A propensity score approach that accounts for environmental differences showed that SMC conveyed a significant protective effect against malaria infection , clinical malaria ), and hemoglobin concentration ) in 2012 and 2014, respectively. Conclusion: SMC significantly reduced frequency of malaria infection, clinical malaria and anemia two years after SMC scale-up in Koutiala.Background: Previous controlled studies demonstrated seasonal malaria chemoprevention (SMC) reduces malaria morbidity by >80% in children aged 3–59 months. Here, we assessed malaria morbidity after large-scale SMC implementation during a pilot campaign in the health district of Koutiala, Mali. Methods: Starting in August 2012, children received three rounds of SMC with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ). From July 2013 onward, children received four rounds of SMC. Prevalence of malaria infection, clinical malaria and anemia were assessed during two cross-sectional surveys conducted in August 2012 and June 2014. Investigations involved 20 randomly selected clusters in 2012 against 10 clusters in 2014. Results: Overall, 662 children were included in 2012, and 670 in 2014. Children in 2014 versus those surveyed in 2012 showed reduced proportions of malaria infection (12.4% in 2014 versus 28.7% in 2012 ( Malaria remains a leading cause of morbidity and mortality, causing an estimated 228 million cases of clinical malaria and 405 thousand deaths worldwide . More thIn 2012, the World Health Organization (WHO) recommended seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine and amodiaquine (SP plus AQ) in the Sahel countries in Africa to reduce malaria among children under 5 years of age . SeasonaSMC is a strategy to prevent malaria in children under 5 years of age across the Sahel sub-region, where most childhood malaria mortality and morbidity occurs during a few months (approximately 4 months). SMC has been associated with a reduction in the incidence of malaria infection, as well as uncomplicated and severe malaria, in the range of 67% to 86% in children . SeveralNo significant rebound in clinical malaria incidence was observed in the year after SMC was given during initial studies conducted in Senegal, Ghana, and Mali ,6,7,11. Plasmodium falciparum is the predominant species, with approximately greater than 85.9% of the parasite formula, followed by Plasmodium malariae (10–15%) and Plasmodium ovale 1% [Malaria is the primary cause of morbidity and mortality in Mali ,14, partovale 1% .Plasmodium falciparum dihydropteroate synthase (Pfdhps) 540E and P. falciparum dihydrofolate reductase (Pfdhfr)-dhps quintuple mutant genotype, in the treated population several months after the last SMC round in 2014 [Our primary observations of SMC showed an increasing prevalence of two resistance markers,  in 2014 . These vThe present study assessed the impact of this intervention on prevalence of malaria infection, clinical malaria, and anemia among children aged 3–59 months in the health district of Koutiala, Mali.Study site: The study was conducted in community health areas located in the district of Koutiala, a region of Sikasso in the south of Mali, with an average rainfall of about 700 mm per year. Koutiala is located at about 420 km south of Bamako, the capital city, with about 575,000 inhabitants in 2012, and 580,453 inhabitants in 2014, according to the National Institute of Statistics of Mali . KoutialSMC delivery: MSF, in collaboration with the NMCP, delivered SMC in 2012 and 2013. The delivery teams used a combination of fixed post and door-to-door strategies for all children aged 3–59 months. The investigators on this study were not involved in the delivery of SMC drugs.Cross-sectional study: Two cross-sectional surveys were conducted, first in August 2012 (a few days before the start of SMC implementation), and second in June 2014 (~8 months after the last doses of SMC drugs were distributed). After identification of eligible children, informed consent was obtained from their parents prior to their interview and inclusion. Children were eligible to participate to the study if they were aged 3–59 months at the time of enrolment, and residents of the study area. Children with the following criteria were excluded: severe or chronic illness, such as acquired immune deficiency syndrome (AIDS), and known adverse reaction to SP or AQ. During the 2012 malaria transmission season, three cycles of SMC with SP plus AQ were provided in August, September, and October. During the 2013 malaria transmission season, four cycles of SMC with SP plus AQ were provided in July, August, September, and October.Plasmodium falciparum malaria (by microscopy) without clinical sign; (ii) prevalence of clinical malaria, defined as the presence of asexual stage of P. falciparum (by microscopy), plus fever or history of fever; and (iii) proportion with anemia (as measured by hemoglobin concentration <11 g/dL) [®,  was conducted, and anemia was measured for all enrolled children. The participants included both symptomatic and asymptomatic children, provided they were aged 3–59 months during the survey. Symptomatic cases were diagnosed with RDT; in case of positivity, they received artemether-lumefantrine (CoartemR), per National Malaria Treatment Guidelines (NMTG) of Mali, and those positive did not receive SMC.The three primary endpoints were (i) prevalence of malaria infection, defined as the presence of asexual stage of 11 g/dL) , before Laboratory methods: Thick blood films were air dried, stained with 3% Giemsa, and examined for malaria parasites by two well-trained technicians; 100 high-power fields under an oil immersion objective were counted before a film was declared negative. Parasite density was determined by counting the number of parasites present per white blood cell (WBC) on a thick smear, assuming a WBC count of 8000 per microliter. In the case of a discrepancy (positive/negative or a difference in parasite density greater than 30%), a third reading was done. Hemoglobin concentrations were measured using a hemoglobin analyzer  on blood obtained by finger prick.p < 0.05.Data management and statistical analysis: Data were collected on standardized forms, double-entered, and verified using MS Access and then exported to R Statistical Software (version 3.0.3) for additional cleaning and analysis. Proportions of children with malaria infection, clinical malaria, and anemia were first compared using a univariate approach with chi-square or Fisher’s exact tests per age category (3–11 months versus 12–59 months) and area . Because exposure to malaria varies due to differences in location, meteorology, and environmental characteristics, a propensity score approach was implemented to reduce confounding biases . PropensAssessment of malaria morbidity and sample size: A random sample of 20 clusters  was selected from all 42 enumerations  in the health district of Koutiala. The 20 clusters were selected on a proportional basis after stratification of urban and rural communes according to the target population of each stratum. In each cluster, at least 32 children were surveyed, and in each household, all target children were sampled. All villages outside county boundaries were considered rural areas, while urban areas were represented by Koutiala city neighborhoods. Each team was accompanied by a representative of the village chief or district, to identify the consensual center of the village/neighborhood. Random selection of households was conducted by flipping a pen, then using its tip to indicate the direction to follow for the investigation. Once a direction was chosen, the investigation began, and focused on families on the right side of the street up to the end, then the opposite side. This process was repeated until the sample size of households was achieved. For each family, a legal guardian of a child volunteer aged 3–59 months provided informed consent, and was interviewed via questionnaire. A total of 20 villages and neighborhoods yielded a sample size of 640 children, sufficient to compare primary parameters before and after SMC implementation. For logistical reasons, in 2014, the number of villages/neighborhoods was changed to 10, from which 64 children were surveyed per cluster. All participants received an initial history and physical examination, followed by blood smear and hemoglobin measurement collected by finger prick. Uncomplicated and severe malaria were also documented for all volunteers at each health center, before, during, and after SMC implementation from 2011 to 2014. The cumulative number of malaria cases was used in the propensity score from January to the survey date. Meteorology and environmental characteristics  were collected at each center.Ethics clearance: The study protocol was reviewed and approved by the Ethical Committee of the Faculty of Medicine and Dentistry and Faculty of Pharmacy/University of Sciences, Techniques and Technologies of Bamako (USTTB), Mali (N°2015/109/CE/FMPOS). Community clearance was obtained after meetings with community authorities, leaders, heads of families, and other community members of each locality prior to the start of the study. Individual, written, informed consent was obtained from a parent or guardian of each child prior to screening. The study was conducted according to current Good Clinical Practices (cGCP) by investigators from Malaria Research and Training Center (MRTC), Bamako, Mali.p = 0.001. The proportion of all children with clinical malaria was 4.2% (28) at baseline vs. 0.3% (2) post-intervention; p < 0.001. The anemia prevalence was 67.4% (446) at baseline, and 50.1% (336) post-intervention; p = 0.001.From 4 to 9 August 2012 and 20 to 30 June 2014, 662 and 670 children aged from 3 to 59 months were enrolled in the study, respectively. The proportion of children aged 3–11 months were 11.4% (N = 662) and 10.3% (N = 670) at baseline and post-intervention, respectively . The proSMC treatment conferred significant reduction in malaria infection, with an incidence ratio of IR = 0.01; 95% CI , using a weighted (propensity score) quasi-Poisson regression . SimilarP. falciparum malaria was lower among children aged 3–11 months than among children aged 12–59 months  compared to the urban area (2.9%) (p < 0.001). No case of severe malaria was observed during this study (table not shown). The proportion of children with anemia (Hb < 11 g/dL) was significantly higher in the rural area, compared to the urban area . Anemia was also more prevalent among children age of 3–11 months, compared to children aged 12–59 months (79.0% vs. 66.0%: p = 0.02). The difference was larger for moderate anemia (Hb < 8 g/dL), with a proportion of 10.8% vs. 1.9% in the rural and urban area, respectively (p = 0.005). Moderate anemia prevalence was lower among the children aged 3–11 months vs. 12–59 months (p = 0.03). A total of four cases of severe anemia (Hb < =5 g/dL) occurred in rural areas among children aged 12–59 months (p = 0.39). No case of severe anemia was observed in urban areas, or in children aged 3–11 months.The proportion of asexual stage P. falciparum was lower among children aged 3–11 months than among those aged 12–59 months . The proportion of anemic children was significantly higher in rural areas compared to urban areas . No difference was observed for moderate anemia (Hb < 8 g/dL) and severe anemia (Hb ≤ 5 g/dL) (p > 0.5). No case of severe malaria was observed during this study (table not shown).The proportion of asexual stage Previous studies determined that during the highest malaria transmission season, SMC provides substantial additional protection against episodes of clinical malaria and severe malaria in children sleeping under long-lasting insecticide-treated bed nets ,23,24,25In our study, the proportion of malaria infection, clinical malaria, and anemia were significantly decreased after two years of pilot implementation of SMC in the health district of Koutiala, Mali. The national survey of parasitemia  with the support of USAID President’s Malaria Initiative (PMI)) among children 6–59 months of age showed an overall parasitemia of 59.2% in the Sikasso region in 2010, with 52% of children aged 6–59 months infected with malaria at the time of interview in Mali , versus P. falciparum was lower in children aged 3–11 months, compared to 12–59 months. This may be due to the fact that the risk of clinical event increases from birth to about 6 months of age, depending on the transmission rate, and begins at around 3 to 4 months of age [Parasitemia peaks in children less than 5 years old, and subsequently declines in an age-dependent manner . Therefos of age .The proportion of anemia within the national survey  showed aLimitations of this population study include (1) the fact that surveys were conducted during different periods (months); and (2) the lack of a comparison region, as SMC was implemented in all neighboring health districts of Koutiala. In 2012, study parameters were measured in August, while in 2014, these were measured in June. This difference in period could affect the parameter comparisons between baseline and post-intervention. The propensity score approach was used to reduce the impact of this confounding bias, rendering the groups comparable. Another limitation of the study is related to the fact that the cross-sectional study design limits the level of evidence collected from the study. Others confounding factors were not accounted for, such as parental behaviors , long-lasting insecticide-treated nets (LLINs), or fumigation), and genetic make-up of the children could account for some of the differences observed in the pre- and post-analysis.Our results are consistent with previous studies that found a substantial reduction in malaria infection, clinical malaria, and anemia ,32,33,34P. falciparum transmission intensity will reduce high-density parasitemia and malaria-associated morbidity and mortality. Therefore, ITNs provide protection against morbidity and mortality attributable to malaria [The current study differs from those prior since this was an observational study, and not a controlled trial. Nevertheless, our study shows that SMC significantly reduces malaria morbidity . SMC implementation in our study was also associated with insecticide-treated nets (ITNs), long-lasting insecticide-treated nets (LLINs), rapid diagnostic tests (RDTs), artemisinin-based combinations (ACTs), and a campaign of sensibility. These methods existed previously, but were not implemented at the population level. These interventions may also have reduced malaria morbidity along with SMC. Several sets of experimental data support the hypothesis  that int malaria ,38, as w malaria  and the  malaria .Malaria infection, clinical malaria, and anemia prevalence were significantly reduced after two years of large-scale implementation of SMC, together with LLIN, RDT, and ACT strategies, in the health district of Koutiala.


Plasmodium falciparum MOI and genetic diversity in a hyperendemic area of Burkina Faso.Investigating malaria transmission dynamics is essential to inform policy decision making. Whether multiplicity of infection (MOI) dynamic from individual infections could be a reliable malaria metric in high transmission settings with marked variation in seasons of malaria transmission has been poorly assessed. This study aimed at investigating factors driving Blood samples collected from a pharmacovigilance trial were used for polymerase chain reaction genotyping of the merozoite surface proteins 1 and 2. MOI was defined as the number of distinct parasite genotypes co-existing within a particular infection. Monthly rainfall data were obtained from satellite data of the Global Precipitation Measurement Database while monthly malaria incidence aggregated data were extracted from District Health Information Software 2 medical data of the Center-West health regional direction.P. falciparum variants. Each allelic family of the msp1 and msp2 genes was present all year round with no significant monthly fluctuation.In the study area, infected people harboured an average of 2.732 (± 0.056) different parasite genotypes. A significant correlation between the monthly MOI and the monthly malaria incidence was observed, suggesting that MOI could be a good predictor of transmission intensity. A strong effect of season on MOI was observed, with infected patients harbouring higher number of parasite genotypes during the rainy season as compared to the dry season. There was a negative relationship between MOI and host age. In addition, MOI decreased with increasing parasite densities, suggesting that there was a within-host competition among co-infecting genetically distinct msp1 and msp2 genes were found in both dry and rainy season. The approach offers the opportunity of translating genotyping data into relevant epidemiological information for malaria control.In high malaria endemic settings with marked variation in seasons of malaria transmission, MOI represents an appropriate malaria metric which provides useful information about the longitudinal changes in malaria transmission in a given area. Besides transmission season, patient age and parasite density are important factors to consider for better understanding of variations in MOI. All allelic families of  Plasmodium falciparum, the most prevalent and most dangerous Plasmodium species infects about 228 million people causing 400,000 deaths worldwide [Malaria is the most common life-threatening disease and nearly half of the world’s population is exposed to malaria infection . Each yeorldwide . In Burkorldwide . In spitorldwide . RestricNew malaria metrics are needed to complement the assessment of malaria transmission dynamics in a context of multiple control interventions. Multiplicity of infection (MOI), the number of different parasite genotypes co-existing within a particular infection, has been suggested as a useful malaria metric describing transmission dynamics –6. WhethP. falciparum MOI but the directionality of the relationship was controversially reported [Furthermore, factors driving the MOI remain poorly understood with controversial findings though this elucidation is crucial to justify its epidemiological significance. For instance, MOI correlation with age and parasite density was reported either positively –9 or negreported , 13. Whireported , 14, othreported , 15. Anoreported , 17. A rreported , while aP. falciparum variants of the msp1 and msp2 allelic families are well adapted to specific seasonal characteristics of the study area than others, is hitherto unknown. In neighboring Ghana, where malaria transmission tends to be perennial, the relative frequencies of msp1 allelic families were found to be dynamic across transmission season [P. falciparum genetic diversity regarding this seasonal pattern are not available.Finally, whether specific n season . In BurkP. falciparum multiplicity of infection and genetic diversity in an endemic area of Burkina Faso with marked seasonality of malaria transmission.This study explored the influence of season, patient age, sex, and parasite density on both P. falciparum is the most common Plasmodium species. The population mostly practice subsistence farming with Mossi being the major ethnic group, and Gourounsi and Fulani representing minor groups.Clinical data were collected from September 2010 to October 2012 at two primary health centers (Nanoro and Nazoanga) of the Nanoro sanitary district in Burkina Faso. Nanoro is one of the sentinel sites for the assessment of antimalarial therapeutic efficacy and is located at 85 km from Ouagadougou, the capital city of Burkina Faso. Malaria is hyperendemic with seasonal variation in transmission which peaked between July/August and October/November. As in other sub-Sahara African countries, Dried blood spots were collected from 724 malaria patients : 2–6, male: female ratio = 381:343) with parasite density ≥ 2000/µl, attending a pharmacovigilance trial over 23 months of the study period. For this investigation, only samples collected on day 0, i.e. from screened participants before the administration of study drugs artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) were considered. Screened participants included any patient attending the two health facilities during the study period with fever or history of fever or clinical suspicion of malaria according to the routine medical staff of the health facilities. Those who met all the criteria for the effectiveness study were included in this cohort and treated with either AL or ASAQ and those who failed were treated with ASAQ, the only available ACT during the study period in the drug stores of the health facilities. Details of the study site and sampling procedures were described previously elsewhere , 20, 21.msp1 block 2 and msp2 block 3 was performed as previously described [msp1 and msp2 using family-specific primers. All PCR rounds were carried out with a total volume of reaction mixture of 25 µl. Fragments were revealed by ethidium bromide stained agarose gel electrophoresis using LabnetTM  and MupidTM-One  electrophoresis machines. Gels were visualized by ultraviolet transillumination and band sizes were calculated using Photo CaptMW (version 11.01).Molecular analysis was performed at the Molecular Biology Laboratory of Centre Muraz of Bobo-Dioulasso, Burkina Faso, from September 2012 and October 2014. Genomic DNA was extracted from dry blood spots using the QIamp DNA Kit  following the manufacturer’s procedure. A nested PCR amplification  and a Biometra thermal cycler  PCR machines of msp1 block 2 gene  and the msp2 block 3 gene (3D7 and FC27) [msp1 revealed 2 (MAD20) + 2 (K1) + 1 (RO33) = 5 genetic variants, while the genotyping of msp2 revealed 3 (3D7) + 1 (FC27) = 4 genetic variants. In other words, MOI for msp1 for each clinical sample represented the number of PCR fragments obtained from K1 + MAD20 + RO33 gels, while the MOI for msp2 represented the number of PCR fragments obtained from 3D7 + FC27 gels. Samples with a single parasite genotype at both msp1 and msp2 loci were classified as mono-infections while samples with more than one parasite genotype at any of the two loci were classified as multiple infections. The final MOI value for each clinical isolate represented the maximum MOI value from both msp1 and msp2 loci i.e. MOI = 3 if MOImsp1 = 3 and MOImsp2 =2.MOI was defined as the number of distinct parasite genotypes co-existing within a given infection based on the genotyping of the 3 gene 3D and FC27nd FC27) , 22. Eacnd FC27) , 24. Formsp1, and 3D7 and FC27 for msp2) was calculated as the ratio of the number of PCR bands obtained for each family to the overall number of gene-specific PCR bands obtained from PCR positive samples. For each patient, the number of fragments observed for a given allelic family was divided by the total number of fragments observed for the msp1 or msp2 genes. For example, if for a given blood sample, 5 K1 fragments, 1 MAD20 fragment and 0 RO33 fragments were observed, the relative frequencies of K1, MAD20 and R033 for this patient were respectively 0.83 (5/6), 0.17 (1/6) and 0 (0/6).The frequency of allelic family  Database . Monthlyvs rainy), sex , parasite density, age and interactions, on P. falciparum multiplicity of infection. For each allelic family , a GLM with binomial errors was used to investigate the effect of time (numeric variable from 1 (September 2010) to 23 (July 2012)) on the relative frequency of the studied allelic family. A GLM with quasibinomial errors was used to explore the effect of season (2 levels: dry vs rainy), sex (2 levels), parasite density, age, and interactions on P. falciparum genetic diversity. 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.All statistical analyses were performed with R (version 3.5.1) software . A genermsp1 yielded a total of 1717 PCR fragments ranging between 142–497 bp for the K1 allelic family, 91–319 bp for the MAD20 allelic family, 95–391 bp for the RO33 allelic family, while 1653 fragments ranging between 170–773 bp for the 3D7 allelic family and 180–719 bp for the FC27 allelic family were obtained from msp2 genotyping. The MOI ranged from 1 to 7 parasite genotypes with a mean of 2.732 (± 0.056) different parasite genotypes co-existing per isolate.A total of 724 samples were successfully genotyped. Of these, 316 (43.65%) samples were collected during the dry season while 408 (56.35%) were collected during the rainy season. In total, The monthly trends of rainfall, MOI, and malaria incidence in the study area are shown in Fig. F = 11, P = 0.004)  = 2.6, P = 0.12)  = 3.9, P = 0.06) , with infected patients harboring a higher number of parasite genotypes during the rainy season, rather than during the dry season  , the negative relationship between MOI and host age was confirmed (LRT P = 0.009). There was no significant sex effect on MOI (LRT P = 0.24)   Fig. b, regardmsp1 genetic variant circulating in infected patients (P < 0.001). Of a total of 1717 observed fragments for msp1, 879 (51%) belonged to the K1 family, 456 (27%) to MAD20 and 382 (22%) to RO33. The 3D7 allelic family was more frequent than the FC27 family (P < 0.001) with 58% (957/1653) of the msp2 genetic variants.From September 2010 to July 2012, the K1 allelic family was the most frequent msp1 gene was present all year round. There was no significant monthly fluctuation of the msp1 allelic families from September 2010 to July 2012 , . However, toward the end of the dry season (May 2011 and May 2012), the over representation of K1 and 3D7 respectively, turns to diminish.Each allelic family of the P = 0.04).The allelic family K1 was more frequent than the MAD20 and RO33 families regardless of the season Fig. a, b. TheThe allelic family 3D7 was more frequent than the FC27 family regardless of the season Fig. c.P. falciparum population structure observed in high transmission settings while low transmission settings are characterized by a predominance of monoclonal infections [versus new methods with higher discriminatory power such as capillary electrophoresis and genome-wide association studies and this represents one limitation of this study.From the start of the study (2010) to the end (2012) no significant difference in MOI values was observed from year to year. The study started just after the large-scale distribution of insecticide-treated nets (ITN) and no major intervention occurred during the course of the study, probably explaining the lack of significant MOI variation between the beginning in 2010 and the end of the study in 2012. The high MOI value observed in this study indicates the endemicity level in the area while highlighting that the expected outcome from the large-scale deployment of ITN was not achieved. High MOI results from high diversity in fections –30. The fections , 31–36. A match between the peak MOI and malaria peak and the period of maximum rainfall was observed, suggesting that MOI could be a reliable indicator describing malaria transmission intensity in high endemic malaria settings as previously suggested . HoweverP. falciparum infections. In contrast to studies in Ghana where an association between highest MOI with low transmission season was reported [Our study reported a strong effect of season on the multiplicity of reported , 34, ourreported  highlighreported .P. falciparum variants. This competition becomes clearly apparent when analyzing the interactions of different allelic families in mixed infections as previously reported [A negative correlation between the MOI and parasite density was observed, suggesting a within-host competition among co-infecting genetically distinct reported . This fireported –41. Thisreported .Similarly, a strong effect of host age on MOI was observed presumably because of the acquisition of antiparasitic immunity in high malaria endemic settings , 43, 44.P. falciparum variants was found in the Nanoro area. All allelic families of msp1 and msp2 genes were found in both the dry and the rainy season. The absence of association between particular msp1 or msp2 alleles and a particular season was previously reported [msp1-K1 and msp2-3D7 allelic families predominated over the others regardless of season. The predominance of the msp1-K1 and msp2-3D7 was reported by previous findings in Burkina Faso [P. falciparum genetic variants was observed. The K1 to MAD20 ratio increased with age in male patients during the rainy season but decreased during the dry season. Similarly, the relative frequency of 3D7 during the dry season decreased with parasite density in female patients only. These findings highlight the multifactorial aspect underlying the development of a particular variant within the host, ranging from genetic  to environmental factors. As the majority of the study population is represented by children under five years-old, no sex-related behavior differences in each season could explain these findings. However, hormonal and immunological mechanisms mediating sex differences in parasite infection has been suggested [A high diversity of circulating reported , 34. Howina Faso , 32. Thiina Faso , as wellina Faso . The reluggested , 17, higmsp1 and msp2 genes were found in both dry and rainy season. However, patterns affecting the distribution of these P. falciparum variants deserve further investigations. The approach offers the opportunity of translating genotyping data into relevant epidemiological information for malaria control.In high malaria endemic settings with marked variation in seasons of malaria transmission, MOI represents an appropriate malaria metric which provides useful information about the changes in malaria transmission longitudinally in a given area. All allelic families of Additional file 1: Table S1. Determinants of the multiplicity of infection (MOI). Effect of season, patient age, sex, parasite density, and interactions on the MOI. Significant effects are emphasized in bold.Additional file 2: Table S2. Determinants of the frequency of the K1 allelic family. Effect of season, patient age, sex, parasite density, and interactions on the proportion of K1 variants in infected patient. Significant effects are emphasized in bold.Additional file 3: Table S3. Determinants of the frequency of the MAD20 allelic family. Effect of season, patient age, sex, parasite density, and interactions on the proportion of MAD20 variants in infected patient. Significant effects are emphasized in bold.Additional file 4: Table S4. Determinants of the frequency of the RO33 allelic family. Effect of season, patient age, sex, parasite density, and interactions on the proportion of RO33 variants in infected patient. Significant effects are emphasized in bold.Additional file 5: Table S5. Determinants of the frequency of the 3D7 allelic family. Effect of season, patient age, sex, parasite density, and interactions on the proportion of 3D7 variants in infected patient. Significant effects are emphasized in bold.Additional file 6: Table S6. Determinants of the frequency of the FC27 allelic family. Effect of season, patient age, sex, parasite density, and interactions on the proportion of FC27 variants in infected patient. Significant effects are emphasized in bold.


Plasmodium falciparum malaria, aiming to determine whether there are reversible effects of malaria infection on vaccine responses.Drivers of lower vaccine efficacy and impaired vaccine-specific immune responses in low-income versus high-income countries, and in rural compared with urban settings, are not fully elucidated. Repeated exposure to and immunomodulation by parasite infections may be important. We focus on We have designed a randomised, double-blind, placebo-controlled, parallel group trial of intermittent preventive malaria treatment versus placebo, to determine effects on vaccine response outcomes among school-going adolescents (9 to 17 years) from malaria-endemic rural areas of Jinja district (Uganda). Vaccines to be studied comprise BCG vaccine on day ‘zero’; yellow fever, oral typhoid and human papilloma virus vaccines at week 4; and tetanus/diphtheria booster vaccine at week 28. Participants in the intermittent preventive malaria treatment arm will receive dihydroartemisinin/piperaquine (DP) dosed by weight, 1 month apart, prior to the first immunisation, followed by monthly treatment thereafter. We expect to enrol 640 adolescents. Primary outcomes are BCG-specific interferon-γ ELISpot responses 8 weeks after BCG immunisation and for other vaccines, antibody responses to key vaccine antigens at 4 weeks after immunisation. In secondary analyses, we will determine effects of monthly DP treatment (versus placebo) on correlates of protective immunity, on vaccine response waning, on whether there are differential effects on priming versus boosting immunisations, and on malaria infection prevalence. We will also conduct exploratory immunology assays among subsets of participants to further characterise effects of the intervention on vaccine responses.Ethics approval has been obtained from relevant Ugandan and UK ethics committees. Results will be shared with Uganda Ministry of Health, relevant district councils, community leaders and study participants. Further dissemination will be done through conference proceedings and publications.ISRCTN62041885.Current Controlled Trials identifier:  This will be the first well-powered trial to evaluate the effect of repeated intermittent presumptive anti-malarial treatment on vaccine response outcomes in adolescents.Effects on both live-attenuated and inert vaccines will be studied.The use of an interventional research design will contribute strong evidence to determine whether malaria has causal and reversible effects on vaccine response outcomes, and our strong immunoepidemiological design and nested immunological studies will make it possible to address specific hypotheses regarding pathways of effects.The use of vaccines most of which are part of Uganda’s national Expanded Programme on Immunisation, makes the findings from this study applicable and could inform the introduction of combined school health programmes for malaria control and vaccine provision.With the high prevalence of malaria in the proposed study area, participants in the placebo arm may notice more frequent episodes of malaria and this could lead to individual self-unblinding and self-treatment, compromising the design of the study.Population differences in vaccine-specific immune responses and efficacy have been documented. In tropical low-income, rural settings, responses to both liveThis study is one of three parallel trials whose designs and cross-cutting analyses are described separately in this journal . The focus of this POPulation differences in VACcine responses (POPVAC) B trial is on infection with malaria parasites. Malaria remains highly prevalent in Africa and Uganda is among the six countries with the highest prevalence of malaria parasites on the continent.et alSeveral studies have previously reported impaired meningococcal, tetanus and typhoid vaccine responses in children infected with malaria parasites.Malaria may impact vaccine responses due to acute immunological changes (associated with fever),The overarching goal of the POPVAC programme is to understand population differences in vaccine responses in Uganda, in order to identify strategies through which vaccine effectiveness can be optimised for the low-income, tropical settings where they are especially needed. For this Trial B, we focus on the hypothesis that malaria infection suppresses responses to unrelated vaccines, and that this effect can be reversed, at least in part, by IPT of malaria in schools in high transmission settings.To determine whether there are reversible effects of malaria infection on vaccine response in adolescents, using an intervention study.Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) reporting guidelinesAttending the selected school and planning to continue to attend the school for the duration of the study.Aged 9 to 17 years and enrolled in primary 1 to 6.Written informed assent by participant and consent by parent or guardian.Agree to avoid pregnancy for the duration of the trial .Willing to provide locator information and to be contacted during the course of the trial.Able and willing (in the investigator’s opinion) to comply with all the study requirements.Clinically significant history of immunodeficiency (including HIV), cancer, cardiovascular disease, gastrointestinal disease, liver disease, renal disease, endocrine disorder or neurological illness.Moderate or severe acute illness characterised by any of the following symptoms: fever, impaired consciousness, convulsions, difficulty in breathing or vomiting; or as determined by the attending project clinician.Family history of sudden death attributable to heart condition in a first-degree relative.Family history of long QT syndrome.Know congenital prolongation of the corrected QT (QTc) interval.History of known heart disease or fainting.Known allergy or history of adverse reaction to DP or to artemether-lumefantrine.History of serious psychiatric condition or disorder.>5 years.Previous immunisation with yellow fever (YF), oral typhoid or HPV vaccine; previous immunisation with BCG or tetanus and diphtheria vaccine (Td) at age Concurrent oral or systemic steroid medication or the concurrent use of other immunosuppressive agents within 2 months prior to enrolment.Current use of medications known to prolong the QT interval.History of allergic reaction to immunisation or any allergy likely to be exacerbated by any component of the study vaccines including egg or chicken proteins.Tendency to develop keloid scars.Haemoglobin less than 80 g/L.Positive HIV serology.Positive pregnancy test.Female currently lactating, confirmed pregnancy or intention to become pregnant during the trial period.Use of an investigational medicinal product or non-registered drug, live vaccine, or medical device other than the study vaccines for 30 days prior to dosing with the study vaccine, or planned use during the study period.Administration of immunoglobulins and/or any blood products within the 3 months preceding the planned trial immunisation date.Further information on recruitment criteria can be found in 10.1136/bmjopen-2020-040427.supp1Supplementary dataWe will individually randomise participants in a 1:1 ratio to an experimental intervention of monthly DP versus a DP-matching placebo in a double-blind, placebo-controlled trial . DP has An independent statistician will generate the randomisation code using a randomly permuted block size. This code will be embedded into a web-based randomisation system in REDCap (Research Electronic Data Capture) software.The trial will be double-blind, and will remain so until data collection and data cleaning are complete and data locked for analysis, unless unblinding is required for individual participants who become pregnant  or as a result of serious adverse events or reactions (at the request of the Data and Safety Monitoring Board).We will study a portfolio of licensed vaccines  expected to be beneficial  to adolescents in Uganda. Our schedule  will comThe schedule of immunisation and sampling is outlined in These will be assessed in all participants.BCG: BCG-specific interferon-γ ELISpot response 8 weeks post BCG immunisation.YF-17D: neutralising antibody titres  at 4 weeks post YF immunisation.Salmonella typhi lipopolysaccharide-specific IgG concentration at 4 weeks post Ty21a immunisation.Ty21a: HPV: IgG specific for L1-proteins of HPV-16/18 at 4 weeks post HPV priming immunisation.Td: Td-specific IgG concentration at 4 weeks post Td immunisation.These will be assessed in all participants and will further investigate estimates of protective immunity (for vaccines where these are available) and dynamics of the vaccine responses, as well as the impact of the interventions on parasite clearance.Proportions with protective neutralising antibody (YF); protective IgG levels (TT);Primary outcome measures  repeated at week 52, and area under the curve analyses.Effects on priming versus boosting will be examined for HPV only, comparing outcomes 4 weeks after the first, and 4 weeks after the second vaccine dose.Will be assessed retrospectively by PCR on stored samples collected on immunisation days and at week 52.Furthermore, our sample collection will offer opportunities for an array of exploratory immunological evaluations on stored samples, focussing mainly on vaccine antigen-specific outcomes. Exploratory assessments will provide further detail on immune response characteristics over the study time-course, and on the role of immunological profiles in malaria-mediated modulation of vaccine-specific responses.Schistosoma mansoni infection status and intensity will be determined by serum/plasma levels of circulating anodic antigen (CAA). This method is quantitative, highly specific for Schistosoma infection and much more sensitive than the conventional Kato Katz method.Current Prior exposure to schistosomiasis will be evaluated by ELISA for IgG to schistosome egg antigen using stored blood samples collected at baseline.The presence of other helminth infections will be determined retrospectively using stool PCR of samples collected at baseline and at weeks 28 and 52. In accord with national guidelines, all participants will be treated with albendazole or mebendazole after collection of samples for primary endpoints at week 8 and 28, and after collection of samples for secondary endpoints at week 52.Malarial fever: Individuals presenting with fever will be investigated using rapid diagnostic tests for malaria and treated based on the results and according to prevailing national guidelines.Prior malaria exposure will be evaluated by ELISA for IgG to malaria antigen using stored samples collected at baseline.10; that responses in rural, high-parasite settings may be 0.3 to 0.4 log10 smaller than in the urban setting, and that effective treatment of parasitic infections may restore responses by approximately 0.2 log10 .10) or (in some cases) smaller. Among 5 to 15 year olds in this study setting, previous studies have reported malaria prevalence of >50% based on microscopy;>60% based on PCR.Based on the literatureBased on these assumptions, we plan to include 640 participants in total (320 DP and 320 placebo); of whom 384 are expected to be malaria infected, giving 192 participants in each trial arm who are infected at baseline.Ethical approval has been granted from the Research Ethics Committees of the UVRI  and the LSHTM (reference: 16033), and from the Uganda National Council for Science and Technology  and the Uganda National Drug Authority . Any protocol amendments will be submitted to ethics committees and regulatory bodies for approval before implementation.Participants will be adolescents and therefore a vulnerable human population. Care will be taken to provide adequate, age and education-status appropriate information and to ensure that it is understood; and to emphasise that participation is voluntary. Participants will be enrolled only when they have given their own assent and when consent has been given by the parent or guardian. No major risks to the participants are anticipated since all the treatments and vaccines to be given are licensed and known to be safe. The main risk to participants will be time lost from school work: we will work with teachers and parents to minimise disruption to classes, and will avoid enrolment of primary seven students since these classes are involved in national examinations. Further risks are discussed in For this trial, we gave particular consideration to the differential provision of malaria treatment. IPT with DP is an experimental intervention that has been shown to reduce the prevalence of anaemia and reduce episodes of clinical malaria in Ugandan schoolsMalaria infection status will be determined retrospectively through assays conducted in bulk on stored samples . These results will not, therefore, be useful to determine management of individual participants. Participants in the placebo arms will receive lower levels of anti-malaria treatment. However, all trial arms will receive a minimum of well-implemented national standard of care. Malaria standard of care will comprise provision of bed nets to minimise malaria exposure for all participants. Rapid diagnostic tests and treatment will be made readily available for participants who develop symptomatic malaria.Study findings will be published through open-access peer-reviewed journals, presentations at local, national and international conferences and to the local community through community meetings. Anonymised participant level data sets generated will be available on request.Concepts involved in this work have been discussed with colleagues at the Vector Control Division and EPI in the Ministry of Health (Uganda). Before commencing the trial, discussions will be held with officials at Jinja District Council, community leaders and Village Health Teams from sub-counties in which selected schools are located. In addition, we will engage teachers and parents in planning the detailed standard operating procedures for the study. Prior to recruitment, we will hold meetings to explain the proposed work to teachers, parents and students and to address their questions. At the end of the study, results will be shared with these stakeholders and with participants.Sociodemographic information and clinical and laboratory measurements will be recorded and managed using REDCap (Research Electronic Data Capture) tools,The effect of monthly IPT for malaria versus no IPT on the outcomes will be analysed using unpaired t-tests, with results presented as a mean difference in vaccine response measure together with 95% CI and p value. In the event of any imbalance in key characteristics between trial arms, multivariable regression models will be used to adjust for these potential confounders. We anticipate that outcomes will be positively skewed, and will apply log transformations to normalise distributions before analysis is conducted. Information on malaria infection status will only be available after randomisation. The primary analysis will be done on individuals identified as infected at baseline ; this will test the hypothesis that treating the infection (and subsequent reinfections) removes parasite-induced effects on vaccine responses. Secondary analyses will include all randomised individuals; this will provide insight into the potential benefit of the interventions as public health measures.Parasites have long been proposed as modulators of vaccine responses;This study aims to determine whether treating malaria with DP before and after immunisation with unrelated vaccines can improve vaccine responses in adolescents. The use of DP in this study is considered an attractive option for preventive treatment and preventive chemotherapy for malaria because of the long half-life of piperaquine (approximately 23 days).A concern arises where treatment of malaria with DP may have potential adverse effects such as cardiac toxicity, particularly dose-dependent prolongation of the QTc interval and associated increased risk of arrhythmias. There has been concern that provision of multiple doses might increase this risk especially when taken with food. However, a recent meta-analysis involving 11 trials of DP  involving 14 628 participants, 3935 of whom received multiple treatments with DP, found no evidence to suggest significant cardiac toxicity.We expect about 60% of the adolescents to have malaria infection at the outset in the proposed study area. This may vary by season and year, and the prevalence could be much lower depending on the timing of the start of the study. If this becomes the case, the two study arms will not be different enough to test our hypothesis. To mitigate this, we will work closely with colleagues monitoring trends in malaria prevalence in Uganda and adjust our study site to optimise the likelihood of achieving the expected prevalence.This interventional study will help us to understand how malaria affects responses to commonly used vaccines, which will inform implementation of public health programmes for immunisation and infection control especially in low-income and middle-income countries.Applications for ethical approval were submitted in May 2018, with approval received in September 2018 (UVRI REC), May 2019 (UNCST), June 2019 (LSHTM) and January 2020 (NDA). Collaborator/investigator/trial steering committee meetings were also held during the initial 12-month planning period. Recruitment to POPVAC B is scheduled to commence in February 2021. Intervention will be up to 12 months, with completion of the project scheduled for April 2022.


Plasmodium infection in the liver and blood stages of the parasite life cycle is composed by both pro- and anti-inflammatory programs. Pro-inflammatory responses primarily mediated by IFN-γ controls the infection, but also induce tolerogenic mechanisms to limit host damage, including the tryptophan (TRP) catabolism pathway mediated by the enzyme Indoleamine 2,3-Dioxygenase (IDO1), an enzyme that catalyzes the degradation of TRP to kynurenines (KYN). Here we assessed total serum kynurenines and cytokine dynamics in a cohort of natural Plasmodium vivax human infection and compared them to those of endemic healthy controls and other febrile diseases. In acute malaria, the absolute free kynurenine (KYN) serum levels and the KYN to TRP (KYN/TRP) ratio were significantly elevated in patients compared to healthy controls. Individuals with a diagnosis of a first malaria episode had higher serum KYN levels than individuals with a previous malaria episode. We observed an inverse relationship between the serum levels of IFN-γ and IL-10 in patients with a first malaria episode compared to those of subjects with previous history of malaria. Kynurenine elevation was positively correlated with serum IFN-γ levels in acute infection, whereas, it was negatively correlated with parasite load and P. vivax LDH levels. Overall, the differences observed between infected individuals depended on the number of Plasmodium infections. The decrease in the KYN/TRP ratio in malaria-experienced subjects coincided with the onset of anti-P. vivax IgG. These results suggest that P. vivax infection induces a strong anti-inflammatory program in individuals with first time malaria, which fades with ensuing protective immunity after subsequent episodes. Understanding the tolerance mechanisms involved in the initial exposure would help in defining the balance between protective and pathogenic immune responses necessary to control infection and to improve vaccination strategies.The human immune response that controls  Plasmodium vivax infects over 20 million people each year , with immunoregulatory effects in both T and myeloid cells . The conP. vivax malaria immunity in an endemic region. We also aimed to explore its potential role for immunomonitoring disease dynamics in the context of natural infection, where multiple exposures to P. vivax are prevalent and define natural protection to the parasite.We therefore aimed to explore the dynamics of the potent IFN-γ-induced cell intrinsic response mediated by the tryptophan catabolic pathway, assessing its potential role during the development of anti-This study was approved by the Universidade Federal do Amazonas (UFAM). All the study participants provided an informed consent prior to enrollment.P. vivax infection by measuring different hematological and immune parameters  in an endemic region.All study participants were recruited at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado  from March 2015 to February 2016. We analyzed the immune response to natural P. vivax-diagnosed patients, with either a first-time malaria episode or more than one episode, n = 81), patients with other febrile diseases , and healthy malaria-negative endemic controls (n = 34)  . The cliP. vivax antibodies.The study was exploratory and the sample size was not pre-defined by effect size, but limited by voluntary participation and enrollment. Previous malaria history and the number of days with fever and other symptoms before diagnosis were assessed by self-reported medical history. The degree of pre-existing immunity was assessed by the presence of serum anti-P. vivax-positive patients after successful anti-malaria treatment and clearance of blood-stage parasites, as recommended by the Brazilian Ministry of Health. However, only 47% of the recruited patients returned for followup after anti-malaria treatment . After treatment, all patients were malaria-negative, confirmed by thick blood smear microscopy.A follow-up assessment was performed to evaluate the dynamics of the hematological and immune response parameters in ®): Hematocrit, Hemoglobin, White blood cells, Red blood cells, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Mean platelet volume, Platelets.Peripheral blood samples were obtained from 136 participants. A total of 5 mL of blood was drawn from each participant  and divided into two parts: one part was used for hematological analysis and a second part was used for blood plasma analysis. Whole blood samples were employed for assessing hematological parameters using an automated hematological analyzer (Sysmex KX-21 N–1 and 20 μL of clear sample was then injected into the HPLC system using an autosampler. TRP was identified at 278 nm and KYN at 360 nm by UV detection (y) against known TRP or KYN concentration (x), respectively. The linearity of the standard curves was confirmed using regression variance analysis and significance of correlation co-efficient (r) checked using Student’s t-test. KYN/TRP ratio was calculated using the formula KYN concentration (μM/L)/TRP concentration (μM/L).After separation from whole blood, plasma samples (200 uL) were treated with 8% Perchloric acid, to precipitate proteins and extract soluble metabolites. After extraction, the aqueous phase was analyzed by High Performance Liquid Chromatography (HPLC) as previously described . For theetection . RetentiPlasma cytokines  levels were quantified using the CBA Th1, Th2, and Th17 kit , following the manufacturer’s instructions. Samples were analyzed on a FACSCanto II  flow cytometer and data analyzed by FCAP ArrayTM software (V3.0.1). The mean fluorescence intensity (MFI) of each bead cluster was determined and forth logistic regression applied to build the standard curves. Cytokine concentrations for each sample were then extrapolated from the standard curves and data was expressed as pg/mL for each plasmatic cytokine.in-house sandwich ELISA to detect P. vivax LDH (PvLDH) in untreated malaria patients as previously described , as previously described  and Rstudio version 1.1.4 with R version 3.5.0 and RPQ, HNP and Tidyverse packages.Differences in means and medians were tested, respectively, using either one-way ANOVA, with Tukey’s P. vivax malaria patients , and (4) P. vivax malaria patients followed-up after successful anti-malarial treatment. Median  parasite load/mm3, estimated by thick blood smear, was 2158 (537.6−4185) in P. vivax malaria patients, and the mean parasite load was not different between patients with a first-time malaria episode, and individuals with previous malaria history  , and out of eighty-one malaria patients, forty-three patients were lost to follow-up after treatment . Table 12-5.75)] .n = 34), P. vivax-malaria patients (n = 81), and patients with other febrile diseases (n = 21). The hematological parameters and serum cytokines levels of these groups are summarized in P. vivax infection induces a significant decrease in multiple circulating blood cell lineages, together with a distinct pro- and anti-inflammatory serum cytokine response.In this cross-sectional study we evaluated healthy subjects . All measured hematological parameters returned to levels comparable to those of the healthy controls . The patients or participants provided their written informed consent to participate in this study.RS, ML, and PL contributed conception and study design. RS, MC, and SL collected and analyzed the patient samples. LO, PN, IS, and FK provided reagents and performed malaria serology. EL assisted with HPLC analysis. AC provided reagents and cytokine analysis. RS, FC, CG, and PL analyzed the data and prepared the figures. RS, CG, and PL wrote the manuscript. All authors contributed to manuscript revision, read 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 parasites cause millions of people worldwide to suffer malaria every year. Drug-resistant Plasmodium parasites and insecticide-resistant mosquitoes make malaria hard to control. Thus, the next generation of antimalarial drugs that inhibit malaria infection and transmission are needed. We screened our Global Fungal Extract Library (GFEL) and obtained a candidate that completely inhibited Plasmodium falciparum transmission to Anopheles gambiae. The candidate fungal strain was determined as Aspergillus aculeatus. The bioactive compound was purified and identified as asperaculane B. The concentration of 50% inhibition on P. falciparum transmission (IC50) is 7.89 µM. Notably, asperaculane B also inhibited the development of asexual P. falciparum with IC50 of 3 µM, and it is nontoxic to human cells. Therefore, asperaculane B is a new dual-functional antimalarial lead that has the potential to treat malaria and block malaria transmission.Mosquito-transmitted  Plasmodium parasites have developed resistance against the most commonly used antimalarial drugs throughout Africa and Asia  were seeded into microplates and incubated for 24 h at 37 °C and 5% CO2. Next, the cells were incubated with 100µL of MTT reagent for four h at 37°C . After removing all but 25 µL of the medium from the wells, 100 µL of DMSO was added to each well and incubated for 10 m to dissolve the purple formazan crystal for measurement. The optical density at an absorbance wavelength of 540 nm was measured with the plate reader.The MTT -2,5-diphenyl tetrazolium bromide) cell proliferation assay (Thermo Fisher Scientific) was used to measure the general cytotoxicity of pure compounds to human embryonic kidney 293 (HEK293) cell line. The HEK293 cells at a concentration of 2 × 10p-value of infection difference between control and experimental groups in each assay with Prism .All the experiments were independently repeated at least twice. As reported previously ,9,10, th


In this article, the effect of hydroxyethyl methyl cellulose (HEMC), which is a polymeric viscosity modifying admixture on the mineral based composites setting processes, was studied. Previous studies available in the literature included the evaluation of the influence of this admixture on the hydration processes of cement or lime pastes. In this paper, the analysis of this issue was extended to include cement-lime composites. The composition of the pastes and mortars differed in the type of binder , as well as the amount and viscosity of the admixture. The study of mortars setting processes and hardening processes using the ultrasonic method was supplemented in the work with calorimetric measurements and phases analysis by the X-ray diffraction method. Finally, it was found that the HEMC reduces the rate of a hydration reaction in cement and cement-lime pastes. The amount of admixture used has a greater influence on the changes taking place during the setting process than the admixture viscosity or the type of binder. The use of chemical admixtures in modern mortar technology is a prerequisite for obtaining materials of high quality and durability that meet the standard requirement. The aim is to ensure that the mortars are characterized by good workability, ease of application, adequate adhesion to the masonry, and lowest possible shrinkage deformation. 2 crystals was reported to reduce its entanglement between chains and, hence, the viscosity of the pastes as well as its water retention ability. Similar results of the influence on the delay of setting time were observed in ceramic tile adhesive mortars by Petit and Wirquin [3A and C3A-sulphate systems. It was observed that this admixture leads to a gradual slowing down of the C3A hydration depending on cellulose ether chemistry. Two types of cellulose ethers were used in their research: hydroxypropyl methyl cellulose (HPMC) and hydroxyethyl cellulose (HEC). They observed that HECs include a higher delay than HPMCs. In their opinion, the substitutions group seems to be more important for controlling factors on C3A hydration rather than molecular mass. Betioli et al. [Cellulose ethers (CE) are a group of polymer admixtures used in dry pre-mix mineral-based binder materials ,2,3. Hyd Wirquin . Initial Wirquin  studied i et al.  observedi et al.  concludei et al.  observedThe correct selection of the admixture, especially its quantity and viscosity, affects the parameters of the finished product. Knowledge of the processes taking place during the setting and hardening of mortars modified with this admixture allows for a correct and comprehensive assessment of its effectiveness and applicability. Based on the publications available in the literature, it can be concluded that the research results presented so far focus mainly on determining the influence of cellulose ethers on the rheological, physical-mechanical properties of cement ,5,6,24 oIn the studies, Portland cement CEM I 42.5 R , hydrated lime deeply separated , two fractions of quartz sand 0.1–0.5 mm and 0.2–0.8 mm , and polymer admixture in the form of HEMC  were used. Cellulose ether used in tests is a hydroxyethyl methyl cellulose with viscosity of 25,000 mPa·s and 45,000 mPa·s (for measurements using a Brookfield rheometer at 20 °C) with low chemical modification. This admixture has the form of white powder with a grain size below 0.063 mm. The pH of its 1% solution in water (at 20 °C) is 7.0. The characteristics of cement and hydrated lime are given in Mortars and pastes differing in their composition with the type of binder, amount, and viscosity of HEMC admixture were prepared for the tests. As a reference material, a cement mortar without admixture, designated as C-0, was used. In addition, cement composites modified with admixture in various amounts and viscosity  and composites modified with cellulose ether, in which cement was replaced with 50% hydrated lime . The MV symbol stands for Medium Viscosity , whereas the HV symbol stands for High Viscosity . The admixture was added in the amount of 0.05%, 0.175%, and 0.3% in the relation to the dry ingredients of the mortar (binder and fine aggregate). The detailed composition of mortars is given in Cement, hydrated lime, and fine aggregate were weighed with an accuracy of 0.1 g, and the chemical admixture was weighed with an accuracy of 0.0001 g. The ingredient mixing procedure was as follows: 90 seconds of mechanical mixing at 45 rpm, 30 s of rest, and 90 s of mechanical mixing at 57 rpm. The time and method of mixing were constant (the same in each case).All mortars were performed, maintaining a constant weight ratio of the binder to the fine aggregate of 1:10. The amount of water was chosen so as to obtain a constant consistency of 165 mm, measured using a flow table, according to the PN-EN 1015-3:2000 standard . All samTesting of setting processes by the ultrasonic method,Calorimetric measurements,Phases analysis by the XRD method.Determination of the effect of hydroxyethyl methyl cellulose on the setting processes of cement and cement-lime composites was carried out using three methods, which included:Using the first method, mortar samples were tested, whereas, using the second method, paste samples were tested. In each case, the experiments were carried out for the first 48 h of material maturation. Additionally, using X-ray diffraction analysis, measurements after 40 days of maturation were made to verify whether there are differences in the leaven hydration products compared to the results obtained after two days.The setting process was determined using the ultrasonic wave velocity method by an IP-8 Ultrasonic Measuring System . The essence of the study consisted in passing the ultrasonic wave through the material from one end to the other and recording its velocity over time. Ultrasonic heads and a temperature sensor were mounted on both sides for all forms. Each time, a sample of fresh mortar was taken , which was placed for the entire duration of the test in the sampler . The uppThe BT2.15CS low-temperature differential scanning microcalorimeter by SETARAM  was used for micro-calorimetric measurements. Binder samples (with admixture or without it) were mixed with distilled water and put into small PE zip-bags and, immediately after mixing, placed in the calorimeter. Calorimetric curves were recorded with an electronic measuring unit, connected to a personal computer.XRD images were obtained in an X-ray diffractometer Empyrean , equipped with a copper lamp. FT scanning was carried out at a 2θ range of 5–65° and the step length was 0.0167°. The PANalytical XRD analysis software HighScore 4.6  with the International Center for Difration Data (ICDD) database PDF-2 was used for phase identification. X-ray diffractometric studies were carried out on powdered samples of pastes after the time of 2 and 40 days of setting.The ultrasonic technique used in the research is based on the registration of the speed of the ultrasonic wave, which is related to the change in the viscosity and elasticity of the medium. The publications ,34,35,36The setting process varies in intensity depending on the binder used and the amount and viscosity of the cellulose ether. It is clearly visible that the use of an admixture in the amount of 0.3% delays the setting process of both cement and cement-lime mortars (C-0.3-MV and CL-0.3-MV mortars). This is especially true when comparing the induction period of individual materials. This parameter is defined as the time counted from the setting of the measurement to the moment of a sudden change in the velocity of the ultrasonic wave. It should also be noted that, in the case of all modified mortars, the maximum velocity of the ultrasonic wave has been significantly reduced, from 2180.1 m/s, even to 423.3 m/s (more than 5 times), which may be related to the different microstructure (among other porosities) of these materials, resulting from the replacement of cement binder on lime and the use of admixtures. Mortar CL-0.3-MV modified with cellulose ether, in which 50% cement was replaced with hydrated lime, was characterized not only by the lowest ultrasonic wave velocity after 48 h of maturation, but also the ultrasonic wave velocity recorded during all setting processes was the lowest. The smallest differences in the setting process, in relation to the reference sample (C-0), are visible for the cement mortar with the admixture of 0.05% (sample C-0.05-MV) and viscosity of 25,000 mPa·s. In other cases, it is clearly visible that both the lime and the admixture significantly reduce the velocity of the ultrasonic wave during every period of time for measurements. The induction period of 10 h 54 min and 7 h 18 min indicates that the setting processes of C-0.3-MV and CL-0.3-MV mortars were delayed due to the use of an admixture in the amount of 0.3% and an admixture viscosity of 25,000 mPa·s. The induction time is visible only in the case of mortars modified with a cellulose ether of 0.175% and 0.3%. Despite the fact that the induction time is clearly visible only in the case of four mortars , a slow The changes in the rate of heat release and the amount of heat released during the hydration of pastes as a function of time are shown in The curve of cumulative heat of hydration  shows thIn Comparing the results obtained on the basis of measurements using the ultrasonic technique with the results of calorimetric tests, there can be a similarity of the course of the curves showing the change in the velocity of the ultrasonic wave in time , Table 4The hydration of the reference paste (cement paste without admixture of cellulose ether) is model-like, which means that, in this case, significant amounts of Portlandite are formed . The larFor cement-lime pastes, the increase of calcite peaks is also characteristic, which may indicate of the partial lime binder carbonation. Due to a binder composition, alite and belite have less intense peaks in these pastes.The diffractograms of the pastes after 40 days of maturation were shown in 3 and the progressive carbonation process. In 2 and CaCO3 were seen in TG analysis. Percent content H20, Ca(OH)2, and CaCO3 after 28 and 91 days was bigger than after 7 days maturation (compared to a reference sample), which confirm the ongoing carbonation processes. On the other hand, percent content of Ca(OH)2 and CaCO3 after 7 days was smaller in the case of the sample with cellulose ether than the reference sample, which may indicate a delay of reaction of carbonation processes caused by the admixture.However, comparing the diffraction patterns after two days and after 40 days, it is possible to notice the different intensity of Portlandite and calcite. A greater amount of Portlandite after 40 days may be caused by the hydration process, and an increased amount of calcite is associated with the formation of calcium carbonate CaCOThe following conclusions could be drawn from the research.The addition of hydroxyethyl methyl cellulose influences the setting processes of cement and cement-lime mortars. The addition of HEMC with a viscosity of 25,000 mPa·s in the amount of 0.3% significantly delays the setting process regardless of the type of binder , which is not so visible in the case of the admixture in the amount of 0.05%.HEMC reduces the rate of the hydration reaction in cement and cement-lime pastes, which has been confirmed in calorimetric tests. The induction time on the heat evolution curve becomes significantly longer (by a maximum of 10 h 10 min). The changes are the most visible with an increasing dosage of the admixture.In the XRD analysis, significant effects of the presence of HEMC were found on the resulting Portlandite peaks. Limiting their intensity during the period of the first two days shows that the hydration process is lower.The differences in the hydration of pastes after 40 days of maturation are much smaller than the hydration of the same samples after two days of maturation. This shows that significant phase changes take place at a later stage of hydration (after 48 h). The addition of HEMC has a greater effect on the hydration of cement and cement-lime pastes in the initial hours of maturation. After a longer time, this effect practically disappears. Therefore, control of hydration kinetics of cement and cement-lime pastes on early stages is important.The effect of HEMC admixture on the inhibition of setting processes is more visible in pastes and mortars containing only cement binder, especially by analyzing the differences in the induction time from the ultrasonic technique and calorimetric measurements.The amount of the admixture used is the most important in the process of setting pastes and mortars. A much-reduced impact is visible when taking into account the viscosity of the admixture or the type of binder used.The ultrasonic technique in combination with calorimetric measurements and XRD method gives the possibility of a more complete and broader assessment of the setting mortars. It also enables the assessment of the influence of a chemical admixture on the hydration and setting processes of pastes and mortars. It can be useful in monitoring and controlling for application plastering mortars.


After a marked reduction in malaria burden in Cambodia over the last decades, case numbers increased again in 2017–2018. In light of the national goal of malaria elimination by 2025, remaining pockets of high risk need to be well defined and strategies well-tailored to identify and target the persisting burden cost-effectively. This study presents species-specific prevalence estimates and risk stratification for a remote area in Cambodia.Plasmodium infection by PCR. Risk of infection was associated with questionnaire-derived covariates and spatially stratified based on household GPS coordinates.A cross-sectional survey was conducted in 17 villages in the high-incidence province Mondulkiri in the dry season (December 2017 to April 2018). 4200 randomly selected participants (2–80 years old) were tested for Plasmodium infection was 8.3% (349/4200) and was more than twice as high for Plasmodium vivax  than for Plasmodium falciparum . 97.8% (262/268) of P. vivax and 92.8%  of P. falciparum infections were neither accompanied by symptoms at the time of the interview nor detected by microscopy or RDT. Recent travels to forest sites  and forest work  were particularly strong risk factors and risk profiles for both species were similar. Large village-level differences in prevalence of Plasmodium infection were observed, ranging from 0.6% outside the forest to 40.4% inside. Residing in villages at the forest fringe or inside the forest compared to outside was associated with risk of infection . Villages inside the forest formed spatial hotspots of infection despite adjustment for the other risk factors.The prevalence of PCR-detectable P. vivax the implementation of radical cure. In villages inside the forest, within-village exposure was indicated in addition to risk due to forest activities. Village-level stratification of targeted interventions based on forest proximity could render the elimination efforts more cost-effective and successful.Persisting pockets of high malaria risk were detected in forested areas and in sub-populations engaging in forest-related activities. High levels of asymptomatic infections suggest the need of better case detection plans and the predominance of  In the last two decades, Cambodia has seen a marked decrease in numbers of reported malaria cases from approximately 140,000 cases in 1999 to 60,000 in 2018 .Plasmodium falciparum and Plasmodium vivax in 2017, they declined for P. falciparum in 2018 but increased further for P. vivax [P. vivax now accounts for almost three-quarters of all malaria cases in Cambodia [This ambitious aim however faces several obstacles. After an all-time low of approximately 20,000 reported cases in 2016, numbers have increased again in 2017–2018 [P. vivax . ConsequCambodia .P. falciparum malaria in Western Cambodia [P. falciparum cases in 2017 and the change of first-line treatment from dihydroartemisinin-piperaquine to artesunate-mefloquine their subsequent decline in 2018. For P. vivax, such resistance is not reported. However, this species has dormant liver-stages called hypnozoites that escape acute blood stage treatment and cause relapsing infections. Therefore, although LLINs do reduce P. vivax transmission and ACT is effective against blood stage P. vivax infection, neither intervention targets the hypnozoite reservoir. Hence, there is a less marked effect of these interventions on P. vivax than P. falciparum.The national malaria control programme (NMCP) is based on mass distribution campaigns of long-lasting insecticidal nets (LLIN), diagnosis by light microscopy (LM) and rapid diagnostic tests (RDT) in the public health sector, RDT-based testing by village/mobile malaria workers (VMW/MMW), and treatment of blood stage infections with artemisinin-based combination therapy (ACT) . The sprCambodia  might exP. vivax radical cure is part of the national treatment guidelines but has not yet been implemented because of concerns over the drug’s potential haemolytic effect in patients with G6PD deficiency [P. falciparum malaria  and referred to a local health care provider for treatment if positive.Based on the census, a random selection of households was drawn oversampling small villages to ensure sufficient coverage , and work in the deep forest were considered part of the multivariate model a priori in order to assess the association of forest exposure with risk of infection. Having slept outdoors or under a bed net last night were kept as part of the model based on causal reasoning or prior knowledge due to their association with infection risk in previous publications, e.g. [The association of covariates with infection by ns, e.g. . Gender ns, e.g. .All questionnaires were applied on tablets using the REDCap (Research Electronic Data Capture) software hosted at Institut Pasteur in Paris , 31. DatFrom among the 10,053 individuals in 2351 households identified in the census, the survey recruited 4200 participants from 1147 households and oversampled smaller villages on average  of participants. The proportion of samples positive for P. vivax was 6.4% (268/4200) compared to 3.0% (125/4200) for P. falciparum  of all infections compared to 36% (125/349) for P. falciparum. Four samples were positive for P. malariae mono-infections and none for P. ovale. Speciation by PCR was unsuccessful in 4 samples which were analysed as negative. Extrapolation to the entire Kaev Seima population yielded an estimated prevalence of 8.9% for Plasmodium infection, 6.8% for P. vivax, and 3.3% for P. falciparum. Estimated prevalence was highly heterogeneous across the villages, ranging from 0.6% (3/594) to 36.3% (54/152) and from 0% (0/594) to 25.1% (27/106) for P. vivax and P. falciparum, respectively  for P. falciparum  and 16.8% (21/125), respectively (p < 0.01). Only 2.2% (6/268) of all P. vivax infections were symptomatic and also positive by RDT or LM , less often than for P. falciparum . Asexual parasite stages were detected in all LM+ samples for P. vivax (37/37) and in most  for P. falciparum , respectively. While gametocytes were found in only one  P. vivax LM+ sample, more than a third  of LM+ samples for P. falciparum were gametocyte-positive with a geometric mean of 51.1 gametocytes/μL.Of all PCR-detected infections, 83.2% (223/268) were sub-microscopic for i.e. 10.4% (188/1969) vs. 3.6% (80/2231) for P. vivax (p < 0.001), 4.8% (83/1969) vs. 1.9% (42/2231) for P. falciparum (p < 0.001), and 13.3% (239/1969) vs. 5.0% (110/2231) regardless of species (p < 0.001). The patterns across age were similarly heterogeneous for both species , regardless of the proximity of the village to the forest , but not across those villages at the forest fringe or inside the forest.Estimated prevalence was more than twice as high in men as in women, P. vivax and P. falciparum in a highly similar fashion  and lowest for urban destinations . Infections of both species were also more prevalent among those who reported work trips to sites in the nearby and deep forest (assessed separately). In particular, a significantly higher prevalence was observed in participants who reported work trips into the deep forest  compared to those who did not . Once clustering at household and village-level was taken into account in univariate mixed-effects logistic regression, work in the nearby forest was no longer significantly associated with risk of infection though . A higher risk in men, at working age, in villages at the forest fringe or inside the forest, and with indicators of lower socio-economic status was also found similarly for both species . Risk of infection was not significantly attributed to the other behavioural covariates that were included in the model a priori, namely having slept outdoors  and under a bed net . Risk of infection was increased in males  and working age . A fitted interaction term of gender and age improved the model . The covariate on recent information on malaria via TV  most likely also acts as a proxy for higher socio-economic status, i.e. being able to afford a TV in the first place.Behavioural covariates related to activities in the forest were statistically significant in the multivariate model of on Table . Having vs. 2.6%, 57/2231, of the women, p < 0.001) and particularly frequent in male adolescents . Forest work was independently assessed for sites of the nearby and deep forest. Only for the latter, a statistically significant association with risk was observed. As for travels, they were reported more frequently by men than by women  and most often by 21–30 years old males . However, working in the deep forest occurred in both men aged between 8 and 60 years and in 8–43 years old women. Forest travels were reported in 3–62 years old men and 3–63 years old women. Both kind of trips occur usually frequently and of short duration. In 81% (217/268) of the times, the travels to forest sites lasted less than a week. Work trips into the deep forest occurred in 75% (108/145) of the times at least weekly. When going for work several times a month or once a month, the reported duration was also most often less than a week . A net was used during overnight work trips into the forest in only 25% (28/113) of the instances. While equally few participants from villages outside the forest, at the forest fringe, and inside the forest reported work in the deep forest , travelling to forest sites was reported more often in forest fringe and forest villages .Work-unrelated overnight travels to forest sites was a predominantly male domain . New advances in the use of multiple antigens to detect antibodies as serological markers for recent exposure and thus for potential hypnozoite carriers are promising in this respect [More asymptomatic infections, lower diagnostic sensitivity for  respect .P. falciparum and P. vivax presented here is encouraging as it suggests that such targeted interventions would appropriately address both of the two species.With as many as 40% of the inhabitants infected in the highest-prevalence village in this survey, it may be necessary to complement the current malaria control efforts by active or reactive case detection schemes , 48 to rThis study emphasizes the predominant role of forest malaria transmission in explaining elevated risk of infection in remote rural areas of Southeast Asia. Previous cross-sectional surveys have also identified an association of infection risk with either work in the forest , 17, 49 2, the prevalence ranged from less than 1% to above 40% in the dry season. The trend towards higher prevalence in villages at the forest fringe and inside the forest compared to those outside the forest is in line with the widely accepted notion for forest-related mosquito abundance and exposure in Cambodia. Other studies have also identified a higher prevalence in villages at close proximity to the forest [On the relatively small spatial scale of 17 villages in an area of 22 × 26 kme forest , 49, 50.Not more than 60% of infections could be attributed to the strong behavioural, forest-related risk factors. In villages outside the forest or at the forest fringe, most infections indeed occurred in working age men. This predominance of this occupational risk group was much lower in forest fringe villages and entirely lost in villages situated inside the forest. Taking gender and age as proxies for risk-related behaviour and occupational activities, this could indicate that in villages outside the forest, risk can be explained almost completely by such occupational exposure. By contrast, for villages within the forest, the more homogeneous presence of infection across all age groups in both males and females indicates additional exposure inside the village. This is in line with other studies in which household-level risk factors in forested areas were important to explain infection risk  or infecThe current WHO report on malaria eradication calls for subnational stratification of intervention programmes , 53. ThiPlasmodium vivax infections were detected at a higher prevalence than P. falciparum infections, more often asymptomatic, and less detectable by RDT and LM. Consequently, novel tools for the identification of hypnozoite carriers could play a key role in the presented setting as well as the roll-out of routine radical cure treatment. The study corroborates the notion of forest malaria in the Greater Mekong Subregion by demonstrating the independent association of travels to the forest, of forest work, and of living in close proximity to forest with malaria infection risk. However, the study also demonstrates that infection risk is less confined to forest-goers in sub-populations that already live in forested areas and suggests within-village transmission therein. A focus of interventions solely on forest-goers could thus be insufficient to reach the goal of nation-wide malaria elimination in due time. In the light of the presented results, interventions could be targeted at whole villages inside the forest, and targeted at stratified risk groups for villages outside the forest.Despite a substantial reduction in malaria burden in Cambodia over the last decades, this study demonstrates that pockets of high malaria prevalence persist in the country. Given that the vast majority of infections were asymptomatic, the study strengthens the argument to enhance malaria elimination efforts by measures of (re-)active detection of also asymptomatic infections. Additional file 1. Additional tables ST1–ST5 and additional figures S1–S3.