years), all with PCR-confirmed knowlesi malaria. Cases of P. knowlesi demonstrated significant seasonal variation, and correlated with rainfall in the preceding three to five months.Plasmodium knowlesi is the most common cause of malaria admissions to Kudat District Hospital. The wide age distribution and presence of family clusters suggest that transmission may be occurring close to or inside people’s homes, in contrast to previous reports from densely forested areas of Sarawak. These findings have significant implications for malaria control. Prospective studies of risk factors, vectors and transmission dynamics of P. knowlesi in Sabah, including potential for human-to-human transmission, are needed. Plasmodium knowlesi has emerged as a common and potentially fatal cause of human malaria in Malaysian Borneo, p = 0.004).Overall the median age of females with PCR-confirmed knowlesi malaria  was not significantly different than that of males , however among adults with knowlesi malaria, females were significantly older than males . Significant seasonality was also demonstrated (p < 0.001), with increased transmission from March to July in 2009 and April to August in 2011. In both years the number of cases peaked in May, with the peak being particularly marked in 2011. Remarkably few cases of knowlesi malaria occurred in 2010.The number of patients diagnosed with PCR-confirmed n Figure, with 13P. knowlesi in the proceeding three to five months  was significantly less than in 2009  and 2011 .Rainfall recorded at Kudat meteorological station was strongly correlated with the number of cases of hs Table. The numP. falciparum mono-infections (excluding the five patients found to be P. knowlesi by PCR) decreased slightly in the last year of the study period, with 26 (0.4%), 29 (0.5%) and 16 (0.2%) cases diagnosed in 2009, 2010 and 2011 respectively . In contrast, microscopy-diagnosed P. vivax mono-infections increased, with 45 (0.7%) cases in 2011 compared to zero and eight (0.1%) in 2009 and 2010 respectively .The number of cases of microscopy-diagnosed P. knowlesi monoinfection were identified. The first consisted of a 37 year-old man with his 10 year-old daughter and 11 year-old son, who all presented on the same day with the same duration of illness. The family lived in town, the children attended the local school, and the father worked as a clerk in a town resort. Although they had not travelled into forested areas, the family reported seeing macaques close to their home. The second family included three brothers aged one, five and 11 years, all presenting on the same day. They lived on the island of Banggi, a forested area with numerous macaques. The 11 year-old boy had chronic myeloid leukaemia for which he took imatinib. His uncomplicated knowlesi malaria was unremarkable except for a notable absence of thrombocytopenia.Two family clusters of patients with PCR-confirmed Plasmodium knowlesi is the most common cause of malaria admissions to Kudat District Hospital and affects all ages from young children to the elderly. Although the greater proportion of males, particularly among adults, is likely to be consistent with occupational forest or plantation exposure as a risk factor for knowlesi malaria, the wide age distribution suggests that this is not the only determinant of transmission. Furthermore, this report of two family clusters, one of which had not travelled outside Kudat town, suggests that transmission may be occurring close to or inside people’s homes. These findings differ from those reported from two studies in Sarawak, where a smaller proportion of knowlesi malaria cases occurred in children  infected mosquitoes found in forest and farm locations[An. latens were found in longhouses, none were infected, suggesting transmission occurs away from homes[P. knowlesi remains a zoonosis, based on an extremely high prevalence (87%) of P. knowlesi in long-tailed macaques, and sequencing of the csp gene and mtDNA showing a greater number of P. knowlesi genotypes per monkey infection than human infection, with genotypes common to both hosts and no genotype exclusive to either[In Kapit, Sarawak, ocations. Althougom homes. A subseo either.P. knowlesi vector may be biting humans close to or inside people’s houses. Previously An. balabacensis was known to be the primary vector of human malaria in Sabah[Anopheles flavirostris also identified as a potent P. falciparum vector on Banggi Island[Anopheles balabacensis have been shown to readily bite monkeys at the canopy level[P. knowlesi-infected An balabacensis was recently found in Ranau[An. balabacensis have been shown to exhibit “learning behaviour” in relation to host preference, and may also demonstrate “habitat loyalty” by returning to previous feeding sites[An. balabacensis has been shown to be a highly efficient vector[Anopheles dirus, the primary malaria vector in the Mekong region, which maintains high levels of malaria endemicity at low population densities[P. knowlesi DNA[In Kudat, the wide age distribution and clustering of cases suggest that the in Sabah, with Ani Island. Anophelpy level, and a Pin Ranau. Of signng sites. Furthert vector, and is ensities, is capaensities, and in lesi DNA.An. donaldi was shown to have replaced An. balabacensis as the primary species in the Kinabatangan region of Sabah[An. donaldi in the Kinabatangan region[More recently however, of Sabah. The pean region, speciesAn. balabacensis and An. donaldi have both been shown to have increased parity rates during the months of greatest rainfall[A strong association was demonstrated between knowlesi malaria cases and rainfall, with a lag time of three to five months. Anopheles mosquitoes depend on rainfall to provide aquatic breeding sites, and rainfall. An assorainfall-29, and rainfall,31. ThisP. vivax and P. falciparum in this study were small, and could only be assessed over a three-year period, the number of P. vivax cases increased during the study period along with P. knowlesi, while a modest decrease was seen in P. falciparum cases. Larger prospective studies will be required to further evaluate the interaction between malaria species, which may have implications for malaria control.Although the numbers of P. falciparum or P. vivax had PCR performed, and microscopy reports were therefore relied on for analyses involving these species. It is possible that some of the patients diagnosed by microscopy as P. falciparum or P. vivax had P. knowlesi. However, despite the potential for this to dilute age-related differences among species, a significant difference in age distribution was noted between P. knowlesi and microscopy-diagnosed P .falciparum or P. vivax, making the estimates of age-related differences conservative. Finally, this study involved the use, prior to June 2011, of a nested PCR assay (using Pmk8-Pmk9 primers) that has been associated with cross-reactivity with P. vivax DNA, and false-positive PCR findings of mixed P. knowlesi/P. vivax in true P. vivax mono-infections[P. vivax mono-infection prior to June 2011, and subsequently found to have mixed P. vivax/P. knowlesi infections by PCR. Cross-reactivity with P. vivax isolates has not been reported with the real-time PCR assay[P. knowlesi/P. vivax mixed infections diagnosed by PCR despite an increase in microscopy-diagnosed P. vivax mixed and mono-infections over this time.This study had several limitations. First, the retrospective design did not allow the collection of information on where patients acquired their malaria infection. This prevented detailed assessment of forest exposure as a risk factor for knowlesi malaria, and may also have influenced the association between malaria cases and rainfall recorded at Kudat Meteorological Station. In particular, information was lacking on which patients came from Banggi Island, which, although only a short distance from Kudat town, may represent a different geographical setting with different transmission dynamics. Second, identification of case clusters was based only on patients with the same family name recorded in microscopy books, and this may have led to an underestimation of case clustering. Third, only a small number of patients with microscopy-diagnosed fections. In thisCR assay, and it P. knowlesi cases appear to be increasing, with the species now accounting for the majority of malaria admissions to Kudat Hospital as well as other district hospitals throughout Sabah and Sarawak[P. knowlesi and the possibility of transmission occurring close to or inside people’s homes may increase the possibility of a switch to the human host. Prospective studies on epidemiological risk factors, vectors and transmission dynamics of P. knowlesi in Sabah, including the possibility of human-to-human transmission, are required in order to develop strategies for knowlesi malaria control.Malaysia has made impressive progress in reducing malaria prevalence in recent years; however,  Sarawak,4,9. TheThe authors declare that they have no competing interests.BEB, NMA, TWY, TW, PD and MJG conceived and designed the study. FA performed the PCR assays at Sabah State Reference Laboratory. BEB collected and analysed the data. BEB and NMA wrote the manuscript. All authors read and approved the final manuscript.


Wuchereria bancrofti) are transmitted by the same vector species of Anopheles mosquitoes, and thus are likely to share common environmental risk factors and overlap in geographical space. In a comprehensive nationwide survey in 2000-2003 the geographical distribution of W. bancrofti was assessed by screening school-aged children for circulating filarial antigens (CFA). Concurrently, blood smears were examined for malaria parasites. In this study, the resultant malariological data are analysed for the first time and the CFA data re-analysed in order to identify risk factors, produce age-stratified prevalence maps for each infection, and to define the geographical patterns of Plasmodium sp. and W. bancrofti co-endemicity.In Uganda, malaria and lymphatic filariasis . High W. bancrofti prevalence was predicted for a much more confined area in northern Uganda, with an overall population adjusted infection risk of 7.2% in the highest risk age-group (14-19 year olds). Observed overall prevalence of individual co-infection was 1.1%, and the two infections overlap geographically with an estimated number of 212,975 children aged 5 - 9 years living in hyper-co-endemic transmission areas.W. bancrofti transmission have been identified to help provide a better informed platform for integrated control.The empirical map of malaria parasitaemia risk for Uganda presented in this paper is the first based on coherent, national survey data, and can serve as a baseline to guide and evaluate the continuous implementation of control activities. Furthermore, geographical areas of overlap with hyper-endemic  Malaria and lymphatic filariasis are two of the most common mosquito-borne parasitic diseases worldwide. Their overall prevalence and health significance have made them top priorities for global control and elimination ,2. To plPlasmodium falciparum is by far the most common of the existing malaria species in Uganda, contributing 90-98% of the parasite population [In Uganda, malaria is highly endemic with temperature and rainfall allowing stable, year-round transmission with relatively little seasonal variability in most parts of the country . Plasmodpulation ,8 Despitpulation ,10,11. Mpulation -16. HoweWuchereria bancrofti. Despite being recognized as a major public health and socio-economic problem, knowledge about the occurrence of lymphatic filariasis in Uganda was scanty, until a school-based survey in 2000-2003, allowed the geographical distribution of lymphatic filariasis throughout the country to be mapped [Besides malaria, several neglected tropical diseases are reported to be co-endemic in Uganda ,17 amonge mapped . During Plasmodium and W. bancrofti parasites share common mosquito vector species [In Uganda, as in many other tropical regions,  species ,20. Thus species ,22, and  species -29. Yet,In the present paper, ecological correlates and risk factors of both malaria and lymphatic filariasis infections in Uganda are investigated, and nation-wide risk maps are developed using solid empirical survey data and model-based geostatistics.i) to derive statistically robust prevalence estimates of malaria and lymphatic filariasis infections separately at un-sampled locations , and ii) to determine the extent of geographical overlap (co-endemicity) of the two parasitic infections. Risk estimates are moreover linked to population data to calculate the number of school-aged children at risk of each infection separately, as well as the number of children living in areas of overlapping hyper-endemic malaria and lymphatic filariasis transmission.The objectives were two-fold: μl) was first collected from each child and examined for W. bancrofti-specific circulating filarial antigens (CFA) using commercial immunochromatographic cards. When possible, and when the child agreed another (100- μl) blood sample was collected and used to prepare a thick smear to examine for infection with malaria parasites. Malaria parasites were identified to the genus level, and will hereafter be referred to as Plasmodium sp. As not every subject allowed a second sample to be collected, the number of children examined for Plasmodium sp. was slightly lower than the number examined for W. bancrofti antigens. In total, malariological data was available from 71 schools , whereas W. bancrofti infection data were available from 76 schools  [The screenings were carried out between October 2000 and April 2003 and included pupils aged 5 - 19 years of age from Ugandan primary schools covering all major topographical and ecological zones of the country. Details about survey design and sampling methods have already been given elsewhere . Briefly pupils) ,30. BoysThe studies which contributed data used in this paper, received ethical clearance from the Uganda National Council for Science and Technology and were approved by the Central Scientific Ethical Committee of Denmark.Satellite sensor data on Normalized Difference Vegetation Index (NDVI) and day- and night-time land surface temperatures from the Moderate Resolution Imaging Spectroradiometer (MODIS) satellite were obtained from the United States Geological Survey U.S. Geological Survey (USGS) Land Processes Distributed Active Archive Center (LP DAAC)  at 1 km The products were downloaded for every 8 - 16 days in the period 2000 to 2003 and average annual composite maps produced by combining all maps in the period 2000-2003 to reduce the variation between climate years. Based on Uganda's two principal rainy seasons, March to May, and August to November, average wet season and dry season composite maps were similarly produced. A climate surface grid at 5 × 5 km resolution consisting of monthly long-term normal rainfall and evapotranspiration estimates was obtained from the IGAD/Nile Minimum Medical Database (MMDb)  and seasEnvironmental data for model fitting were extracted using the satellite positions of each school, as the average of the environmental conditions found within a circular area of 1 km buffered around each school position.2 resolution was obtained from LandScanTM 2002 Global Population Database [A gridded population surface at 1 kmDatabase . The perDatabase .For the purpose of predicting infection risk at unobserved locations, a grid covering Uganda at 2 × 2 km resolution was constructed,  and the average environmental data extracted for each grid cell. Environmental grid data was managed with ArcGIS version 9.3  with the Geospatial Modelling Environment (GME) extension School-children were classified into three age groups of approximately equal sizes  following Onapa et al. .2-test was used to assess associations between sex and age with Plasmodium sp. and W. bancrofti, single and mono-infections. Single infection refers to infection with a particular species irrespective of other infections that may be present in the individual, whereas mono-infection refers to infection with only the particular species of parasite in question.The Pearson χBivariate logistic regression models for each parasitic infection were developed to investigate the relationship between the outcome variable (single infection status) and covariates . To account for clustering at the school-level, a location-specific random effect was included in the bivariate models. To reduce confounding arising from correlated environmental variables, significant variables within the same environmental themes were ranked according to the goodness of fit Akaike Information Criteria (AIC)  and the p < 0.15 were then built into Bayesian logistic regression models, using the Bayesian statistical software OpenBUGS  [i) an exchangeable model assuming that random effects arise from an independent normal distribution with mean 0 and variance quantifying extra binomial variation, and ii) a geostatistical model assuming that random effects arise from a Gaussian spatial process quantifying geographical correlation. Model fit was implemented via Monte Carlo methods which allow flexibility in fitting complex models and avoid asymptotic inference and the computational problems encountered in likelihood-based fitting [Environmental and demographic covariates with significance level don, UK) . For eac fitting . FurtherModel-based predictions of prevalence at unobserved locations were obtained via Bayesian predictive distributions for the models , using RTo assess the best performing prediction model, model fit was also carried out on a randomly selected subset (~80%) of the schools (training set). The remaining locations, comprising a simple random sample, were used for validation . The 95% credible interval of the posterior distribution at the test localities were calculated, and the best performing model for each age group was considered the one with the highest proportion of test locations having observed prevalences within this interval.A complete mathematical description of the models used is given in the Additional file The observed infection status with single- or mono-infections of school-children, stratified by sex and age can be seen in Table Plasmodium sp. infection was 35.2%, ranging from 7.4% in sites in the Southern and North-West of Uganda, to 75.5% on the eastern shore of Lake Victoria  prevalence was 3.9% ranging from 0% in sites in the southern parts in the country to 30.7% in the West Nile area and North of Lake Kyoga. Prevalence was lowest in the 5-9 year olds (2.9%) and peaked in the oldest school-children (6.3%), but there was no significant difference between boys and girls.Overall observed CFA . In general, more boys than girls had co-infections . The age group with the highest level of co-infection was the 10-14 year olds ). Figure The summary results of the model validation can be seen in Table W. bancrofti the geostatistical models were able to predict more test locations correctly within the 95% BCI (87-93%) than the exchangeable models, and also showed the narrowest overall BCIs.For For both malaria and lymphatic filariasis the linear models performed as good as or better than the categorical models, with the linear models predicting as many or more of the test locations correctly, and with overall narrower BCI's for the linear models.W. bancrofti antigenaemia risk with demographic and environmental factors resulting from the bivariate and the Bayesian multivariate exchangeable and geostatistical models with linear terms can be seen in Table Associations of malaria parasitaemia and Plasmodium sp., the non-spatial bivariate logistic regression analysis and likelihood ratio tests showed that age, sex, season of data collections and all remotely sensed factors were significant at the 15% significance level. Males and younger children had a significantly higher risk of parasitaemia than females and older children. Diurnal temperature range and precipitation and NDVI composited over the wet season performed better than the other seasonal composites as measured by AIC (results not shown), and was thus selected for inclusion in Bayesian multivariate models. However, only the positive relationship with NDVI remained significant in these models.For the W. bancrofti infection revealed that age, season, elevation and all remotely sensed environmental factors were significant risk factors. High risk was related to high average annual rainfall and low dry season NDVI. The positive associations with day- and night-time temperatures and season were no longer significant in the Bayesian multivariate models. Older children had a significantly higher risk of being CFA positive, whereas sex was not significant at the 15% significance level and thus not included in the Bayesian models. The spatial decay parameter in the geostatistical model had a posterior mean of 1.00 which in the current exponential setting corresponds to a minimum distance for which the spatial correlation becomes (less than 5%) of 3 km ), indicating the presence of spatial autocorrelation in the filariasis data.The non-spatial bivariate analysis of The best performing models according to the model validation procedure were employed to predict infection risk at the un-sampled locations represented by the app. 60,000 grid cells across Uganda. The predicted risk maps of single malaria and lymphatic filariasis infections are shown in Figure Plasmodium sp. was lower for girls and older children, and the proportion infected with W. bancrofti lower for younger children. Risk maps for all age-sex groups are available from the corresponding author upon request.Since age remained a significant risk factor for both malaria and lymphatic filariasis after adjusting for confounding factors and spatial correlation, prediction maps were developed for each age group separately. For malaria this was also the case for gender. The spatial patterns were identical for each age-sex group, though the proportion infected with 2 and summarizing across the whole study area gave the total number of infected children in each age-sex group in Uganda. The estimated number of children at risk in the respective age-sex groups is presented in Table 2 with either malaria or filarial parasites is illustrated in Figure The age and sex stratified 2002 population surfaces was multiplied with gridded surfaces of the prediction models representing the mean and the lower and upper 95% BCI of the predicted prevalence in each age-sex group, for each parasite infection separately. The resulting layers gave maps of the predicted number of infected children per kmW. bancrofti single-infections were superimposed to identify geographical areas where hyper-endemic malaria (> 50%) and hyper-endemic lymphatic filariasis (> 10%) overlap in Uganda. Figure The predicted prevalence maps for malaria and With recent advances in geospatial tools and spatial statistics, studies analysing the relationship between infection dynamics and ecological factors have given new insights into the ecology and epidemiology especially of malaria ,15,42 - In the present study, nationally representative survey data of malaria and lymphatic filariasis in Uganda were applied to identify significant predictors associated with parasitaemia and antigenaemia risk. Model-based infection risk maps for 2002 were produced and the number of infected children estimated for three different age groups.The map of malaria is the first for Uganda based on empirical data sampled in a standardised and coherent manner. The survey locations are randomly chosen and the data are available at individual level allowing for estimation of age-specific risk, as well as adjusting for seasonality. Even though the data were collected during 2000-2003, the maps produced still represent the most up-to-date and detailed maps of malaria and lymphatic filariasis risk in the country. On-going LF and malaria control initiatives in Uganda are likely to have changed the patterns, thus the maps can serve as an important baseline to guide and evaluate the continuous control implementation.et al [et al [Prior to the present analysis, historical malariological data were compiled by the MAP   and MARAet al  developel [et al  as part et al [The first risk map of lymphatic filariasis in Uganda was produced by Onapa et al  based onet al . Here, tet al . FurtherBayesian geostatistical modelling has previously been applied to lymphatic filariasis data in Haiti ,48, WestMore commonly, Bayesian geostatistical modelling has been applied to malaria data and used to estimate parasitaemia risk for a number of countries and regions in Africa, including amongst others West and Central Africa -52, AngoAn important advantage of Bayesian geostatistical analysis is that it takes the spatial correlation often present in many epidemiological data sets into account and model the effects of the co-variates and spatial correlation, if present, simultaneously. If spatial correlation is present the independence assumption central to generalized linear model theory is violated, which could potentially lead to imprecise risk estimates, prediction errors and wrong estimation of the significance of the risk factors.W. bancrofti infection estimated a spatial correlation with a minimum distance at which spatial correlation becomes negligible of ~ 3 km. In accordance with this the spatial models also outperformed the non-spatial LF models in terms of number of test locations captured within the 95% posterior credible intervals and their widths. In line with our findings, Alexander et al [W. bancrofti in Papua New Guinea was reduced by half over a distance of 1.7 km and Boyd et al found that the distance beyond which remaining spatial correlation is assumed to be negligible to be 2.15 km for W. bancrofti in school-children in Leogane, Haiti [The geostatistical model of er et al  found the, Haiti .et al [et al found the risk of malaria varying by a factor of 6 over 500 m [et al found distance over which the correlation reduces by half to be only 14 meters [The malaria data analysed in the present study on the other hand, showed signs of very weak spatial correlation. Thus attempting to model the spatial correlation could lead to increased number of model parameters while decreasing the precision of the estimates. The Bayesian geostatistical model developed estimated a lower spatial correlation than the pixel size used for prediction (2 × 2 km) indicating that spatial correlation is only present at very local scales. Validation confirmed that the fitted non-spatial malaria models were superior to the fitted spatial models. A similar result was found by Riedel et al  when mapet al ,60,61. Fer 500 m , while A4 meters .W. bancrofti infections, which also last much longer and the risk of infection per mosquito bite is much smaller [The much smaller scale of variation of malaria could be related to its rapid variation in infection over time. One infective mosquito bite results in patent infection about two weeks later. By comparison, the patent period is about one year for  smaller .Besides from mapping, important common environmental risk factors for malarial and filarial infections in Uganda have been highlighted. For both malaria and lymphatic filariasis, the bivariate analysis indicated that the season the data was collected in was important with the wet season months having higher disease risk. However, in the multivariate models this relationship only remained significant for malaria reflecting the stronger seasonality of malaria transmission.et al [Plasmodium sp. infections, with the youngest age-group being significantly more at risk. This was also the findings of Pullan et al [As also presented by Onapa et al  older agan et al  in a recIn contrast to the well-characterized patterns of single parasite infections, we know surprisingly little about the patterns and risk factors of co-endemicity and co-infection of e.g. malaria and helminth infections, despite poly-parasitism being common among populations in developing countries . Co-endeIn Uganda, the same species of Anopheles mosquitoes transmit malaria and lymphatic filariasis ,30 and cand lymphatic filariasis (blue area in Figure A substantial geographical overlap was observed, with an estimated 212,975 children between five and nine years of age living in areas that are hyper-endemic for malaria W. bancrofti antigenaemia is not fully overlapping with hyper-endemic malaria areas (Figure The geographical distribution of lymphatic filariasis in Uganda was expected to overlap with that of malaria, in particular in very high malaria transmission areas where the density of the transmitting mosquitoes is expected to be high. Surprisingly however, the distribution of s Figure , despiteA possible explanation for this pattern could be that in some locations, even though data were collected during wet season months, those years and locations were found to be exceptionally dry for the season. This could have impacted the observed prevalence of malaria resulting in lower than usual number of infections; whereas lymphatic filariasis with its much longer prepatent period would remain unchanged even in a dry year. It could also be partly due to the difference in "high prevalence age groups" between the two infections, with far the majority of CFA positives found in age group 15-19 years, the age group with the lowest observed malaria infection.P. falciparum is generally lower in microfilaremic individuals compared to amicrofilaraemic individuals [Some studies have also shown that the intensity of ividuals  and thatividuals . Thus, tividuals ,68 and uThe age-sex stratified risk maps of malaria and lymphatic filariasis presented here for Uganda are based on national, coherent survey data collected during 2000-2003 and rigorous Bayesian geostatistical modelling and prediction. They can serve as an important baseline to guide and evaluate the continuous implementation of single infection control activities. The maps were furthermore used to identify areas of hyper- and co-endemicity to help provide a better informed platform for integrated control.Parasitic co-infection and interaction phenomena in human populations are complex and our understanding of the mechanisms by which interactions between parasites occur and the public health implications is still limited. Further studies on malaria-filarial co-infection, co-occurrence, prevalence and species interactions in various geographical settings are clearly warranted. Besides the immediate public health implications, identifying high-prevalence zones of human parasitic infections and their spatial overlap may also help generate new insights and hypotheses regarding their interrelationships and transmission.LF: Lymphatic filariasis; CFA: circulating filarial antigens; NDVI: Normalized difference vegetation index; MCMC: Markov Chain Monte Carlo; LST: land surface temperature; BCI: Bayesian credible interval.The authors declare that they have no competing interests.AS and AWO conceived the idea for the present study. AS analysed the data and drafted the manuscript. PV was responsible for the conception and design of the statistical analysis and supervised the implementation. AWO, PES and EMP designed and carried out the parasitological surveys and provided important intellectual content to the study. CR and TKK gave critical input and re-appraisal in the manuscript. All authors read and approved the final manuscript.Additional information regarding model formulation. Supporting information on Bayesian model formulation for malaria and lymphatic filariasis,Click here for file


Although several studies have investigated the impact of reduced malaria transmission due to insecticide-treated bed nets (ITNs) on the patterns of morbidity and mortality, there is limited information on their effect on parasite diversity.Plasmodium falciparum vaccine candidate antigens, the 19 kilodalton blood stage merozoite surface protein-1 (MSP-119kDa) and the Th2R and Th3R T-cell epitopes of the pre-erythrocytic stage circumsporozoite protein (CSP) in a large community-based ITN trial site in western Kenya. The number and frequency of haplotypes as well as nucleotide and haplotype diversity were compared among parasites obtained from children <5 years old prior to the introduction of ITNs (1996) and after 5 years of high coverage ITN use (2001).Sequencing was used to investigate the effect of ITNs on polymorphisms in two genes encoding leading 19kDa haplotypes were detected in 1996 and 2001. The Q-KSNG-L and E-KSNG-L haplotypes corresponding to the FVO and FUP strains of P. falciparum were the most prevalent (range 32–37%), with an overall haplotype diversity of > 0.7. No MSP-119kDa 3D7 sequence-types were detected in 1996 and the frequency was less than 4% in 2001. The CSP Th2R and Th3R domains were highly polymorphic with a total of 26 and 14 haplotypes, respectively detected in 1996 and 34 and 13 haplotypes in 2001, with an overall haplotype diversity of > 0.9 and 0.75 respectively. The frequency of the most predominant Th2R and Th3R haplotypes was 14 and 36%, respectively. The frequency of Th2R and Th3R haplotypes corresponding to the 3D7 parasite strain was less than 4% at both time points. There was no significant difference in nucleotide and haplotype diversity in parasite isolates collected at both time points.A total of 12 MSP-1High diversity in these two genes has been maintained overtime despite marked reductions in malaria transmission due to ITNs use. The frequency of 3D7 sequence-types was very low in this area. These findings provide information that could be useful in the design of future malaria vaccines for deployment in endemic areas with high ITN coverage and in interpretation of efficacy data for malaria vaccines based on 3D7 parasite strains. Plasmodium falciparum continues to place a heavy burden on the health and economic development of populations in endemic areas [Malaria caused by ic areas . Over thic areas  ITNs havic areas ,4 and thic areas . Previouic areas . Based oic areas . Studiesic areas ,9. The fic areas -14.P. falciparum parasites. Malaria transmission intensity has been shown to play an important role in determining the genetic diversity of malaria parasites [However, there is limited information on the impact of transmission reduction as a result of sustained use of ITNs on the genetic diversity of arasites . This isarasites . In areaarasites ,17. Thisarasites .P. falciparum after five years of high ITN coverage except for a significant MS locus specific change associated with gametocytes [P. falciparum vaccine candidate antigens during the same study period and in the same study area in western Kenya. This study assessed single nucleotide polymorphisms (SNPs) in genes encoding the relatively conserved B-cell epitopes of the blood-stage 19-kDa region of the merozoite surface protein (MSP-1 19kDa) and the highly polymorphic pre-erythrocytic-stage circumsporozoite protein (CSP) Th2R and Th3R T-helper cell epitopes. These two genes were chosen for assessment because their polymorphism is thought to be partly driven by the host immune pressure and are likely to be subjected to the diversifying effects of changes in malaria transmission [P. falciparum vaccine candidate antigens over time will help better understand the relationship between transmission intensity as malaria interventions are scaled up and the diversity of parasite antigens that are under selection pressure. In addition, these data will be useful in interpreting efficacy data of malaria vaccines that are based on 3D7 sequences.A previous study conducted by this same group using neutral microsatellite (MS) markers has shown an overall stability in genetic diversity of etocytes . The cursmission . Monitor19kDa) adhering on the surface of merozoites during erythrocyte invasion [19kDa contains two cysteine-rich epidermal growth factor (EGF)-like motifs that play an important role in erythrocyte invasion, making it an ideal target of inhibitory antibodies for blocking parasite invasion of erythrocytes [19kDa[19kDa have undergone field trials with varied results [The merozoite surface protein-1 (MSP-1) is the major protein on the surface of asexual blood stages of the malaria parasites . The MSPinvasion . The MSPhrocytes  and theres [19kDa,22. Seve results .P. falciparum csp gene encodes a protein of approximately 420 amino acid residues with a molecular weight of 58 kDa consisting of two non-repetitive regions at the 5' and 3' ends and a variable central region that encodes immunodominant B-cell epitopes with multiple repeats of four amino acid motifs [csp gene and malaria transmission, with the highest diversity observed in parasite populations from Africa [The CSP is the predominant protein found on the surface of sporozoites . The P. d motifs . The C-td motifs . The CSPd motifs . The cend motifs ,28. Studm Africa .P. falciparum polymorphic vaccine candidate antigens in 1996 before and 5 years after the deployment of ITNs in an area of western Kenya holoendemic for malaria. Prior to the ITN trial, malaria transmission in this area was intense and perennial with an estimated entomologic inoculation rate (EIR) of 60 to 300 infective bites per person per year [The current study investigated the impact of transmission reduction due to the use of ITNs on the genetic diversity of genes encoding two per year . It was This study was conducted in the context of a community-based cluster randomized controlled trial of ITNs in western Kenya that consisted of two phases. Phase 1 was designed to assess the impact of ITNs on all-cause mortality in young children while phase 2 consisted of an extended surveillance to monitor whether sustained use of ITNs during infancy increased all-cause morbidity and mortality in older children. The characteristics of the study area, design of the ITN trial and the two-year surveillance have been described in detail elsewhere ,30. BrieAnopheles in the houses with ITNs and in the second phase, villages with ITNs experienced a 77% reduction in Anopheles densities compared with villages in an adjacent area [At the end of the two-year trial period, ITNs were also distributed to all control villages and monitoring continued for an additional two years until March 2001. Throughout the two phases, annual cross-sectional surveys were conducted in 60 villages to collect information on malaria morbidity indicators. During the surveys finger stick blood samples were taken among children aged below five years. In addition, entomologic indices were collected throughout the two phases of the trial to estimate malaria transmission. During phase 1 of the trial there was a 72% reduction in the density of indoor-resting blood-fed ent area . In addient area ,31. The ent area .The blood samples used in the current study were collected in October and November 1996, before phase 1 of the ITN trial and in May and June in 2001, 5 years after introduction of ITNs in phase 1. Microscopically confirmed malaria positive blood samples were randomly selected from those collected in the 1996 cross-sectional survey and matched by village with samples collected in the 2001 survey.-70°C until the day of DNA isolation.During each survey, 250-500 μl of blood was collected from each child by finger-stick into EDTA microtainers . Blood samples were transported in cool boxes to the central laboratory in Kisian, about 50 km from the field site and stored at 4°C until further processing. All samples were labelled with unique identification numbers that could not be linked to the randomization status of the sampled children. Erythrocytes were separated from plasma by centrifugation at 700 G for 5 min. Packed red blood cells (RBCs) were aliquoted into sterile vials and stored at 2, 250 μM dNTPs, and 2.5 U Taq DNA polymerase . The cycling conditions were as follows: 95°C for 5 min, 58°C for 2 min and 72°C for 2 min for 1 cycle, then 94°C for 1 min, 58°C for 2 min and 72°C for 1 min for 32 cycles followed by 10 min extension at 72°C. The primers were designed to hybridize to conserved regions covering the Th2R, Th3R and the universal T-cell epitopes, resulting in a 354 base-pair fragment. The MSP-119-kDa gene was amplified in a 25 μl reaction mixture containing 1 μl of DNA, 0.2 μM of two forward primers- 5’ CGTTTTATCTAATTTACTTGATGGAA 3’(K16F), specific for K1 and 5’ CCTAATACAATAATATCAAAATTAATTGA 3’ (M16F), specific for MAD-20 and a common reverse primer 5’ TTAAGGTAACATATTTTAACTCCTAC 3’ (C3flaR), 2.0 mM MgCl2, 200 μM dNTPs, and 1.25U Taq DNA polymerase with the following cycling conditions: 91°C for 1.5 min, then 91°C for 30 sec, 50°C for 40 sec and 70°C for 40 sec for 40 cycles followed by 5 min extension at 72°C. PCR was carried out in a GeneAmp® PCR System 9700 .Parasite genomic DNA was extracted using the QIAamp® DNA Mini Kit  according to the manufacturer’s instructions. The CSP gene was amplified by polymerase chain reaction (PCR) in a 25 μl reaction mixture containing 1 μl of DNA, 0.2 μM of forward and reverse primers CSP-F: ACAATCAAGG TAATGGACAAGG and CSP-R:GGATTAATAATGGTATTATCCTTCT, 1× TBE buffer, 2.0 mM MgClThe PCRs products were cleaned using CentriSep spin columns  according to the manufacturer instructions. Sequencing reactions were carried out using forward and reverse primers in separate reactions by the BigDye® Terminator v3.1 Cycle Sequencing Kit and analysed in an ABI 3100 DNA Sequencer . The ChromasPro, version 2.31 software  was used to align the sequences. Each sequence was manually checked for true base calling and samples with doubtful sequencing results were repeated.19-kDa sequences were aligned with known sequences from the P. falciparum genome database at the National Center for Biotechnology Information (NCBI) using the P. falciparum clone 3D7 as the reference sequence while the CSP Th2R and Th3R sequences were aligned using the 3D7 and 7G8 clones. Molecular analyses were conducted using MEGA version 4 [Pfmsp-119-kDa) and BankIt1630320: KF158556 - KF158712 .The MSP-1ersion 4  and DNA ersion 4  softwareersion 4 , MEGA veersion 4  and ARLEersion 4 . NucleotThe study was approved by the Ethical Review Committee of the Kenya Medical Research Institute, Nairobi, Kenya and the Institutional Review Board of the Centers for Disease Control and Prevention (CDC) Atlanta, Georgia, USA.Written informed consent was obtained from the patient’s guardian/parent/next of kin for the publication of this report and any accompanying images.19-kDa, 58 at baseline and 78 in 2001. Overall, seven non-synonymous amino acid replacements were detected at positions 1644 (Q-E), 1691 (K-T), 1695 (E-K), 1699 (S-N), 1700 (S-N), 1701 (G-R) and 1716 (L-F). Based on the amino acid replacements, a total of 12 MSP-119-kDa haplotypes were detected in the study area in 1996 and 2001. Out of these, 3 haplotypes, Q-TESNGL, E-TESSGL and E-TESNGF are new and have not been reported before. Out of the 12 haplotypes identified, 8 haplotypes were observed in the 1996 and 2001 surveys while 4 haplotypes were observed at only one time point, 2 in 1996 and 2 in 2001. The E-KSNGL and Q-KSNGL haplotypes corresponding to the FUP-Uganda PA and FVO-Wellcome strains of P. falciparum had the highest prevalence in 1996, 37 and 32% respectively, and in 2001 it was, 37% for both strains  of 0.0038 and 0.0033 respectively  out of 175 parasite samples yielded interpretable sequences and were included in the analysis. Overall, there was a high diversity on the CSP C-terminal domain containing immunodominant T-cell Th2R and Th3R epitopes  in areas of high transmission and high EIRs ranging from 50 to 600 infective bites person per year, [19-kDa haplotypes in 1996 and 2001. These two haplotypes have been reported at high frequencies in previous studies conducted in this area and other regions in Africa. In a study conducted in the same area in 1998, Qari et al reported frequencies of 42 and 21% for the E-KSNGL and Q-KSNGL haplotypes respectively [19-kDa haplotypes was very low.For the gene encoding the blood stage MSP-1er year, ,22,36 coer year, ,38. The ectively . In the 19-kDa haplotypes, haplotype diversity and π. The stability in the frequency of haplotypes and the genetic diversity overtime, suggests that the MSP-119-kDa gene could be under balancing selection as documented in previous studies [MSP-119-kDa gene was relatively stable for a period of 15 years [19-kDa haplotypes reported in a study conducted in an area of low malaria transmission [MSP-119-kDa haplotypes observed in this study over a period of 5 years suggests that the rates of meiotic recombination in parasite populations in this area remain high despite the significant reduction in transmission associated with the deployment of ITNs.There was no significant change overtime in the frequency, the number of MSP-1 studies -42. Simi15 years . This cosmission . This dismission . TherefoP. falciparum was not detected at baseline and was less than 4% in 2001. Low frequencies of MSP-1 3D7 sequence-types has been reported in previous studies conducted in different malaria transmission areas [P. falciparum could compromise the efficacy of MSP-1 vaccines that are based on the 3D7 clone. For example, a recent phase IIb trial of an MSP-142-based vaccine candidate derived from the 3D7 clone of P. falciparum in children aged 12-27 months in a nearby study area reported an overall vaccine efficacy of 5.1% [42 gene in this area [P. falciparum antigens [The frequency of the MSP-1 haplotype (E-TESSRL) corresponding to the 3D7 strain of on areas ,36,43,44 of 5.1% . The poohis area . Similarantigens . TherefoP. falciparum and comprises part of the repeat, Th2R and Th3R regions of the CSP. Although there is no evidence that the observed efficacy of the RTS,S vaccine is sequence-dependent [For the highly polymorphic Th2R and Th3R T-helper cell epitopes, there were 26 and 34 Th2R haplotypes at baseline and 5 years post-ITNs respectively and 14 and 13 Th3R haplotypes at the two time points. These findings are consistent with results of an earlier study conducted in the same area that reported 29 and 16 haplotypes for the Th2R and Th3R epitopes respectively . This suependent ,50 the iCSP gene was also apparent when measured by haplotype diversity and π. These findings are consistent with previous studies that have documented high genetic diversity in ThR2 and Th3R regions of CSP in parasite populations from regions of high malaria transmission [MSP-119-kDa gene, there was no significant difference in genetic diversity of the two T-cell epitopes in parasite populations at baseline and 5 years post-ITN intervention. The relative stability in the diversity of Th2R and Th3R regions has also been documented over a period of 5 years in a region of low transmission in Vietnam [CSP gene.The high genetic diversity in the T-cell epitopes of the smission ,51,52. Tsmission ,53, Indismission  and Iran Vietnam . Taken tP. falciparum genes. A few factors could explain this observation. Firstly, despite a decline in transmission intensity following the deployment of ITNs, parasite prevalence remained relatively high and 50% of children sleeping under ITNs were parasitaemic during phase 1 of the ITN trial (1996-1998) [Several factors have been suggested to play a role in the generation and maintenance of parasite diversity including transmission intensity, immune-mediated pressure, parasite positive rates, rates of multiple-genotype infections and patterns of antimalarial drug use . In the 96-1998) , and > 396-1998) . In phas96-1998) . It is a96-1998) . The sho19kDa haplotypes have remained stable overtime and transmission reduction due to ITNs had no impact on the diversity of MSP-119kDa and the Th2R and Th3R domains of the CSP. The results also show that the CSP Th2R region is more diverse than the Th3R region and could be under strong selective pressure unrelated to the intensity of malaria transmission. The information on low frequency of 3D7 clone could be useful in interpreting data on the efficacy of malaria vaccines based on the 3D7 parasite strain in areas of stable malaria transmission and in informing the development of future malaria vaccines for deployment in areas of high transmission.Data obtained in this study show that the MSP-1The authors declare they have no competing interests.SK, YPS, FK, DT, WH, PP-H, BL KL, MH, and LS designed the study. SK, JN, AM, and GM collected the molecular biology data. JW, SK, YPS, WG, JN, AM participated in the data analysis. SK and YPS wrote the paper. All authors read and approved the final manuscript.