Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are affected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation.Plasmodium falciparum (markers of exposure) was explored. The effects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed.The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from a malaria endemic area of southern Mozambique were compared between 2 years of different MTI: 2010  and 2013 . The effect of the year on the correlations between cytokines, chemokines and growth factors and IgGs to P. falciparum-infected and uninfected subjects. Age and neighbourhood showed an effect on analyte concentrations.An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors, regardless of their immune function, had higher concentrations in 2010 compared with 2013 in The results show a different regulation of the cellular immune response in 2010 vs 2013 which could be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance. The burden of malaria disease has experienced significant changes in endemic areas in the 21st century. Between 2000 and 2015, an expansion of malaria interventions helped to reduce malaria incidence by 37% globally, and by 42% in Africa. However, between 2015 and 2017 no significant progress was made, with an estimated 219 million cases and 435 000 related deaths in 2017 [Plasmodium parasite dynamics. Thus, the expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors affected by rapid changes in MTI.Geographical and temporal changes in malaria transmission intensity (MTI) can affect disease burden and Plasmodium falciparum infection. NAI is suggested to be comprised of two main components: (i) an anti-parasite component, resulting in control of parasite replication and parasite clearance, which takes years to be acquired and is never sterilizing [In endemic areas of Africa, naturally acquired immunity (NAI) to malaria is developed with age and exposure to rilizing , 3; and rilizing , 5.Tolerance is a less understood phenomenon. From the immunological perspective, it is defined as any endogenous mechanism by which a potentially injurious immune response is prevented, suppressed, or shifted to a non-injurious response . In malaEpidemiological and immunological data suggest that anti-parasite immunity and tolerance wane in the absence of continuous exposure to the parasite , and chaP. falciparum appeared to be reduced [P. falciparum would result in a lesser development of NAI in children, and a possible partial loss of previous immunity and tolerance in the older population if exposure was very low or discontinued, which may enhance malaria pathogenesis.Temporally, immune adults who migrate to non-endemic areas are at significant risk of contracting malaria upon return to an endemic area, even increased susceptibility to severe malaria . Previou reduced . Thus, aP. falciparum-specific cytokine responses have been associated with protection against clinical malaria, including interferon gamma (IFN-γ) [Cytokines, chemokines and growth factors mediate cellular responses and are responsible for the symptoms and pathological alterations during disease. The outcome of infection depends on the regulation of pro-inflammatory and anti-inflammatory responses, leading to protection or immunopathology . Thus, b (IFN-γ) –17, inte (IFN-γ) , 19. Oth (IFN-γ) , 20. Ser (IFN-γ) .P. falciparum infected but also in uninfected subjects. This could be indicative of a loss of immune-tolerance after years of decline in MTI, and a re-establishment of the tolerance as a consequence of a more continuous exposure due to a rise in MTI starting before 2013. The possibility of cytokines as surrogates of loss of parasite tolerance upon changes in MTI deserves further investigation.In this study, changes in cellular immune mediator profiles in 2 years of different MTIs were assessed in individuals of all ages from a malaria endemic area of southern Mozambique. To address this, a comprehensive panel of 30 cytokines, chemokines and growth factors, several of them known to vary upon malaria infection and/or exposure , 24, 25,P. falciparum malaria is perennial and of moderate intensity, with two different seasons, a warm and rainy season from November to April, and a cool and dry season the rest of the year [http://chg.geog.ucsb.edu/data/chirps/). The cumulative rainfall levels between the months of September 2009 and January 2010 was 310.76 mm, and doubled to 625.26 mm between the months of September 2012 and January 2013. Individuals of all ages were selected by random age-stratified sampling from the demographic surveillance system census and were invited to participate in the study. Weighted sampling was done according to the following age groups: Infants < 1 year; 1 ≤ 2 years; 2 ≤ 3 years; 3 ≤ 4 years; 4 ≤ 5 years; 5 ≤ 10 years; 10 ≤ 20 years; 20 ≤ 40 years; 40 ≤ 60 years; ≥ 60 years. Participants were recruited from six different neighbourhoods: Malavele, Manhiça and Maragra, historically considered of low MTI; and Ilha Josina, Palmeira and Taninga, of moderate MTI [Plasmodium parasitaemia according to standard, quality-controlled procedures [This study was developed in the context of yearly cross-sectional surveys performed to monitor changes in malaria burden and MTI in the Manhiça District, Maputo Province, in southern Mozambique, which started in 2010 and are still on going. The characteristics of the study area have been described in detail elsewhere . The clithe year . The crorate MTI . A standocedures .P. falciparum parasites in blood detected qPCR, together with fever or reported fever during the previous 24 h. Febrile infections with parasites also detected by microscopy were considered clinical malarias. Anemia was defined as haemoglobin < 11 g/dL. Participants presenting with parasitaemia, anaemia, fever or history of fever in the previous 24 h were treated according to standard procedures. All malaria infections were treated with the first-line anti-malarial treatment (Coartem), and anaemia cases received ferrous sulphate, according to national guidelines. Participants presenting signs/symptoms of severity were transferred to the Manhiça District Hospital.Haemoglobin was measured using HemoCue portable devices . A blood aliquot was collected in an EDTA microtainer and plasma separated by centrifugation and cryopreserved at − 80 °C for immunological analyses. Blood was also collected onto filter paper for IgG quantification by ELISA , and forPlasma samples analysed in this study were from 377 participants from the surveys performed in 2010  and 2013 . Years 2010 and 2013 were defined as lower and higher MTI periods, respectively, based on the trends reported in Mozambique, with a decrease in malaria prevalence from 2007 to 2011, and an increase since 2012 , 33.All plasmas from infected individuals (161 participants qPCR positive) and the plasmas from 216 non-infected participants (qPCR negative) were analysed separately to assess cytokine profiles in 2010 and 2013 during an infection and at baseline, respectively. The plasmas from non-infected individuals were randomly selected from the surveys, balancing between years and stratifying by specific age groups , with the last two groups enriched to have a sample size powered to address older age effects.The Cytokine Human Magnetic 30-Plex Panel from Life Technologies™ was used to measure the concentrations (pg/mL) of the following cytokines, chemokines and growth factors in plasma: epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF), interferon (IFN)-α, IFN-γ, interleukin (IL)-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40/p70), IL-13, IL-15, IL-17, IFN-γ induced protein (IP-10), monocyte chemoattractant protein (MCP-1), monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation normal T cell expressed and secreted (RANTES) and eotaxin. This panel has been used in previous studies showing several analytes varying upon malaria infection and/or exposure , 24, 25.® 100/200 instrument and analysed in xPONENT® software 3.1. The concentration of each analyte was obtained by interpolating the median fluorescent intensity (MFI) (after blank MFI subtraction) to a 5-parameter logistic regression curve automatically calculated by xPONENT® software. Any value below the lower limit of detection (mean of blanks + 2 standard deviations) was assigned half the expected concentration at the low limit of quantification for that analyte.Twenty-five microlitres of all plasmas were tested in single replicates applying a modification of the manufacturer’s protocol that implies using half the volume of each reagent except for the washing buffer ; this moAntibody data obtained in a separate study of seroconversion rates performed in the context of the Manhiça cross-sectional surveys were analysed in relation to the cytokine data. Briefly, 2.5 mm disks of dried blood (≈ 1.5 µL of blood) were cut from filter paper spots and incubated overnight at room temperature (RT) with 150 mL of PBS/0.05% Tween 20/0.01% sodium azide (w/v) .42) (3D7 strain) or apical membrane protein-1 (AMA-1) (FVO strain) produced at the Walter Reed Army Institute of Research , at 0.5 mg/mL of carbonate buffer. PBS with 0.05% Tween (PBS-T) was used to wash plates between incubations. Plates were blocked with 1% skimmed milk powder  in PBS-T (blocking buffer) for 3 h at RT. Reconstituted antibodies were transferred to the ELISA plates  and incubated overnight at 4 °C. HRP-conjugated rabbit anti-human IgG  was applied at 1/5000 in PBS-T and incubated for 3 h at RT. OPD development substrate was applied and incubated in the dark at RT for 20–25 min, stopping development with the addition of 2 M H2SO4. Plates were read on Bio-Tek ELx 50 plate reader using KC Junior software package . Normalized optical density (OD) was calculated as the mean background-adjusted OD divided by the mean of a hyperimmune plasma. The same standard dilution was used for normalizing each plate in the study. Test samples were assayed in duplicate and included in the analysis if the coefficient of variation was less than 50% for all values greater than 0.1 OD.Reconstituted sera were stored at − 20 °C until use. The ELISA assay was performed as previously described . High-bi10 transformed for further analysis.Plasmas from malaria-infected and non-infected volunteers were analysed separately. The studied population was categorized into 6 age groups  according to commonly observed immunological patterns. Demographic continuous variables were analysed using the non-parametric Wilcoxon rank-sum test. Comparisons between groups for categorical variables were done using Fisher’s exact test. Concentrations of cytokines, chemokines, growth factors, parasite densities and IgG levels (OD) were logComparison of cellular analyte concentrations between two or more groups was performed through Wilcoxon rank-sum test or Kruskall–wallis test, respectively. Comparisons of IgG levels between years were performed by Wilcoxon rank sum test with continuity correction for all age groups together or into each age category separately. Trends of IgG levels along age groups were assessed by Spearman correlations (p-trend). The correlations of IgG levels and analyte concentrations were assessed with univariable linear models, with IgG levels as independent variable, separate for each analyte. The effects of year, age group, neighbourhood, sex and parasite density (predictors) on analyte levels (outcome) were assessed through univariable and multivariable (adjusted models) linear regressions, separate for each analyte. Interaction tests were performed to determine if there was an interaction of age group, neighbourhood, sex, parasite density and antibody levels with year on the analyte levels. When interactions were statistically significant, a stratified analysis was performed.P-values were adjusted for multiple testing to control the false discovery rate using the Benjamini–Hochberg approach in each study endpoint separately  except for the comparison of antibody levels between years into age categories for which the Holm method was used. In all endpoints evaluating cytokine associations of this exploratory study, the Benjamini–Hochberg approach was used because it has more power than family-wise-error rate methods, such as Bonferroni or Holm, and allows identifying as many significant associations as possible while incurring a relatively low proportion of false positives [ggplot2 package [compare Groups package was used for computing p-trend values [fmsb package [radarchart function. The reshape and dyplr packages were used for data manipulation purposes [ReporteRs package for exporting data tables in Microsoft Word documents [All p-values were considered statistically significant when < 0.05. ositives . For theositives . The ggp package  was usedd values . The fms package  was usedpurposes  and the ocuments .P. falciparum infection in 2010 and 2013 were 1.1% and 5.2% by microscopy, and 12.6% and 23.9% by qPCR, respectively; and the numbers of febrile infections were 15 and 43, being 3 and 18 clinical malarias, respectively  individuals were tested by microscopy and 970 (99%) by qPCR; while 944 (96.3%) and 808 (82.4%) individuals were tested by microscopy and qPCR, respectively, in 2013. Weighted prevalence of 42 were higher in 2013 compared to 2010 (p < 0.001 for both IgGs) mirroring the MTI trends between 2010 and 2013, and increased with age within each cross-sectional , reflecting the continuous exposure to P. falciparum infection  and parasitaemia [Recent studies suggest that repeated exposure to olerance , which molerance , αβ T ceolerance –52, B ceolerance , and myeolerance  that shoolerance , 56. A rsitaemia . Accordisitaemia .P. falciparum and an increase of malaria adverse consequences after the decline of MTI [P. falciparum infection at the time of the survey, suggesting that the rate of previous exposures could shape the basal immunological status. This difference in the basal immune system agrees with the observation by Tran et al. [Plasmodium infection. This study also suggests that previous exposure to malaria, and possibly other pathogens or commensals, shape the basal immune system.Another study showed diminished cytokine response with age in children, in part probably due to increased exposure, which could also be indicative of tolerogenic mechanisms . A decree of MTI . Interesn et al.  of a marP. falciparum [Plasmodium species susceptibility [In the same line, a previous study by our group in semi-immune African adults, migrants and European travellers, showed that naïve adults had stronger cytokine responses upon infection than semi-immune adults; and that migrants, in the absence of continuous exposure, presented higher concentrations of cytokines and chemokines than semi-immune individuals, but lower than individuals with a first infection . There, lciparum . Similarlciparum , 47, 62.tibility , 64 and tibility , being dtibility . This imDespite the lower cellular immune mediators observed in 2013, infected individuals showed higher levels of several cytokines and chemokines compared to the uninfected ones, differences that were not observed in 2010; among them, the pro-inflammatory cytokines IFN-α, IL-1β and IL-6, the chemokines IL-8, MCP-1, MIP-1α and MIP-1β and the growth factor VEGF. This may be related to the lower basal levels of cellular immune mediators in 2013 compared to 2010. In 2010, basal levels of cellular mediators were so high that infections might have not been able to induce further increases; whereas in 2013, cytokine, chemokine and growth factor basal levels were low and infections increased them significantly, though still below the basal levels from 2010. This observation supports the hypothesis of a more controlled, non-harmful cellular immune response upon re-infection in 2013.Despite the overall decrease in cellular immune mediators in 2013, a higher prevalence of clinical malaria (12%) was registered compared to 2010 (3%) among the infected individuals, similar to the prevalence in the surveys (18/162 vs 3/106). However, the total number of clinical malaria cases was low, and clinical malaria depends not only on tolerogenic responses but also on anti-parasite immunity. During lower MTI periods there is a delay in the acquisition of immunity in younger children and a reduction in premunition in older children and adults; thus, in the context of a sudden increase of MTI, anti-parasite immunity may be acquired slower than tolerogenic responses. It is likely that a higher number of clinical cases would have been observed in 2013 had some level of tolerance not been developed.As expected, among the infected individuals the level of parasitaemia affected several analytes, some of them similar to previous reports , 66. IntH1 cytokines IL-2, IL-12 and IL-2R and the TH1 related IL-15 decreased continuously with age; while eotaxin, IP-10 and the TH2 cytokines IL-13, IL-4 and IL-5 presented a U-shape, i.e. higher levels in children and the elderly and lower levels in young adults. Previous studies have shown that some cytokines increase in the elderly [Age-related differences in immunity are suggested to also explain the different susceptibility to malaria disease in children vs adults  independ elderly , while o elderly , 73, but elderly .Previous studies have also shown that the severity of malaria infection differs between males and females , with meP. falciparum IgG levels in 2013 compared to 2010 across all age groups and negative correlations with cellular immune mediators suggests that P. falciparum exposure could be driving the changes described. Future studies should have a longitudinal design and comprise different MTI periods to address the role of cellular immune mediators as surrogates of MTI and tolerance/susceptibility to malaria. In addition, controlling for other factors influencing the basal immune system would also be important. Understanding the mechanisms of longevity of immunity and tolerance may help in developing approaches to promote long-lasting anti-disease immunity, even after sustained interrupted exposure, which could be used in areas of low MTI and in elimination campaigns.There are some limitations in this study that could have affected the results. First of all, the different storage times for plasma samples collected in 2010 vs samples collected in 2013, which may have differently affected the cytokine concentrations. However, storage time tends to decrease the concentration of most of the cytokines . TherefoP. falciparum infected and non-infected individuals, which could be related to a loss of immune tolerance when MTI declines and a rapid re-establishment of immune tolerance after an increase in P. falciparum exposure. This finding warrants further investigations on plasma cellular immune mediators as potential surrogate candidates of MTI and the associated host susceptibility and tolerance.Cytokine, chemokine and growth factor profiles varied between years of different MTIs in Additional file 1: Table S1. Monthly mean rainfall in the study area in the 5 months previous to the 2010 and 2013 surveys. Table S2. Effect of infection on analyte concentrations and the interactions with year. Table S3. Interaction of age with year on analyte levels in infected and uninfected individuals. Table S4. Effect of sex on analyte concentrations and the interactions with year in infected and uninfected individuals. Table S5. Interaction of neighborhood with year on analyte levels in infected and uninfected individuals.Additional file 2. Differences in cellular immune mediator concentrations between 2010 (lower MTI) and 2013 (higher MTI) in P. falciparum-infected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in infected subjects recruited in 2010 and in 2013. Levels between both years were compared by Wilcoxon rank-sum test and p-values were adjusted for multiple testing by Benjamini–Hochberg approach.Additional file 3. Differences in cellular immune mediator concentrations between 2010 (lower MTI) and 2013 (higher MTI) in uninfected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in uninfected subjects recruited in 2010 and in 2013. Levels between both years were compared by Wilcoxon rank-sum test and p-values were adjusted for multiple testing by the Benjamini–Hochberg approach.Additional file 4. Correlations of VEGF, MCP-1 and IFN-α with P. falciparum antibody levels stratified by year. Data are presented in scatter plots with trend lines, with analytes concentrations in the Y axes and antibody levels in the X axes. R2 and p-values were obtained through linear regression models. Only analytes that had a statistically significant interaction with year for the correlation with antibodies are shown.Additional file 5. Cellular immune mediator concentrations in 2010 (low MTI) and 2013 (high MTI) stratified by age group. Box plots representing the median and interquartile range of analytes concentrations (log10 pg/mL) in P. falciparum infected (a) and uninfected (b) subjects. Only analytes in which age and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.Additional file 6. Cellular immune mediator concentrations in 2010 (low MTI) and 2013 (high MTI) stratified by sex. Box plots representing the median and interquartile range of analyte concentrations (log10 pg/mL) in P. falciparum infected (a) and uninfected (b) subjects. Only analytes in which sex and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.Additional file 7. Differences in cellular immune mediator concentrations between areas in P. falciparum infected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in infected subjects stratified by neighborhood. Levels between areas have been compared by Kruskal–Wallis test.Additional file 8. Effect of parasitema on IL-10 and GM-CSF concentrations stratified by year. Scatter plots with trend line representing the distribution of analytes concentration by parasitemia stratified by year. Only analytes in which parasitemia and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.


Plasmodium vivax infections are now the major challenge for malaria elimination in this country. Since children have higher risk of contracting malaria, this study investigated the dynamics of Plasmodium spp. infections among children including the associated risk factors of residual P. vivax burden.Following the scale-up of intervention efforts, malaria burden has decreased dramatically in Solomon Islands (SI). Submicroscopic and asymptomatic Plasmodium spp. infections and illness. Parasites were detected by quantitative real-time PCR (qPCR) and genotyped. Comprehensive statistical analyses of P. vivax infection prevalence, molecular force of blood stage infection (molFOB) and infection density were conducted.An observational cohort study was conducted among 860 children aged 0.5–12 years in Ngella . Children were monitored by active and passive surveillances for Plasmodium vivax infections were common , whereas Plasmodium falciparum infections were rare (0.3%) but persistent. Although children acquire an average of 1.1 genetically distinct P. vivax blood-stage infections per year, there was significant geographic heterogeneity in the risks of P. vivax infections across Ngella . Malaria incidence was low (IR: 0.05 episodes/year-at-risk). Age and measures of high exposure were the key risk factors for P. vivax infections and disease. Malaria incidence and infection density decreased with age, indicating significant acquisition of immunity. G6PD deficient children (10.8%) that did not receive primaquine treatment had a significantly higher prevalence  and increased risk of acquiring new bloodstage infections , underscoring the importance of anti-relapse treatment.P. vivax infections, alongside sporadic P. falciparum infections. Implementing an appropriate primaquine treatment policy to prevent P. vivax relapses and specific targeting of control interventions to high risk areas will be required to accelerate ongoing control and elimination activities.Residual malaria transmission in Ngella exhibits strong heterogeneity and is characterized by a high proportion of submicroscopic and asymptomatic The online version of this article (10.1186/s12936-019-2727-9) contains supplementary material, which is available to authorized users. Plasmodium spp. infections becoming more heterogeneous within countries, between regions, among villages and even within households , p = 0.035; Additional file aCoefficient: 0.91, CI95 [− 1.37 to − 0.45], p < 0.001; Additional file P. vivax infection density.Reduction of parasitaemia was observed to be significantly associated with IRS univariately , (2) presumptive malaria , and (3) potential malaria (febrile and positive detection of Plasmodium spp. by qPCR). Since the qPCR TaqMan detection method is a highly sensitive and more accurate as compared to RDT or light microscopy, all malaria species were corrected based on qPCR species typing to reduce the possibility of misinterpreted RDT results at field sites . In Solomon Islands, only patients with RDT or light microscopy-confirmed malaria receive anti-malarial treatment. However, the infected species is not generally recorded in the patient’s health book. The combination of confirmed (Definition 1) and presumptive (Definition 2) malaria is thus most likely to reflect the true overall burden of clinical malaria episodes reliably.Due to unforeseen staffing and reporting problems, the quality of passive case detection was highly variable during follow-up. To overcome this issue, three distinct definitions of malaria episodes were used: (1) P. vivax accounted for most of the clinical infections . The PATR  and the number of new infections acquired (molFOB: 2.31 (cube root-transformed), CI95 [1.77–3.02], p < 0.001) were associated with increased risk of confirmed and presumptive malaria (i.e. definitions 1 and 2), while age  decreased the risk. The same association of PATR and molFOB with increased risk were also found for confirmed cases only (Definition 1) and all confirmed, presumptive and potential cases (Definition 1–3), whereas the decreased incidence with age was found only for the latter . Although the occurrence of P. falciparum was observed to be sporadic in Ngella, four individuals with repeated P. falciparum infections during follow-up visits were residing in the Bay region . Of these four individuals, only one had a febrile episode once. Gametocyte detection by qPCR was not conducted but no P. falciparum gametocytes were detected during the three ACD visits’ microscopy examinations.Only 27 Plasmodium spp. infection dynamics in Solomon Islands. As recognized in earlier cross-sectional studies in Ngella [Plasmodium spp. infections detected from the cohort were submicroscopic and asymptomatic P. vivax infections. This is in line with the general trend of an increasing proportion of submicroscopic Plasmodium spp. infections as malaria transmission decreases [Plasmodium falciparum infections which was previously thought to be nearly eliminated in Ngella [This study presents the first prospective cohort study focusing on n Ngella , and in n Ngella  and Isabn Ngella , most Plecreases , 7, 41. n Ngella  remainedP. vivax accounted for most cases. This contrasts with the annual national malaria report by the National Vector-borne Disease Control Programme, Ministry of Health and Medical Sciences of Solomon Islands, which reported that approximately half of the malaria incidence in Central Islands was due to P. falciparum  [P. falciparum episodes were more common among cases treated at the health facilities as compared to those diagnosed during active case detection. However, it has been previously recognized that the accuracy of clinical malaria case reporting in primary health care facilities in Solomon Islands is often relatively poor [P. vivax increased from 33% in 2008 to 84% in 2013 [P. falciparum and P. vivax to the burden of clinical malaria in Ngella, data of this study make it clear that the very high burden of asymptomatic, low-density P. vivax infections is the key challenge to future malaria elimination in Ngella.The incidence of clinical malaria was low among cohort children and when species determination was possible, ctively) . Althougely poor . In addi in 2013 . While fP. vivax infections were found throughout the study area, a very high degree of spatial heterogeneity in transmission was observed among the study villages. Prevalence varied up to 40-fold (range 1.2–47.4%) while the molFOB varied up to 90-fold (range 0.05–4.57) between villages that were not more than 5–25 km apart. Such strong variation in malaria transmission levels at small geographical scales has been described before in both moderate to high transmission areas [Although on areas , 45 and on areas , 47. Whion areas , 48.P. vivax infections varied during follow-up, seasonal patterns, while present, were modest. After a small drop in prevalence from ACD1 to ACD2, which was due to the fact that children with LM-positive infections detected at baseline (n = 52) received antimalarial treatments, prevalence and incidence of infections peaked in August (i.e. ACD 3) and then decreased during further follow-up. This coincided with a significant decrease in infection densities during the rainy season , indicating that infection during the rainy season could be due to relapsing rather than newly acquired P. vivax infection. According to a previous environmental study in Northern Guadalcanal (west of Ngella), rainfall has a negative effect on Anopheles farauti larvae numbers as heavy rainfall flushes away the breeding soil ridges by the stream [Although the burden of e stream , thus exe stream –52, as pP. vivax infection prevalence among cohort children. Distribution of LLINs is undertaken every 3 years as the main vector control intervention in Solomon Islands and a 1.5 people/net ratio/population was reported for 2013 in Central Islands province [P. vivax blood-stage infections. It remains to be confirmed if this is a consistent effect. In Solomon Islands, IRS is considered to be a complementary intervention to LLINs [P. vivax infections and lower parasite densities. This supports the continued use of IRS not merely as a complementary intervention in high risk areas but also in elimination areas where interruption of Plasmodium spp. transmission is the ultimate goal [Despite more than two-thirds of Anopheline mosquitoes in Solomon Islands bites outdoor and early before 21.00 h) , 53, LLI.00 h , 5province . High LLto LLINs  and onlyate goal .P. vivax. Although the prevalence of infections, the incidence of new infections, and thus exposure (molFOB), all increase with age and exposure is a major risk predictor of clinical P. vivax malaria and parasite density, the incidence of clinical P. vivax episodes itself is not only very low but also decreases with age as does the mean density of P. vivax infections. These observed trends are signs of significant natural acquisition of clinical immunity against P. vivax. Similarly rapid acquisition of immunity to P. vivax was not only observed in a high transmission setting in PNG, where children start acquiring immunity to P. vivax in their 2nd year of life [P. vivax illness is virtually absent by age five [P. vivax is largely due to the higher molFOB [Despite relatively low levels of transmission, children in Ngella acquire substantial levels of clinical immunity to  of life  and clinage five , but alsage five . Althougr molFOB , 58 drivr molFOB .P. vivax infection in children with PATR suggests that treatment policy for P. vivax malaria in Solomon Island is sub-optimal. Although primaquine radical cure is part of the Solomon Islands’ standard treatment guidelines, it is often not prescribed due to the lack of G6PD testing and when prescribed, it is taken by the patient without supervision. A recent analysis of > 60,000 patients in Indonesian Papua found that unsupervised PQ was associated with only a minimal reduction in the risk of clinical P. vivax recurrences in the next 12 months [P. vivax blood-stage infections [P. vivax infections in children with PATR underlines that PQ treatment was either not given or not successful in removing hypnozoites from the liver, leading to future relapses. This effect is likely to be confounded by significant inter-individual heterogeneity in P. vivax infection risk as indicated by the high degree clustering of P. vivax infections among individuals. Having had a previous P. vivax infection is thus not only an indication that the child may carry further hypnozoites in its liver, but also indicates children with past exposures are significantly more likely to be exposed to new infectious mosquito bites in future. While molFOB is a marker for current exposure to P. vivax infection, PATR is thus also a marker of (recent) past exposure.Having suffered an episode of malaria in the previous 12 months  was found to be a strong risk predictor for infection prevalence, infection density, new infection, and malaria incidence. A higher risk of 2 months . As relafections , 60, theP. vivax is further highlighted by the observation that G6PD deficient children had a higher prevalence of P. vivax infection and increased risk of acquiring new infection. This observation conflicts with previous reports that G6PD deficiency confers protection against severe P. falciparum malaria and clinical P. vivax infections and P. vivax parasitaemia [P. vivax parasitaemia and thus clinical disease, the higher risk among G6PD deficient children is more likely due to the fact that these children represented a group that could not be treated with PQ, and therefore only received blood stage treatment. This again underlines the importance of providing efficient anti-relapse treatment and the need to find alternative treatment or prevention strategies for G6PD-deficient individuals.The important contribution of relapses to the burden of sitaemia –63. WhilP. falciparum prevalence that are sporadic and largely asymptomatic occurring throughout the study areas and follow-up period is intriguing. Even more so as the National Vector-borne Disease Control Programme [P. falciparum  carry the same msp2 FC27 allele as the five P. falciparum infections detected in the previous year’s cross-sectional survey [P. falciparum populations may persist in Ngella, similar to that previously observed in elimination province Temotu [P. falciparum population genetics in Ngella and comparisons to neighbouring populations, as well as a detailed assessment of clinical isolates compared to asymptomatic infections will be required to better understand the nature of residual P. falciparum transmission in Ngella. In particular, it will be essential to understand what proportion and what infections are imported from neighbouring islands, which have much higher endemicity.Observation of low rogramme  reports rum see “” above. l survey . Likewise Temotu . FurtherP. vivax infection and the sporadic occurrence of P. falciparum in Ngella, largely asymptomatic and sub-microscopic. The Solomon Islands malaria control programme currently relies almost entirely on vector control measures (such as LLINs and IRS) and management of clinical cases. While these approaches which have jointly achieved an overall 95% reduction in malaria cases in the last two decades remain effective, they are unlikely to achieve elimination of malaria transmission in Solomon Islands on their own. Alternative approaches that are better able to identify and target these silent reservoir Plasmodium spp. infections will be required. Higher sensitivity diagnosis tools such as loop-mediated isothermal amplification (LAMP) or PCR may be required to track these infections, and using active surveillance method rather than relying solely on passive surveillance method may be required to aid malaria elimination in Solomon Islands [P. falciparum transmission, further progress with eliminating P. vivax will crucially depend on attacking the hidden, hypnozoite reservoir [P. vivax infected individuals [P. vivax infections.This study has confirmed the presence of a significant burden of  Islands –67. Whil Islands , 68, 69 eservoir . A firstividuals . FurtherAdditional file 1: Figure S1. Schematic overview of the paediatric cohort study in Ngella, Solomon Islands.Additional file 2: Table S1. Characteristics of children that met inclusion criteria vs excluded children.Additional file 3: Table S2. Retention of cohort participants and malaria prevalence during active case detection.Additional file 4: Table S3. Detailed demographic characteristics of 860 children included in the cohort study.Additional file 5: Figure S3. Frequency of Plasmodium vivax infections among cohort participants (n=860).Additional file 6: Figure S4.Plasmodium falciparum genotypes variants that were successfully characterized by msp2, TA81 and Polyα markers.Additional file 7: Figure S5. Asymptomatic and submicroscopic Plasmodium spp. infections during ACD1, ACD8 and ACD11 visits.Additional file 8: Table S5. (i) Association between selected risk factors and Plasmodium vivax infection density by qPCR detection of the 256 participants with at least 1 P. vivax infection during cohort and household spraying data. (ii) Detailed estimates for village and visit interval effects for (A) Plasmodium vivax infection prevalence, (B) new P. vivax infection, and (C) P. vivax infection density by qPCR detection.Additional file 9: Table S4. Univariate analysis of risk factors associated with (A) Plasmodium vivax infection, (B) new P. vivax infection and (C) P. vivax density by qPCR detection.Additional file 10: Figure S2. Prevalence of Plasmodium vivax infection in villages during ACD visits.Additional file 11: Table S6. Risk factors for incidence of clinical episodes: (A) Confirm malaria, presumptive malaria and potential malaria, (B) Confirm malaria.


Plasmodium knowlesi causes severe and fatal malaria, and incidence in Southeast Asia is increasing. Factors associated with death are not clearly defined.P. knowlesi cases was conducted. Case fatality rates (CFRs) during 2010–2017 were calculated using incidence data from the Sabah Department of Health.All malaria deaths in Sabah, Malaysia, from 2015 to 2017 were identified from mandatory reporting to the Sabah Department of Health. Case notes were reviewed, and a systematic review of these and all previously reported fatal P. knowlesi. Median age was 40  years; 4 cases (67%) were male. Three (50%) had significant cardiovascular comorbidities and 1 was pregnant. Delays in administering appropriate therapy contributed to 3 (50%) deaths. An additional 26 fatal cases were included in the systematic review. Among all 32 cases, 18 (56%) were male; median age was 56  years. Cardiovascular-metabolic disease, microscopic misdiagnosis, and delay in commencing intravenous treatment were identified in 11 of 32 (34%), 26 of 29 (90%), and 11 of 31 (36%) cases, respectively. The overall CFR during 2010–2017 was 2.5/1000: 6.0/1000 for women and 1.7/1000 for men (P = .01). Independent risk factors for death included female sex , and age ≥45 years .Six malaria deaths occurred in Sabah during 2015–2017, all from Earlier presentation, more rapid diagnosis, and administration of intravenous artesunate may avoid fatal outcomes, particularly in females, older adults, and patients with cardiovascular comorbidities. Plasmodium knowlesi malaria in Sabah, Malaysia in 2015–2017 and 26 previously reported deaths were reviewed. Associated factors included female sex; age ≥45 years; cardiovascular-metabolic comorbidities; and delays in presentation, diagnosis, and treatment.Six deaths from  Plasmodium knowlesi, a zoonotic parasite of long-tailed and pig-tailed macaques, is an important cause of human malaria in Southeast Asia [P. knowlesi now the most common cause of malaria in Malaysia [ast Asia . In someMalaysia  and partMalaysia .P. knowlesi is at least as high as from Plasmodium falciparum in coendemic areas [Risk of severe disease from ic areas , 9, occuic areas , 11, andic areas . Indepenic areas , 17; howPlasmodium knowlesi case fatality rates (CFRs) in Sabah during 2010–2017 were calculated using previously reported mortality and incidence data [To delineate factors associated with knowlesi malaria mortality, we reviewed malaria deaths in Sabah, Malaysia, from 2015 to 2017, and conducted a systematic review of all previously reported fatal cases. nce data , 18.Plasmodium species confirmation by polymerase chain reaction (PCR).All malaria deaths in Sabah from 2015–2017 were identified from the Sabah Department of Health. In Sabah, hospital admission and notification to the Department of Health are mandatory for all malaria cases, as is reporting of deaths. Since 2015 it has also been mandatory for a blood sample from all malaria cases to be sent to the state public health laboratory for Case notes were retrieved from relevant health facilities and reviewed. Approval was obtained from the ethics committees of the Ministry of Health, Malaysia, and the Menzies School of Health Research, Australia.We searched Medline, PubMed, and Science Direct for articles published up to August 2018, containing the words “knowlesi AND death,” “knowlesi AND fatality,” or “knowlesi AND outcome,” including synonyms and Medical Subject Heading terms. Relevant references cited within these articles were also reviewed.Plasmodiumknowlesi CFRs in Sabah were calculated from 2010 to 2017 using malaria incidence and fatality data from the Sabah Department of Health [f Health , 18. MalP. knowlesi monoinfections. Baseline demographic and clinical details are shown in P. knowlesi in 2 (33%) cases; the others were reported as Plasmodium malariae (n = 2) and P. falciparum (n = 2). Median time from admission to death was 29  hours.Six malaria deaths were reported to the Sabah Department of Health during 2015–2017, all confirmed by PCR as P. knowlesi, with 22 400 parasites/µL. Oral artemether-lumefantrine and doxycycline were given, and tertiary hospital transfer was arranged for IV artesunate; however, the patient had a cardiac arrest while awaiting transfer. Admission blood cultures were negative.A 32-year-old pregnant woman at 35 weeks’ gestation (G6P5) presented with reduced fetal movement, 14 days of fever, and 2 days of intermittent abdominal pain and dyspnea. She was hypotensive, tachycardic, hypoxic, and tachypneic. She had hyponatremia, thrombocytopenia, and acute kidney injury (AKI). She was intubated, ventilated, and given IV ceftriaxone, sodium bicarbonate, and ionotropic support. A chest radiograph showed diffuse interstitial infiltrates consistent with acute respiratory distress syndrome (ARDS). A blood film taken 4 hours after admission was reported as P. knowlesi with 71 939 parasites/µL. Chest radiography showed generalized interstitial infiltrates. He was diagnosed with severe malaria and commenced on IV artesunate, and a tertiary hospital transfer was arranged; however, he arrested en route. This case has been previously documented [A 50-year-old male Indonesian palm-oil plantation worker presented to a district hospital with 1 week of fever, rigors, headache, epigastric pain, and diarrhea. Physical examination revealed hepatomegaly (3 cm), but was otherwise unremarkable. He had severe AKI, hyponatremia, and thrombocytopenia. A blood film was reported as cumented .P. malariae with 2285 parasites/µL. It was later noted that an admission blood film had been reported as “P. falciparum 3+.” No antimalarial treatment had been given. Blood cultures were negative.A 37-year-old woman with a history of gestational hypertension presented to a district hospital with 4 days of fever, rigors, epigastric pain, dizziness, and vomiting. She was hypotensive, tachycardic, and had epigastric tenderness. The initial diagnosis was dyspepsia, and she received ranitidine, prochlorperazine, and fluid resuscitation. AKI was noted 2 hours after admission, and IV ceftriaxone was initiated for presumed sepsis. Twelve hours after hospitalization, she became tachypneic, with reduced consciousness , and severe metabolic acidosis . She underwent intubation, ventilation, and hemodialysis and received IV sodium bicarbonate and imipenem. The patient deteriorated further on day 2, with transaminitis , metabolic acidosis, and coagulopathy  requiring fresh frozen plasma. The patient arrested 43 hours after admission. Blood films taken prior to death were reported as P. malariae 1+” with AKI, hyponatremia, and thrombocytopenia also noted. Oral artemether-lumefantrine was given. Twenty-two hours after admission, the patient had hemoptysis with tachypnea and hypoxia. A chest radiograph showed generalized interstitial infiltrates. A diagnosis of severe P. malariae with ARDS was made and IV artesunate was initiated. After further hemoptysis, a bedside ultrasound showed pleural and pericardial effusions and noninvasive ventilation was initiated. At 48 hours, massive hemoptysis occurred. He required intensive care unit admission, intubation, ventilation, inotropic support, hemodialysis for metabolic acidosis and uremia, and red cell and fresh frozen plasma transfusions. Echocardiography (ECG) showed severe mitral stenosis. Admission blood cultures were negative. He died on day 4, with cause of death recorded as severe malaria with pulmonary hemorrhage and underlying mitral stenosis.A 32-year-old Filipino man presented with 5 days of fever, cough, and coryzal symptoms. He was tachycardic, but physical examination was otherwise unremarkable. He was given fluids and acetaminophen (paracetamol). A blood smear was reported as “Plasmodium species was not specified. On admission he was tachycardic, hypotensive, and hypoxic. ECG showed atrial fibrillation at 160 beats per minute. He received IV artesunate, fluid resuscitation, and high-flow oxygen. He was hyponatremic, with AKI, hyperglycemia, and a compensated metabolic acidosis. A blood film was reported as “P. malariae 4+.” He required intubation, hemodialysis, IV insulin, digoxin, and inotropic support. Seven hours postadmission, ECG showed cardiac ischemia, and at 15 hours the patient arrested. Admission blood cultures were negative. Cause of death was recorded as (1) malaria with hyperparasitemia, and (2) decompensated cardiac failure, with likely underlying cardiomyopathy.A 42-year-old fisherman with morbid obesity, obstructive sleep apnea, and congestive cardiac failure was referred to a district hospital from a peripheral clinic with 9 days of fever, and 3 days of dyspnea, epigastric pain, and vomiting. The clinic blood film was reported as “complicated malaria with hyperparasitemia”; P. falciparum.” He was anemic, hyperbilirubinemic, and hyponatremic, with AKI and metabolic acidosis. He was commenced on ionotropic support and transferred to a tertiary public hospital for IV artesunate. There he also received ceftriaxone; hydrocortisone; insulin infusion for hyperglycemia; cardioversion for ventricular tachycardia; and, later, intubation, ventilation, and continuous venovenous hemofiltration. A blood film on day 1 was reported as P. knowlesi with 246 100 parasites/µL. On day 2, he had worsening metabolic acidosis , coagulopathy , and transaminitis , with a bilirubin of 247 μmol/L, and was anuric. He died on day 3, with cause of death recorded as severe knowlesi malaria with multiorgan failure.A 58-year-old Caucasian expatriate man with a history of ischemic heart disease, left bundle-branch block, hypertension, and paroxysmal atrial fibrillation presented to a private hospital with 9 days of fever, palpitations, and lethargy. He was hypotensive, hypoxic, and tachypneic. A blood smear was reported as “heavy infection, likely P. knowlesi fatalities were identified  as P. falciparum, and 2 (7%) as Plasmodium vivax. Thrombocytopenia was universal on presentation, as was elevated creatinine , including 20 (69%) diagnosed as eatinine . All patP. knowlesi CFR in Sabah was 1.70/1000  during 2015–2017. For women this was 3.6/1000 , compared to 1.4/1000  for men (P = .24). Incorporating previously reported data from 2010 to 2014  [P = .01). The higher CFR in women remained significant after adjusting for age . The CFR for patients aged ≥45 years was 5.8/1000  compared to 1.1/1000  for those aged <45 years (P < .001). Age ≥45 years remained a significant risk factor for death after adjusting for sex .The P. knowlesi deaths. Plasmodium knowlesi accounted for all recent malaria deaths in Sabah, in contrast to previous reports during 2010–2014 where 13 of 29 (44%) deaths were attributed to P. knowlesi [P. knowlesi is consistent with the decline of falciparum and vivax malaria in Sabah, and the ongoing increase in cases of P. knowlesi [P. knowlesi deaths per year has remained relatively stable despite this increasing incidence is consistent with previously reported declining CFRs in Sabah [We describe 6 malaria deaths in Sabah during 2015–2017, and review 26 previously reported knowlesi . This inknowlesi . The facP. falciparum and P. vivax in the pediatric age group [It is notable that all reported fatal cases were adults. This is consistent with the absence of severe knowlesi malaria in children , 19, andge group . Older age group , 10, 17,ge group , 22. We ge group . Howeverge group . Furtherge group .P. knowlesi compared with other species [Respiratory distress and AKI were the most common severity criteria on presentation in fatal cases. Abdominal pain, previously reported as a risk factor for severe knowlesi malaria , was pre species . Hyponat species , 10, 27, species , 17, andComorbidities are a notable feature in fatal knowlesi malaria. Half of the newly described cases had underlying cardiovascular-metabolic disease , and hypertension and hyperglycemia or diabetes mellitus were also common in the previously reported cases. Cardiovascular disease and metabolic syndrome are known to be associated with increased systemic inflammation, endothelial activation, and microvascular dysfunction , all feaPlasmodium knowlesi is relatively rare in pregnancy, with only 5 cases reported previously [P. knowlesi in pregnancy, 1 had severe malaria with intrauterine death, 2 had moderate anemia, and 1 delivered a preterm low-birth-weight infant [P. knowlesi in pregnancy have resulted in severe maternal malaria and fetal death. Prospective studies are required to further evaluate the true risks and consequences of knowlesi malaria in pregnancy.This series includes the first report of fatal knowlesi malaria in pregnancy, occurring at 35 weeks’ gestation. eviously , 30. Preeviously . Of the t infant , 30. WitPlasmodium species diagnosed on admission microscopy. This is consistent with the inability of routine microscopy to reliably distinguish P. knowlesi from other species [Several health-system issues contributed to deaths in this newly reported series. In 3 cases, the diagnosis of severe malaria was delayed despite the presence of compatible clinical features, and in another 2, IV artesunate was not available. Half of the cases were non-Malaysian citizens, which, together with the long duration of fever in this series , suggests possible barriers to accessing healthcare. These findings highlight the need to evaluate strategies to improve healthcare access in endemic areas, in addition to ensuring availability of appropriate antimalarials at all facilities. In the combined analysis, 90% of fatal cases had an alternative  species , which,  species , 33, hig species , 34.Plasmodium knowlesi parasitemia is known to increase with age; however, age has been shown to be an independent risk factor for severe knowlesi malaria [P. knowlesi are notified, so the CFRs reported in this manuscript may overestimate the true CFRs.A limitation of this study was the retrospective nature of the case series, resulting in either incomplete or unvalidated data. In particular, parasite counts were surprisingly low in some cases (G4 and G3), and it is possible that blood films may have been misreported. Second, the Department of Health data used to calculate CFRs did not include parasite counts, and hence the contribution of parasitemia to age and sex as risk factors for death could not be evaluated.  malaria , 17, and malaria , 17, an In conclusion, this review highlights the potential for poor outcomes from knowlesi malaria, and identifies female sex, age ≥45 years, and comorbidities as important associated factors. With the near-elimination of falciparum and vivax malaria in Sabah, Malaysia, health systems must maintain efforts to promptly diagnose and treat patients with knowlesi malaria to avoid fatal outcomes, particularly in these at-risk groups. This will require ongoing surveillance and efforts to increase awareness among communities and health providers, as well as improving access to healthcare and ensuring availability of appropriate antimalarials at all health facilities.


Plasmodium falciparum and Plasmodium vivax infections, varying travel history, and varying disease severity. Variation in immunity in hospital settings is particularly understudied.Naturally acquired immunity to malaria across the globe varies in intensity and protective powers. Many of the studies on immunity are from hyperendemic regions of Africa. In Asia, particularly in India, there are unique opportunities for exploring and understanding malaria immunity relative to host age, co-occurrence of P. falciparum or P. vivax or both species, and with different clinical severity were applied to an established protein array system with over 1000 P. falciparum and P. vivax antigens. Differential binding of patient IgG to different antigens was measured.A US NIH ICEMR (South Asia) team examined the level of immunity in an Indian malaria patient population visiting or admitted to Goa Medical College and Hospital in Goa, India. Sera from 200 patients of different ages, in different seasons, infected with P. vivax than P. falciparum, IgG reactivity towards P. falciparum antigens was very strong and comparable to that seen in regions of the world with high P. falciparum endemicity. Of 248 seropositive P. falciparum antigens, the strongest were VAR, MSP10, HSP70, PTP5, AP2, AMA1, and SYN6. In P. vivax patients, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 were the strongest IgG binders. Both P. falciparum and P. vivax patients also revealed strong binding to new antigens with unknown functions. Seropositives showed antigens unique to the young  or to non-severe malaria (MSP3.8 and PHIST).Even though Goa itself has much more P. falciparum and P. vivax in a low malaria transmission region with much migration. In addition to markers of transmission, the data points to specific leads for possible protective immunity against severe disease. Several, but not all, key antigens overlap with work from different settings around the globe and from other parts of India. Together, these studies confidently help define antigens with the greatest potential chance of universal application for surveillance and possibly for disease protection, in many different parts of India and the world.Seroreactivity at a major hospital in Southwest India reveals antibody responses to The online version of this article (10.1186/s12936-019-2771-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum dominates in sub-Saharan Africa and accounts for the very high prevalence rates associated with this continent. Plasmodium vivax is mostly prevalent in the American continent and Asia [Nearly half of the world’s population lives in malaria endemic zones. and Asia . Prevaleand Asia .Within India, individuals are exposed to varying levels of endemicity and many develop partial protection against infection –4. This P. falciparum and P. vivax in a cost-effective manner [NAI is mediated by acquired immune mechanisms, primarily directed against blood stages of malaria parasites. Such immunity involves circulating immunoglobulins, antibody-producing plasma cells, and memory B cells –9. Knowle manner , 11. A le manner –19. The e manner , 17, 18.P. vivax and no local P. falciparum. Yet, importantly, asymptomatic carriers revealed higher reactivity to 19 P. falciparum antigens compared to symptomatic patients and they included PHISTc, MSP11, RH2b, RON2, SERA4, two VARs, RESA, ETRAMP5, MSP2, SEMP1, HSP70-x, GEXP18, GCN5, LSA3, ETRAMP14, MSP4, ring infected erythrocyte surface antigen and a conserved protein.Immunity against malaria in India is less well understood compared to in Africa, SE Asia, or neighbouring islands. The effort above with the first protein arrays included a promising start to genome-scale probing of malaria immunity in India. An early comparative analysis of the seroreactivity profile from three sites, Raurkela, Nadiad, and Chennai in India indicated varied immune responses across the epidemiologically diverse endemic settings . RaurkelP. falciparum and P. vivax. Most importantly, Goa Medical College and Hospital offers free health care to all and attracts a very wide spectrum of malaria patients. Many participants involved in the present study were immigrants from different parts of India, with different genetic backgrounds, and different histories of prior exposure to malaria [To assess the generality of the early findings from the studies in India and the world, and to understand and measure the level of exposure in low transmission areas, protein array-based immune surveillance was performed in a low epidemiological setting in Southwest India (Goa). While Goa is considered a non-malaria endemic region of India, it has significant local seasonal transmission of both  malaria . These cP. falciparum and P. vivax antigens of symptomatic malaria patients in Goa and (ii) the relationship of IgG antibody levels of this population in Goa to multiple factors that could contribute to differential immunity. These include their age, disease severity status, and season of sample collection. Such insights can potentially identify markers of exposure and disease severity that would be useful for malaria surveillance. The present results compare favourably with reactivity from non-urban areas of India and to the other ICEMR studies from around the world. This suggests that such antigens may have value as general sero-surveillance tools in very different epidemiological settings.In summary, the present study reveals, (i) seroreactivity profiles against both The present study was a part of the US National Institutes of Health-sponsored Program Project entitled Malaria Evolution in South Asia-International Center of Excellence for Malaria Research (MESA-ICEMR). Samples were collected at Goa Medical College and Hospital (GMC), Bambolim, Goa, India. The sample collection process was approved by the Institutional Review Boards (IRB) of the Division of Microbiology and Infectious Diseases (DMID) at the US National Institute of Allergy and Infectious Diseases (NIAID), GMC, and the University of Washington (UW). Detailed ethics statements have been previously reported . After sP. falciparum, 100 P. vivax, and 4 mixed infections) were analysed by hybridization to protein microarrays. Parasite density (parasites/µL) was determined by microscopy for 199/200 individuals. Patients were further classified based on age , hospitalization status, signs/symptoms of disease severity and time of sample collection , year-round for 4 years 2012–2015). Written informed consent was obtained from all the volunteers during enrolment. A detailed description of the study site, enrolment and sample processing protocols has been published elsewhere [12–2015. P. falciparum and P. vivax proteins was studied using a protein microarray  displaying 515 P. vivax and 500 P. falciparum proteins . At ADI, the proteins were expressed as polypeptide fragments and printed from cell-free in vitro transcription translation (IVTT) reactions by the manufacturing team. A few large proteins such as dynein beta chains, rhoptry neck proteins, NOT family proteins, conserved Plasmodium proteins, reticulocyte binding proteins, erythrocyte membrane proteins (PfEMP1) and a few transcription factors were printed as overlapping peptides or exon fragments. The antigens were down-selected from larger arrays (Pf4528 and Pv4441) based on the seroreactivity of different patient populations from other malaria endemic regions [http://www.plasmodb.org).Twenty microlitre (µL) aliquots of sera from 200 patients were transported to IITB in cryovials labelled with unique numerical codes, one for each patient. Antibody reactivity to  regions . AntigenHybridizations were carried out at the Indian Institute of Technology Bombay (IITB). Each slide was composed of eight microarray pads. Multiple slides were used in each large experiment. Highly reactive sera from a single patient was used on one pad of every slide as a positive control. The rest of the microarray pads were used to probe sera from different patients. To track possible variations during array printing on individual pads on slides, batches of slides, or even assay performance during each experiment, select patient sera were probed across all runs and their signal intensities were compared for quality control and reproducibility. To account for non-specific reactivity of the IVTT expression system, each array pad contained 24 “No DNA-control” reaction spots, lacking a DNA template in the plasmid vector . Normalization was performed as previously described . Median P. falciparum infected and P. vivax infected based on the Plasmodium species detected by microscopy. Analysis of these two populations would provide information on immune status of a patient with respect to responses to infection by each of the two human malaria parasite species seen. In addition, both study groups  were sub-divided into individual age distributions as shown in Fig. P. falciparum and P. vivax groups, respectively, Fig. In order to correlate immune responses with aspects of the local clinical manifestations of malaria, the participant data were grouped into two major classes; P. falciparum and 66 P. vivax) who were all out-patients. It is important to reiterate that not all hospitalized individuals exhibited severe symptoms.Patients were also sub-divided according to the severity of disease, as immune patterns that correlate with low severity might, in principle, give an indication of disease protecting immunity. According to the clinical information that was documented for each patient, 90/200 patients were hospitalized (in-patients), while 110 participants did not require hospitalization (out-patients) [P. falciparum malaria, patients with parasite densities > 10% have severe infection, while there is no parasite density threshold for severe P. vivax malaria. A majority of the patients in this study presented with moderate to low parasite densities  except two with parasite densities of 192,153  and 147,809 parasites/µL , estimated by microscopy. Importantly, there was no correlation between parasite density, hospitalization status and severity, thus making study of immune status important , while a separate group of 68 patients were admitted during the dry season (December to April). In order to eliminate sampling bias, the sample details were kept blind to those handling sera hybridizations or microarray slides until after data analysis.P. falciparum and 100 P. vivax positive patients were probed on a protein array having 500 P. falciparum and 515 P. vivax polypeptides . The magnitude and breadth of the Ab response, determined by the number of features recognized, was greater in case of P. falciparum than P. vivax malaria  were found to be seroreactive in 30–40 patients, while only a few antigens  were seroreactive in more than 80 P. falciparum and 90 P. vivax patients. Two P. falciparum antigens and 8 P. vivax antigens showed lowest reactivity among all other seroreactive antigens. These were reactive in 10–20 P. falciparum and 20–30 P. vivax patients, respectively.As detailed in "ria Fig. a. Two huP. falciparum antigens while one P. vivax patient had high reactivity to 265 out of 515 P. vivax antigens. Despite the large breadth of antibody responses, these patients were not excluded from the study. The gene IDs of the top antigens showing seroreactivity in more than 60% of the patients in both groups are listed in Table The antibody breadth  is shown as a box and whisker plot in Fig. P. falciparum seroreactive antigens indicated that a majority of the seroreactive P. falciparum antigens were conserved Plasmodium proteins (17.9%) , transcription and translation factors (3.5%), proteins involved in ATP-binding (3.9%), ion-binding (3.5%) and proteins involved in ubiquitination (1.5%) also elicited high IgG responses despite their intracellular locations in the nucleus and the cytosol. Antigens with features and functions similar to P. falciparum antigens were also seroreactive in P. vivax patients, along with hypothetical proteins that constituted the major group (33.3%).An analysis of the features/functions of the 248 P. falciparum and P. vivax antigens but all of the patterns are not easy to understand. One hundred and seventeen seroreactive P. falciparum antigens (Sr) from P. falciparum patients were also recognized by P. vivax patients , including non-human infecting parasites. However, 29 P. falciparum antigens did not have orthologs in P. vivax-Sal1 strain. On the contrary, 98 P. falciparum antigens which were reactive only in P. falciparum group (not crossreactive in P. vivax patients) had orthologs in P. vivax . These antigens could represent markers of cumulative exposure. Alternatively, certain 88 P. falciparum antigens were found to be seroreactive in all age-groups; they included transmembrane emp24 domain-containing protein, ApiAP2, several conserved Plasmodium proteins, MSPs, PF70, members of ETRAMP family, liver stage antigens, rhoptry neck proteins, SYN6, PIESP2, members of the PfEMP1 family, enzymes and proteins involved in translation and transcription, rifin and a few cytoskeleton proteins like dynein heavy chain. In case of P. vivax, 41 antigens were seroreactive in all age groups, most of which were hypothetical proteins and merozoite surface proteins, while other hypothetical proteins and other known proteins such as MSPs and HSPs were exclusively seroreactive in older adults . The sample size differed in each age group as follows: The number of falciparum patients were 5, 36, 36 and 10, while vivax patients were 9, 30, 34 and 27 patients in age groups A, B, C and D, respectively. Clearly, the study involved some groups with very small numbers, especially children aged 1 to 5 years. Hence, a comparison of seroreactivity between groups was not performed. Moreover, there was no correlation between the number of seroreactive proteins and patient age, when scatter plots depicted antibody breadth for individual patients  and P. vivax (p = 0.01) study groups, respectively. SAM analysis using Log2 FOC values revealed a list of five candidate P. falciparum antigens with differential seroreactivity among the two P. falciparum sub-groups sensitive to hospitalization . In case of P. vivax, a hypothetical protein and ubiquitin-like protein . It is possible that some of these antigens could offer protection from severe disease, although many more diverse clinical events will have to be characterized to fully test this hypothesis. Several conserved Plasmodium proteins, MSP 3.8, PHIST a, NOT family protein and a few others were found to be seroreactive only in non-severe patients  and non-severe (NS), non-severe falciparum patients displayed significantly higher Ab levels compared to severe falciparum patients . Most of these antigens were seroreactive in both seasons, but a few P. falciparum antigens , had higher Ab responses in select patients during the dry season,  were seroreactive in both wet and dry seasons and 31% were seroreactive only during peak wet season.Goa is characterized by seasonal malaria transmission, yet many clinical cases are also reported during the dry season. To study the dynamics of IgG Ab responses accompanying changes in intensity of malaria incidence and transmission, patients were segregated based on time of the year during sample collection. A marked increase in seroreactivity was observed during the dry season  in on, Fig. . The reaPlasmodium species. To an extent, it is also specific to different life stages of the parasite [Naturally acquired immunity (NIA) depends on previous infection and builds with every pathogen encounter . It is gparasite . To diseparasite . In prinPlasmodium species and vectors, (c) changing climatic patterns that may have an impact on malaria transmission and (d) emergence of anti-malarial drug resistance [P. vivax and 373 P. falciparum antigens. A significant difference in the levels of seroreactivity and breadth of antibody response was observed across the three study sites [P. falciparum, but not P. vivax. These findings suggest that indicators of exposure may vary across diverse endemic settings and there may be a need for establishing pan-specific general antigens that may be of use for surveillance campaigns in India [India contributes significantly to the overall global malaria burden. It is important for the study of malaria immunity due to (a) the diverse eco-epidemiological profiles across the country, (b) co-existence of multiple sistance . India hsistance . As a pady sites . Additioin India . Of partin India .P. vivax (77%), while 21% had P. falciparum malaria. Of these 1000 confirmed malaria cases at GMC, 96 falciparum and 100 vivax patient sera were used in the present serology study.The current study focuses on patients from Goa, a small prosperous coastal state in western India where the epidemiology of malaria includes the presence of migrant workers and transient communities. Goa is classified as a non-endemic region with low transmission intensities where parasitaemia are usually low (< 1%). Using the shared ICEMR protein array platform, the present study describes the profiles of NAI in malaria-positive patients residing in Goa. The primary goal was to explore species-specific and pan-species serological correlates of exposure. Further, differential seroreactivity was identified in severe versus non-severe malaria patients to help identify antigens likely to offer protection from severe manifestations. This cross-sectional serological study was performed using a small subset of the total number of malaria-positive individuals referred to the MESA-ICEMR study team . Of overP. falciparum patients showed a remarkably stronger immune response compared to P. vivax. The reasons for this could be many, the major one being the significantly different biology of the two parasites [P. falciparum 3D7, P. vivax Sal1), allowing the introduction of several important biases. Genetically, P. vivax is extremely diverse and the protein array is made based on a South American Strain. This may explain the lower general reactivity seen in Goa. Overall, 248 P. falciparum and 73 P. vivax seroreactive proteins were identified. Interestingly, most of the top seroreactive antigens of P. falciparum , a hypothetical protein (PVX_118705) and MSP10 could discriminate between naïve and semi-immune individuals [P. vivax recently published using a protein array with 1936 genes encoding P. vivax proteins identified 151 highly seroreactive P. vivax antigens, a few of which  were also recognized in our study [Consistent with previous ICEMR protein array data from around the world, arasites . Secondllatforms , 28, 29.latforms . Similarur study . In concP. falciparum and P. vivax. Individual proteins, or even different peptides in a protein, can elicit varying levels of IgG reactivity. Protein-specific features such as subcellular location, protein abundance, degree of polymorphism and presence of human orthologs may influence the magnitude of antibody responses during natural malaria infections [Importantly, the malaria patients in Goa showed differential levels of IgG reactivity to different polypeptides from the same protein, in both fections . OverallP. falciparum and 73 P. vivax seroreactive antigens identified were understandably either proteins normally exported to the erythrocyte cell surface during the parasite life cycle or present on the merozoite surface. Yet, some nuclear, cytoskeletal and cytoplasmic proteins also triggered high IgG binding in the majority of the infected population. A 1990s study from India, provided early evidence for immune reaction against intracellular proteins [P. falciparum growth in culture and correlated with protection in mice [A majority of the 248 proteins . A diffe in mice . Most imP. falciparum and P. vivax proteins printed on the chip, but it is not that simple. Of 123 P. falciparum antigens exclusively seroreactive in the P. falciparum study group, 93 were found to share orthologs in P. vivax . The biggest benefit is the ease with which antibodies can be stored using dried blood spots, making sample collection and storage relatively simple .To build on present advances, future follow-up studies may improve in four areas: first, more effort is required to enroll children and adolescent patients. Second, longitudinal serology studies will track exposure to malaria, protective immunity, and waning of such immunity after an infection is resolved. Third, more detailed information will be sought on the origin of migrant workers and prior history of infection in their place of origin. Finally, the array platform itself will evolve to test sera against some shorter exon fragments on the array, some full-length proteins, all accepted blood-stage antigens, and antigens from non-erythrocytic stages of malaria parasites to assess their contribution to local transmission.Malaria exposure is largely underestimated in several countries due to limitations in microscopy-based diagnosis. The present study provides insights into antibody-based immune responses against parasite antigens in low transmission settings in India, using data collected over 4 years during both peak and dry malaria seasons. The work complements global efforts by ICEMR programs to compare variations in malaria across the globe, including possible variations in immunity to malaria. The overall immunity patterns in Southwestern India mirror what has been seen in other parts of India and the world. The contrasting immunity in individuals with varying disease presentations at a tertiary care hospital offer powerful new avenues to achieve protective immunity against severe disease.Additional file 1. Patient data for parasite density, hospitalization status and severity.Additional file 2. Seroreactive P. falciparum and P. vivax antigens.Additional file 3. Functional classification of seroreactive P. falciparum and P. vivax antigens.Additional file 4. Details of cross-reactive P. falciparum and P. vivax antigens.Additional file 5. Data for orthology and synteny.Additional file 6. Age-dependent seroreactivity of P. falciparum and P. vivax antigens.Additional file 7. Scatter plots for antibody breadth in patients.


Plasmodium falciparum malaria contributes to major complications of severe malaria. This study explored EC permeability in malaria, and evaluated the potential use of FTY720 to restore EC permeability. ECs were incubated with sera from malaria patients . Cellular permeability was investigated using a fluorescein isothiocyanate (FITC)-dextran permeability assay. FTY720, an analogue of sphingosine-1-phosphate (S1P), was tested for its potential action in maintaining EC integrity. ECs incubated with sera from malaria patients with complicated P. falciparum showed higher fluorescein leakage compared with ECs incubated with sera from P. vivax (p < 0.001) and uncomplicated P. falciparum (p < 0.001). ECs pretreated with FTY720 before incubation with malaria sera had significantly decreased fluorescein leakage compared with no FTY720 treatment. In addition, FTY720 treatment significantly reduced fluorescein leakage for both uncomplicated (at 45 min) (p = 0.015), and complicated P. falciparum malaria (15 min) (p = 0.043). The permeability increase induced by complicated P. falciparum sera was significantly reversed and prevented by FTY720 in vitro. FTY720 may have clinical applications to protect against endothelial barrier dysfunction in severe P. falciparum malaria.Increased endothelial cell (EC) permeability in severe  In Thailand, malaria periodically occurs in high risk areas, especially along the international borders of Thailand-Myanmar and Thailand-Cambodia. Severe complications are caused by the interaction between malaria parasites and the host, resulting in mechanical, immunologic, and humoral responses. The process of cytoadhesion between endothelial cells (ECs) and parasitised red blood cells (PRBCs) is an important factor in the pathogenesis of severe Plasmodium falciparum malaria. Cytoadhesion of PRBCs to the vascular ECs of different host organs along with rosette formation is considered the central mechanism of severe malaria2. Signalling events after cytoadhesion can cause injury to host tissues and trigger cellular changes such as apoptosis and cellular junctional changes4.Malaria is an important global health problem, especially in Africa and Asia. In 2016, the World Health Organization estimated there were 216 million cases of malaria with the highest incidence in the African region (90%)5. S1P has an important role in controlling EC permeability by promoting cytoskeleton arrangement and restoring adherens junctions6. Previous studies on the use of S1P for the treatment of scalds and burns7 and acute lung injury/acute respiratory distress syndrome9 demonstrated S1P restored EC permeability. To date, no study has reported in detail on cell junctions between ECs in severe malaria. The present study explored the EC permeability in P. falciparum severe malaria. In addition, FTY720, an S1P analogue was evaluated for its potential use in protecting and restoring EC integrity and subsequently preventing fluid leakage caused by P. falciparum malaria.Sphingosine-1-phosphate (S1P) is a bioactive molecule that regulates cell growth, and suppresses apoptosis and survivalp > 0.05, media only = 243.08 ± 4.33, media + FITC-dextran = 237.17 ± 5.68, normal serum + FITC-dextran = 245.40 ± 7.96, P. vivax + FITC-dextran = 238.13 ± 9.64, P. falciparum (uncomplicated) + FITC-dextran = 248.20 ± 10.39, P. falciparum (complicated) + FITC-dextran = 246.25 ± 10.66). A flat fluorescence reading from T0–T120 was obtained from the media alone group, providing a good negative control for fluorescence recording . ECs induced by uncomplicated or complicated P. falciparum showed significant FITC-dextran leakage , and at 45 min in uncomplicated P. falciparum . The highest FITC-dextran leakage was noted in ECs induced by complicated P. falciparum sera compared with media alone, normal serum, P. vivax serum, and P. falciparum uncomplicated groups . At the recording endpoint of T120, FITC-dextran leakage in ECs induced by complicated P. falciparum was increased more than 3-fold compared with normal sera and P. vivax sera, and was increased 1.6-fold compared with uncomplicated P. falciparum.This study investigated the changes in permeability of an EC monolayer exposed to malaria sera. Figure P. vivax, uncomplicated P. falciparum or complicated P. falciparum. Results showed no difference in FITC-dextran leakage between FTY720 treatment and without FTY720 treatment in normal sera , adherens junctions (AJ), and gap junctions that are complexes located at the intercellular junction20. In malaria, alterations in junctional proteins, vascular endothelial (VE)-cadherin (a component of AJ)21, and zonula occludens (TJ protein)22 have been documented. Vascular barrier disruption inflicted on these junctional proteins in malaria infection causes sequential damage to cytoskeleton components including actin, intermediate filaments and microtubules.EC dysfunction is a major factor in the development of vascular damage, an important consequence of complicated 23 and interferon (IFN)-γ24 are present in high concentrations and are associated with clinical severity, as well as adhesion receptors such as intercellular adhesion molecule (ICAM)-1, endothelial-leukocyte adhesion molecule (ELAM)-1 and E-selectin26. These mediators might contribute to the disruption of vascular integrity, cytoskeleton damage and subsequent increase in EC permeability.In severe malaria, the process of cytoadhesion between PRBCs and ECs can cause direct damage to ECs resulting in the loss of barrier integrity. Chemical mediators dissolved in malaria sera are also important factors that enhance permeability changes. During malaria infection, soluble mediators such as tumour necrosis factor (TNF)-α, interleukin (IL)-6, 10P. falciparum sera. FTY720 had beneficial effects against damage induced by malaria sera on the EC barrier, in addition to its usefulness as a protective factor. In ECs of blood vessels, FTY720 maintained barrier integrity and prevented permeability leakage28. In experimental cerebral malaria, FTY720 prevented vascular leakage, inhibited neurological signs and prolonged animal survival30. Previous studies have shown that FTY720 induced the translocation of VE-cadherin to the contact sites at the EC junction28, and promoted cortical actin formation thereby stabilizing the AJ32. It would be interesting to assess the disruption of junctional molecules, which are complexed in TJ and AJ, i.e. occludin, claudins, junctional adhesion molecule (JAM), VE-cadherin, β-catenin, and the cytoskeleton changes in ECs exposed to malaria parasites/sera. In this study, the therapeutic efficacy and preventive effects of FTY720 to restore EC permeability exposed to malaria sera were demonstrated. A positive result with experimental FTY720 serves as a proof of concept to further explore the benefit of FTY720 as an adjuvant drug to prevent severe malaria complications such as cerebral oedema and pulmonary oedema.An important focus of this study was the impact of FTY720 on EC permeability in malaria. As a treatment target, the study evaluated the benefit of FTY720 on barrier integrity by exposing ECs with malaria sera to induce an increase in vascular permeability, prior to treatment with FTY720. Here, the effect of FTY720 on permeability leakage was particularly evident in decreasing FITC-dextran leakage in ECs treated with severe 2 vented cap flasks  (4 ml per seeding vessel). The seeding vessels were coated with 2% bovine gelatine  diluted to 1% gelatine in 1× phosphate buffered saline solution (PBS). HUVEC cultures were monitored under an inverted microscope daily. The EC media was changed the day after seeding and every other day thereafter. Cells were allowed to grow to >90% confluence (4–6 days), then HUVECs were subcultured. ECs used in the experiments were from passages 4–6. To grow ECs on the transwell filter, confluence adherent cells were trypsinised using trypsin-EDTA solution (1×) , and seeded on the upper well of the Costar transwell filter (pore-size 0.4 μm)  coated with gelatine. Approximately 300,000–400,000 cells in 500 µl of cell suspension were loaded to the upper well. One cc of pre-warmed complete EC media was added to the lower well. Cells were maintained at 37 °C, 5% CO2 in a humidified incubator for 3–5 days or until >90% confluence.Primary human umbilical vein endothelial cells (HUVECs) were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB), Japan, and cultured according to the manufacturer’s instructions with modifications. One cryovial of primary HUVECs (1 ml) re-suspended in complete EC media (12 ml)  was used to seed three gelatine-coated T25 cmP. vivax and P. falciparum malaria patients were used in this experiment. Controls were matched to patient sex and age. Malaria patients enrolled in the study were admitted at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand. Age range was from 16–58 years old. Sera used were from the day of admission before treatment. The clinical manifestations of severe malaria were based on WHO criteria33. Prior to storage, collected blood samples were left to stand at room temperature (RT) for 15–30 min to allow for clot formation. After centrifugation at 1,500 × g for 5 min, the sera were collected and stored at −80 °C until analysis. The study protocol was approved by the Ethics Committee, Faculty of Tropical Medicine, Mahidol University . All methods used during the investigation were performed in accordance with the relevant guidelines and regulations of the above institutional committee. The informed consent was obtained from all subjects.Stored sera from normal volunteers, 34. Media in the lower well was replaced by 1,000 µl fresh media only. To record the amount of fluorescein leakage, 100 µl of lysate was taken from the lower well immediately after incubation (Time (T) 0), then every 15 min until 2 hr . One hundred µl of fresh complete media was added back thereafter. Special care was taken not to agitate the plate. The plate was constantly covered with aluminium foil to protect against the degradation of fluorescein by light. Lysate from the lower transwell was used to determine fluorescence at wavelengths of 485 nm and 528 nm35, using a Synergy H1 Hybrid Reader . Experimental groups consisted of P. vivax sera with FITC-dextran, uncomplicated P. falciparum sera with FITC-dextran and complicated P. falciparum sera with FITC-dextran (5 sera samples per groups with 2–4 replicates per group). Control groups included complete media alone , complete media with FITC-dextran , and normal sera with FITC-dextran .When ECs reached >90% confluence, complete EC media was removed from the upper well and replaced with 500 µl of new complete media containing malaria sera (10%) + Fluorescein isothiocyanate (FITC)-dextran conjugate 7 was added to ECs on the upper transwell for 15 min. Then, FTY720 was removed and replaced with malaria sera (10%) containing FITC-dextran. The fluorescein leakage was determined as above at different time points.FTY720  at a final concentration of 1 µMECs on the upper transwell filter were incubated with malaria sera for 45 min. Then, FTY720 containing FITC-dextran was added at a final concentration of 1 µM (at T45). The fluorescein leakage was assessed as above at different time points.t-test was used to compare paired effectiveness of FTY720. A p value < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS, version 18.0 .Original data were expressed as the mean ± standard error of the mean (SEM). Fluorescence readings were quantified as ratiometric results relative to the control group. The normality of distribution was determined by the Kolmogorov-Smirnov test. Differences between groups were analysed by one-way ANOVA and the LSD multiple comparison test. Paired All data generated or analysed during this study are included in this published article.


Background and objectives: Diabetes mellitus represents a metabolic disorder the incidence of which has been on the increase in recent years. The well-known long-term complications of this disease encompass a wide spectrum of renal, neurological and cardiovascular conditions. The aim of the study was to investigate the serum concentration of endothelial microparticles (EMPs) as well as selected noninvasive parameters of the ascending aorta stiffness calculated with echocardiography. Materials and Methods: 58 patients were enrolled in this study—38 subjects diagnosed with type 2 diabetes mellitus (T2DM) and 20 healthy controls. The analyzed populations did not differ significantly with respect to age, renal function, systolic and diastolic blood pressure. Results: The patients with T2DM and concomitant hypertension presented higher levels of EMPs in comparison with diabetic normotensive subjects. Among patients with T2DM and hypertension, aortic stiffness assessed with the elasticity index (Ep) was higher and the aortic compliance index (D) lower than in the diabetic normotensive group. No correlation between the amount of EMPs and lipid profile, C-reactive protein (CRP) level and glycemia, was observed in the studied group. There was, however, a statistically significant positive correlation between the creatinine level and amount of EMPs, while the negative relationship was documented for EMPs level and the estimated glomerular filtration rate (eGFR). Conclusions: Considering the elevated number of EMPs in diabetic patients with hypertension as well as the positive correlation between EMPs and serum creatinine level, EMPs assessment could be useful in identifying patients who are at high risk of organ damage due to diabetes mellitus. Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and long-lasting disorders in the carbohydrate, lipid, and protein metabolism, which leads to chronic progressive dysfunction and failure of the nervous, renal and cardiovascular systems . There aVascular endothelium is an active tissue covering the interior surface of blood vessels and it constitutes an interface between circulating blood and other tissues of the body. The endothelium plays a crucial role in blood pressure regulation, angiogenesis and hemostasis by secreting various biologically active substances . ChronicEndothelial microparticles (EMPs) are the cellular markers of endothelial dysfunction. EMPs are membrane vesicles derived from activated or apoptotic endothelial cells . Their fThe aim of this study was to noninvasively assess endothelial function in patients with T2DM by measuring the EMPs level in the blood serum and the ascending aorta stiffness parameters in transthoracic echocardiography.The authors obtained consent for conducting this study from the Bioethics Committee of the Medical University of Lublin. The patients gave written informed consent to participate in the study after being presented with the study protocol.In this study, 58 patients admitted to the Department of Internal Diseases were included. The studied group consisted of 38 patients with T2DM diagnosed earlier or during present hospitalization. The diagnosis of T2DM was based on WHO criteria . The conThe identification of EMPs was performed with flow cytometry ,13. The max) when the aortic valve was open and the minimal aortic diameter (Aomin) at the peak of R wave in ECG performed at the same time [The ascending aorta stiffness was assessed during transthoracic echocardiography performed with a Vivid 4 device  and 2.2 MHz convex transducer. Electrocardiography (ECG) was also performed during the examination. The maximal aortic diameter —pressure-related vessel elasticity index, which shows the value of the blood pressure change necessary to increase the vessel diameter by 100%, was calculated as in equation below ,17,18:D [mmA1c) expressed as a percentage (%).The blood samples were drawn from the patients 12 h after their last meal. The following parameters were measured: The lipid profile , triglycerides; the low-density lipoprotein (LDL) level was calculated in accordance with Friedewald’s equation ; creatinp < 0.05.The statistical analysis was performed using Statistica 10 PL computer software. The results are presented as the mean value ± standard deviation in the case of quantitative variables and as a percentage is the case of qualitative variables. The statistical significance was set for The studied parameters/quantitative variables convergence with a normal distribution was tested with Shapiro-Wilk test. The student’s t-test and ANOVA analysis of variance was used for the variables and groups of variables convergent with a normal distribution. When the variables distribution was significantly different from normal, the Mann-Whitney U test and one-way ANOVA on ranks  were applied. The correlations between the studied parameters were explored using the Pearson correlation coefficient or Spearman’s rank correlation coefficient for variables with a distribution different from normal.p = 0.840). Men constituted 42.1% of studied group and 40% of the control group and the difference was not statistically significant (p = 0.461). The high BMI values were observed both in the studied and control group . A majority of patients in both groups were hypertensive − 81.6% of the studied group and 80% of the control group (p = 0.895).The age of the patients in studied and the control group did not differ significantly . There were no significant differences in the lipid profile, renal function parameters and CRP level between the studied and the control group . The level of EMPS was not significantly higher among patients with T2DM than among those in the control group .The beta index was 3.52 ± 0.45 in the studied group and 3.30 ± 0.50 in the control group. The elasticity index (Ep) in patients with T2DM was 1160.0 ± 573.4 mm Hg versus 905.4 ± 501.2 mm Hg in the control group, this difference was not statistically significant (p = 0.094). The aortic compliance D was 1.03 ± 0.51 × 10−3 mm Hg−1 in the studied group and 1.45 ± 0.79 × 10The patients with T2DM were divided into two subgroups . The patA1c. The EMPs level, β, Ep and D indexes did not significantly differ between the subgroups with HbA1c higher and lower than 8%  and between the amount of EMPs and PP . However, there was no correlation between the amount of EMPs and DBP  nor between the EMPs level and MAP . There were statistically significant positive correlations between the Ep index and DBP as well as MAP , Ep index  and compliance D  among patients with T2DM.In the presented study, there were no statistically significant correlations observed between the EMPs level and β index . There was also a significant negative correlation between the amount of EMPs and eGFR . There were no statistically significant correlations between the aortic stiffness parameters and the lipid profile, renal function parameters, CRP level and glycemia in studied group.There was no correlation found between the amount of EMPs and lipid profile components, CRP level and glycemia in the studied group. There was a statistically significant positive correlation between the creatinine level and the amount of EMPs (r = 0.411; The assessment of early atherosclerotic lesions in the arteries would be useful in establishing cardiovascular risk in patients without clinical symptoms . This isThe level of EMPS was not significantly higher among patients with T2DM than among those in control group. Koga et al. reported a significantly higher amount of CD144+ EMPs in patients with T2DM in comparison with the control group. This study also suggested that increased EMPs level is an important cardiovascular risk factor . HoweverThis study also observed that, except for hypertension, renal function disorders are connected with higher amount of EMPs, which may lead to the acceleration of atherosclerotic process in course of diabetes. Huang et al. found CD31+ EMPs level to be higher among hypertensive patients with albuminuria in comparison with hypertensive patients without this condition . The autIn this study, hypertensive patients with T2DM presented significantly increased aortic stiffness measured with the Ep index and significantly decreased aortic compliance D in comparison with normotensive diabetic patients. Furthermore, moderate positive correlations were found between the Ep index and SBP, DBP and MAP as well as significant negative correlations between the aortic compliance D and SBP, MAP. Zapolski et al. obtained similar results in their study patients with T2DM had increased aortic stiffness β assessed with echocardiography in comparison with healthy individuals . In anotThis study did not find any significant correlation between the amount of EMPs and aortic stiffness parameters. These results are contradictory to some other research results. Wang et al. observed that increased arterial stiffness assessed with brachial-ankle PWV is positively correlated with CD31+/CD42- EMPs level in healthy individuals . SimilarThe authors of the presented study are aware of its limitations. The research was conducted on a small number of patients and only in one clinical center. The lack of standardized EMPs detection methods among publications on this subject constituted a significant limitation to this work. Thus, the EMPs level results are difficult to compare with other studies. Taking into consideration the fact that amount of CD31+/CD42b- and CD62E+ EMPs rises in −80 °C temperature , the EMPA1c with aortic stiffness parameters and the EMPs level. Furthermore, this study did not find any significant correlation between the amount of EMPs and the aortic stiffness parameters. Thus, the EMPs level in patients with T2DM seems to constitute information complementary but not identical to aortic stiffness parameters. Considering the elevated number of EMPs in diabetic patients with hypertension as well as the positive correlation between EMPs and serum creatinine level, EMPs assessment could be useful in selecting patients at high risk of organ damage in diabetes. Additional investigations in this field are necessary to fully define its effect.In conclusion, this study observed that hypertension coexisting with T2DM had a negative impact on the endothelium state assessed with the EMPs level. In addition, the EMPs level in diabetic patients increased along with renal failure progression. Hypertension coexisting with T2DM was also connected with increased aortic stiffness. In contrast, this study did not find any correlation of the lipid profile, CRP level and glycemic control measured with Hb