Severe acute malnutrition (SAM) has been associated with low polyunsaturated fatty acid (PUFA) status. However, investigations regarding PUFA status and correlates in children with moderate acute malnutrition (MAM) from low-income countries are scarce. The aim of this study was to describe whole-blood PUFA levels in children with moderate acute malnutrition (MAM) and to identify correlates of PUFAs.We conducted a cross-sectional study using baseline data from a prospective nutritional intervention trial among 1609 children with MAM aged 6–23 months in Burkina Faso,West Africa. Whole-blood PUFAs were measured by gas chromatography and expressed as percent of total whole-blood fatty acids (FA%). Potential correlates of PUFAs including infection, inflammation, hemoglobin, anthropometry (difference between children diagnosed as having MAM based on low mid-upper-arm-circumference (MUAC) only, low MUAC and weight-for-height z-score (WHZ), or low WHZ only) and diet were assessed by linear regression adjusted for age and sex.Children with MAM had low concentrations of whole-blood PUFAs, particularly n-3 PUFAs. Moreover, children diagnosed with MAM based only on low MUAC had 0.32 , 0.14; 0.50) and 0.40  FA% lower arachidonic acid (AA) than those recruited based on both low WHZ as well as low MUAC and those recruited with low WHZ only, respectively. Infection and inflammation were associated with low levels of all long-chain (LC)-PUFAs, while hemoglobin was positively associated with whole-blood LC-PUFAs.While PUFA deficiency was not a general problem, overall whole-blood PUFA concentrations, especially of n-3 PUFAs, were low. Infection, inflammation, hemoglobin, anthropometry and diet were correlates of PUFAs concentrations in children with MAM.http://www.isrctn.com (ISRCTN42569496).The trial is registered at  Approximately, 33 million children worldwide suffer from moderate wasting . The curTissue concentrations of PUFAs are reflected in the fatty acid composition of plasma and erythrocytes and the amount of various PUFAs in these pools has been shown to be low in children with severe malnutrition , 8. LimiPUFAs play an important role in early life because they are needed to ensure optimal growth and development of all organ systems . More spPrevious studies investigating blood PUFA composition in Burkina Faso, West Africa, have been undertaken in populations not suffering from MAM and in the central region –20, an aThis is a cross-sectional study using baseline data from a prospective nutritional intervention trial among 1609 children with MAM aged 6–23 months. The study took place in the Province du Passoré, Burkina Faso, at five local governmental health centers . The recruitment area covered a total of 143 villages and a population of approximately 258,000. Recruitment took place from September 2013 to August 2014.Children, aged 6–23 months, residing in the recruitment area were invited to participate if they were diagnosed with MAM; based on a WHZ between -3 and -2 based on the 2006 WHO growth standard [A local nurse carried out a clinical examination and collected data on sociodemographic characteristics as well as 2 week retrospective morbidity. Fever was defined as an axillary temperature ≥37.5 °C . The nurPlasmodium falciparum malaria , and a second drop for determination of hemoglobin level by Hemocue . A third drop was used to saturate 1 cm2 of a chromatography paper strip treated with 50 μg 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene) and 1000 μg deferoxamine mesylate salt  as described previously [During the medical examination up to 2.5 mL of none fasting venous blood were collected by phlebotomy using needle and syringe. One drop of blood was used to carry out rapid antigen test for eviously . The blo1-acid glycoprotein (AGP) were determined using a simple sandwich enzyme-linked immunosorbant assay [Serum was stored at -20 °C until shipment to a laboratory in Germany  for analysis. Serum C-reactive protein (CRP) and αnt assay . The thrnt assay  and AGP nt assay .The fatty acid composition of whole-blood was analysed as previously described , 31. In Due to lack of cut-off points for whole-blood Mead acid and n-6 docosapentaenoic acid (DPA), the ratios Mead acid:AA, n-6 DPA:DHA or n-6/n-3 PUFAs were used to define PUFA deficiency. Based on data in healthy well-nourished Danish infants , we considered a Mead acid:AA ratio > 0.02 as an indicator of PUFA deficiency and a ratio of n-6 DPA:DHA > 0.2 and a n-6/n-3 PUFA ratio > 10.5 was taken to indicate low n-3 PUFA status.Anthropometric measurements were done by trained staff, after standardisation sessions. Weight was measured in duplicate to the nearest 100 g with an electronic scale  with double weighing function. Length was measured in duplicate to the nearest 1 mm with a wooden length board. MUAC was measured to the nearest 1 mm at the midpoint between the olecranon and the acromion process using a standard measuring tape.p < 0.05.The data were doubled entered into Epidata 3.1 software . The statistical analysis and tests were performed in Stata 12 . A WHO WHZ table was used during recruitment at sites, but for data analysis WHZ scores were recalculated using the STATA package “zscore06”. Variables were tested for normality using Shapiro-Wilk tests and histograms. Results are shown as mean ± standard deviation (±SD) or median (interquartile range (IQR)). The difference between groups was analyzed by one-way analysis of variance (ANOVA) and p-values from post-hoc pairwise comparisons (three comparisons for each ANOVA test) were Bonferroni adjusted. Multivariable linear regression analysis was performed to assess correlates of individual PUFAs in children with MAM adjusted for age and sex. Statistical significance was set at Among the 1609 recruited children, a total of 1572 (97.7%) children had whole-blood fatty acids determined and 37 (2.3%) did not either due to failure to draw blood or problems during blood analysis at the lab. Upon recruitment, 457 (29%), 786 (50%), and 329 (21%) were diagnosed with MAM based on “MUAC only”, “MUAC and WHZ”, and “WHZ only”, respectively, as previously reported , co-morbThe majority (85.2%) of the children had consumed cereals, roots and tubers in the previous 24 h, while 285 (18.1%), 237 (15.1%), and 454 (28.9%) had consumed liquid oils and fats, legumes and nuts or vitamin A-rich fruits and vegetables, respectively (Table The mean weight percent of whole-blood LA and ALA were 16.22 (±2.26) and 0.21 (±0.09) FA%, respectively, while that of their respective LC-PUFAs, AA and DHA, were 7.08 (±1.53) and 1.64 (±0.53) FA%, respectively (Table p = 0.007) and WHZ only , but higher ALA than those recruited based on WHZ  (Table p = 0.040). Furthermore, multiple regression analyses, adjusted for age and sex, revealed that the children who were recruited based on MUAC only had 0.32 , 0.14; 0.50) FA% lower AA than those recruited based on both MUAC and WHZ and 0.40  FA% lower AA than those recruited based on WHZ only. Recruitment based on MUAC only was also associated with 0.09  FA% lower DHA than those recruited based on WHZ only, whereas recruitment based on both MUAC and WHZ was associated with a higher ratio of n-6 DPA relative to DHA  and higher Mead acid  compared to those recruited with WHZ only. Being recruited based on MUAC only compared to WHZ only was positively associated with Mead acid:AA ratio . Although median age differed between children recruited based on MUAC only, both MUAC and WHZ, or WHZ only, the difference in AA, DHA and Mead acid:AA across recruitment criteria was not confounded by age, sex and breastfeeding.Comparisons using ANOVA showed that children recruited based on MUAC only had lower AA than those recruited based on both MUAC and WHZ . Children with elevated CRP had 0.91  and 0.23  FA% lower AA and DHA, respectively, but 0.04  FA% higher ALA. Elevated AGP was also associated with lower AA and DHA  but higher ALA . A positive malaria test was associated with lower AA and DHA  and higher ALA . Overall, elevated serum CRP and AGP, and a positive malaria test were associated with a higher Mead acid:AA ratio. A positive malaria test was also associated with a higher n-6 DPA/DHA ratio. For each 1 g/dl increase in hemoglobin, LA and ALA decreased by -0.22  and -0.02  FA%, respectively. However, for each 1 g/dl increase in hemoglobin, AA and DHA increased by 0.39  and 0.11  FA%, respectively.Morbidity and biochemical markers of infection were also correlates of whole-blood PUFA content after adjustment for age and sex Table . ChildreWhile we found that PUFA deficiency was not a general problem in children with MAM, overall PUFA concentrations, especially of n-3 PUFAs, were low. There could be a number of explanations for this. Firstly, the low levels of whole-blood n-3 PUFAs found in children with MAM could be linked to their diet . The chiAs expected, breastfeeding, flesh foods, vegetable oils and fats were found to be correlates of PUFAs in children with MAM. Breast milk is recognised as a good source of LC-PUFAs , althougWith regard to anthropometry, children recruited by MUAC only seem to have lower whole-blood n-6 PUFAs and n-3 PUFAs compared to those recruited by WHZ only. Specifically, recruitment by MUAC only was associated with low AA and DHA and this could be related to a low muscle mass. Low MUAC only has been shown to be related to low arm muscle mass , which iMarkers of infection and inflammation were negatively associated with whole-blood PUFAs. This finding could be explained by an infection-induced supression of appetite and could be a direct effect of dietary intake, but may also be related to impaired absorption, poor nutrient utilization and/or increased nutrient catabolism caused by the inflammatory state , 47. We The current study is the first to investigate whole-blood PUFA composition and correlates among a large sample of children with MAM. Also, this is the first study to show a relationship between low MUAC and PUFAs. The diversity of correlates such as infection, inflammation, diet, and anthropometry highlight the complexity of factors which may affect whole-blood PUFA composition in children with MAM, and these factors should be taken into account in studies investigating PUFAs among malnourished children. However, the study had some limitations, in particular the lack of a control group from the same study setting, which makes it difficult to differentiate between potential effects of diet and the overall low nutritional status. Differences in diet could confound differences in nutritional status even if we had been able to collect data from a well-nourished group of children in Burkina Faso. The diet information was based on a single qualitative 24-h recall which may have reduced accuracy and thus, reduced the association between diet and PUFAs. The observed associations were however meaningful. Furthermore, PUFA status was only based on a single non-fasting whole-blood sample and may not provide a measure of long-term status as is the case for example in adipose tissue samples , but whoIn conclusion, although PUFA deficiency based on the definition used here was not common, children with MAM in Burkina Faso have low whole-blood n-3 PUFA concentrations. Low n-3 PUFA levels are likely to be, to some extent, explained by a low intake of animal based foods, vegetable oils and fat sources of n-3 PUFA, or by other correlating factors such anemia, infection and inflammation. Children with low MUAC seem to have lower whole-blood PUFAs compared to children with MAM based on WHZ. This observation needs to be confirmed in future investigations among children with MAM.


Plasmodium vivax in a border area between Brazil and French Guiana. The cross sectional study took place in Oiapoque, a municipality of Amapá, on the Amazon border. Malaria was diagnosed using thick blood smears, haemoglobin dosage by an automated method and coproparasitology by the Hoffman and Faust methods. The anti-PvMSP-119 IgG antibodies in the plasma were evaluated using ELISA and Th1 , and Th2  cytokine counts were performed by flow cytometry. The participants were grouped into those that were monoinfected with vivax malaria (M), vivax malaria-enteroparasite co-infected (CI), monoinfected with enteroparasite (E) and endemic controls (EC), who were negative for both diseases. 441 individuals were included and grouped according to their infection status: [M 6.9% (30/441)], [Cl 26.5% (117/441)], [E 32.4% (143/441)] and [EC 34.2% (151/441)]. Males prevailed among the (M) 77% (23/30) and (CI) 60% (70/117) groups. There was a difference in haemoglobin levels among the different groups under study for [EC-E], [EC-Cl], [E-M] and [Cl-M], with (p < 0.01). Anaemia was expressed as a percentage between individuals [CI-EC (p < 0.05)]. In terms of parasitaemia, there were differences for the groups [CI-M (p < 0.05)]. Anti-PvMSP-119 antibodies were detected in 51.2% (226/441) of the population. The level of cytokines evaluation revealed a large variation in TNF-α and IL-10 concentrations in the co-infected group. In this study we did not observe any influence of coinfection on the acquisition of IgG antibodies against PvMSP119, as well as on the profile of the cytokines that characterize the Th1 and Th2 patterns. However, co-infection increased TNF-α and IL-10 levels.Malaria-enteroparasitic co-infections are known for their endemicity. Although they are prevalent, little is known about their epidemiology and effect on the immune response. This study evaluated the effect of enteroparasite co-infections with malaria caused by  Plasmodium vivax has been the most prevalent species in the country, accounting for approximately 80% of episodes, while Plasmodium falciparum is responsible for approximately 20%, and Plasmodium malariae is rarely detected. Historically, severe clinical P. vivax cases have been rarely reported[In Brazil, over 99% of reported malaria cases occur in the Amazon region ,2. The c reported. However reported–7.Currently, it is estimated that more than a third of the world's tropical and sub-tropical population is infected with one or more enteroparasites . Human iPlasmodium and helminth co-infections are prevalent in tropical countries, the effect of their interactions remains unclear [Plasmodium infection [P. vivax and P. falciparum-mixed infections and that parasite diversity may be greater in patients who are infected with helminths [Although  unclear . Some stnfection ,16, resunfection , a reducnfection , the supnfection  and an infection ,20,21. Ielminths ,23. The elminths .P. falciparum. However, the protective effect against malaria from P. vivax has also been observed in two studies outside Brazil [P. vivax malaria episodes in a population of children in the city of Manaus, Amazonas State [vivax malaria [These associations between helminthiases and malaria are well documented in malaria-endemic regions of Africa and Asia, especially where malaria is caused by e Brazil ,19. In tas State . In addi malaria ,27, but  malaria .Plasmodium vivax in this region of the Brazilian Amazon.The municipality of Oiapoque is in the French Guiana border region, and it has a close relationship with local social health determinants. It also has an intense population flow. The objective of this study was to evaluate the effect of enteroparasite co-infection on malaria caused by A cross-sectional study was conducted in the municipality of Oiapoque, Amapá State, in northern Brazil, on the western border of the Amazon region. It has an Annual Parasite Index that designates it as at high risk of malaria transmission, and it is located in the northern part of the state of Amapá. Due to the border with the municipality of Saint Georges de l’Oyapock in French Guiana, there is an intense population flow related to trade, mining, tourism and social life, which also facilitates the spread of communicable diseases.Participants were required to meet the following inclusion criteria: (1) signed consent form, (2) being native to the study area, (3) being over seven years of age, (4) consenting to blood collection and (5) providing a faecal sample.After compliance with the inclusion criteria, in addition to the median, subjects were grouped according to age following the World Health Organization categories, as follows: “children” were those aged 0 to 9 years, “adolescents” those between 10 and 19 years of age, “adults” individuals aged 20 to 59 years and “seniors” those over 60 years of age.A total of 441 participants formed the study population, with the sample collection taking place over one year between the months of November 2014 and November 2015. Data collection was performed by passive detection in the municipality's Basic Health Units, following phenotypic diagnoses using thick blood smears and prior to treatment. For the uninfected group, samples were also collected in the same municipality so that the participating individuals were froma similarbackground exposure conditions. After a parasitological evaluation, the subjects were divided into the following four groups: 1) patients with an intestinal parasite and malaria co-infection (n = 117); 2) patients with malaria who were negative for intestinal parasites (n = 30); 3) patients without malaria who were positive for enteroparasites (n = 143); and 4) patients who were negative for both (n = 151). Epidemiological data such as the age, gender and number of previous episodes of malaria were obtained during interviews and from medical records.Plasmodium vivax and Plasmodium falciparum on the Franco-Brazilian border", which was certified by the Research Ethics Committee of the Federal University of Amapá - CEP/UNIFAP, on December 20, 2013, protocol n° 18740413.7.0000.0003, as being in accordance with the Ethical Principles in Human Experimentation, adopted by the National Committee of Ethics in Research—CONEP. The research subjects were invited to participate by Free and Informed Consent signature while the inclusion of those under 18 years of age was conditioned to their parents or guardians signature of a Free and Clarified Consent. Fingerprinting was used to group non-literates. All patients identified as positive for malaria received chemotherapy treatment asprescribed by the medical staff of the present study, provided by the Brazilian Ministry of Health, while those diagnosed with intestinal parasitoses were referred to medical treatment in one of the Oiapoque mucicipality Health Units.The present work is an integral part of the project "Coinfection of intestinal helminthiasis and susceptibility to infection by Blood samples were collected by venipuncture. Ten millilitres of venous blood was collected from each patient. Four millilitres was dispensed into a tube containing EDTA (ethylenediamine tetraacetic acid)  to perform haematological analysis and to prepare thick blood smear slides, and 6 mL was dispensed into a tube  with no anticoagulant for immunological analysis. After this collection, each slide was maintained at room temperature to dry the blood drop. The slides were subsequently stained and analyzed using light microscopy  according to the protocol described by the World Health Organization.The slides were prepared by following the Walker technique (methylene blue and Giemsa)  and eval3, and the gametocytes were counted directly on the slide (N° gametocytes X N° of leukocytes (8000)/100 = gametocytes per μL). This procedure is similar to the diagnostic methods recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines of the United States Centers for Disease Control (CDC) and the World Health Organization (WHO) guidelines. The count was subsequently confirmed in LAFRON/AP and sent to LACEN/AP's endemic quality control section.For the sexual form count (gametocytaemia), the thick blood slides were reviewed for gametocyte quantification in 100 leukocytes. The conversion to gametocytes/μL was performed by calculation using a reference value of 8000 leukocytes/mmIn order to confirm parasitological diagnosis, DNA was extracted from whole blood using the Easy-DNATM kit  and the QIAamp® DNA Blood Kit  followed by nested PCR, as previously described by Snounou et al. (1993) .Anemia was evaluated according to the diagnostic standards recommended by the World Health Organization through hematocrit (hemocytes and hemoglobin) indices with slight variations according to age and sex. The haemoglobin concentration was measured in venous blood using the Oiapoque Hospital's automated equipment (Mindray-BC-3000plus). Anemia was defined with haemoglobin reference values. The haematological parameters evaluated were the total number of erythrocytes  and haemoglobin levels . Individuals were considered anemic when their haemoglobin blood levels were ≤ 13 g/dL for males, ≤ 12 g/dL for females and ≥ 11 g/dL for the children.P. vivax MSP-1 (Belém strain) was expressed in Escherichia coli. The 96 wells Enzyme Linked Immuno Sorbent Assay (ELISA) plates  were sensitized with 50 μL of His-MSP-119 protein (2 μg/mL) diluted in 0.05M carbonate buffer pH 9.0, for 16 hours at room temperature (RT). Subsequently, the plates were washed with PBS/Tween 0.05% and blocked with 200 μl of 5% PBS skin milk. After 2 hours at 37°C, the plates were washed and 50 μl of the patient’s plasma, diluted 1:100 in PBS/milk, were added in each well, in duplicate, and the plates were incubated for 16 hours (RT). After rinsing the plates with PBS 0.05% Tween, 50 μl of peroxidase-linked human IgG  conjugate diluted 1:10,000 in 5% milk PBS was added to each well, and the plates were incubated for 2 hours at room temperature. After a final wash with PBS 0.05% Tween, 100 μl of OPD (ortho-phenylenediamine) (1 mg/ml) diluted in phosphate-citrate buffer  containing 0.03% hydrogen peroxide were added to the plates. This reaction was kept in the dark and quenched after 10 minutes by the addition of 25 μl of H2SO4 4 N in each well. Optical density (OD) was quantified in ELISA reader  at a wavelength of 490 nm. The cutoff point was established by the mean OD of 56 plasma samples from individuals with no history of malaria, from the Center for Hemotherapy and Hematology of Pará (HEMOPA) residing in Belém-Pará, plus three standard deviations. The reactivity index (IR) was determined by dividing the OD value of the sample by the cutoff point . IR values were used to estimate the concentration of IgG antibodies.The evaluation of total IgG antibodies against MSP-119 was performed at the Federal University of Pará, following the protocol of Cunha et al. (2001) . The recA CBA (cytometric bead array) Kit (BD) was used in this study to quantify cytokines IL-2, INFγ and TNF (Th1) and IL-4, IL-5, and IL-10 (Th2) in the same sample. Six bead populations with distinct fluorescence intensities were conjugated with a capture antibody specific for each cytokine. They were mixed to form the CBA and read on a FACSCanto II-type flow cytometer  that was previously calibrated with "setup beads", incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) according to the manufacturer's recommendations. A standard curve was performed for each cytokine and analyzed using FACSDiva software . The bead populations were displayed according to their respective fluorescence intensities, from dimmer to brighter. In the CBA, the cytokine capture beads were mixed with detection antibody conjugated with PE fluorochrome and then incubated with the test samples to form a "sandwich" test. The acquisition tubes were prepared with 50 μL of sample, 50 μL of bead mix and 50 μL of Th1/Th2 PE detection reagent . The same procedure was performed to obtain the standard curve. The tubes were homogenized and incubated for three hours at room temperature in the dark. The results were presented on graphs and in tables using FCAP Array 3 software . Raw MFI (media fluorescence intensity) values were quantified for each cytokine. The values were expressed in pg/mL for each cytokine in comparison to the standard curve. Three hundred events were considered for each cytokine.All individuals were provided with two sterile plastic containers and asked to provide faecal samples to be collect in the morning as follows: one without any solution and another with a preservative solution of 10% formaldehyde. For the negative cases three fecal samples were requested on alternate days to increase the detection sensitivity and to also to double check parasite nullity by negative slides. Fecal samples were prepared using the technique and/or methods of Hoffman-Pons-Janer and Faust. For each sample, two slides were examined for detection of parasites by two investigators with identification experience, using optical microscopy  with magnifications of 100X and 400X. All fecal analyzes were performed in a private laboratory in the municipality of Oiapoque/AP.Each volunteer's epidemiological data results, which were obtained from the questionnaire, was stored in an Epi-Info 3.5.1 database . The values for each group , as well as the subgroups , were expressed as a percentage. The age, residence time in Oiapoque (years) (RT), number of previous malaria episodes (NPE), period since last malaria (months) (PLM), haemoglobin levels (g/dL), parasitaemia (parasites/L) and gametocytes were expressed as medians (1st and 3rd Quartile) using BioEstat 5.3 statistical software. The differences between groups with regard to age, RT, NPE, PLM and haemoglobin were calculated using Tukey's test based on a one-way analysis of variance (ANOVA). The Kruskal-Wallis-Dunntest was used to calculate the degree of anaemia.Wilcoxon-Mann-Whitney test. For the PvMSP119 reactivity index (RI) among the studied groups, multiple correlations were performed using the non-parametric Kruskal-Wallis test, followed by Dunn's post-test. The data were expressed in box plot format . Significant differences were estimated using the median values for each group; those with p ≤ 0.05 were considered significant.The statistical tests used here were chosen by considering the size and type of each variable and number of evaluated groups. The differences in the parasitaemia and the gametocytes between the malaria and co-infected groups were calculated using the For the cytokine expression analysis, the significance level was obtained by comparing the cytokine concentrations of the studied groups using the GraphPad Prism program, version 6.0 . An analysis to uncover the correlation between the groups was performed using the non-parametric Kruskal-Wallis test followed by Dunn's post-test. The data were expressed in box plot format . Significant differences were estimated using the median values for each group, and those with p ≤ 0.05 were considered significant.We also observed differences in the mean residence time (RT) for groups E, CI and M (p < 0.05) when compared to the EC group. Regarding the number of previous malaria episodes (NPE), there were nodifferences between groups (p = 0.0716). When analyzing the period since last malaria (PLM) variable, we observed differences between the CI and EC groups (p < 0.01) and between the E and CI groups (p < 0.01).Plasmodium vivaxparasites), there were differences between the CI and M groups (p = 0.0152), as calculated by the Wilcoxon-Mann-Whitney test. However, when using the same test to quantify the Plasmodium vivax gametocytes, no statistically significant differences were observed between the CI and M groups (p = 0.0819) , EC and CI (p < 0.01), E and M (p < 0.01) and M and CI (p < 0.01). However, there was no significant difference when comparing groups EC and M and E and CI. Anaemia was expressed as a percentage, and the Kruskal-Wallis-Dunntest found a difference between the CI and EC individuals . With regard to parasitaemia (count of  0.0819)  Tables.19 IgG-1 antibodies is shown in  in the CI group. The lowest prevalence was in the EC group, with 33.1% (50/151)  . To supp(50/151) , each in(50/151) .19-specific IgG antibodies as distributed across the four study groups as well as the reactivity index, which specified and subdivided the CI and E groups. (19 reactivity index (RI) among the studied groups structured as in , CI (n = 50), E (n = 50) and EC (n = 100). The TNF-α concentration was higher in the CI group than in the other groups. The median values differed for the CI-E, M-CI and CI-EC groups (p = 0.001)  S2 Fig)S2 Fig).Additionally, the IFN-γ levels were higher in group M than in the other groups. Comparisons of the M-EC (p = 0.001), M-E (p < 0.05), E-CI (p < 0.05) and EC-CI (p = 0.001) groups were statistically significant . IncreasThe IL-4 concentration was higher in the CI group, and there was a statistically significant difference for CI-E (p = 0.05). Comparisons between the remaining groups were not statistically significant . IL-5 shP. falciparum malaria transmission and its correlation with anaemia [vivax malaria and intestinal parasite co-infections [P. vivax in a malaria-endemic area located on the Brazilian border with French Guiana.Microorganism co-infections are largely unexplored, and their effects can be either favourable or harmful to the human host. Some studies have explored the effects of helminth co-infections on the dynamics of  anaemia ,27. In Bfections –27 in twThe parasitological findings of this study show a higher prevalence of individuals who were infected only with E (32.4%), followed by the CI group (26.5%). Group M had the lowest prevalence (6.9%). The CI and M groups were predominant in the male population, emphasizing their geographical peculiarities and aspects related to the primary constraints and health determinants in the border region related to work activities, more specifically, the illegal mining (gold mining) and agricultural activities prevalent in the region where contact with the vector and soil is frequent.Of all individuals included in this study, the minority was composed by children  and seniors , while 37% (163/441) and 59.4% (262/441) were adolescents and adults, respectively. Likewise, the wide age range of our study population probably did not affect the observed results, since children and seniors, expected to suffer greater variations in relation to the immunological response, only represented 3.6% of the total number of subjects. The present study’s research group was mainly composed of adults and young adolescents, belonging to a socioeconomically disfavored population stratum, mainly working in in activities related to mining.Plasmodium infection [The results show that individual haemoglobin levels differ between the CI and M groups. Anaemia was more prevalent in the CI and E individuals, with significant differences in the plasmodial parasite load between the CI and M groups. This haematological change observed between the groups emphasizes that anaemia is multifactorial and is a frequent early manifestation of malaria that contributes to disease severity, especially in the context of concurrent infections –34. Thisnfection  as well nfection .vivax malaria still requires further investigation. These differences may be explained by the fact that in a co-infection, there may be a negative interaction between parasites, characterized by a protective effect on the individual and a suppressive effect on one of the pathogens or a positive interaction in which an infection is facilitated by the presence of the other parasite [P. vivax and intestinal parasite co-infection could modify the immune response profile against certain specific Plasmodium antigens.The molecular mechanisms underlying malarial anaemia are largely unknown, although it is accepted that the disease is complex and multifactorial and has characteristics in common with acute malaria ,43. In tparasite . Thus, ivivax malaria immune response in naturally exposed populations, in both Brazil [P. vivax antigens for use in creating an anti-malarial vaccine. The differences between antibody profiles reported in the present study indicate the importance of performing immunoepidemiological studies in different malaria-endemic areas, where transmission intensities and human genetic backgrounds are very distinct [Seroepidemiological studies to investigate the type and magnitude of the h Brazil –48 and ah Brazil –52, havedistinct .Plasmodium antigens offer a key process in the discovery and development of anti-malarial immunotherapy. Several studies have reported high antibody responses to P. vivax antigens in individuals who have been exposed to malaria infections [19 IgG antibodies were detected in 51.2% (226/441) of the studied population, indicating the presence of an immunogenic protein, with results similar to those of other studies in the Amazon region [19 appears as an immunogenic molecule during naturally acquired malaria infections [Studies on antibody responses to fections ,54. In tn region ,52–56. Hfections –60.vivax malaria-co-infected individuals was higher than that detected in the Oiapoque municipality, where geohelminths were more prevalent.Corroborating the findings in the Western Brazilian Amazon, the presence of enteroparasites also did not affect antibody responses to this antigen . The redP. vivax co-infection is very important for predicting the emergence and distribution of different strains of the parasite, which may differ in their virulence and drug resistance [These results support those of other studies ,62 that sistance . This fiIn these regions, individuals from socio-economically disadvantaged populations frequently migrate between the two countries, especially those involved in illegal mining. This populational flow contributes to the practice of malaria self-medication and may influence plasmodial resistance to some antimalarial drugs. However, we did not find references in relation to the individual's immune response to the parasite, which, therefore, does not suggest important biases. It is worthy of note that in the Oiapoque municipality there are no de-worming policies, which could interfere in our study analysis.Plasmodium vivax populations between the two countries [P. vivax's complex geographical pattern is important, both for evaluating diversity when encoding candidate antigens for vaccines and when formulating and structuring surveillance measures to control malaria.Due to Oiapoque's local genetic diversity and the combined effects of the geographical structure of the border between Brazil and French Guiana in a regional, national and international context, there is a dispersion of ountries . EstabliP. vivax, as previously shown in another Brazilian Amazon region [In fact, helminths were the most prevalent enteroparasites in the CI  and E  groups. The reduction in the antibody prevalence and reactivity index in group E may be explained by the association (protozoa-helminths) and/or presence of intestinal protozoa. However, in the CI group, the IgG prevalence and reactivity index were not reduced and were similar to those in infected group M. These data confirm the non-importance of protozoa in the immune process during co-infection with n region . HoweverThe general trend in co-infection studies  is convergent and points to the possibility of increasing incidence and prevalence of malaria ,66 and aPlasmodium due to the anti-inflammatory effect of helminth-induced cytokines [In malaria, protective immunity is gradually acquired through the natural exposure of people living in areas where the disease is endemic, but this immunity is rapidly lost when exposure ceases, indicating that malaria's immunological memory is short term and requires constant exposure to the parasite ,69. Althytokines .19 immunogenic molecule being present in response to malaria, other mediators, such as pro- and anti-inflammatory cytokines, also act in the elimination of plasmodial infections.Understanding the Th1/Th2 response pattern is of great importance for understanding the host's defense, and these antagonistic responses allow for the homeostasis of the immune system, which is important for parasite containment . In caseThe individual analysis of mean serum cytokine levels in the studied population showed a broad variation in the serum concentrations of all the Th1 inflammatory profile cytokines  in the studied groups. Initially, we found that the expression of these cytokines exhibited a very similar profile between groups, with high levels for M and CI individuals. This observation suggests that only malarial infection, and not intestinal parasites, affected the increase in these levels. Elevated serum cytokine levels during infection have been reported previously, both in uncomplicated and severe malaria ,72. Howevivax malaria immune response [Some studies have shown the importance of the cytokine pattern and its interactions in the response . Increasresponse . Inflammresponse . This obresponse .In the literature, IL-4 and IL-5 concentrations have been shown to confer protection against extracellular pathogens, including helminths . Some stP. vivax, with a shifting pattern inchanges associated with malaria and intestinal parasite co-infection. They seem to act in inflammatory processes in a nonspecific way, not exerting any effect on co-infection. However, only the IL-10 cytokine levels were affected in cases of malaria and intestinal parasite co-infection. Further wide-ranging studies are needed to evaluate the intestinal parasite species present in malaria-endemic areas and their relationship to the immune response of infected individuals.Variations in the cytokine production and high Th1 (TNF-α) and Th2  profile levels in the co-infected group therefore suggest that cytokines exhibit marked increases in their plasma concentrations when individuals are infected with Plasmodium vivax malaria. These studies will be essential in the development of vaccines and new immunotherapeutic targets.These results may reflect the fact that the number of parasites in the bloodstream at a particular time may not always show the maximum immunogenic potential capable of inducing a host's immune response because some parasite antigens may be in immune system tissue and not just in the blood . The oveThe present study has some limitations, we acknowledge. First the cross sectional design is prone to bias and confounding and does not allow to infer causality. Secondly, the small sample size, notably in the malaria alone group, may not have been sufficient to detect some differences with other groups. Morevover, this evaluationdid not allow to look at the respective effects of different nematode species which may have contrasting effects. Likewise, the current results highlight the possibility that the enteroparasite and vivax malaria co-infection on the Brazil-French Guiana bordermark biological differences, until unexplained, towards the differential immune response and haematological profile of the host-parasite interaction in vivax malaria in Oiapoque, Amapá state. Aditionaly, provide additional information about the influence that enteroparasite could have in a malaria vivax development essential for diagnostic and preventive strategies.P. vivaxis endemic in the region studied and its coinfection with intestinal parasitosis is present in the municipality. Fecal findings demonstrate a diverse enteroparasitic prevalence among the groups, highlighting the particularities and geographic aspects related to the determinants of health in the border area. In addition to this context, there was a predominance of male individuals in the adult age group people with low purchasing power and who were engaged in activities related to goldmining. Hemoglobin levels appeared to be strongly related to malaria infection, especially in the context of concomitant infections, with coinfected individuals more likely to be anemic than single infected malaria patients. In this study we did not observe the influence of coinfection on the acquisition of IgG antibodies against PvMSP119, as well as on the profile of the cytokines that characterize the Th1 and Th2 patterns. However, the co-infection between Plasmodium vivax and enteroparasitoses increased levels of TNF-α, and IL-10 in comparison to those with single malarial infection. Longitudinal studies including a greater number of patients are needed to better characterize the potential effects of this coinfection in the Brazilian Amazon population’s immune response.The malaria by S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(XLSX)Click here for additional data file.S2 Fig(XLSX)Click here for additional data file.


A meta-regression model confirms that LLINs are significantly more effective than ITNs in the prevention of malaria, when controlling for other covariates. For both types of nets, protective efficacy was greater in high transmission areas when nets were used for an extended period. However, cross-sectional studies may overestimate the effect of the nets. The results surprisingly suggest that nets are less effective in protecting children under the age of five, which may be due to differences in child behavior or inadequate coverage. Compared to a previous meta-analysis, insecticide-treated nets appear to have improved their efficacy despite the risks of insecticide resistance. These findings have practical implications for policymakers seeking effective malaria control strategies.Long-lasting insecticidal nets (LLINs) have been widely used as an effective alternative to conventional insecticide-treated nets (ITNs) for over a decade. Due to the growing number of field trials and interventions reporting the effectiveness of LLINs in controlling malaria, there is a need to systematically review the literature on LLINs and ITNs to examine the relative effectiveness and characteristics of both insecticide nettings. A systematic review of over 2000 scholarly articles published since the year 2000 was conducted. The odds ratios (ORs) of insecticidal net effectiveness in reducing malaria were recorded. The final dataset included 26 articles for meta-regression analysis, with a sample size of 154 subgroup observations. While there is substantial heterogeneity in study characteristics and effect size, we found that the overall OR for reducing malaria by LLIN use was 0.44 (95% CI = 0.41–0.48,  With approximately 200 million cases per year, malaria is responsible for a large proportion of the global burden of disease and is a serious public health issue in Africa . The figWhile the effectiveness of conventional ITNs in controlling malaria has been well established ,6,7, theAs a simpler solution to the problems of conventional ITNs, long-lasting insecticide-treated nets (LLINs) have been developed using novel fabric technologies . LLINs, Many field-based studies examining the efficacy of mosquito nets in reducing the incidence of malaria look at LLINs. However, most analyses combined all insecticide-treated nets, including both ITNs and LLINs. Some studies examined LLIN efficacy alone, but it is difficult to get a sense of large-scale performance of LLINs from individual studies. No study has assessed the overall effectiveness of LLINs in preventing malaria infection rates compared to ITNs. An early meta-analysis of ITN efficacy, published in 1995 by Choi et al., reported that ITNs reduced malaria cases by around 24% . In his Meta-regression is a form of meta-analysis that has grown in popularity in recent years. This is in part due to the increasing numbers of systematic reviews and meta-analyses published in the biomedical literature . Meta-reUsing five databases  a comprehensive review of the literature published since 2000 was performed. Publications were searched for with keywords including malaria, long-lasting treated bed net, insecticide-treated bed net, LLIN, ITN, prevalence and incidence. This process identified 2051 articles. Experimental studies that reported on the protective efficacy of either LLINs or ITNs in human populations that adopted cross-sectional, cohort, or randomized controlled trial study designs were included. Articles were excluded if they met any of the following three criteria: they were review articles; they were articles reporting only the impact on mosquito populations; they were articles reporting ITN/LLIN interventions combined with untreated nets, IRS, or immunization. After this step, a total of 215 articles were identified for full review. The full texts of the 215 articles were read to confirm they qualified for inclusion in the meta-analysis. Articles were excluded that did not include sufficient numerical information for calculating the effectiveness of ITNs/LLINs, such as the numerical incidence of malaria before and after intervention (for cohort studies) or between treatment and control groups . After this step, a total of 39 articles qualified for data collection and analysis.Data from the 39 articles were recorded with quantitative measures for the following covariates: endemicity, chemical class, malaria species, intervention duration, group age, net types, study design and study location. The costs of treated nets were not recorded because almost all nets were supplied by social distribution campaigns, usually at a subsidized price. This approach promotes and distributes the socially beneficial intervention rather than commercializing the product. Thirteen articles were excluded due to a lack of quantitative data for at least one of the covariates listed above. The final sample for meta-analysis includes 54 data points from 12 LLIN (long-lasting insecticide-treated nets) articles and 100 data points from 14 ITN (insecticide-treated nets) articles. Plasmodium falciparum only, and 0 represents both P. falciparum and Plasmodium vivax. A dummy variable for the study location was created to assess the effectiveness of treated nets in Africa, the continent bearing the highest burden of malaria. To represent the studies undertaken in Africa, 1 was used, and 0 represented the studies undertaken in Asia. Due to a concern over the long-term durability of treated nets and the fact that ITNs need to be retreated at least once a year to ensure continued insecticidal effectiveness, a dummy variable was created for the duration of the study, where 1 represented a study duration longer than 12 months and 0 represented a study duration of less than one year. Additionally, two dummy variables were created to indicate cohort and cross-sectional study designs, compared with randomized controlled trials as baseline. A dummy variable was created for children under the age of five to investigate the marginal impact of treated nets on this vulnerable group [Odds ratios (ORs) were estimated for the effectiveness of treated nets. If the OR was not reported in a study, it was calculated based on malaria incidence before and after the intervention (cohort studies), or malaria incidence between the treatment and control groups . New variables were created from quantitative information synthesized from studies to enable a more comprehensive meta-regression analysis. Covariates for sample size, pesticide type, malaria parasite, location, study duration, study design, and participants under the age of five were incorporated into the model. Sample size was created by summing the total number of individuals in study sub-groups. Two dummy variables were created for the insecticides deltamethrin and alphapermethrin. These were compared with the insecticide permethrin as a baseline. A dummy variable was created for the type of malaria parasite, where 1 represents le group . There wle group  and two le group ,26 that To conduct a meta-analysis of the odds of malaria prevalence, we implemented a regression meta-analysis using the statistical package Stata . The adjusted R-Square value for the regression is calculated in the statistical software following the formula of the unadjusted R-Square multiplied by a ratio of the degrees of freedom to the residual degrees of freedom.P. falciparum or P. vivax, and two articles did not note the species [P. falciparum only [P. falciparum accounted for about 80–90% of total infections. Of the 14 ITN articles, three were conducted within Asia [Of the 14 ITN articles, six were randomized controlled trials ,27,28,29 species . Of the rum only ,32,33,34rum only ,7,28,29,hin Asia ,28,29 . p = 0.0001). This meta-regression analysis reveals that the efficacy of both ITNs and LLINs in reducing the prevalence of malaria is strong and statistically significant (p = 0.0001). This study also suggests that LLINs are more effective than ITNs in reducing the prevalence of malaria , representing the relative odds of contracting malaria between a treatment group and a control group. Studies were subdivided into two groups for analysis based on the type of net, either ITN or LLIN. Even though a meta-regression analysis revealed that LLINs have a greater protective efficacy than ITNs, there remains a high level of heterogeneity between individual studies. As shown in P. falciparum only, and cohort and cross-sectional study designs. An OR of significantly less than 1 or a negative log OR implies that an increase in a covariate is associated with a decrease in the risk of contracting malaria. Use of LLINs, the log transformation of the initial prevalence prior to intervention, whether the study lasted longer than 12 months and whether children less than five years of age were present in the study are variables that were hypothesized to be negatively related to the OR. A model using the African covariate had a lower adjusted R-square, as well as an insignificant coefficient for Africa. Therefore, the African covariate was not included in the final model. The best-fit model is shown in In the meta-regression model, the dependent variable is the log transformation of the OR for net usage in regard to malaria prevention. The primary independent variables are whether an LLIN was used, the log-transformed proportion of study population with malaria before the intervention, the total sample size, whether the study duration lasted longer than 12 months, children under the age of five, the use of deltamethrin and alphapermethrin, P. falciparum and P. vivax), and different insecticides . There was no difference in net efficacy for these covariates.As hypothesized, LLIN usage, initial malaria prevalence, and the study duration were shown to be strong and statistically significant negative predictors of the OR for malaria prevalence. Overall, the meta-regression results can be interpreted as follows: Firstly, the negative relationship of initial malaria prevalence to the log transformation of the OR indicated that the use of LLIN/ITN confers a stronger protection against contracting malaria for people living in malaria-endemic areas. Second, the negative relationship of the log transformation of the OR to whether the study lasted longer than 12 months implies that insecticidal nets were effective over longer periods of time if they are either long-lasting or routinely re-treated. As noted, conventional ITNs needs to be re-treated at least once a year to maintain insecticidal effectiveness. Third, contrary to expectation, the coefficient for children under the age of five was positive and significant, indicating that treated nets are less effective in preventing malaria for this age group compared to children aged five years or over. However, nets were somewhat effective in reducing malaria prevalence in both age categories. Fourth, cross-sectional study design was negatively related to the OR, as compared to cohort study and randomized controlled trial study designs, which could suggest that cross-sectional studies overestimated the effect of mosquito nets . Fifth, In the meta-analysis study published Choi et al., ITNs were shown to avert around 24% of malaria cases . The preThere are some differences between Choi et al.’s study and the present study. In the 1995 study, eight of the ten total field trials included in the analysis limited the study population to children less than 15 years old. The other two studies included in Choi et al.’s analysis contained both children and adults. Choi et al.’s analysis used two sub-groups based on the type of control. These were an ITN intervention group versus a group that received an untreated bed net, and a group that received permethrin-impregnated bed nets as compared to a group that received no net. Each sub-group used small samples of six field trials, comparing measures of the incidence of parasitemia as a function of person-weeks at risk for parasitemia. In this analysis, the sample study size increased to 26 studies (published since the year 2000), in which 14 studies reported on the effectiveness of ITNs and 12 reported on the effectiveness of LLINs. This analysis focused on the protective efficacy of both LLINs and ITNs and their protective efficacy when used in conjunction with each other. This study used the malaria prevalence odds ratio as the main indicator of infection prevention. In this analysis and unlike Lengeler’s [Meta-analyses, such as the one presented here, are particularly useful for policymakers in strategically allocating resources in disease control. The main finding of this meta-analysis is the outstanding protective efficacy of LLINs over ITNs over long periods of time. LLINs are designed so that their insecticidal activity could last as long as the nets themselves, a claim which is supported by this meta-analysis. An additional finding is that even though some studies show that vulnerable groups  sleep under insecticidal bed netting more often than others ,47, the The limitations of this study are common to meta-analyses. A meta-analysis of the literature only examines published studies. The comprehensive effectiveness of LLINs and ITNs might not be captured if most published studies focus on success stories, while insignificant impacts are not published. Several studies were excluded due to missing selection criteria for the adopted meta-regression model, which may have led to bias. Also, the studies used in the meta-analysis vary by approach. For example, most reported that the distribution of netting was free, though some studies involved the promotion and/or marketing of nets. The price of nets that users have to pay may affect proper use and deployment . More brThere remains high global demand for sustainable malaria control and elimination. Provided that people use treated nets properly and consistently, LLINs and ITNs are highly effective at preventing malaria ,4. ReplaThe meta-regression model presented in this paper confirms that LLINs are statistically more effective than ITNs in preventing malaria, although the effectiveness of both has been substantially improved during the past decade. The findings support the importance of treated nets, and the improvement from LLINs, in malaria control.


We therefore compared the CD4+ T cell phenotypes of Ghanaian children with complicated malaria, uncomplicated malaria, asymptomatic Plasmodium falciparum (Pf) infection or no infection. Using flow cytometric analysis and automated multivariate clustering, we characterized the expression of the co-inhibitory molecules CTLA-4, PD-1, Tim-3, and LAG-3 and other molecules implicated in regulatory function on CD4+ T cells. Children with complicated malaria had higher frequencies of CTLA-4+ or PD-1+ CD4+ T cells than children with uncomplicated malaria. Conversely, children with uncomplicated malaria showed a higher proportion of CD4+ T cells expressing CD39 and Granzyme B, compared to children with complicated malaria. In contrast, asymptomatically infected children expressed only low levels of co-inhibitory molecules. Thus, different CD4+ T cell phenotypes are associated with complicated versus uncomplicated malaria, suggesting a two-sided role of CD4+ T cells in malaria pathogenesis and protection. Deciphering the signals that shape the CD4+ T cell phenotype in malaria will be important for new treatment and immunization strategies.The immune response of malaria patients is a main factor influencing the clinical severity of malaria. A tight regulation of the CD4 An infection with Plasmodium falciparum (Pf), the most prevalent malaria parasite in Africa, can lead to a wide spectrum of disease manifestations, ranging from severe, life-threatening malaria through mild, febrile illness  to asymptomatic infections. The immune response of the infected host is one important factor that influences the course of a Pf infection. In individuals with little prior exposure, an infection with Pf activates a strong, pro-inflammatory response3, which induces fever and contributes to the development of malaria complications5. In endemic areas, regularly exposed children gradually develop a partial immunity which protects from severe and febrile disease and is associated with an increasing incidence of asymptomatic infections.Malaria remains one of the leading causes of morbidity and mortality among children in Sub-Saharan Africa+ T cells are an important player of the adaptive immune response to plasmodia and can provide protection but also have detrimental effects and contribute to disease complications9. Several observations support the idea that qualitative changes of the T cell response occur during acute malaria11. Murine models with chronic Plasmodium infection show that the initial strong pro-inflammatory response is downregulated during the course of the infection12. Previous studies in humans and mice by us and others have shown that acute malaria induces an upregulation of co-inhibitory molecules, such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1), lymphocyte-activation gene-3 (LAG-3) or T-cell immunoglobulin and mucin domain-3 (Tim-3) on CD4+ T cells which leads to impaired cytokine production of the CD4+ T cells18. The blockade of the co-inhibitory receptors CTLA-4, PD-1, Tim-3 and/or LAG-3 leads to an enhancement of the pro-inflammatory T cell responses and a more severe course of disease in murine malaria models, but can also improve parasite clearance, indicating the double-edged role of CD4+ T cells in malaria17.CD422. Other activation and effector molecules which are expressed on regulatory as well as activated T cells and have been shown to modulate immune responses to infectious pathogens include Granzyme B (GrzB), CD39 or CD3825.Co-inhibitory molecules such as PD-1, LAG-3 or Tim-3 are also preferentially expressed on regulatory T cells including Type 1 regulatory T cells (Tr1 cells), Tr27 and other peripherally induced regulatory T cell subsets, which are expanded during natural exposure or in experimental infection models of malaria+ T cell analyses conducted so far have been in murine models or in experimental human malaria infections. It still remains unclear which T cell profiles are associated with clinical protection upon natural exposure in endemic areas. In our study, we therefore compared T cell phenotypes in children with different clinical severities of malaria in an endemic setting and focused on T cell markers with regulatory capacity, using multi-colour flow cytometry analysis and automated multivariate clustering.Most of the CD4+ T cell signatures. Children with complicated malaria showed higher frequencies of CTLA-4+ and PD-1+CD4+ T cells, whereas children with uncomplicated malaria had higher percentages of CD39+, as well as GrzB+CD4+ T cells, suggesting that distinct regulatory mechanisms are activated and might shape the clinical picture of acute malaria.Children with complicated versus uncomplicated malaria expressed different CD4Pf  and 41 were asymptomatically infected with Pf, detected by HRP2 -rapid diagnostic test . 30% of the screened children at the school were positive for Pf. At the hospital, 66 children with acute, symptomatic malaria between 1–12 years of age were enrolled, of whom 32 were treated as outpatients with oral artemisinin combination drug for uncomplicated malaria (outpatient = OP) and 34 were treated as inpatients with iv Artesunate for clinically diagnosed complicated malaria (inpatient = IP). As a consequence of the collection strategy at the school, healthy controls and asymptomatically infected children were on average older than children with acute malaria  or no microscopically detectable parasitemia by thin blood smear (n = 26)  Table .Table 1Cex-vivo expression of the co-inhibitory molecules PD-1 and CTLA-4 on CD4+ T cells in our four study groups. Both groups of children with acute malaria showed higher frequencies of CTLA-4+CD4+ T cells than uninfected or asymptomatically infected children. The highest percentage was seen in the inpatient group with complicated malaria , which allows for an automated identification of stratifying subpopulations. The automated hierarchical clustering of the flow cytometry data of the two groups showed two differing cluster groups of the co-inhibitory molecules analyzed Fig. . Cluster+ T cells in our four study groups. Contrary to the observations with co-inhibitory molecules, the expression of these markers was not overall increased in the two children groups with acute malaria. Only children with uncomplicated malaria, but not children with complicated malaria, showed an increase in the proportion of CD39+, CD69+, and GrzB+ CD4+ T cells  and the markers for recent activation and proliferation, CD69 and Ki-67, on CD4Pf infected children  positive, asymptomatic children. Multiple factors are involved in the induction of these markers during an acute infection. However, it is known that the expression of CTLA-4 is generally induced upon T cell receptor activation27 and CTLA-4 levels remained low in the children with asymptomatic Pf infection. We therefore propose that the T cell activation levels in these children remain beneath a certain threshold that is required for the induction of co-inhibitory molecule expression. This diminished T cell activation is associated with the asymptomatic clinical picture of the Pf infection in this group. This supports the notion that a strong T cell activation during malaria is an important trigger of the clinical symptoms of malaria.We initially anticipated that children with asymptomatic 18 (and unpublished observation) and a longer time elapse between onset of disease and hospital presentation could be a cause for higher CTLA-4 and PD-1 expression and severer symptoms in the inpatient group. However, the higher frequency of GrzB+ CD4+ T cells in children with mild malaria argues against purely kinetic reasons for the differential expression pattern of inhibitory molecules in both groups. High levels of pro-inflammatory cytokines have also been proposed to induce CTLA-4+ and PD-1+ CD4+ T cells39. But we did not detect any differences in plasma cytokine levels in our two acute malaria groups nor did cytokine levels correlate with expression of any of the co-inhibitory molecules.Due to the descriptive, cross-sectional nature of our study, factors influencing the expression of the investigated co-inhibitory molecules could not be fully elucidated. Age groups did not differ in the expression pattern of co-inhibitory molecules. CTLA-4 and PD-1 but none of the other markers correlated moderately with parasitemia. The type of anti-malarial treatment had no effect as the T cell analysis was conducted before initiation of treatment. The duration of the ongoing infection might be an influencing factor that we cannot exclude, which is a limitation of the study. We know that levels of CTLA-4, PD-1, Tim-3 or GrzB expression further increase after initiation of treatment42. Specifically, we would have expected higher levels of IL-10 in our group with uncomplicated malaria. But measurement of cytokines in plasma is a very broad and unspecific method, influenced by different subsets of leukocytes and their cytokine production. A more specific approach is the intracellular staining of cytokines. In a prior study with adult malaria patients, we detected IFN-γ+/IL-10+ and IL-10-single positive CD4+ T cells which expressed co-inhibitory molecules in a T cell subset that exerted suppressor function in vitro. Yet, percentages of T cells with Pf-specific cytokine production were very low18. It would be of interest to further clarify the function and Pf-specific cytokine production of the different CD4+ T cell populations identified in this study.The similar cytokine levels in our two groups with acute malaria are in contrast to several prior studies that observed differences in pro-inflammatory and anti-inflammatory cytokine levels and their ratio between patients with mild and severe malaria+ T cell response is an important factor influencing the clinical outcome of malaria. Our results identify specific clusters of CTLA-4+PD-1+CD4+ T cells which are associated with malaria complications. Additional longitudinal and functional studies are urgently needed to further investigate if and how CTLA-4+PD-1+CD4+ T cells display a detrimental effect in malaria or rather prevent a worse course of disease.In conclusion, our study results support the hypothesis that the quality of the CD4Pf malaria . The remaining 41 children in the second subgroup tested positive in the rapid diagnostic test but were healthy and afebrile and therefore labeled as asymptomatically infected children (AS). Recent malaria treatment or hospital visits were excluded via questionnaire. Children with acute malaria were enrolled at St Michael´s Hospital at time of diagnosis and before initiation of treatment. Malaria patients were also divided into two subgroups. One subgroup included children with fever and mild symptoms who were treated as outpatients with oral artemisinin combination drugs for uncomplicated malaria (“outpatients” = OP). The second group comprised of children with more severe symptoms, that required inpatient treatment with iv Artesunate and were clinically diagnosed with complicated malaria according to the WHO bedside clinical classification of severe malaria (“inpatients” = IP)43.The study was conducted as a cross-sectional study in the village Jachie-Pramso in the Bosomtwi District, Ashanti Region, Ghana, an area with perennial malaria transmission, between June-August 2015. In total, 82 healthy children and 68 children with acute malaria between 1–12 years of age were enrolled at two different study sites, Jachie D/A Primary school and St. Michael’s Catholic Hospital, Pramso. At the primary school, 82 healthy, afebrile children were recruited and divided into two subgroups. The first subgroup included 41 children who tested negative in a HRP2 rapid diagnostic test for ex vivo flow cytometry staining.Blood was drawn into heparinized tubes (1–2 mL) before initiation of treatment or directly at the school and processed within 5 hours of collection for Pf infections were determined using CareStart™ Malaria HRP2 (PF) rapid diagnostic test. Implementation was carried out according to the manufacturer’s protocol.Present Parasitemia was determined with thin blood smears stained with 4% Giemsa and examined under oil immersion .+ T cells were further gated for single cells and the expression of CD4 and CD8. Finally, gates were set on the co-inhibitory molecules and other molecules implicated in regulatory function mentioned above. Fluorescence minus one (FMO) controls were used for setting the gates. The gating strategy and exemplary flow cytometry stainings can be seen in Supplementary Figures 100 μL of whole blood was incubated for 30 minutes at 4 °C with the following surface antibodies: PD-1 PerCP/Cy5.5 (clone EH12.2H7), CD8 AF700 (RPA-T8), Tim-3 BV421 (F38–2E2), CD4 BV510 (OKT4), CD69 FITC (FN50), CD38 PE (HB-7), CD39 BV421 (A1) , and LAG-3 APC (3DS223H) (eBioscience). Subsequently, the samples were lysed and fixed for 15 minutes at room temperature using RBC Lysis/Fixation Solution (BioLegend) and washed with cold PBS. Thereafter, samples were stained intracellularly with Ki-67 PE/Cy7 (clone Ki-67), CTLA-4 PE (L3D10), CD3 APC/Cy7 (HIT3a), and Granzyme B AF647 (GB11)  using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. After washing, samples were stored at 4 °C until reading on a LSR II Cytometer . Where possible, 100 000 cells were collected in the lymphocyte gate. Results were analyzed using FlowJo X 10.0.7r2 (Treestar). Cells were first gated on CD3, followed by a forward/side scatter to exclude cell debris. CD344. CITRUS enables an automated discovery of statistically significant, differing cell signatures between two groups. By unsupervised hierarchical clustering, cell clusters are identified, phenotypes of the cells are characterized, and differences between groups are determined. Flow cytometry data of children with complicated and uncomplicated malaria were analyzed after using the same gating strategy as before  (BioLegend) according to the manufacturer’s protocol. Samples were read on a BD Accuri™ C6 Flow Cytometer.The statistical significance of differences between the four groups was defined using One-way ANOVA with Tukey’s Multiple Comparison Test (Graph Pad Prism 5). Correlation analysis was conducted using Pearson Correlation Test. The correlation matrices were composed using Excel correlation analysis. * was used when P <0.05, ** for P <0.01 and *** for P <0.001 unless stated otherwise.Ethical approval was obtained from the Committee on Human Research, Publication and Ethics, School of Medical Sciences/Komfo Anokye Teaching Hospital, Kwame Nkrumah University of Sciences and Technology, Kumasi, Ghana. Written informed consent was given from a legal guardian/parent of all participants prior to inclusion in the study. The study was carried out in accordance with the principles laid down in the Declaration of Helsinki.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplemental information


Young children are at greatest risk for malaria-associated morbidity and mortality. The immune response of young children differs in fundamental ways from that of adults, and these differences likely contribute to the increased susceptibility of children to severe malaria and to their delayed development of immunity. Elevated levels of pro-inflammatory cytokines and chemokines in the peripheral blood during acute infection contribute to the control of parasitaemia, but are also responsible for much of the immunopathology seen during symptomatic disease. Clinical immunity to malaria may depend upon the ability to regulate these pro-inflammatory responses, possibly through mechanisms of immunologic tolerance. In order to explore the effect of age on the immune response to malaria and the development of clinical immunity, cytokines and chemokines were measured in the plasma of children at day 0 of an acute malaria episode and during convalescence.Younger children presenting with acute malaria exhibited much higher levels of TNF, IL2, and IL6, as well as increased Th1 associated chemokines IP10, MIG, and MCP1, compared to older children with acute malaria. Additionally, the regulatory cytokines IL10 and TNFRI were dramatically elevated in younger children compared to older children during acute infection, indicating that regulatory as well as pro-inflammatory cytokine responses are dampened in later childhood.Together these data suggest that there is a profound blunting of the cytokine and chemokine response to malaria among older children residing in endemic settings, which may be due to repeated malaria exposure, intrinsic age-based differences in the immune response, or both. Plasmodium falciparum malaria is responsible for one in every five childhood deaths in Africa  and six chemokines  were analysed using a custom R&D Magnetic Luminex Screening Assay Human Premixed Multi-Analyte Kit . Plasma was diluted 1:2 as per the manufacturers recommendations. All samples were analysed in duplicate. Protocols were performed as indicated by the vendor: Briefly, 50 μl of provided standard or test sample, along with 50 μl of the mixed microparticles, were added to each well of a 96-well micro-titer plate. After 2 h of incubation and washing at room temperature, 50 μl of diluted biotin antibody cocktail was added to each well. Samples were incubated for another hour. After washing, 50 μl of diluted Streptavidin-PE were added to each well. The plate was incubated for 30 min, washed, and the microparticles were resuspended in 100 μl of wash buffer. The plate was read on a Luminex MAGPIX CCD Imager and data were analysed using xPONENT software (v4.2). A minimum of 50 beads per region were analysed. A curve fit was applied to each standard curve according to the manufacturer’s instructions and sample concentrations were interpolated from the standard curves. MFIs were background subtracted. Variability between duplicates was minimal, with an average coefficient of variance of 5.2%. Age groups and time points were randomly distributed between plates to minimize batch effects. The limit of detection for each analyte was as follows: TNF: 14.83 pg/ml, TNFR1: 220.58 pg/ml, IFNγ: 7.55 pg/ml, IL1B: 17.75 pg/ml, IL2: 30.62 pg/ml, IL4: 18.26, IL5: 17.57 pg/ml, IL6: 14.03 pg/ml, IL7: 5.84, IL8: 8.13 pg/ml, IL10: 13.64 pg/ml, IL12p70: 278.45 pg/ml, IL17a: 21.42 pg/ml, IL23: 239.36, MIG: 396.06 pg/ml, MDC: 59.01 pg/ml, MIP1α: 60.39 pg/ml, MCP1: 32.70 pg/ml, IP10: 3.78 pg/ml, TARC: 9.06 pg/ml. Samples below the limit of detection were either given a value at the limit of detection, or half the limit of detection when log-transformed for statistical analysis.Human IFNγ BD OptEIA ELISA II Kits  were used to quantify IFNγ levels in plasma. Samples were diluted 1:2 and run in duplicate using the protocol provided by the manufacturer. Plates were read on a SpectraMax M2 (Molecular Devices) and analysed using SoftMax Pro (v) software to fit a curve to the standards supplied by the vendor. Background absorbance was subtracted from sample measurements. The limit of detection for this assay was interpolated from the standard curve to be 5 pg/ml.P < 0.05 was considered significant.All statistical analyses were performed using Prism 7.0a (GraphPad) or STATA SE version 13.1 (College Station). Comparisons of malaria between age groups, as well as comparisons of cytokine and chemokine concentrations between age groups and timepoints, were done using the Wilcoxon rank sum test or the Wilcoxon signed-rank test for paired data (i.e. day 0 vs. week 3). Associations between parasite densities and cytokine/chemokine concentrations were assessed using Spearman correlation. All multivariate statistics were derived by linear regression models with transformed variables following log or square root-transformation of any non-normal variables. Two-sided P values were calculated for all test statistics and Children who presented at the time of an acute malaria episode were enrolled to undergo an initial blood draw and a follow up draw 3 weeks after treatment. Subjects were recruited as part of a nested sub-study within an ongoing longitudinal cohort study of childhood malaria in Eastern Uganda. Details of this study have been published elsewhere . The stuParasite density was assessed by blood smears performed on-site. Subjects were removed from the study if convalescent samples tested positive for parasitaemia by blood smear at 3 weeks after treatment. As expected, the median parasite density at day 0 for children in the 1–3 year age group was significantly higher than for children in the 7–10 year age group  and 3 weeks after treatment. Plasma cytokine levels were measured using a multiplex bead array on a Luminex platform. Pro-inflammatory cytokines, including TNF and IL6, were significantly higher in the younger children than in older children at presentation Fig. a. IndeedBecause parasite density also varied with age, the relationship between cytokine concentrations and parasite density was examined, both independently and as a covariate with age Table . A positSurprisingly, the concentration of IFNγ, as well as the other pro-inflammatory cytokines IL12p70 and IL1β, were below the limit of detection for all samples. Because this lack of detectable IFNγ was in conflict with some previous reports , 20–22, Because regulatory responses are important for limiting the pathology associated with malaria infection, we also quantified concentrations of IL10, sTNFRI, IL4, IL5, IL7, IL17 and IL23. As with the pro-inflammatory cytokine levels discussed above, levels of the regulatory cytokines IL10 and sTNFRI were markedly higher in young children compared to older children during acute malaria. IL10 levels were significantly higher in both age groups at day 0 as compared with convalescence . This relationship was significant both in the cohort as a whole and when limiting the analysis to the younger age group Fig. , suggestThe Th2 cytokine IL4, while present at detectable levels in many children, showed no relationship with age or time after infection. IL17, IL23, IL5 and IL7 were undetectable in all samples.In order to determine whether the diminished regulatory responses seen in older children could be explained by proportionally diminished pro-inflammatory responses in this age group, the relative ratios of TNF, IL2 and IL6 to IL10 at day 0 were compared between younger and older children. For both TNF and IL2, but not IL6, older children exhibited higher ratios of these pro-inflammatory cytokines to IL10 Fig. , indicatIn addition to cytokines, concentrations of several key chemokines were measured. These molecules are crucial regulators of leukocyte trafficking; increased levels of numerous chemokines have been observed in severe malaria . SeveralParasite density trended toward being a significant predictor for these chemokines in univariate analysis, but only age remained significant in multivariate analysis. There were no significant interactions between age and parasite density, and stratifying by age group to look at the influence of parasite density on chemokine concentrations did not reveal any further relationships. Table . Thus, sPlasma concentrations of chemokines known to bind CCR4, a receptor associated with Th2 CD4+ T cells, exhibited a different pattern from that described above for CXCR3-binding chemokines. These CCR4-binding chemokines include thymus activation regulated chemokine (TARC)/CCL17, human macrophage derived chemokine (MDC)/CCL22, and macrophage inflammatory protein (MIP1α). TARC concentrations were significantly elevated during convalescence as compared to day 0 in both younger and older children suffering an acute malaria episode Fig. b.MDC was elevated at both day 0 and week 3 in younger children as compared to the older age group; however, there were no significant differences between timepoints for either age group Fig. b, thoughThis study sought to determine the effect of age on peripheral blood cytokine and chemokine levels during acute malaria infection in a highly endemic setting. The resulting data indicate that older children respond with diminished levels of the pro-inflammatory cytokines TNF, IL2 and IL6, as well as Th1-biased chemokines when compared to younger children at the same stage of infection. Moreover, older children had strikingly lower plasma concentrations of the regulatory cytokines IL10 and sTNFRI than younger children. Together these data suggest that there is a profound blunting of the cytokine response to malaria among older children residing in endemic settings, which may be due to repeated malaria exposure, to intrinsic age-based differences in the immune response, or to a combination of both factors.These observations are consistent with the idea that repeated malaria may drive the host towards a disease tolerance state in order to reduce the negative impacts of infection-related pathology . While tOf note, the striking difference in malaria incidence between age groups following sampling, evident in Table P. falciparum much more rapidly than children [The main limitation of this study is its inability to uncouple the influence of prior malaria exposure from that of biological age in this cohort of children. Differences in cytokine production may reflect either the fact that the younger children have sustained less prior exposure to malaria than older children, or instead, intrinsic age-related differences in immune function. Several prior studies support the notion that biological age profoundly influences the development of immunity to malaria. In an elegant study of Javanese transmigrant families emigrating together from an area of little to no malaria transmission to an area of high malaria transmission, adults were found to develop immunity to children , 4; howechildren . These cchildren –7. This The complete lack of detectable IFNγ or IL12p70, both in younger and in older children, was somewhat surprising. These findings are at odds with some prior descriptions of plasma cytokines during acute infection , 20–22, P. falciparum infection, in some circumstances biasing the immune environment towards a Th2-driven response [P. falciparum-infected mosquitoes, the cytokine responses of children from high- and low-transmission settings could not be compared.This study had additional limitations, including the inability to record the exact timing of infection, although all samples were obtained within 48 h of the onset of symptoms. Thus, when comparing these data to cytokine concentrations measured during controlled human malaria infection, the timing of sample collection in the present study was less uniform and individual measurements more variable. Helminth co-infection has been shown to impact cytokine responses to response , 44. As response , this maTogether, these findings provide new evidence that despite age-related differences in immune responses, young children respond with a largely pro-inflammatory Th1-type cytokine and chemokine response, similar to naïve travellers; however, they also show increased levels of the regulatory cytokines IL10 and sTNFRI, which may limit their ability to develop robust immunity to subsequent malaria infections. This inflammatory response is dampened in older children, suggesting both differences in age-related immune function and the development of partial clinical tolerance.


As the prevalence of the malaria has been decreasing in many endemic countries including Myanmar, malaria elimination in Greater Mekong Region was targeted not later than 2030. The relevance of molecular and serological tools to identify residual transmission remains to be established in this setting.One-year cohort study was conducted and sera samples were collected in every 3 months with active and passive case detection for clinical malaria episodes by RDT, microscopy and molecular method. The sera were used to detect the malaria antibody against PfMSP1-19, PvAMA1, PvDBPII and PvMSP1-19 by protein microarray.Among the recruited 1182 participants, there was no RDT positive case for malaria infection although two vivax infections were detected by microscopy in initial collection. Molecular methods detected the asymptomatic cases of 28/1182 (2.37%) in first, 5/894 (0.42%) in second, 12/944 (1.02%) in third, 6/889 (0.51%) in fourth collection, respectively. Seropositivity rates against the PfMSP1-19, PvMSP1-19, PvAMA1 and PvDBPII were 73/270 (27.0%), 85/270 (31.5%), 65/270 (24.1%) and 160/270 (59.3%), respectively. PfMSP1-19 and PvMSP1-19 showed high and stable antigenicity in acute and subacute samples but declining in 1-year history samples. No cross reactivity of PfMSP1-19 and PvMSP1-19 between the two species and higher seropositivity among the asymptomatic carriers were observed. Mapping data indicated serological surveillance can detect the geographical pattern of malaria infection under low transmission setting.These findings support that PfMSP1-19 and PvMSP1-19 are suggested for serosurveillance of the malaria especially in low transmission setting for further necessary actions have to be carried out to eliminate the malaria. Under t malaria . Current malaria . Althoug malaria . AlthougUnlike many other infectious diseases, malaria antibodies against the parasite antigens are widely diverted and some may last for long duration , 6. As tAlthough malaria serological analysis was reported, there are no validated serological marker(s), no standardized high accuracy detection platform and no interpretation approach . Many stOne-year longitudinal follow-up sample collections were done in Shwegyin Township , one of the Tier I areas of Myanmar Artemisinin Resistance Containment (MARC) areas in 2015. Because of the nearby gold mines, many migrant workers were working in these areas and malaria was notified as one of the leading diseases in Shwegyin and evidence on drug resistant-malaria was documented [Required sample size has been calculated by PASS sample size software . As this study was carried out as a longitudinal observational cohort, group sequential Log-rank test has been applied assuming 90% power to detect the hazard rate 0.76 when the proportions sero-conversion in each group were 0.3 and 0.4 at a significant level  of 0.05 using a two-sided Log-rank test for the four sequential tests in each active sample collection. The required minimal sample size is 884. Loss to follow-up had to be assumed 20% in each time and the sample size had to be 1060 in minimum.The participants in this study were recruited by randomized cluster sampling in Shwegyin Township. Inclusion criteria included minimum age of 5 year, both sex and local residents in the study area for more than 3 years. As the study aimed to conduct a follow-up longitudinal study up to 1 year, the migrant or mobile populations were excluded. The person who currently showed the severe sign and symptoms of malaria was also not included in this study.At each visit, 1 mL of the whole blood was collected from the fore arm of the participants under aseptic condition. The sera was extracted from the blood and kept at − 80 °C until analysis. The remaining whole blood was used for malaria parasite detection as described below.Randomized cluster sampling method was used in this study. According to the distribution of the local health centers, two out of the four centers were randomly selected. Then, all villages were listed, from which seven villages were randomly selected. Among them, sampling interval was calculated to get the required samples in the villages i.e., at least 151 per village. Household visit or public meeting places were used to collect the samples based on the convenience of the participants. Active case detection was done in every 3 months until 1 year and passive case detection was carried out by local health personnel.For negative control, healthy sera were collected from the people of the Republic of Korea who had no exposure to malaria. A total of 96 healthy malaria-naïve individuals were collected and they were 6–13 years old with mean age of 10 years.Rapid diagnostic test (RDT) , microscopy and molecular method using nested PCR amplifying the small subunit ribosomal RNA, were used for detection of asymptomatic malaria infection in all samples collected in every 3 months as described details previously , 17.All malaria infections were treated according to the National Malaria Treatment Guideline of Myanmar. Briefly, artemisinin-based combinational therapy (ACT) with primaquine stat dose for falciparum infection, chloroquine followed by 14 days course of primaquine in vivax infection and chloroquine for malaria infection.Among the collected samples, 685 (57.95%) attended all four visits. Among them, 270 samples were selected after excluding of the possible confounding factors, i.e., two cohorts in which composed of less than 15 years-old and more than 15 years-old samples in which equal distribution of male and female, and history of malaria  data were normalized and transformed by vsn method with asinh (hyperbolic arc sine) using R-program [Data was checked and analysed by using Microsoft excel and IBM SPSS Statistics . Pearson’s Chi squared test was used to determine association with a -program . The cut-program . CirculaThe study was conducted only after receiving the ethical approval from Institutional Ethical Committee of the Department of Medical Research, Myanmar . It was also registered with ClinicalTrial.gov (Identifier: NCT02708199). Written informed consents were taken in all of the participants. Participation in this study was entirely voluntary. All detected malaria cases were treated according to the national anti-malarial treatment guideline in Myanmar. The personal information collected in this study was kept confidential.In this study, a total of 1182 local residents were recruited to study. Passive case detection was carried out by local health authorities and local malaria volunteers to detect the clinical malaria episodes. Active case detection and field blood sample collections were conducted one in every 3 months until 1 year. A total of 685 (58.0%) attended all four visits . Among the participants, 39 showed asymptomatic infection by PCR and 12 were detected vivax infection in more than one visit  and 98 healthy sera were included to analyze. Among the candidate serological markers, PvDBPII showed the highest seropositivity: 160/270 (59.3%) in at least one visit of sample collection, followed by PvMSP1-19 antigen 85/270 (31.5%), PfMSP1-19 antigen 73/270 (27.0%) and PvAMA1 antigen 65/270 (24.1%), respectively.Similarly, overall seropositivity in four times visits (V1-V4) data showed the highest rate in PvDBPII (31.7%), followed by PvMSP1-19 (17.7%), PfMSP1-19 (14.5%) and PvAMA1 (9.9%), respectively. PvDBPII showed the highest seropositivity and stable level among the study population in all four visits. Similarly, PfMSP1-19 showed the stable antigenicity among the population. However, PvMSP1-19 seropositivity showed the highest antigenicity in V4. Interestingly, PvAMA1 seropositivity was lowest in V4 Fig. .Fig. 2Sep = 0.000 and p = 0.004). However, there was no association between the seropositivity of the antigens and sex of the participants (Table When the age group (< 15 and > 15 year) was considered as a factor that may contribute to the seropositivity of the candidate antigens, only PfMSP1-19 and PvDBPII showed higher seropositivity in old age group (ts Table .Table 2Sp = 0.0025). All antigens showed high seropositivity among the asymptomatic infection. PfMSP1-19 showed 3/4 (75.0%) seropositivity among asymptomatic falciparum infection while PvMSP1-19 showed 39/51 (76.5%) seropositivity among the asymptomatic vivax infection. Similarly, PvDBPII and PvAMA1 showed 37/51 (72.5%) and 38/51 (74.5%), respectively among the asymptomatic vivax infection  showed co-seropositivity against PvMSP1-19 and PfMSP1-19 antigens. On the other hand, PvMSP1-19 seropositivity alone accounted for 57/270 (21.1%) and PfMSP1-19 seropositivity alone for 45/270 (16.7%). When PfMSP1-19 and PvDBPII seropositivity rates were compared, 49/270 (18.2%) showed co-seropositivity. While PfMSP1-19 seropositivity alone accounted for 24/270 (8.9%), PvDBPII seropositivity alone was 111/270 (41.1%). Similarly, seropositivity rates of PfMSP1-19 and PvAMA1 were compared and 13/270 (4.8%) were seropositive against both antigens. Similar seropositivity rate against the PfMSP1-19 alone  and that against PvAMA1 alone  was observed.p = 0.0034).Among the three vivax antigens, seropositivity rates of PvMSP1-19 and PvDBPII were compared as these two candidates showed the highest seropositivity rate. Only 50/270 (18.5%) were seropositive against both antigens. While 110/270 (40.7%) were seropositive against PvDBPII only, 35/270 (13.0%) of the samples showed seropositivity against PvMSP1-19 elimination area where previously occupied by both falciparum and vivax malaria. However, a limited number of the PCR confirmed asymptomatic cases were not allowed to analyze the correlation of the seropositivity rate between asymptomatic cases and non-infected samples.Moreover, MSP1-19 antibody responses were similar geographical distribution to the parasitological based detection using PCR. Both falciparum and vivax MSP1-19 antibodies were found to be stable up to 9 months after infection, then decreased significantly indicating the promising candidates to be used to assess local transmission of malaria under low transmission setting.One limitation of this study is that the strain specific antibody responses in the community against the strain specific antigen(s) were not determined. This may affect the overall seroprevalence of the antigens. Moreover, this study was conducted as a longitudinal observational study under low transmission setting for only 1 year period. To validate the evidence, a multisite cross sectional study including control site should be conducted in a different endemic setting.In summary, estimation on local transmission of malaria is a fundamental information for priority assessment, strategy making, planning, implementation and evaluation of the interventions in all malarious areas. Routine methods based on phenotypic and genotypic characteristics are not sensitive enough to estimate the local transmission of malaria in the community. Serological surveillance using the recombinant PfMSP1-19 and PvMSP1-19 may provide an alternate tool for estimation on local transmission of malaria under low transmission settings.