As The Gambia aims to achieve malaria elimination by 2030, serological assays are a useful surveillance tool to monitor trends in malaria incidence and evaluate community-based interventions.Pf antigens on a multiplexed bead-based assay. Village-level antibody levels were quantified as under-15 sero-prevalence, sero-conversion rates, and age-adjusted antibody acquisition rates. Antibody levels prior to MDA were assessed for association with persistent malaria infection after community chemoprophylaxis.Within a mass drug administration (MDA) study in The Gambia, where reduced malaria infection and clinical disease were observed after the intervention, a serological sub-study was conducted in four study villages. Spatio-temporal variation in transmission was measured with a panel of recombinant Pf prevalence, respectively). At the end of the malaria season, short-lived antibody responses to Etramp5.Ag1, GEXP18, HSP40.Ag1, EBA175 RIII-V, and Rh2.2030 were lower amongst 1–15 year olds in the West Coast compared to the Upper River, reflecting known differences in transmission. Prior to MDA, individuals in the top 50th percentile of antibody levels had two-fold higher odds of clinical malaria during the transmission season, consistent with previous findings from the Malaria Transmission Dynamics Study, where individuals infected before the implementation of MDA had two-fold higher odds of re-infection post-MDA.Seasonal changes in antibodies to Etramp5.Ag1 were observed in children under 15 years in two transmission settings—the West Coast and Upper River Regions (4.32% and 31.30% Serological markers can serve dual functions as indicators of malaria exposure and incidence. By monitoring age-specific sero-prevalence, the magnitude of age-stratified antibody levels, or identifying groups of individuals with above-average antibody responses, these antigens have the potential to complement conventional malaria surveillance tools. Further studies, particularly cluster randomised trials, can help establish standardised serological protocols to reliably measure transmission across endemic settings. In malaria elimination settings, heterogeneity and hotspots of transmission are increasingly prevalent as disease burden declines –5. At loThe Gambia has a long history of research on the heterogeneity of malaria in West Africa. Entomological and clinical data have shown spatial variation in malaria infections within 2 km of mosquito breeding sites, where immune differences between households were hypothesised to drive differences in clinical outcomes . StudiesPlasmodium falciparum (Pf) malaria exposure that are able to predict clinical infection in the previous year with a high degree of accuracy [Serological studies in The Gambia have described country-wide heterogeneities in malaria transmission. School surveys have found strong correlations between sero-prevalence and microscopy-detectable parasitaemia . More reaccuracy . These mPf antigens was used to quantify antibody levels with a multiplexed immuno-assay. In our previous analysis in The Gambia, a number of targets in this antigen panel were found to be highly correlated with clinical and asymptomatic infection at the individual, household, and village level [While serology has been used in research settings to measure malaria transmission for some time, standardised antigen panels for routine surveillance have yet to be established. Furthermore, gaps in our understanding of how antibody levels reflect changes in transmission still remain. Therefore, this study evaluates the use of antibody responses as a measure of malaria exposure and incidence, with the aim to inform the future design of surveillance strategies. We used serological markers to measure spatio-temporal variation in transmission in a subset of four study villages from the Malaria Transmission Dynamics Study in The Gambia, which assessed epidemiological trends in six village pairs , 19, folge level , and werPf prevalence as measured by polymerase chain reaction (PCR) ranged from 3.61% in the West Coast Region to 19.6% in the Upper River Region. As previously described by Mwesigwa et al. [Pf infection measured by PCR, and factors associated with infection post-MDA.A prospective cohort study was conducted from 2013 to 2015 in six village pairs across five administrative regions from the West Coast Region (WCR) to the Upper River Region (URR). Pf, Orchid Biomedical System, India).Blood samples were collected by finger prick for haemoglobin measurement, blood smear for malaria diagnosis by microscopy, and molecular and serological analysis by dried blood spot (DBS) on filter paper . Clinical malaria cases were identified by passive case detection (PCD) at local health facilities or in villages by study nurses; clinical malaria was defined as history of fever in the previous 24 h or axillary temperature ≥ 37.5 °C and a positive rapid diagnostic test (RDT) result (Paracheck N = 530) and at the end of the season in December 2013 (N = 522). In the Upper River Region, analysis included all samples collected in Njayel and Madina Samako in July 2013 (N = 769), December 2013 (N = 626), April (dry season) 2014 (N = 797), and December 2014 (N = 733). These regions are at the extreme of transmission intensity found in The Gambia—low transmission in the West Coast Region and moderate transmission in the Upper River Region. Samples from clinical PCD cases were linked by study participant identification code to samples from the same individuals collected during routine monthly surveys. Samples collected as part of the Malaria Transmission Dynamics Study and subset of samples for serological analysis are described in Fig. Serological analysis included all available samples from the West Coast Region in Besse and N’Demban from surveys at the start of the malaria transmission season in July 2013 .For diagnostic PCR, DNA was extracted from three 6-mm DBS  using the automated QIAxtractor robot (Qiagen). Negative and positive (3D7) controls were included to control for cross contamination and DNA extraction efficiency, respectively. Extracted DNA was used in a nested PCR, amplifying multi-copy Antibody responses were summarised at village level and used to describe geographical differences in transmission between the West Coast Region (low transmission) and the Upper River Region (moderate-high transmission). Within each region, temporal changes were assessed using antibody responses at the start and end of transmission season (July and December 2013) and the dry season (April 2014), represented with the methods as summarised below.P. falciparum merozoite surface antigen 1 19-kDa carboxy-terminal region (PfMSP119), P. falciparum apical membrane antigen 1 (PfAMA1), and P. falciparum glutamate rich protein (PfGLURP.R2)—the distribution of MFI values for all serological samples was characterised with a two-component Gaussian mixture model to estimate the distributions of negative and positive antibody levels. Sero-positivity thresholds were defined as the mean log MFI plus two standard deviations of the negative distribution [For antigens associated with longer-lived antibody responses –29—P. faribution . For antribution , 31—earlPfMSP119, PfAMA1, PfGLURP.R2). A reverse catalytic model was fitted to age-adjusted sero-positivity data using maximum likelihood methods [z test, where standard errors were based on a pooled sample population size and differences in sero-prevalence with a significance level of 0.05 reflected by a 95% CI range that excluded zero.Age-adjusted sero-conversion rates (SCRs), the annual rate at which sero-negative individuals become sero-positive, were estimated for antigens associated with long-lived antibody response .Antibody acquisition models , 34 werePf infection (actively detected using PCR from monthly survey samples) during the transmission season following MDA (October to December 2014) amongst individuals below and above median residual dry season antibody levels, compared to individuals at or below expected antibody responses, allowing for clustering at the household/compound. Multivariate analyses also adjusted for age, LLIN use, and MDA adherence using generalised linear mixed-effects models with the ‘glm’ and ‘glmer’ functions in the ‘lme4’ v1.1-21 package in R version 3.6.1.Individual antibody levels in April 2014 prior to MDA were assessed by comparing observed and expected MFI values as predicted by the antibody acquisition fit for individuals in the Upper River Region. For individuals with above-average MFI values for their age group, the difference between the observed and expected log MFI was calculated and classified to below or above median of residual MFI antibody response. Logistic regression was used to estimate the odds of clinical malaria  or asymptomatic PfMSP119, by 2.9% (95% CI 2.3–3.6) for PfGLURP.R2, and by 2.2% (95% CI 0.9–3.5) for GEXP18 , sero-prevalence to Etramp5.Ag1 in children aged 1–15 years in the West Coast Region increased by 4.8% (95% CI 4.0–5.6), by 3.1% 95% CI 2.7–3.5) for .7–3.5 foPfMSP119, PfAMA1, and PfGLURP.R2 were higher in the Upper River Region than the West Coast Region. Seasonal differences in sero-conversion rate within the same region were also observed. In the West Coast Region, PfMSP119 sero-conversion rate was three times higher in December 2013  than in July 2013 , while in the Upper River Region, PfMSP119 sero-conversion rate was four times higher in December 2013  than in July 2013 . However, for PfAMA1 and PfGLURP.R2, seasonal differences in sero-conversion rates were only observed in the Upper River Region, where PfAMA1 sero-conversion rate in December 2013 was 0.0913 (95% CI 0.0735–0.1135) compared to 0.0616 (95% CI 0.0511–0.0743) in July 2013 and PfGLURP.R2 sero-conversion rate was 0.0708 (95% CI 0.0605–0.0829) in December 2013 compared to 0.0499 (95% CI 0.0427–0.0584) in July 2013. Sero-conversion rates for all three antigens were similar between April and December 2014. Increases in sero-prevalence in older age groups are not pronounced for markers associated with short-lived antibody responses, which is an underlying assumption when using sero-catalytic models. Therefore, antibody acquisition models are used in place of sero-conversion rates to assess age-adjusted changes in antibody responses based on continuous data for markers of short-lived antibody responses.Differences in sero-conversion rate between geographical region and transmission season varied by antigen     showed higher antibody levels in 1–15 year olds in the Upper River Region compared to the West Coast Region at the end of the transmission season , differences in age-adjusted antibody acquisition amongst 1–15 year olds were observed between the West Coast Region and Upper River Region at the end of the 2013 transmission season (December)  .For antigens associated with long-lived antibody responses , PfGLURP.R2 , Rh2.2030 , PfMSP119 , and Etramp5.Ag1    after MDA , EBA175 , HSP40.Ag1 , and GEXP18   and PfGLURP.R2  , HSP40.Ag1 , PfMSP119 , Etramp5.Ag1 , and EBA175   were observed for nearly all antigens. The largest decreases were for Pf antigens, likely reflecting changes in malaria incidence as well as residual antibodies due to prior infection. In moderate to high transmission settings, higher antibody levels in the dry season due to prior exposure are common. However, the rate at which protective antibodies develop varies by setting. Immunity to clinical malaria has been observed in children as young as 5 years [In this study, we build on previous studies evaluating individual- and household-level associations between concurrent clinical and asymptomatic malaria infection with serological responses to a panel of antigenic targets. This study extends upon these findings to develop serological methods for monitoring seasonal and geographical trends in transmission, tailored for potential integration into routine surveillance or trial evaluation. Spatio-temporal variations in serological responses were observed for several  5 years , 37, whi 5 years , 39. ChiPfMSP119, PfAMA1, and PfGLURP.R2 were observed between geographical regions and between the start and end of the 2013 transmission season in the Upper River Region. However, seasonal differences in sero-conversion rates were not detected in the West Coast Region. Long-lived antibody responses typically reflect past exposure rather than incidence during a single transmission season. However, these markers have been shown to be sensitive to changes in transmission over longer periods of time in The Gambia [Pf antigens screened found to be most associated with recent episodes of clinical malaria in Uganda and Mali [Differences in sero-conversion rates to e Gambia . Antibodand Mali .PfAMA1, PfMSP2, PfMSP1, EBA175), with antibody half-lives estimated between 10 and 50 days [PfMSP119. However, these studies focus on a limited number of antigens. Estimates of antibody longevity for a larger range of antigens can help inform optimal sampling strategies for future serological surveillance using multiplex assay platforms. For example, studies in Mali have quantified antibody responses to over 2000 Pf antigens on protein microarray, observing rapid declines in antibody levels within 6 months [Between December 2013 and April 2014, antibody levels did not decline significantly, indicating that antibody decay probably occurs over a period longer than 4 months. This may be due to persistent asymptomatic parasitaemia or infections occurring late in the transmission season. In The Gambia, antibody decay in children has been observed for several antigens associated with protective immunity  compared to clinical cases (n = 87). Results may also indicate that dry season antibody levels for individuals who are later at risk of asymptomatic infection, while still higher than average, may be lower relative to those who develop clinical malaria. The threshold antibody levels that indicate increased risk of subsequent clinical malaria warrant further study. Given that this study was not designed specifically for the serological evaluation of MDA, analysis of a larger set of antigens or a grouping of antibody data based on antigen expression or function during the parasite life cycle may help delineate or categorise the wide range of responses with respect to malaria exposure, incidence, or immunity. Prospective cohort studies measuring persistent asymptomatic parasitaemia throughout the dry and wet seasons across different transmission intensities and populations would further elucidate the dynamics between parasite density, immunity, and serological responses.Above-average antibody levels in the dry season before MDA were associated with increased odds of clinical malaria following MDA. Associations were moderate for post-MDA . While rPf antigens identified in this study in future diagnostic platforms. Evaluating their agreement and consistency with other diagnostic outcomes in intervention trials, including cluster randomised trials and surveillance studies, can help establish standardised serological protocols usable across a variety of epidemiological settings.This study demonstrates that a diverse panel of serological markers can be used to monitor malaria exposure (observed through long-lived antibody responses) and recent infection incidence (through moderate to shorter-lived antibody responses). By monitoring antibody responses in a variety of ways, such as age-specific sero-prevalence, the magnitude of age-stratified antibody levels, or identifying areas with clustering of above-average antibody responses, these antigens have the potential to complement conventional malaria surveillance tools. Serological measures could be used as secondary endpoints alongside clinical and molecular diagnostics in the evaluation of community interventions. They could also be used for baseline stratification of individuals or communities according to their risk of exposure, or to evaluate the degree to which historical or recent malaria exposure influences study outcomes. For example, a subset of the serological markers used in this study have been used to stratify individuals in a controlled human malaria infection (CHMI) study in The Gambia, where individuals with serological evidence of higher malaria exposure were found to have higher parasite growth inhibitory activity and control infection more effectively. Microarray studies evaluating the combined use of multiple serological markers suggest that panels of at least five antigens confer greater diagnostic accuracy compared to a single marker, while panels of more than 20 confer only minor improvements in accuracy , 44. BetSerological markers can serve dual functions as indicators of malaria exposure and incidence. By monitoring age-specific sero-prevalence, the magnitude of age-stratified antibody levels, or identifying groups of individuals with above-average antibody responses, these antigens have the potential to complement conventional malaria surveillance tools. Further studies, particularly cluster randomised trials, can help establish standardised serological protocols to reliably measure transmission across endemic settings.Additional file 1: Supplementary methods and tables. Supplementary methods describe the antibody acquisition model used in Figs. Additional file 2. Dataset.


Plasmodium-infected erythrocytes (pRBCs) to host brain microvasculature beds via P. falciparum erythrocyte membrane protein 1 (PfEMP1). Under normal conditions, activated protein C (APC) bound to endothelial protein C receptor (EPCR) has cytoprotective properties via the activation of protease-activated receptor 1 (PAR1). During malaria infection, pRBCs transports PfEMP1 to the membranes to bind EPCR in the same region as APC. As a result, APC is less capable of inducing cytoprotective effects via PAR1. Two studies involving adult malaria patients revealed that EPCR rs867186-GG allele is associated with protection against severe malaria, while three other studies involving child malaria patients could not show association between EPCR rs867186-GG genotype and severe malaria or increased mortality among children with CM.Cerebral malaria (CM) is characterized by the sequestration of PROCR gene (NM 006404) and were used to amplify a 660 bp product as described before. PCR products were purified, and DNA sequences were determined by Sanger Sequencing . Nonparametric tests were used to compare the groups. To analyze differences in allele frequencies, we used chi-squared or Fisher's exact tests for categorical variables if the expected values were less than 5. P-value <0.05 was considered statistically significant.We examined the association between the EPCR rs867186-GG genotype and the protection against cerebral malaria. Peripheral blood samples were collected from 47 malaria patients and 34 healthy individuals from a study conducted from 2004 to 2007 at the NSCB Medical College Hospital in India. CM and malaria-associated complications were defined based on WHO criteria. Genomic DNA was isolated from the peripheral blood mononuclear cells. Primer sequences were designed to contain rs867186 of the Our results showed significantly higher rates of AG and GG genotypes in CM patients compared to mild malaria (P = 0.0034). Our results indicate that rs867186-GG or rs867186-AG genotypes are not associated with protection against HCM. PfEMP1 t a time . The cyst a time  are locat a time . PfEMP1  (UM/MM) , and to  domains . Recentl domains . By comp domains , and lea domains . Shabani malaria . The abutargeted . Solublegenotype . Two stugenotype , while tgenotype  involvinSamples used in this study were collected and stored at −80°C as part of an NIH-funded research project (R21TW006804-01) from 2004 to 2007 at the NSCB Medical College Hospital and at the Jabalpur and Civil Hospital Maihar, District Satna, both in the state of Madhya Pradesh, Central India. This study was approved by the ethics research committee of the National Institute of Malaria Research New Delhi, Regional Medical Research Center for Tribals, Jabalpur, Center for Disease Control and Prevention, CDC, Atlanta, GA, and Morehouse School of Medicine, Atlanta, GA, USA.P. falciparum positive asexual stage parasitemia on blood smear were enrolled after obtaining written informed consent from their parents or close relatives. Consent was also obtained for long-term storage and later use of samples for assessment of biomarkers of disease severity. Patients satisfying the enrollment criteria were enrolled as healthy control (HC), mild malaria (MM), and cerebral malaria (CM) following the definition given below. Malaria associated complications were defined using WHO criteria (Adults and children (< = 14 years) with only criteria .Healthy controls (HC) included members of the community who did not have malaria or other febrile illness 60 days before enrollment, and with no history of mental/metabolic illness, tuberculosis, meningitis, or accidental head injury. Thirty-four healthy control samples were left for this current project since the samples used in this study were collected for another project in India in 2007.P. falciparum parasitemia of <25,000 parasites/μl of blood  and no evidence of severe malaria and no history of mental/metabolic illness, tuberculosis, meningitis, or accidental head injury.Mild malaria group included patients who had a fever with P. falciparum parasitemia, and have no other clinically evident cause of impaired consciousness, no history of mental/metabolic illness, tuberculosis, meningitis, or accidental head injury.Cerebral malaria group included patients who have a Glasgow coma score of ≤10 , have a Genomic DNA was isolated from the peripheral blood mononuclear cells of healthy individuals and malaria patients using the DNeasy Blood and Tissue kit .PROCR gene (NM 006404.3) which were described previously .Primer sequences were designed to contain rs867186 of the eviously . The foleviously . GenotypWe used two-sided Fisher's exact test to estimate power for comparing two binominal proportions. The significance level of the test is 0.05. Group sample sizes of 23 in the group of mild malaria (MM) and 24 in the group of cerebral malaria (CM) achieved 69.174% power to detect a difference between the group proportions of 0.3600. The proportion in the group of MM is assumed to be 0.0900 under the null hypothesis and 0.4500 under the alternative hypothesis. The proportion in group of CM is 0.0900.Chi-Square test or Fisher's exact test were used to compare the rs867189 genotypes in three groups as well as pairwise comparisons. If the expected value of any cell count was less than 5, Fisher's exact test was used. P < 0.05 was considered statistically significant. SAS 9.4 software was used for all the stiatistical test.A total of 81 subjects  was included in this study, and the details of patients' clinical characteristics and clinical parameters are shown in We assessed DNA rs867186 polymorphism among 81 subjects. post hoc analysis for each of the two groups. There are significantly higher rates of rs867186-AG+GG in CM patients compared to mild malaria patients . Our results indicate that rs867186-G variant (rs867186-GG or rs867186-AG genotypes) are not associated with protection against HCM. The difference in genotype AA and G-variant (AG+GG) of rs867186 is likely due to pressure exerted by parasites of the genus P. falciparum genome holds about 60 PfEMP1-encoding var genes. PfEMP1 containing DC13-variants are capable of binding to both the EPCR /activated protein C (APC), factor VIIa, T-cell receptor, and PfEMP1. Under physiological conditions, Protein C is activated by the thrombin-thrombomodulin complex, the activated protein C (APC) then cleaves the protease-activated receptor (PAR1) at Arg46, which triggers anti-apoptotic, anti-inflammatory reaction and inhibits thrombin production and stabilization of endothelial barrier function . In SM, the EPCR  and ICAMthe EPCR , where b malaria , while E malaria .The rs867186-GG genotype of EPCR was analyzed in patients with mild malaria and SM by some groups; they found that the rs867186-GG genotype is significantly more frequent in patients with mild malaria than in those with SM . By contvice versa, namely, where sEPCR levels were higher with the increased presence of the G variant. Unfortunately, in the current study, we were unable to examine sEPCR since the matched serum plasma were all used by the primary research project. sEPCR is shed from memberanous EPCR on the surface of the endothelial cells, where it can bind to PfEMP1-DC13 and DC8 variants and free membranous EPCR, (A soluble EPCR (sEPCR) circulates in plasma and is released from the surface of the endothelium by metalloprotease ; it is aus EPCR, , therefous EPCR,  would inus EPCR, . TherefoMethods section).In the present study, we analyzed the samples that were collected in 2007 for another project. Although the sample size available for investigations is fixed, an estimate of statistical power with the anticipated available sample size is low but acceptable (see The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Written informed consents were obtained from all participating subjects. This study was approved by the ethics research committee of the National Institute of Malaria Research New Delhi; Regional Medical Research Center for Tribals, Jabalpur; Center for Disease Control and Prevention, CDC, Atlanta, GA, and Morehouse School of Medicine, Atlanta, GA, USA.FY and ML performed statistical analyses. ML and JS conceptualized and wrote the manuscript. JC performed experiments including PCR amplication and PCR product purication for direct sequencing with the help of ML. SK and PB collected blood samples and extracted DNA from human blood samples. ML, JH, and JS participated in the design and coordination of the study. All the authors read and approved the final manuscript.This work was supported by NIH/MIMHD MD007589 and NIH/NINDS R01 NS091616.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum variants. The influence of genetic multiplicity and whether some specific genetic variants are more or less likely to invest into gametocyte production is not clearly understood. This study explored host and parasite-related risk factors for gametocyte carriage, and the extent to which some specific P. falciparum genetic variants are associated with gametocyte carriage.Multi-genotype malaria infections are frequent in endemic area, and people commonly harbour several genetically distinct msp1 and msp2 genetic variants and gametocyte carriage was also explored.Gametocytes and asexual forms were detected by light microscopy on thick smears collected between 2010 and 2012 in Nanoro, Burkina Faso. Merozoite surface protein 1 and 2 were genotyped by nested PCR on clinical samples. Associations between gametocyte carriage and factors, including multiplicity of infection, parasite density, patient age, gender, haemoglobin (Hb) level, and body temperature were assessed. The relationship between the presence of a particular P. falciparum and successfully genotyped, gametocytes were found in 48 samples (6.63%). There was no effect of patient gender, age and body temperature on gametocyte carriage. However, the probability of gametocyte carriage significantly increased with increasing values of multiplicity of infection (MOI). Furthermore, there was a negative association between parasite density and gametocyte carriage. MOI decreased with parasite density in gametocyte-negative patients, but increased in gametocyte carriers. The probability of gametocyte carriage decreased with Hb level. Finally, the genetic composition of the infection influenced gametocyte carriage. In particular, the presence of RO33 increased the odds of developing gametocytes by 2 while the other allelic families K1, MAD20, FC27, and 3D7 had no significant impact on the occurrence of gametocytes in infected patients.Of the 724 samples positive to This study provides insight into potential factors influencing gametocyte production in symptomatic patients. The findings contribute to enhance understanding of risk factors associated with gametocyte carriage in humans.Trial registration NCT01232530. Plasmodium parasites that are transmitted through the bites of infected female Anopheles mosquitoes. It is a life-threatening disease claiming about 405,000 lives each year worldwide [Malaria is an infectious disease caused by orldwide . Burkinaorldwide . ChildrePlasmodium species infecting humans, Plasmodium falciparum is the most dangerous involved in about 99% of overall malaria-related deaths [Plasmodium species in tropical settings, including in Burkina Faso. Multi-genotype malaria infections are frequent in endemic area and people commonly harbour several genetically distinct P. falciparum variants [msp1 and msp2) are commonly used as polymorphic markers to distinguish parasite genetic variants [Of d deaths . This isvariants –5. Multivariants , 7. In hvariants , 9. Usinvariants , 9–12.Anopheles mosquitoes. Although crucial for the understanding of the infectivity cycle and disease transmission, the ultimate and proximate mechanisms leading to gametocyte differentiation are not fully elucidated [During blood stage in humans, malaria parasites undergo either asexual or sexual differentiation leading to gametocyte production. Gametocytes are the sexual forms of the parasite assuring the continuity of the parasite life cycle in ucidated –16.versus reproduction trade-off [P. falciparum infection in patients and their ability to produce gametocytes [Contrasting predictions can be made regarding the relationship between gametocyte production and multiplicity of infection (MOI). First, the existence of within-host competition among distinct genotypes of malaria parasites , 9, 17 crade-off . In malaetocytes –25. Accoetocytes , 26, andetocytes , 28. Theetocytes .Plasmodium berghei with different capability to produce gametocytes were previously described [P. falciparum clones have a relatively higher capacity to produce gametocytes [msp1 (chromosome 9) and msp2 allelic families. Therefore, there is a good reason to postulate that allelic variability in msp1 and msp2 genes could influence gametocytogenesis in humans. This study explored parasite- and host-related factors associated with gametocyte carriage and the extent to which some specific P. falciparum variants are associated with gametocyte carriage in symptomatic patients in Burkina Faso.Besides genetic complexity, other factors generally associated with stressful conditions and pointing to deterioration in host conditions can drive gametocyte differentiation , 15. Theescribed . Geneticescribed , 37 and escribed . In natuetocytes . Causal etocytes , 41. VarPlasmodium species identification; (ii) positive versus negative results; and, (iii) if the higher count divided by the lower count was ≥ 2. In that case, the two closest readings among the three were considered.Samples for this report were collected between 2010 and 2012 during a pharmacovigilance for artemisinin-based combination therapy study carried out in Nanoro, Burkina Faso. The latter was described in detail elsewhere , 43. Mal+ . Undiluted blood obtained from finger prick was drawn into the microcuvette, which was inserted in the analyzer and Hb value in g/dL was read immediately. Electronic digital thermometers  were used for the measurement of axillary temperature. The tip of the thermometer was inserted under the armpit and numeric value of axillary temperature in degree Celsius was recorded.Haemoglobin (Hb) level was measured using Hemocue® 301msp) 1 block 2 gene  and msp2 block 3 gene (3D7 and FC27) genotyping by nested PCR as previously described. Briefly, 5 µL of DNA extract was used to initiate the first PCR round using a Mastercycler® Gradient,  and a Biometra thermal cycler  PCR machines. For the primary multiplex PCR round, the cycling conditions consisted of an initial denaturation step of 5 min at 94 °C, followed by 36 cycles of 1 min at 94 °C, 2 min at 58 °C and 2 min at 72 °C, and a final extension step of 10 min at 72 °C. One µl of this PCR product was used as DNA template to launch the nested round. For the nested msp1 round, the cycling conditions consisted of an initial denaturation step of 5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 2 min at 59 °C and 2 min at 72 °C, and a final extension step of 10 min at 72 °C. For the nested msp2 round, the cycling conditions consisted of an initial denaturation step of 2 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 45 s at 58 °C and 1.5 min at 72 °C, and a final extension step of 10 min at 72 °C. PCR amplicons were detected under UV light transillumination after ethidium bromide-stained agarose gel electrophoresis. DNA fragments sizes were calculated using Photo CaptMW (version 11.01) regarding fragments sizes of the reference which was a 100 Pb molecular weight marker from Thermo Scientific Fermentas® . Details about the genotyping procedure were previously reported [msp1 and msp2 and the final MOI value for each clinical isolate represented the maximum MOI value from both msp1 and msp2 loci as previously described in detail [msp1 and 3D7 or FC27 for msp2) on the basis of an occurrence of at least one band after DNA amplification using the family specific primers [Parasite genomic DNA was extracted from dried blood spots collected during the screening process of patients, i.e., before treatment administration using QIamp DNA Kit  following manufacturer’s instruction. DNA extract was used for merozoite surface proteins  on gametocyte carriage was tested using a GLM with binomial errors. Finally, a GLM with negative binomial errors was used to investigate the effect of MOI, parasite density, patient age, gender, Hb level, body temperature, and two-way interactions on gametocytaemia. Model simplification used stepwise removal of terms, followed by likelihood ratio tests (LRT). Term removals that significantly reduced explanatory power (p < 0.05) were retained in the minimal adequate model. Odds ratios and 95% confidence interval (CI) of the binomial models were also computed. Collinearity between explanatory variables was checked using the “vif” function of the “car” package.Clinical data were entered in an ACCESS database while molecular data were captured in a separate EXCEL file. All statistical analyses were performed with the R (version 3.5.1) software . A generP. falciparum and successfully genotyped, gametocytes were found along with asexual forms in 48 samples, i.e., a gametocyte carriage prevalence was 6.63% (48/724) with a mean gametocyte density of 186.9 ± 35.1 gametocytes/µl . This represents a minimum gametocyte prevalence since the sensitivity of microscopy is relatively low. All 724 positives samples were P. falciparum mono-infection.Of the 724 samples positive to LRT X21 = 5, P = 0.025, Fig. LRT X21 = 4.78, P = 0.029, Fig. The probability of gametocyte carriage significantly increased with increasing values of MOI  at low parasite densities; and, (ii) with host anaemia [versus asymptomatic including sub-patent parasite carriers) and parasite detection method, i.e., light microscopy against qPCR [Plasmodium species. In addition, reading malaria slides against 500 leucocytes rather than 1,000 leucocytes for gametocyte detection could have underestimated detection power.Results of this study support two predictions made by a model of transmission strategies in the rodent malaria parasite  anaemia . This st anaemia , 49. Thi anaemia . The difnst qPCR . GametocIn gametocyte-negative patients, MOI was lower in patients with heavy infections than in patients with mild infections, thus supporting the existence of within host competition with suppressive effect for disadvantaged strains . HoweverFurthermore, as pointed out by several other authors, this study identified host anaemia as a risk factor for gametocyte carriage, such that for every 1-point increase in Hb level, the odds of being gametocyte-positive decreased by 19% , 48, 52.There was no main effect of patient gender, age, body temperature, and other two-way interactions on gametocyte carriage, yet some of these host factors such as gender and body temperature were previously described as associated risk factors of gametocytaemia , 48. Thimsp1 and msp2 as polymorphic markers which are under immune pressure due to their involvement in the merozoite invasion [msp1 and msp2 genotyping. Nevertheless, the genetic composition of the infection influenced gametocyte prevalence. Previous reports suggested that different P. falciparum parasite variants may influence gametocyte carriage differently. A study reported that individuals carrying both 3D7 and FC27 allelic types of parasite had a higher risk of harbouring gametocytes compared to single 3D7 or FC27 allelic types [dhfr allele was previously reported [This study used invasion . This reic types . In addireported . In thisreported . TherefoThis study provides insight into potential factors influencing gametocyte production in symptomatic patients. The findings contribute to enhance understanding of risk factors associated with gametocytes carriage in humans.Additional file 1: Table S1. Determinants of the frequency of the K1 allelic family.Additional file 2: Table S2. Determinants of the frequency of the MAD20 allelic family.


Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone.Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species.Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens.MMSR detected the presence of any malaria marker in 50.2% of all tested samples with Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings. Plasmodium falciparum is responsible for 99% of malaria infections in Africa, while P. vivax causes the majority of infections in other parts of the world. Plasmodium knowlesi, which until recently was believed to infect only monkeys, was found a common cause of human infections in Southeast Asia [Plasmodium ovale and Plasmodium malariae are less commonly diagnosed species of malaria parasite found mostly in Africa but are also detected elsewhere [Plasmodium vivax and P. ovale require use of specialized anti-malarials to eliminate dormant stages that can cause relapses [Of the more than 200 million malaria cases estimated to occur each year, the overwhelming majority (90%) take place in sub-Saharan Africa . Sierra lsewhere , 4. Plasrelapses . It is tP. falciparum and/or P. vivax, but not P. malariae or P. ovale, and sensitivity for the latter species is significantly lower [P. malariae and P. ovale  may be significantly underestimated [P. falciparum with deletion of histidine-rich protein 2 and 3, which are relatively common in certain regions [Giemsa-stained thick and thin film microscopy is still considered the gold standard in malaria diagnostics, as it is relatively sensitive, quantitative, and allows for species identification. Since many resource-poor environments lack widespread expertise in microscopy and high quality equipment, the World Health Organization (WHO) recommends the use of rapid diagnostic tests (RDTs) in these settings . RDTs arly lower , 7–9. Asstimated . Another regions , 12.Plasmodium species [DNA-based diagnostics, also referred to as Nucleic Acid Amplification Tests (NAATs), such as DNA/RNA hybridization, conventional and real-time PCR, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA) and others can potentially fill this gap by providing a sensitive and accurate identification of multiple  species , 13. DNA species  compared species , 18 and  species , 19. Dep species .P. falciparum and P. vivax, and also detect presence of less common Plasmodium species via a universal malaria gene target. The results of this assay were compared with a well-characterized RDT assay and locally performed microscopy. The obtained results were consistent with previous observations that PCR-based tests have a significantly higher sensitivity when compared with both microscopy and RDTs [P. falciparum and P. vivax malaria species. Further analysis of these P. falciparum/P. vivax-negative samples revealed the presence of other Plasmodium species  circulating within the tested population.A novel, room temperature-stable, multiplex real-time PCR assay was used in this study to test blood samples from febrile patients in Bo, Sierra Leone. The Malaria Multiplex Sample Ready (MMSR) PCR assay can identify and RDTs , 16, 17.All patients seeking treatment at Mercy Hospital in Bo, Sierra Leone between February 5 and November 4, 2018, who had a clinically confirmed or self-reported fever starting not earlier than 10 days before the date of the interview, were invited to participate in the study, which was part of a bigger study testing the performance of experimental diagnostic devices. The research protocol used in this study was approved by the Sierra Leone Ethics and Scientific Review Committee and the institutional review boards of the US Naval Research Laboratory (NRL.2012.0007) and George Mason University (477605). Informed consent from patients  was obtained and documented prior to collection of clinical data or biological specimens. A total of 534 subjects consented to participate in the study. Samples of venous blood were collected from all study participants. In addition each subject participated in a survey which collected basic demographic information and also information related to previous infections, potential exposures and use of antibiotics before the enrolment. The participants were offered free standard-of-care testing at Mercy Hospital and referred for appropriate treatment when needed.P. falciparum, P. vivax, Plasmodium spp.  and RNaseP (sample extraction control).For PCR-based detection, DNA extracted from venous blood  was analysed using MMSR , a TaqMan-based real-time PCR assay. Developed and validated by researchers at Walter Reed Army Institute of Research (WRAIR) , 22, MMSIn parallel, venous blood samples were tested using the Malaria Ag Pf/Pan RDT  performed according to manufacturer’s instructions. Samples were also analysed by microscopy using Giemsa-stained thick smears according to current World Health Organization (WHO) standard operating procedures . At leasP. falciparum and P. vivax in the MMSR assay but with evidence of amplification of the universal malaria marker were retrospectively tested using a malaria speciation PCR assay [P. falciparum (Pf r364), P. vivax (Pv dhfr), P. malariae (Pm dhfr), P. ovale (Po dhfr) and P. knowlesi (Pk r140). The obtained 18S rRNA amplicons were sequenced for confirmatory identification of the detected species. All DNA sequencing was performed by Eurofins Genomics .Specimens negative for both CR assay . This reP. falciparum, while none were positive for P. vivax. The 87% concordance between P. falciparum-specific and universal malaria marker (Plasmodium spp.) detection was observed with the universal malaria marker identified in a noticeably smaller population of positive specimens . Only eight samples (1.5%) were positive for the universal malarial marker, but negative for both P. falciparum and P. vivax, suggesting the presence of other Plasmodium species.Table As expected, the distribution of malaria-positive samples as detected by MMSR assay was not uniform among the tested subject age groups. A significantly larger percentage of children under age 15 were positive compared to other age groups (p < 0.001), Table P. falciparum. Twenty-nine (22.5%) of these 129 were positive for both P. falciparum and Plasmodium spp.  antigens. No samples tested positive for Plasmodium spp. alone.Among specimens tested by the commercial RDT, 524 of 534 (98.1%) produced valid results. Slightly less than one-fourth of them  were positive for Plasmodium spp. Species identification based on microscopic images of the parasites was not attempted.A total of 523 samples were analysed by light microscopy (Giemsa-stained thick blood smears). Among these samples, sixty-seven (12.8%) were found positive for P. falciparum detection showed a 71.2% concordance between the two methods. The low concordance could be mostly attributed to the lower apparent sensitivity of the RDT as compared to the MMSR PCR  with slightly more than 10% (65 of 514) positive by all three methods Fig. . One samP. falciparum and P. vivax. An additional eight P. falciparum- and P. vivax-negative specimens showed evidence of universal malaria marker amplification, but did not reach the pre-determined threshold of detection (Ct < 35). All sixteen samples were retrospectively tested for the presence of other Plasmodium species using a real-time SYBR green-based PCR assay. The speciation PCR utilized six separate PCR amplifications to target markers specific for P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi as well as a universal malaria marker (Plasmodium spp.). Positive results were obtained in nine of the sixteen samples tested. Amplicons with the expected melting temperatures indicated the presence of P. falciparum in five samples, P. malariae in two samples, and P. ovale in two samples. None of these nine samples were positive for more than one species of Plasmodium. DNA sequencing of the 16S  amplicons detected in the same nine samples confirmed the Plasmodium species identification results.Among MMSR-tested specimens, eight were clearly positive for universal malaria marker but negative for both Plasmodium species and to identify the two predominant species responsible for human malaria, P. falciparum and P. vivax [This study explored the utility of a room temperature-stable, pre-aliquoted molecular assay for detection of malaria in a remote, low-resource location in West Africa. The MMSR PCR test was developed to detect multiple P. vivax , 22. TheP. falciparum parasites, as determined by PCR [Plasmodium vivax was not detected in any patients. The absence of P. vivax cases was expected, as this species is rarely encountered in West Africa [P. vivax DNA and/or antibodies against P. vivax proteins in some West African individuals; therefore, testing for this species has significant value [This study confirmed the earlier reports that approximately 50% of Mercy Hospital patients with a fever harbour d by PCR , 26. Plat Africa . Howevernt value , 28.P. ovale and P. malariae was detected, each of these species accounting for at least 0.8% (2 of 264) of all malaria infections identified by the MMSR assay. The prevalence of P. ovale and P. malariae parasites is likely to be higher since only the 16 samples positive (or nearly positive) for the universal malaria, but negative for P. falciparum and P. vivax were tested, which excluded detection of any mixed infections in P. falciparum-positive samples. Mixed infections have been documented at low but significant numbers in West Africa [Plasmodium species, which may improve the effectiveness of treatment regimens  and lessen the burden of long-term morbidity  [On the other hand, the presence of less common t Africa –31. Thisalariae) .The MMSR assay detected much greater numbers of malaria-positive samples than commercial RDT and microscopy. These results were consistent with earlier studies and the current knowledge on diagnostic sensitivity these techniques , 16, 17;One of the analysed specimens was positive by microscopy only. It is unclear whether this microscopic observation was a false positive related to difficulties with slide preparation and examination or was due to the presence of other  parasites . The accP. falciparum by the PCR used for species identification but negative by MMSR shows that a small proportion of P. falciparum parasites in the tested population may evade detection by MMSR. An alternative explanation may be that the RDT detected residual malarial antigens that can circulate within the bloodstream for several weeks following clearing of the infection [A small number of samples that were malaria-positive by RDT were found negative by both microscopy and MMSR (n = 12). These samples may represent true malaria positives for which both MMSR and microscopy failed to detect the malaria parasites or they may be RDT false-positives. The presence of MMSR false-negatives is possible as a result of sequence variability in the PCR primer binding sites used by MMSR assay. The observation of five samples (distinct from these 12 RDT-positive/MMSR-negative samples) that were positive for nfection . Additionfection –41.Plasmodium species in a single reaction) and capability of measurement of the parasite density with room temperature stability and simple setup requiring just one pipetting [® Malaria PCR Kits by Altona diagnostics) require transport and storage at − 20 °C and involve complex multi-step assay preparation. LAMP, a recently developed isothermal DNA amplification technology, holds promise of exceeding sensitivity of PCR-based malaria assays without the need for expensive and time consuming thermal cycling [Plasmodium species in ~ 60 min and require minimal hands-on manipulations for sample prep [The MMSR assay used in this study is only one of many NAATs developed specifically for malaria detection but has several unique advantages. While costly (14.50 USD each), it offers wide coverage (detection of all and identification of two most prevalent ipetting , 22. Mos cycling . At leasple prep –45. Howeple prep , 47. WhiPlasmodium than can be assumed based only on microscopy and RDT testing results [Plasmodium-positive persons should be treated [The high percentage of febrile patients at Mercy Hospital PCR positive for malaria in this and previous studies—combined with seasonal increases—suggest that a much larger part of the patient population is infected with  results , 26. App treated . However treated .Due to their cost and relative complexity, the WHO and malaria diagnostic community in general understandably do not consider NAATs—including PCRs—a viable alternative for routine diagnostics in most countries . HoweverPlasmodium species—represents a valuable tool for detection and epidemiological surveillance of malaria especially in low-resource areas directly affected and most impacted by this disease.Although not yet approved for diagnostic use, the MMSR—and indeed any sensitive molecular test capable of detecting multiple


Atherosclerotic remodeling starts early in life and can accelerate in the presence of cardiovascular risk (CV) factors. Regular physical activity (PA) can mitigate development of large and small artery disease during lifespan. We aimed to investigate the association of changes in body mass index (BMI), blood pressure (BP), PA behavior and retinal microvascular diameters with large artery pulse wave velocity (PWV) in prepubertal children over 4 years.The school-based prospective cohort study included 262 children initially aged 6–8 years, assessing the above CV risk factors and retinal vessels by standardized procedures at baseline (2014) and follow-up (2018). PWV was assessed by an oscillometric device at follow-up.P = 0.002). In contrast, increased vigorous PA corresponded to a lower PWV at follow-up . Progression of retinal arteriolar narrowing and venular widening were linked to a higher PWV after 4 years .Children with increased systolic BP over 4 years showed higher PWV at follow-up (β [95% CI] 0.006 [0.002 to 0.011] mmHg per unit, Increase in systolic BP and progression of microvascular dysfunction were associated with higher PWV after 4 years. Children with increasing levels of vigorous PA were found to have lower PWV at follow-up. Habitual vigorous PA has the potential to decelerate the process of early vascular aging in children and may thus help counteract CV disease development later in life.ClinicalTrials.gov, Identifier: NCT03085498. Both richildren  and may children .Preclinical measurements are required to determine CV health at early life exposure to initiate targeted preventive measures before clinical manifestations. Arterial stiffness is a non-invasive surrogate biomarker of the atherosclerotic process and of high clinical relevance given its predictive value of CV events and mortality in adults independent of other CV risk factors . An incrPhysical activity and fitness across the life-span impede the process of arterial stiffening , whereashttp://www.clinicaltrials.gov: NCT03085498).The Sportcheck Follow-up study is a large cohort study in primary school children from the City of Basel, Switzerland with baseline measurements in 2014. The children were between 6–8 years old at baseline and were reexamined 4 years later in 2018. The measurements took place in school settings and were divided into medical and physical fitness assessments. To participate in the medical screening, written consent from the parents was required. The physical fitness tests were performed during regular physical education and were mandatory for all children. The recruitment and screening process of the participants is illustrated in the flow chart . The stuAn oscillometric device  was used to assess central PWV. The technique represents a simple and valid method  with gooHeight and weight were assessed by a wall-mounted stadiometer  and a bioelectrical impedance analyzer  in light clothes and without shoes. BP measurements were performed according to the guidelines of the American Academy of Pediatrics . An autoA 20 m shuttle run was performed to assess CRF. It is a valid field test to estimate maximal oxygen uptake in children and adults . The parStatic retinal vessel diameters were assessed by a fundus camera  and analyzed with the semiautomated software Visualis . Two valid images from each eye were taken at an angle of 45 degrees with the optic disk at the center. Arteriolar and venular vessel diameters were identified in the area of 0.5 to 1 disk diameter from the optic disk margin. The Parr-Hubbard formula was used to average the central retinal arteriolar (CRAE) and venular (CRVE) equivalent . The AVRt-test of dependent samples. To determine the association of BMI, BP, CRF, PA, and retinal vessel diameters with PWV, a mixed linear regression model was performed considering schools as random effects  and standard deviation was calculated for the baseline and follow-up data and compared by a simple  effects . The ass effects . The firiameters . A poweriameters . A signi2), BP , and CRF (Δ 1.8 ± 1.7 stages) within childhood development. Regarding daily PA, the participants were less engaged in total PA (Δ 45.1 ± 83.3 min/day) and spent more time sedentary (Δ 32.7 ± 50.3 stages) at follow-up. However, the time spent in vigorous PA remained stable from baseline to follow-up with approximately 55 min per day. In comparison to baseline, narrower CRAE (Δ−6.3 ± 8.6 μm) and no significant changes in CRVE (Δ−0.2 ± 7.9 μm) were detected at follow-up. A mean PWV of 4.7 ± 0.3 m/s was observed at the age of 10–12 years.In total, 391 children were included at baseline aged 6–8 years and, 4 years later, 262 children were reexamined. 50 children were ill or relocated at the day of examination. The lost to follow-up rate accounts for 33%. Participants demographics at baseline and follow-up are presented in 2 increase, P = 0.860). The comparison of weight categories also revealed little evidence  that children with overweight or obesity  at baseline had a higher PWV at follow-up compared to their lean peers  at follow-up. A 0.01 unit decrease of retinal AVR over 4 years was associated with a higher PWV by 0.014 m/s , reflected by microvascular narrowing of CRAE .P = 0.047), independent of BP and weight . However, increase in CRF levels over 4 years did not correlate with lower PWV  at follow-up. Interestingly, the subgroup analysis of children with elevated or high systolic BP at baseline (n = 62) revealed a significant lower PWV at follow-up in those children who increased their total PA  and not vigorous PA. No association between increases in total PA  or vigorous PA  and PWV at follow-up was found in children with overweight or obesity.The association of CRF, PA behavior with PWV at follow-up are demonstrated in We investigated the association of 4 years changes in BMI, BP, PA behavior and CRF as well as retinal microvascular diameters with large artery PWV at follow-up in 262 prepubertal children. Increase in systolic BP during the observation period was associated with higher PWV at follow-up. A lower AVR over 4 years, reflected by retinal arteriolar narrowing was related to a higher PWV. In contrast, an increase in vigorous PA over 4 years contributed to a favorably lower PWV at follow-up.The results of our study indicate an positive association between prospective increase in systolic BP and PWV at follow-up. These results are in line but go beyond our previous report of an inverse correlation between BP and PWV at the cross-sectional level . In chilOur findings demonstrate no significant association between BMI at baseline and PWV at follow-up. In 9 years old children, body fat but not BMI was related to increased arterial stiffness in mid-adolescence . An adveWith respect to the cross-talk between the macro- and microvascular beds, narrowing of CRAE and widening of CRVE over 4 years, resulting in an unfavorable lower AVR, were related to a higher PWV at follow-up. However, the association of CRAE with PWV was not independent of BP and no direct association between CRVE and PWV was found. It was, therefore, the low retinal AVR which was inversely and independently associated with higher PWV at follow-up. From a physiological perspective, the BP-dependent modulation of the cross-talk between microvascular CRAE and large artery PWV seems comprehensible. In a simple model, blood flow, determined by pressure gradient and vascular resistance, is the decisive link between the micro- and macrovascular bed . The macOn the other hand, increase in vigorous PA over 4 years was related to lower arterial stiffness at follow-up independent of weight and blood pressure. The amount spent in total PA was not associated with improved arterial stiffness except for the subgroup of children with either elevated or high BP. However, this finding has to be verified in a larger cohort of children with increased risk of high blood pressure. Vigorous PA increases blood flow and laminar shear stress and leads to an increased nitric oxide production and pronounced vasodilation. An increased stimulus of shear stress and nitric oxide bioavailability over time, induced by vigorous PA, may lead to a favorable vascular wall integrity and may explain why increases in total PA were not sufficient to improve PWV at follow-up. A detailed investigation of the underlying mechanisms was beyond the scope of this study. Similar results were found in preschool children, indicating that higher levels of moderate to vigorous PA can contribute to a slower age-related progression of PWV . The retWe found little evidence for an association between changes in CRF at baseline and arterial stiffness at follow-up. In childhood, more than half of the individual differences in maximal oxygen uptake are determined by genetic factors . HeritabSome limitations need to be addressed. 33% of the included participants did not participate in the follow-up measurements due to illness at the day of examination, relocation or personal reasons. However, the population characteristics of the loss to follow-up group did not differ notably form the follow-up group  and, thun = 235). Two retinal vessel images of each eye were semi-automatically analyzed to guarantee high accuracy. Reference values for the retinal vessel diameters in children and adults are not available yet.The clinical relevance of quantifying arterial stiffness in childhood is unclear and requires future research to establish evidence-based recommendations for the assessment of arterial stiffness in children. Furthermore, we carefully assessed BP status with five separate measurements for each participant. However, the measurements were taken on a single occasion and did not allow to identify children with hypertension or white coat hypertension. PA behavior was assessed by a parental questionnaire and entails the risk of response and recall bias. Nevertheless, the chosen method allowed to measure a large sample size and we achieved a relatively high response rate of 80% . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.GL conceptualized and designed the study, collected data, performed the statistical analysis, prepared, and revised the manuscript. CH and SK revised the manuscript. DI performed the statistical analysis, reviewed, and revised the manuscript. KE conceptualized and designed the study and revised the manuscript. OF critically reviewed the manuscript. HH conceptualized and designed the study, helped perform the statistical analysis, prepared, and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium species circulating within the population over a period can inform appropriate treatment recommendations. This study monitored variations in the prevalence of four common human Plasmodium species carried by children with asymptomatic malaria infections over a two-year period.  Monitoring changes in the composition of the μl of finger-pricked blood was used to prepare thick and thin blood smears as well as spot Whatman® #3 filter paper. Genomic DNA was extracted from the dried blood spots and used in PCR to amplify the 18S rRNA gene from four different human Plasmodium parasites.  Two cross-sectional studies were conducted in November 2017 and December 2019. A total of 210 children aged between 4 and 13 years were recruited in 2017, and 164 similarly aged children were recruited in 2019. Approximately 150 P. falciparum detected by PCR was relatively similar at the two time points . However, the prevalence of PCR detectable P. malariae increased by 8.5-fold, whilst P. ovale increased from 0 to 9% in the children sampled in 2019 relative to the children sampled in 2017. The only parasite species identified by microscopy in this study was P. falciparum, and no P. vivax was identified by either microscopy or PCR in the study population during the study period.  Parasite prevalence by microscopy and the prevalence of P. malariae and P. ovale, whose population is expanding in parts of Ghana including Simiw. There is the need to implement molecular diagnostic tools for malaria parasite surveillance in Ghana. This will enable the identification and treatment of all circulating malaria parasites including  All human malaria parasite species are capable of causing symptomatic as well as asymptomatic infections . SymptomPlasmodium parasites contained in asymptomatic infections [P. malariae and P. ovale, is greater than previously reported [P. malariae and P. ovale most often present as low-density asymptomatic infections that require molecular tools for accurate detection [The gold standard for the diagnosis of symptomatic malaria in endemic areas is microscopy. However, the inability of microscopy to detect low-density infections –7 does nfections . A numbereported , 10. Infetection , 12.falciparum Plasmodium parasites including P. ovale and P. malariae is less common than P. falciparum in sub-Saharan Africa. Although P. falciparum is the leading cause of symptomatic malaria in sub-Saharan Africa, including Ghana [P. malariae and P. ovale can cause chronic infections [P. falciparum prevalence to be as high as 31%, 32%, and 44%, respectively [The prevalence of the non-ng Ghana , P. malafections  and havefections . Some stectively , 9.P. falciparum-endemic country, Tanzania, has suggested that a decrease in the prevalence of P. falciparum can result in an increase in the prevalence of non-falciparum malaria parasites [P. falciparum in Ghana over the years could result in an increase in the prevalence of the non-falciparum malaria parasites due to a decline in the prevalence of P. falciparum. In this study, we sought to determine the changes in the prevalence of four common human malaria parasite species carried by asymptomatic children living in Simiw, a community in the Central Region of Ghana over a two-year period.Successful malaria control interventions are expected to reduce the prevalence and density of malaria parasites circulating within a population over a period of time. The effectiveness of malaria control interventions implemented in a country or community can therefore be evaluated by determining changes in the prevalence and density of malaria parasites contained within infected individuals over time. An earlier report from a arasites . This suThe Institutional Review Board of the Noguchi Memorial Institute for Medical Research granted ethical clearance for this study . Permission to conduct this study was also obtained from the Komenda Edina Eguafo Abrem (KEEA) Municipal Director of Education. The study was explained to the parents and or guardians of the school children as well as the school children through a series of interactive meetings. Parents/guardians who were willing to enroll their children into the study provided written parental consent prior to the child being recruited into the study. Children aged 12 years and above were made to endorse a child assent form.Two cross-sectional studies were conducted over a two-year period. The first was conducted in November 2017, where 210 children aged between 4 and 13 years were enrolled. The second study was conducted in December 2019 where 164 children aged between three and 13 years were recruited. The study was conducted in Simiw, a community in the KEEA District of the Central Region of Ghana . Recruitμl of finger-pricked blood was obtained from each child. The blood was used to prepare a thick and thin blood film as well spot a strip of Whatman® #3 filter paper . The blood films were processed for microscopy, whilst the blood spots were air-dried and stored in a Ziplock® bag containing silica gel beads. The samples were then transported to the NMIMR for onward processing and malaria parasite identification.Approximately 150 Plasmodium parasite density (PD) was estimated by multiplying the number of parasites identified per 200 WBCs by 40 [Plasmodium parasites if no parasite was observed in 200 high-powered fields. Discordant slides in terms of presence or absence of Plasmodium parasites were re-examined by a third malaria microscopist. Discordant results agreeing with the third reading were considered final.The blood films were processed using the standard WHO protocol  and readCs by 40 . A thickμl aliquot of 6% Chelex-100 was then added to each spot and heated at 95°C for 10 minutes. Finally, tube containing the heated mixture was centrifuged at 12,000 × g for 1 minute, and 120 μl of the supernatant containing the extracted DNA was transferred into a new sterile labeled 0.5 ml microcentrifuge tube. The DNA was stored at −20°C or used immediately.Genomic DNA was extracted from the dried filter paper blood spots (DBS) using the Chelex-saponin extraction method as previously described  with ver18s rRNA gene of P. falciparum, P. malariae, and P. ovale were amplified from extracted genomic DNA using a previously described PCR protocol [18s rRNA gene of P. vivax was amplified using a similar protocol [μl primary reaction mixture contained 20–40 ng of DNA, 80 nM of the genus specific primer rPLU5 (forward) and rPLU6 (reverse), 1X PCR buffer, 167 nM dNTP mix, 2.5 mM MgCl2, and 1 U of OneTaq DNA polymerase.The conserved regions of the protocol . The 18sprotocol . The 15 μl of the primary reaction product, and the primer set used was one of the four species-specific primers rFAL1/rFAL2 (133 nM), rMAL1/rMAL2 (333 nM), rOVA1/rOVA2 (333 nM), and rViv1/rViv2 (333 nM) (Supplementary Four independent nested (secondary) reactions were performed, with each set up containing a similar composition of reagents as the primary reaction; however, the template used was 0.5 The primary and nested PCR reaction cycling conditions included an initial denaturation at 95°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, 55°C (for primary) or 58°C (for nested) for 1 minute, 68°C for 1 minute, and a final extension at 68°C for 5 minutes. The nested PCR products were resolved on a 2% agarose gel prestained with ethidium bromide and subsequently visualized on a Vilber gel documentation system .P. falciparum, P. malariae, and P. ovale (Fisher's exact test) in the two sites.IBM SPSS ver 22 was employed in the descriptive statistics such as frequency and also to identify significant difference between parasite prevalence and PCR-detectable T-test was used to identify differences in the mean age and parasite densities. Differences in the prevalence of males as well as the prevalence of the different Plasmodium species detected by PCR were determined using Fisher's exact test. p values less than 0.05 were considered statistically significant.The results were either described as a whole or categorized into young children aged below 10 years and older children aged 10 years and above. The independent samples There were a total of 374 children aged between 3 and 13 years recruited into this study. Although the age range of the children recruited in 2017 and 2019 were similar, the children recruited in 2019 had a higher mean age than those recruited in 2017 . There wp=0.56)  . Althougp=0.56) . The onlN = 150 in 2017 and N = 35 in 2019) and older children aged 10 years and above (N = 60 in 2017 and N = 129 in 2019), the parasite density in the children aged below 10 years in both 2017 and 2019 years was similar. However, a significant increase in parasite density was observed in children aged 10 years and above in 2019 relative to similarly aged children in 2017  and 57.9 (95/164) of the children recruited in 2017 and 2019, respectively, to have asymptomatic infections . There was a significant increase in the number of children aged 10 years and above that were infected with P. falciparum in 2019 relative to similarly aged children 2017  of children from 2017 and 20.7% (34/164) of children from 2019 to harbor asymptomatic infections. In 2017 as well as 2019, a significantly higher number of older children (≥10 years old) tested positive for P. malariae relative to the younger children (<10 years)  and 3% (1/34) of the children from 2017 and 2019, respectively  . Plasmodectively . The remlciparum .P. ovale species-specific PCR; however, 9.1% (15/164) of the children tested in 2019 had P. ovale. A majority  of the P. ovale infections were contained in mixed infections with P. falciparum.None of the samples collected in 2017 was identified as positive by the The difference in parasite prevalence between children under 10 years old and those 10 years old and above was similar over the study duration, although smaller in 2017 (22.6%) than in 2019 (21.9%) . The chiThe National Malaria Control Program of Ghana has implemented numerous vector control interventions including enhanced distribution of insecticide-treated materials and adulticiding and larviciding of mosquitoes in addition to parasite control interventions including adapting the T3: Test, Treat, Track Initiative. These interventions have resulted in a reduction in the incidence and prevalence of severe manifestations of malaria and uncomplicated malaria but not asymptomatic malaria . The escP. falciparum in asymptomatic infections to have remained stable over the two-year study period. This supports an earlier report that malaria control in several parts of sub-Saharan Africa, including Ghana, remains a substantial challenge [This study determined the prevalence of children harboring hallenge . Behaviohallenge , 25 and hallenge , have behallenge  as well P. malariae over the two-year period is likely a result of P. malariae presenting primarily as asymptomatic infections, which are not treated or symptomatic infections that are treated with alternative antimalarials other than the first-line ACTs. The WHO recommended treatment for P. malariae and P. falciparum is artemisinin-based combination therapy (ACTs), which when taken appropriately should clear both P. falciparum and P. malariae. The WHO recommended treatment for P. ovale or P. vivax is an ACT plus primaquine to clear the hypnozoites [P. ovale on the Ghanaian market [P. ovale in Simiw. The hypnozoite stage of P. ovale is known to become active after several days to months of dormancy. Infections with P. malariae and P. ovale most often present as asymptomatic infections [P. ovale and P. malariae [falciparum malaria parasites across Ghana and not just in Simiw. The trend of an increase in parasite carriage by older children relative to younger children has also been identified in studies from other parts of Ghana including the Eastern Region [The significant increase in the prevalence of nozoites . The absn market  is likelfections , which afections . A previmalariae , suggestn Region  and in on Region  and Ugann Region . Youngern Region .Plasmodium malariae and P. ovale parasites were predominantly identified in mixed species infections containing P. falciparum. Malaria infections containing P. falciparum in addition to a non-falciparum parasite is a common occurrence in many tropical regions where Plasmodium falciparum is endemic [ endemic  includin endemic  and Ugan endemic .P. malariae and P. ovale, whose population is expanding in parts of Ghana including Simiw.There is the need to implement the use of molecular diagnostic tools for malaria parasite surveillance. This will enable the identification and treatment of all infecting malaria parasites including


Plasmodium falciparum is widespread in adults and children living in malaria-endemic countries. This study identified the prevalence of malaria parasites and the corresponding levels of naturally acquired anti-parasite antibody levels in afebrile adults living in two communities in the Greater Accra Region of Ghana.Asymptomatic carriage of Two cross-sectional studies conducted in January and February 2016 and repeated in July and August 2016 recruited subjects aged between 6 and 75 years from high parasite prevalence (Obom) and low parasite prevalence (Asutsuare) communities. Whole blood (5 ml) was collected from each volunteer, plasma was aliquoted and frozen until needed. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells and each stored at − 20 °C until needed. Anti-MSP3 and Pfs230 antibody levels were measured using ELISA.Asexual parasite and gametocyte prevalence were higher in Obom than Asutsuare. Antibody  responses against the asexual parasite antigen MSP3 and gametocyte antigen Pfs230 were higher in Obom during the course of the study except for IgM responses against Pfs230, which was higher in Asutsuare than in Obom during the rainy season. Antibody responses in Asutsuare were more significantly associated with age than the responses measured in Obom.The pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season. Plasmodium parasites [Asymptomatic carriage of malaria parasites has been associated with the development of immunity to malaria mainly due to continuous exposure of the host’s immune system to the arasites . Low pararasites . The levarasites .Plasmodium parasites within the human erythrocyte, which is critical for the survival of the parasite is initiated by the merozoite. The merozoite is one of several daughter cells released from a mature schizont, which proceed to invade a new host erythrocyte and continue the asexual erythrocytic cycle of the parasite [Plasmodium falciparum merozoite, including the merozoite surface protein 1 (MSP1) and 3 (MSP3) have been validated as malaria vaccine candidates [The propagation of parasite . Merozoindidates , 5 due tndidates , 7. Durindidates , 9. Pfs2ndidates –12 and mndidates . Antibodndidates , 15. Sucndidates –18. Thesndidates  and are Plasmodium infections as they have associated with protections against infection [Immunoglobulin G (IgG) antibodies have been shown to be a very important component of humoral immunity in the fight against nfection –22 and tnfection , 24. Cytnfection –29. Moninfection . A few enfection  or in a nfection . Other snfection , asexualnfection , 33 or onfection  antibodyThe Institutional Review Board (IRB) of Noguchi Memorial Institute for Medical Research granted ethical approval for the study (Study number 089/14-15). Written informed consent was obtained individually from all participants before they were enrolled into the study.This study was carried out in Obom and Asutsuare Fig. , both inVolunteers aged between 6 and 75 years old and without any symptom of malaria were recruited into a cross-sectional study in February 2016 (dry season), which included 264 volunteers from Obom and 230 volunteers from Asutsuare. In July 2016 (rainy season) a second set of volunteers was recruited, 192 from Obom and 174 from Asutsuare, which included 120 volunteers from the off-peak season sample collection for both sites. Venous blood (5 ml) was collected from each volunteer into acid dextrose vacutainer tubes on the day of recruitment. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells via centrifugation and independently stored at − 20 °C until used.P. falciparum parasites [The thin and thick blood smears were processed and used to identify and quantify the presence of arasites . Briefly® Green 2X master mix RT-PCR kit (Applied Biosystems). The reactions were run in triplicate on a QuantStudio 3™ Real-Time PCR System . The primer validation and all positive and negative controls used in this experiment have been previously described [RNA was isolated from the Trizol-preserved whole blood using the Quick RNA miniprep kit  according to manufacturer’s instructions and subsequently converted into cDNA using the Protoscript II first strand cDNA synthesis kit  as previously described . Genomicescribed , 40.P. falciparum sexual stage and asexual stage antigens were quantified using an indirect ELISA protocol [Lactococcus lactis. Briefly Pfs230 antigen was diluted to 1 µg/ml in carbonate buffer [2SO4 and optical densities (OD) read at 450 nm using a 96-well ELISA plate reader .Antibody responses including IgG, IgM, IgG1, and IgG3 against recombinant protocol . The antprotocol  and MSP3protocol  producede buffer , 15 and The cut-off for positivity for gametocyte presence by RT-PCR as determined by the no template control was ‘undetermined’. Any sample that yielded a CT value other than ‘undetermined’ was scored as positive.For each measured antibody, OD data were normalized against the standard calibrator. OD data were converted into concentration in arbitrary units using the 4-parameter logistic curve-fitting program, ADAMSEL . Log10-transformed OD data from naïve malaria volunteers from the two seasons were used to define a common cut-off from which seroprevalence was calculated as the population of sample with Log10-transformed ODs higher than the common cut-off. Analysis of data and graphics was performed using Kruskal–Wallis test, Spearman correlation and other statistical tests (Graph Pad Prism version 7). Statistical significance was set as P ≤ 0.05, unless otherwise stated.The study recruited 230 and 174 volunteers from Asutsuare during the dry (January–February) and rainy (July–August) seasons, respectively, and 264 and 192 volunteers from Obom during the dry (January–February) and rainy (July–August) seasons, respectively. Volunteers aged 10 years and below were the least represented in both sites at both sampling time points, and participants aged above 15 years were the most represented in Asutsuare at both sampling time points  and Spearman r = 0.2633, p = 0.0001 (IgG3), whilst in Asutsuare, the correlations although higher than Obom were still weak; Spearman r = 0.3341 (IgG1), Spearman r = 0.4057 (IgG3), p < 0.0001 for both)  Fig. a, b signAnti-IgG3 responses measured in Obom against MSP3 remained significantly lower in the young children (≤ 10 years) compared to the adults (≥ 16 years) , however, IgG, IgG3 and IgM levels across the three age groups were similar during the rainy season . All variance inflation factors (VIFs) for all the analyses were less than 1.8  could not explain the variations observed in total IgG responses in Obom and could only account for 3.3% of the variations in total IgG observed in Asutsuare Table . In the Within the community, all antibody responses measured against Pfs230 in Obom were significantly  higher than that measured in Asutsuare. Moving from the dry to the rainy seasons, naturally induced IgG responses against Pfs230 decreased significantly   very weak negative correlation was observed between age and IgM responses in Asutsuare.During the rainy season, age positively  correlated with anti-Pfs230 total IgG in volunteers from Obom, whilst age correlated significantly  with IgG3 responses in Asutsuare. Anti-IgG1 responses against Pfs230 from both sites correlated negatively with age but the correlation was not significant .2 increased to 0.455 (p < 0.001 for Asutsuare) and 0.313 (p < 0.001 for Obom)  responses. There were too few samples with microscopic densities of parasites in Asutsuare to do any statistical analysis  forms of the parasite. Inhabitants of malaria-endemic countries, especially children, are highly at risk of being infected with the malaria parasite and have previously been the focus of earlier studies to understand and identify differences in asymptomatic parasite carriage in high and low malaria transmission communities in Ghana [Asymptomatic carriage of in Ghana , 40. In P. falciparum parasite prevalence has been reported to be very low for over a decade [As anticipated, the prevalence of asymptomatic carriers was significantly higher in Obom, the high parasite prevalence setting, than in Asutsuare where a decade , 36. No a decade . Howevera decade . The absa decade .P. falciparum parasites remained the same in moving from the dry to rainy season. One possible explanation for the increase in IgM in rainy season could be an increase in the prevalence of sub-microscopic density infections in the rainy season that was not captured by microscopy but has been reported using more sensitive tools [A significant increase in the community-wide levels of anti-MSP3 IgM was anticipated and confirmed in moving from the dry to the rainy season in Obom, where there was a subsequent increase in asexual parasite prevalence. Interestingly, a similar significant increase in anti-MSP3 IgM was observed in Asutsuare, although asymptomatic carriage of microscopic densities of ve tools . In Asutve tools .Cytophilic (IgG1 and IgG3) responses against MSP3 in Asutsuare were similar in both the dry and rainy season, where the adults had significantly higher levels than both groups of children. This result suggests that more frequent exposure is likely to be required to mature cytophilic antibody responses , buttresThe reduction in the levels of Pfs230 IgG in moving from the dry to the rainy season observed in both Obom and Asutsuare could be due to a reduction in the number of gametocyte carrier, especially in Obom were fewer participants with active gametocyte infections were identified in the rainy relative to the dry season. The higher levels of Pfs230 IgM observed in Asutsuare relative to Obom in the rainy season could be due to very recent gametocyte infections in some participants in Asutsuare as antibody responses to gametocyte antigens have been suggested to develop rapidly after exposure . SimilarThe decrease in anti-Pfs230 IgG and IgG1 with increased exposure was not unexpected as antibody responses to gametocyte antigens have been suggested to be influenced more by recent exposure compared with cumulative exposure . AlthougThe pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen, MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites likely due to requirement of IgG1 to clear gametocytes from circulation. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season.Additional file 1. Statistical analysis of antibody responses obtained at each site during both the dry and rainy season.Additional file 2. Regression analysis of antibody responses obtained at each site during both the dry and rainy season.Additional file 3. Correlation analysis of antibody responses obtained at Obom with age and parasite density.