Plasmodium vivax infection accounts for around 80% of malaria cases. This infection has a substantial impact on the productivity of the local population as the course of the disease is usually prolonged and the development of acquired immunity in endemic areas takes several years. The recent emergence of drug-resistant strains has intensified research on alternative control methods such as vaccines. There is currently no effective available vaccine against malaria; however, numerous candidates have been studied in the past several years. One of the leading candidates is apical membrane antigen 1 (AMA1). This protein is involved in the invasion of Apicomplexa parasites into host cells, participating in the formation of a moving junction. Understanding how the genetic diversity of an antigen influences the immune response is highly important for vaccine development. In this study, we analyzed the diversity of AMA1 from Brazilian P. vivax isolates and 19 haplotypes of P. vivax were found. Among those sequences, 33 nonsynonymous PvAMA1 amino acid sites were identified, whereas 20 of these sites were determined to be located in predicted B-cell epitopes. Nonsynonymous mutations were evaluated for their influence on the immune recognition of these antigens. Two distinct haplotypes, 5 and 16, were expressed and evaluated for reactivity in individuals from northern Brazil. Both PvAMA1 variants were reactive. Moreover, the IgG antibody response to these two PvAMA1 variants was analyzed in an exposed but noninfected population from a P. vivax endemic area. Interestingly, over 40% of this population had antibodies recognizing both variants. These results have implications for the design of a vaccine based on a polymorphic antigen.In Brazil,  Plasmodium vivax is the most abundant Plasmodium species in Brazil. While this species has been neglected for many years, the recent emergence of drug-resistant strains and the absence of a vaccine intensified the efforts for a better control method. Naturally acquired immune response analysis is a useful tool for understanding the antigenicity of Plasmodium proteins and evaluating the potential of a vaccine candidate. In this study, the genetic variability of one of the leading P. vivax vaccine candidates (PvAMA1) was analyzed. Two distinct variants were expressed and the antibody response was evaluated in infected and noninfected individuals in the Brazilian Amazon. This improved understanding of the magnitude and dynamics of the antibody response will contribute to the knowledge of a vaccine candidate and open new perspectives in vivax malaria vaccine development. P. vivax accounts for over 70% of malaria cases, thereby representing the most prevalent Plasmodium species. In Brazil, approximately 174,000 cases of vivax malaria were reported last year, which corresponds to 89.2% of the total number of malaria cases [Malaria remains one of the greatest global public health problems, with approximately 3.3 billion people being at risk of infection. In South and Central America, P. vivax infection can be treated with chemotherapy; however, resistance is rising and alternative therapies are increasingly desirable [esirable . No vaccesirable , 5. AmonPlasmodium species. AMA1 is involved in the process of parasite invasion into host cells [ama1 of P. falciparum and Toxoplasma gondii have shown that AMA1 has a central role in merozoite invasion, indicating that this protein might be essential to parasite survival [P. berghei sporozoites showed that without AMA1, parasites can invade and develop in hepatocytes, but subsequently formed merozoites cannot invade erythrocytes. These results suggested that AMA1 has a fundamental role in the blood stage cycle, which could be involved in the connection, redirection and stabilization of erythrocyte binding [AMA1 is expressed in the microneme of Apicomplexa parasites, present in all st cells , 7, and,st cells . Moreovest cells . This ansurvival , 10. Stu binding . Taking  binding , 13.Rhesus monkeys with the native protein purified from P. knowlesi, which induced significant protection against infection [P. chabaudi or P. fragile, respectively, was able to induce significant protection [P. vivax AMA1 is highly immunogenic in natural infections [The immunogenic potential of AMA1 was first observed after immunization of nfection . Furtherotection , 16. In fections .P. falciparum and P. vivax [AMA1 contains four regions: 1) pro-sequence, 2) rich cysteine ectodomain, 3) transmembrane domain and 4) cytoplasmic region. Within the ectodomain, the disulfide bridges formed by cysteine residues enable them to be separated into three domains (D), referred to as DI, DII, and DIII . The higP. vivax , being rP. vivax . AlthougP. vivax AMA1 (PvAMA1) on the reactive profile of two distinct haplotypes in an endemic malaria region. The hypothesis is that genetic polymorphisms might direct the immune response.This study examined the impact of the genetic diversity of Ethical permission for the study was obtained by the local ethical committee in Manaus, AM, Brazil (CAAE-0044.0.114.000–11) and Itaituba, PA,  and of Instituto Evandro Chagas (Protocol no. 1.219.346/2015). All samples were collected after obtaining written consent from all individuals.Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas state, a low transmission region located in northern Brazil from 2011 to 2012. Blood samples were collected using BD Vacutainer with sodium citrate anticoagulant. Blood analysis was carried out just after blood collection using a Sysmex KX21N .Blood samples for DNA extraction and sequence analysis were collected from patients with malaria who were seeking medical care at Plasmodium spp. infections were detected and excluded from analysis after a PCR species-specific diagnostic was performed as previously described [A thin blood smear was prepared from each blood sample to determine species of malaria parasites. Any mixed escribed .o06’26”S; 60001’34”W) and in Itaituba, Pará, Brazil , a gold mining area, located in the most southwestern part of the state of Pará. Plasma from Itaituba were collected during the year of 2016. Samples from Manaus were from non-severe patients currently infected with P. vivax (n = 33). P. vivax samples from Itaituba were from two distinct groups: 1) non-severe patients acutely infected with P. vivax (n = 79), 2) individuals who were previously, but not currently, infected with malaria (n = 92). After collection of peripheral blood, patients received treatment following national guidelines.Plasma samples were collected in Manaus (2011–2012), Amazonas-Brazil . In contrast, Manaus presents low malaria transmission (5.8 cases/1000 inhabitants per year). In northern Brazil, the predominant species is malariae .Genomic DNA from malaria infected peripheral blood was extracted using the phenol:chloroform method . Four ovpvama1 from 40 isolates from Manaus were analyzed. Fragments of each sample were analyzed and assembled using the software Phred [pregap4, present in the Staden Package [pvama1  was analyzed using MEGA 7.026 software. These sequences were aligned together with the reference sequence PvAMA1 from the P. vivax Sal-I strain (PVX_092275) to identify single nucleotide polymorphisms (SNPs) [Sequences of re Phred  and preg Package . Sequencn PVX_0925 to idenpvama1 gene using the same software. The frequency of polymorphic sites in DI, DII and DII was compared through Chi-square test. Public sequences from previous studies [Each of the 40 sequences was parsed in the 3 functional domains of the protein, which were analyzed as its own individual set of data. The DNA polymorphism analysis tool (DnaSP 6.12.03) was used to calculate the number of polymorphisms, number of haplotypes, gene diversity and nucleotide diversity (π). Tajima's D test was performed to assess the selective pressure on the  studies , 28–35 wThe values of synonymous substitution (dS) and nonsynonymous substitution (dN) were obtained by computing the overall mean distance of the sequences pairwise with the substitution type syn-nonsynonymous, using Nei-Gojobori  with Jukhttp://www.atgc-montpellier.fr/phyml/) was used. The Bayesian information criterion (BIC) was applied to select a final model with 1000 bootstrap resampling.To obtain a dendrogram showing genetic proximity, the online program PhyML 3.0  were conducted at BepiPred-2.0: Sequential B-Cell Epitope Predictor [B-cell epitope predictions for PvAMA1 from the reference redictor . An epitredictor .E. coli Arctic Express (DE3) competent cells (Agilent Technologies) for protein expression.Sequences corresponding to the PvAMA1 ectodomain (nucleotides 130 to 1490) from two PvAMA1 haplotypes (H5 and H16) were cloned into the pGEX 4T-1 vector and transformed into 600 of 0.6. Cells were induced by the addition of 0.08 mM IPTG for 16 h at 12°C. The culture was collected by centrifugation , and resuspended in 20 mL of lysis buffer  in the presence of 1X Complete Protease Inhibitor Cocktail  and incubated for 1 h on ice. The culture was then disrupted using an M-110L Pneumatic High Shear Fluid Processor (Microfluidcs). Cell fragments were pelleted by 30 min of centrifugation at 10000 x g. The supernatant was collected and tested by SDS-PAGE and western blotting to confirm the recombinant protein expression. The synthesized recombinant proteins were purified using Glutathione Sepharose 4B (GE Heatlhcare) according to the manufacturer’s instructions.Bacteria were cultured according to the manufacturer’s instructions. Briefly, 100 mL of overnight culture was transferred into 3000 mL of LB containing gentamicin (20 μg/mL) and ampicillin (100 μg/mL). The culture was placed at 37°C and grown until reaching an optical density (OD)CD (Circular dichroism) spectroscopy was performed to predict the secondary structure of the two PvAMA1 variants. CD spectrum was recorded on a JASCO J-815 CD spectrometer. PvAMA1V5 and PvAMA1V16 proteins were buffer exchanged into 15mM Potassium phosphate pH 8.0. and concentrated to 0.16 mg/mL. Proteins were scanned from 260 to 190 nm using a 0.1 cm path length cuvette. Data were corrected for the baseline with respect to buffer and analyzed using Spectra manager version 2. The ellipticity (θ) values were calculated according to Kelly et al., 2005 .Naturally acquired IgG antibodies against two rPvAMA1 variants were measured in plasma samples by direct enzyme-linked immunosorbent assay (ELISA). First, plasma samples from infected patients from Manaus were evaluated. Subsequently, samples collected in gold-mining regions in Itaituba from infected and noninfected individuals who had previously been exposed to vivax malaria were analyzed to evaluate the persistence of antibodies against the PvAMA1 variants.490 values were normalized using the values of the anti-GST control. Plasma samples of 20 healthy individuals from a non-endemic area were used as negative controls. The cutoff value was calculated as the mean plus three standard deviations of the negative control. The reactivity indexes were obtained from the ratio of absorbance values of each sample and the cut off value. IgG reactive indexes greater than one were considered to be positive. Bias produced by a possible reactivity of the GST tag was evaluated as described by Bittencourt et al., 2018 [Briefly, high binding plates were coated with 50 μl of each rPvAMA1 variant (PvAMA1V5 and PvAMA1V16) at 5 μg/mL in coating buffer (0.05M carbonate-bicarbonate pH 9.6), overnight, at room temperature. Plasma samples diluted 1:100 were added to each well and incubated for 1 h at room temperature. Goat anti-human IgG conjugated to peroxidase (Sigma-Aldrich) was used for detection at a dilution of 1:2000. The presence of IgG antibodies was detected by O-phenylenediamine diluted in phosphate-citrate buffer containing 0.003% hydrogen peroxide. The OD was measured at 490 nm using CLARIOstar data analysis. All ODl., 2018 .The plasma cytokines IL-6, IFN-γ, IL-10 and TNF-α were quantified by flow cytometry using IL-6, IL-10, IFN-γ, TNF-α Human Flex Set  following the manufacturer’s instructions. The mean fluorescence intensity of each bead cluster was determined, and the forth logistic regression was applied to build the standard curve. Data analyses were performed using the FACSDiva software .rho) was computed to examine the relationship between PvAMA1V5 and PvAMA1V16 reactivity indexes and between these variables and age, parasitemia, platelets, total lymphocytes, number of red blood cells, hematocrit and hemoglobin. To assess if rho was statistically different from zero, we applied t-test. Additionally, paired comparison between PvAMA1V5 and PvAMA1V16 reactivity indexes was evaluated through Wilcoxon signed-rank test. A significance level of 0.05 for all statistical tests were applied.Spearman’s correlation coefficient  as a reference sequence.A total of 40 sequences of Among the 1689 nucleotides analyzed 51 SNPs were found. The overall nucleotide diversity was π = 0.0062 . The higp = 0.356).Thirty-three polymorphic sites were found at the amino acid level with 12 of them being singleton sites. Twenty-nine dimorphic sites and four trimorphic sites were found  across the PvAMA1 sequence . Within Among the 40 isolates analyzed, 19 distinct haplotypes were found with haplotype diversity (Hd) of 0.923 . The freAll haplotypes detected were genetically distributed in a dendrogram . Two of Predicted B-cell epitopes are listed in Haplotypes 5 and 16 differed in 12 polymorphic sites across the ectodomain. Nine polymorphic sites were located in DI, two sites in DII and only one in DIII. Among those sites, seven sites are located in predicted B-cell epitopes , Fig 1B.Recombinant proteins were characterized in terms of purity and quality by SDS-PAGE and structure by CD analysis . The CD To understand the importance of sequence variability, IgG responses towards both variants were evaluated in a subset of the population previously characterized for genetic diversity. Most of the population from Manaus (87.9%) had antibodies against PvAMA1V5 and 60.6% had antibodies towards PvAMA1V16. All patients who had antibodies to PvAMA1V16 also had antibodies to PvAMA1V5 .p< 0.001 for Wilcoxon signed-rank test, rho = 0.867 and p< 0.0001) (All samples that contained haplotype PvAMA1H5 (n = 9) also had antibodies recognizing this sequence (PvAMA1V5). Nevertheless, only 5 out of 9 of individuals containing PvAMA1H5 had antibodies against PvAMA1V16 . On the  0.0001) .Although the sample size was notably limited, individuals infected with parasites belonging to haplotypes H2, H6 and H14 had no antibodies to either of the variants analyzed. Individuals infected with H7, H13 and H18 did not have antibodies to PvAMA1V16. On the other hand, individuals infected with parasites containing H1, H10, H11, H15, H16 and H19 had antibodies towards PvAMA1V5 as well as PvAMA1V16.rho = 0.408 and p = 0.010) and PvAMA1V16 (rho = 0.318 and p = 0.038). While a moderate negative correlation was observed between the immune reactivity and neutrophils (rho = -0.339 p = 0.027 and PvAMA1V16 rho = -0.299 p = 0.045). However, no other association was found when hematocrit, platelets, total number of white blood cells or age were analyzed , 81/171 (47.4%) individuals had antibodies to both variants while 45/171 (26.3%) of the studied population had no antibodies to these variants.The importance of these polymorphisms for antibody response was also evaluated in a geographically distinct population in Itaituba, state of Pará. Among the 171 individuals evaluated, 70.8% (n = 121) had antibodies to PvAMA1V5 and 50.3% (n = 86) had antibodies to PvAMA1V16 . The higIn the infected group, 51.9% (n = 41) had antibodies to both variants while 29.1% (n = 23) had no antibodies to either variant. In this population, 65.8% (n = 52) of individuals had an immune response against PvAMA1V5 and 57% (n = 45) against PvAMA1V16. In the noninfected population, 43.5% (n = 40) had antibodies to both variants, and 23.9% (n = 22) of individuals did not have antibodies to either variant. In this group, 75% (n = 69) had antibodies against PvAMA1V5 while 44.6% (n = 41) had antibodies against PvAMA1V16 . Similarrho = 0.249 and p = 0.001); for PvAMA1V16 antibody response, this correlation was lower and with no statistical significance at 0.05 level (rho = 0.117 and p = 0.064). When the analyses were performed according to infected and noninfected groups, similar results were observed, with statistical significance only for correlation between PvAMA1V5 antibody response and age for the latter group (rho = 0.2913 and p = 0.0024)  . No corr 0.0024) .P. vivax isolates were identified and the influence of two different PvAMA1 variants was evaluated for the specific antibody responses.AMA1 plays an essential role in erythrocyte invasion and is one of the major potential vaccine candidates against malaria. This protein is exposed to the immune system and is capable of inducing an antibody response that blocks parasite invasion into host cells , 44. A mP. vivax isolates [P. falciparum [P. vivax [In this study, 18 amino acid changes sites were identified in PvAMA1 DI, 8 sites in DII and 4 sites in DIII compared to PvAMA1 . The highest degree of polymorphism was found in DI, as previously observed in isolates , 46. In isolates , 47, 48 isolates  that is isolates . Therefolciparum  and P. vP. vivax .B-cell epitopes were predicted on the protein structure by taking into account the importance of SNP localization to the immune response and selective pressure . NotablyFive mutations were detected at DI and two at DII, localized at B-cell epitopes in H5 or H16. Although the consequences of these mutations in each variant are not known, it is important to consider that changes in protein sequence can affect the immunorecognition of those epitopes.Plasmodium antigens with the highest vaccine potential [AMA 1 is one of the otential . Studiesotential , 54, 55.otential . AMA1 isotential .P. falciparum were tested against different AMA1 alleles. Most individuals had not only a specific response to conserved regions but also raised specific antibodies against polymorphic regions of the antigen [Immunization with only one allele may not protect against parasites expressing different AMA1 alleles , 58. Pla antigen . This faP. falciparum and P. vivax during natural malaria infection was also observed in earlier studies [In this instance, the immunoassay results showed that the variants of PvAMA1 (V5 and V16) are highly reactive. However, a greater naturally acquired immune response against PvAMA1V5 was found among different haplotypes in individuals from Manaus, including the isolate belonging to H16. Manaus is a low transmission region. Furthermore, the naturally acquired immune response was also analyzed in a population from gold mining areas, representing a high endemic area. The prevalence of antibodies against PvAMA1V5 is higher in Manaus compared to Itaituba. Since the haplotypes were identified in samples from Manaus, the immune response of individuals living in this region would be higher due to circulating target strains. On the other hand, H5 is probably not distributed with the same frequency in Itaituba. This possibility highlights the importance of vaccine formulations containing frequent haplotypes from different malaria endemic areas. The high immunogenicity of AMA1 for most individuals exposed to  studies .The immune response of infected individuals was also compared to noninfected individuals who had previous episodes of malaria. Interestingly, although there was no significantly higher prevalence of antibodies in either group, noninfected individuals had a higher IgG reactivity index compared to infected patients. Thus, a possible maturation of the immune system over time can be inferred.Plasmodium antigens [In addition, individuals from Itaituba showed a statistically significant correlation between PvAMA1V5 reactivity index and age. Nevertheless, the correlation between age and PvAMA1V16 reactivity index was not statistically significant. This correlation has been previously described for several antigens –63 and santigens  and becoantigens . In the P. vivax strains [For vivax malaria, there are few studies to date testing PvAMA1 as a vaccine candidate –67. A va strains . However strains , 66.Multi-allele vaccine inducing a broad inhibitory antibody response are aimed at an effective malaria vaccine. Therefore, we suggest that vaccine trials based on polymorphic antigens be designed considering strain frequencies at individual study sites. The high reactivity and the possible maintenance of the immune response over time indicate that PvAMA1V5 may be a promising component of a vaccine formulation.S1 Fig(TIF)Click here for additional data file.S2 FigA) SDS-PAGE analysis of ~77kDa rPvAMA1V5 and PvAMAV16 stained with Comassie Brilliant Blue. B) Circular Dichroism spectra of PvAMA1 variants. Dotted line (PvAMA1V16) and solid line (PvAMA1V5). MM: Molecular marker PageRuler Prestained Protein Ladder, 10-180kDa (Thermo Scientific).(TIF)Click here for additional data file.S3 Figp < 0.0001, n = 171). B) Spearman correlation coefficient (rho) of IgG antibodies against each PvAMA1 variant and t-test p-value (p). C) Prevalence of responders and non-responders towards PvAMA1V5 and PvAMA16.A) Paired PvAMA1V5 and PvAMA1V16 IgG responses comparison Click here for additional data file.S4 Figrho) between PvAMA1V5 and PvAMA1V16 IgG antibodies and t-test p-value (p) against each variant in acutely infected individuals. B) Prevalence of immune response towards PvAMA1 variants in infected individuals, C) rho between PvAMA1V5 and PvAMA1V16 IgG antibodies and t-test p-value (p) in noninfected individuals. D) Prevalence of immune response towards PvAMA1 variants in noninfected individuals.A) Spearman correlation coefficient ((TIF)Click here for additional data file.S5 Figrho) between PvAMA1 variants reactivity indexes and age from full Itaituba sample. B) rho between PvAMA1 variants reactivity indexes and age for infected individuals, C) rho between PvAMA1 variants reactivity indexes and age for noninfected individuals. T-test p-value (p).A) Spearman Correlation coefficient ((TIF)Click here for additional data file.S6 Figr values are indicated in the figure.A) Correlation matrix of the infected group. B) Correlation matrix of the noninfected group. Positive correlations are represented with red, and negative correlations are represented with blue squares. Pearson correlation was applied to verify associations between the PvAMA1 variant reactivity index and hemoglobin, hematocrit, platelets and plasmatic cytokines . p<0.05 was considered significant. (TIF)Click here for additional data file.S1 TableAmino acids colored red indicate polymorphisms.(DOCX)Click here for additional data file.


The precursor peptide of vasopressin, copeptin, has previously been linked to increased risk of developing diabetes mellitus, coronary artery disease and cardiovascular mortality. Whether elevated copeptin is associated with markers of atherosclerosis and arteriosclerosis in the general population is not known.In this population-based, cross-sectional study, coronary artery calcium score (CACS), carotid-femoral pulse wave velocity (c-f PWV) and fasting plasma copeptin were measured in 5303 individuals in the Swedish cardiopulmonary bioimage study (SCAPIS). Multivariable logistic regression models were used to analyze the associations between copeptin and high CACS (>100) and high c-f PWV (>10 m/s), respectively.The number of individuals with high CACS and c-f PWV increased across increasing tertile of copeptin . The top tertile of copeptin was, compared with reference tertile 1, significantly associated with both high CACS and high c-f PWV after adjustment for age, sex, hypertension, diabetes mellitus, HDL, triglycerides, BMI, smoking status, creatinine and high sensitive CRP with an odds ratio (OR) of 1.260 : 1.022–1.555) for CACS and OR 1.389 (95% CI: 1.069–1.807) for PWV.Copeptin is associated with both coronary atherosclerosis and increased arterial stiffness in the general population. Our data indicates that copeptin may be a useful marker in the assessment of cardiovascular risk. With broadened knowledge about its vastly distributed three receptors, vasopressin has lately been acknowledged for its importance in several other physiological systems . The vasCardiovascular disease (CVD) is the leading cause of death world-wide , and is CACS is an estimation of coronary artery calcification. CACS is identified as areas of hyper-attenuation on computed tomography (CT)  and is hPWV is a marker of arterial stiffness. Loss of vessel distensibility results in an increased PWV, which is calculated by dividing the distance between two sites of an arterial segment by the arterial pressure wave transit time. When assessing carotid-femoral PWV (c-f PWV), the right common carotid and right femoral arteries are used . C-f PWVEven though a link between copeptin and several cardiovascular conditions has been established, a possible association between copeptin and objective measures of subclinical CVD has not been investigated. This study aims to determine whether copeptin is associated with markers of atherosclerosis and arteriosclerosis measured by CACS and PWV.22.1SCAPIS is a collaborative project between six Swedish universities investigating the cardiopulmonary health of a random selection of individuals aged 50–64 years, residing in the six respective cities . Between 2013 and 2018, 30,154 men and women were recruited. The participants completed an extensive questionnaire and underwent a health examination including anthropometry, blood sampling and CT with measurement of CACS. The overall participation rate was 50%. PWV measurement was included in the baseline examination for participants examined in Malmö. A total of 6251 individuals were examined at this site. Out of these, complete data including PWV and CACS, copeptin and cardiovascular risk factors were available in 5303 individuals. The research was conducted in accordance with the Declaration of Helsinki. The multicenter study was ethically approved by the Umeå University Ethical Review Board whereas the present study was approved by the Lund University Ethical Review Board. All participants provided written informed consent .2.2C-f PWV was measured with Sphygmocor Xcel  in supine position after 5 min of rest with cuffs on the upper left arm and on the right thigh approximately 10–20 cm below the groin. The carotid-femoral distances were measured from the femoral pulse to the upper edge of the thigh cuff and from the carotid pulse to the upper edge of the thigh cuff. A carotid tonometer was then used simultaneously with the leg cuff to capture blood pressure waveforms at the carotid and femoral sites. Two measurements of c-f PWV were then obtained. If the difference between the measurements differed more than 0.5 m/s, a third measurement was performed. The mean value of the measurements was used in further analysis. The calcium content in each coronary artery, visualized by CT  was measured and summed to produce a total CACS  accordin2.3A venous blood sample was collected from participants after an overnight fast, and laboratory analyses , triglycerides (TG), calculated low-density lipoprotein (LDL), high sensitive c-reactive protein (hsCRP) and creatinine) were performed at the Department of Clinical Chemistry, Skåne University Hospital, Malmö, Sweden.Biobanked (- 80 °C) fasting plasma samples were used for analysis of copeptin. Copeptin was successfully analyzed in 5779 individuals using a KRYPTOR Compact Plus device and a commercially available chemiluminescence sandwich immunoassay copeptin ProAVP kit with coated tubes from samples stored at –80 °C .2.4Baseline characteristics were expressed as absolute counts and proportions for categorical variables and as means ± standard deviations (SD) for continuous variables. The distribution of copeptin was not normally distributed and was therefore transformed using the natural logarithm. Tertiles of copeptin, with sex-specific tertile limits, were used throughout. PWV and CACS were examined as categorical variables. HsCRP was categorized into quartiles due to a considerable number of participants with values below the lower limit of quantification . Chi-square test was used to assess between group differences of categorical variables and Student’s t-test was used to investigate between group differences of continuous variables. Multivariable logistic regression models were used to assess the associations between copeptin and high CACS (>100) and PWV (>10 m/s), respectively. All models were adjusted for age and sex and in the next step additionally adjusted for prevalent hypertension, prevalent diabetes mellitus, BMI, smoking status, HDL, TG, creatinine and hsCRP.p < 0.05. All analyses were performed using the SPSS statistical software .The level of significance was set at 3versus low CACS   .p < 0.001), and the proportion of high PWV was 6.9%, 8.5% and 10.6% (p = 0.001). Increasing tertile of copeptin was significantly associated with both high CACS and high PWV, after adjustment for age and gender  years and 47% were men. Data on CACS were available in 703 individuals out of which 14.1% had high CACS, whereas data on PWV were available in 623 individuals out of which 9.7% had high PWV.4In this population-based cross-sectional study with high participation rate, the key finding is that elevated copeptin concentration in plasma is significantly associated with CACS and PWV above the cut-off values considered as pathological, also after adjustment of several cardiovascular risk factors. The median copeptin concentration was significantly higher in the groups of high CACS and PWV, except for PWV among women. The latter could be a consequence of loss of power, since the number of women in the group of high PWV was relatively small (n = 122).The mechanisms behind the link between vasopressin/copeptin and CVD are not known. Previous studies have shown that elevated copeptin predicts development of CVD, cardiovascular mortality, incidence and prognosis in heart failure as well as several cardiovascular risk factors –8,10,11.Elevated copeptin is shown to predispose for new onset diabetes mellitus ,31, and Another possible role of vasopressin in CVD development is promoting inflammation. Vasopressin is a pro-inflammatory peptide and stimulates the release of several cytokines and other inflammatory peptides, hereby inducing an inflammatory cell response. Interestingly, immunoneutralization of serum vasopressin in rats has been shown to diminish inflammation . Many st4.1Both CACS (>100) and PWV (>10 m/s) can be used as risk assessment tools for CVD and are suggested to be of value in the risk reclassification of moderate CV risk patients . However4.2This cross-sectional cohort study has several limitations. First, due to its cross-sectional design, it is not possible to draw any conclusions regarding causality. Secondly, complete data was lacking in a substantial number of individuals (N = 948), which could possibly obscure the results. However, baseline characteristics of this group  were comparable to baseline characteristics of the study sample. Thirdly, the cut-offs used (PWV >10 and CACS >100) is not equal to established CVD, and many individuals with high CACS or PWV never develop CVD. As well, coronary plaques that are not calcified does not elevate CACS. Whether copeptin can discriminate CV risk better than CACS and/or PWV is an interesting area of further studies.4.3Copeptin is associated with markers of atherosclerosis and arteriosclerosis. We propose a possible role of copeptin as a non-invasive, low risk and cost-effective method of evaluating CV risk.Supplementary material


Malaria in pregnancy (MiP) has been associated with adverse pregnancy outcomes. There is limited information on MiP in low transmission regions as Colombia. This study aimed to describe the epidemiology of MiP through active surveillance of infections by microscopy and polymerase chain reaction (PCR). Plasmodium spp was made in peripheral and placental blood samples by microscopy and PCR.  A cross-sectional study was conducted between May 2016 and January 2017 in five municipalities  in Colombia. Pregnant women self-presenting at health centers for antenatal care visits, seeking medical care for suspected malaria, or delivery, were enrolled. Diagnosis of P. falciparum (78.5%), and 46% were afebrile (asymptomatic). Women in the first and second trimester of pregnancy were more likely to be infected . To live in the urban/peri-urban area , to have a history of malaria during last year , and the infrequent bed net usage  were associated with the infection. Pregnant infected women had a higher risk of anaemia  and fever .  A total of 787 pregnant women were enrolled; plasmodial infection was diagnosed by microscopy in 4.2%  or by nPCR in 5.3%  in peripheral blood. Most of the infections were caused by Plasmodium spp. infection in pregnancy and as an important activity for the surveillance of asymptomatic infections in the control of malaria. The screening for malaria during antenatal care in endemic areas of Colombia is highly recommended due to the potential adverse effects of  Plasmodium falciparum and 2.9 million with Plasmodium vivax . The study locations were selected based on the number of cases reported during previous years by the National Institute of Health in Colombia .P. vivax caused 90% of cases. Turbo is located in the Urabá region (8.0926° N 76.7282° W), API of 1.2 in 2017 (moderated risk), P. vivax caused 78% of cases. El Bagre is located in the Bajo Cauca region , API of 25.2 in 2016 (high risk), P. vivax caused 70% of cases.The municipalities of Apartadó, Turbo, and El Bagre were included in the study; the transmission of malaria is in rural areas. Apartadó is located in the Urabá region , API of 1.0 in 2017 (moderated risk), P. falciparum caused 70% of cases. There is malaria transmission in rural and peri-urban settings.The municipality of Quibdó is located in the Pacific region , API of 100.8 in 2016 (high risk), P. falciparum caused 90% of cases. There is malaria transmission in rural and peri-urban settings.Tumaco is located on the Pacific coast , API of 16 in 2016 (high risk), Pregnant women, self-presenting at health centers for antenatal care (ANC) visits, were seeking medical care for suspected malaria or delivery. Each participant was enrolled only once, in one of the two study groups (ANC or delivery).n = (Z2pq)/d2, where n is the sample size, N is the population size, Z is the confidence level, p is the expected proportion of MiP, q is the proportion of the population without MiP (1-p), and d is the margin of error. An expected prevalence of MiP of 4% was assumed, according to previous data from studies performed among different regions in South America [Sample size was calculated for a proportion assuming an unknown population size, using the following equation:  America –14. A coTo obtain the expected prevalence of MiP detected by microscopy, data from 10 reports of seven countries of Latin America were reviewed –20. The Inclusion criteria were the following: aged ≥15 years old, at any gestational age, resident in areas of the municipality with malaria transmission, and without previous antimalarial drug intake in the past 48 hours. When pregnant women resulted positive by microscopy, samples were taken before the treatment. Eligible pregnant women were enrolled after gave their written informed consent. All women diagnosed with malaria by microscopy were treated, according to the Colombian Ministry of Health recommendations .The study protocol and informed consent form were reviewed and approved by the Ethics Committee of the School of Medicine, Universidad de Antioquia (Record 005 of 31-03-2016). For women younger than 18 years old, informed assent was obtained from the parents or legal guardian.Socio-demographic, reproductive history, and clinical information were recorded using an interview-based questionnaire. Axillary temperature was measured using a digital thermometer. A total of 8 mL of peripheral blood was collected by venipuncture using heparin tubes. When the participants were enrolled at delivery, new-born data were recorded, and 8 mL of placental blood was collected. Briefly, a small incision was made on the maternal side of the placenta, near the cord insertion area, placental blood accumulated at the place of insertion was collected by aspiration. Blood samples were processed immediately at the hospital's laboratory. Hemoglobin was measured by HemoCue AB® . Thick and thin blood smears were prepared from fresh blood, and PCR was performed from blood spotted on Whatman filter paper #3 .μL, standard value) and was expressed as parasites/μL (p/μL). P. falciparum parasitaemia was calculated by counting ring forms, while P. vivax parasitaemia was calculated by counting all asexual forms  and gametocytes. The gametocitaemia was calculated by counting the number of gametocytes of P. falciparum and P. vivax in 200 leukocytes and was expressed as gametocytes/μL (g/μL). As a quality control, a second reading was performed in all PCR positive samples and 10% of PCR negative samples.A qualified malaria microscopist read Field-stained thick and thin blood slides, following the national guidelines . A thickμL of blood) using QIAamp DNA Mini Kit , according to manufacturer's instructions. The nPCR was performed as a two-step procedure, using 2 μL of DNA template and following the protocol described by Singh et al. 1999 [P. falciparum, P. vivax, and Plasmodium malariae (nest 2). Amplified products were revealed in a 1.5% agarose gel stained with GelRed ™ . The sequences of primers used in the nPCR are shown in the supplementary table μL and was estimated using in vitro cultures of P. falciparum, preparing several dilutions with parasites densities ranging from 0.09 to 1000 parasites by microliter; DNA was extracted and PCR performed.DNA was extracted from half blood-spot filter  or history of fever in the last three days; this definition was used as a reference to symptomatic infections. Afebrile infections are infections detected by any method in pregnant women without fever or history of fever in the last three days; this definition was used as a reference to asymptomatic infections. Anaemia is define as hemoglobin level <11 g/dL, mild (10.0-10.9 g/dL), moderate (7.0-9.9 g/dL) and severe (≤6.9 g/dL), according to WHO guidelines [Submicroscopic infections are idelines . LBW indidelines .p value <0.25 in the bivariate analysis [U test was used. All analyses were carried out using SPSS-v25 software. p values <0.05 were considered significant.The information was entered into an Access® database. Descriptive analysis was performed to summarize the baseline characteristics, using proportions and median (interquartile range—IQR). The proportion and its 95% confidence interval (CI) of infections detected by microscopy and nPCR were calculated. Chi-square test was used to compare the frequency of MiP and anaemia between ANC and delivery groups. The association of potential risk factors with MiP was analysed using a binary logistic regression. Crude and adjusted odds ratio (OR and aOR) and their 95% CI were calculated. The associated factors were selected based on the biological plausibility and a analysis . Additiop = 0.005). Other baseline characteristics are shown in A total of 787 pregnant women were enrolled: 289 (37%) in Antioquia, 242 (31%) in Chocó, and 256 (32%) in Tumaco . The overall frequency of parasite presence in peripheral blood was 4.2%  by microscopy and 5.3%  by nPCR (p < 0.001), and nPCR . All peripheral blood samples positives for Plasmodium infection by microscopy were also positive by nPCR, and 9 (1.2%) with negative smear were positive by nPCR . Most infections were caused by P. falciparum .A total of 46/787  of pregnant women were infected by  by nPCR . The fren = 33) was 2480 parasites/μL (IQR 1130-4018); 1952 parasites/μL for P. falciparum mono-infection (n = 26) and 6180 parasites/μL for P. vivax mono-infection (n = 6). A total of 40.7% (11/27) of pregnant women with microscopic infection by P. falciparum had gametocytes with a median (IQR) of 32 gametocytes/μL (24-88), while a total of 87.5% (7/8) of pregnant women with microscopic infection by P. vivax had gametocytes with a median (IQR) of 328 gametocytes/μL (120-1000).The median of parasite density by microscopy  of placental samples using microscopy and 9 out of 329  using nPCR, from those eight were caused by Maternal age, marital status, education level, socioeconomic status, and occupational exposure to mosquito bites were similar between infected and noninfected pregnant women . Most ofThe proportion of primigravidae was similar between infected and noninfected pregnant women (39.1% vs. 38.3%). Gestational age and being in the first and second trimester were associated with infection . Among ip = 0.009), and the proportion of neonates small for gestational age (SGA) was higher in the infected pregnant women.Infected pregnant women had lower hemoglobin levels than uninfected women . InfectiBeing in the first or second trimester , to live in the urban/peri-urban area , to have history of malaria during last year , history of malaria in a cohabitant , and the infrequent usage of bed net  were associated with infection .μL); also, the proportion of submicroscopic infections were higher in afebrile as compared with febrile women (The frequency of afebrile (asymptomatic) infections was 46% (21/46). The characteristics of afebrile and febrile infections are shown in le women .P. falciparum infections had gametocytes, and most of them were asymptomatic.The frequency of malaria among pregnant women attending ANCs and delivering at the study site hospitals was 4.2% detected by microscopy. The infection during pregnancy was associated with negative consequences for maternal health like anaemia that may have a negative impact on neonatal health. Importantly, most asymptomatic infections were undetected with microscopic examination, which is the routine test used to diagnose malaria in Colombia. Additionally, 40% of P. vivax infections are most frequent. In this region, there was an incidence of MiP of 94 cases/100.000 pregnant women in 2014, which varies among countries [Less is known about the burden and impact of infection in low transmission scenarios, particularly in the Americas where ountries . The preountries –29. The ountries , 20. Howountries .P. vivax [In Colombia, MiP has been mainly studied in the north-west region of the country during 2005-2011, detecting a prevalence ranged between 1 and 13%; most of the cases were caused by P. vivax , 19, 28.P. vivax , low birP. vivax . There aP. vivax .P. falciparum and P. vivax were associated with MA [Similar to the current findings, recent studies indicated that microscopy infections by  with MA  as well  with MA , 33, red with MA , 33, a h with MA , 33. In  with MA . It is i with MA –36.A significant proportion of infected pregnant women carries subpatent parasitaemias that are not detected by routine test , 27, 28.In this study, it was observed that previous malaria episodes during last year and the infrequent usage of bed nets were risk factors for MiP. Previous episodes of malaria may reflect a continuous vector exposure explained by residence conditions and preventive measured adopted, among others. In a previous study conducted in Antioquia and Chocó, it was reported that 86% and 93% of participants had at least one mosquito net, respectively; however, 72-73% used it daily . TogetheP. falciparum parasite strains [There was no statistical association between gravidity and infection. Studies from high transmission areas in Africa have shown a gravidity-dependent susceptibility to malaria, which has been attributed to the acquisition of antibodies to pregnancy-associated  strains . Similar strains .Women in the first and second trimester of pregnancy were more likely to be infected. At the end of pregnancy, a reduction of microscopic infections and an increase of subpatent infections have been reported; this could be explained because of previous infections treated, and possible acquisition of immunity as the pregnancy progressed . There iMost of the studies have used the absence of fever as a standard definition of asymptomatic infection, especially in pregnant women, due to the fact that the signs and symptoms associated with malaria are common during pregnancy. Most of the malarial clinical symptoms had low positive predictive values (less than 40%), and only the fever or history of fever during last 72 hours has been associated with infection . In thisThis study had some limitations. First, the cross-sectional design that provides an estimate of the burden of the infection in a single period. A longitudinal follow-up of pregnant women would have allowed us better quantify the burden and consequences of infection. Second, the sample size was not calculated for each geographic area; thus, the findings of this study should be interpreted with caution. Third, this study was focalized on women attending health facilities and did not evaluate women who did not attend the ANC or had community delivery. Fourth, the evaluation of gestational age by ultrasound was not available in most of cases, and the Ballards method was used, which is not as reliable as ultrasound is. Finally, the number of infected pregnant women detected was modest, which limited the power to detect associations.Plasmodium across study sites but suggests that asymptomatic and submicroscopic infections are common. The submicroscopic infections were higher in some areas, like Tumaco (south Pacific Coast), which may have implications for the diagnosis of malaria with the currently used test. Routine screening of malaria during ANC visits is recommended due to the fact that infection is associated with maternal anaemia, which may have a negative impact on neonatal and infant health. Furthermore, the high proportion of afebrile infections supports the importance of the screening in all pregnant women regardless of the presence of symptoms. Also, we found a significant proportion of infected pregnant women that carry gametocytes, and their role as parasite reservoir must be considered in surveillance activities towards malaria elimination.This study shows a low frequency of pregnant woman infected by


Plasmodium falciparum histidine-rich protein 2 (PfHRP-2), parasite lactate dehydrogenase (pLDH), aldolase, glutamate dehydrogenase (GDH), and the biocrystal hemozoin. The models that demonstrate a potential for field application have been discussed, looking at the fabrication and analytical performance characteristics, including (but not exclusively limited to): response time, sensitivity, detection limit, linear range, and storage stability, which are first summarized in a tabular form and then described in detail. The conclusion summarizes the state-of-the-art technologies applied in the field, the current challenges and the emerging prospects for malaria biosensors.The impact of malaria on global health has continually prompted the need to develop more effective diagnostic strategies that could overcome deficiencies in accurate and early detection. In this review, we examine the various biosensor-based methods for malaria diagnostic biomarkers, namely;  Anopheles mosquitoes. The greatest burden of the disease is in the tropical and subtropical regions of the world ..74].Until date, most malaria biosensors have been principally confined to the established markers. Some other likely candidate targets have been speculated; heat-shock protein 70 (Hsp70), dihydrofolate reductase (DHFR)–thymidylate synthase (TS), heme-detoxification protein (HDP), glutamate-rich protein, hypoxanthine phosphoribosyl transferase, and phosphoglycerate mutase ,104,105.2–108 cells/mL of infected RBCs  antibodies directed against both antigens as low as 6–50 pg/L (concentrations in the range of 10–15 M) by means of EIS. Overall, the method demonstrated a promising approach to real-time probing of antibodies in serum directly without the need for sample preparation . S. SP. vivP. falciparum [P. falciparum and P. vivax [P. falciparum DNA fragments is suitable for automation and integration into portable molecular POCTs [P. falciparum DNA target sequences is then concentrated for SERS measurement. The assay produced a LoD of 100 attomoles DNA (Nucleic acid markers have been exploited as reliable alternatives to proteins and antibodies in malaria diagnostics . A DNA blciparum . The poslciparum  and distP. vivax . Both ofar POCTs . The assoles DNA .Simultaneous analysis of biomarkers maximizes the use of samples and gives a wide range of results that significantly improves test accuracy. Increased throughput, reduced reagents/assay setup and less labor are among features that could decrease assay errors and make parallel testing ideal for healthcare delivery. Moreover, the benefit directly translates to convenient sampling and low cost to providers and patients at the same time ensuring optimum outcome. A combination of antigens and genes have been reported in ICT and nucleic-acid-based methods respectively and are discussed elsewhere ,112,113.The drawbacks of diagnostics in spite of the vast array of tests require more effective alternatives that is crucial in the long-term fight against/and probable elimination of malaria. Taking the tremendous success of glucometers into account, biosensors are a potential driving force in the emerging medical demands for onsite diagnostics in resource-limited settings. The growing interests in biosensor research and the progress made with the prototypes from published data implies that the technology could have a significant impact on malaria diagnosis regardless of being in nascent stages. These concepts, so far have offered ideal and desirable characteristics like high performance, low fabrication costs and easy operation with the aim of practicality in clinical diagnostics. The sensors discussed in this review present with impressively low detection limits that are superior to immune chromatographic dipstick which is usually between 2–16 nM. Overall, it can be argued that the slight increments in LoDs observed in serum samples relative to buffer are attributable to the complexity of serum that can hinder interactions between biorecognition molecules and target biomarkers. Given that a major challenge to malaria diagnosis is the lack of highly sensitive tools POCTs at low parasitemia, the impressive analytical performances that biosensors have demonstrated would allow for ultra-sensitive diagnosis of asymptotic cases and monitoring treatment with antimalarial drugs. To put this in perspective, randomized control trials that apply these technologies to a large number of clinical samples are needed in order to evaluate the practical usefulness. For easy translation of malaria biosensors to the field, aspects related to complexity and instrumentation which minimizes user intervention while ensuring low-cost are key areas which remains unclear. Studies are required to model self-contained devices as most biosensors presented rely on traditional immunoassay principles and involve multiple assay steps. Innovative technologies should take advantage of 3-D printing techniques and microfluidics to integrate prototype sensors into self-contained lab-on-chip models in readiness for field deployment.The increase in preference for probing LDH in blood seems an amelioration in malaria diagnostics since its concentration correlates with parasite density and implies metabolically active parasites. This is in contrast to HRP-2 that either has delayed clearance or is absent in certain mutant strains. Furthermore, considering the advantages of electrochemical systems in POCTs, efforts towards improved system complexity could propose appropriate electrochemical systems for enzyme biomarkers like LDH, GDH, and aldolase that could be easily integrated into enzyme-based biosensors. For instance, substrates that are specific to these enzymes could be incorporated for direct enzymatic reaction detectable through electrochemical methods. Undoubtedly, the future of biosensors is being changed by the growing demands for novel biotechnologies in POC diagnostics. These innovations coupled with the capacity of multiplexing are necessary in fulfilling a major requirement of detection and species differentiation in the clinical diagnosis of malaria.


As malaria transmission declines, sensitive diagnostics are needed to evaluate interventions and monitor transmission. Serological assays measuring malaria antibody responses offer a cost-effective detection method to supplement existing surveillance tools.Plasmodium falciparum  antigens—Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175 RIII-V, PfMSP119, PfAMA1, and PfGLURP.R2—were quantified using a multiplexed bead-based assay. The association between antibody responses and clinical and parasitological endpoints was estimated at the individual, household, and population level.A prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions in The Gambia. Serological analysis included samples from the West Coast Region at the start and end of the season (July and December 2013) and from the Upper River Region in July and December 2013 and April and December 2014. Antigen-specific antibody responses to eight PfMSP119 (aOR 4.09 95% CI 2.60–6.44), PfAMA1 (aOR 2.32 95% CI 1.40–3.85), and PfGLURP.R2 , while asymptomatic infection was associated with sero-positivity to all antigens. Village-level sero-prevalence amongst children 2–10 years against Etramp5.Ag1, HSP40.Ag1, and PfMSP119 showed the highest correlations with clinical and P. falciparum infection incidence rates. For all antigens, there were increased odds of asymptomatic P. falciparum infection in subjects residing in a compound with greater than 50% sero-prevalence, with a 2- to 3-fold increase in odds of infection associated with Etramp5.Ag1, GEXP18, Rh2.2030, PfMSP119, and PfAMA1. For individuals residing in sero-positive compounds, the odds of clinical malaria were reduced, suggesting a protective effect.Strong associations were observed between clinical malaria and concurrent sero-positivity to Etramp5.Ag1 (aOR 4.60 95% CI 2.98–7.12), At low transmission, long-lived antibody responses could indicate foci of malaria transmission that have been ongoing for several seasons or years. In settings where sub-patent infections are prevalent and fluctuate below the detection limit of polymerase chain reaction (PCR), the presence of short-lived antibodies may indicate recent infectivity, particularly in the dry season when clinical cases are rare. Serological responses may reflect a persistent reservoir of infection, warranting community-targeted interventions if individuals are not clinically apparent but have the potential to transmit. Therefore, serological surveillance at the individual and household level may be used to target interventions where there are foci of asymptomatically infected individuals, such as by measuring the magnitude of age-stratified antibody levels or identifying areas with clustering of above-average antibody responses across a diverse range of serological markers. As malaria transmission declines, the prevalence of infection becomes increasingly heterogeneous and focal. In low transmission settings, highly sensitive diagnostics are needed to measure subtle changes in malaria incidence, evaluate the effectiveness of community-based interventions, and monitor potential re-introduction after elimination. Evidence suggests that infections below the detection limit of microscopy and rapid diagnostic tests (RDTs) contribute to on-going transmission, but the magnitude of their public health importance is yet to be determined –3. NucleSerological assays measuring antibody responses may be a more stable diagnostic alternative, offering a cost-effective detection method to supplement existing surveillance tools. A number of studies in The Gambia have observed spatio-temporal variations in antibody responses to malaria over time , 7, withUsing a suite of novel serological markers of malaria exposure, this study aimed to determine the correlation of antibody responses with clinical and parasitological endpoints at the individual, household, and population level in rural Gambia over 2 years and estimate the strength of association between serological markers and gold standard metrics of active malaria infection. These findings can support the application of serology in measuring recent malaria transmission and allow selection of markers most suitable for use in future surveillance, particularly for reactive detection strategies in pre- and post-elimination settings.Plasmodium falciparum (Pf) prevalence measured by polymerase chain reaction (PCR) ranged from 2.27 to 19.60% in the Central River and Upper River Regions respectively .To understand the dynamics of malaria infection and the impact of annual mass drug administration (MDA), a prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions—West Coast (WCR), North Bank (NBR), Lower River (LRR), Central River (CRR), and Upper River (URR) Regions- as described by Mwesigwa et al. [n = 4599) from a selection of monthly surveys in four villages, totalling 1795 individuals  and at the end of the season in December 2013 (N = 524). In the Upper River Region (URR), serological analysis included all samples collected in Njaiyal and Madina Samako in July 2013 (N = 778), December 2013 (N = 628), April (dry season) 2014 (N = 799), and December 2014 (N = 737) . Verbal consent was first obtained at village sensitisation meetings, followed by individual written informed consent for all participants. Parents/guardians provided written consent for children less than 17 years, and assent was obtained from children between 12 and 17 years.Escherichia coli (E. coli) as glutathione S-transferase (GST)-tagged fusion proteins [PfAMA1 expressed in Pichia pastoris as a histidine-tagged protein [E. coli was used in assay buffers to eliminate background reactivity to E. coli proteins that were not specific to malaria target proteins.Antigens were selected from an initial screen of 856 candidates on an in vitro transcription and translation (IVTT) protein microarray based on correlation with malaria infection in children . Antigenproteins –22, with protein . ProteinAdditionally, to account for potential non-malaria reactivity against GST-tagged fusion proteins, GST-coupled beads were included to quantify GST-specific immunoglobulin (IgG) responses and correct for non-specific binding. After laboratory processing, there were 71 participant samples with GST antibody responses above 1000 median fluorescence intensity (MFI), which was defined as the threshold to indicate potential non-malaria-specific binding, and were excluded from further analyses. Tetanus toxoid  was also included as an internal positive control, assuming that vaccinated Gambians would show antibody responses to this protein target. A summary of antigen constructs and coupling conditions are detailed in Table E. coli extract). Negative and positive controls were also incubated 1 day prior in Buffer B, with negative controls prepared at a 1:500 dilution and Gambian pooled positive controls in a 6-point 5-fold serial dilution . The positive control was based on a pool of 22 serum samples from malaria hyper-immune individuals in The Gambia, and ten individual plasma samples from European malaria-naive adults were used as negative controls.Antigen-specific antibody responses were quantified using the Luminex MAGPIX protocol described in Wu et al. 2019 . Plasma Plasmodium ribosomal RNA gene sequences using genus and species specific primers [Samples were prepared for diagnostic PCR as described by Mwesigwa et al. . Briefly primers . All PCRP. falciparum merozoite surface antigen 1 19-kDa carboxy-terminal region (PfMSP119), P. falciparum apical membrane antigen 1 (PfAMA1), and P. falciparum glutamate-rich protein, region 2 (PfGLURP.R2)—were included as a comparison, which have historically been used to assess sero-conversion rates over time [PfMSP119, PfAMA1, PfGLURP.R2), individuals residing in an endemic region previously exposed to malaria, but not recently infected, may still have residual antibody levels that are significantly higher than malaria-naïve individuals in non-endemic regions. Therefore, a two-component Gaussian mixture model was used to define distributions of negative and positive antibody levels, expressed in units of median fluorescence intensity (MFI). Sero-positivity thresholds were defined as the mean log MFI values plus two standards deviations of the negative distribution [normalmixEM’ function in the ‘mixtools’ package v1.0.4 in R version 3.6.1. For antigens associated with shorter-lived antibodies where statistical evidence of a bimodal distribution of antibody responses in the population was not strong given the more rapid decay of antibody levels post-infection—Etramp5.Ag1, GEXP18, HSP40.Ag1, EBA175, and Rh2.2030—the sero-positivity threshold was defined by the mean log MFI plus three standard deviations of 71 malaria-naïve European blood donors used as negative controls.Data analysis was based on total IgG levels to five antigens as potential markers of sero-incidence —early trver time , 9 and hver time , 7. For ribution . MixtureP. falciparum infection (actively detected using PCR from monthly survey samples) were assessed using generalised estimating equations (GEE). Analysis was adjusted for age group  and use of long-lasting insecticide-treated nets (LLINs) in the last 24 h and allowed for clustering at the compound level, where compound is a collection of households centrally located around a main residence. The magnitude of the association between antibody response and odds of infection was evaluated for interaction with age group. Based on a subset of longitudinal samples, individual-level association between antibody response and recent infection in the previous 4 months (as opposed to current infection at the same time point) was also assessed using a GEE model, adjusted for age group, LLIN use, and random effects at the compound level, as above.Individual-level association between antibody response and the concurrent odds of clinical malaria  or asymptomatic Using a GEE model, individual-level odds of clinical malaria or asymptomatic infection was assessed for association with residing in the same compound as a sero-positive individual. Similarly, the association between individual odds of infection and compound-level sero-prevalence (< 50% or > 50%) was assessed using a mixed-effects generalised linear model adjusted for age group and LLIN use and random effects at the compound level, which also accounts for the potential effect of geographical differences in transmission intensity. To ensure that estimates are not biased by the sero-prevalence of small compounds, analysis was weighted by the number of individuals in the compound (whose sero-status was assessed) and only included compounds with at least four individuals. Models were fit using the ‘geeM’ and ‘lme4’ packages in R version 3.14.n = 1001) was compared against all-age clinical and P. falciparum infection incidence rates from the same months, the latter of which were previously reported by Mwesigwa et al. [P. falciparum infection incidence rates were defined respectively as the number of new clinical cases or P. falciparum infections  divided by total person years at risk (PYAR). This age range was selected to align with other routine surveillance metrics, such as annual parasite index (API), commonly using this age group as a sentinel population. The strength of the relationship between village-level incidence rates and sero-prevalence for each antigen was assessed using Pearson’s correlation coefficient. Data from April and December 2014 were not included in the analysis because clinical incidence and Pf infection rates from these surveys have not yet been reported.Village-level sero-prevalence amongst children aged 2–10 years in the West Coast Region and Upper River Region in July and December 2013  was three times higher than at the start of the transmission season in July 2013 (0.23 95% CI 0.13–0.39)  was five times higher than July 2013 (0.56 95% CI 0.42–0.74) (Table PfMSP119 in July 2013 to 36.3% (95% CI 30.2–42.3) for GEXP18 in December 2013 , HSP40.Ag1 (ρ = 0.99), PfMSP119 (ρ = 0.99), and Rh2.2030 (ρ = 0.94). Incidence of clinical malaria had the strongest correlations with Etramp5.Ag1 (ρ = 0.97), PfMSP119 (ρ = 0.95), PfGLURP.R2 (ρ = 0.92), and HSP40.Ag1 (ρ = 0.90). After excluding December 2013 data from the Upper River Region as a potential outlier, correlations with both clinical incidence and P. falciparum infection rates remained high for Etramp5.Ag1  and PfMSP119 . These correlations are based on few data points, with only three observations when excluding the Upper River Region in December 2013.In both regions in July and December 2013, correlations between PfMSP119, PfAMA1, and PfGLURP.R2) , while odds of asymptomatic infection were strongly associated with sero-positivity to all antigens. Amongst individuals sero-positive for Etramp5.Ag1, there was increased odds of both clinical malaria  and asymptomatic infection  , PfGLURP.R2 , and PfAMA1 . Odds of asymptomatic infection in individuals sero-positive to GEXP18 , Rh2.2030 , PfAMA1 , and PfGLURP.R2  was similar to Etramp5.Ag1. Additionally, a subset of longitudinal samples were used to explore the dynamics of antibody responses following infection. After adjusting for age group and LLIN use, antibody responses to six out of eight antigens were significantly associated with infection  in the previous 4 months. Antibody levels against Etramp5.Ag1 were most strongly associated with previous infection , followed by PfAMA1 , EBA175 , GEXP18 , PfMSP119 , and Rh2.2030 .The odds of clinical malaria were higher in individuals sero-positive to six (out of eight) of the antigens analysed  to a 95% reduction if residing in the same compound as someone sero-positive for PfAMA1 . Conversely, the odds of asymptomatic infection were positively associated with residing in the same compound as sero-positive individuals for five antigens . Adjusted odds of asymptomatic infection was nearly 3-fold higher if residing with an individual sero-positive for Etramp5.Ag1  and for GEXP18 . If residing in a compound with individuals sero-positive to Rh2.2030, PfMSP119, or PfAMA1, the odds of asymptomatic infection were all nearly twice as high as individuals living in compounds sero-negative to these antigens  in July 2013 and 39.8% (95% CI 23.6–56.0) in December 2013, while prevalence of compounds sero-positive for PfAMA1 and PfGLURP.R2 ranged between 77.0% (95% CI 66.9–87.1) in July 2013 and 80.7% (95% CI 71.5–89.9) in December 2013  in July 2013 to 100% in December 2014, but prevalence was slightly lower for PfMSP119, which was between 70.4% (95% CI 55.9–84.9) in July 2013 and 88.6% (95% CI 80.7–96.5) in December 2014. By contrast, prevalence of sero-positive compounds for short-lived antibody responses were lower in both regions, ranging from 41.4% (95% CI 25.3–57.5) for Etramp5.Ag1 to 63.2% (95% CI 50.5–76.0) for EBA175 in July 2013 in the West Coast Region and from 72.2% (95% CI 58.2–86.3) for Etramp5.Ag1 in July 2013 to 97.1% (95% CI 93.2–100) for GEXP18 in December 2014 in the Upper River Region.The prevalence of sero-positive compounds for long-lived antibody responses varied by antigen and region but remained generally stable throughout the transmission season. For PfMSP119)   in compounds with less than 50% sero-prevalence to aOR 8.17  in compounds with sero-prevalence greater than 50%, compared to sero-negative compounds as the baseline reference  to 8.85   to aOR 8.75   suggests that clinical malaria and asymptomatic infection is localised near households with Etramp5.Ag1 sero-positive individuals Fig. . There aPfMSP119, PfAMA1, and PfGLURP.R2, while asymptomatic infection was correlated with sero-positivity to all antigens included in this study. The strongest associations were between asymptomatic infection and sero-positivity to Etramp5.Ag1 and PfAMA1. Village-level sero-prevalence amongst children 2–10 years against antigens Etramp5.Ag1, HSP40.Ag1, and PfMSP119 showed the highest correlations with clinical incidence and Pf infection rates. In addition to current infection, antibody responses to several antigens, particularly Etramp5.Ag1, were associated with prior infection in the last 4 months, highlighting the potential for cross-sectional serological surveillance to identify areas of recent malaria exposure, even after parasites have been cleared and are no longer detectable by PCR. To better inform how these antigenic targets can be used to estimate time since infection, future research is needed to quantify the half-life of antibody responses and determine the sensitivity and specificity for identifying previous infection across varying epidemiological settings. This will require more detailed cohort studies with intensive follow-up for extended periods  and with sufficient sample sizes to capture individual heterogeneity [We investigated the association between antibody responses and clinical and parasitological endpoints at the individual, household, and population level across The Gambia. At the individual level, clinical malaria was highly correlated with concurrent sero-positivity to Etramp5.Ag1, ogeneity .PfMSP119. While these results cannot confirm a causal relationship between serological responses and clinical outcomes or asymptomatic infection, they indicate that infected individuals tend to be concurrently sero-positive to most of the serological markers assessed. There was strong evidence of reduced odds of clinical malaria for individuals residing in the same compound as an individual sero-positive to any of the antigens, suggesting a protective effect. In fact, previous studies suggest that several of the antigens investigated in this study—EBA175, Rh2, PfGLURP.R2, PfAMA1, and PfMSP1—have a functional role in mediating protective immunity if antibodies are acquired at a sufficiently high magnitude [At the household level, individuals had increased odds of asymptomatic infection when residing in a compound with greater than 50% sero-prevalence to all antigens, with the highest odds associated with sero-prevalence to Etramp5.Ag1, GEXP18, and agnitude –29.PfMSP119 and PfAMA1 have been used extensively to measure medium- and long-term trends in transmission intensity, these markers may be less sensitive to changes in transmission at higher endemicities or to short-term fluctuations due to the rapid acquisition of antibodies and the long half-life of sero-positivity for some blood-stage antigens [PfAMA1, PfMSP119, and EBA175. In settings where most infections are sub-patent and fluctuate above or below the detection limit of PCR, detecting the short-lived antibody responses in individuals or households could imply recent infectivity, particularly in the dry season when clinical cases are rare.While antigens –33. Indiantigens . The celWhile factors such as the method for determining sero-positivity cut-off values could affect estimates of sero-prevalence or the magnitude of association between endpoints , the obsImmuno-epidemiological studies will be subject to variations in antigen-specific antibody dynamics, as well as the antigenicity of recombinant constructs. Differences in sequence selection and expression systems may result in recombinant proteins that differ slightly from natural epitope conformation or exposure. It is unclear the degree to which these subtle variations affect the strength of antibody detection between recombinant antigens. While this study focuses primarily on total IgG responses, quantification of antibody levels using a combination of isotypes or subclasses may allow for improved detection of sero-incidence. For example, some studies have found that IgG3 antibodies bind more weakly or have a reduced serum half-life relative to IgG1 , 38, sugChanging household sero-prevalence throughout the season may influence the precision of estimates, which could be overcome with more frequent sampling of study participants, such as 3-month intervals depending on antibody decay rates. The findings in this study suggest that household sero-prevalence may be a robust proxy for past malaria exposure. It is simultaneously associated with increased risk of asymptomatic infections, but reduced risk of clinical malaria . However, the biological relationship between household serological status and the risk of clinical or parasitic infection could be better investigated and confirmed through controlled observational cohort studies.Our study findings indicate that a number of new serological markers of malaria exposure could be useful for epidemiological surveillance in highly seasonal malaria transmission areas. There is potential for several of the antigenic targets explored to be used for population-wide screening, all of which have also been optimised for use on a multiplexed bead-based platform in research settings. Further translating this to a lateral flow device may offer opportunities for widespread application if clear use-cases can be established . In lighAs more countries move towards malaria elimination, including several in sub-Saharan Africa, sensitive surveillance tools will be needed to further understand the dynamics between the parasite, host, and vector at very low levels of transmission. Integrating serological markers of recent exposure into existing field diagnostics and case reporting will become increasingly important in estimating the human reservoir of malaria infection, understanding patterns of transmission, and designing targeted elimination strategies.Additional file 1. Sero-prevalence amongst individuals aged 2–10 year, prevalence of sero-positive compounds, and population size and age distribution by compound.Additional file 2. Odds of clinical malaria and asymptomatic infection for sero-positive individuals and individuals residing in sero-positive compounds. Unadjusted and adjusted odds ratios for eight malaria antigenic targets - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175, PfMSP119, PfAMA1, and PfGLURP.R2.Additional file 3. Map of household geolocation of PfAMA1 sero-positive, clinical malaria and Pf infections across four villages during dry and wet transmission seasons. Spatial distribution of infections shown for lower transmission (N’demban) and higher transmission (Besse) villages in the West Coast Region (WCR) and lower transmission  and higher transmission (Madina Samako) villages in the Upper River Region (URR). Infections at the start of the wet season (June – July 2013) shown on the left and during the wet and transmission season (August – December 2013) on the right.Additional file 4. Gambia serology endpoints study data.


Malaria is a major public health risk in Rwanda where children and pregnant women are most vulnerable. This infectious disease remains the main cause of morbidity and mortality among children in Rwanda. The main objectives of this study were to assess the prevalence of malaria among children aged six months to 14 years old in Rwanda and to identify the factors associated with malaria in this age group. This study used data from the 2017 Rwanda Malaria Indicator Survey. Due to the complex design used in sampling, a survey logistic regression model was used to fit the data and the outcome variable was the presence or absence of malaria. This study considered 8209 children in the analysis and the prevalence of malaria was 14.0%. This rate was higher among children aged 5–9 years old (15.6%), compared to other age groups. Evidently, the prevalence of malaria was also higher among children from poor families (19.4%) compared to children from the richest families (4.3%). The prevalence of malaria was higher among children from rural households (16.2%) compared to children from urban households (3.4%). The results revealed that other significant factors associated with malaria were: the gender of the child, the number of household members, whether the household had mosquito bed nets for sleeping, whether the dwelling had undergone indoor residual spraying in the 12 months prior to the survey, the location of the household’s source of drinking water, the main wall materials of the dwelling, and the age of the head of the household. The prevalence of malaria was also high among children living in houses with walls built from poorly suited materials; this suggests the need for intervention in construction materials. Further, it was found that the Eastern Province also needs special consideration in malaria control due to the higher prevalence of the disease among its residents, compared to those in other provinces.  Malaria is one of the main public health problems in the world and is an important cause of morbidity and mortality among children in Sub-Saharan Africa. Worldwide, malaria incidence has significantly decreased between 2010 and 2017, from 71 to 57 cases per 1000 of the population at risk [In east African countries, the prevalence of malaria in children under five years has decreased in general, but it is still high. For instance, the prevalence of malaria from the last two recent studies in Burundi was 22% in 2013 and 27% in 2016–2017; in Kenya it was 5% in 2015; in Rwanda it was 2% in 2015 and 7% in 2017; in Tanzania it was 14% in 2016 and 7% in 2017; in South Sudan it was 32% in 2017 and in Uganda it was 30% in 2015 and 30.3% in 2016 [Anopheles mosquito. It is most prevalent among children and among pregnant women in malaria-prone areas such as the Eastern Province and Southern Province which together accounted for 79% of malaria cases in Rwanda in 2017 [mutuelle de santé which has helped the citizens of Rwanda to access private or public health centers, clinics and hospital services. In addition, the government has implemented various strategies to prevent malaria, such as distributing insecticide-treated mosquito ets (ITNs) and employing indoor residual spraying (IRS) within the households. As a result, the prevalence of malaria among the population of Rwanda dropped drastically up to 2011 [In Rwanda, malaria is also a public health concern because the entire population is at risk of contracting this infectious disease. Malaria has been the main cause of morbidity and mortality in Rwanda for several years . It is a in 2017 .The GoveDespite the above-mentioned strategies, the prevalence of malaria among children under five years old in Rwanda has increased by 6% from 2010 to 2017 ,10. FurtThis study used cross-sectional secondary data from the 2017 Rwanda Malaria Indicator Survey (RMIS). This survey was conducted from October to December 2017, at the peak of malaria season. The survey used the sampling frame from the 2012 Rwanda Population and Housing Census . The datn = 8209 children aged six months to 14 years old were considered in this study.The response variable of interest in this study is the malaria rapid diagnostic test (RDT) result. The RDT is immune-chromatography form of test which assists in diagnosing malaria by detecting specific antigens (proteins) produced by malaria parasites in the blood of infected person. Some RDT can detect only one species  while others detect multiple species. If malaria antigens are present, then the person tests positive. If malaria antigens are not present, the person tests negative . TherefoThe potential explanatory variables considered in this study were demographic and socio-economic factors and factors such as age of the head of the household (continuous), age of the child , sex of the child , number of household members (or size of the family); socio-economic factors such as wealth index of the household , ownership of radio , ownership of television , use of electricity , wall material of dwelling , roof material of dwelling , floor material of dwelling , number of living rooms ; environmental and sanitation factors such as use of mosquito net for sleeping , whether the dwelling was sprayed for mosquitoes in the twelve months prior to the survey , place of residence , province of residence  and location of drinking water source . The justification as to the choice of these variables from a rigorous theoretical framework was that these variables have been found to be statistically significant in the existing literature ,13,14,15The surveys, were done were based on multi-stage sampling, stratified and cluster sampling with unequal probability of selection for elements to be included in the survey, known as complex survey design. When modelling the data collected from these surveys, the complexity of the sampling design must be taken into consideration. Therefore, in order to account for the effect of stratification, clustering, sampling weights and to relax the assumption of independence of observation of the ordinary logistic regression model, the present study used a survey logistic regression model for the data analysis. Failure to account for clustering and sampling weights may lead to underestimation of the variability and, consequently, to wrong inference. In general, the theory of ordinary logistic regression and survey logistic regression is the same; they differ only in variance estimation. They are both members of the generalized linear models, and a maximum likelihood is used to estimate the parameters. The model formulation used in this study is discussed in detail as follows:i from stratum j and cluster k, with i has malaria and Let The current study assumes that the outcome variable The analysis in this study was done using SAS Proc Surveylogistic from SAS software version 9.4 . The Taylor series method was used as a variance estimator. The model fit statistics were done based on Akaike information criteria (AIC) and −2 Log-Likelihood (−2LogL) principles. The model test was done based on the likelihood ratio, score and the Wald test principles.The current study considered 8209 children aged six months to 14 years; 4053 of them were female, whereas 4156 of them were male. Of the sample, 14.0% tested positive for malaria infection and 86.0% tested negative. It is observed from The association between RDT results and various potential factors was tested by the chi-square statistical test at a 5% level of significance and the results are summarized in The prevalence of malaria among children was highest in the Eastern Province, 31.1% compared to other provinces and it was lowest in the Northern Province at 2.0%. It was observed that the prevalence of malaria detected by RDT was higher among children from rural residential areas—16.2%—than their urban counterparts—3.4%. The results from the cross-tabulation showed that the prevalence of malaria among children was highest among children from the poorest households (19.4%) and lowest among children from the richest families (4.3%). The prevalence of malaria was lowest among children under five years (11.7%) and highest among children aged five to nine years old (15.6%) .p-value < 0.001)  . The pre< 0.001) .The current study considered the individuals’ available media and it was found that the prevalence of positive malaria RDT outcomes was lessened by the ownership of media such as a radio and a television. The prevalence of malaria was 10.7% among the children from households that owned a radio and 16.4% among the children from households that did not have a radio. Children from households that had a television were less associated with the prevalence of malaria (4.4%) and the prevalence of malaria was 15.1% among children from families which did not have a television.The model fit is presented in p-value < 0.0001), which means that at least one of the parameters is not equal to zero.The global null hypothesis states that all parameters are equal to zero. This was tested, with the results presented in The results from multiple survey logistic regression are summarized in In order to avoid possible confounding effects, the two-way interaction between potential variables was considered, but none were found to be significant. The results from multiple survey logistic regression are summarized in p-value = 0.0175). It was found that the probability of having malaria was higher among children living in rural areas than among their urban counterparts (Adjusted Odds ratio (OR) = 2.792, CI = 1.199, 6.498). The present study revealed that the type of wall materials was significantly associated with the risk of malaria among children aged six months to 14 years. It was observed from the results that children living in houses with re-used wood as the main wall material were 2.234 times more likely to have malaria than children living in houses with wall materials other than bamboo with mud or uncovered and a covered adobe. However, this study did not find a significant difference between children living in houses of bamboo with mud or an uncovered adobe and children living in houses with main wall materials other than reused wood.The place of residence was significantly associated with a risk of malaria in children aged six months to 14 years in Rwanda . The location of source of drinking water was also found to be a significant factor associated with malaria among children aged six months to 14 years . The results showed that children from a household where they used water from their dwelling or yard were less likely to have malaria, as compared to children from a household where they used water from locations other than the dwelling or yard. The risk of malaria was 0.106 times less among children from a family where they used water from their dwelling compared to children from households where they used water from other locations. The age of the head of the household was also found to be a significant risk factor associated with malaria among children aged six months to 14 years . The results revealed that one unit increase in age of the head of the household increased the odds of a positive malaria test by 0.9%  among children of that household.The number of household members was also found to be a significant risk factor associated with malaria among children in Rwanda . The resp-value = 0.0325) times less likely to have malaria than children from a household with one living room. The current study did not find a significant difference between children from a household with three or more living rooms and children from a household with one living room.The province of residence was also found to be a significant risk factor for malaria. The risk of having malaria was 2.490 times more likely among children from the Eastern Province compared to children from Kigali City. It was also found that children from the Northern Province and from the Western Province were, respectively, 0.097 and 0.145 times less likely to have malaria compared to children from Kigali City. The present study did not find a significant difference among children from the Southern Province and children from Kigali City, but the results showed a positive association. The number of living rooms was also found to be a significant risk factor associated with malaria prevalence among children aged six months to 14 years in Rwanda. Children from a household with two living rooms were 0.7 . Children from the age group 5–9 years were 1.357 times more likely to have malaria than those from age group six months to 59 months. However, the present study did not find a significant difference between children aged less than five years and those aged from 10 to 14 years.The age group of the children was found to be significantly associated with factors of malaria among children . The results from this study showed that the risk of malaria among children lessened with the use of mosquito bed nets for sleeping. Children from households where mosquito nets were not used for sleeping were 1.5 times more susceptible to malaria infection (OR = 1.499) compared to their counterparts from households where they used mosquito nets for sleeping. It was also observed that IRS was significantly associated with the risk factors of malaria . Children from households that had not been sprayed against mosquitoes in the 12 months prior to the survey were 2.266 times more likely to have malaria than those from households which had been sprayed against mosquitoes in the same time period.It was observed from Malaria is still the main cause of morbidity and mortality among children in Sub-Saharan African countries. The knowledge of the risk factors associated with malaria among children aged six months to 14 years provides an insight into the methods and policies that can be used to combat this public health problem effectively.The government of Rwanda has invested great efforts to improve healthcare programs, particularly at the level of primary healthcare. It has implemented several strategies for monitoring and evaluating malaria control on a regular basis, where the focus is mainly to reduce malaria morbidity and mortality.The present study was carried out based mainly on data from the 2017 Rwanda Malaria Indicator Survey. The main objective of this study was to identify the factors associated with malaria status among children aged six months to 14 years and to determine the prevalence of malaria among the children from this age group. In the current scientific setting we have used the gender of the child, the age of the child, the age of the head of the household, the number of household members, the number of living rooms, wall material of dwelling, roof material of dwelling, floor material of dwelling, province of residence, place of residence , use of ITNs for sleeping, location of source of drinking water.It was found that the likelihood of malaria increases with the increasing size of the household. This is in line with the findings of other studies such as ,14. The Umuganda (compulsory collective work done on the last Saturday of every month), which happens at the same time countrywide in all grass root administrations, or through Abajyanama Bubuzima  and this may be the reason why the media does not have an effect. This is confirmed by the high rate of malaria awareness in women aged 15 to 49 years old (81%) [It was found in this study that exposure to media such as a radio and a TV was not significant. The government of Rwanda sometimes communicates messages regarding malaria to the population through ld (81%) ,20,21,22The use of mosquito bed nets was found to play an essential role in preventing malaria infection among children aged six months to 14 years in Rwanda. The present study found that children from the families where mosquito bed nets were not used when sleeping were more vulnerable to malaria infection than children from families where they were used. While this is consistent with other findings from similar studies ,23,24,25The current study found that the malaria infection rate among children increases with their increasing age. A similar finding has been observed in previous studies ,27,28,29The results from this study revealed a higher infection rate of malaria among children from poor families compared to children from the richest families. This finding is consistent with previous studies ,30,31,32The results of this study showed a significant association between the type of house wall materials and malaria prevalence. This finding is consistent with similar studies ,36. It wThe province of residence was shown to be significantly associated with the risk of malaria, where children from the Northern Province and from Western Province had a low risk of contracting malaria compared to children from Kigali City. This finding is consistent with that of . This isIn the present study, survey logistic regression was used to account for the complexity of the design which led to appropriate statistical inference with accuracy. The findings from this study have provided insights into the demographic, socio-economic and environmental factors such as age of the household head, size of the household, wealth index of the household, whether the household was sprayed for mosquitoes in the 12 months prior to the survey, use of mosquito bed nets for sleeping, the dwellings’ main wall materials, location of source of water for drinking, place of residence, province of residence, and age of the children, which were significant factors associated with malaria prevalence among children aged six months to 14 years. This study found that the use of IRS and insecticide treated nets is an important intervention to prevent malaria infection among children aged six months to 14 years. It was found that the provinces had different risk levels, with the Eastern Province being highest risk. This suggests special consideration should be given for malaria control, such as the provision of ITNs, in this province. Therefore, the findings from this study may help public health planners, policy-makers and other related institutions in effective decision-making related to malaria control, especially as it pertains to children aged six months to 14 years. The higher prevalence of malaria was among children from low income households, which suggests the need for an intervention in households with lower income levels. The results from this study revealed a high prevalence of malaria among children aged five to nine years, which suggests the need for an intervention in this age group: for instance, increasing the use of mosquito nets among children of school-going age because it is in this age group that the use of mosquito nets for sleeping is low, compared to other age groups.The limitation of this study is that the data are cross-sectional and more meaningful trends and patterns could be identified from a longitudinal analysis. Future research can possibly be undertaken through incorporation of spatial information.