Intermittent preventive treatment (IPT) is a well established malaria control intervention. Evidence that delivering IPT to schoolchildren could provide community-level benefits is limited. We did a cluster-randomised controlled trial to assess the effect of IPT of primary schoolchildren with dihydroartemisinin-piperaquine (DP) on indicators of malaria transmission in the community, in Jinja, Uganda.ClinicalTrials.gov, number NCT02009215.We included 84 clusters, each comprising one primary school and the 100 closest available households. The clusters were randomly assigned 1:1 to receive IPT with DP or standard care (control) by restricted randomisation to ensure balance by geography and school type. Children in intervention schools received IPT monthly for up to six rounds . We did cross-sectional community surveys in randomly selected households at baseline and in January to April, 2015, during which we measured participants' temperatures and obtained finger-prick blood smears for measurement of parasite prevalence by microscopy. We also did entomological surveys 1 night per month in households from 20 randomly selected IPT and 20 control clusters. The primary trial outcome was parasite prevalence in the final community survey. The primary entomological survey outcome was the annual entomological inoculation rate (aEIR) from July, 2014, to April, 2015. This trial is registered at vs 23%, adjusted risk ratio 0·85, 95% CI 0·73–1·00, p=0·05). The aEIR was lower in the intervention group than in the control group, but not significantly so .Among 23 280 students registered in the 42 intervention schools, 10 079 (43%) aged 5–20 years were enrolled and received at least one dose of DP. 9286 (92%) of 10 079 received at least one full course of DP (three doses). Community-level parasite prevalence was lower in the intervention clusters than in the control clusters (19% IPT of schoolchildren with DP might have a positive effect on community-level malaria indicators and be operationally feasible. Studies with greater IPT coverage are needed.UK Medical Research Council, UK Department for International Development, and Wellcome Trust. Over the past decade, reductions in malaria burden have been documented worldwide after heavy investment in control measures.Intermittent preventive treatment (IPT) is a well established malaria control intervention, recommended for use in pregnant women and infants in specific settings, and for children younger than 5 years in areas of seasonal transmission. The malaria burden in school-aged children has been underappreciated,Evidence before this studyWe searched PubMed for original articles published in English between Jan 1, 2000, and Oct 1, 2017, with the term “intermittent, preventive treatment AND malaria OR Plasmodium falciparum AND schoolchildren NOT infant NOT pregnancy”. We assessed titles and abstracts and found no studies that had investigated the effects of intermittent preventive treatment (IPT) for malaria in schoolchildren on malaria transmission at the community level. One systematic review had assessed the efficacy and safety of IPT in schoolchildren. This review included five studies, including four individually randomised trials and one cluster-randomised trial, which were done in 2002–12 in Kenya, Mali, and Uganda, but none reported population-level health effects. Three later studies of school-based malaria interventions included one individually randomised trial of IPT for malaria and helminths in Ghana, one individually randomised trial of IPT for malaria in the Democratic Republic of Congo, and one cluster-randomised trial of IPT for malaria plus distribution of long-lasting insecticide-treated nets in Mali. Again, none assessed population-level outcomes. One stepped-wedge, cluster-randomised trial in Senegal assessed community-level outcomes but with seasonal malaria chemoprevention in schoolchildren and not IPT. In that trial, children younger than 10 years received amodiaquine and sulfadoxine-pyrimethamine, administered by community health workers via health posts. No reduction in all-cause mortality (the primary outcome) was seen, but the incidence of confirmed malaria in community residents too old to receive seasonal malaria chemoprevention reduced significantly by 26%. These results were encouraging, but the intervention, method of assessment, and outcome measures differed substantially from those in our trial, precluding direct comparisons.Added value of this studyThis study in Jinja, Uganda, makes an important contribution to the limited evidence on use of IPT as a tool to reduce malaria transmission. The use of dihydroartemisinin-piperaquine for IPT of malaria in schoolchildren was associated with reductions in parasite prevalence and annual entomological inoculation rate, but these were of borderline significance. Intervention coverage, however, was lower than expected.Implications of all the available evidenceEvidence on the effects of providing IPT of malaria to schoolchildren is limited, but promising. Schoolchildren are major contributors to the infectious reservoir, and providing IPT in schools offers an operationally attractive and potentially sustainable intervention that could be integrated with currently deployed malaria control methods. Additional studies are needed to explore these findings further, including assessments of the population-level effects with greater intervention coverage.In the School-based Treatment with ACTs to Reduce Transmission of malaria (START-IPT) trial, we investigated whether IPT for malaria with dihydroartemisinin-piperaquine (DP) in schoolchildren would affect community-level indicators of malaria transmission in Jinja district, Uganda. DP is highly efficacious and well tolerated, and is more effective than other artemisinin-based combination therapies in preventing new infections because of the long half-life of piperaquine, making it an attractive option for IPT.START-IPT was a cluster-randomised controlled trial. We obtained ethics approval from the Ugandan National Council for Science and Technology (reference HS 1530); Makerere University School of Medicine Research and Ethics Committee, Kampala, Uganda (SBS 145); the London School of Hygiene & Tropical Medicine Ethics Committee, London, UK (6509); the School of Biological and Biomedical Sciences Ethics Committee, Durham University, Durham, UK; and the University of California, San Francisco Committee on Human Research, San Francisco, CA, USA (074826). Sponsorship and insurance was provided by the London School of Hygiene & Tropical Medicine Clinical Trials Sub-Committee (reference QA380). The trial was overseen by an independent data and safety monitoring board and a trial steering committee. We obtained verbal consent from schools and written consent from students and community residents (with assent from children aged ≥8 years).Jinja district is an area of perennial malaria transmission in eastern Uganda. The district has 11 subcounties that range from periurban areas of Jinja town , height, and weight. We recorded participants' fingerprints to facilitate future identification for DP treatment.The DP preparation was full-strength Duo-Cotexcin tablets , containing 40 mg dihydroartemisinin and 320 mg piperaquine and taken orally. IPT was delivered to students in intervention schools by study personnel, with doses given once daily for 3 days per month (one round of treatment), according to weight-based guidelines , for up To assess safety we monitored all intervention participants for serious adverse events, and a subset of participants selected by convenience sampling for cardiac monitoring. The relationship of serious adverse events to DP was assessed by study staff, and all serious adverse events were reported to the institutional review boards and the trial's data and safety monitoring board. Detailed results on safety will be reported separately.Cross-sectional community surveys were done in households randomly selected from each cluster at baseline  and after the intervention . The sampling frame for the baseline survey was generated from the digital enumeration done to generate clusters, and for the final survey from a census of clusters. Each survey was done in a different random sample.Study personnel visited households for both community surveys to identify those with an adult resident available to do the survey who met the following eligibility criteria: a usual resident present on the night before the survey, age 18 years or older, and agreement to provide written informed consent. Households were excluded if the dwelling could not be found or was vacant or if no adult resident was at home on more than three visits. If selection criteria were met, a household questionnaire was administered to the head of the household or their designate to gather information on all residents, ownership and use of all bednets, and proxy indicators of wealth.The target sample sizes of age groups varied because of age-related differences in parasite prevalence. Thus, recruitment of household residents into the community clinical surveys was stratified by age. For the baseline survey, all members of a household were eligible for inclusion. For the final survey, all members of a household aged 4 months to younger than 5 years or older than 15 years were eligible for inclusion, but for residents aged 5–15 years, only one per household could be included, and was chosen at random by random number tables. To participate in the clinical survey, household residents were reviewed for selection criteria, including being a usual resident of the household on the night before the survey, ability to locate the resident, appropriate age, provision of written informed consent for adults or parents or guardians of children, and provision of written assent from children aged 8 years and older.Plasmodium falciparum  in participants who had fever (tympanic membrane temperature ≥38·0°C) or history of fever in the previous 48 h.At the time of the survey, we measured each participant's temperature and obtained a finger-prick blood sample that was used to prepare a thick blood smear to measure haemoglobin concentration with a portable spectrophotometer  in children younger than 5 years. Samples were stored on filter paper for future molecular testing. Rapid diagnostic testing was done with CareStart Malaria HRP2 for We did an entomological survey in 200 households, five per cluster in 20 of the IPT clusters and five per cluster in 20 of the control clusters. The clusters and households were randomly selected before the survey started. Selection criteria were as follows: the ability to locate the household; residence in the subcounty for the previous 6 months; and provision of written informed consent by the head of the household (or designate). Participating households were sampled one night per month with miniature Centers for Disease Control and Prevention light traps . Traps were positioned 1 m above the floor at the foot end of the bed where a person slept under an insecticide-treated net.9/L). We classified the result as negative if examination of 100 high-power fields revealed no asexual parasites or gametocytes. For quality control, all slides were read by a second microscopist, and a third reviewer designated discrepant readings.Thick blood smears were stained with 2% Giemsa for 30 min and read by experienced laboratory technologists who were unaware of study group assignments. Parasite and gametocyte densities were calculated from thick blood smears by counting the number of asexual parasites and gametocytes, respectively, per 200 leucocytes , assuming a leucocyte count of 8000 cells per μL  and analysed by loop-mediated isothermal amplification (LAMP) for detection of The primary outcome was prevalence of asexual parasitaemia in the final community survey, as measured by microscopy of individual blood smears. Secondary outcomes were prevalence of parasitaemia by microscopy and LAMP, prevalence of gametocytaemia, prevalence of anaemia,The target sample size for the baseline survey was based on age-stratified estimates of parasite prevalence from Walukuba subcounty in Jinja district.vs 15%). Additionally, we weighted the sample sizes of age groups to ensure 80% power to detect a relative difference in parasite prevalence of 30·0% between study groups for children younger than 5 years (n=64), 34·5% for children aged 5–15 years (n=14), and 40% for individuals older than 15 years (n=27).Sample size calculations for the final community survey were informed by data collected in the baseline survey. Calculations accounted for correlation among clusters by calculating the coefficient of variation (k).For the entomological survey, we calculated a k value of 0·57 for variation between houses, based on preliminary data from Uganda . With this level of variation, we calculated that five households in 20 clusters in each study group would be sufficient to detect a 50% reduction in aEIR in the IPT group with 80% power and 5% significance.We did all analyses by intention to treat. Thus, all community residents were classified as participating in the intervention or control irrespective of whether they (or their children) received the intervention. For all analyses, we used statistical methods that allowed for within-cluster correlations, and analysed data with STATA/IC version 12.1. All outcomes were assessed at the individual level because of the large number of clusters per study group.For binary prevalence outcomes, we used generalised linear Poisson models with log link function. We compared the effects of the intervention between groups with prevalence risk ratios (RRs) and 95% CIs. The probability of selection for the final community survey was related to the sampling frame, which was determined by the expected parasite prevalence in the age categories younger than 5 years, 5–15 years, and older than 15 years. Therefore, the age structure of the study population was not representative of the community population overall. To account of this discrepancy, we assigned individuals one of three weights based on their age, calculated as the inverse of the percentage of people in each age group in the census survey. Population estimates of prevalence were obtained with svy commands in STATA, with the cluster as the primary sampling unit.Anopheles spp mosquitoes captured/number of house-nights of collection × 365·25 days per year. The sporozoite rate was calculated as the number of mosquitoes positive for sporozoites / the number of mosquitoes tested. The aEIR was the product of the annual human biting rate and the sporozoite rate. We compared aEIRs between study groups with negative binomial regression (to account for overdispersion), with random effects for clusters and repeated measures. The number of mosquitoes positive for sporozoites was used as the outcome with an offset variable of the natural log of the number of house-nights of capture and having a coefficient constrained to 1. Households selected for the entomological survey were randomly selected and, therefore, needed no weighting. We did a subgroup analysis to assess whether the effect of the intervention changed over time in three time periods after the intervention was first implemented: 1–3 months , 4–6 months , and 7–10 months . This trial is registered at ClinicalTrials.gov, number NCT02009215.The annual human biting rate was calculated as total number of female The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Of 86 eligible clusters 84 were included in the study : 42 wereThe baseline community survey was done from March to June, 2014, and included 2994 households  and 10 031 participants  of anophelines collected were A gambiae sensu lato, 277 (4%) A funestus, 51 (1%) A arabiensis, and one (<0·5%) another species. The number of female Anopheles spp mosquitoes, the human biting rate, sporozoite rate, and aEIR were all lower in the intervention group than in the control group (In the entomological survey, 27 034 mosquitoes were collected over 1936 nights, of which 6651 were ol group . OverallSchool-aged children are important contributors to the human infectious malaria reservoir.Despite an erratic history, chemoprevention is likely to be an important addition to existing malaria control strategies, partly because of renewed interest in treating asymptomatic individuals to reduce transmission. Chemoprevention can be delivered in various ways to communities, including mass or intermittent screening and treatment, mass drug administration, and IPT of specific high-risk populations. Mass drug administration reduces malaria parasitaemia and transmission,Achieving high coverage is key to the success of chemoprevention programmes.We saw no effect of IPT on the secondary outcomes, including prevalence of parasitaemia assessed by microscopy and LAMP, gametocytaemia, or anaemia. Our approach to selecting samples for LAMP assessments, which was done in only 25% of samples negative on microscopy in children younger than 5 years and 50% of negative samples in those older than 15 years, might have affected our results. Although we accounted for our sampling approach in our analysis of the data, the selected samples might not have been representative of the population overall. The lack of effect of IPT on low-density infections identified by LAMP might also reflect that IPT works by preventing incident infections rather than by treating chronic low-density infections. Gametocytaemia was determined from thick blood smears, which is less sensitive than molecular amplification of specific sexual stage genes,This study had several other limitations. First, because of the challenges related to recruitment, the number of children participating in the intervention group increased gradually. Ideally, enrolment would have been completed before IPT was started to enable high coverage for all six rounds of treatment. Second, attendance at cluster schools by children in the school catchment areas was surprisingly low, possibly because of travel to school and absenteeism. In 2006, 82% of primary-school-aged children in Uganda were reported to attend school, but absenteeism, particularly in rural areas, was an issue.Targeted IPT of schoolchildren with DP as an approach to malaria control at the community level in Jinja district, Uganda, might have a positive effect on malaria indicators. Coverage in our study, however, was lower than expected and differences between groups were only of borderline significance. This study makes an important contribution to the evidence for the use of IPT as a tool to reduce malaria transmission. Schoolchildren are major contributors to the infectious reservoir. In high transmission areas, innovative approaches to malaria control will be needed to reduce and ultimately eliminate the burden of malaria. IPT of schoolchildren offers an operationally attractive and potentially sustainable intervention that could be integrated with currently deployed malaria-control methods. Future studies should investigate how to achieve high coverage, methods to integrate IPT with other school-based programmes and control measures, the potential effects on naturally acquired immunity and risk of rebound, and the risk of accelerating antimalarial drug resistance.


Pulse-wave velocity (PWV) measures aortic stiffness. It is an independent predictor of cardiovascular events and mortality, yet there is paucity in the literature on its association with the severity and extent of coronary artery disease (CAD).To examine the utility of PWV in predicting CAD burden in men and women the PWV was determined in 344 patients  presenting for invasive coronary angiography for the assessment of suspected CAD. Pearson correlations and multivariate analysis were used to evaluate the relationship between these coronary scores, PWV and traditional cardiovascular risk factors.Compared to men, women with chest pain had lower mean Extent scores  and Gensini scores . PWV was similar between men and women  and correlated with Extent score  but not Gensini or vessel score . PWV was associated with Extent score in men . It was not a predictor of Gensini score .PWV correlates with the extent of CAD, as measured by the ‘Extent’ score in men more than women. However, it does not correlate with the severity of obstructive CAD in either gender. The diagnosis of coronary artery disease (CAD) involves risk factor assessment, combined with stress testing and imaging to select patients for coronary angiography. It is known that the incidence of coronary artery disease in people presenting with chest pain is dramatically different between men and women with significant CAD absent in up to 60% of women and 30% of men. –3 FurtheCarotid—femoral pulse wave velocity (PWV) is currently considered the current gold-standard method for assessing arterial stiffness. , 8 It is1680 participants were recruited for the AHES from January 2010 and January 2012. These were people who presented to Westmead Hospital, Sydney, Australia for invasive coronary angiography for the assessment of chest pain due to suspected CAD. Patients were referred for investigation by a cardiologist for outpatient or inpatient investigation. The decision to pursue angiography was made by the referrer who was not involved in the subsequent recruitment of the patients to the study. Participants were consented to the study prior to or following invasive coronary angiography. Exclusion criteria were presentation with acute myocardial infarction, unstable angina, cardiogenic shock. Of the 1680 examined in the AHES study, a total of 489 participants were excluded because they had a previous history of coronary artery bypass grafting (n = 191) and/or previous coronary artery stent (n = 298). Of the remaining 848 in the study, 343 agreed to have carotid-femoral PWV assessments performed as part of the study .The protocol was approved by the Westmead Hospital  ethics committee, and was performed in accordance with the Declaration of Helsinki. All patients provided written informed consent.Aortic stiffness was assessed with carotid-femoral pulse-wave analysis using applanation tonometry  that allowed on-line pulse wave recording and automatic calculation of PWV. Measurement was repeated over 10 different cardiac cycles, and the mean value was used for the final analysis. –14 The cDiagnostic coronary angiography was performed via a femoral or radial approach. Selective coronary injections were filmed in standard projections with a Siemens Bi-Plane radiographic unit . Cine runs were stored at the time of acquisition in DICOM format.All angiograms were analyzed offline by a cardiologist (author J.C.) blinded to the medical history and adjunctive investigations. Two orthogonal views were examined in end-diastole and the image with the most severe stenosis was used. Each lesion that was visually scored as greater than 50% luminal obstruction in a vessel that was ≥1.5mm diameter was further analyzed by quantitative coronary analysis (QCA) using validated computerized edge-detection software  . CoronarVessel score: was calculated based on the number of vessels with significant obstructive coronary disease defined as greater than 50% stenosis. [tenosis.  This deftenosis.  Left maiGensini Score [ni Score : This dini Score –22Extent Score: First published by Sullivan et al. [n et al.  this indt-test was used for the comparison of demographic data that were continuous variables and chi-squared analysis for categorical variables. Pulse-wave velocity was adjusted for waist:height ratio and systolic blood pressure. Relationships between PWV, gender, age, cardiovascular risk factors and the coronary artery scores were assessed using Pearson correlations. Risk factors were analyzed as binary variables for the correlation models. The effect of each independent clinical variable on the Extent and Gensini scores were examined using multiple regression models both as a grouped population as well as divided into gender. Data is provided as unstandardized coefficients (B) with corresponding p-values. PWV results were further dichotomized into <10 meters/second and ≥10 meters/second as recommended [Data were analyzed using SAS 9.2 . All categorical data were reported as percentages and continuous variable expressed as mean ± standard deviation (SD). Student’s ommended  and t-teThe study cohort was predominantly men (77%) with an average age of 59.4 years and Caucasian background . There wGender was the most significant factor for the Gensini score , followed by smoking . PWV was not associated with Gensini score for this cohort . By contrast, several risk factors were positively correlated with Extent score. Sex was the most correlated , followed by PWV , diabetes  and age . PWV was not correlated with the Vessel score .Multiple regression analyses revealed that gender was the most significant risk factor for both the Gensini and Extent score. When examining the factors by gender, only diabetes and PWV were significantly associated with Extent score in men . However, this association was not significant in women. The vessel score was not associated with PWV in either the grouped cohort or when examined by gender.Tables In a cohort of people with suspected ischemic chest pain referred for coronary angiography, we found an independent association between carotid-femoral PWV and atheroma burden and showed that PWV correlated with the extent of coronary atheroma but not with stenosis severity as assessed with Gensini score in either gender. To the best of our knowledge, this is the first study to examine potential gender differences in using PWV to predict angiographic coronary artery disease.Our study used the Extent score to determine the burden of coronary atherosclerosis in a large population presenting for the evaluation of suspected angina. The Extent score provided anatomical perspective on the diffusion of atherosclerosis independent of stenosis severity reflected by the Gensini score. , 23 It hIn our study, only the Extent score was correlated significantly with age and the presence of diabetes. It is known that diffuse coronary atherosclerosis increases with age and is also significantly more prevalent in men.  Further,Pulse-wave velocity has been shown in a recent meta-analysis to predict future cardiovascular events and risk stratification, independent of the presence of other risk factors.  PWV may Our study suggests that PWV can be used to predict the extent of coronary atheroma but not stenosis severity. Interestingly, PWV was not a predictor of Gensini score in women. The borderline lack of association of Extent score with PWV in women probably reflects their lower number in the study and therefore may have underestimated the overall association as illustrated by the significant association in the grouped cohort.The study by Coutinho et al.  found thWomen with suspected ischemic chest pain undergoing invasive coronary angiography have less extensive epicardial atheroma, also presenting with less obstructive epicardial stenoses and often with microvascular dysfunction.  Our resuPWV may be associated with diffuse atheroma. This positive correlation between PWV and Extent score suggests that PWV may be predictive of future cardiovascular events due to the established link between Extent score and cardiovascular risk. Although aortic stiffness is an independent marker for future cardiovascular risk, our study shows the carotid-femoral PWV cannot predict the underlying obstructive coronary disease in symptomatic individuals presenting for the investigation of suspected ischemia.Our study represents a group of patients from a single center with a broad demographic, all of who were symptomatic and being investigated and medically treated for suspected ischemia. This limits the generalizability of our results to similar care settings and the general population. Given the numbers of people who were not included in the analysis due to having exclusion criteria or inadequate coronary angiograms and reluctance to consent to having the PWV assessment done, there is some selection bias. The low proportion of women recruited in the study could have influenced the outcomes. Importantly, the association of PWV with Extent scores in women may be under-estimated. This does highlight the lower number of women presenting for coronary angiography as well as the perceived lower risk of ischemic heart disease. Selection bias cannot be completely excluded even after adjusting for a wide range of patient characteristics. Last, we did not collect information on microvascular or endothelial dysfunction or long-term cardiovascular outcomes.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Plasmodium spp. partially depend on cholesterol from the host, we hypothesize that these PCSK9 genetic polymorphisms could influence the course of malaria infection in individuals who carry them. Here we examined the frequency distribution of one dominant (C679X) and two recessive  hypocholesterolemic polymorphisms as well as that of one recessive hypercholesterolemic polymorphism (E670G) among healthy and malaria-infected Malian children.Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a hepatic secretory protein which promotes the degradation of low-density lipoprotein receptors leading to reduced hepatic uptake of plasma cholesterol. Non-synonymous single-nucleotide polymorphisms in its gene have been linked to hypo- or hyper- cholesterolemia, depending on whether they decrease or increase PCSK9 activity, respectively. Since the proliferation and the infectivity of PCSK9 polymorphisms using Taqman assays. Associations of genotype distributions and allele frequencies with malaria were evaluated.Dried blood spots were collected in Bandiagara, Mali, from 752 age, residence and ethnicity-matched children: 253 healthy controls, 246 uncomplicated malaria patients and 253 severe malaria patients. Their genomic DNA was extracted and genotyped for the above P = 0.035). The association proved to be stronger when allele frequencies between healthy controls and severe malaria cases were compared ; P = 0.031).The minor allele frequency of the A443T, I474V, E670G, and C679X polymorphisms in the study population sample was 0.12, 0.20, 0.26, and 0.02, respectively. For each polymorphism, the genotype distribution among the three health conditions was statistically insignificant, but for the hypercholesterolemic E670G polymorphism, a trend towards association of the minor allele with malaria severity was observed (Carriers of the minor allele of the E670G PCSK9 polymorphism might be more susceptible to severe malaria. Further investigation of the cholesterol regulating function of PCSK9 in the pathophysiology of malaria is needed. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a secretory glycoprotein discovered in 2003 and initially termed Neural Apoptosis-Regulated Convertase 1 (NARC-1) .PCSK9 is primarily expressed in the liver. It is biosynthesized as a 692-amino acid preproPCSK9 within hepatocytes and cleaves itself after the prodomain, forming an enzymatically inactive heteroduplex made of the propeptide and the mature PCSK9. After release into the bloodstream, it acts as an escort protein for low-density lipoprotein receptor (LDLR) to which it binds at the surface of hepatocytes; the binding pair is internalized and directed towards lysosome-like compartments where it is degraded . LDLR mePCSK9 SNPs are c.426C> G and c.2037C> A, which cause the nonsense p.Y142X and p.C679X mutations, respectively. The two mutations are found at a rate of 1 in 40 African Americans and are 100-fold less frequent in European Americans [Two of the most effective LOF mericans . In a lamericans . In Westmericans .Plasmodium parasite scavenges endogenously produced or LDLR-captured cholesterol for its proficient replication in hepatocytes [Until recently, CHD was relatively uncommon in sub-Saharan Africa, primarily because of lower prevalence of lifestyle risk factors . Althougatocytes . Merozoiatocytes . From a atocytes , 29.PCSK9 SNPs with a documented cholesterolemia phenotype in sub-Saharan African children of Mali enrolled in a case-control study evaluating risk and protective factors for severe malaria.We wanted to assess whether by affecting cholesterol metabolism, PCSK9 could influence the pathophysiology of malaria. As a first step, we have determined the frequency of four common The study protocol was reviewed and approved by the Review Board of the University of Mali Faculty of Medicine, Pharmacy and Dentistry as well as the Research Ethics Committee of the Clinical Research Institute of Montreal. The original case control study protocol and the use of archived samples for malaria studies was approved by the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from parents or guardians of all study participants.P. falciparum parasitemia and an axillary temperature ≥ 37.5 °C detected by active surveillance or parasitemia and symptoms leading to treatment-seeking behavior in the absence of other clear cause of fever on passive surveillance. Children were enrolled as HC if they were asymptomatic for acute illness, had no evidence or history of chronic illness, and if the result of their thick blood smears was negative for malaria. All the subjects gave blood samples that were blotted onto Whatman filter papers FTA Classic cards and dried.The subjects were Malian children (n = 752), aged 3 months to 14 years, enrolled in a case-control study evaluating risk and protective factors for severe malaria . Index cGenomic DNA was isolated from punctures of the FTA cards (6 mm in diameter) incubated in 0.4 mL of the phosphate buffered saline (1X PBS), pH 7.4, for 20 min at room temperature with gentle agitation. Following centrifugation, the supernatant was discarded and the paper was treated with 40 μL of a solution containing 10 mM NaOH, 200 mM NaCl, and 0.05% sodium dodecyl sulfate for 6 min at 95°C. After centrifugation for 3 min, the supernatant was collected. Aliquots were diluted 12.5 X in water and used for PCR. TaqMan assays were used for genotyping the SNPs. The assay is based on the presence of fluorescence due to degradation of allele-specific fluorochrome-conjugated probes after annealing to SNP-containing PCR amplicon . It was www.oege.org). Chi-square tests or Fisher’ exact tests were used to compare genotype distributions or allele frequencies between cases and controls. The strength of allelic association with malaria was expressed as odds ratios (OR) and 95% confidence intervals (95% CI). A P value of < 0.05 was set for significance in all analyses. The data were analyzed by GraphPad Prism 5 software.A preliminary quality control of all data was conducted (Supporting information: G>A), rs562556 (A>G), rs505151 (A>G), and rs28362286 (C>A) SNPs leading to A443T, I474V, E670G, and C679X PCSK9 polymorphisms, respectively. Their genotype distribution in this population sample did not deviate from HWE and their MAFs were 0.12, 0.20, 0.26, and 0.022, respectively , suggesting that hypercholesterolemia may render individuals relatively more susceptible to severe malaria.The percent of heterozygotes for the dominant 679X variant were 5.9%, 3.2, and 3.2% among HC, UM, and SM groups, respectively. Although the difference was not significant, the trend would be expected if low cholesterol offered relative protection against malaria. A similar trend was observed for homozygotes for the recessive 474V variant (HC/UM/SM: 5.5/4.1/3.2%), but not for homozygotes for the recessive 443T variant (HC/UM/SM: 3.58/0.81/3.16%). Interestingly, an opposite trend was noted for the recessive GOF 670G variant  (P = 0.024)  , and a s= 0.024) .P < 0.0001), not in females . Indeed, 15 out the 20 homozygous carriers of the 670G variation (75%) were males. No such gender dichotomy was observed for the A443T polymorphism in relation with uncomplicated malaria  of PCSK9. This domain is required for the LDLR-degrading activity of the protein . The subPCSK9 locus, Ding and Kullo [It is intriguing that the risk allele of the E670G polymorphism should be found at such a high frequency (MAF: 0.26) if it is associated with child morbidity and mortality due to endemic malaria. As in the case of balanced polymorphisms , it coulnd Kullo  have obsC>A) nonsense SNP would be less frequent among severe malaria patients than among healthy controls. It was not to a statistically significant extent. This SNP was initially identified among African-Americans at MAFs of 0.018 [It was expected that minor allele of the rs28362286  and rs562556 (A>G) LOF SNPs are found in many populations at varying frequencies [The rs28362263 Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 FigA>G) PCSK9 SNP (E670G) in uncomplicated and severe malaria. No significant difference was observed among genotype within either malaria conditions.Blood parameters per genotype of the rs505151 ((TIF)Click here for additional data file.


Sickle cell anemia is due to a mutations on the betaglobin gene, inducing abnormal hemoglobin. In West Africa the main mutations lead to S or C types of hemoglobin. Patients with homozygote mutations seem protected against severe malaria, but not against mild disease. The prevalence of abnormal hemoglobin among patients attending dispensaries for mild malaria is thus unknown. A retrospective study was conducted to update data on the prevalence of S and C hemoglobin among patients attending dispensaries with mild malaria. Enrolment of patients was conducted during in vivo malaria treatment efficacy survey following the 42 days WHO protocol. A group of non-infected pregnant women and a group of patients with fever different from malaria, were also recruited in the same dispensaries.794 blood samples were included. S and C genotypes were found in all the regions of Ivory Coast with the highest prevalence in the Northern region . In non-infected patients, prevalence of mutations was higher than in malaria patients.A high proportion of patients with mild malaria carried genetic hemoglobin disorder. This population of high risk must be better investigated to control treatment efficacy and to manage complications.The online version of this article (10.1186/s13104-018-3296-7) contains supplementary material, which is available to authorized users. Nearly 5% of the world population carries mutations in genes of hemoglobin, either as sickle-cell anemia or thalassemia . In the The overlapping maps of sickle cell anemia and malaria sustain the hypothesis of the role of malaria in the maintaining of sickle cell trait , 3: i.e.The objective of this retrospective study was therefore to update data on the prevalence of S and C alleles among patients attending dispensary with mild malaria. For this purpose we implemented a FRET (fluorescence resonance energy transfer) molecular method for the detection of mutations on codon 6 of betaglobin gene  which alParasitaemia of patients can vary with the malaria transmission level, which correlates with ecological and climatic condition. Sites were as follow.Abidjan is the economic capital of the country accounting for almost 5 million inhabitants. It is located in the South of the country, with a sub-equatorial climate. Ayamé is located 100 km North–East of Abidjan, in a region of lakes. It is a tropical area where rainfalls occurred almost all over the year and with a very high malaria transmission level. Yamoussoukro is the administrative capital, 248 km North from Abidjan, located in a region of lakes with an equatorial climate. Transmission of malaria is lower with rainfalls accounting nowadays for less than 900 mm/year. Man is the main town of the Western part of the country, 150 km far from Yamoussoukro, surrounded by hills and dense forests. The climate is tropical and transmission of malaria is intense. Bouake is a 1 million inhabitant area, 200 km North from Yamoussoukro, located in a region of savannah. Korhogo is at the frontier with Mali and Burkina Faso. In these two places, the climate is typically sub-Sahelian (dry and hot) and malaria transmission is less intense than in other part of the country.The patients included in this study were recruited in six sites during standard in vivo survey of drug efficacy conducted on behalf of the Health Ministry. Protocol of enrolment followed the 42 days WHO  protocol . Briefly® paper or as venous blood.Blood samples were collected according to WHO recommendations , either ® gel following manufacturer recommendations. In these places blood samples were collected and analyzed in the same time and data were already available in the data bases. For blood samples collected on paper, typing was done retrospectively using FRET approach [® Blood Minikits following manufacturer’s recommendations. For amplification, initial denaturation was carried out at 95 °C for 5 min followed by 32 cycles of denaturation (30 s), hybridization (30 s) and elongation (1 min). Finally, a 5 min elongation was carried out. Hybridization of FRET probes was conducted on RotorGene Q. Denaturation of amplicons was done at 95 °C for 10 min, followed by incubation with probes . For hybridization of probes, the temperature was raised from 45 to 95 °C at a rate of 1 °C/cycle with a stop of 5 s between each step. For each sample the speed of raise of fluorescence was recorded according to the temperature (dfluo/dt curves)  and Kruskal Wallis (KW) tests respectively. Statistical analysis and graphics were done using Statistica 7.0 software. The size of the control groups is calculated to give a 5% confidence interval around a hypothetical prevalence of abnormal hemoglobin of 25%. Thirty-six persons are sufficient and 40 are selected.From patients with malaria 794 samples were genotyped including 422 women (53%) and 372 men Table . IndividA total of 545 (68%) samples with malaria were genotyped using electrophoresis and 249 (32%) by molecular method (FRET). All the 90 samples without malaria were genotyped using FRET. Out of 545 samples studied by electrophoresis 483 AA, 24 AS, 34 AC, 1 SS, 2 SC and 1 CC were determined. Out of 249 samples analyzed with FRET, 182 AA, 28 AC, 24 AS, 5 CC, 6 SC and 4 SS . Different alleles are identified according to the temperature of the peak of fluorescence in reference with standards. Dissociation curves showing: A/ one peak for a homozygous genotype AA with the allele A1 (Tm = 61 °C). The Tm of the allele A is between 60 °C and 64 °C; another allele A2 can be identified with a Tm between 52 and 54 °C. B/ Two major peaks for an heterozygous genotype AS with the alleles A1 (Tm = 61 °C) and S . C/ A single peak for homozygous sickle cell genotype SS with the S allele (Tm = 66 °C). D/ Two peaks for a patient with sickle cell genotype SC with the alleles S (Tm = 66 °C) and C .Additional file 2: Figure S2. Age and parasitaemia for patients from the different regions of Ivory Coast. Parasitaemia are expressed as the number of trophozoites per microliter of blood, counted on Giema stained thick smears. Patients from Korhogo harbored lower parasitaemia.


Plasmodium vivax is the most widely distributed human malaria parasite. Outside sub-Saharan Africa, the proportion of P. vivax malaria is rising. A major cause for concern is the re-emergence of Plasmodium vivax in malaria-free areas. Oman, situated in the south-eastern corner of the Arabian Peninsula, has long been an area of vivax malaria transmission but no locally acquired cases were reported in 2004. However, local transmission has been registered in small outbreaks since 2007. In this study, a local outbreak of 54 cases over 50 days in 2014 was analyzed retrospectively and stained blood slides have been obtained for parasite identification and genotyping. The aim of this study was to identify the geographical origin of these cases, in an attempt to differentiate between imported cases and local transmission. Using circumsporozoite protein (csp), merozoite surface protein 1 (msp1), and merozoite surface protein 3 (msp3) markers for genotyping of parasite DNA obtained by scrapping off the surface of smears, genetic diversity and phylogenetic analysis were performed. The study found that the samples had very low genetic diversity, a temperate genotype, and a high genetic distance, with most of the reference strains coming from endemic countries. We conclude that a small outbreak of imported malaria is not associated with re-emergence of malaria transmission in Oman, as no new cases have been seen since the outbreak ended. P. vivax results in considerable morbidity and mortality [P. vivax is currently the most widely distributed human malaria parasite with an estimated 2.5 billion people at risk [P. vivax accounts for more than half of all malaria cases in Latin America, the Middle East, Asia, and the Western Pacific. Outside sub-Saharan Africa, the proportions of P. vivax malaria are rising, a clear indication of the resilience of this parasite to control measures [P. vivax has long been considered a neglected parasite while its socio-economic burden in endemic areas is huge. P. vivax exist in a temperate form with a long latency period up to nine months and a tropical form with a short latency period [Malaria due to ortality , 11, 16. at risk . P. vivameasures , 40. P. y period , 28, 39.y period , 38.P. vivax into previously malaria-free areas is related to population movements [P. vivax malaria is of the highest importance in order to achieve effective control measures in malaria-endemic areas.The risk of introducing ovements , as receovements , 8, 34. https://www.ncsi.gov.om/Pages/NCSI.aspx). Oman has long been an area of P. vivax malaria transmission , but control aiming at eradication was started in 1991 and in 2000 the annual parasite incidence had been reduced to 1 per 10,000 population [P. vivax, is endemic.Oman is situated in the south-eastern corner of the Arabian Peninsula, bordering the Kingdom of Saudi Arabia, the United Arab Emirates, and Yemen. The summer is hot and humid and the winter is colder with some rain. The population in 2017 was estimated to be 2.5 million Omani nationals plus an expatriate population of 2.12 million  [msp1), and merozoite surface protein 3 (msp3) have been used extensively [Plasmodium vivax parasites circulating in areas with low and very low endemicity or causing outbreaks in areas free of malaria.Several molecular markers have been tested to examine parasite population diversity . Microsacsp) , merozoiensively , 32, 35.2. It is important to determine whether this malaria transmission was introduced into Oman repeatedly from migrant workers coming from malaria-endemic areas, or was locally re-established.This study is a retrospective analysis of DNA samples collected in Oman from Giemsa-stained blood films obtained from 45 patients diagnosed with malaria in 2014 and collected during a short period of less than 50 days in a focus area of 16 kmThis study did not impact the diagnosis, treatment, or follow-up of patients since samples were obtained after the end of the outbreak. Ethical clearance was obtained from the Ministry of Health, Sultanate of Oman.The first case was registered on 22 September 2014 and the last case on 9 November 2014.All cases were found within a 4 × 4 km area of Mabela, in the Seeb district of Muscat governorate, Oman. This is an area with intensive construction of new buildings.Anopheles larvae. The malaria vector in Oman is Anopheles culicifacies.Environmental investigation was initiated and seven open water tanks related to building construction sites and 15 pools used for irrigation were sampled. Three water tanks and five pools were identified with P. vivax circulating blood stages as detected by experienced microscopists. One patient  had recently traveled to Pakistan and was excluded from the study. The remaining 53 patients were all expatriates from India (n = 14), Pakistan (n = 6), Bangladesh (n = 32), and Egypt (n = 1). The epidemiological classification of cases was based on the field: it took into consideration the travel history of the patients, date of onset, and entomological data. From the travel history, one out of 54 patients had no travel history to a malaria-endemic country. The transmission of malaria most likely occurred near that patient.Fifty-four cases were registered from the area, 52 men and 2 women (mean age = 32 ± 8). All patients had All patients were treated with chloroquine and received a regimen of 14 days of primaquine to kill hypnozoites.The classification of malaria cases into 1-Imported case, 2-Indigenous case, 3-Introduced case, and 4-Locally acquired case, was performed according to the 2017 WHO definitions .Dry blood films from 45 different patients were obtained by scrapping off the surface of the smear with a scalpel and re-suspending in 100 μL of phosphate-buffered saline (PBS) . GenomicPlasmodium vivax species detection, as previously described [Pvcsp, Pvmsp1 (fragment 1), and Pvmsp3 alpha block II.Extracted DNA was first submitted to polymerase chain reaction (PCR) for the plasmodium genus and escribed . ConsidePvcsp and Pvmsp1 or 0.1 μM of each oligonucleotide primer for Pvmsp3 alpha and 2.0 μL of LightCycler FastStart DNA Master SYBR Green 1 reaction mix.All amplification reactions were carried out in a total volume of 20 μL and the presence of 250 nM of each oligonucleotide primer for Pvmsp1 and Pvcsp PCRs or 30 s for Pvmsp3 alpha PCR, extension step at 72 °C for 2 min for Pvmsp1 and Pvcsp or at 68 °C for 2.5 min for Pvmsp3 alpha PCR, and a denaturation step at 95 °C for 1 min for Pvmsp1 and Pvcsp PCRs or 30 s for Pvmsp3 alpha PCR.Primary amplification reactions were initiated with 5.0 μL of the template genomic DNA prepared from Giemsa-stained thin blood smears and 1.0 μL of the product of these reactions was used to initiate the secondary amplification reactions. The cycling parameters for PCR were as follows: an initial denaturation step at 95 °C for 10 min preceded the cycles of annealing at a temperature defined for each primer pair for 2 min for Pvmsp1 and Pvcsp PCRs, reaction mixtures from each capillary were collected and stored at 4 °C until secondary PCR or sequencing analysis.After a final annealing step followed by 5 min of extension only for Pvcsp and Pvmsp3 alpha was performed in both directions using 3730 XL DNA analyzer (Applied Biosystems).The sequences were determined directly from the PCR-purified templates using a Qiagen DNA purification kit, according to the manufacturer’s instructions. Direct sequencing of the full length of To carry out the study of the potential origin of the samples collected in Oman, nucleotide sequences with the same gene fragment were extracted from GenBank. These reference strains were selected either because they were from eight countries known to be the origin of frequent imported cases in Oman , or because they had the three genes available for the same strain. The accession codes and exact positions of the selected parts of the genes are reported in Pvmsp1 and Pvmsp3 alpha, and had to be done manually for csp due to its repetitive-patterns nature. To calculate the nucleotide diversity of the genes among the Omani sequences compared to the potential diversity of the genes, Oman samples and the reference strains were analyzed using DnaSP, version 5.10.01.Each Omani and reference sequences were trimmed to conserve only the highest quality sequence part, using BioEdit sequence alignment editor, version 7.2.5. The alignments were done through Muscle  for PvmsKY629006–KY6290023 for pvcsp, KY629024–KY629045 for pvmsp1, and KY629046–KY629070 for pvmsp3.These sequences have been deposited in GenBank under Submission Numbers P. cynomolgi using the maximum likelihood method based on the Tamura-Nei model [P. cynomolgi is the closest known relative of P. vivax. Initial trees for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with the superior log likelihood value [Phylogenetic inferences from concatenated amino acid sequences of Omani samples and reference strains were rooted on ei model . P. cynood value .Plasmodium vivax DNA. These 35 samples were submitted to PCR for the Pvcsp, Pvmsp1, and Pvmsp3 alpha genes, and amplicons were double-stranded sequenced. After manual cleaning of INDEL or sequencing errors, sequences from 18, 22, and 25 samples were selected for Pvcsp, Pvmsp1, and Pvmsp3 alpha, respectively  and the average number of nucleotide differences (k) of Pvcsp, Pvmsp1, and Pvmsp3 alpha were π = 0.00065, 0.00000, and 0.00100 and k = 0.333, 0.00000, and 0.920, respectively. In comparison, those of the reference strains were π = 0.04016, 0.143, and 0.02967 and k = 9.800, 39.6, and 27.619, respectively.The nucleotide diversity .The circumsporozoite protein gene of  domains . The repP. vivax, located on chromosome 7, encodes a protein of 190 kDa with 7 interallele conserved blocks and 6 variable blocks. Three segments  have been described for P. vivax genotyping [The merozoite surface protein 1 gene of notyping . The F1 P. vivax is a member of the msp3 family , located on chromosome 10, which encodes a protein with a predominant central alanine-rich domain [Pvmsp3 alpha have been studied, block I (residues 104–396) and block II (434–687), which is known to be relatively well conserved. Its main non-random polymorphisms appear in motif I from amino acid positions 533 to 538 and in motif II from 580 to 587 [The merozoite surface protein 3 alpha of h domain . This ge0 to 587 , 27, 29.Pvcsp, the reference sequence from Iran showed the lowest divergence with the Omani samples  and migrant workers (approx. 46% of the population). One of the major concerns for the Omani Health Authorities is that migrants mainly originate from countries in Southeast Asia with endemic Pvcsp, Pvmsp1, and Pvmsp3 alpha, respectively.An outbreak of malaria cases was detected by staff at the Department of Malaria, Ministry of Health, Oman, and stained blood smears were obtained from finger pricks and used for the diagnosis. Venous blood samples were not collected at the time of diagnosis and patients were not followed up. Thus, the only DNA material available to investigate the outbreak was the stained thick blood smears. The low quality and quantity of this material preclude a deep genetic analysis and it was not possible to use microsatellite analysis or whole-genome sequencing. DNA was obtained from 35 slides, and PCR sequencing made it possible to obtain 18, 22, and 29 sequences of the conventional markers π) and average number of nucleotide differences (k) showed that all 35 samples were very closely related and thus most probably originated from a single index case. This is in agreement with the hypothesis of a series of secondary locally acquired cases contracted from a gametocyte carrier infected outside Oman. These carriers may have been in the pre-patency or patency period if recently infected, or in a long-term patency period if infected with a temperate parasite genotype.The measures of genetic polymorphism using nucleotide diversity  does not lead to the definitive conclusion that the Omani cases were imported from Iran. The lack of available sequences of Pvmsp1 and Pvmsp3 alpha from Iran precludes more detailed analysis. Migrant workers from Iran are numerous in Oman, but most of them are not exposed to the poor environmental conditions that the freelance construction workers from the Indian subcontinent are subjected to. There were no Iranian migrant workers in the area where the malaria outbreak occurred. Samples collected from Iran should be compared to Omani sequences to confirm this hypothesis.The proximity of P. vivax circulating in the country and causing small outbreaks like this one is highly unlikely, given that no new cases have been seen since the outbreak ended.Alternatively, the hypothesis of an Omani http://publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by the Editor-in-Chief.The Associate Editor of Parasite, Stéphane Picot, is one of the authors of this manuscript. COPE (Committee on Publication Ethics,


Diagnosis of malaria in pregnancy is problematic due to the low sensitivity of conventional diagnostic tests (rapid diagnostic test and microscopy), which is exacerbated due to low peripheral parasite densities, and lack of clinical symptoms. In this study, six potential biomarkers to support malaria diagnosis in pregnancy were evaluated.Blood samples were collected from pregnant women at antenatal clinic visits and at delivery. Microscopy and real-time PCR were performed for malaria diagnosis and biomarker analyses were performed by ELISA . A placental biopsy was collected at delivery to determine placental malaria.p < 0.001). Both markers were also positively associated with parasite density (p < 0.001 and p = 0.003 for IL-10 and sTNF-RII respectively). IL-10 levels at delivery, but not during pregnancy, were negatively associated with birth weight. A prediction model was created using IL-10 and sTNF-RII cut-off points. For primigravidae the model had a sensitivity of 88.9% (95%CI 45.7–98.7%) and specificity of 83.3% (95% CI 57.1–94.9%) for diagnosing malaria during pregnancy. For secundi- and multigravidae the sensitivity (81.8% and 56.5% respectively) was lower, while specificity (100.0% and 94.3% respectively) was relatively high. Sub-microscopic infections were detected in 2 out of 3 secundi- and 5 out of 12 multigravidae.IL-10 and sTNF-RII were significantly higher at all time-points in malaria-infected women  contains supplementary material, which is available to authorized users. Plasmodium falciparum can have severe health consequences, such as maternal anemia, low birth weight, preterm birth, or even abortions and stillbirth [P. falciparum-infected erythrocyte. However, during malaria in pregnancy a unique surface antigen on P. falciparum-infected erythrocytes is expressed, against which immunity is lacking [P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of surface antigens. With this pregnancy specific antigen, P. falciparum parasites can sequester in the placenta by binding to chondroitin sulphate A on syncytiotrophoblasts, thereby evading splenic clearance [In sub-Saharan Africa, an estimated 28 million pregnant women are at risk of contracting malaria . Malariaillbirth . In Afriillbirth . Normallillbirth , 6. This lacking . This sulearance . This colearance –14. The learance , 11, 15.P. falciparum infections during pregnancy.Currently, one of the main methods to prevent malaria in pregnancy and its related morbidity is intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP), which consists of systematically administering SP at antenatal care (ANC) visits from second trimester onwards, regardless of malaria infection status . AlthougIn the current diagnostic landscape, there is increasing interest in identifying and using host biomarkers to diagnose infection; for malaria in pregnancy, several potential biomarkers have been suggested  . Based oThe study was conducted in the Nanoro health centre catchment area, situated approximately 85 km North-West of Ouagadougou, the capital of Burkina Faso. Malaria is highly seasonal with intense transmission during the rainy months (June – October), with a peak towards the end of the rainy season.This study aimed to identify host biomarkers that can be used to support malaria diagnosis. The study was nested within a cluster-randomized controlled trial . Full details of the trial have been reported elsewhere [At each ANC visit, women received standard antenatal care, including intermittent preventive treatment with sulfadoxine-pyrimethamine according to national guidelines and as advised by the World Health Organization.  FurthermP. falciparum real-time PCR results from peripheral blood spots collected at delivery. By using a random number generator, 79 malaria positive (cases) and 81 malaria negative (controls) pregnant women were selected. Blood samples were analysed using commercial ELISA kits for the 6 selected biomarkers . For statistical analyses of biomarker results the case-control definition was abandoned, instead the following definitions of malaria infection were used: peripheral malaria infection  or placental malaria infection .The current study was set up as a case-control study. Initial cases and controls were defined by 18S P. falciparum DNA was detected by real-time PCR as previously described with some minor adjustments in primer concentrations and probe sequence [P. falciparum FCR3 culture was included (104 parasites/μL – 1 parasites/μL), as well as positive  and negative extraction controls .Blood spots on filter paper collected at ANC visits and delivery were air dried, sealed in bags with silica and transported to the central laboratory  where they were stored at ambient temperature until shipment to the Netherlands  for molecular analyses. For each woman at each of the three time-points one blood spot per collected filter paper was punched using Acu-punch skin biopsy punchers  and transferred to a 5 mL polystyrene tube. DNA extraction was performed using the Nuclisens EasyMag  system as previously described . Samplessequence –27. In eBlood slides collected at ANC visits and delivery were Giemsa stained (3%) for 45–60 min. Slides were read independently by two microscopists. Parasites were counted against 200 leukocytes, or 500 leukocytes if the count was less than 10 parasites/200 leukocytes. Slides were considered negative if no parasite was detected in 100 high power fields. Any discrepancies in the two readings were resolved by a third independent reader.3 were collected from the maternal side of the placenta and preserved in 10% neutral buffered formalin. The tissue was subsequently embedded in paraffin wax and stored at 4 °C until shipment to the Medical Research Council The Gambia (MRCG) for further evaluation. Four millimeter thick paraffin embedded tissue sections were stained with hematoxylin-eosin after which they were read by a trained microscopist. Spot checks were done by a senior lab technician on a random sample of biopsies. Histology results were categorized into: acute infection , chronic infection (both parasites and pigment present), past infection (only pigment present) and no infection  [Placental biopsies of ±1 cmpigment) .Venous blood samples collected at ANC visits and at delivery were stored and transferred in cooling boxes to the central laboratory in Nanoro within one day. The exact time and date of blood collection as well as the time and date of further processing of the sample in the laboratory were documented to enable estimation of storage and transfer time. Blood samples were centrifuged at 1000 g for 15 min after which the plasma was aliquoted in 1.5 mL cryotubes and stored at −80 °C. Plasma samples were shipped to the Netherlands  on dry ice for further analyses. Commercially available quantikine ELISA kits for human samples were used according to the manufacturer’s instructions to test the following biomarkers: IL-10, TNF-α, sTNF-RII, sFlt-1, leptin and Apolipoprotein-B . The sensitivities of the quantikine ELISA kits for the respective biomarkers were 3.9 pg/mL, 5.5 pg/mL, 2.3 pg/mL, 13.3 pg/mL, 7.8 pg/mL and 9.97 ng/mL. Measurements below the detection limit were given half the concentration of the detection limit.p values are reported, unadjusted for multiple comparisons.Data were analysed using Stata 14.1. Differences in baseline characteristics such as age, haemoglobin, gravidity, infection status at enrolment and delivery and (low) birth weight were analysed by linear or logistic regression with robust standard errors to account for clustering of women in villages. Regression analyses with robust standard errors were also used to associate the biomarkers with peripheral and placental malaria infection, (low) birth weight, parasite density and storage time. Sub-analyses were performed for trimester of sample collection and gravidity. A diagnostic model was created by selecting the biomarkers with clinically relevant and significant differences between malaria-infected and uninfected women. The dataset was randomly divided into a test and validation set. Based on the distributions of the test set in both malaria positive and malaria negative women, cut-off points were manually determined for the biomarkers of interest. Sensitivity and specificity for the different end scores in the validation set were calculated using logistic regression analyses with robust standard errors. Confidence intervals (95%) and n = 155), samples collected during 84 days prior to delivery as third trimester (n = 148) and those collected more than 84 days prior to delivery as second trimester samples (n = 127). The mean age was 25.4 ± 6.0 years and none of the participants showed signs of preeclampsia during follow-up. Hemoglobin levels at delivery were equal in cases and controls. There were slightly more primigravidae among cases (25.3%) than controls (19.8%), but the difference was not statistically significant (p = 0.371). There were no differences in infection rates at enrollment between cases and controls, both by real-time PCR  and microscopy . At delivery, only 43% of cases (34/79) detected by real-time PCR were also positive by microscopy. In contrast, most real-time PCR negative samples at delivery were also microscopy negative . Only 11.9% (8/67) of women with a peripheral infection detected by real-time PCR had a concurrent active placental infection, while 4.5% (3/67) of real-time PCR negative controls had an active placental infection . In multivariate analyses including storage and transport time, none of the associations between biomarkers and peripheral or placental malaria infection were affected.The influence of the storage and transport time of EDTA samples to the central laboratory on the markers was assessed  and 34 (7.9%) samples respectively that fell outside the range of the standard curve. Repeated measurements were not conclusive as the adjusted mean difference was >10%. Therefore these measurements were excluded from the initial analyses, but included for sensitivity analyses (see below).At each time point  the levels of the biomarkers were compared between women with and without peripheral malaria infection Fig. . Periphep < 0.001) . Multivariate analyses, including age and gravidity, did not substantially change the associations between any of the markers and malaria infection. Sensitivity analyses including the primary or repeated measurements of the excluded sTNF-RII and sFlt-1 samples, did not result in different conclusions, except that p values for the decrease in sFlt-1 levels in second trimester were significant .Both inflammatory markers IL-10 and sTNF-RII were significantly higher in infected than in uninfected women at all time-points (01) Fig. . AlthougSub-analyses were performed for differences in biomarker concentrations between women with sub-microscopic peripheral infections compared with uninfected women. Only IL-10 and sTNF-RII remained significantly increased in second and third trimester in women with sub-microscopic infections compared with uninfected women. There was no longer a difference in these cytokine levels between infected and uninfected women at delivery Fig. .Fig. 3Bip values <0.001 and 0.006 respectively) . Multivariate analyses, including age and gravidity, did not change the results.IL-10 and sTNF-RII were significantly higher in women with an active placental infection (both acute and chronic) than in those without (ly) Fig. . These tp = 0.001; p = 0.025; p < 0.001 and p = 0.001 respectively). In the third trimester, higher levels of IL-10 and sTNF-RII were associated with an increased risk of chronic PM infection at delivery (p = 0.002 for both).Regression analyses were also performed for biomarker levels during pregnancy compared with a chronic malaria infection (active chronic or past chronic) at delivery. Increased IL-10, TNF-α, sTNF-RII and Apo-B levels in second trimester were significantly associated with chronic PM infections at delivery (n = 44) and without (n = 22), as determined by placental histology. None of the markers showed significant differences . Multivariate analyses adjusting for gravidity and age did not change these associations. Repeating the analysis using real-time PCR estimations of parasite densities (including sub-microscopic infections) produced similar results, except that sFlt-1 was no longer significantly associated with parasite density (p = 0.344) (data not shown).Regression analyses for the association between each of the biomarkers and parasite density were based on microscopy results Table . IL-10, p = 0.184) (Table p = 0.873 and p = 0.488 respectively). An inverse correlation was seen for IL-10 levels and birth weight (p = 0.006) at delivery, but not for IL-10 concentrations in second and third trimester (Table p = 0.027) and also in second and third trimester (p = 0.020 and p = 0.076 respectively). However, in multivariate analyses adjusting for age, gravidity and malaria infection no associations were observed for any of the biomarkers and birth weight. Also, none of the markers were significantly associated with low birth weight (<2500 g) at any time point.  and a validation set (n = 80). Based on the distributions of the individual biomarkers in malaria positive versus negative women  in the test set  for the model in the validation set resulted in a sensitivity of 88.9% (95% CI 45.7–98.7) and specificity of 83.3% (95% CI 57.1–94.9) in primigravidae; 81.8% (95% CI 48.8–95.50) and 100.0% (95% CI 71.5–100.0) for secundigravidae; and 56.5% (95% CI 41.7–70.2) and 94.3% (95% CI 86.3–97.8%) for multigravidae  sub-microscopic infections would be detected by the model, without any false positive results. For multigravidae 5 out of 12 (41.7%) women with sub-microscopic infections would be identified, while 3 out of 51 (5.9%) women would have a false positive test result.Two inflammatory markers were useful as biomarkers for malaria in pregnancy: the cytokine IL-10 and soluble cytokine receptor sTNF-RII. Both markers were significantly higher throughout pregnancy and at delivery in malaria infected women  than in uninfected women, as was shown previously –33. FurtAs a novelty to previous research, the discriminative properties of the two markers were translated into a model for the diagnosis of malaria during pregnancy, with a high sensitivity  and fair specificity (83.3% (95% CI 57.1–94.9) for primigravidae. For secundi- and particularly multigravidae the model was less adequate in terms of sensitivity, possibly because IL-10 and sTNF-RII responses are negatively influenced by the immunity acquired during previous pregnancies and by lower parasite densities. As primigravidae are at highest risk of malaria infection and of malaria-related morbidity , the modA model using host biomarkers would be most likely of use as an addition to current malaria diagnostics. For example, given the limited sensitivity of RDT and field microscopy, the biomarkers model might assist in identifying women with malaria infections not detected by these tests. Therefore, the additional value of the model was assessed for sub-microscopic infections (RDTs were not performed) in the current study. Unfortunately, the number of sub-microscopic infections were relatively small and none were found in primigravidae in the validation set. In secundi- and multigravidae, it seems that the model results in few false positive diagnoses of malaria, yet, the sensitivity for detecting sub-microscopic infections can still be improved.Initially, the idea to generate sufficient sensitivity and specificity of the biomarkers model was the inclusion of markers from different groups or pathways involved in PM, e.g. sFlt-1, leptin and Apo-B, and not solely (anti)inflammatory markers. As these other markers could not be included in the model, it is unclear whether the combination of IL-10 and sTNF-RII is sufficiently specific for diagnosing malaria. Both markers are involved in inflammatory processes and may increase during infectious or inflammatory diseases other than malaria. IL-10, produced by numerous immune cells, has both anti- and pro-inflammatory effects: it can inhibit antigen presentation and pro-inflammatory Th1 cytokine expression, yet it can also have stimulatory effects on B-cells and natural killer cells . ItThis study has some limitations. Inherent to this type of explorative biomarker study is the risk of overfitting when creating a diagnostic or predictive model. Furthermore, there was no information on concurrent infectious diseases other than chorioamnionitis that may have influenced the level of biomarkers in the study population. As stated before, the model should be evaluated in other populations, preferably in a prospective manner. Another limitation is the uncertainty in estimations of the gestational age by considering all births full-term, which may have resulted in some samples being allocated to the wrong trimester. However, because for most biomarkers there were no large differences in the results of samples collected in second or third trimester, it was not considered to be of substantial impact to the results. Nevertheless, the uncertainty in gestational age estimations made it impossible to relate the biomarkers to outcomes such as preterm birth or small for gestational age. For future surveys, one of the recommendations would be to perform ultrasound imaging throughout pregnancy to have reliable estimates of gestational age and, more importantly, to enable more direct comparisons of fetal growth with malaria infections and biomarker levels.This study has shown that both IL-10 and sTNF-RII are biomarkers of peripheral and placental malaria infection. More importantly, a new approach of combining these biomarkers in a prediction model resulted in high sensitivity and specificity for detecting microscopically proven malaria infections in primigravidae. However, as confidence intervals were large and there is a risk of overfitting, the potential of this model should be confirmed in other studies. Furthermore, as a biomarkers model is probably of most value for sub-microscopic infections, an increase in sensitivity for detecting (sub-)microscopic infections should be pursued, while at the same time specificity might need improvement if other infectious diseases are prevalent. The current model can therefore serve as a step-up for future pursuits in creating a diagnostic model for malaria in pregnancy. Addition of one or two other markers to the model, such as haptoglobin, C5a, angiopoietins, HDL/LDL, or IP-10 should be considered. In this era of rapidly changing malaria epidemiology, rapid spread of drug resistance and the increased demand for reliable diagnostics for malaria in pregnancy, the field of biomarkers could play an extremely important role.Additional file 1: Table S1.Table S2. Regression analyses of the association between chorioamnionitis infection and each of the biomarkers at delivery. Table S3. Association of biomarker levels with low birth weight by regression analyses with robust variance estimators. (PDF 468 kb)Regression analyses of the association between storage and transport time (in hours) of EDTA samples and each of the biomarkers. Additional file 2: Figure S1.Distribution of IL-10 and sTNF-RII in malaria infected versus uninfected women during pregnancy. (PDF 36 kb)