Plasmodium falciparum has been poorly documented in Senegal, and limited data are available from the Kedougou Region. This study examines the spatial and temporal variation of the genetic diversity and complexity of P. falciparum infections in acute febrile patients in Kedougou, southeastern Senegal. A total of 263 sera from patients presenting with acute febrile illness and attending Kedougou health facilities between July 2009 and July 2013 were obtained from a collection established as part of arbovirus surveillance in Kedougou. Samples identified as P. falciparum by nested PCR were characterized for their genetic diversity and complexity using msp-1 and msp-2 polymorphic markers.Genetic analyses of the malaria parasite population and its temporal and spatial dynamics could provide an assessment of the effectiveness of disease control strategies. The genetic diversity of P. falciparum accounted for 60.83% (160/263) of the examined samples. All three msp-1 allelic families  and two msp-2 allelic families (FC27 and 3D7) were detected in all villages investigated over the 5-year collection period. The average genotype per allelic family was comparable between villages. Frequencies of msp-1 and msp-2 allelic types showed no correlation with age  or gender , and were similarly distributed throughout the 5-year sampling period  and across villages . Mean multiplicity of infection (MOI) for both msp-1 and msp-2 was highest in Kedougou village  and among younger patients aged ≤ 15 years . The mean MOI was highest in 2009 and decreased progressively onward.Samples containing only P. falciparum infections in Kedougou revealed no spatio-temporal variation in the genetic diversity of P. falciparum isolates. However, mean MOI varied with time of sera collection and decreased over the course of the study (July 2009 to July 2013). This suggests a slow progressive decrease of malaria transmission intensity in Kedougou Region despite the limited impact of preventive and control measures implemented by the National Malaria Control Programme on malaria morbidity and mortality.Characterization of the genetic diversity and complexity of  According to the World Health Organization (WHO), the incidence of malaria and associated mortality respectively decreased by 30 and 47% globally between 2000 and 2013  was found to be significantly higher  than those harboring only the FC27 allelic family [29.37% (47/160)] ] Fig. . Dimorph0)] Fig. .msp-1 and msp-2 allelic types were categorized within two arbitrarily defined age groups: ≤ 15 years and > 15 years  and two msp-2 allelic types (FC27 and 3D7) were similarly distributed with no significant differences between age groups   Fig. . Within msp-1 and msp-2 allelic families showed similar distribution between males and females with no significant difference observed . The frequencies of all msp-1 and msp-2 allelic families were also comparable within a given gender  throughout the 5-year sampling period  between villages with a consistent low frequency of all allelic types in Ninefesha , in patients aged ≤ 15 years (MOI = 2.12 and 2.00), and those diagnosed in 2009 (MOI = 2.21 and 2.50) than P. falciparum isolates originating from others villages, patients aged older than 15 years, and those diagnosed from 2010 onward, respectively , age groups (≤ 15 years and > 15 years), and sampling periods (2009 to 2013). The mean MOI for both P. falciparum parasite population among febrile patients in Kedougou Region. This bridges an important gap in the understanding of the molecular characteristics of the parasite population in this region where Plasmodium transmission is still active and could inform efforts to monitor and control Plasmodium transmission in this region.The present study provides information about the spatial and temporal dynamics of the msp-1 and msp-2 allelic families were represented in all villages in Kedougou Region where samples were collected and elevated frequencies of msp-1 and msp-2 genotypes were reported with MAD20 and 3D7 being the most prevalent, particularly in Bandafassi and Kedougou villages. However, the comparable average number of genotypes for each locus between villages suggested that the large number of genotypes in Bandafassi potentially reflects an elevated number of samples rather than higher genetic diversity of P. falciparum isolates in this village.All Plasmodium-infected individuals [P. falciparum isolates from both asymptomatic and symptomatic individuals to comprehensively establish the potential relationship between specific allele carriage and disease outcome. High to moderate mean MOI was observed among P. falciparum isolates across all the studied villages with the highest mean MOI being observed in Kedougou village. A high MOI is a common feature in most malaria hyperendemic areas [msp-1 and msp-2 loci from 2009 to 2013 in Kedougou Region could suggest a progressive decrease of Plasmodium transmission although a lack of data prior major interventions by the NMCP in Kedougou Region does not allow for a thorough comparison.FC27 allelic type has been shown to be more prevalent in asymptomatic rather than symptomatic ividuals , suggestividuals , 13 and ividuals  but contividuals , 27, empic areas –30 and hic areas , 28, 30.Plasmodium population structure in relation to changing malaria transmission in Senegal, thus precluding a meaningful comparison with the situation observed in Kedougou Region. The application of molecular barcoding genomic surveillance on P. falciparum parasites collected from Thies region  between 2006 and 2013 revealed reduced genetic diversity of P. falciparum population along with a decline of Plasmodium transmission from 2006 to 2010, a period transcending the introduction of insecticide-treated bednets in 2008 [Plasmodium transmission documented in Senegal [To date, only a few studies have applied genetic surveillance tools to evaluate  in 2008 . The mas Senegal , 33 and  Senegal .P. falciparum genetic diversity conducted in Senegal failed to establish a link between MOI and age groups [The finding that MOI decreased with age supports studies conducted in other countries , 36 thate groups –12, thusP. falciparum infections in acute febrile patients in Kedougou, a Senegalese region with active malaria transmission. The findings revealed that P. falciparum isolates of Kedougou Region are highly diverse with limited spatio-temporal variation in the region. However, MOI decreased over time, which may be an indication of a progressive reduction in Plasmodium transmission in the region despite the limited impact of control measures on malaria morbidity and mortality in the area.This study reports the spatial and temporal variation of the genetic diversity and complexity of P. falciparum parasite populations, considering factors such as the malaria transmission intensity based on entomological inoculation rates and the immune status of P. falciparum-infected individuals would tremendously improve our understanding of P. falciparum parasite diversity in Kedougou Region and contribute to guide malaria interventions.Additional studies that examine the dynamics of the genetic diversity of


Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection.Iron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint.This observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children  participating in an iron supplementation trial  in which they received iron (12 mg/day) as part of a micronutrient powder for 84 days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based P. falciparum in vitro (~ 10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49 days of iron supplementation relative to baseline (p < 0.001), paralleling increases in erythropoiesis.Anemia substantially reduced the invasion and growth of both laboratory and field strains of falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African descent may reflect past genetic selection by malaria.These results confirm and quantify a plausible mechanism by which anemia protects African children against National Institute of Child Health and Development, Bill and Melinda Gates Foundation, UK Medical Research Council (MRC) and Department for International Development (DFID) under the MRC/DFID Concordat. •P. falciparum laboratory and field strains invade and grow less efficiently in RBCs from anemic children.•P. falciparum are reversed when RBCs are used from anemic children receiving iron supplementation for 49 and 84 days.Deficits in invasion and growth for erythrocytic stage •The population level impact of protection against malaria from anemia was greater than that for sickle-cell trait.in vitro P. falciparum assays. P. falciparum invasion and growth was decreased in anemic RBCs and increased after 49 days of iron supplementation relative to baseline (p < 0.001), paralleling increases in young RBCs, which the parasite prefers. The parasite growth protection from anemia was substantial, providing greater population level impact than sickle-cell trait.The long-term consequences of anemia are severe, and it is easily treatable. However, concerns remain about the safety of iron supplements, particularly for children in malaria-endemic countries lacking adequate access to health services. We used RBCs from Gambian children before, during, and after 12 weeks of daily iron supplementation for  Acute malaria can cause severe anemia due to hemolysis of infected and uninfected RBCs, and chronic or subclinical malaria can induce anemia of inflammation . There iMultiple studies have raised concern that iron supplementation in malaria-endemic areas may put people at increased risk of acquiring malaria . Most imP. falciparum growth in vitro in RBCs from anemic African children before, during, and after 12 weeks of iron supplementation.In an effort to assess the magnitude of protection from anemia and the safety of iron supplementation in a malaria endemic area where sickle-cell trait is common, we have systematically characterized 22.1The blood samples for the parasite assays were taken from children enrolled in the control arm of a randomized trial testing the efficacy and safety of a hepcidin-guided screen-and-treat strategy for combatting anemia (see published protocol for full details) . Note w.in vitro P. falciparum growth characteristics to model malaria susceptibility in anemic subjects before and after iron supplementation. We compared subject characteristics of those whose blood was and was not able to be used for growth rate data to ensure no sampling bias occurred . For a full description of this embedded observational study, please see the published protocol . Field workers visited children daily in order to supervise the micronutrient powder administration and check the children's health status. For baseline population characteristics, see Supplemental Table 1. Fresh RBCs were obtained from these anemic (Hgb < 11 g/dl) but otherwise healthy children (6–23 m) living in rural Gambia . Blood wprotocol .2.2Parasite lines FCR3-FMG  and 3D7  were routinely cultured in RBCs from healthy donors using standard methods . Parasit2.3In vitro growth was assessed in fresh, washed RBCs as in  and isotype control , and analyzed by flow cytometry  for reticulocyte percent relative to non-anemic control.Reticulocyte (CD71 +) levels in fresh subject RBCs were assessed using PE-conjugated 2.6t-test, one-way ANOVA, and/or 95% CI values using GraphPad Prism 5.All experiments were done in triplicate. Growth rates, invasion assays, and hematological data were compared by two-tailed Student's 2.7in vitro parasite growth rates. First, bivariate associations and their respective 95% CI were calculated between growth rates and hematological and patient characteristics at Day 0. We then used multivariate linear regression. We used directed acyclic graphs to identify potential confounders and controlled for them in our modelling approach .We employed linear regression to estimate the effect of hematological characteristics on approach . An a pr2.8in vitro data on the erythrocytic stage growth of the malaria parasite as a proxy measure for malaria susceptibility, we compared the relative protection offered by sickle-cell trait carriage and anemia using the following formula: pp(RG-1)/RG, where pp is the percentage of the population exposed to the protective factor and RG is the relative in vitro parasite growth rate associated with that factor. The RG values for sickle-cell trait and hemoglobin were based on the standardized β coefficients from our multivariate modelling results. In this population of Gambian children, the pp for anemia is 0.75  (Using our n Study)  and the n Study) . This ca2.9The trial from which children were recruited was approved by the MRCG Scientific Coordinating and The Gambia Government/MRC Joint Ethics Committees (SCC 1358) and the UNC IRB (14-1551) which conform to Declaration of Helsinki standards. Parents/guardians were given a full description of the study in their native language and provided written signed consent.33.1in vitro parasite growth in RBCs from anemic children at baseline, we consistently found lower parasite growth rates than in RBCs from iron replete individuals. Furthermore, growth was lower in RBCs from those donors with the lowest hemoglobin concentrations (Hgb 7–9 g/dl = mean relative growth rate (GR) 32.6%; Hgb 8.1–10 g/dl = GR 45.9%; Hgb 10.1–11 = GR 55.9%; p < 0.05 by ANOVA) (e.g. with inflammation or without) and severity of iron deficiency, with no significant differences between groups  A. Iron py ANOVA) . Howevern groups .P. falciparum in vitro growth, hematological, iron, and inflammatory data obtained for subjects prior to iron supplementation to determine which variables influenced parasite growth in anemic children , as other β-globin genotypes (homozygous sickle-cell anemia (SS), hemoglobin C (AC), and a heterozygous combination (SC)) were rare. Hemoglobin concentration, hemoglobin genotype, and mean corpuscular volume (MCV) all significantly influenced parasite growth. G6PD status  did not significantly affect parasite growth, nor did age, sex, ferritin, hepcidin, or CRP  from healthy African-American children   and anemchildren ; using tchildren .3.3P. falciparum isolates  to ensure the observed decreased parasite growth in anemic RBCs was not solely a phenomenon of laboratory adaptation. These field isolates assayed in parallel in RBCs from 5 randomly chosen anemic subjects at baseline  all exhibited decreased growth compared to RBCs from non-anemic individuals  versus non-anemic donors. Susceptibility Indices (SI) of RBCs from the anemic donors were significantly decreased using both strains   also exhibited decreased invasion into RBCs from anemic donors   C. P. fal49–0.75) D. These ent RBCs .3.5in vitro parasite growth 49d and 84d after daily iron supplementation compared to baseline. The children were monitored daily for changes in health status and underwent weekly malaria testing. Consistent with the fact that malaria incidence is now low in The Gambia , increased markedly by Day 49 , and then by Day 84 decreased back to levels closer to those seen in non-anemic individuals . One-way ANOVA confirmed significant differences in parasite growth rates across Days 0, 49 and 84 (p < 0.0001) and post-hoc analysis using Tukey's test indicated significant differences between Days 0 and 49 (p < 0.001), Days 0 and 84 (p < 0.01), and Days 49 and 84 (p < 0.001) (p < 0.001)  A. Restri< 0.001) A.n = 8). The mean SI values of these donors before iron supplementation  and post iron  were significantly different by student's t-test (p < 0.01)  B.3.6p < 0.01), and Tukey's test showed significant difference between Days 0 and 49 only (p < 0.01)  was comparable to non-anemic controls (standardized as 100%), and increased at Day 49  and Day 84 . Means were significantly different by one-way repeated measures ANOVA ( < 0.01) C.n = 8). We examined changes in surface expression of: glycophorin A (GPA), a sialoglycoprotein affecting RBC charge; CD47, an anti-phagocytic RBC marker; C3b deposition on RBC surfaces; CD35, complement receptor 1; CD55, a decay accelerating factor regulating complement on the cell surface; CD147; and sialic acid, all of which can reflect RBC age and overall membrane integrity and/or have been implicated in malaria merozoite invasion. We found significantly increased GPA, CD47, CD35 and CD147 levels and significantly decreased C3b deposition at Day 49   . We were4in vitro growth assays as our primary outcome provided a rare opportunity to systematically examine the cellular determinants of parasite growth in anemic and iron-supplemented children. We demonstrate here that blood stage in vitro P. falciparum growth is decreased in RBCs from anemic children and this effect is reversed by iron supplementation.Use of Defining iron deficiency in children with ongoing infections or inflammation is difficult, and has confounded previous clinical studies trying to determine the protective effect of iron deficiency on malaria susceptibility. Here we show protection offered by anemia is substantial (~ 10% per standard deviation shift in hemoglobin), and RBCs from children with iron deficiency – no matter the definition criteria nor the presence of potential confounders such as inflammation – consistently reduce parasite growth compared to RBCs from non-anemic individuals. Additionally, the use of clinical parasite isolates from The Gambia confirms that this is not merely an artefact of laboratory strains. Notably, at the population level, anemia was estimated to confer at least four times as much protection against blood stage parasite growth than sickle-cell trait. Taken together, this data is evidence that anemia exhibits a profound natural influence on parasite growth beyond even the mostly commonly studied and referenced RBC polymorphisms which evolved due to malaria pressure.vs 120 days, on average) and exhibit physiological differences such as microcytosis, decreased deformability, and increased oxidative membrane stress, among other effects – similar to changes in aged RBCs (P. falciparum's preference for young RBCs (Furthermore, we demonstrate parasite growth increases dramatically relative to baseline in RBCs taken from children during iron supplementation, transiently rising at Day 49 to exceed growth rates in non-anemic controls and remaining elevated at Day 84 relative to baseline. Iron deficient RBCs have a shorter circulation lifetime (90 ged RBCs . As paraged RBCs , we asseung RBCs . The stuP. falciparum strains and that iron supplementation increased invasion susceptibility. Our RBC surface marker data corroborating a shift towards younger, healthier RBCs corresponds with our hypothesis that changes in RBC population structure influence overall malaria risk.We also examined merozoite invasion into RBCs from anemic and non-anemic individuals, as our previous work found invasion differences contributed significantly to reduced malaria pathogenesis in iron deficient RBCs . We expain vitro parasite growth might not translate directly to malaria susceptibility. Yet there are no other viable alternatives for addressing this safety aspect regarding iron supplementation in malarious regions. While our system only examined the RBC impact of anemia on malaria growth, eliminating the impact of serum iron or immune cells, the fact that we still observe such profound growth effects highlights the protection afforded by anemia and the need for caution regarding iron supplementation. Furthermore, our results provide insight into why other clinical studies on this topic produce such variable results – given we find increased malaria susceptibility is transient, other studies may miss the window of enhanced susceptibility. We detect significant changes in parasite growth rates despite relatively small changes in hemoglobin levels, emphasizing the impact of iron and RBC population dynamics on P. falciparum pathogenesis. Our data clearly show that the safety of iron supplementation must be addressed, even if additional unknown mechanisms contribute to increased malaria susceptibility. We thus advocate temporary malaria prophylaxis should always accompany iron supplementation for anemic children in malaria endemic areas, though the period of enhanced susceptibility has not been accurately identified by this study. Finally, quantifying the sizeable contribution of anemia to population level protection against malaria, our research raises the question of whether consistently reduced hemoglobin and MCV values in people of African descent are genetic signatures of evolution under significant malaria pressure, much like the hemoglobinopathies.The public health implications of our study are significant, shedding light on the overarching question of whether iron supplements cause harm. We acknowledge that Supplementary materialImage 1The following are the supplementary data related to this article.None of the funding sources had a role in study design, data collection or interpretation, writing of the manuscript, or the decision to submit for publication. The corresponding author had full access to all the data included in the study and assumed final responsibility for the decision to publish; all authors reviewed the report and agreed to submit for publication.P. falciparum isolates. JCP provided statistical support for data analysis. All authors reviewed and approved the final version.MMG, RW, AB, AMP, and CC designed the study and were involved in data analysis and interpretation, as well as writing. MMG, BD, ED, and DG participated in data collection. MA provided clinical We declare that we have no conflicts of interest.


Using the data of a trial on cotrimoxazole (CTX) cessation, we investigated the effect of different antiretroviral therapy (ART) regimens on the incidence of clinical malaria.+ at least 250 cells/μl were randomized to receive either CTX prophylaxis or placebo and were followed for a median of 2.5 years. Blood slides for malaria microscopy were examined at scheduled visits and at unscheduled visits when the participant felt unwell. CD4+ cell counts were done 6-monthly. Malaria was defined as fever with a positive blood slide. ART regimens were categorized as nucleoside reverse transcriptase inhibitor (NRTI) only, non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing or protease inhibitor containing. Malaria incidence was calculated using random effects Poisson regression to account for clustering of events.During the cotrimoxazole cessation trial (ISRCTN44723643), HIV-infected Ugandan adults with CD4+ cell count.Malaria incidence in the three ART regimen groups was 9.9 (3.6-27.4), 9.3 (8.3-10.4), and 3.5 (1.6-7.6) per 100 person-years, respectively. Incidence on protease inhibitors was lower than that on the other regimens with the results just reaching significance . Stratification by CTX/placebo use gave similar results, without evidence of an interaction between the effects of CTX/placebo use and ART regimen. There was no evidence of an interaction between ART regimen and CD4+ cell count. The antimalarial properties of protease inhibitors may have clinical and public health importance.There was some evidence that protease inhibitor-containing ART regimens may be associated with a lower clinical malaria incidence compared with other regimens. This effect was not modified by CTX use or CD4 In adin vitro–5. In chin vitro. Howeverin vitro. Studies+ cell count.The present study undertook a subanalysis of data collected in the COSTOP (cotrimoxazole cessation) trial (ISRCTN44723643), a study designed to investigate the safety of stopping cotrimoxazole (CTX) in HIV-infected adults stable on ART, to assess whether malaria incidence differed between participants receiving different ART regimens and whether any such effects were modified by CTX use or CD4+ cell counts at least 250 cells/μl.The study was conducted among COSTOP trial participants in Uganda, a country with high malaria endemicity ,9. COSTO+ cell counts were done 6-monthly. Participants diagnosed with malaria were treated in accordance with the national guidelines [Participants were followed every month for the first 3 months and 3-monthly, thereafter, for a median of 2.5 years. At enrolment participants were provided with an insecticide-treated bed net. Blood samples for malaria microscopy were collected at scheduled visits and unscheduled visits if malaria was suspected. CD4idelines . ParticiInformation on participants’ ART regimens at ART initiation and at enrolment was obtained from the records of the ART providers from where participants were recruited. During the trial, participants continued to receive ART from their usual providers, but trial staff ensured an uninterrupted supply of ART in case of unexpected shortages. Most participants were on an NNRTI-containing regimen (recommended first-line regimen in Uganda) , the rem+ cell counts and measured using a FACS-count system .Blood samples were used to prepare thick and thin films on a glass slide. Specimens were processed using Leishman's stain and examined by microscopy. Venous blood samples were taken for CD4+ cell count (<500 or ≥500) to assess whether the effect of ART regimen on malaria differed by CD4+ cell count.Data were double entered and verified in MS Access and analysed using Stata, release 13 . Person years at risk were calculated from enrolment until the date last seen or end of trial. After each malaria episode, participants were considered to be not at risk for another episode until the episode resolved, or for 28 days, if a resolution date was not available. The time to the first malaria episode was examined using Kaplan–Meier methods and log rank tests to compare between groups. The incidence of malaria and rate ratios comparing ART regimens were calculated using random effects Poisson regression to account for multiple episodes within the same participant. Models were adjusted for treatment group (CTX or placebo), enrolment site, and time since enrolment as a priori confounders. ART regimens were categorized as NRTI (nucleoside reverse transcriptase) only, if a regimen containing only NRTIs was used; NNRTI containing, if one of the drugs was an NNRTI and none was a protease inhibitor; or protease inhibitor containing, if at least one drug was a protease inhibitor. ART regimen was analysed as a time-updated exposure. The effect of ART regimen on malaria incidence was examined overall and separately for both treatment groups; stratified rate ratios were obtained from a model containing a term for interaction between treatment group and ART regimen. Analyses were also stratified by current CD4+ cell count less than 350 cells/μl at enrolment, and 569 (26%) were male. In total, 1721 (79%) participants had been on ART for at least 2 years. At the time of ART initiation, 2114 (97%) started on an NNRTI-containing regimen, 58 (3%) on an NRTI-only regimen, and three on a protease inhibitor -containing regimen; data were missing for five participants. At enrolment, 2046 (94%) were on an NNRTI containing regimen, 22 (1%) on an NRTI-only regimen and 86 (4%) on a protease inhibitor-containing regimen; enrolment ART information was missing for 26 participants (1%). Of those on a protease inhibitor-containing regimen, 75 (87%) were on lopinavir/ritonavir, 4 (5%) on atanazavir and 7 (8%) on another protease inhibitor. Of those on an NNRTI, 2066 (88%) were on nevirapine, 255 (12%) on efavirenz, and two on etravine. In total, 10 participants changed ART regimen during follow-up ; all changes were to a protease inhibitor-containing regimen .Baseline characteristics have been described previously . BrieflyP = 0.05; Fig. P = 0.05; Table In total, 2154 participants contributed data to the analysis; 447 malaria episodes were observed during follow-up, of which 40 (8%) were diagnosed outside the study clinic. There was some evidence that time to the first malaria episode was shorter for participants on an NNRTI-containing or NRTI-only regimen compared with those on a protease inhibitor-containing regimen  (CTX) and 1.6 (0.5–5.0) (placebo) times higher than those on an NNRTI-containing regimen, whereas that in participants on a protease inhibitor-containing regimen was 0.5 (0.1–2.3) (CTX) and 0.4 (0.2–1.0) (placebo) times lower, respectively. There was no evidence of interaction between the effect of treatment group and ART regimen  and this is consistent with findings of a study in children [We found that NRTI-only regimens provided the least protection against malaria followed by NNRTI-containing regimens. This is consistent with in-vitro studies that showed no antimalarial activity from NRTIs and some activity from NNRTIs but at levels which were not achievable d dosing ,16. Use We have previously reported that CTX use is associated with reduced incidence of malaria , an assoPlasmodium proteases rather than drug–drug interactions with lumefantrine.In a study in children, the effect of protease inhibitors on malaria was partially attributed to a reduction in malaria recurrence as a result of increased lumefantrine levels after treatment, owing to cytochrome P450 enzyme inhibition by lopinavir/ritonavir . These fPlasmodium falciparum liver stage parasites [These findings are potentially of benefit. It has been suggested that the antimalarial prophylactic effect of protease inhibitor-containing ART regimens could reduce the cost of care in malaria endemic countries because of a potential reduction in malaria treatment costs , could harasites , furtherarasites ,30. HoweOur study benefited from a well described study population that was followed regularly for up to 3.5 years and routinely assessed for malaria. However, although this offered an opportunity to investigate the effect of ART regimen on malaria, the COSTOP trial was not specifically designed to address this question. The number of participants on protease inhibitors was small, the observed protective effect just reached statistical significance, and therefore we cannot exclude the possibility that the effects might have occurred because of chance. As participants on protease inhibitors were on second-line therapy, their clinical condition may be different from those on first-line therapy; we were not able to adjust for potential confounders relating to individual health status. A history of fever (within previous 2 weeks) and parasitaemia were the basis for malaria diagnosis, this could have resulted in overestimation of cases, however 92% of cases were diagnosed by the study team, allowing for ascertainment of most diagnoses. Adherence to ART in our study population was high . HoweverIn conclusion, among HIV infected adults on ART, protease inhibitor-containing regimens were associated with reduced clinical malaria incidence compared with NNRTI-containing or NRTI-only regimens. The antimalarial properties of protease inhibitors may have clinical and public health importance.We are grateful to all the study participants and to the staff from the COSTOP study sites for their contribution, and to our ART providing partner institutions for their support in participants’ enrolment (The AIDS Support Organisation (TASO), Kisubi Hospital, Kitovu Mobile, Entebbe Hospital, Masaka Hospital, and Katabi Military Hospital). We acknowledge the valuable work of COSTOP Trial Monitors, the independent Trial Steering Committee, the independent Data Monitoring Committee, and the independent Endpoint Review Committee. Our work was supported through MRC (UK) grant number G0902150. This award was jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 programme supported by the European Union. KB receives support from the MRC UK and DFID (MRC grant number G0700837).There are no conflicts of interest.


In Vietnam, malaria persists in remote forested regions where infections are spatially heterogeneous, mostly asymptomatic and with low parasite density. Previous studies in Vietnam have investigated broad behavioural concepts such as ‘engaging in forest activities’ as risk factors for malaria infection, which may not explain heterogeneity in malaria risk, especially in malaria elimination settings.A mixed methods study combining ethnographic research and a cross-sectional survey was embedded in a 1-year malariometric cohort study in three ethnic minority villages in South Tra My district, Quang Nam Province in Central Vietnam. Qualitative data collection included in-depth interviews, informal conversations and participant observations over a 2-month period, and the findings were used to develop the questionnaire used in the cross-sectional survey. The latter collected data on evening activities, mobility patterns and household characteristics. The primary outcome, recent exposure to malaria, was defined using the classification and regression tree method to determine significant changes in antibody titres during the year preceding the survey. Risk factor analyses for recent exposure to malaria were conducted using logistic regression.Plasmodium falciparum malaria was estimated at 22.9 and at 17.1% for Plasmodium vivax. Ongoing malaria transmission appears to be maintained by activities that delay or disrupt sleeping in a permanent structure in which a bed net could be hung, including evening drinking gatherings, fishing, logging in the forest and outdoor TV watching.22 in-depth interviews and numerous participant observations were recorded during the ethnographic research (April to June 2015), and 160 adults (86% response rate) responded to the cross-sectional survey (November to December 2015). Recent exposure to Vector control tools for outdoor evening activities in villages as well as at farms, forest and river locations should be incorporated into current malaria elimination efforts in Central Vietnam. Micro-epidemiology studies using mixed-methods designs can provide a comprehensive understanding of the malaria risk at fine spatial scales and better inform the implementation of targeted interventions for malaria elimination.The online version of this article (10.1186/s12936-018-2262-0) contains supplementary material, which is available to authorized users. The burden of malaria in the Greater Mekong Subregion (GMS) has declined substantially over recent decades . The VieAnopheles dirus senso stricto demonstrates a preference for outdoor and early evening biting [Studies from Central Vietnam have previously shown that age, bed net use and spending nights at farms and fields located in forested areas away from the village were important risk factors for malaria infection –6. Howevg biting , 15, whiTherefore, this study aimed to gain a detailed understanding of human behaviours during vector biting times that may increase the risk of malaria infection in a low-transmission setting, to inform further improvement of malaria elimination activities in this region.The study was based in Tra Cang Commune, in Nam Tra My district, Quang Nam Province, Central Vietnam. Tra Cang had a population of approximately 4000 people at the time of the study, almost all Xe Dang, an ethnic minority population mostly living in the central mountainous inland regions of Vietnam. Tra Cang commune comprises seven administratively-defined villages each comprising several hamlets of Xe Dang households. Most Xe Dang families also maintain a house or hut at their farms and rice fields, and reside there according to seasonal work requirements.Plasmodium falciparum and Plasmodium vivax malaria transmission can occur year-round, with two peaks of transmission in June/July and October/November. Since 2005, malaria cases had declined substantially in Tra Cang, but a local outbreak occurred in 2012 and 2013, followed by a steady decrease since 2014 . Health care services in Tra Cang commune includes village health workers (one to two per village), a commune health centre (CHC) staffed by Xe Dang and Kinh (majority Vietnamese ethnic group) staff, and the district hospital, which is about half an hour by motorbike from the CHC. Malaria control activities include distribution of long-lasting insecticidal nets (LLINs), as well as provision of malaria testing (rapid diagnostic test and microscopy) and treatment  at the CHC and at two village malaria posts in Village 5 and Village 7. Indoor residual spraying of houses had not been systematically undertaken at the time of the study, though public buildings such as schools were sprayed during the last outbreak in 2012/13. Intensive treatment-based control efforts were conducted between 2012 and 2014 in response to the outbreak, including treating entire households with the first-line treatment where one malaria-positive case was detected by rapid diagnostic test (RDT), and treating entire hamlets when RDT-positive cases were detected in two or more households .Both Plasmodium spp. infection in all six surveys, as well as for serological analysis of antibody levels to two recombinant P. falciparum antigens , and twns (AMA1  and MSP1A1 [1–19 ) from thIn the mixed methods study, the qualitative ethnographic strand aimed to explore the local context and identify possible risk factors for malaria that had not previously been identified, and the cross-sectional survey aimed to test whether these risk factors were associated with recent malaria infection.Data collection and analysis for the qualitative and quantitative strands are described separately below, and reported according to the STROBE statement for reporting observational epidemiological research , supplemThe field research team comprised the first author, the local principal investigator (NXX) and research assistants from the National Institute of Malariology, Parasitology and Entomology (NIMPE), Hanoi, previously trained in qualitative ethnographic research methods. The study team resided in Tra Cang for 2 months (April to June 2015) supported by the health staff at the Tra Cang CHC. The research team from NIMPE had extensive field experience in similar settings, and had previously visited the study setting on several occasions during the MAPARES study.An open-ended research design centred on ethnographic methods guided the fieldwork. A purposive and adaptive sampling strategy was used to select informants, based on criteria such as history of malaria infection, mobility patterns, age, sex, and occupation. Emerging findings led to refinement of the sampling strategy and topic guides in order to maximize both variation and depth of information in the sample.Data collection methods included audio-recorded in-depth interviews, informal conversations , informal group discussions, and participant observation. Participants were usually first visited at their hamlet houses, but the study team also accompanied participants to their farms and fields, especially those who slept overnight in their plot huts. Interviews were also conducted in the general store, schools and CHC located in the administrative centre of Village 5. All interviews were conducted in Vietnamese, with local Xe Dang guides assisting as translators for some interviews. Field diaries and memos were maintained to aid data interpretation.Data were intermittently analysed in the field concurrent with data collection, leading to refinement of topic guides and further sampling. At the end of the fieldwork period, all data were imported into NVivo  for data management. Descriptive open coding and analysis focused on agricultural and social activities occurring at times and locations that may be associated with exposure to local vectors’ bites, but also describing the general socio-cultural context, health-seeking behaviour and local understandings of malaria.A simple random sample of 186 individuals aged 16 years and over was drawn from the population census of two hamlets (Xe Xua and Tu Nak), where malaria infections had been detected during the MAPARES study. The sample size calculation was based on detecting, with 80% power and at 5% significance, a minimum 20% difference in the odds of recent malaria exposure (defined by serology), for individuals staying overnight at forest farms and fields, compared to individuals who do not. As malaria sero-prevalence was unknown at the time of the cross-sectional survey, the maximum sample size  was used.The survey comprised a close-ended structured paper-based questionnaire. The questionnaire was piloted and revised, and the final version back-translated into English and checked for clarity. Trained surveyors from NIMPE administered the questionnaire at respondents’ households. Surveyors made up to three attempts to reach selected individuals. Data were double-entered and cleaned using Epi Info .P. falciparum antigens  and two P. vivax antigens  were defined for all participants in the first and last (sixth) MAPARES surveys. Subsequently, these continuous variables were categorized into four groups indicating the extent of seropositivity  zone, negative) using the classification and regression tree (CART) method [® software . Cut-off points were defined using CART regression on each antibody distribution in the first survey, and applied to the sixth survey  with a minimum absolute increase in PP in Survey 6, for one or more antibodies;Individual was weak or strong positive in Survey 1, and had either a minimum absolute increase in PP in Survey 6, or less than the relative minimum decrease, for one or more antibodies;Individual was strong positive in Survey 6 for one or more antibodies, if missing from Survey 1;Individual was strong positive in Survey 1 for one or more antibodies, if missing from Survey 6.The primary outcome was defined as recent exposure to malaria infection, estimated by serology and defined below. Serology was used because only four PCR-positive cases were detected during the six surveys, but also because serology outcomes may be more robust markers of exposure to malaria infection since it reflects cumulative changes in antibody titres over time, while PCR only detects malaria infections present at the time samples were taken –28. Indi) method  and impligens AMA, GLURP2 The research focused on identifying activities that may increase exposure to vector bites as risk factors for recent exposure to malaria infection. Based on the qualitative strand, the questionnaire included items about time spent in locations of presumed differential exposure to biting vectors , as well as evening and night time activities in these different locations that delay usual sleeping time and/or disrupt bed net use . Several household-level variables were also included, such as household size , number P. falciparum and P. vivax. In line with the causal modelling approach, the emphasis was on appropriate adjustment for confounding rather than statistical significance alone, with the number of exposure variables balanced against data scarcity, which was assessed by inflation of standard errors upon inclusion of additional variables. All exposure variables with p values less than 0.2 in the unadjusted analyses were included in the multi-variable models. Given the small sample size relative to the number of exposure variables, individual-level variables with p value exceeding 0.2 were removed from the multi-variable models, before household-level variables were included. Household-level variables were then retained if their inclusion meaningfully shifted the effect estimates of exposure variables without substantially increasing standard errors. Age and sex were forced (confounding) variables in all models. Sub-group analyses were conducted for respondents who slept overnight at their forest farms and fields, as their overall exposure profile was considered to differ from respondents who always slept in their hamlet. Sensitivity analyses restricted to individuals with serological data in both Survey 1 and Survey 6 were run. All regression analyses were conducted in Stata/IC 13 .Logistic regression models with robust standard errors to account for clustering by household were used to calculate crude and adjusted odds ratios for the association between exposure variables and recent malaria exposure, separately for The study was approved by the institutional review boards of the Institute of Tropical Medicine, Antwerp, Belgium (ITM 1043/15), and the National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam. Participants were included in the study if they were aged 16 years and over (the age of majority in Vietnam) and verbal consent was given. Ethical review and consent procedures were additional to those in the MAPARES study, which had separate approvals (ITM-UZA 958/14).In the qualitative strand, 22 in-depth interviews were transcribed and translated, 10 informal conversations and numerous observations were recorded. Informants included health care workers, community leaders and officials, farmers, loggers and secondary school students. In the quantitative strand, 160 (86%) participated in the survey. All survey respondents were of Xe Dang ethnicity, 52% were males and 48% were females, with a mean age of 35 years. Most (92%) were farmers, and 62.5% had not been educated beyond primary school level  respondents with stable or increasing P. vivax antibody levels who met the criteria for ‘recent exposure to malaria infection’.Serological data was available for 140 of the 160 cross-sectional survey participants, including 125 who participated in the first screening, 119 for the sixth screening, and 104 for both screenings. The proportion of respondents categorized as weak or strong positive declined overall between Survey 1 and Survey 6 for all antibodies , and 75% reported always sleeping under a bed net Table , howevern Tables , 3.Table, transactionally (“paying for favours in wine”), and for a range of traditional ceremonies. Rice wine consumption delayed sleeping time; the median sleeping time was 8 pm if not drinking (IQR 7–9 pm), 9 pm after drinking (IQR 9–10 pm). Evening drinking hindered bed net use as, when asked generally, 88.5% of survey respondents reported to always sleep under a bed net, but only 65.4% reported to sleep under a bed net after drinking in the evening. There was a non-significant trend towards increased risk of P. falciparum exposure amongst daily drinkers in multivariable analyses   (Table Consumption of rice wine in the evenings in the hamlets was very common amongst men 84%) and common amongst women (44%), and 16% of men and 8% of women  reported to drink every evening  (Table Watching television/DVDs (hereafter ‘TV’) in the evenings was a common social activity. Most often, people watched TV together in a central house 26.5%), particularly in Tu Nak, many or all of whom sit outside as there is not enough space in the house. Only 17.5% watched TV at home (thus indoors), reflecting low TV ownership  (Table There were 72 45%) survey respondents who slept overnight at their forest farms and fields. The longest duration of stay was mostly less than 1 week and was not associated with recent malaria, but 10% of overnight sleepers stayed for 1 week to more than 1 month continuously, which was associated with recent exposure to 5% surveyP. vivax exposure  . Not sleeping under a bed net at the farm was associated with recent P. falciparum or P. vivax exposure , but intermittently rather than regularly, and was not associated with e Tables , 3.P. falciparum  but also for P. vivax . Insecticide-treated hammock nets were rarely taken to the forest .46% of respondents reported spending evening hours in the forest, for collecting wild plant foods (24%), hunting (32.5%), collecting firewood 11.3%) and logging (3.8%)  (Table Evening fishing was reported by 20 (12.50%) respondents, mostly men (75%) and mostly in Xe Xua hamlet where the river is located. Men went fishing in small groups of 4–6 people and returned late in the evening . Evening fishing was weakly associated with recent P. falciparum, less so for P. vivax. Females were at higher risk of recent malaria exposure in univariable analyses, an effect that strengthened after adjusting for evening and outdoor activities, which were more prevalent in males.Increasing age was associated with increased risk of recent exposure to P. falciparum and P. vivax exposure. Household bed net ratio or other seropositive members were not associated with P. falciparum or P. vivax exposure in adjusted analyses. Having a hamlet house built on stilts rather than ground level was not associated with P. falciparum or P. vivax exposure overall, but was protective against P. falciparum exposure amongst non-farm sleepers  (Table Higher numbers of children living in a household was associated with decreased risk of 1) Table .P. falciparum exposure (OR attenuated from 10.9 to 3.3), but this may be affected by having only three loggers in the model with complete serology data. Participation in screening surveys was not associated with recent malaria exposure  led to minor fluctuations in effect sizes for most variables, except the effect of logging for e Tables , 3. DiffP. falciparum and P. vivax exposure, respectively, but were not associated with malaria exposure amongst farm sleepers. Instead, farm sleepers, who spend variable proportions of time residing at their farms away from the hamlet throughout the year, were more likely to be recently exposed to P. falciparum malaria if they went fishing or logging in the evenings. At farms, TV watching was unavailable and drinking occurred much less frequently, whereas fishing and logging were more common amongst farm sleepers than non-farm sleepers. Apart from bed net use, no evening activities specifically conducted whilst staying overnight at forest farms and fields could be identified as risk factors.This study used a mixed-methods study design to identify and further explain local risk factors for malaria infection in Central Vietnam. Evening outdoor activities such as drinking and TV watching were the main hamlet-based activities linked to recent P. vivax exposure. Thus, this study provides detailed information on human behaviours that contribute to residual transmission, in contrast to previous literature in which residual transmission has been largely defined and characterized with respect to vector behaviour [The main findings of this study point to the importance of a micro-epidemiological approach in pre-elimination settings with shifting epidemiology. Broadly categorized behaviours previously recognized as risk factors, such as overnight sleeping at forest farms and bed net use in the village –6, were ehaviour , 35 withehaviour , 37).Plasmodium parasites [Females were more likely to be classified as recently exposed by serology, and the effect of female sex became more pronounced after adjustment for behavioural-related risk factors, a finding that is in contrast to previous studies in this area that found males to be at higher risk of malaria when measured by PCR or microscopy rather than serology , 6, 38. arasites –43. It iarasites , 25, 44.arasites , no diffarasites , 47), anarasites , 25 as wP. falciparum are considered more reliable than for P. vivax to detect risk factors for recent exposure to new infections as the latter could not be separated from recrudescence for P. vivax.Though serological markers can be used to describe fine-scale heterogeneity in malaria infection in both high and low transmission settings , 48, 49,P. falciparum and 12.3% for P. vivax), largely reflecting the exclusion of children in the cross-sectional survey, who had lower malaria antibody levels .This study was limited by the sample size, which in the cross-sectional survey was not sufficiently powered for the various sub-group analyses, and complete serological data was only available for 104 of 160 (65%) respondents, reflecting incomplete participation in the cohort study wherein complete serological data was only available for 58.6% of respondents. The sampling frame was restricted to adults as it was anticipated that there would be a high non-response rate from children requiring parental consent to participate as many children reside at school during the week and then accompany their parents to the farms on the weekend. Nonetheless, this restricts the generalizability of findings from this study, including no association between hamlet bed net use and recent malaria exposure, to adult populations in this setting only. Furthermore, a complete net integrity assessment was not undertaken, which restricts the validity of the bed net condition variable. The proportion considered recently exposed in this analysis was higher than in the MAPARES cohort overall  that limit statistical power to detect important risk factors and calculate meaningful confidence intervals. Triangulating quantitative against qualitative findings partly ameliorates this challenge, as in-depth interviews and observation of the setting provides additional context that can support or refute the quantitative results.As residual transmission appears to be maintained by outdoor evening activities that disrupt or delay sleeping, long-lasting insecticidal hammock nets may be of limited utility for achieving further reductions in malaria transmission, despite being designed for similar forest malaria settings in Vietnam . DecliniPersistent malaria transmission in Tra Cang, Central Vietnam, appears to be maintained by outdoor evening activities that delay or disrupt sleeping in a permanent structure in which a bed net could be hung, including evening drinking gatherings, fishing, logging in the forest and outdoor TV watching, as well as non-use of bed nets at forest farms and fields. In addition to existing malaria control efforts, vector control tools adapted for use during outdoor evening activities in villages as well as at farms, forest and river locations should be incorporated into malaria elimination efforts in Central Vietnam. Effectively targeting residual malaria transmission remains an area of active research, to which studies using mixed-methods designs can contribute by identifying context-specific determinants of persisting malaria risk, which may guide further research and application of appropriate interventions in settings in which residual transmission occurs.Additional file 1. Mean percent-positivity (PP) cut-points used to define seropositivity categories in 1st and 6th survey, and minimum changes in mean PPs between surveys used to define stable or increased antibody level.Additional file 2. Seropositivity categories at first and sixth screening survey, by antibody.


In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria.Plasmodium falciparum , one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA.A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d’Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 −3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations.High prevalence (7–93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity. Plasmodium falciparum malaria remains a major threat in tropical and sub-tropical regions, with nearly 50% of the world population exposed to infective bites by Anopheles mosquitoes and almost half million deaths annually .The Magnetic Bead-based MAGPIXescribed ,12,17. 2ie a pool of 25 sera from clinically immune adults living in the village of Dielmo  and a pool of European and/or African non-immune, respectively. Results were expressed as OD ratio = OD sample / OD naive serum pool ; (ii) elevated [104–5x104]; (iii) high [5x104–105]; (iv) very high [>105] trophozoite per μl. Levels of Ab responses were not significantly different as function of levels of parasitemia for any Ag in 2010. Analysis of 2012 cohort showed significant high Ab responses against CSP and LSA141 (P<0.01) in individual with the lowest level of parasitemia compared to the highest one (<104vs 5x104-105 trophozoite per μl). For patients recruited in 2013, similar result was found for Ab responses against schizont extract Ag (P<0.01).Further analysis was done by stratifying levels of parasitemia in 4 classes: (i) positive [<10Analysis for the group of individuals recruited in 2011 was done after stratification according to RDT: negative, positive with/without circulating parasite. As illustrated on Multiple linear regression adjusted on age-groups including all years but 2011 showed no Ab responses significantly (P<0.05) associated with lower parasitemia level .When analyzing year 2011 only, Ab response to GLURP were significantly associated with lower parasitemia level (P = 0.03).When regression model included all years and was adjusted on age and years of follow-up, Ag targets associated with lower parasitemia were: GLURP (P = 0.03), MSP4p20 and PF13 (P = 0.01).Analysis of interrelation between antibody responses and age of individuals showed significant positive correlations  for schizont extract CSP, SALSA, GLURP and PF13 Ags (rho from 0.2 to 0.3).Profiles of Ab responses by age-groups are shown on Figs 41, LSA3 and gSG6 salivary peptide.Results from systematic year by year dichotomy comparisons (Mann Whitney test) are summarized in There is a high level of malaria endemicity in Côte d’Ivoire  and a coThe scaling up of integrated interventions strategies, as reported for Abobo, showed clearly a substantial positive result between 2010 and 2013 in Abobo , underliPlasmodium species-specific antibodies as indicators for exposure when analyzing multi-target Ab responses in acute malaria patients from sentinel health center. Malaria transmission in these settings including Abobo was high; profile of antibody responses was found different according to the location of the setting, accurately reflecting the immune background [A pilot study conducted in 3 different settings of Côte d’Ivoire highlighted the potential of ckground . A similDespite the observed decrease of malaria burden, analysis of the antibody responses to such array of antigens in this study could not sustain any substantial decrease of immune background level in those patients. Indeed, antibody responses were high as expected in individuals where parasite invasion induced clinical symptoms. The age-related significant increase of antibody responses found here witnesses a profile of a site with high ongoing endemic transmission, whose actual decrease is not yet reflected by detectable variation of the immune background. Several factors can contribute to this observation: firstly the recruitment of symptomatic individuals with high antibody boosting, secondly the short time frame of only two years between the strong implementation of prevention measures conducted in 2010–2011 and the latest measure in 2013 and thirdly the lack of biomarkers adapted to precise exposure estimates .Studies using serological analyses as indicators of malaria transmission generally involve recruitments in a relatively stable state for host-parasite interaction, usually of asymptomatic individuals in different settings and/or in longitudinal manner. Several antigens have been used as markers of exposure and malaria risk ,26, inclNumerous results and data underline antibodies to recombinant merozoite antigens as valuable biomarkers of immunity ,31. TherAnopheles salivary peptide gSG6-P1 antigen was the only indicator of a significant trend to decrease, a relevant marker of risk for malaria transmission already underlined [Altogether, analysis of antibody responses in case of longitudinal symptomatic malaria recruitment would rather require testing a multiplex array of antigens as a potential valuable tool. Antibody responses to different parasite targets and other plasmodium species were useful: in this study, IgG response to derlined ,35.vs 88%, P<10−3 by fisher exact test). Whether this observation relates with a lower efficient level of immunity for parasite clearance requires further longitudinal investigation on a larger cohort of individuals.Regarding parasitemia, no trend of differential susceptibility was evidenced in the different groups. Levels of parasitemia were comparable , in line with the almost similar levels of immunity measured in 2010, 2012 and 2013. Interestingly, young individuals harbored higher parasitemia and a significant relationship between high levels of Ab to GLURP, PF13 and MSP4p20 and lower parasitaemia was found. These results confirm that antimalarial antibodies may play an important supportive role in the therapeutic response to antimalarial drugs during acute falciparum malaria as already observed ,37,38. I5 trophozoite per micoliter of blood without any clinical symptoms. In these children, elevated antibody levels and prevalence against the array of antigens were found reaching higher levels than symptomatic individuals for SALSA, GLURP, PF13 and MSP4p20. In non-parasitized RDT positive individuals, there was a trend towards highest levels of antibody (Taken together, this study lacked of power for analyzing potential decrease of immunity expected to parallel the observable decrease of malaria burden due to effectiveness of control measures. However, this study underlined several interesting information regarding analysis of immune responses in high endemic setting. The 2011 particular recruitment shows clearly that immune background and natural immunity are substantially high. A proportion of 27% of young schoolchildren had positive RDT, in these individuals 16% had circulating parasitemia over 10antibody , indicatIndeed, recruitment of symptomatic patients in sentinel centers is a convenient possibility for investigation and follow-up of malaria immune responses which is a mandatory public health structure that supports patients and relevant bio-clinical follow-up. However, this study showed clearly that active additional recruitment of asymptomatic individuals is needed for accurate analysis in such areas where malaria remains strongly endemic.This longitudinal immuno-epidemiological study conducted in peri-urban endemic setting of Abobo shows that immunity profiling using biomarker has the potential for immunological mapping of intervention and help for prevention measures towards malaria elimination. However, analysis of longitudinal variations of immunity in high endemic setting requires further studies delineating the relationship between seroprevalence and transmission intensity. The limited but useful snapshot picture of the high immune background from this study underlines the need for multiple sero-surveys in enlarged cohort of individual among populations in Côte d’Ivoire with different levels of exposure for accurate immune profiling.


Cerebral malaria is still a deleterious health problem in tropical countries. The wide spread of malarial drug resistance and the lack of an effective vaccine are obstacles for disease management and prevention. Parasite and human genetic factors play important roles in malaria susceptibility and disease severity. The malaria parasite exerted a potent selective signature on the human genome, which is apparent in the genetic polymorphism landscape of genes related to pathogenesis. Currently, much genomic data and a novel body of knowledge, including the identification of microRNAs, are being increasingly accumulated for the development of laboratory testing cassettes for cerebral malaria prevention. Therefore, understanding of the underlying complex molecular basis of cerebral malaria is important for the design of strategy for cerebral malaria treatment and control. Cerebral malaria (CM) is a life-threatening disease that represents a global health problem particularly in tropical countries. According to a report of the World Health Organization (WHO) in the year 2015, malaria transmission still occurs in approximately 97 countries and territories, mostly in Sub-Saharan Africa, Southeast Asia, and South America. In the year 2013, the estimated incidence of malaria infection was 198 million cases (range 124–283 million) worldwide. Over 575,000 cases of CM have been reported. Approximately 584,000 cases  died from malaria. African children are the most affected case of CM. Most of the malaria-related deaths, approximately 90 %, occurred in Africa .Tab.TabPlasmThe development of CM is biologically complex and involves multiple mechanisms such as sequestration, immunopathology by the pro-inflammatory cytokine interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and apoptosis. Red cell sequestration is an important step in the development of CM. The binding of PfEMP1 on infected red blood cells to host receptors such as intercellular adhesion molecule-1 (ICAM-1) and CD36 on brain endothelial cells mediates sequestration , 13. ParATP-binding cassette transporter A1 (ABCA1) gene is involved in the process of microparticle release. ABCA1 gene deletion or knockout is associated with protection from CM in mice [Microparticles are cellular membrane-derived vesicles generated by cytoskeletal alterations as a result of cellular membrane remodeling and loss of phospholipid asymmetry. Under physiological conditions, microparticles derived from platelets, white blood cells, red blood cells, and endothelial cells are expressed at normal levels. The overexpression of microparticles is associated with cell activation and apoptosis . The ATP in mice , 19. Mor in mice . This ev in mice .The roles of CD8+ T cell in malaria infection are highlighted in many studies. CD8+ T cell acts as (1) effector cell in hepatic and erythrocytic phase for malaria vaccine development  and (2) Gene regulation is a process responsible for controlling the rate and manner of gene expression. In humans, dysfunctional gene regulation has been demonstrated in various pathological processes such as inflammatory responses and metabolic processes. Researchers have exerted extensive efforts to uncover the genetic regulation of infectious diseases to develop novel therapeutic agents that target the critical components, which are essential for the survival or amplification of the infective agents or which directly regulate the host immune system. Human gene expression is mainly regulated at the transcriptional and translational levels. Moreover, the mechanisms of epigenetic regulation of gene expression such as chromatin remodeling, histone modification, and non-coding RNAs, particularly microRNAs (miRNAs), have been shown to play important roles in the pathogenesis of complex diseases .miRNAs are 21–25 nucleotide single-stranded non-coding RNA molecules. miRNAs are transcribed from miRNA genes by RNA polymerase II into long pri-miRNAs and then cleaved by the enzyme Drosha into smaller pre-miRNAs. This precursor is subsequently exported to the cytoplasm by the exportin-5/Ran-GTP complex and then further processed by Dicer into double-stranded RNAs. One of the strands is degraded, and the mature miRNA is incorporated with RISC (RNA-induced silencing complex). miRNAs regulate gene expression through complementary binding to the 3′ untranslated region (3′ UTR) of the targeted messenger RNA (mRNA). If the complementary paring pattern between the miRNA and the targeted mRNA is perfect, then the mRNA is cleaved by RISC, whereas if the paring is imperfect, then translational repression occurs .Anopheles gambiae showed the differential expression levels between blood meal and after infection by P. berghei [P. falciparum and inhibited parasite growth [A role for miRNAs in malaria pathogenesis has been increasingly documented. Transcriptome-wide analysis of miRNAs in  berghei . In sicke growth . The stue growth . miRNAs e growth . The apoe growth . NF-kB se growth –31. Hypoe growth . In addie growth . TherefoThe early diagnosis and urgent treatment of complicated malaria is a good practice for the prevention of CM. The complicated malaria case must be considered as a medical emergency and high priority of treatment. The recommended antimalarial treatment for complicated malaria including CM is water-soluble artemisinin derivative artesunate. The supportive management of malaria complication is essential such as fluid and electrolyte imbalance and convulsion. Many clinical trials studied on adjunctive therapies for complicated malaria using exchange blood transfusion, anti-TNF, etc. However, no effective adjunctive therapies demonstrated the improvement of severity outcome .P. falciparum resistance to multiple drugs has been widely detected in endemic areas. The failure of artemisinin treatment occurred in five countries of the Greater Mekong sub-region: Cambodia, Myanmar, Thailand, Vietnam, and the Lao People’s Democratic Republic [P. falciparum dihydrofolate reductase (Pfdhfr) [The control programs of malaria are environmental insecticide-spraying programs, insecticide-treated bed nets, drug treatment in the case of clinical infection, and prophylactic measure for travelers. However, the major problems of the control programs include multidrug resistance in parasites, insecticide (pyrethroids) resistance in mosquitos, and the lack of effective vaccines. Republic . Drug re(Pfdhfr) –38.P. falciparum  is in phase III efficacy trials [The current approach for malaria vaccine development is based on the use of recombinant proteins or the attenuated whole organism. However, there is no practical or effective vaccine in clinical use. The key considerations are the delivery system, evolutionary changes in the parasites, and the development of resistance. After vaccination, humans have two main responses of antiparasite and antitoxic immunity. Vaccines are designed for various stages of the malaria life cycle: the pre-erythrocytic-stage vaccines for the prevention of infection, the blood-stage vaccines for the prevention of clinical illness and death, and the sexual stage vaccines for blocking transmission. The most advanced candidate vaccine for y trials .TNF-α gene are significantly associated with susceptibility to CM in the Gambia, Africa, Myanmar, and Thailand [The risk factors of CM are age, immunological status, overall health status of the host, variation in host genotype, and parasite diversity . The speThailand , 42. HowThe human genetic variations/phenotypes associated with malaria infection are mainly demonstrated in the host red blood cells, and the immunological components include (1) red blood cell/hemoglobin defects such as thalassemia, sickle cell trait, HbC, and HbE; (2) enzyme defects such as G6PD deficiency and PK deficiency; (3) membrane defects such as ovalocytosis; (4) the Duffy blood group; (5) immunogenetic variants such as HLA alleles; and (6) immunological components such as complement receptor 1, NOS2, TNF-α, and the chromosome 5q31–q33 region (the cytokine region). These genetic variants are highly detected in malaria-endemic areas as a result of natural selection. The mechanism of protection is not yet clear. The reduction or failure of parasite invasion/multiplication in the red blood cells, induction of clearance by the immune system, and increased oxidative stress are responsible for malaria protection , 44.P. falciparum transforms host red cell membranes by incorporation of parasite-derived proteins with the erythrocyte cytoskeleton. The key virulence proteins are P. falciparum erythrocyte membrane protein 1 (PfEMP1) and knob-associated histidine-rich protein (KAHRP), which involve the formation of a knob structure linked to the red blood cell cytoskeleton. The development of PfEMP1 initiates from the trophozoite stage, and the knob structure that is formed adheres to the endothelial cell of the host [var” is extremely diverse and encodes PfEMP1 that binds to various receptors on the host. The var gene contains two exons encoding Duffy binding-like (DBL), cysteine-rich interdomain region (CIDR), N-terminal sequence (NTS), and acidic terminal sequence (ATS) domains [Csp (circumsporozoite protein) and Msp-1 and Msp-2, which encode the merozoite surface proteins 1 and 2, respectively [Parasite genetics may also be involved in CM because the host , 46. The domains , 48. Theectively , 50. MorCM is a fatal multifactorial disease, and approximately 20 % of patients will die from coma and seizure if no effective treatment is available. Multidrug resistance is still a problem in endemic areas, and effective vaccines are not fully developed. Now, geneticists are exerting extensive efforts to investigate the causative genes/markers of CM. Because more than 60 % of human protein-coding genes are predicted to be regulated by miRNAs, miRNAs may be involved in the mechanism of CM. The utilization of quantitative and qualitative analyses of miRNAs in CM will facilitate the discovery of new interventions for diagnostic and therapeutic purposes.


Plasmodium falciparum sporozoites play a key role in human immunity to malaria. However, antibody mechanisms that neutralize sporozoites are poorly understood. This has been a major constraint in developing highly efficacious vaccines, as we lack strong correlates of protective immunity.Antibodies targeting P. falciparum sporozoites in vitro, and identified targets of functional antibodies. In children and adults from malaria-endemic regions, we determined the acquisition of complement-fixing antibodies to sporozoites and their relationship with antibody isotypes and subclasses. We also investigated associations with protective immunity in a longitudinal cohort of children (n = 206) residing in a malaria-endemic region.We quantified the ability of human antibodies from malaria-exposed populations to interact with human complement, examined the functional effects of complement activity against P. falciparum sporozoites, which consequently inhibited hepatocyte cell traversal that is essential for establishing liver-stage infection, and led to sporozoite death in vitro. The natural acquisition of complement-fixing antibodies in malaria-exposed populations was age-dependent, and was acquired more slowly to sporozoite antigens than to merozoite antigens. In a longitudinal cohort of children, high levels of complement-fixing antibodies were significantly associated with protection against clinical malaria.We found that antibodies to the major sporozoite surface antigen, circumsporozoite protein (CSP), were predominately IgG1, IgG3, and IgM, and could interact with complement through recruitment of C1q and activation of the classical pathway. The central repeat region of CSP, included in leading vaccines, was a key target of complement-fixing antibodies. We show that antibodies activate human complement on These novel findings point to complement activation by antibodies as an important mechanism of anti-sporozoite human immunity, thereby enabling new strategies for developing highly efficacious malaria vaccines. We also present evidence that complement-fixing antibodies may be a valuable correlate of protective immunity in humans.The online version of this article (10.1186/s12916-018-1054-2) contains supplementary material, which is available to authorized users. Plasmodium falciparum, the main cause of malaria morbidity and mortality ..15, but n = 59) compared to adults (n = 16) living in a P. falciparum holoendemic area in western Kenya when transmission was high. We also compared complement-fixation responses to a major merozoite surface protein, MSP2, which is an established target of complement-fixing antibodies in blood-stage immunity [p < 0.001 for both tests) , which were higher than those observed in younger children (p < 0.001). Taken together, these results suggest that complement-fixing antibodies to CSP are poorly acquired during early childhood compared to the merozoite antigen MSP2, and that C1q-fixing antibodies to CSP are overall acquired more slowly than those to blood-stage antigens.The bulk of malaria morbidity and mortality occurs in endemic areas where transmission is stable and relatively intense, particularly in children aged less than 5 years. We, therefore, investigated the acquisition of C1q-fixing antibodies to CSP among young children (immunity . In thists) Fig. . In contN = 206, 5–14 years) who were resident in a malaria-endemic region of PNG [p < 0.001). When stratified into four age groups , there was no significant association between age and C1q-fixing antibodies (p = 0.211)   Fig. . However01) Fig. , reflectp = 0.020), which remained significant after adjusting for potential confounders of age, location of residence, and parasitemia at baseline . A significant association was not observed for children with low C1q-antibodies compared to those who were negative . Furthermore, there was no significant association between IgG to CSP and risk of malaria . Although the prevalence of naturally acquired functional anti-CSP antibodies was generally low, these data suggest that high levels of C1q-fixing antibodies are associated with naturally acquired anti-malarial immunity and protection against disease.To obtain epidemiologic evidence supporting the importance of antibody-mediated complement-fixation to CSP in acquired immunity to malaria, we investigated whether the minority of PNG children with high levels of functional antibodies had a reduced risk of clinical malaria. Children were treated for malaria and then actively monitored for the duration of the study, enabling us to establish any association between C1q-fixing antibody levels at enrolment and the subsequent risk of developing clinical malaria. Children were divided into three response groups based on C1q-fixation reactivity: (i) negative (AU < 1), (ii) low positive , and (iii) high positive  , whereas substantial traversal inhibition of NF166.C8 sporozoites was seen only when antibodies were tested with active complement present. Inhibiting sporozoite traversal may be important, as it is essential for establishing liver-stage infection in animals [P. berghei, reported that glycan-specific antibodies activated the mouse complement and inhibited the traversal and invasion of sporozoites [P. berghei, which has substantial biological and functional differences from P. falciparum, including in the major target antigen CSP, as P. berghei does not have the major NANP epitope of P. falciparum CSP. Furthermore, complement activity in laboratory mice is much lower than for human complement, which plays an important role in immunity [Plasmodium infection are typically dominated by murine IgG1, which does not fix complement, whereas human IgG1 and IgG3 dominate human responses and both subclasses potently activate complement. The future development of new animal models of malaria that better represent antibody–complement interactions may be valuable for vaccine development. Future research to define further the antibody titers, types, and specificity required for optimal complement fixation and functional effects would be valuable to help inform vaccine development.We demonstrated that immune antibodies promote the deposition and activation of human complement against whole in vitro –19, high animals . Mouse srozoites . Interesimmunity . Murine P. falciparum sporozoites [Antibodies alone cannot kill target cells, but our findings suggest that they can mediate this effect by recruitment and activation of complement. We directly showed that incubation with antibodies and complement can lead to sporozoite death. It has previously been proposed that murine antibody–complement interactions lead to morphological changes and bleb formation in rozoites . Howeverrozoites . Interesin vitro [P. falciparum [P. falciparum/P. berghei sporozoites [The NANP-repeat sequence is a known B-cell epitope of CSP, and an important target of RTS,S-induced antibodies . We founin vitro , and canlciparum  or mice rozoites , althougWe identified a subset of individuals from PNG and Kenya with high anti-CSP IgG reactivity that demonstrated a weak ability to fix C1q. This highlights that total IgG reactivity is not wholly reflective of functional activity, and may explain why measuring anti-CSP IgG alone is a poor correlate of protective immunity. Anti-CSP IgG can directly fix C1q, and IgM is also likely to play some role, as we observed high levels of anti-CSP IgM in selected individuals that strongly fixed C1q. Anti-CSP IgG was predominately the IgG1 and IgG3 subclasses, which are known to active complement. However, IgG2 and IgG4 were also detected in some individuals, which may negatively impact C1q-fixation by outcompeting the complement-fixing subclasses; therefore, the overall isotype and subclass profile is important for functional activity. Other antibody factors, such as epitope specificity, affinity, and glycosylation, are also important in determining function.A striking new finding is that children with high levels of C1q-fixing antibodies to CSP had a significantly reduced risk of clinical malaria compared to children who had no detectable functional antibodies. Previously, no association between anti-CSP antibodies and protection in malaria-exposed populations has been found , 45. In The cell-free high-throughput assay to detect and quantify functional, complement-fixing antibodies to CSP, which we developed in this study, could be applied in large-scale clinical trials for vaccine evaluation. Although this may not wholly represent all interactions that occur on the sporozoite surface, CSP is a well-validated antigen that is known to be the major target of naturally acquired immunity to sporozoites , 46, 47 P. falciparum sporozoites, which inhibited sporozoite traversal and lead to sporozoite death. These data provide key knowledge on antibody functional mechanisms against sporozoites, and help us to identify which antibody types and epitopes are important in mediating anti-malarial immunity. We also obtained promising evidence that a strong level of C1q-fixing antibodies to CSP is associated with protection against clinical malaria, and may be a valuable correlate of protective immunity. This is the first evidence of a functional antibody response to sporozoites associated with protection against malaria in children. The future application of these approaches to quantify functional immunity in vaccine trials may greatly aid our understanding of how vaccines work and why efficacy has been suboptimal in trials to date. The antibody C1q-fixation assay may be a suitable correlate of protective immunity in vaccine development and trial evaluation, and studies of acquired human immunity. Defining the functional mechanisms that confer protective immunity against malaria is essential for developing and evaluating much-needed highly efficacious malaria vaccines. These findings have strong translational significance by revealing a mechanism of anti-sporozoite immunity, and thereby enabling the development of strategies to maximize this effector mechanism in the development of highly efficacious malaria vaccines.In summary, this study demonstrates for the first time that immune antibodies fix and activate human complement against CSP and Additional file 1:Supplementary figures: Supporting data. (DOCX 1232 kb)


Malaria prevalence continues to decline across sub-Saharan Africa as a result of various intervention strategies. However, the diseases still poses a public health concern in the region. While symptomatic malaria is recognized and treated, asymptomatic infections become increasingly important for interrupting transmission. A cross-sectional survey was conducted to assess malaria prevalence in symptomatic and asymptomatic children in Kiwangwa ward in Bagamoyo District in Tanzania.Plasmodium falciparum prevalence using rapid diagnostic test (RDT), light microscopy (LM) and reverse transcription quantitative PCR (RT-qPCR) targeting transcripts of A-type 18s rRNA of P. falciparum. Gametocytes were detected by LM and RT-qPCR targeting transcripts of gametocyte specific marker, Pfs25.Four hundred school-aged children in Kiwanga ward were recruited in the study; 200 from Kiwangwa dispensary and 200 from nearby schools. Primary health parameters were examined and blood samples collected and examined for P. falciparum prevalence was 73.3, 40.8 and 36.3% by RT-qPCR, RDT and LM in the study area, respectively (P < 0.001). As expected symptomatic children had a significantly higher prevalence of 89, 67.5 and 64.5% by qPCR, RDT and LM, compared to 57.5, 14 and 8% in the asymptomatic group, respectively. However, gametocyte prevalence in asymptomatic individuals was higher by both LM (2%) and qPCR (14%) than in symptomatic individuals LM (0.5%) and qPCR (3%).Overall A substantial difference in prevalence of symptomatic and asymptomatic infections observed in Kiwangwa ward underpins the use of molecular tools in malaria surveillance aiming at estimating prevalence and transmission. Notably, the higher gametocytaemia observed in asymptomatic children indicates the reservoir infections and points to the need for detection and treatment of both asymptomatic and symptomatic malaria. Plasmodium falciparum can result into asymptomatic carriage, uncomplicated or severe malaria [Plasmodium infections particularly older children and adults where the different manifestation of malaria cases is a function of immunity with respect to age and exposure to the parasite [Infection with  malaria . In endeparasite . In highparasite . On the parasite . Asymptoparasite  where moAsymptomatic infections in many endemic areas have become highly prevalent , 7 whichP. falciparum infections and gametocytes carriage both in asymptomatic and symptomatic individuals following reduction of malaria burden. It is well known that asymptomatic infections go undetected and untreated with little or no clinical manifestation [The prevalence of symptomatic and asymptomatic infections in malaria endemic areas has been well documented  but, theestation . Howeverestation .Malaria surveys that use only LM and RDT as diagnostic tools, usually fail to detect low‐level parasitaemia  that is This was a cross-sectional study conducted in Kiwangwa ward in Bagamoyo. This is a study was a subset of a main study which was conducted in Bagamoyo district. The study was conducted during high malaria transmission seasons. A total of 400 primary school children aged 6–14 years in Kiwangwa ward of Bagamoyo district were recruited during high malaria transmission season between June and August while other groups were recruited during the second high season from October to December 2014. Two hundred blood samples were collected from symptomatic children with uncomplicated malaria seeking health care at Kiwangwa health facility. At health center, all children aged 6–14 years with malaria and consented to be recruited to the study were included, whereas the remaining 200 blood samples were obtained from asymptomatic school children using a simple purposively randomly selection from Msinune (n = 40), Mwavi (n = 43), Fukayosi (n = 37) and Kidomole (n = 80) primary schools. The number of participants from each school was based on recruitment of only consented participants and parent(s)/guardians(s). The study site was selected based on a previous survey on malaria prevalence conducted in 2011/2012 in the sentinel district of Bagamoyo villages. The survey identified Kiwangwa as a high malaria endemic area in Bagamoyo district  and logistically it supported sample collection and transportation of molecular samples to the Bagamoyo laboratory which is located 30–45 min away from the study sites. A semi-structured interview guide combining both closed and open-ended questions was used to collect demographic data of the participants. The sampling of symptomatic and asymptomatic children was done at the health facility and at the schools, respectively.n is sample size, Z is a Z statistic value of 1.96 at confidence level of 95%. P is considered prevalence at 10% [d is a 5% relative precision. To account for dropout from school during the study, 20% of the calculated sample size was added to account for missing samples. Therefore, a design effect of two to account for clustering within health facility and nearby schools was used and the targeted sample size of 400 children was obtained to assess malaria prevalence in symptomatic and asymptomatic children in the study area.The sample size was calculated using a standard formula for prevalence studies  as folloe at 10%  at 95% cBoth symptomatic and asymptomatic malaria cases were referred and offered appropriate treatment according to malaria treatment guidelines , 18. ExcThis study received ethical approval from Institutional Review Board No. IHI/IRB/No: 34-2013 of Ifakara Health Institute and Medical Research Coordinating Committee of the National Institute for Medical Research No. NIMR/HQ/R.8a/Vol.IX/1705. Regional, district and community authorities in the study area were contacted and granted a written approval of the study. Prior to participation, a written consent of each respondent was obtained based on Informed Consent Forms (ICFs) designed for the study. Additionally, the confidentiality of information obtained from study participants was assured by using unique identifiers. Each parent or guardian was given a form that explained risks, benefits and confidentiality that would have been taken. In case a parent or guardian was illiterate, school teachers and/or heath workers provided assistance to acquaint such parent/guardian with the study. Children were only included in the study after their parents and/or guardians, consented, signed and returned the ICFs to the study team through schools under study.® 3MM filter paper as dried blood spots (DBS) direct from the syringe before putting the rest of the blood sample into heparin vacutainer tubes. RDT  was done on the spot in the field and at the dispensary for quick malaria screening as described elsewhere [Three millilitres of venous blood was collected by a syringe from each participant for rapid diagnostic test (RDT), LM and RT-qPCR. Fifty microlitre of the sampled blood was spotted onto Whatmanlsewhere . A totallsewhere . As a qu®reagent  and stored at −80 °C until RNA extraction.Fifty microlitre of whole blood was collected and spotted on the filter papers and air dried but not on direct sunlight for molecular analysis. The dried filter papers were stored into different plastic zipped bags labelled with unique identification number, study number and date together with desiccants. Then each dried blood spot (DBS) was cut into 10–12 small pieces and transferred into microcentrifuge tubes containing 250 µL TRizol® plus mini kit) and incubated for 15 min at 30 °C on a shaker at 1400 revolution per minute (rpm). After incubation, the cocktail was centrifuged for 30 s at 14,000 rpm and the aqueous phase was transferred to a gDNA eliminator column, following manufacturer’s instructions with minor modifications. Lastly RNA was eluted using 50 µL RNase-free sterile water and stored at −80 °C.RNA was extracted from all blood samples collected on 3MM filter papers stored in TRIzol reagent regardless their RDT status. The filter papers were transferred from TRIzol into 606 µL RLT analysis buffer mixed with β-mercaptoethanol  assay for targeting the A-type 18S rRNA of the parasite was performed to determine parasite prevalence [Pfs25 mRNA transcripts were detected in extracted materials using quantitative nucleic acid sequence reverse transcription polymerase chain reaction (RT-qPCR). Pfs25 is expressed only in P. falciparum in late stage gametocytes which shows limited polymorphism and is currently used to quantify gametocytes from field isolates [Pfs25 transcripts were reverse transcribed and the resulting cDNA was amplified by RT-qPCR whereas the assay was performed on extracted RNA samples from asymptomatic individuals after complete gDNA removal. Quantification of P. falciparum parasites and gametocytes was calculated in copy numbers obtained from each individual sample per µL of blood using standard curves of known parasite concentrations.The evalence . All samisolates . Pfs25 t® excel file and transferred to STATA version 11 for data cleaning and analysis. The main outcome variable was malaria prevalence for asymptomatic and symptomatic children obtained using LM, RDT and qPCR. Comparison of these outcomes between the school children and diagnostic test was performed. Other variables were age and sex of children. The two sample test for proportional was used to test for differences of malaria prevalence between symptomatic and asymptomatic children. Sensitivity and specificity analysis on RDT and LM in comparison with qPCR a reference method was conducted based on the parasite density of the children in two groups.Data collected using questionnaires were entered into MicrosoftThe characteristics of study participants recruited from Kiwangwa ward health facility as well as from schools are shown in Table Overall, all three diagnostic methods; LM, RDT and qPCR detected significantly high malaria infections in symptomatic children from health facility  compared to asymptomatic children from schools , respectively Table . The aveThe asymptomatic children had lower risk of infection by asexual stage parasites  and  when tested for qPCR and LM, respectively Table .Table 4TThe sensitivity and specificity of RDT was 84.8% (95% CI 68.1–94.9) and 75.8% (95% CI 66.8–83.2) while that of LM was 87.9% (95% CI 71.8–96.6) and 73% (65.8–79.4) for asymptomatic and symptomatic children respectively . Likewise, a sixfold increase on gametocyte prevalence was detected in symptomatic children when the same methods were compared  compared to asymptomatic ones (57.5%). However, gametocytaemia was high in asymptomatic school children (14%). These findings further support several studies conducted in other malaria endemic countries including; Tanzania [This study assessed malaria prevalence in school-aged children in Kiwangwa ward in Bagamoyo. Three different malaria diagnostic methods, LM, RDT and qPCR were used to detect Tanzania , 24, EthTanzania  and SoutTanzania . These oThe sensitivity of molecular methods in detecting malaria has been reported in many studies conducted in various malaria settings , 27, 28.It was within our expectation that school-age children from the health center to have high malaria infections , 31. HowThe findings from this study indicate the need for conducting a larger or country-wide study to support development and designing more effective elimination programmes in the country. Additionally, the association of mean body weight and malaria infection as it was observed in this study should be considered to further support the explanation of gradual weight loss following prolonged sub-patent illness or infection .The strength of this study lies in the fact that the study assessed malaria prevalence both in asymptomatic and symptomatic school aged children at health facility and schools. Studies conducted in children younger than 10 years in Tanzania focused on either clinical malaria cases  or symptHowever, there were some limitations of the current study encountered. The recruitment of participants involved only four villages, which were easily accessible what may have restricted the sample size. The study recruited only children aged 6–14 years. The age restriction deprived the study of diversity on age group representation of malaria status both from symptomatic and asymptomatic individuals. Lastly, most of commercially available RDT kits operate by targeting the presence of circulating antigens something which limits differentiation of active infections from resolved ones. Hence, the presence of false positives cannot be completely eliminated.Malaria prevalence is high among school aged children, with high malaria cases in symptomatic children. Contrary, gametocytes carriage was high in seemingly healthy children recruited at schools. The use of microscopy for parasite detection usually holds but the use of high sensitive malaria diagnostic tools is important in low malaria settings and where submicroscopic infections are prevalent. The study further established that school-age children are good reservoirs of gametocytes and submicroscopic infections. Hence control efforts should focus on identifying and treating asymptomatic population. Therefore, it is important to identify asymptomatic individuals who are the infective reservoirs in the community in order to achieve malaria elimination in these geographical settings.