Genetic polymorphisms in the human immune system modulate susceptibility to malaria. However, there is a paucity of data on the contribution of immunogenetic variants to malaria susceptibility in infants, who present differential biological features related to the immaturity of their adaptive immune system, the protective effect of maternal antibodies and fetal haemoglobin. This study investigated the association between genetic variation in innate immune response genes and malaria susceptibility during the first year of life in 656 infants from a birth cohort survey performed in Nanoro, Burkina Faso.Plasmodium falciparum infection and clinical disease were documented by active and passive case detection. Case–control association analyses for both alleles and genotypes were carried out using univariate and multivariate logistic regression. For cytokines showing significant SNP associations in multivariate analyses, cord blood supernatant concentrations were measured by quantitative suspension array technology (Luminex).Seventeen single nucleotide polymorphisms (SNPs) in 11 genes of the immune system previously associated with different malaria phenotypes were genotyped using TaqMan allelic hybridization assays in a Fluidigm platform. P. falciparum infection and clinical malaria. Furthermore, heterozygote individuals with rs1801274 SNP in FcγRIIA/CD32 showed higher IL-1RA levels compared to wild-type homozygotes (P = 0.024), a cytokine whose production is promoted by the binding of IgG immune complexes to Fcγ receptors on effector immune cells.Genetic variants in IL-1β (rs1143634) and FcγRIIA/CD32 (rs1801274)—both in allelic, dominant and co-dominant models—were significantly associated with protection from both These findings indicate that genetic polymorphisms in genes driving innate immune responses are associated to malaria susceptibility during the first year of life, possibly by modulating production of inflammatory mediators. Plasmodium protozoan parasites and transmitted by Anopheles mosquitoes. Despite global malaria control and elimination efforts, which reduced the number of malaria-related deaths by 50% since 2000, malaria remains a major public health problem, particularly in pregnant women and children from sub-Saharan Africa [Malaria is a life-threatening infectious disease caused by n Africa , 2.Individual risk for malaria infection and disease is complex and multifactorial, and is influenced/modulated by host genetic background , 4. QuanIn Burkina Faso, genetic epidemiology has demonstrated that the wild-type R131 allele rs1801274) of the FcγRIIA (CD32) and tumour necrosis factor (TNF)-238G allele (rs361525) were associated with protection from clinical malaria and high parasitaemia, respectively, in infants and children until 10 years of age 4 of the . In contPrevious studies by our group in Burkina Faso described that malaria infections and disease during the first year of life is high and has a marked age and seasonal-dependency , that inThe study was conducted in the rural health district of Nanoro (Burkina Faso), located in the central-west region at 85 km from the capital Ouagadougou. Nanoro health district (NHD) has approximately 166,683 inhabitants and comprises 21 peripheral health centres. Malaria transmission is seasonal and hyperendemic with peaks between July and December and overlapping with the rainy season (June to November). Malaria burden in infants is high and strongly dependent on age and season . BetweenA birth-cohort study (n = 734) was nested within the COSMIC clinical trial (NCT01941264) to investigate factors that modulate the risk of malaria during the first year of life . COSMIC P. falciparum var genes acidic terminal sequence  in StepOne Plus thermocycler (Applied Biosystems), as previously described [Parasite and human genomic DNA were extracted from three punches (5 mm in diameter) of dried blood spots on filter papers with QIAamp 96 DNA blood kit  and eluted in 150µL of water, following the manufacturer’s recommendations. Five microliters of DNA were used as template for qPCR analysis targeting escribed . The limescribed . Sampleshttp://phase3browser.1000genomes.org/), and DNA obtained from Coriell Cell Repositories. Both GWD controls and duplicate samples were included in all dynamic arrays. PCR and image processing were carried out on the BioMark® HD080 system (version 4.1.2) to determine SNP genotypes from clusters in each assay. Plots generated by the Fluidigm analysis software were subsequently visually revised to check the cluster profile for each assay and how samples with different genotypes were separated. Allelic calls for all GWD controls were consistent across all runs, and all duplicate samples within each plate had identical allele calls. The genotyping success and the overall call rate was determined per SNP.The SNPs selection for typing was performed based on existing published data. A SNP was included for analysis if: (i) had been previously associated with a malaria-related phenotype , (ii) had an expected minor allele frequency (MAF) ≥ 5% in African populations, and (iii) had a functionally tested and/or validated TaqMan® SNPs genotyping assay. The final SNPs selected for analysis were: TLR1 (rs4833095), TLR4 (rs4986790), TLR9 , interleukin (IL)-4 (rs2243250), IL-10 (rs1800890 and rs1800896), IL-17F (rs4715291), IL-1β (rs1143634), TNF (rs1800629 and rs3093664), interferon-γ receptor 1 , nitrogen oxide synthase 2  and FcγRIIA/CD32 (rs1801274). TaqMan® SNPs genotyping assays were obtained from Applied Biosystems, and genotyping assays performed in GT192.24 dynamic arrays using the BioMark® platform (Fluidigm) at the Department of Genomics of Common Disease, Imperial College . All samples underwent 16 cycles of specific target amplification (STA) in a total reaction volume of 10µL. Eight control samples containing known combinations of mutations at selected loci were identified from the Gambian in Western Division (GWD) population based on data from 1000 Genomes Project (Haemoglobin genotypes were determined by high resolution melting (HRM) analysis, adapting previously described methods . Reactio2, and then frozen at − 80 °C. IL-1β and IL-1RA were quantified with the fluorescent bead-based multiplex immunoassay  and analysed on a Luminex® 100/200™ instrument using Xponent 3.1 software.Levels of IL-1β and IL-1RA by cord blood cells unstimulated and stimulated with TLR7/8 agonist (a stimulant that demonstrated higher induction of cytokine production than TLR3 and TLR9 agonists in the same cohort) were determined as described previously . BrieflyP. falciparum infection during the first year of life, regardless of symptoms; controls were infants who remained uninfected during the first 12 months of life. Uninfected infants who did not complete the 12-month follow-up were excluded from the analysis.The association between polymorphisms in immune genes and (a) malaria infection or (b) clinical malaria was investigated using case–control analysis. For malaria infection analysis, cases were infants who had at least one P. falciparum of any density by qPCR and an axillary temperature ≥ 37.5 °C or history of fever within the past 24 h) from birth to 12 months of age, whereas controls were who did not develop a clinical episode, irrespective of infection status.Clinical malaria cases were infants who experienced at least one clinical episode  was examined using Pearson’s chi-square statistical test. SNPs were excluded from the analysis if they had more or equal 10% of genotype calls missing or a significant genotypic deviation from HWE (P < 0.001). Case–control association analyses for both SNPs alleles and genotypes were assessed using logistic regression in co-dominant, dominant, over-dominant and recessive models [t-test. Benjamini–Hochberg method was applied to adjust P-values for multiple comparisons [P-value < 0.05 was considered statistically significant.Data was analysed using R statistical package version 3.2.3 . Baseline models . Maternae models ), ethnicparisons . A P-valP. falciparum infection during the first year of life, out of which 78% (411/527) developed at least one clinical episode. The baseline characteristics of the study participants (cases and controls) are presented in Table P = 0.021 and P < 0.001, respectively). In addition, the proportion of infants born during the malaria high-transmission season was significantly higher among clinical malaria controls than cases (P = 0.031). In contrast, the proportion of infants born with LBW was significantly higher among clinical malaria cases than controls (P = 0.015). There was an ethnic homogeneity among the study participants with most infants (90%) belonging to the Mossi ethnic group. The proportion of children whose mother received 3 or more SP doses during pregnancy was significantly higher among controls than cases for both malaria infection and clinical episode . There were no significant differences in haemoglobin genotypes between cases and controls. All the participants were breast feed during the first year of life.Of the 694 infants whose DNA samples were genotyped, 656 (94.5%) completed the 12 months follow-up and were included in the analysis. Overall, 80.3% (527/656) of infants experienced at least one P < 0.001). Among the 15 SNPs included in the final analysis, SNPs rs4833095 (TLR1), rs3093664 (TNF-α) and rs2297518 (NOS2A) showed a minor allele frequency below 10% , which had > 10% missing data and was therefore removed from the analysis  and CR Table .Table 3SP = 0.003) and the mutant allele A in FcγRIIA/CD32  were more likely to be protected against malaria infection than infants with the wild type G allele. The genotypic analysis (univariate and multivariate) confirmed that polymorphisms in IL-1β  and iFcγRIIA/CD32  were significantly associated with protection from malaria infection. Furthermore, protection was enhanced among homozygotes individuals in co-dominant models when comparing the genotypes carrying the mutant allele versus the wild-type genotype for both IL-1β  and FcγRIIA/CD32  SNPs .Both univariate and multivariate allele-based analysis showed that infants carrying the mutant allele A in IL-1β  = 0.52, 95%CI .34–0.80, P = 0.032) and mutated allele A in FcγRIIA/CD32 rs1801274 SNP  were associated with protection from clinical malaria. The same associations were observed in the genotype-based analysis showing that individuals with AA genotype in IL-1β rs1143634 SNP  and those with GA/AA genotypes in FcγRIIA/CD32 rs1801274 SNP  were more likely to be protected against clinical malaria , while both AA mutant homozygote and GA heterozygote individuals for FcγRIIA/CD32 rs1801274 SNP were significantly protected   .Next it was assessed whether polymorphisms in IL-1β (rs1143634) were associated with immune effector phenotypes. Therefore, IL-1β levels were quantified in a sub-group of infants) for whom un-stimulated and TLR7/8-stimulated cord blood samples were available (N = 313). Figure P = 0.016, Fig. P = 0.024), while no significant difference was observed for individuals carrying the AA genotype (P = 0.487). Differences of IL-1RA levels between genotypes in un-stimulated cord blood samples was not significant . InThis study investigated associations between genetic variants in immune genes and malaria susceptibility in infants in Burkina Faso. The study focused on cytokine, TLR, FcγRIIA/CD32 and NOS2A genes given their key role in innate immune responses against pathogens in early infancy. SNPs in FcγRIIA/CD32 (rs1801274) and in IL-1β (rs1143634) were significantly associated with protection from both malaria infection and clinical disease during the first year of life and FcγRIIA/CD32 SNP rs1801274 genotypes were associated with IL-1RA levels, a cytokine involved in inflammatory responses to malaria infection.vs GG and GA vs GG) and dominant (GA/AA vs GG) models after adjusting by potential confounding factors.A number of studies have explored the association between FcγRIIA/CD32 rs1801274 SNP and malaria susceptibility resulting in contradictory findings , 54–59. P. falciparum infection through production of inflammatory mediators [Fcγ receptors  bind to the Fc domain of IgG on immune cells and thereby mediate the initiation of a variety of immunological responses including antigen presentation, phagocytosis, cytotoxicity, release of inflammatory mediators and the modulation of immune responses , 62. Theediators , 62, 64.ediators , 53. Sevediators –68, whicediators . While tediators . TherefoThe second genetic variant in immune genes associated with malaria protection in this study was the IL-1β rs1143634 SNP. The carriage of IL-1β rs1143634 mutated allele A (both in homozygosity and heterozygosity) was associated with a decreased risk of malaria infection . In addition, AA homozygote carriers (but not AG) had a significantly reduced risk of clinical malaria, suggesting an enhanced protective effect of AA homozygosity. This is the first study to report a protective effect of IL-1β rs1143634 SNP against malaria infection and uncomplicated disease in infants. Previous studies assessing associations of IL-1β rs1143634 and malaria susceptibility reported heterogeneous findings in children including an association with higher peripheral parasitaemia in Ghanaian children  , an assoIL-1β is a pro-inflammatory cytokine that is implicated in the first line of defence against pathogens, including the hepatic and erythrocytic stages of malaria parasites –75. HoweHowever, these findings should be interpreted with caution due to very low number of individuals carrying the AA genotype among the subset of infants that could be analysed (n = 4) and, therefore, potential functional changes in IL-1β production by IL-1β rs1143634 SNP cannot be ruled out. Likewise, it is worth noting that IL-1β production is likely to be influenced by other SNPs in IL-1β gene, as well as polymorphisms in inflammatory mediators such as TNF –84 that P = 0.057 and P = 0.052, respectively). IL-10 is a key immuno-regulator of immunity to infections [Other genes coding for major TLRs  and cytokines  did not show significant association with malaria infection and clinical episodes, although rs1800890 SNPs in IL-10 gene showed a trend towards increased susceptibility to malaria infection or clinical episode in multivariate analysis (fections  and has fections –91. In afections , 92–95. This study showed that the mutated A allele of IL-1β rs1143634 and mutated A allele of FcγRIIA/CD32 rs1801274 are associated with protection against both malaria infections and uncomplicated disease during the first year of life. These findings suggest that genetic polymorphisms in genes driving innate immune responses could condition malaria susceptibility during the first year of life, possibly by modulating production of inflammatory mediators. Future investigations on the epistatic effects of immune genes polymorphisms described here and functional studies may contribute to further understand how host genetic variation influences malaria susceptibility in infancy.Additional file 1: Table S1. Genotypic-based association analysis for malaria infection using co-dominant models.Additional file 2: Table S2. Genotypic-based association analysis for clinical malaria using co-dominant models.


The results showed that 349 (46.29%) were infected with a single parasite. Infants and preschool-aged children infected with double, triple, and quadruple parasites are 268 (35.54%), 122 (16.18%), and 15 (1.99%), respectively. The prevalence of polyparasitism is 405 (53.71%). Although females (54.07%) were more infected than males (45.93%), there was no significant difference (p > 0.05) observed. Significantly (p < 0.05) more preschool children (65.93%) harbour more infections than the infants do (34.07%). Ara community (14.81%) had the highest cases of polyparasitized IPSAC, but no significant difference (p > 0.05) was observed across the communities. Double parasitic infection of Plasmodium falciparum and Ascaris lumbricoides (30.12%) and triple parasitic infection of P. falciparum, A. lumbricoides, and T. trichiura (14.81%) were the most common forms of polyparasitism encountered in the study. This study showed that polyparasitism is a burden in IPSAC and needs further investigation.Polyparasitism is widespread in many communities in Sub-Saharan Africa. However, there is paucity of data on polyparasitism in infants and preschool-aged children (IPSAC), to inform policy developments. Therefore, a survey of 1110 consented IPSAC was undertaken in Egbedore Local Government Area (LGA), Osun State, Nigeria, to determine the prevalence of polyparasitism in IPSAC in ten randomly selected rural communities. Fresh stool and blood samples were collected and processed for intestinal parasites and malaria infection. Mothers/caregivers were interviewed using a structured questionnaire to obtain demographic data of their IPSAC and to document knowledge, attitude, and practice (KAP) on parasitic infections. Data obtained through the questionnaire were analyzed using EpiData version 3.1, while parasitological data were analyzed using Statistical Package for Social Sciences (version 20.0). Descriptive statistics were computed for demographic data and association which were tested using bivariate analysis at a 95% confidence level while significance was set at  Polyparasitism is widespread in many communities in the tropics and subtropics. Coinfection of malaria and intestinal parasites is among the most common polyparasitic infections in Sub-Saharan Africa (SSA), where significant proportions of the populations including infants and preschool-aged children, school-aged children, and adults are exposed to these infections , 2. The Ascaris lumbricoides infections, Trichuris trichiura infections, and the hookworm infections caused by Necator americanus and Ancylostoma duodenale, and in less-developed countries, it is common that children are parasitized with more than one species at the same time, “with resultant impairments in physical, intellectual, and cognitive development” [In the tropics, intestinal parasites constitute a major public health problem, as these areas are often characterised by all the conditions  favouring transmission of these infections. Helminthiasis is a major cause of morbidity, especially in resource-limited settings . The inclopment” . In addilopment” . The tralopment” , 11.Malaria is thought to be responsible for 35% of mortality in children under the age of five, 25% of maternal mortality, and 60% of hospital admissions for children under five . NationaIn Nigeria, these infections are a major public health problem among infants and preschool-aged children .Children coinfected with these parasites have less than optimal development, have reduced learning and school achievements , 15, andInfants and preschoolers comprise about 10-20% of the 3.5 billion people living in malaria and intestinal parasite endemic areas, and the evidence on the prevalence of these infections among these groups and the infantile is emerging, with a lot of gap in Nigeria , 22, 23.There is paucity of data on polyparasitism with malaria and intestinal parasites in IPSAC. Therefore, the present study was undertaken to determine the prevalence of malaria and IPI coinfection, on infants and preschoolers.2 with a population of 74,435 people as at 2006 census and projection of about 101,900 in 2016, out of which 21,231 were children between 0 and 5years. The main ethnic group is the Yorubas with Yoruba language as a means of communication. Majority of the inhabitants are engaged in small-scale farming; only few of the residents were civil servants. The inhabitants are mainly a Yoruba-speaking tribe that is made up of many towns, villages, districts, and 10 political wards headed by councilors. A Primary Health Center (PHC) is present in each of the communities. Data collection was carried out from May 2016 to February 2017 in ten rural communities  in Osun State, Southwest Nigeria. Its headquarters is in Awo town located at 7°46N and 4°24E. It has an area of 270 kmmunities .Approval for the study was obtained from Osun State Health Research Committee (OSHREC) Ministry of Health (Ref. Number: OSHREC/PRS/569T/66). The director of public health services in the LGA and coordinators of the Primary Health Center in each community sampled were notified about the study; they assisted in the sensitization and mobilization of mothers/caregivers. Informed consent forms were distributed to mothers/caregivers; the forms were verbally translated to the parents and caregiver in their local language; only mothers/caregivers who consented by signing the consent form were recruited into the study.The survey employed a design involving the administration of a structured questionnaire for mother/caregivers and the collection of blood and stool samples from consented infants and preschoolers for laboratory analysis to check for malaria parasite and intestinal parasite infections. The inhabitants of the communities were sensitized prior to field surveys by the research team through their leaders, Community Development Association (CDA) meetings, Primary Health Care (PHC) staff, and community mobilizers. A total sampling of households within the communities was done, and consenting parents with children belonging to age category 0–6 years were recruited into the study in accordance with Ekpo et al. (2012).Enumeration of households within the communities was done to enlist households with at least one or more infant and preschooler. Enumeration was followed simultaneously with recruitment of consenting parents/guardians and children .Children whose parent consented to the study were enrolled into the study and given enrolment numbers (household ID) for easy identification and monitoring.Sterile universal bottle(s) were given to consenting parents/guardians for submission of their wards fresh stool samples. Eligible households were visited in the morning hours of the next day for collection of stool samples, which were later transported to the laboratory where assessment was carried out. Stool samples were analyzed using the sodium-acetate acetic-acid formalin-ether (SAF-ether) concentration method for the presence of intestinal parasite cysts, eggs, and ova. The SAF-ether method was employed to increase the sensitivity of detecting helminth and protozoan ova in stools as well as to preserve the sample prior the time of laboratory assessment. Samples were analyzed within 24 hours of collection.Each bottle containing a stool sample was covered and agitated vigorously to efficiently suspend the stool in the solution. The stool suspension was strained into a centrifuge tube using double gauze of about 13 mm diameter placed in a funnel. The residue was discarded while the filtrate was centrifuged at 2000 rpm for 1 minute. The supernatant was discarded after centrifuging, and 7 ml of normal saline was added to the sediment and left to resuspend. 3 ml of ether was added to the suspension; a stopper was placed on the tube and the mixture shaken vigorously to mix before centrifuging for 5 minutes at 2000 rpm. The first three layers of the suspension observed after centrifuging was pipetted out using Pasteur's pipette leaving the last layer of sediment. The sediment was pipetted onto a clean, oil-free microscope slide and examined for ova of intestinal parasites using the Swiss TPH 2010 Diagnostic Bench Aids for species identifications using shapes of parasite eggs, cysts, and ova.μl of blood; the collected blood was added to the round well on the test kit cassettes and absorbed by the embedded pad.Blood samples were collected by hand pricking and examined microscopically for the presence of malaria parasites using malaria Rapid Diagnostic Tool (RDT). Infants and preschoolers were arranged in a census form and a corresponding number written on each test kit using permanent marker for identifications. The alcohol swab pack was used to clean the finger to be pricked using a circular motion for 10 seconds; the swab was allowed to air dry and a lancet was used to prick the finger of the respondents for a drop of blood; the lancet was discarded into the biohazard-labelled sharps container. The capillary pipette provided in the test kit was used to collect 10 Parents/caregivers whose children provided adequate stool specimens were carefully interviewed using a closed-end structured questionnaire in their different households. Demographic data of parent/caregiver and each infant and preschool, household sanitary, and personal hygiene conditions were documented using the questionnaire. Knowledge about malaria and intestinal parasitic infection and its transmissions, knowledge about coinfections, and attitude and practice of mothers/caregivers on parasitic infections and treatments were also documented using the structured questionnaire.Data obtained from the questionnaire and parasitological investigations were entered into Microsoft Excel version and analyzed using IBM Statistical Package for Social Sciences (SPSS) version 20.0. Descriptive statistics such as proportion and percentages was computed for demographic data while associations and relationship between risk factors and disease outcome was tested using chi-square and regression analysis at 95% confidence level.A total of 1110 IPSAC were recruited for the study; 1060 (95.50%) met the inclusion criteria by providing stool samples and blood specimens and 516(48.68%) were males and 544(51.32%) were females between the age 0 and 72 months .Plasmodium falciparum (61.89%) was the single most prevalent parasitic infection recorded among IPSAC across the communities. There were no significant difference (p > 0.005) observed in Plasmodium falciparum infection in the communities; 23.21%, 7.16%, 4.87%, 0.29%, and 1.45% for A. lumbricoides, T. trichiura, E. histolytica, Taenia spp, Giardia lamblia, and Hookworm spp, respectively, were the intestinal parasitic infections (IPIs) observed in this study. A significant difference (p < 0.005) was observed between malaria parasite infections and intestinal parasitic infection  than soil-transmitted helminths (STH) and other protozoan parasites (38.11%).nfection . InfantsPlasmodium falciparum and A. lumbricoides (30.12%); P. falciparum, A. lumbricoides, and T. trichiura (14.81%); and P. falciparum, A. lumbricoides, T. trichiura, and E. histolytica are the most prevalent double, triple, and quadruple parasitic infections recorded in this survey. No significant difference (p > 0.005) was observed in polyparasitic infections across the communities, but a significant difference (p < 0.005) was observed between double and triple infections  = 2.043, p = 0.041 (p = 0.340) was observed between polyparasitism and gender (p = 0.001) (A  = 0.041 . The pred gender . The pre= 0.001) .χ2 = 6.843, df = 3, p = 0.023) while ownership and utilization of LLIN is significantly associated (p < 0.005) with malaria infection; households that sleep regularly under the treated net had a low prevalence of malaria infection compared to those that did not. There were no associations between malaria infections, intestinal parasites, and risk factors such as water source, walking barefooted, causes of malaria or STH, deworming activities, and handwashing (p > 0.230).The risk factor of polyparasitic infection is shown in dwashing . There wdwashing  while thPolyparasitism is widespread in rural communities in Sub-Saharan Africa where a significant proportion of people are exposed to coinfections of more than one parasite. Polyparasitic infections are more prevalent in the rural communities and are closely associated with poverty  and poorp > 0.230).According to , parasitp < 0.001). This reflects the exposure patterns in view of the fact they are more active, adventurous, and mindless of hygienic habits. Therefore, the infection rates in 0-24-month children were due to reduced exposure to the infective stages of the parasite as a result of less active adventures. The prevalence of polyparasitism was observed to be higher in females than in males in the study area which was statistically insignificant. The difference appears to confirm the possible roles of female activities that enhance relative contact with the infective stages of the parasites.The risk of polyparasitism increased with age; older children (25-72 months) were more affected compared to younger children (0-24 months). The differences were statistically significant by bivariate and multivariate regression analyses , use of mosquito nets, and proper treatment of infected persons in the communities; this measure should be promoted and advocated by government and stakeholders in the health sector.Infants and preschoolers represent a reservoir for intestinal helminths and protozoan parasitic infections; therefore, routine screening for these infections in health facilities should be included as part of their health care packages.


Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients’ sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target.  Plasmodium vivax attributable illness is the most geographically widespread morbidity of all human malaria with approximately 3.5 billion people in tropical and subtropical regions at risk of infection. The presence of difficult-to-treat hypnozoites responsible for chronic relapsing ailments and the emergence of chloroquine-resistant strains have made infections with P. vivax difficult to control and eliminate by current intervention strategies1. As an alternative means, vaccine development is a challenging approach to control malaria.P. falciparum known as acidic-basic repetitive antigen (ABRA) or merozoite surface protein 9 (PfMSP9) forms a co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP119) that binds to erythrocyte band 3 during the process of erythrocyte recognition/invasion4. PfMSP9 is peripherally associated with the merozoite surface and is found within the parasitophorous vacuole of P. falciparum6. Importantly, PfMSP9 possesses chymotrypic-like proteolytic activity akin to malarial proteases implicated in erythrocyte entry7 while synthetic peptides derived from this protein could bind to erythrocytes8. Antibodies raised against MSP9 could interfere with merozoite invasion of erythrocytes9 and parasite growth in vitro10. Meanwhile, immunization of mice with the P. berghei MSP9 homologue has conferred protection against the parasite challenge11. Furthermore, antibodies to P. vivax MSP9 (PvMSP9) were associated with protection against symptomatic infections among children in Papua New Guinea12, supporting the role of MSP9 as a promising vaccine candidate.Proteins that play crucial roles during merozoite invasion of erythrocytes are attractive vaccine targets. Of these, a 101 kDa protein of P. falciparum and other simian malaria species13, characterized by (1) the N-terminal signal peptide, (2) conserved cysteine residues at the N-terminal part and (3) repetitive amino acid sequences intervening conserved domains in which the locations of repeats seem to be variable across species14. It is noteworthy that the N-terminal conserved domain I of PvMSP9 shared 82% and 77% amino acid sequence identity with its orthologs in P. cynomolgi (PcyMSP9) and P. knowlesi (PkMSP9), respectively  of purified recombinant proteins from the N-terminal part of PvMSP-9 (TMS102 and CT1186) measured approximately 39 kDa, corresponding to the expected molecular mass. No shift in the migration pattern was observed for the N-terminal antigens under reducing and nonreducing SDS-PAGE, suggesting that the 4 cysteine residues in the recombinant proteins neither formed intermolecular nor intramolecular disulfide bridges . For the C-terminal antigens, the mean optical density (O.D.490) values were lower than those for the N-terminal antigens  . It is noteworthy that 154 patients (62.60%) had IgG antibodies to either or both variants of the N- and the C-terminal antigens while sera from 74 patients (30.08%) were reactive to either the N- or the C-terminal antigens  from Tak and Ubon Ratchathani Provinces has shown that 216 patients (87.80%) had antibodies to one or both variants of the N-terminal antigens, i.e. TMS102 and CT1186 . Likewise, reactivity of all serum samples to the antigens CT1750 and CT1756 has shown highly significant pairwise correlation of the OD values  .A pairwise comparison of the ODPlasmodium infections could not be completely obtained, vivax malaria has been most prevalent in Thailand since the past two decades and about 80% of recurrent malaria episodes were caused by the same species22. Based on this categorization, the seropositivity rates of anti-PvMSP9 antibodies to antigen TMS102 seem to increase with increasing prior malaria episodes  while no significant trend was observed for antibody reactivity to other antigens   no history of previous malaria infection (n = 91), (2) a single prior malaria episode (n = 53) and (3) two or more previous episodes of symptomatic malaria (n = 30). Although the precise species of U test, p > 0.05). Likewise, the reactivity index values for antibodies to the N-terminal antigen have a tendency to increase with increasing malaria exposure . However, such tendency was not observed for antibodies to the C-terminal antigen . However, the mean difference was not statistical significant  in both endemic provinces of Thailand  may suggest the presence of common epitopes recognized by these antibodies. The remaining 39 patients (18.06%) had antibodies exclusively reactive to either one or the other N-terminal antigen, implying variant-specific antibody responses. Our previous analysis of pvmsp9 among Thai isolates has revealed that substitution at codon 122 (D122G or N) has been under positive selection in which a consistent result was obtained by both codon-based and amino acid property-based detection methods15. Meanwhile, host immune responses to infections could elicit selective pressure on pathogens leading to pathogen adaptations to evade host immunity. Although the precise B cell epitopes in the N-terminal PvMSP9 remain to be elucidated, at least five T cell epitopes  have been mapped in this conserved domain but outside residues 122, 215 and 21725. It is likely that the 3 amino acid substitutions in antigens TMS102 and CT1186 could be the targets for variant-specific antibody recognition based on in silico linear B-cell epitope prediction using the Bepipred 2.0 server26 15  or PvMSP9(E795-A808) in domain R2 has been associated with extended time period since the last malaria episode whilst no such correlation was observed for antibodies to the C-terminal antigen encompassing domains R2 and R323. Therefore, it is likely that the C-terminal domain of PvMSP9 contain both protective and non-protective B cell epitopes. Meanwhile, the linear B-cell epitope in the C-terminal domain of PvMSP9, designated PvMSP9(E795-A808), could be a target for specific memory B cells resulting in stable levels of IgG antibodies over 6 months in the absence of re-infection32. It is therefore likely that the association between seropositivity rates as well as the levels of anti-PvMSP9 antibodies and the number of prior malaria episodes observed in this study could partly stem from a cumulative effect from specific memory B cells during repeated malaria infections.For the C-terminal antigens, a higher seropositivity rate of antibodies to antigen CT1756 than to antigen CT1750 was observed among patients from both Tak and Ubon Ratchathani Provinces. It is likely that more repeat units in the former antigen could contribute to more B epitopes that could be recognized by the patients’ sera  have been reported to confer clinical protection from subsequent infection43 and exert inhibitory effects on merozoite invasion of erythrocytes44. By contrast, antibodies against other merozoite surface proteins of P. falciparum seem not to be associated with the risk of acquiring malaria45. While the reasons behind these discrepancies remain unknown, it could be that biological function exerted by specific antibodies besides the magnitude of antibody avidity may be the crucial determinant for effective immunity against malaria. For example, high avidity antibodies to the conserved C-terminal domain of P. falciparum circumsporozoite protein evoked by the RTS,S/ASO1E vaccine have been associated with vaccine efficacy whilst high avidity antibodies to the tetrapeptide repeats failed to confer clinical protection against malaria in the same cohorts46. While the biological relevance of high avidity antibodies to PvMSP9 requires further investigation, a significant positive correlation between the magnitudes of antibody avidities to both N- and C-terminal antigens of PvMSP9 and prior malaria episodes has supported avidity maturation upon repeated exposure.Although it has been well perceived that high avidity antibodies play an important role in neutralization or inactivation of viruses and bacteria including toxins, inconsistent results have been found in malarial infections. High avidities of antibodies against 47. A remarkable increase in the levels of anti-malarial antibodies has been reported to occur within 1 or 2 weeks after the onset of symptoms among individuals who experienced previous malaria exposure50. However, no seroconversion was also observed in some patients in this study despite 3 weeks of follow-up period. Although a delay in seroconversion may occur beyond the follow-up period, it is possible that some patients may be immune unresponsiveness to PvMSP9. Polymorphism in the Human Leukocyte Antigens (HLA) class II loci has been found to influence humoral immune responses to PvMSP9 among people in the Brazilian Amazon. A high prevalence of seropositivity rates of anti-PvMSP9 antibodies was observed among individuals carrying HLA-DRB1*04 and HLA-DQB1*03 alleles whereas most non-responders to the C-terminal repeats had HLA-DRB1*0124. Meanwhile, the allele frequencies of HLA-DRB1*04, HLA-DQB1*03 and HLA-DRB1*01 among Thai population were 0.0830, 0.3288 and 0.0043, respectively51. Therefore, it is likely that the HLA-DRB1*01-related non-responders to the C-terminal PvMSP9 antigen may not be prevalent among Thai population.The cross-sectional prevalence of seropositivity rates of antibodies to specific malarial proteins among acutely infected patients could be potentially underestimated because seroconversion could occur during a short-term follow-up period as shown in the present study as well as in our previous reportP. vivax infection has been determined among Thai patients. Most IgG antibodies to these antigens belonged to cytophilic isotype. The majority of antibodies were directed against conserved or cross-reactive epitopes while variant-specific antibodies could be detected at low frequencies. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies seem to be influenced by previous malaria episodes. As immunity against malaria requires both humoral and cellular immune responses, further investigations of both B and T cell epitopes in PvMSP9 are required for subunit vaccine development.In conclusion, the immunogenicity of both N- and C-terminal portions of PvMSP9 upon natural P. vivax-infected patients who attended malaria clinics or district hospitals in Tak  and Ubon Ratchathani Provinces  during 2013 and 2014. Data on demography and malaria infections are shown in Supplemental Table A total of 246 blood samples were obtained from symptomatic 15 and PvMSP9N1R . Amplification was performed in a total volume of 30 µL containing template DNA, 2.5 mM each deoxynucleotide triphosphate, 2.0 µL of 10X EX Taq PCR buffer, 0.3 µM each primer, and 0.5 units of Ex Taq DNA polymerase . The cycling condition for amplification contained 38 cycles of 94 °C 40 s, 55 °C 30 s and 72 °C 60 s, followed by a final extension at 72 °C 5 min. Likewise, the 3′ portion of pvmsp9 was amplified under the same condition except that the PCR primers were replaced with PvMSP9C1F  and PvMSP9C1R . The PCR products were fractionated in 1% agarose gels and purified by QIAquick Gel Extraction Kit . After digestions of the purified PCR products with NdeI and XhoI , they were ligated to pET19b vector and transformed into E. coli JM109. The recombinant plasmid DNA was verified by direct sequencing prior to transformation into E. coli BL21 Star (DE3) pLysS expression host . Protein expression comprising induction with isopropyl-β-D thiogalactopyranoside (IPTG), solubilization by sonication, refolding by multistep dialysis and purification using His-Bind Resin  were essentially followed the procedures previously described47 . The N-terminal proteins comprised two variants derived from isolates TMS102 and CT1186 in which the former was identical with the Belem sequence and the latter contained amino acid substitutions at D122G, E215Q and R217H490) values plus 3 standard deviations (S.D.) of negative control sera from 50 healthy individuals living in Bangkok without history of malaria infection. To detect subclasses of human IgG antibodies, ELISA was performed essentially as described except that secondary antibodies were biotin-conjugated isotype-specific mouse anti-human IgG subclasses  with 1:1000 dilution. The cut-off values of antibody titres for each recombinant antigen are shown in Supplemental Table After the optimal antigen concentration and the dilution of the primary and secondary antibodies were determined by checkerboard cross-titrations, the plasma samples were analyzed by ELISA for the detection of specific IgG antibodies against the N- and the C-terminal antigens of PvMSP-9. The assays were performed in duplicate wells using 0.5 µg of the affinity purified antigen per well and 100 μL of 1:100 dilution of individual plasma samples. Details on the procedures were in Supplemental Method 52. All tests were repeated twice. The avidity index was determined from the percentage of the ratio of mean OD490 value of urea-treated samples to that of untreated samples. The magnitudes of antibody avidity were classified as low, intermediate and high when the avidity indices were < 30%, 30%-50% and > 50%, respectively53.An ELISA for detection of total anti-IgG antibodies was carried out in duplicates, one followed the protocol essentially as described above whereas the other was assessed with a urea elution-based method as previously reported490 values of tested serum samples by the cut-off values for respective antigens. Comparisons between the seropositivity rates were determined by the chi-square test. Associations between antibody responses and prior malaria episodes were performed by using the chi-square test for trend analysis and the Kruskal–Wallis test. Statistical significance was considered as p < 0.05. Prediction of linear B cell epitope was performed with Bepipred Linear Epitope Prediction server using the default option26.Quantitative data were analyzed in terms of percentage, mean and standard deviation. To compare the levels of antibodies to different antigens, the reactivity indices or RI were computed by dividing the mean ODThis study was reviewed and approved by the Institutional Review Board in Human Research of Faculty of Medicine, Chulalongkorn University, Thailand (IRB No. 322/59 and COA No. 041/2016). Prior to blood sample collection, informed consent was obtained from all participants or from their parents or guardians. All procedures were performed in accordance to the relevant guidelines and regulations.Supplementary information 1.


Indicators of successful malaria control interventions include a reduction in the prevalence and densities of malaria parasites contained in both symptomatic and asymptomatic infections as well as a reduction in malaria transmission. Individuals harboring malaria parasites in asymptomatic infections serve as reservoirs for malaria transmission. This study determined the prevalence of asymptomatic malaria parasite carriage in afebrile children attending six different schools in two districts, the Cape Coast Metropolitan Assembly (CCMA) and the Komenda Edina Eguafo Abirem (KEEA) of the Central Region of Ghana. Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax in DNA extracted from the filter paper spots. The multiplicity of P. falciparum infection was determined using merozoite surface protein 2 genotyping.  This cross sectional study recruited afebrile children aged between 3 and 15 years old from six randomly selected schools in the Central Region of Ghana. Finger-pricked blood was collected and used to prepare thick and thin blood smears as well as spot a strip of filter paper (Whatman #3). Nested PCR was used to identify Plasmodium parasites in asymptomatic infections, whilst microscopy identified malaria parasites in 10.6% of the children. The overall PCR estimated prevalence of P. falciparum and P. malariae was 26.6% and 1.3%, respectively, with no P. ovale or P. vivax identified by PCR or microscopy. The RDT positivity rate ranged from 55.8% in Simiw to 4.5% in Kuful. Children from the Simiw Basic School accounted for 87.5% of all the asymptomatic infections. The multiplicity of P. falciparum infection was predominantly monoclonal and biclonal.  Out of the 528 children sampled, PCR identified 27.1% to harbor P. ovale and P. vivax was identified in the afebrile children sampled from the selected schools. The low prevalence of asymptomatic malaria parasite carriage by the children living in the Cape Coast Metropolis suggests that the malaria control interventions in place in CCMA are highly effective and that additional malaria control interventions are required for the KEEA district to reduce the prevalence of asymptomatic malaria parasite carriers. No molecular evidence of  Plasmodium parasite carriage is a dire setback to malaria control and elimination efforts, particularly as they serve as potential reservoirs for malaria transmission [μl of blood at which parasites in a blood smear would be detected [μl of blood [μl [Asymptomatic smission . Microscsmission , has a tdetected  of betweof blood  but a molood [μl . The widlood [μl , 5. MajoPlasmodium falciparum  is the predominant Plasmodium species in the World Health Organization (WHO) Africa Region, that accounts for more than 90% of the malaria-associated morbidity and mortality in this region [Plasmodium malariae and P. ovale have been identified in some sub-Saharan African countries including Ghana at a very low rate [Plasmodium vivax was previously thought to be restricted to Asia and South America due to the absence of the Duffy antigen, which has been indicated as a requirement for erythrocyte invasion by P. vivax merozoites [P. vivax in West African countries including Benin, Mali, Senegal, and Nigeria [P. malariae and P. falciparum is artemisinin combination therapy (ACT), whilst the recommended treatment of P. ovale and P. vivax requires the supplementation of the ACT with a hypnozoticidal agent such as primaquine (PQ), for the clearance of dormant hypnozoites  [P. ovale and P. vivax are accurately identified and provided with a radical cure treatment. This study used a sensitive molecular tool to identify and determine the prevalence of four human Plasmodium species circulating in afebrile school children living in the Central Region of Ghana.Although s region . Plasmodlow rate –11. The low rate , 12. Plarozoites  among th Nigeria –16 that  Nigeria . The WHOal cure) , 19. It Six basic schools were selected from two municipalities in the Central Region of Ghana: Presbyterian Basic School (Presby), Church of Christ Basic School (Ewim), and St. Lawrence Catholic Basic School (St Lawrence) from the Cape Coast Municipality (CCMA) and Kuful Basic School (Kuful), Ntranoa Basic School (Ntranoa), and Simiw Basic School (Simiw) from the Komenda Edina Eguafo Abrem District (KEEA) .The Cape Coast Municipality lies within latitudes 50.07′ to 50.20′ North of the Equator and between longitudes 1°.11′ and 1°.41′ West of the Greenwich Meridian and is bound to the south by the Atlantic Ocean and to the West by the Komenda Edina Eguafo Abrem District.The Komenda Edina Eguafo Abirem (KEEA) District lies between longitude 1° 20′ West and 1° 40′ West and latitude 5° 05′ North and 15° North.Vegetation, rainfall, and temperatures in both areas are similar, with the vegetation consisting of mainly secondary forest with thickets and shrubs. The temperature ranges between 24°C and 32°C with relative humidity ranging from 60% to 80%. The peak rainfall season is between May to June and the peak malaria season from June to August .The Institutional Review Board of the Noguchi Memorial Institute for Medical Research granted ethical clearance for this study . Permission to conduct this study was also obtained from the KEEA and CCMA Municipal Directors of Education. Parents and guardians, teachers of the school children as well as the school children were engaged during a number of encounters at each school premise where the study's aims and objectives were explained. Willing parents/guardians provided written parental consent for the children prior to being recruited into the study, and children aged 12 years and above were also made to endorse assent forms.µl) were collected from 528 afebrile children in October 2017. The blood was used to prepare thick and thin blood smears and dried blood spots (DBS) [™ malaria HRP2 rapid diagnostic test kit (RDT) following the manufacturer's instructions. Each DBS was stored individually in a Ziploc bag containing a desiccant.All children aged between 3 and 15 years whose parent/guardian endorsed the parental consent forms were recruited into the cross-sectional study. Two drops of finger-pricked blood (∼100 ts (DBS)  as well Thick and thin blood smears were processed for malaria parasite identification and quantification according to the WHO protocol , 22 as pµl of a 10% saponin solution. After an overnight incubation, the PBS/saponin solution was decanted and the discs were washed with 1 ml of ice-cold PBS and left at 4°C. After a 30-minute incubation, the tubes were centrifuged at 10,000 ×g for 30 seconds and the supernatant was discarded. A freshly prepared solution of 20% Chelex-100 (60 µl) and 140 µl of distilled water were then added to each tube. The tubes were heated for 10 minutes at 95°C and then centrifuged at 13,000 rpm for 6 minutes. The supernatant (∼120 µl) containing the extracted DNA was then transferred into a new sterile labeled 0.5 ml microfuge tube and either used immediately or stored at −20°C for later use.Genomic DNA was extracted from DBS using the Chelex extraction method , 24. Briµl) of DNA template supplemented with 1X PCR buffer, 167 nM dNTPs, 2.5 mM MgCl2, and 1 U of OneTaq DNA polymerase. The secondary reaction used 0.5 µl of the primary reaction product as template and either of the four sets of species-specific primers , 333.33 nM of rMAL1/rMAL2 , rOVA1/rOVA2 , and rVIV1/rVIV2 (P. vivax-specific) in addition to a reaction mixture similar to that described for the primary reaction ), gDNA from a P. ovale field isolate (787 bp), gDNA from a Pf/Pm mixed infection (field isolate) used as an in-house P. mal control (144 bp), and gDNA from P. vivax (obtained as part of the 2019 WHO NAAT EQA Scheme (117 bp)).A nested PCR protocol adapted from Snounou et al.  and simiP. falciparum infection was determined by genotyping the msp2 locus using nested PCR as previously described . Briefly, the two allelic families (FC27 and IC3D7) of the msp2 gene were differentiated in the central polymorphic region. Block 3 of the msp2 gene was amplified from about 50 µg of parasite DNA using the M2-OF and M2-OR primer set . The primary PCR product (0.5 µl) was then amplified using two sets of primers, N5 rev and S1w-f  and M5 rev and S1w-f  (200 nM of each) in a PCR mixture containing 200 nM of dNTP, 1.8 mM of MgCl2, and 0.5 U of OneTaq DNA Polymerase. Positive control samples including gDNA extracted from the K1 (MRA-159G) and 3D7 (MRA-102G) parasite strains and a negative no template control samples were included in all PCR amplifications.The MOI of The prevalence of asymptomatic infections was calculated as the proportion of children in each school who tested positive for malaria parasites (irrespective of the species contained in the infection).P. falciparum parasite clones detected in a sample. Such that samples containing a single amplified product after msp2 PCR were considered as clonal, samples containing 2 amplified products were considered biclonal, and those with more than two amplified products were considered as multiclonal.Multiplicity of infection was defined as the number of different P values of less than 0.05 were considered statistically significant.Statistical analysis was performed using GraphPad Prism version 5  and IBM SPSS version 22. The Kruskal–Wallis test followed by Dunn's multiple comparison was used to determine significant differences between the median age of the children, RDT positivity rate, parasite density, and the prevalence of malaria parasites across the different schools. Descriptive statistics including mean and its 95% confidence interval (95% CI), frequency (estimated as a percent), and Pearson chi-square analysis was used to identify significant differences in the distribution of males in the different schools. P=18.44, P=0.002), with the proportion of males in the study population ranging between 33% (33/100) in children from St. Lawrence and 57.5% (69/120) in children from Simiw. Children from Ewim, Simiw, and Presby Basic schools had similar ages, whilst children from Ntranoa Basic school were significantly older than children from all the other schools , and children from Kuful Basic school were significantly younger than children from all the other schools .The study population comprised of 528 children between 3 and 15 years  recruitePlasmodium infections using microscopy, an HRP2-based malaria RDT and PCR.Samples from all the 528 children were tested for the presence of P. falciparum monoinfections accounting for 95.9% of the infections and P. malariae accounting for the remaining 4.1%  of the schools, with ing 4.1% . Childreand Ewim .P. falciparum infections. The overall RDT positivity rate amongst the entire population of school children was 21.2% (112/528). Children from Simiw had the highest RDT positivity rate of 55.8% (67/120), which was significantly higher than the positivity rates detected in children from all the other schools . Two schools, Presby and Ewim, had RDT positivity rates below 5% and three schools  had positivity rates of between 11% and 20%, but the differences between the positivity rates in the five schools were not statistically significant  (The HRP2-based RDT kits provided an estimate of the positivity rates for only  > 0.05) .P. malariae as monoinfections and four children harboring both P. falciparum and P. malariae as mixed-species infections. Plasmodium falciparum was identified in 26.5% (140/528) of the children. No Plasmodium parasites were identified in children from the Ewim and St. Lawrence Basic Schools  of the children carried P. falciparum parasites  than P. falciparum carriage identified in children from all the other five Basic Schools. No significant difference was observed in the prevalence of P. falciparum parasite carriage in four of the Basic Schools: Presby, St Lawrence, Ewim, and Ntranoa . However, parasite carriage in Kuful was significantly higher than parasite carriage in Presby  but similar to that identified in Ntranoa .The overall prevalence of asymptomatic children estimated by PCR was 27.1% (143/528), which included three children harboring  Schools . The higarasites . PlasmodP. malariae was identified  in children from two of the Basic Schools, Ntranoa at 2.9% (2/70) and Kuful at 5.6% (5/90). The five Plasmodium malariae infections identified in Kuful were primarily (80%) contained in mixed infections with P. falciparum, with only 1/5 (20%) identified as a P. malariae monoinfection. In Ntranoa, both (100%) of the P. malariae infections were identified as monoinfections. No molecular evidence of P. vivax or P. ovale was identified in any of the children screened  and a specificity of 91.8% (356/388) were obtained  overall and ranged from 32.7% in Simiw to 12.5% in Ntranoa . The speP. falciparum-positive samples at the merozoite surface protein 2 (msp2) locus identified 70% (98/140) of the samples as positive. A majority  of the msp2-positive samples contained infections with low multiplicity of infection . Monoclonal infections represented 56.9% (33/58) of the low MOI infections. Overall, monoclonal infections represented 33.7% (33/98) of the msp2-positive samples and 23.6% of the P. falciparum-positive samples whilst biclonal samples represented 25.5% (25/98) and 17.9% of the msp2- and the P. falciparum-positive samples, respectively (P. falciparum/P. malariae coinfected samples identified by Plasmodium species-specific PCR were amongst the msp2-positive samples whilst all the three P. malariae monoinfected samples tested negative after msp2 amplification (Genotyping of ectively . All thefication .P. falciparum, P. malariae, P. ovale, and P. vivax can present as asymptomatic infections [Plasmodium falciparum is known to cause the most lethal form of malaria; however, both P. ovale and P. malariae are capable of causing fatal disease, with reports suggesting an association of P. malariae with hematological morbidity [P. ovale with acute respiratory distress syndrome [Infections containing any of the human malaria parasites including fections . A largefections . More imfections . Plasmodorbidity  and P. osyndrome . It is tsyndrome  and effesyndrome . This stPlasmodium infections identified in this study. A possible reason for the lower parasite prevalence in the CCMA compared with the KEEA district is that the CCMA is the district capital of the Central Region and is more urbanized than the KEEA, which comprises of more semirural settlements [Children from the KEEA district generally had higher parasite prevalence than children from the CCMA, with children from the Simiw Basic School accounting for almost 90% of the total tlements . The restlements . The vertlements  as well tlements , which atlements , 37. Enttlements , but suptlements .P. malariae supports findings from a number of studies from Ghana that have reported the presence of P. malariae in some infections [P. vivax or P. ovale was identified by PCR and microscopy. The absence of P. vivax is not surprising as until recently, no P. vivax infections were identified in sub-Saharan Africa due to the absence of the Duffy antigen on erythrocytes in this population [P. vivax in Ghana.The presence of asymptomatic infections containing fections , 39. No pulation  and therP. malariae was not surprising as a number of studies from Ghana have reported an increase in the prevalence of P. malariae infections [P. vivax in this study population is not surprising as there have been no reports of the presence of P. vivax in Ghana to date. However, P. vivax has recently been identified in a number of sub-Saharan African countries including Mali, Senegal, Nigeria, and Cameroon [P. vivax invasion into erythrocytes. This finding in neighboring countries calls for enhanced surveillance of P. vivax in Ghana.The presence of asymptomatic infections containing fections , 11, 40.Cameroon , 41, 42 P. falciparum by PCR but tested negative by RDT could be a result of low parasite densities, which are common during the off-peak malaria season. Sample collection for this study was conducted during the off-peak malaria season where parasite prevalence and densities are expected to be low, especially in the Central Region of Ghana [Although the HRP2-based malaria RDT kits are the most sensitive and widely used RDT for malaria diagnosis, they are noted to have low specificity due to the persistence of the HRP2 antigen in the blood. The HRP2 antigen has been found in samples several days to weeks after the treatment of malaria and clearance of the parasites , 44. Anyof Ghana  and fromof Ghana . The accof Ghana . The preof Ghana ; howeverfalciparum species are encountered [falciparum species are usually encountered at a low density and or in a mixture with P. falciparum [P. falciparum infections were misclassified as P. malariae, likely due to inadequate smear preparation or the presence of some unanticipated mature forms of P. falciparum in the smear, which prevented the microscopists from efficiently identifying the correct Plasmodium species. This is a major concern for malaria microscopy that has been previously reported in countries including Ghana [Plasmodium parasites at very low densities [Microscopic evaluation of thick blood smears for microscopy is subjective and accuracy reduces as the parasite density of the sample reduces  and alsoountered , 51, esplciparum , 53. A fng Ghana , 50 and ng Ghana . Althougensities , it has ensities .Plasmodium falciparum 18S rRNA gene amplification in this study could be due to the low parasite densities contained in the samples. Secondly, the msp2 locus is a single copy locus relative to the 18s rRNA gene which is a multicopy locus [The reduced amplification efficacy obtained after performing the msp2 genotyping reactions relative to the py locus , which wpy locus . The disGhana is still in the WHO malaria control phase; as such, most malaria control interventions target the reduction of clinical malaria, with very little attention paid to asymptomatic manifestations of the disease . AsymptoThis study focused only on children, who are the most likely to be affected by malaria episodes due to them having a less matured immune system  as well The low prevalence of asymptomatic malaria parasite carriage in the Cape Coast Metropolis suggests that malaria control interventions in place in CCMA are highly effective. Similarly, additional malaria control interventions are required for the KEEA district to reduce the prevalence of asymptomatic malaria parasite carriers.


Plasmodium falciparum infections are widespread in many areas. However, the contribution of these infections to symptomatic malaria is not well understood. This study evaluated whether participants with submicroscopic P. falciparum infections have higher prevalence of fever than uninfected participants in southern Malawi.Submicroscopic P. falciparum detection by microscopy and PCR. Participants without malaria parasites detected on microscopy but with a positive PCR for P. falciparum were defined as having submicroscopic infection. Fever was defined as having any one of: reported fever in the past 2 weeks, reported fever in the past 48 h, or a temperature of ≥ 37.5 °C measured at the time of interview.A total of 16,650 children and adults were enrolled in the course of six cross-sectional surveys during the dry season (October–November) and after the rainy season (April–May) between 2012 and 2014 in three districts in southern Malawi. Demographic and socioeconomic data were collected in conjunction with data on clinical histories, use of malaria preventive measures, and anti-malarial medication taken within 2 weeks of the survey. Axillary temperatures were measured, and blood samples were collected for P. falciparum infections and fever were both detected in 9% of the study population. In the final analysis adjusted for clustering within household and enumeration area, having submicroscopic P. falciparum infection was associated with reduced odds of fever in the dry season ; the association in the rainy season did not achieve statistical significance . The association between submicroscopic infection and fever was consistent across all age groups. When the definition of fever was limited to temperature of ≥ 37.5 °C measured at the time of interview, the association was not statistically significant in either the rainy or dry season.Submicroscopic P. falciparum infection was not associated with increased risk of fever. Submicroscopic detection of the malaria parasite is important in efforts to decrease transmission but is not essential for the clinical recognition of malaria disease.In this series of cross-sectional studies in southern Malawi, submicroscopic  Plasmodium falciparum infections remain widespread [P. falciparum infections act as transmission reservoir of malaria [Despite major public health efforts to eliminate malaria worldwide, the incidence of malaria illnesses is still high in many areas, particularly in sub-Saharan Africa . Althougdespread –8. These malaria –11, howeP. falciparum infections in many geographical areas has been difficult because microscopy, the gold standard for the clinical diagnosis of malaria illnesses, and rapid diagnostic tests (RDTs) are not sensitive enough to detect low-density parasitaemia [P. falciparum infections [Accurate estimation of the incidence and prevalence of sitaemia , 13. Thefections , 15. Howfections , 17.P. falciparum infections and clinical malaria [P. falciparum infections may be associated with symptoms [P. falciparum infection and fever in different transmission settings and seasons, and across all age groups. This study tested the hypothesis that submicroscopic P. falciparum infection would be positively associated with fever and that the association would differ by season, transmission intensity and age. These are all factors that affect force of infection and acquired immunity that would modulate the likelihood of fever in the presence of low-level parasitaemia.Others have evaluated the association between submicroscopic  malaria , 18–21, symptoms . This reP. falciparum infection and to understand factors that influence parasite carriage. The three districts were: Blantyre, an urban and peri-urban highland with low transmission; Chikhwawa, a lowland with high transmission; and Thyolo, a highland with moderate transmission. The surveys were carried out twice a year for a period of 6 weeks: soon after the rainy season (April and May) and in the dry season (October and November).Six household-based cross-sectional surveys were conducted in three districts in southern Malawi between 2012 and 2014 to characterize the burden of The surveys included all individuals over six months of age from households that were randomly selected in Blantyre, Chikhwawa and Thyolo districts. Briefly, 10 enumeration areas (EA) were randomly selected from each district. The 10 EAs per district were divided into compact segments containing 30 households each. One segment was selected from each EA to be included in the study and the same compact segments were visited for each survey. All households in the segment selected were eligible for the study; however, if one of the households did not meet the criteria for inclusion in the study it was replaced by the nearest household . Same hoThe surveys targeted all members of the household who had slept in the house for at least 2 weeks of the month prior to the survey. The analysis included household members aged 6 months old and above. Household visitors and individuals from households where the eldest person present at the time of the survey was a minor were excluded from the survey. Consent to participate in the study was sought from the head of the household and from each adult present. Parents or guardians were asked to provide consent on behalf of the children below the age of 18 years old and assent was sought from children who were 13–17 years old. Once consent and assent were granted, interviewers collected data on demographic and socioeconomic status (SES) variables, malaria preventive measures used, and any anti-malarial medication taken within 2 weeks of the survey. In addition, axillary temperature was measured using digital thermometers, and peripheral blood samples were collected on glass slides (thick and thin films) for malaria microscopy and on Whatman 3MM filter paper  for molecular testing as described previously .P. falciparum infection was defined as a positive result by real-time PCR targeting P. falciparum lactate dehydrogenase gene and a negative malaria microscopy result. Details of the procedures for P. falciparum detection using real-time PCR and microscopy have been previously described [The primary outcome of interest was fever. Three fever measures were collected: reported fever in the past two weeks, reported fever in the past 48 h and temperature ≥ 37.5 °C measured at the time of specimen collection. In this study, for the primary analysis, fever was defined as having a fever using at least one of the three fever measures outlined above. A secondary analysis was conducted with objective fever as the outcome, and this was defined as axillary temperature of ≥ 37.5 °C. Submicroscopic escribed , 23. Indescribed . The surP. falciparum infection and proportion of individuals with and without fever were computed within strata of district, season, and age groups. Mixed effects logistic regression models were used to estimate the association between submicroscopic P. falciparum infection and fever within strata age groups, districts, SES quartile and season, while accounting for clustering within household and EA. In the final model comparing the log odds of fever between submicroscopic P. falciparum infection and no parasitaemia, district, participant age and season were included as covariates. In addition, a term for the interaction between submicroscopic infection and season (submicroscopic infection*season) was included in the final model. To determine if the association between submicroscopic infection and fever would be different when using broad  or narrow (objective fever) definition of fever, we conducted a sensitivity analysis using objective fever (axillary temperature of ≥ 37.5 °C measured at the time of the interview) as the outcome of interest. All data analyses were done using SAS 9.4  statistical software and were conducted at alpha level of 0.05.Median age in years and proportion of individuals were estimated within strata of sex, SES quartile, season and age group in each district. Proportion of individuals with and without submicroscopic A total of 16,650 people participated in the six surveys conducted between 2012 and 2014 in Blantyre, Chikhwawa and Thyolo districts. The median age of survey participants was 13 (IQR: 6–27) in Blantyre, 12 in Chikhwawa (IQR: 6–28), and 13 in Thyolo (IQR: 6–30). The distribution of participants within sex, age groups, and seasonal categories was similar in the three districts  had microscopic infection and 13,563 (81%) had no parasitaemia  had submicroscopic ia Table . The limia Table .Table 2DOverall, 1500 of 16,650 (9%) participants had recent or concurrent fever by objective measurement or self-report. Fever was most common among residents of Chikhwawa District 11.2%), young children aged 6 months to 5 years (12.6%), people in the lowest SES quartile (10.8%), and rainy season participants (10.8%)  had submicroscopic P. falciparum infections and 1032 (7%) had fever by objective measurement or self-report. Submicroscopic P. falciparum infection was significantly associated with a 49% decrease in odds of fever in the dry season , and a non-significant increase in the odds of fever in the rainy season .The prevalence of fever among individuals with submicroscopic P. falciparum infection and fever within strata of either district or age groups. Only 1.2% of the population had fever when defined by objective measure of body temperature ≥ 37.5 °C at the time of survey. In the sensitivity analysis limited to objectively measured fever, the association between submicroscopic infection and objective fever was not statistically significant within strata of either season, district or age group, though the directions of the association were generally consistent with those of the primary analysis ; no statistically significant association was observed in the rainy season . In the sensitivity analysis limiting the definition of fever to objective measurement of axillary temperature ≥ 37.5 °C at the time of the assessment, the estimated ORs were similar but not statistically significant in either season  , they disitaemia . That stsitaemia , 27. ImpThe observation of reduced odds of fever in participants with submicroscopic malaria in the dry season was unexpected, though it has been reported previously . It is pP. falciparum infection and fever cannot be established, thus limiting the conclusions that can be drawn about the role of these infections in causing clinical malaria over time. History of fever was included as one of the definitions of the outcome and this may have introduced poor recall of fever episodes by participants. However, this would not bias the findings as the inaccuracies in the reporting of fever was similar in participants with submicroscopic infections and participants without infection. In this study, it was impossible to identify alternative sources of fever in participants. Assuming the distribution of non-malaria febrile illness was equally distributed among those with and without submicroscopic P. falciparum infection, this should not have led to a bias in the results.Because this study was cross-sectional, the temporal relationship between submicroscopic P. falciparum infections are common in many malaria endemic populations [P. falciparum infection did not have higher prevalence of fever compared to uninfected people. However, these submicroscopic infections may persist for many months in human host and harbour transmission stages (gametocytes) of the malaria parasite [Submicroscopic ulations –6. Findiparasite , 23, andP. falciparum infections are common in most malaria endemic areas but the utility of more sensitive diagnostics to identify individuals with clinical malaria disease has not been definitively established. This study demonstrates that across all age groups and a range of transmission settings, individuals with submicroscopic P. falciparum infections do not have higher prevalence of fever compared to those without infection. Molecular diagnostic tests should not replace microscopy and RDTs for clinical diagnosis of malaria or for treatments aimed to reduce clinical malaria. However, molecular methods remain important for identifying reservoirs of P. falciparum transmission.Submicroscopic


The World Health Organization (WHO) promotes long-lasting insecticidal nets (LLIN) and indoor residual house-spraying (IRS) for malaria control in endemic countries. However, long-term impact data of vector control interventions is rarely measured empirically.Surveillance data was collected from paediatric admissions at Tororo district hospital for the period January 2012 to December 2019, during which LLIN and IRS campaigns were implemented in the district. Malaria test positivity rate (TPR) among febrile admissions aged 1 month to 14 years was aggregated at baseline and three intervention periods  and compared using before-and-after analysis. Interrupted time-series analysis (ITSA) was used to determine the effect of IRS (Bendiocarb + Actellic) with the second LLIN campaign on monthly TPR compared to the combined baseline and first LLIN campaign periods controlling for age, rainfall, type of malaria test performed. The mean and median ages were examined between intervention intervals and as trend since January 2012.Among 28,049 febrile admissions between January 2012 and December 2019, TPR decreased from 60% at baseline (January 2012–October 2013) to 31% during the final period of Actellic IRS and LLIN (June 2016–December 2019). Comparing intervention intervals to the baseline TPR (60.3%), TPR was higher during the first LLIN period , and lower during the Bendiocarb IRS  and Actellic IRS  periods. ITSA confirmed a significant decrease in the level and trend of TPR during the IRS (Bendicarb + Actellic) with the second LLIN period compared to the pre-IRS (baseline + first LLIN) period. The age of children with positive test results significantly increased with time from a mean of 24 months at baseline to 39 months during the final IRS and LLIN period.IRS can have a dramatic impact on hospital paediatric admissions harbouring malaria infection. The sustained expansion of effective vector control leads to an increase in the age of malaria positive febrile paediatric admissions. However, despite large reductions, malaria test-positive admissions continued to be concentrated in children aged under five years. Despite high coverage of IRS and LLIN, these vector control measures failed to interrupt transmission in Tororo district. Using simple, cost-effective hospital surveillance, it is possible to monitor the public health impacts of IRS in combination with LLIN. Indoor Residual Spraying (IRS) has regained attention as a means to prevent malaria infection in high transmission areas of Africa when used in combination with insecticide-treated nets, largely as a result of funding made available by the US President’s Malaria initiative (PMI) . CarbamaIn Uganda, between 1959–1964, IRS using dichloro-diphenyl-trichloro-ethane (DDT) with three rounds of mass drug administration (MDA) with single doses of chloroquine-pyrimethamine was piloted in Kigezi and parts of Masaka districts . During Lambda-cyhalothrin IRS was introduced in 2004 to tackle localized epidemics in Kabale, Kisoro and Kanungu districts. In 2005, Uganda became one of the first countries in sub-Saharan Africa to benefit from PMI funding, supporting a sustained IRS campaign in different parts of the country. From 2006, IRS with lambda-cyhalothrin was introduced to Kabale and Kanungu districts . PMI proIn 2009, both DDT and pyrethroid resistance were detected at IRS districts  and the Between April and October 2014, IRS was scaled down in the northern districts (Acholi sub-region) and switched to seven mid-northern districts  and eight eastern districts  initially using Bendiocarb and later switching to Actellic 300 CS (organophosphate pirimiphos-methyl). Cessation of IRS in the Northern Districts resulted in large-scale epidemics from April 2015 through to 2016 affecting over 1 million people [Unlike the introduction of IRS during the late 1950s, the rapid expansion of IRS in Uganda from 2007 was not accompanied by a comprehensive, longitudinal epidemiological impact analysis plan across all districts where IRS was implemented . There hAnopheles gambiae sensu stricto (s.s.) (82%) and Anopheles arabiensis (19%) [An. arabiensis (99.5%) became the dominant vector [Tororo District is in Bukedi sub-region, a historically highly malaria endemic, rural area of eastern Uganda bordering Kenya. The 2014 national census put the population of Tororo District at 517,082 . The areis 19%) . Post-20% . Post-The public health care system in Tororo district covers four tiers of out-patient services including 61 level II and III health centres (HCs) providing the most basic services and three level IV HCs providing more advanced levels of care in the form of some in-patient care. There are two hospitals providing general in-patient care, including the main Tororo district hospital with 52 in-patient paediatric beds. The fee-paying St Antony’s mission hospital, also located in Tororo town, admits few patients, and does not serve as a major competitor to the government hospital. The nearest Regional Referral Hospital is at Mbale, 46 km south of the district. Since 2006, artemether-lumefantrine (AL) has been used as the first-line treatment for uncomplicated malaria in Uganda. In 2011, artesunate-amodiaquine was introduced as an alternative first-line treatment to AL . Since 2The Government of Uganda has maintained routine distributions of long-lasting insecticide-treated nets (LLIN) since 2007 through antenatal clinics . Mass ca, An. gambiae sensu lato (s.l), has been 100% susceptible to Actellic [IRS began in Tororo district in December 2014-February 2015 and was repeated in June-July 2015, and November–December 2015 using Bendiocarb . Details of the programme are described elsewhere –36. In bAnalysis was limited to children resident in Tororo district at the time of hospitalization between January 2012 and December 2019. To determine the physical location of individual households, relevant address information  recorded on patient MRFs was used to map residences of all admissions to the hospital. Where insufficient information was available on the MRF, missing address details were crosschecked against original hard copy admission registers. If residential information was still missing, admissions were excluded from analysis. Age was calculated in months from reported/documented date of birth and admission date. Neonates were excluded from the analysis and therefore analysis was restricted to children aged 1 month to 14 years.Between 2012 and 2015, complete MRFs were entered into an Access database  and transmitted to a central database located in Kampala, Uganda on a monthly basis. From 2016, the data management system was upgraded to a web-based data management system hosted on the District Health Information System (DHIS-2), allowing for real time data entry. Data were analysed using STATA . Monthly malaria test positivity rate (TPR) measured over time formed the basis of the primary analysis and was defined as the number of hospitalized children with a fever (temperature ≥ 37.5 °C and/or a history of fever in the presenting illness) and a positive malaria test result (RDT or microscopy) among all hospitalized febrile children with a malaria test result. The period of observation ranged from January 2012 to December 2019 and was divided into four time-periods. First, the baseline period, starting January 2012 to October 2013, characterized by routine malaria control interventions. Second, from November 2013 to November 2014, representing the period immediately after the first universal LLIN campaign and before the start of IRS in the district. Third, from December 2014 to May 2016, representing a period of three rounds of IRS with bendiocarb, implemented at six monthly intervals. Fourth, from June 2016 to December 2019, representing four rounds of IRS with Actellic, implemented annually, with a second universal LLIN distribution campaign in the district. Two analytical approaches were used to determine the impact of interventions on TPR during the observation period; before-and-after analysis and interrupted time series analysis (ITSA).For before-and-after analysis, the impact of interventions on TPR was assessed by calculating the absolute difference between the aggregated baseline TPR (before) with the three intervention intervals (after): (1) first universal LLIN campaign, (2) IRS with Bendiocarb, and (3) IRS with Actellic and second LLIN campaign. The chi square test was used to assess if differences in TPR were statistically significant. The significance level, alpha, was set at 0.05. The 95% CI was determined based on the absolute difference in TPRs plus or minus the margin of error; a product of the standard deviation of the difference between periods and the critical value (Z = 1.960). To explore more immediate effects of interventions on TPR, data were also repeated limited to children aged 1–11 months.ITSA –40 was uAge (months) of hospitalized children with malaria was summarized as median (inter quartile range) and mean (± standard deviation) for the baseline period and different intervention periods stratified depending the malaria test result. A Kruskall-Wallis test was used to test for differences between age during different intervals. The linear temporal change in mean age among test positive hospitalized were estimated using linear regression adjusted for 1 month lagged rainfall and type of test done.Across the 96 months of observation, January 2012 to December 2019, 33,643 admissions aged ≥ 1 month to 14 years were recorded at Tororo District hospital. 1553 (4.6%) admissions were residents of neighbouring districts or Kenya, 194 (0.6%) children had insufficient address information to define district of residence. Among the 31,896 hospitalized children aged 1 month to 14 years resident in Tororo District, 28,049 (90%) had a fever defined as a temperature ≥ 0.5 °C or reported history of fever, and 25,884 (93%) of these children had a malaria test performed at, or during, admission , TPR was higher during the first LLIN campaign period , but was lower during the Bendiocarb IRS  and Actellic IRS  , monthly rainfall, age and test type adjusted TPRs among children aged 1 month–14 years did not change significantly ; lowest during the baseline period; median 17 months , mean 23.7 months (SD 21.6) and highest during the last intervention period of IRS with Actellic; median 30 months , mean 30 months (SD 30.9). The regression analysis, adjusting for lagged rainfall and test type showed a significant monthly increase from January 2012 to December 2019 in the mean age of febrile test-positive admissions  and 2014–2017  among a cohort of children aged 6–30 months in Nagongera sub-county, Tororo , 32. A cThroughout the time-series the mean age of paediatric febrile admission malaria test positives increased from 24 months in 2012–2013 to 46 months in 2019 (Fig. Despite the entomological inoculation rate in Nagongera sub-county declining from 129 infective bites per person per year in 2013 to an apparent zero in 2017 , findingThe WHO recommends that IRS or universal LLIN coverage should be selected as district-wide vector control, not both . The avaThe use of fever TPR has avoided the attribution of admissions to a specific admission diagnosis of malaria, in part because admissions have multiple causes, but largely because fever surveillance for infection is a much simpler, less ambiguous surveillance outcome. Unlike routine health information systems in Uganda which provide only crude age groupings above and below 5 years of age, it has been possible in the hospital analysis presented here to explore in more detail the precise age of test positives with time. However, long-term hospital surveillance suffers from several intrinsic problems that require contextualization, for example changing diagnostic protocols, access to diagnostics and patient access. Furthermore, TPR is less sensitive to measuring the true magnitude of the impact of interventions as it ranges from 0–100% and cannot represent incidence expressed by the number of people at risk of hospitalization. Defining incidence through routine data, affected by access and use of services, is complex  and the The findings from this study have important policy implications regarding malaria control in Uganda. The combination of IRS with LLIN distribution has a larger impact than LLIN alone. Sustained vector control increases the mean age of febrile test-positive admissions to hospital a likely result of delayed immune acquisition. However, even after many years of IRS the majority of febrile test positive are still among young children. LLINs and IRS used in combination will not be enough to achieve elimination in Uganda. Combinations of interventions that target vectors and the parasite might be required to interrupt transmission including MDA and vaccines. Routine enhanced health facility-based malaria surveillance data offers a cost-effective means of monitoring the impact of malaria control interventions on malaria infection rates over-time. Expanding the adoption of a standardized MRF for paediatric admissions across the country by the Ministry of Health, and regular interrogation of hospital data will provide insights into intervention impacts, failures and an epidemiological understanding of the changing clinical patterns of disease.Additional file 1: Table S1. Summary of key indicators among hospitalized children at Tororo hospital: 2012 to 2019.