The global decline of malaria burden and goals for elimination has led to an increased interest in the fine-scale epidemiology of malaria. Micro-geographic heterogeneity of malaria infection could have implications for designing targeted small-area interventions.P. falciparum and P. vivax infections. After confirming the spatial dependence of malaria episodes with Ripley’s K function, SatScanTM was used to identify purely spatial and space-time clusters (hotspots) of annual malaria incidence for 2 years follow-up: year 1 (July 2008-June 2009) and year 2 (July 2009-June 2010).Two-year longitudinal cohort study data were used to explore the spatial and spatio-temporal distribution of malaria episodes in 2040 children aged < 10 years in 16 villages near the Gilgel-Gibe hydropower dam in Southwest Ethiopia. All selected households (HHs) were geo-referenced, and children were followed up through weekly house-to-house visits for two consecutive years to identify febrile episodes of P. falciparum episodes (in 492 HHs) and 385 P. vivax episodes (in 290 HHs) were identified, representing respectively incidence rates of 14.6 (95% CI: 13.4–15.6) and 8.2 (95% CI: 7.3–9.1) per 1000 child-months at risk. In year 1, the most likely  and secondary  clusters of P. vivax incidence were found respectively in southern and north-western villages; while in year 2, the most likely cluster was located only in north-western villages . Instead, most likely spatial clusters of P. falciparum incidence were consistently located in villages south of the dam in both years: year 1  and year 2 . Space-time clusters in southern villages for P. vivax were found in August-November 2008 in year 1 and between November 2009 and February 2010 in year 2; while for P. falciparum, they were found in September-November 2008 in year 1 and October-November 2009 in year 2.In total, 685 P. falciparum incidence in children were more stable at the geographical level and over time compared to those of P. vivax incidence during the study period.Hotspots of The online version of this article (doi:10.1186/s13071-017-2124-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum (63.7%) over P. vivax cases (36.3%). Oromia is the second regional state of Ethiopia with the highest malaria incidence, accounting for about 20%  of total reported clinical cases in the country , but the first one in terms of malaria mortality, representing about one third (214 deaths) of total malaria-related deaths in Ethiopia [Despite a decline in the global malaria burden over the past 15 years, about 3.5 billion people were at risk worldwide in 2015, and millions of them are still not accessing the services they need to prevent and treat malaria k. Of 438,000 registered malaria deaths in 2015, approximately 80% of the deaths were concentrated in just 15 countries, mainly in Africa [Anopheles arabiensis  [Malaria transmission is mainly seasonal and unstable throughout the country and varies due to differences in altitude, season, and population movement , 4. A gon pools) , 12 or rn pools) . Conditin pools) , and humn pools) .P. falciparum and P. vivax [P. falciparum and P. vivax malaria episodes in 2040 children aged < 10 years living in 16 villages around the Gilgel-Gibe hydropower dam. The study was conducted as part of several other studies, intended to assess the impact of the Gilgel-Gibe hydroelectric dam on health and other sectors  following its starting operation in 2004 [A previous analysis of malaria surveillance data based on passive case detection (PCD) in villages located around the Gilgel-Gibe hydroelectric power dam in Southwest Ethiopia suggests different spatial and temporal variations of malaria episodes for both P. vivax . Until nThe study was conducted in Gilgel-Gibe dam area, in Jimma zone Fig. , which iA longitudinal 2-year malaria cohort study was conducted in children under 10 years old, living in the 16 selected villages around the Gilgel-Gibe hydroelectric power dam. A total of 2040 children aged less than 10 years were enrolled in July 2008 and completed weekly follow-ups until June 2010 . Each chPlasmodium species.Active case detection (ACD) through weekly household visits was conducted to identify and register all febrile malaria episodes in the study population during the two-year follow-up period. During the household visits, axillary body temperature was taken, and the caregiver was asked about fever history. If a child had a fever (temperature ≥ 37.5 °C) or reported a history of fever in the past 24 h a finger-prick blood sample was taken for immediate diagnosis by LM in the same site, or at Omo-Nada District Health Center Laboratory. Trained laboratory technicians conducted LM diagnosis. Thick smears were used to confirm the presence or absence of parasites, whereas the thin smear was used to identify the P. vivax, and artemether-lumefantrine (AL) for P. falciparum twice daily according to the body weight as follows: 5–14 kg, one tablet per dose; 15–24 kg, two tablets per dose; 25–34 kg, three tablets per dose; and adults, four tablets per dose. Treatment adherence was monitored during household visits by asking for the medication packages and the remaining pills. Absent children were followed in the next visits, and their caregivers were asked about the occurrence of symptomatic episodes and the confirmation of malaria at the health facility. In addition, all the health facilities near the study communities were visited monthly to verify their clinical records, checking if any enrolled children had presented a confirmed malaria episode in the past month, not being detected during the weekly visits.All children with malaria confirmed by LM were treated according to the national treatment guidelines . Treatmeh of a given child with episodes), while a bivariate K-function explored the spatial independence between children classified into two groups according to specific conditions (determination of the expected number of children with condition 1 within distance h of children with condition 2) [Data were double entered, validated and cleaned in Excel . A univariate Ripley’s K-function was used to assess whether children with malaria episodes tended to be near other children with episodes (determination of the expected number of children with episodes within distance ition 2) .Ho). The univariate K-function tested the Ho that the children with malaria episodes were randomly spatially distributed , while the bivariate K-function tested two separated Ho: a) the children with and without malaria episodes were independently spatial distributed, and b) the children with malaria episodes, younger than 3 years old and older than 3 years old, were independently spatial distributed.For each malaria species and year of follow-up (first or second year), a file was created with the coordinates of children’s households. The file was read in R software as a table, and a grid was created using the maximum and minimum values of latitude and longitude coordinates. After creating an object of class “ppp” for the point pattern distribution of children in a polygonal window, the Kest and Lest functions from the package Spatstat were applied to plot both observed and expected K-values over a range of distances . Expecteh indicated random distribution of the children with malaria episodes for that distance h, while those larger than the high CE values or those smaller than the low CE values at specific distance h indicated respectively significant spatial clustering or spatial dispersion of children with malaria episodes for that distance h. In the bivariate analysis, observed difference of K values between the children group 1 and group 2 which were between low and high CE at a specific distance h indicated spatial independence between the children groups for that distance h. Instead, a difference of K values larger than the high CE values at specific distance h indicated that the children group 1 tended to be more clustered than children group 2 for that distance h, while a difference smaller than the low CE values indicated that the household group 1 tends to be more dispersed than household group 2. In all analyses, distances h ranged from 0 to the maximum distance between the two closest children’s households (about 5 km).In the univariate analysis, observed K-values between low and high CE at specific distance P. falciparum and P. vivax malaria episodes using the Bernoulli probability model [The QGIS software v.2.16   was usedty model . The BerP-value < 0.05 [The spatial analysis tests the null hypothesis of no clustering of children with malaria episodes. Different windows with varying size, from zero to a maximum radius of less than 15% of the total children, were allowed to move across the study area. This maximum radius was selected to avoid large non-populated areas within the identified malaria clusters by SaTScan. More details about the selection of maximum windows size can be found in Additional files e < 0.05 . Once thThe space-time analysis was performed under the null hypothesis that the risk of having malaria episodes was the same in all households and over time, with cylindrical windows having a circular geographic base and height corresponding to the time scale in weeks. The radius of each circular base was allowed to vary in size, to include up to as many as 15% of the total children. Comparably, the height of the cylinder varied in size up to a maximum of 50% of the study period with a time precision of one week. An unlimited number of overlapping cylinders with different dimensions were obtained, each cylinder corresponding to a possible space-time cluster. For each space-time cluster, the LLR was calculated and the most likely cluster defined as the cylinder with the maximum LLR. The statistical significance of the clusters was tested through 999 Monte Carlo simulations  to achieve strong power, and the null hypothesis was rejected when the resulting p-value was below 0.05.P > 0.05)  were due to P. vivax in 290 HHs. P. falciparum incidence rates were respectively 15.5 and 13.7 episodes/1000 child-months in the first and second year of follow-up (14.6 episodes/1000 child-months thorough the study period), while P. vivax incidence rates were respectively 12.6 and 3.8 episodes/1000 child-months in the first and second year of follow-up (8.2 episodes/1000 child-months thorough the study period). Additional files 3 and 4 show videos with the spatio-temporal distribution of species-specific malaria incidence by HH. The visual inspection from first video suggests seasonal spatial distribution of P. falciparum incidence, with increased occurrence of P. falciparum episodes in households located at south of the dam mainly in the last months of the long rainy season (August and September), as well as, in the first months of the dry season (October and November). Instead, the second video shows a decreasing trend in the occurrence of P. vivax episodes along the study period, with a less clear spatial and seasonal pattern.Of the total 2040 followed-up children, 981 (48.1%) were females and 1059 (51.9%) males. The mean age at enrollment was 4.9 ± 2.0 years, not varying significantly across villages Additional file 4: Spatio-temporal distribution of households presenting P. vivax episodes. Each yellow point represents an individual household. When a P. vivax episode is found in a child in the household, the point becomes red. (MP4 769 kb)P. falciparum episodes in the first year were significantly more clustered than children without episodes only at distances greater than 4.0 km . In year 2, the most likely cluster of P. falciparum episodes was also located south of the dam with a radius area of 3.1 km, including 133H of mainly Kara and Yasso villages and accounting for 67 episodes   and 5 weeks in year 2 (October 11th - November 14th 2009).Purely spatial analysis by SaTScan confirmed that malaria episodes due to both species were not randomly distributed. The most likely spatial cluster of des Fig. ; Table 201) Fig. ; Table 2ear Fig.  and the P. vivax incidence were a 4.1 km radius area located south of the dam in year 1  while the second one included 88 HHs in Buddo village presenting 16 episodes . The re-analysis of the children within the most likely clusters for P. vivax did not identify a further hotspot in both years. In addition, two small secondary clusters were identified west of the dam only in year 1; one of them had a similar location to the most likely cluster identified in the following year (year 2) , and 13 weeks in year 2 (November 15th 2009 - February 13th 2010).The most likely spatial clusters of r 1 Fig. ; Table 2r 1 Fig. ; Table 2P. falciparum episodes predominated over P. vivax episodes with P. vivax showing a sharp decrease in annual incidence rates during the study period. Comparatively, hotspots of P. falciparum incidence in children were more stable at a geographical level and over time than those of P. vivax incidence, with consistent locations at the south of the dam in the two successive study years.Species-specific malaria episodes in children under 10 years old detected by ACD between July 2008 and June 2010 were clustered in groups of HHs (hotspots) in selected villages around the Gilgel-Gibe hydropower dam in Southwest Ethiopia. Like in other endemic Ethiopian regions , 26, P. P. vivax were mostly observed in younger age groups, while P. falciparum episodes were mainly seen in older children) [The level of statistical significance is an important factor in determining whether a certain geographical area forms a plausible hotspot of malaria transmission. In this study, the global clustering K-function test suggested the existence of clustering of children with malaria episodes without pinpointing specific locations, while its variant, the bivariate K-function, was able to demonstrate that children with malaria episodes tended to be more aggregated than children without episodes. Although a previous study using recurrent-event models to analyse incidence data in the same study children showed contrasting associations between the age of children and the species-specific malaria incidence (i.e. hildren) , the ageP. falciparum incidence suggested a higher exposure to infectious mosquitoes in southern villages, especially after the long rainy season . After rains, intermittent streams could create pockets or pools of water which can serve as potential breeding sites for mosquitoes Anopheles arabiensis, contributing to an increase in mosquito density and vector-human contacts, and consequently to a greater number of malaria episodes during the dry season [In contrast to global clustering tests, local clustering tests  were able to identify the most likely location of hotspots of malaria incidence in the two consecutive years of study. Indeed, hotspots of y season , 27. They season , as welly season , 30, wouy season , the GilP. falciparum, the hotspots of P. vivax incidence were less stable in place and time during the study period, suggesting that the occurrence of P. vivax clinical episodes are less sensitive to seasonal and environmental changes than P. falciparum, and that other factors should be considered to understand the spatial-temporal heterogeneity of infections due to this species [P. vivax infections may also influence the spatial distribution of the disease, particularly the ability of parasites to relapse weeks or months after a primary parasitaemia [P. vivax episodes in the study received chloroquine (CQ) but not primaquine (PQ), following the national guidelines for areas where the glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is not known, and where tests to detect that condition are not available [In contrast to  species . The biositaemia . Howeversitaemia , 33. Thivailable .P. vivax than with P. falciparum. Taking this into account, the fast development of clinical immunity in areas with the highest P. vivax exposure and incidence (i.e. southern villages) identified during the first study year may also explain why the hotspots did not remain in the same location in the following year. Similarly, immunity would be a factor to be considered in the analysis of the reduction of P. vivax clinical incidence rates during the study period [The immunity to the malaria infection is another factor that should be considered when interpreting the spatial and spatial-time patterns of malaria clinical episodes in study children. As previously hypothesised in a recent article , differey period .P. falciparum and P. vivax incidence (despite being composed of most households of a village or a group of contiguous villages). Other limitations of the study may be related to the absence of data on other potential risk factors for malaria infection  preventing the evaluation of their influence on the spatial distribution of the clinical malaria incidence. Gender and distance to the Gilgel-Gibe dam were not considered as covariates for the spatial analysis since those variables were not significantly associated with both P. vivax and P. falciparum malaria incidence in a longitudinal modelling approach [The main limitations of our study may be related to the malaria metrics and the geospatial analysis used for the identification of clusters of malaria transmission in the area. The spatial analysis of clinical malaria incidence obtained through rigorous weekly active case detection of symptomatic episodes in enrolled children may be the best method for detecting malaria hotspots if most malaria infections occurred in those children were symptomatic and microscopically detected . Howeverapproach ; while tapproach .P. falciparum incidence in children were more stable at the geographical level and over time than P. vivax incidence in selected villages around the Gilgel-Gibe hydropower dam in Southwest Ethiopia. Different malaria spatial-time patterns due to Plasmodium falciparum and Plasmodium vivax should be taken into account to better design and deliver targeted interventions.Hotspots of Additional file 1:Selection of window size for SaTScan analysis. (DOCX 17 kb)Additional file 2:Spatial scan statistics of the most likely cluster of malaria episodes using different maximum window sizes. (DOCX 16 kb)Additional file 5:P. falciparum: a (first year), b (second year); P. vivax: c (first year), d (second year). The red line represents the expected K function values under the null hypothesis of complete spatial randomness, the solid black line represents observed K function values, and the grey area represents confidence envelopes for expected K-function values calculated from 999 simulations. (TIF 7910 kb)Ripley’s K function analyses showing significant spatial clustering of children with malaria episodes. Additional file 6:P. falciparum and P. vivax malaria episodes by year of study. (DOCX 15 kb)Spatial scan statistics of the secondary clusters of


The inverse association between socioeconomic status (SES) and cardiovascular disease (CVD) risk is well documented. Aortic stiffness assessed by aortic pulse wave velocity (PWV) is a strong predictor of CVD events. However, no previous study has examined the effect of SES on arterial stiffening over time. The present study examines this association, using several measures of SES, and attained education level in a large ageing cohort of British men and women.Participants were drawn from the Whitehall II study. The sample was composed of 3836 men and 1406 women who attended the 2008–2009 clinical examination (mean age = 65.5 years). Aortic PWV was measured in 2008–2009 and in 2012–2013 by applanation tonometry. A total of 3484 participants provided PWV measurements on both occasions. The mean difference in 5-year PWV change was examined according to household income, education, employment grade, and father's social class, using linear mixed models.PWV increase [mean: confidence interval (m/s)] over 5 years was higher among participants with lower employment grade (0.38: 0.11–0.65), household income , and education , after adjusting for sociodemographic variables, BMI, alcohol consumption, smoking, and other cardiovascular risk factors, namely SBP, mean arterial pressure, heart rate, cholesterol, diabetes, and antihypertensive use.The present study supports the presence of robust socioeconomic disparities in aortic stiffness progression. Our findings suggest that arterial aging could be an important pathophysiological pathway explaining the impact of lower SES on CVD risk. In industrialized countries, cardiovascular diseases (CVD) are the leading causes of mortality  and geneAortic pulse wave velocity (PWV) is a measure of the intrinsic stiffness of the aortic wall and a novel surrogate marker of CVD risk. Recent meta-analyses have reported a 15% increased CVD risk for each unit increase in PWV (1 m/s)  and an iThere is sparse evidence to date on the relation between SES and arterial stiffness, and none examining progression. Ethnicity, low education, family income, and neighborhood deprivation were associated with higher PWV in a small sample of American adolescents . Less edThe present study aims to examine the association between SES and PWV progression, using several measures of SES, and attained education level in a large ageing cohort of British men and women.n = 4379) or 2012–2013 (n = 4347) assessments, using the same protocol. A total of 3484 participants provided PWV measurements on both occasions.The Whitehall II study is a longitudinal study of 10 308 male and female civil servants  based in London and set up in 1985. The civil service refers to branches of public service concerned with all governmental administrative functions. The baseline response rate was 73%. Details on this cohort of white-collar workers have been published . ParticiThree indicators were used to measure SES: father's social class, employment grade, and household income. In addition, participants reported their highest level of educational attainment.Father's social class is a frequently used indicator of SES in childhood . It was Employment grade was measured using current or last known employment grade. The civil service identifies 12 nonindustrial grades: clerical assistant, clerical officer, executive officer, higher executive officer, senior executive officer, and seven ‘unified grades’ of administrator. Other professional and technical staff were assigned to these grades on the basis of salary. Unified grades 1–6 were combined into one group and the bottom two clerical grades into another, producing six categories.Annual household income in 2008–2009 included the ‘total annual household income from any source, including personal income’. Categories were £100 000 or more, £70 000–99 999, £50 000–69 999, £35 000–49 999, £25 000–34 999, £20 000–24 999, £15 000–19 999, £10 000–14 999, <£9999.Education was assessed as the highest qualification attained while in full-time education. It was grouped into five categories: No academic qualification, ordinary level, advanced level, BA/BSc, and higher degrees. Ordinary level is the basic level of the General Certificate of Education, whereas the advanced level is a higher and more in depth qualification, usually required for university admission.N = 1666, 22, 696, and 207 for father's social class, employment grade, household income and education, respectively).A number of participants with missing data on SES and education variables were further excluded in the corresponding analyses . Path length was determined with a tape measure by subtracting the carotid-sternal notch distance from the femoral-sternal notch distance. In each participant, PWV was measured twice, and if the difference in velocity between the two measurements was larger than 0.5 m/s, a third measurement was taken. The average of all of the measurements was used in the analyses. PWV measurements were repeated within 30 days in 125 participants in 2008–2009 and 114 participants in 2012–2013 to assess short-term reproducibility. Median intraindividual difference in PWV was respectively 0.83 m/s (interquartile range 0.43–1.40) and 0.89 m/s (interquartile range 0.41–1.47).Ethnicity was classified as white/nonwhite. Weight, height, and waist circumference were measured according to standard protocols . SmokingA slope index of inequality (SII) was computed for each socioeconomic indicator . IndividA SII was computed for each socioeconomic indicator . Each inLinear mixed models were used to measure the effect of SES on baseline PWV 2008–2009) and PWV longitudinal change between 2008–2009 and 2012–2013. These models use all available data over the follow-up, handle differences in length of follow-up, and account for correlation between repeated measures on the same individual. The linear mixed models included a term for time . The main effect estimates the effect of SES on PWV at baseline (2008–2009), whereas the SES × time interaction term estimates the mean difference in the 5-year changes in PWV. The effect of SES on PWV was estimated with categorical SES indicators and with the slope indexes of inequality. For clarity purposes, each SES indicator was regrouped into three categories. Analyses using all available categories and longitudinal analyses using categorical SES are presented in the supplementary file. The base model was adjusted for age, sex ethnicity, and MAP. Models were then further adjusted for BMI, smoking status, and alcohol intake; SBP, heart rate, total cholesterol, high-density lipoprotein cholesterol, diabetes, and antihypertensive use; and all of the foregoing, following the approach adopted in a recent meta-analysis examining the independent effect of PWV on cardiovascular risk  and PWV .The Whitehall II study was reviewed and approved by the University College London Ethics Committee (85/0938). Written informed consent was obtained from each participant at each phase. The study was conducted according to the principles of the Helsinki Declaration.Table Table Figure The present longitudinal study, conducted among a large sample of men and women, showed that lower educational attainment and adult SES are associated with more rapid progression of aortic stiffening. These associations were observed for household income, employment grade, and educational attainment, but not childhood circumstances, as assessed by father's social class. The associations were largely robust to adjustment for demographics, lifestyle-related risk factors, and other cardiovascular indicators. In the cross-sectional analysis, father's social class was associated with baseline PWV.r = 0.56). Our findings suggest that household income cover additional dimensions of socioeconomic circumstances as supported by the stronger observed effect. According to our results, the economic dimension of SES could be of particular importance to explain social disparities in aortic stiffening at older ages.Household income was the SES indicator most strongly associated with PWV progression. Low family income was previously found to be associated with higher PWV among a small sample of American adolescents . Income The magnitude of the social gradient in PWV might also be sensitive to the proximal/distal nature of the employed socioeconomic indicator . A measuIn the present study, employment grade was also associated with PWV progression. Studies have established a consistent relation between employment grade and CVD ,28. ThisLow educational attainment was also robustly associated with PWV increases despite the weaker magnitude of the observed effect. The effect of education on PWV was previously reported in two previous cross-sectional studies ,11. EducSocioeconomic disparities in aortic stiffness progression might also be mediated through biological pathways. For example, low employment grade was previously linked with coronary artery calcification . StructuIn the present study, father's social class was found to be associated with baseline PWV but not with PWV change over 5 years. This result suggests that alteration to arteries attributable to adverse social conditions in childhood might have already occurred at an earlier stage of life. This hypothesis is supported by Thurston and colleagues , who demThere is a debate about the optimal model parameterization for modelling change. High baseline PWV may be the consequence of faster increases before the study's baseline measurement in low SES individuals. This effect, described as the horse-racing effect, is most likely to occur in observational cohort studies such as ours . In thisOur study has limitations. First, there was considerable missing data on father's social class (32%) and education level (13%). We conducted a supplementary analysis comparing those who had missing values on those indicators with those who participated in the study, based on demographics and other socioeconomic variables (Tables S3 and S4 in the supplementary file). These analyses suggest that missingness was related to nonwhite ethnicity and low SES. We can therefore not exclude the possibility of selection bias, which underestimates effects of education and SES. Second, the Whitehall II cohort is composed of relatively healthy participants and does not include blue-collar workers, limiting generalizability of our findings. However, the cohort covers a wide socioeconomic range, as shown by the distribution of the sample in each SES indicators categories.Our study has important strengths. It was conducted among a large sample of men and women. Aortic stiffness was measured using the same gold standard tonometry method at baseline and follow-up, using a rigorous protocol. Moreover, multiple indicators of SES were examined, each of them showing different strength of relationship with PWV. Finally, a large number of covariates have been considered, including demographics, lifestyle-related risk factors and cardiovascular indicators, which support the robustness of social disparities in aortic stiffening.Aortic stiffness is an independent risk factor for hypertension, CVD, and stroke ,43. PrimThe present longitudinal study supports the presence of socioeconomic disparities in aortic stiffness progression at older ages. Our findings suggest that arterial aging could be an important pathophysiological pathway explaining the impact of SES on CVD risk.Part of the work from this article has been previously presented at the 2015 Canadian Hypertension Congress and at the 2016 scientific meeting of the Quebec Hypertension Society.The work was supported by a fellowship from the Canadian Institutes of Health Research, the British Heart Foundation (RG/13/2/30098), British Medical Research Council (K013351), the British Health and Safety Executive, the British Department of Health, the British Stroke Association (TSA 2008/05), the US National Heart, Lung, and Blood Institute (R01HL036310), and the US National Institute on Aging (R01AG013196 and R01AG034454).There are no conflicts of interest.


Plasmodium falciparum populations collected from two southeastern areas (Shwekyin and Myawaddy bordering Thailand) and one western area (Kyauktaw bordering Bangladesh) of Myanmar.The genetic diversity of malaria parasites reflects the complexity and size of the parasite populations. This study was designed to explore the genetic diversity of P. falciparum infections during 2009 and 2010 were used for genotyping at the merozoite surface protein 1 (Msp1), Msp2 and glutamate-rich protein (Glurp) loci.A total of 267 blood samples collected from patients with acute Msp1 and Msp2, respectively. The Glurp allele with a bin size of 700–750 bp was the most prevalent both in Shwekyin and Myawaddy, whereas two alleles with bin sizes of 800–850 bp and 900–1000 bp were the most prevalent in the western site Kyauktaw. Overall, 73.91% of samples contained multiclonal infections, resulting in a mean multiplicity of infection (MOI) of 1.94. Interestingly, the MOI level presented a rising trend with the order of Myawaddy, Kyauktaw and Shwekyin, which also paralleled with the increasing frequencies of Msp1 RO33 and Msp2 FC27 200–250 bp alleles. Msp1 and Msp2 genes displayed higher levels of diversity and higher MOI rates than Glurp. PCR revealed four samples (two from Shwekyin and two from Myawaddy) with mixed infections of P. falciparum and P. vivax.One hundred and eighty four samples were successfully genotyped at three genes. The allelic distributions of the three genes were all significantly different among three areas. MAD20 and 3D7 were the most prevalent alleles in three areas for Msp1, Msp2 and Glurp loci, which revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations at these sites. The results indicated that malaria transmission intensity in these regions remained high and more strengthened control efforts are needed. The genotypic data provided baseline information for monitoring the impacts of malaria elimination efforts in the region.This study genotyped parasite clinical samples from two southeast regions and one western state of Myanmar at the The online version of this article (doi:10.1186/s13071-017-2254-x) contains supplementary material, which is available to authorized users. P. falciparum is one of the major human infectious diseases affecting millions of people worldwide. Despite enormous control efforts over several decades, malaria still remains an important public health problem. In 2015, there were about 214 million new malaria cases and 438,000 deaths [Plasmodium species [P. falciparum parasites still maintained a high level of genetic diversity in northeastern Myanmar [Malaria caused by The genotypic and phenotypic diversity of malaria parasites enhances their ability to counteract control measures such as therapeutic drugs. In hyperendemic areas, each individual host may harbor multiple malaria parasite strains with different genotypes, including those that confer drug resistance . Sexual P. falciparum parasites from three areas in southeast and western Myanmar using three polymorphic markers: merozoite surface protein 1 (Msp1), Msp2, and the glutamate-rich protein (Glurp).In this study, we assessed the genetic diversity of Plasmodium falciparum clinical samples were collected in 2009–2010 during efficacy studies of artemisinin combination therapies conducted in three locations in Myanmar  following the manufacturer’s instructions. Nested polymerase chain reaction (PCR) was used to identify the knowlesi –12. The escribed , 13, 14.Alleles were identified based on the type and the fragment size of PCR products and assigned to different size bins. If an isolate had one allele at each of the three loci, this sample was considered to have a monoclonal infection. The multiplicity of infection (MOI) was defined as the largest number of alleles among three loci detected in the sample. This measure is conservative and it likely underestimates the number of clones present . Yet, thMsp1, Msp2 and Glurp allele frequencies were expressed as percentages. The proportions of alleles observed in each gene among three areas were compared using the Chi-square test. Pairwise comparison of MOI was done by Student’s t-test. The correlation between MOI and percentages of gametocytemic patients was analyzed by using Spearman’s correlation. The P value less than 0.05 was considered statistically significant.The P. falciparum infections by microscopic examination, nested PCR identified four samples as P. falciparum and P. vivax mixed infections (two from each of the southeast sites). Other Plasmodium species  were not detected.The demographic and clinical characteristics of the study populations are presented in Table Msp1, Msp2 and Glurp genes. The PCR fragment sizes and allelic frequencies for Msp1, Msp2 and Glurp in three areas are shown in Table Msp1, Msp2 and Glurp genes were significantly different among three areas  = 3.096, P = 0.002). In addition, the MOI did not show significant correlation with the proportion of gametocytemic patients in each site (Spearman correlation: r(184) = 0.1, P = 0.2).Of the 184 samples successfully genotyped for all three genes, only 26.09% samples were mono- allelic at each of the three genes Table . AnalysiP. falciparum parasites impacts malaria transmission and malaria control strategies. Therefore, it is important to resolve the genetic population structure of P. falciparum parasites in epidemic areas. Here we compared the genetic diversity of P. falciparum populations in two southeast regions and one western state of Myanmar using three polymorphic antigen markers. Consistent with earlier findings, genotyping the two Msp markers Msp1 and Msp2 identified significantly higher numbers of allele variants than genotyping Glurp [Msp1, the MAD20 type was more prevalent than the RO33 and K1 alleles, as reported in other areas of the GMS including southwestern China [Msp2, the 3D7 type was more abundant than the FC27 type, which was similar to what has been reported from the Thai-Myanmar border [Msp2 allele . For Glurp, Shwekyin and Myawaddy both had the 700–750 bp allele as the most frequent, whereas the 800–850 bp and 900–1000 bp alleles were the most common ones from Kyauktaw. The genetic similarities of the analyzed P. falciparum may reflect the geographical proximity of the study areas and possible population mixing from neighboring areas of the GMS. For example, the genetic diversity of parasites from the southeast site Myawaddy described in this study was similar to that from the Thai-Myanmar border [The genetic diversity of ng Glurp , 17. Forrn China , northearn China  and the rn China . For Mspr border  and Laosr border . InteresMsp1, Msp2 and Glurp, respectively. Such a high level of mixed infections indicates that transmission intensity was still high in these regions. Moreover, the trend for increased MOI to parallel the increased frequencies of the Msp1 RO33 and the Msp2 FC27 200–250 bp alleles suggested that these allelic types may be used as molecular markers for malaria prevalence or burden assessment in the region. Whereas this study and our earlier work in northeastern Myanmar using the same antigenic markers identified similarly high levels of genetic diversity of the parasites in spite of recent drastic reduction in malaria cases, the northeastern region had significantly lower MOI [The level of MOI usually reflects the degree of transmission intensity, although the association is not linear , 20. Genower MOI . As the P. falciparum/ P. vivax infections. These four misdiagnosed samples were not in the same area. Misdiagnosis of the mixed infections is most likely due to lower parasitemia normally associated with P. vivax infections. Given that the missed P. vivax co-infections would preclude treatment with primaquine for anti-relapse therapy, it is likely that some of these patients would experience P. vivax relapses. Therefore, more detailed epidemiological studies involving all human parasites are needed to clarify the prevalence, dynamics and genetics of the parasites in the endemic regions of Myanmar.It is interesting to note that although this study was not designed to detect the prevalence of all malaria parasites, PCR analysis did identify four cases of mixed P. falciparum from two southeast regions and one western state of Myanmar during 2009–2010. Successful genotyping of 184 clinical samples at Msp1, Msp2 and Glurp revealed high levels of genetic diversity and MOI, indicating that P. falciparum transmission remained relatively intense in these regions. These results can serve as the baseline information to monitor the effectiveness of the malaria elimination efforts. We can compare the MOI with future’s data, to assay the effect of the prevention measures.This study described the genetic diversity of Additional file 1: Table S1.The information of all samples. (XLS 376 kb) Additional file 2: Table S2.Msp1, Msp2 and Glurp genes. (XLS 83 kb) Allele sizes and types of


Serological markers for exposure to different Plasmodium species have recently been used in multiplex immunoassays based on the Luminex technology. However, interpretation of the assay results requires consideration of the half-life of specific antibodies against these markers. Therefore, the aim of the present study was to document the half-life of malaria specific serological makers, as well as assessing the sensitivity of these markers to pick up recent changes in malaria exposure.A recently developed multiplex immunoassay was used to measure the intensity of antibody (Ab) responses against 19 different Plasmodium specific antigens, covering different human malaria parasites and two vector saliva antigens. Therefore, 8439 blood samples from five cross-sectional surveys in Ratanakiri, Cambodia, were analysed. These involve a random selection from two selected surveys, and an additional set of blood samples of individuals that were randomly re-sampled three, four or five times. A generalized estimating equation model and linear regression models were fitted on log transformed antibody intensity data.Results showed that most (17/21) Ab-responses are higher in PCR positive than PCR negative individuals. Furthermore, these antibody-responses follow the same upward trend within each age group. Estimation of the half-lives showed differences between serological markers that reflect short-  and long-term (year round) transmission trends. Ab levels declined significantly together with a decrease of PCR prevalence in a group of malaria endemic villages.Plasmodium falciparum, antibodies against LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be a reflexion of recent (range from 6 to 8 months) exposure in the Mekong Subregion. PvEBP is the only Plasmodium vivax Ag responding reasonably well, in spite of an estimated Ab half-life of more than 1 year. The use of Ab intensity data rather dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance.For The online version of this article (doi:10.1186/s12936-016-1576-z) contains supplementary material, which is available to authorized users. Despite a considerable decline, the malaria burden in the Greater Mekong Subregion is still high and has a major impact on public health, especially in some specific regions within countries . With 68In malaria elimination areas, detection and surveillance of persisting malaria transmission is key for focussing interventions . TraditiPlasmodium transmission intensity in low endemic areas [Alternatively, serology is believed to be more informative in obtaining epidemiological information in malaria control programmes. In particular, serological markers can be used as a proxy for ic areas  where paic areas . More spic areas . Where tic areas  serologyic areas , 13, 14.From an immunological point of view, partial protective immunity to malaria is built up after recurrent infections, typically over a period of several years of exposure. Hence, different types of memory are expected based on the frequency of short-  and long-lived plasma cells (years) . In areaPlasmodium falciparum, Plasmodium vivax, Plasmodium malariae) and saliva of vectors as well as assessing the sensitivity of the markers to pick up recent changes in malaria exposure. Methods to estimate the half-life of serological markers are still in its infancy, therefore, to reach this goal, three different approaches [The aim of the current study is to document the half-life of IgG-Abs against 21 Ags from specific malaria parasites  [Ratanakiri is the highest malaria endemic province of Cambodia. Malaria transmission occurs during the rainy season, between April/May and October/November. Over the years a major decline in malaria transmission had been observed in this province: In 2000, the recorded annual parasite incidence in Ratanakiri was 11/1000 inhabitants, whereas , 24 in 2Plasmodium parasites. Furthermore, all samples of individuals that were randomly re-sampled three , four  or five times  during the entire MalaResT project were also included  were included. All peptides were chemically synthesized with an added N-terminal cysteine residue and BSA  [The selection of Ags was based on their availability and tested in a previous study that focused on the implementation of the multiplex bead-based immunoassay . The curis, USA)  by GeneC6 beads to 35 × 106 paramagnetic beads/beadset  [Each Ag was covalently coupled at a concentration of 4 μg Ag/10TX, USA) . BSA  .P. falciparum and two for P. vivax [+-conjugated AffiniPure F(ab’)2 fragment of goat anti-human IgG, Jackson Immuno Research Laboratories) at a dilution of 1:500 was added [® system with a minimum amount of 400 beads per spectral address. Results were represented as the median fluorescent intensity  from the median value of the Ag-coupled beads (MFIAg), defined by ∆MFI = MFIAg−MFIBSA [A scheme of the data analysis can be found in Fig. g−MFIBSA . The MFIentclass1pt{minima‘OutlierD’ [Further differences between duplicate samples were explored using a quantile regression on an MA-plot using R package utlierD’ . M is thutlierD’ , 31. SamutlierD’ .As age influences Ab-responses , 32, agePlasmodium species. Generalized estimating equation (GEE) models were used (geeglm function in the R package ‘geepack’ [Towards selecting markers that pick up contact with the parasite (recent of not recent), ln(MFI) values of PCR positive and PCR negative samples were compared per geepack’ –37), takgeepack’ . The modgeepack’ , 39. Dif‘confint’ function in R [To estimate the half-life of the Abs after effective treatment of malaria, the presence of Abs in the same individuals (642 persons) followed up over time (±600 days) was examined . Therefo-ln(2)λ) , 41. Theion in R . This anPlasmodium infections, the ln(MFI) values were plotted against PCR prevalence at cluster level. Therefore, forest plots . Each cluster was examined twice, once for survey 2 and once for survey 4. Then, the prevalence data were ordered from low to high prevalence  were cr ‘Hmisc’ ). For ea [‘lme4’ ) were coAlthough PmCSP was included in the battery of Ags, the results for PmCSP are not shown as the MFI signal (mean MFI < 600) did not surpass the background signal for the BSA.A total of 8654 field blood samples were analysed distributed over 108 96-well plates. Each plate was analysed in duplicate. After quality control, out of the 216 plates 30 plates were repeated  , but that the magnitude of the difference depends on the age group. These Ags can be divided into two different groups. First PvEBP shows a significant difference (P < 0.01) between MFI values of PCR positive and PCR negative persons only in the younger age groups (2–5 and 6–15 years old), whereas for STARP.R, LSA1.41, GLURP and Pf.GLURP.R2 the differences in MFI values between PCR-positive and PCR-negative persons are significant (P < 0.01) in the older age groups (6–15 and 16–50 years old) only.P. vivax Ags, clearly depend on the serological marker used  and long-term (year round) transmission trends. The model with time since infection and age groups as dependent variables (without interaction) was selected as the best model for all 20 Ags, suggesting that age does not have an effect on the slope of the Ab decay, although the Ab titres at time point zero differ between age groups Figs. , 4. For sed Fig. . Abs agaFor this analysis for some Ags  samples from young children (2–5 years) were not included, due to a limited amount of samples available. For the same reason the age group of 16–50 years old was excluded for LSA3.RE.Plasmodium data (mono and mixed infections) were used, whereas the P. falciparum and P. vivax Ags were analysed species specific (only mono infections).To test whether Abs pick up recent malaria exposure in a population, the relation between Ab-titres (ln(MFI)) and the PCR prevalence of malaria in the province of Ratanakiri was explored. Therefore, the cluster PCR prevalence data from survey 2 and 4 was divided into different groups  was selected. This model shows that when the PCR prevalence in groups of villages becomes lower, MFI levels also decrease  the following three criteria were used: (1) the outcome of the estimated Ab-responses based on the differences in ln(MFI) between PCR positive and PCR negative individuals, (2) the assessed half-lives and 3) the estimated Ab-responses in relation to high-, medium- and low levels of  the estiPlasmodium Ags [Plasmodium have generated knowledge about past and recent changes in malaria transmission within communities [P. falciparum [P. vivax due to possible relapses in the latter one [Plasmodium parasites including P. vivax. However, in this study age did not show any interactions on the outcomes.Several research groups recently developed multiplex serological assays for the detection of Abs against dium Ags , 45–48. munities , 40, 49.munities . At thislciparum , 52, 56,tter one . Howevertter one , 58 and tter one –61. As stter one –62. In tP. falciparum exposure at individual  and population level . Studies based on longitudinal data mainly use approaches such as linear regression models [The present study is to our knowledge the first study in Southeast Asia that uses multiplex serological measurements to document the half-life of IgG-Abs. Previous studies declare that Ab-levels follow a predictable pattern, in which every few months the Ab-level drops by half in the absence of re-infection , 41, 63.n models , 20, genn models , or Ab-in models  to analyPlasmodium infected people was compared to non-infected people. In agreement with results of previous studies, the present study shows that most Ab-responses (17/20) within each age group rise similarly by an infection. In contrast, in infected individuals Ab-responses to STARP.R, LSA1.41, GLURP, Pf.GLURP.R2 (among persons between 6 and 50 years) and PvEBP (among children from 2 to 15 years) seem to be influenced by age. [A first criteria for selecting serological markers indicative for recent infection, is that the marker can at least pick up current infection. Therefore, the Ab intensity in  by age. . The dif by age. . In thisIn a next step, antibody decay rates  were estimated by using linear regression models that were fit on Ab-responses obtained from a cohort at different time points. In summary, the obtained results showed that the used serological markers could be divided in four groups with a wide variety of half-life estimates Fig. ; Table 3Finally, the cross-sectional data were examined to investigate whether the serological markers were capable of picking up current differences in malaria prevalence. Overall, for a lower PCR prevalence in a group of villages, the observed MFI levels were also lower within all age groups. This trend was similar as what was observed by Ambrosino et al. , who obsP. falciparum exposure were identified. Moreover, Ags, CSP, Pf13, STARP.R, SALSA2, SR11.1, LSA1.41 and LSA1.J also seemed to respond well to a lesser extent. Therefore, these Ags should not be ruled out for future research in different settings. PvEBP was selected as the better candidate for P. vivax surveillance.Based on the three different criteria i.e. (1) sensitive to infection in individuals, (2) sensitive to the level of endemicity in communities, and (3) a short half-life of IgG-Abs, the best serological markers  that reflect recent P. falciparum LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be reflective for recent exposure, whereas PvEBP is the only Ag from P. vivax that responds reasonably well, in spite of a half-life of more than 1 year. Therefore, it is essential to explore other Ags from P. vivax. The only available P. malariae antigen did not provide a good response. It is remarkable that the best reflective Ags are mainly liver- and blood stage Ags, whereas in contrast to high endemic areas sporozoite level Ags are not useful in this setting (low-endemic). It is worth noting that all 20 Ags exhibit a direct link with the endemicity. In short, these Ags should certainly not be excluded for further in-depth analyses related to malaria control programmes. Another important aspect is that the use of Ab intensity data rather than dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance.Given the broad utility of serology, identifying serological markers for recent exposure seems a worthwhile investment . In summ


Plasmodium falciparum. Differentiation between species is however crucial for a correct approach. The objective of this study is to report the cases of this often missed aetiology of malaria in a tertiary hospital in Portugal.Non-falciparum malaria (NFM) has been reported to be responsible for around 25% of imported malaria cases in Europe but is often neglected due to its less severe clinical course when compared to ®) and/or PCR. Epidemiologic and clinical aspects were reviewed.Data were retrospectively analysed from patients admitted from January 2006 to August 2016 with a NFM diagnosis based on microscopy, rapid diagnostic tests (RDT)  of the cases, Plasmodium ovale for 32% (n = 6) and Plasmodium vivax for 17% (n = 3). In three (16%) patients, morphology was suggestive of P. vivax or P. ovale, but precise species identification was not possible. Regarding presentation, fever was the most reported symptom, and the most frequent laboratory finding was thrombocytopaenia. Quinine–doxycycline was prescribed in eleven patients (58%), chloroquine in six cases (32%) and artemether–lumefantrine in two (11%). All of the patients showed clinical improvement.A total of 19 NFM cases were diagnosed, corresponding to 8.4% of the total 225 cases of malaria. Seventeen (89%) were male with a median age of 41 years. All but one case were imported from sub-Saharan Africa, with 12 (63%) of the cases returned from Angola. Microscopy was positive for all patients and correctly identified the species in 12 (63%) patients. BinaxNOWP. falciparum. Access to PCR techniques facilitates diagnosis, as low sensitivity from RDTs and microscopy are to be expected.NFM remains an important cause of imported malaria in patients from sub-Saharan Africa, alone or as mixed infection with  Malaria remains one of the most important infectious diseases responsible for a high morbidity and mortality burden worldwide. Nearly half of the world’s population lives in endemic regions , and in Plasmodium. Plasmodium falciparum is responsible for the majority of cases and is also associated with a more severe course of illness. As such, it is the most studied species in scientific literature. Non-falciparum malaria (NFM), i.e., Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi, is less well documented, although it has been reported to be responsible for around 25% of the imported malaria cases in Europe [Plasmodium vivax is the most geographically widespread species, accounting for 6% of estimated cases globally [P. vivax comprises about half of the estimated cases [Plasmodium falciparum. In sub-Saharan Africa, P. vivax infections are uncommon because of the presence in a high proportion of the population of Duffy antigen-negative red cells which cannot be invaded by this parasite [Plasmodium malariae and P. ovale are relatively common in many parts of sub-Saharan Africa and comprise <10% of isolates [Plasmodium knowlesi has recently been identified as the cause of human malaria in Malaysian Borneo, but seems so far to be limited to Southeast Asia [P. vivax and P. ovale have a similar presentation to P. falciparum, although usually less severe. These two species have dormant liver stages (hypnozoites), and can induce re-activation of malaria several months after the initial infection. Plasmodium malariae (quartan fever) can result in long-lasting infection if not well treated. Plasmodium ovale and P. malariae are also believed to be responsible for asymptomatic cases [P. falciparum infection [®, a RDT available for use in Portugal, detects the histidine-rich protein 2 (HRP-2), specific to P. falciparum, and aldolase, a pan-malarial antigen common to all Plasmodium species [Plasmodium spp. and differentiating species [The disease in humans is caused by several species of the genus n Europe –5. Plasmglobally . Outsideed cases , with arast Asia . Clinicaic cases . Diagnosic cases . Rapid dnfection , but see species , and as  species , 16, altDue to current incomplete data on artemisinin combination therapy (ACT) efficacy for NFM in non-endemic settings and the need for effective elimination of hypnozoites, differentiation between species is crucial, as well as understanding the epidemiology of NFM. Therefore, the objective of this study was to describe the clinical and epidemiological features of imported cases of NFM in a tertiary hospital centre in Porto, which is the reference centre for malaria and imported diseases in the north of Portugal.Plasmodium antigens (BinaxNOW®) or by molecular techniques  based on the 18S subunit of the parasite’s ribosomal DNA) which became available in 2013. The different Plasmodium species were identified morphologically by the laboratory when possible. Demographic, clinical and epidemiological data of the patients were collected. The delay before diagnosis was defined as the time between the onset of symptoms and the day of diagnosis. SPSS was used on data analysis. The study was approved by the SJCH Ethics Committee.Data were retrospectively analysed from patients admitted at São João Hospital Centre (SJHC) from January 2006 to August 2016 with a NFM diagnosis. Parasitological diagnosis was done either by light microscopic observation of Giemsa-stained thin blood smear, immunochromatographic assay for the qualitative detection of Plasmodium malariae accounted for 37% (n = 7) of the cases; P. ovale occurred in 32% (n = 6), and P. vivax in 16% (n = 3). In three (16%) patients the diagnosis of NFM was made by microscopy because of morphology suggestive of P. vivax or P. ovale, but precise species identification was not possible. During the same period there were 2 cases of mixed infection with P. falciparum .A total of 19 NFM cases were diagnosed at SJHC, corresponding to 8.4% of a total 225 cases of malaria diagnosed in the same period. P. malariae and one patient as P. ovale. In the three remaining patients, species identification was not possible, but BinaxNOW® was positive for the pan-malarial antigen aldolase for two of them, while the other one had a negative RDT. BinaxNOW® was performed in all 19 patients. It was positive for the pan-malarial antigen in 11 (58%) patients. The test was negative in eight (38%) cases, showing a sensitivity of 58% . PCR was performed in nine patients and was positive in eight cases . In one patient, PCR was negative for P. falciparum, P. ovale and P. vivax , although microscopic examination of thin blood films identified the species as P. ovale. Out of the seven P. malariae cases, four (57%) were not identified by microscopy, needing PCR for the species identification.Microscopy was positive for all 21 patients. Species identification was made by microscopic examination of Giemsa-stained thin blood films in 12 (63%) patients. Out of the seven cases where species identification was not possible by microscopy, PCR was diagnostic in four patients: three patients as P. vivax infection in a patient returning from South America. The remaining case was imported from the Democratic Republic of Congo. Demographic and diagnostic features are resumed in Table Of the studied sample, 89% n = 17) were male with a median age of 41 years (range 25–68 years). None of the patients was under chemoprophylaxis for malaria at time of infection, and six (32%) patients had a previous history of malaria infection. Seventeen (89%) patients were Portuguese, and the remaining two were from Brazil and Angola. Most of the cases were imported from Angola , followed by Equatorial Guinea with three patients and Mozambique with two. One case was imported from outside of Africa, diagnosed as 7 were maP. malariae infection. The median of delay before diagnosis was seven (2–14) days, with P. malariae associated with a longer delay. Clinically, fever was the most reported symptom, present in all patients, followed by chills, headaches and myalgia with 63, 52 and 53%, respectively. One patient presented with acute respiratory distress syndrome (ARDS), a severe malaria according to WHO criteria [The median number of days between individuals arriving in Portugal and the time of symptom onset was two, ranging from 0 to 30 days in a patient with criteria , and wasAll patients presented with thrombocytopaenia (defined as platelets below 150 cells/cu mm) with a mean platelet count of 94 cells/cu mm. Five (26%) had platelets below 50,000, and 47% (n = 9) had platelets below 80,000. Mean haemoglobin levels at admission was 14.5 g/dL with eight patients (42%) presenting with anaemia (defined as haemoglobin below 13 g/dL). Eleven patients (58%) presented with elevation of liver enzymes with a mean alanine aminotransferase (ALT) of 63 U/L (range 17–357), but only seven (37%) had total bilirubin elevated. All cases at admission had elevated C-reactive protein (CRP) (mean level of 118 mg/L), and three patients (24%) had coagulopathy. The mean duration of hospitalization was 5 days (2–15), with no significant differences between species.P. vivax, P. ovale, mixed infection, or undetermined species infection, nine (69%) received terminal prophylaxis with primaquine, after exclusion of deficiency of glucose-6-phosphate dehydrogenase (G6PD). The patients who did not receive eradication treatment were either lost to treatment or returned to endemic areas. There were no fatalities and all of the patients showed clinical cure at the time of hospital discharge.Quinine–doxycycline (QD) was the treatment used in 11 patients (58%), followed by chloroquine (CQ) prescribed in six cases (32%) and artemether–lumefantrine (AL) used in two patients (11%). In four patients (21%), QD or AL was initially prescribed before switching to CQ. The patient admitted to the ICU was treated with intravenous QD. Of the 13 patients with Most scientific literature has focused on falciparum malaria over the years. Due to a lower prevalence and morbidity, data on NFM, especially imported cases, are limited. However, globalization and the current growing migration movements have forced non-endemic countries to become familiar with these diseases, changing the perspective on its ‘lower prevalence’. The clinical differences between falciparum and non-falciparum malaria, as well as the need for terminal prophylaxis in some cases, make the differential diagnosis vital for a correct medical approach of these imported cases. As such, diagnostic difficulties must be overcome to achieve a correct identification of these species.P. falciparum incidence. Plasmodium vivax has consistently been reported as the most frequent imported NFM infection in Europe [P. malariae was the most frequently identified NFM species, accounting for 37% of the cases. Considering that 95% of the cases were imported from Africa the higher prevalence of non-vivax species is comprehensible. The Plasmodium species identification remained to be known in three cases (14%) as microscopy could not differentiate between P. vivax and P. ovale and PCR was not available in these patients.Scientific literature has reported NFM to be responsible for around 25% of the imported malaria cases in Europe –5. In thn Europe , 19, as P. vivax/ovale differentiation was not possible and most P. malariae cases (57%) were not correctly identified by microscopy probably due to the more difficult microscopic identification of this species. BinaxNOW® showed a sensitivity of 58% for NFM infection, concordant with published data [P. malariae 57%, P. vivax 66%, P. ovale 50%). PCR has only been available since 2013 and is usually performed when doubts about the microscopic diagnosis are raised either by the microbiologist or the clinician. It was performed in only nine patients and it was positive in eight patients. The one case with negative PCR was identified as a P. ovale infection by microscopy, but PCR was negative for P. falciparum, P. vivax and P. ovale. At that time, PCR for P. malariae was not performed at SJHC, meaning that this case could have been a P. malariae infection misdiagnosed by microcopy as P. ovale, as normally we would expect a higher sensitivity with PCR techniques [Microscopic diagnosis was positive in all patients, but it was able to correctly identify the species in only 63% of the cases. This is explained by different experienced technicians involved at the time of diagnosis. In three cases hed data , 20. Thechniques , 16. TheP. malariae infection. Late onset of symptoms may also contribute to a longer delay to diagnosis, since the association between disease and tropical setting fades over time in the minds of the patient and the physician. Haematological complications were frequent, and thrombocytopaenia was the most frequent haematologic laboratory finding, present in all patients, while anaemia was only present in 42% of the cases. In this study, only one patient presented with severity criteria defined by the World Health Organization (WHO) for P. falciparum [The most frequently reported symptoms were fever, chills, headaches, and myalgia, meaning the clinical presentation is indistinguishable from falciparum malaria, although the less severe course of illness could suggest a NFM infection. The longer delay to diagnosis is also suggestive of NFM infection, as it is probably due to less severe clinical symptoms, especially when considering lciparum , most liP. falciparum, QD was the most prescribed treatment in this study. This probably also reflects that physicians are less experienced in NFM, and thus more comfortable in treating falciparum malaria. AL, the ACT regimen available at SJHC since 2013, will probably be more frequently used in the future due to its pan-malarial efficacy. Countries with CQ-resistant P. vivax, such as Indonesia, are already defending an ACT-based treatment for all species. A recent systematic review showed efficacy comparable to CQ, but cost-effectiveness studies are needed [CQ remains the recommended treatment for NFM . Howevere needed . AlthougPlasmodium species is not always achieved. In the future, ‘one treatment for all’ strategies based on ACT regimens may simplify therapeutic options, but due to the ability of some species to produce dormant liver stages and cause reactivation of the disease, correct diagnosis and identification of species remains crucial to reduce morbidity. Access to PCR techniques facilitates diagnosis, as microscopy (with low parasitaemia) and RDTs have lower sensitivity than in P. falciparum infection.NFM remains a neglected tropical disease due to its lesser severity compared to falciparum malaria. However, globalization has forced non-endemic countries to become more familiar with the correct diagnosis and treatment of this infection. In Portugal, NFM is an important cause of imported malaria, even in patients from sub-Saharan Africa. There are still many obstacles to overcome, as correct identification of


Plasmodium falciparum over the past decade without a corresponding decline in Plasmodium vivax incidence. It is unknown to what extent local transmission is sustained by a chain of clinical and sub-clinical infections or by continued re-introduction via migration. Using an ultrasensitive molecular technique, 20 villages in western Cambodia were surveyed to detect the low season prevalence of P. falciparum and P. vivax and local treatment records were reviewed.Cambodia has seen a marked reduction in the incidence of Plasmodium infections by rapid diagnostic test (RDT), microscopy and high volume (0.5 ml packed red blood cell) quantitative polymerase chain reaction (uPCR). Positive samples were analysed by nested PCR to determine the Plasmodium species. Malaria case records were collected from the Provincial Health Department and village malaria workers to determine incidence and migration status.During March to May 2015 cross-sectional surveys were conducted in 20 villages in Battambang, western Cambodia. Demographic and epidemiological data and venous blood samples were collected from 50 randomly selected adult volunteers in each village. Blood was tested for Plasmodium infection by uPCR, seven (0.7%) by microscopy, and two (0.2%) by RDT. uPCR P. vivax prevalence was 6.6%, P. falciparum 0.7%, and undetermined Plasmodium species 1.8%. Being male ; being a young adult <30 years ; recent forest travel ; and, a history of malaria  were independent risk factors for parasitaemia. Of the clinical malaria cases diagnosed by village malaria workers, 43.9% (297/634) and 38.4% (201/523) were among migrants in 2013 and in 2014, respectively. Plasmodium vivax prevalence determined by uPCR significantly correlated with vivax malaria incidences in both 2014 and 2015 , whereas no relationship was observed in falciparum malaria .Among the 1000 participants, 91 (9.1%) were positive for any Plasmodium prevalence correlated with subsequent malaria incidence. The association was attributable chiefly to P. vivax infections, which were nine-fold more prevalent than P. falciparum infections. In the absence of a radical cure with 8-aminoquinolines, P. vivax transmission will continue even as P. falciparum prevalence declines. Migration was associated with over a third of incident cases of clinical malaria.There was heterogeneity in the malaria parasite reservoir between villages, and Trial registration clinicaltrials.gov (NCT01872702). Registered 4 June 2013The online version of this article (doi:10.1186/s12936-017-1703-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum, whereas Plasmodium vivax declined by only 17%. This progress was due to the roll-out of rapid diagnostic tests (RDTs), treatment of confirmed malaria cases with artemisinin combination therapy (ACT) provided by village malaria workers (VMWs), and the distribution of insecticide treated bed-nets (ITNs) [Malaria incidence in Cambodia has decreased by approximately 42% from 2009 to 2015 [P. falciparum to chloroquine emerged from this area before spreading to Asia and Africa and causing millions of deaths [P. falciparum in western Cambodia has become resistant to artemisinins and more recently to the partner drugs used in ACT, such as piperaquine [Plasmodium parasites were found on the Thai-Myanmar border and other parts of Southeast Asia [P. falciparum it is necessary to completely interrupt P. falciparum transmission [The Thai-Cambodian border area around Pailin Province in western Cambodia has been a cradle of anti-malarial drug resistance. In the 1960s, resistance of f deaths . Since 2eraquine –6. Subseast Asia . ACT offast Asia , 9. Thersmission –12.Plasmodium infections [Plasmodium parasitaemias and thus characterize in detail the micro-epidemiology of malaria in areas of low transmission. These sub-clinical parasitaemias may play an important role in the transmission of infection, especially in areas of low and seasonal transmission [Previous surveys have described an asymptomatic reservoir of malaria in Cambodia , 14. Micfections , 15, 16.smission , 18.There are large seasonal population movements in and out of Samlout District in Battambang Province where the study was conducted. People from the district, predominantly men, spend time working outside the district and some return infected with malaria parasites. Agricultural labourers from other provinces come into the district to work during harvests and may harbour sub-clinical malaria infections acquired in other parts of the country. Seasonal work in forests collecting fruit, vines and hunting small game puts local people at risk of infection. These may contribute disproportionately to the transmission of malaria parasites. Battambang Provincial Health Department reports that approximately 83% of all malaria cases were aged 15–49 years, and 82% were males in 2014. The proportion of malaria cases among migrant workers in Battambang increased from 19% in 2009 to around 40% in 2014. In Cambodian border areas, the role of local, national and international migrants in malaria transmission has been acknowledged; however, the attributable fraction of infections has not been determined [P. falciparum infections over the past decade in western Cambodia, it is unknown to what extent local transmission is sustained by a chain of clinical infections, a reservoir of sub-microscopic malaria, or by continued introduction via migration. 20 villages in western Cambodia were surveyed to detect the sub-clinical prevalence of Plasmodium infections in an area where transmission is low but persistent and analysed the data with reference to available treatment records.Accurate malaria risk stratification is important for targeting control and elimination activities. Following the decline in During the low season of malaria in March to May 2015, cross-sectional surveys were conducted in 20 villages in Samlout District, Battambang Province, western Cambodia. All available VMW records and provincial malaria data were reviewed. Samlout District lies to the south of Pailin Province, north of Pursat Province and the Cardamom Mountains, and east of Thailand . Unless the total number of households in a village was fewer than 50, no more than one person per household was selected. For the villages with fewer than 50 households, more than one participant was randomly selected from the list. In cases of refusal or a person being absent on the survey day, replacement participants were selected randomly from the same household or the next household on the list. Pregnant women were excluded from the randomization as they would be ineligible to join a mass anti-malarial treatment study which followed this survey.Public meetings were conducted in Khmer by a dedicated Community Engagement Team (CE-Team) to inform the commune authorities, VMWs and village leaders about the study. All residents were invited to a meeting prior to the survey where the consent form was read aloud and questions were answered. Individual written informed consent was obtained immediately prior to sample collection and interview. A fingerprint was obtained from participants who could not read and write and a witness countersigned their consent form.Anthropometric and demographic data were collected, including tympanic temperature, height, weight, health status, malaria history, bed net use, travel history, and occupation; 1.5 ml of venous blood was collected in an EDTA tube. Samples were kept in a cool box and sent to a nearby hospital laboratory on the same day. If a participant was febrile (defined as tympanic temperature ≥37.5 C°) an RDT  was performed immediately, and if positive, treatment was provided by the VMW according to national guidelines.® Hb 201+Analyzer SE-262 71 Angeltholm, Sweden). Blood was tested for malaria by RDT and microscopy ). The remaining blood was centrifuged and plasma, buffy coat and 500 µl of packed red blood cells (pRBCs) for uPCR were separated into three different cryo-tubes and stored at −80 °C. Ten per cent negative controls of total participant samples were added for each village. All the samples were transported to the Mahidol University Faculty of Tropical Medicine molecular laboratory in Bangkok.The samples were separated; 200 µl of whole blood was used to prepare a dried blood spot on filter paper, a blood smear for microscopy, and to perform haemoglobin assay  of parasite per millilitre from each sample. The 18S rRNA-targeting primers and hydrolysis probes used in the assay have been validated and are highly specific for Plasmodium species. The lower limit of accurate quantitation of this method is 22 parasites/ml of whole blood [A detailed description, evaluation and validation of the high-volume ultrasensitive (uPCR) methodology has been reported previously . In briele blood .®  was used, and the PCR mixture and the cycling conditions were as per manufacturer’s instructions. For samples containing parasite DNA by uPCR analysis, Plasmodium species detection was attempted using real time PCR protocols specific to 18sRNA of P. falciparum and P. vivax. Samples for which Plasmodium species were not determined were reported as ‘unknown species’.A Quanti-Tect Multiplex PCR No ROXTo detect polymorphisms associated with reduced susceptibility to artemisinin derivatives the open-reading frame of the PF3D7_1343700 kelch propeller domain was amplified using a nested PCR protocol [Data were collected on case record forms and entered on smartphones before being exported into Open Clinica. Survey data were analysed by univariate and multivariate methods to identify risk factors for parasitaemia. Odds ratios with 95% confidence intervals were calculated to explore which variables were independently associated with subclinical infection. The Pearson Chi squared test was used to test for the significance of associations. Variables that were independently associated with sub-clinical parasitaemia at the level of p < 0.05 were explored by logistic regression to produce adjusted odds ratios. Incidence rate was calculated by dividing total malaria cases in a given year with the total village population and multiplied by 1,000. Malaria incidence and prevalence were compared by linear regression. The analysis was done in STATA 14.0 .Malaria case reports were obtained from the Provincial Health Department and VMW records. Available treatment records were reviewed by cross-checking with the VMWs to confirm the identity of patients. The incidence of clinical malaria for 2014 and 2015 was compared to the prevalence of malaria parasites detected by uPCR in March and April 2015 for each village. Villages with no Health Department records were excluded from this comparison. For villages where only a hamlet  was selected for the prevalence survey, the incidence rate of the whole village population was used for the analyses. All prevalence survey participants were from registered households and were considered to be residents.). The median age of the study participants was 33 years (range from 18 to 78 years). The median age of male and female participants were 34 years (18–72 years) and 32.5 years (17–78 years), respectively; 11.7% (117/1000) subjects had a temperature of 37.5 °C or higher; 65.4% (654/1000) had a history of malaria at any time prior to the survey.Population size and malaria incidence in 2014-2015 differed significantly among the 20 study villages. Population size ranged from 147 (in Ou Nounong: ONG) to 1012 (in Ouda: ODA). Malaria incidence per thousand ranged between two and 224 among villages in 2014 and 2-166 in 2015. Two villages  in 2014 and two villages  in 2015 were not listed in Provincial Health Department records, so no incidence data were available for these villages  by microscopy, and two (0.2%) by RDT. By uPCR, P. vivax prevalence was 6.6% (66/1000), P. falciparum 0.7% (7/1000), and 1.8% (18/1000) had undetermined Plasmodium species. In Table Among 1000 participants, 91 (9.1%) were positive for any . Plasmodium falciparum parasites were detected by uPCR in four of the 20 villages, with P. falciparum prevalence ranging from 2 to 6% (p = 0.010). Plasmodium vivax infections were detected by uPCR in 16 villages, with P. vivax prevalence ranging between 2 and 26%, (p < 0.0001) and the undetermined Plasmodium species were detected in nine villages, prevalence ranging from 2 to 10% (p = 0.011 for heterogeneity). Three out of 20 villages with had no parasite prevalence by uPCR.The prevalence in the villages ranged from 0 to 30% by uPCR (test for heterogeneity p < 0.001)Plasmodium species by uPCR are shown in Table Plasmodium species prevalence among males was 14.1% (66/402) versus 4.7% (25/532) among females p < 0.001; 11.5% (51/443) among those aged 18-29 versus 7.2% (40/557) among those aged 30 or above p = 0.018. Plasmodium prevalence was 6.8% (8/117) among those with temperature of 37.5 °C or higher versus 9.3% (83/883) among those with a temperature below 37.5 °C (p = 0.365). Among participants with previous history of clinical malaria, 12.5% (82/654) were parasitaemic versus 2.6% (9/346) among those who said they had never had malaria (p < 0.0001).Factors associated with any positive Plasmodium parasites among participants spending one or more nights in a forest within the same period was 22.4% (35/156) versus 6.6% (56/844) among those without any forest travel (p < 0.001). Among participants with parasitaemia, 27/91 (29.7%) had recently stayed overnight in the forest in the same province, and 8/91 (8.8%) had stayed in a forest in a different province; 56/91 (62%) did not have a history of overnight stay in the forest (p < 0.001). Parasite prevalence among participants with a history of travel outside the village within the previous 3 months was 10.2% (25/245) versus 8.7% (66/755) among those without any travel history (p = 0.489). Among those with parasites, 66.7% (66/91) did not travel in comparison to 27.3% (25/91) who travelled outside the village. Among the 25 positives who travelled outside the village, 44% (11/25) had stayed overnight in the forest.Prevalence of Prevalence of parasitaemia was 8.0% (68/849) among those who used ITNs every night, 15.2% (20/132) among those who sometimes used ITNs, and 15.8% (3/19) among those who never used ITNs, p = 0.004. Parasite prevalence varied between the strata used for sampling: 19.9% (39/196) among young men, 9.9% (27/272) among older men, 4.8% (12/247) among young women, and 4.6% (13/285) among older women. Men were more likely to use ITNs irregularly or never  versus women  p < 0.0001. Men were also more likely than women to stay overnight in forests 28.4% (133/468) versus 4.3% (23/532), p < 0.0001. Young men  used ITNs irregularly or never, and 36.7% (72/196) had spent a night in a forest within the past 3 months.After adjustment, being male , younger adults , recent forest travel , and a prior history of malaria  remained significantly associated with parasite prevalence.P. falciparum-positive individuals than those with P. vivax. Parasitaemia was lowest among people with undetermined Plasmodium species where the species were not determined. Tympanic temperature was not associated with the level of parasitaemia for any Plasmodium species . Four had stayed in a local forest overnight in the previous 3 months, of whom two reported travel outside the village in the same period. Two were positive both by RDT and by microscopy. Only one of seven falciparum positives had a history of travel to another province within the previous 3 months. Five reported everyday ITN use.Among participants with P. falciparum parasite strains detected by uPCR could be sequenced for PfKelch 13 mutations. All six P. falciparum isolates obtained Pfkelch13 C580Y mutation.Six of seven Plasmodium vivax prevalence correlated with P. vivax incidence (p = 0.001 and p = 0.002), respectively. No correlation was observed between P. falciparum prevalence and incidence (p = 0.36 and p = 0.59). The combined prevalence of P. vivax and undetermined Plasmodium parasites (p < 0.001) was also correlated with P. vivax incidence. This was done as previous surveys found approximately 90% of undetermined Plasmodium species detected by uPCR in western Cambodia were later identified as P. vivax during 12 months of prospective follow-up , respectively. P. falciparum and mixed P. falciparum and P. vivax cases from 88 (13.9%) and 237 (37.4%) in 2013, to 36 (7.3%) and 66 (13.0%) in 2014, respectively. The number and proportion of P. vivax cases increased from 309 (48.7%) in 2013 to 417 (79.7%) in 2014 respectively  were among people aged 45 years and above, 842 (69.6%) aged 15–45, 189 (15.6%) aged five to 15, and (2.1%) aged <5 years. People recorded as migrants (no permanent address in the village) accounted for (297/634) 43.9% of all malaria cases in 2013, and (201/523) 38.4% in 2014; 914/1,210 (75.5%) of subjects were male and 295/1,210 (24.5%) were female. There was an approximately three-fold reduction in the number of Variations observed in the prevalence of malaria in the study villages were consistent with a recent study in the northeast of Cambodia, which also found evidence of heterogeneity of malaria prevalence in nearby villages.P. vivax in the infections detected in the survey is consistent with local treatment records. A significant proportion of clinical falciparum cases were among migrants. More detailed information on travel history and places of overnight stay in malaria-endemic areas would be helpful to better explain where malaria infections were acquired.In the low season nearly one in ten people had malaria parasites. Sub-clinical vivax parasitaemia had a higher prevalence than sub-clinical falciparum parasitaemia. The predominance of Young men were at greater risk of clinical malaria and had a higher prevalence of sub-microscopic malaria parasites compared to older men or to adult women. Young men were also more likely to spend time in forests and use ITNs irregularly or not at all, consistent with other studies in Cambodia , 21.Plasmodium vivax prevalence determined by uPCR significantly correlated with vivax malaria incidence in each of 2014 and 2015, whereas no relationship was observed for P. falciparum. Due to the shorter duration of P. falciparum infections, it is unlikely that a single snapshot of P. falciparum by uPCR during the dry season would be able to describe the picture of P. falciparum infections throughout the whole year. Whereas for P. vivax, the higher persistence of sub-clinical infections means a survey in the dry season reveals a large reservoir of infections. In a previous longitudinal study a large proportion of sub-clinical P. vivax persisted through the year and seasonality had little effect on the number of detectable infections [P. vivax.There was a significant association between a prior history of malaria and having vivax parasitaemia. These self-reported histories (which often dated back many years) are not a promising target to guide screening or treatment as the absolute risk difference was 3%  and two-thirds of all participants reported a history of clinical malaria. However, clinical treatment records of confirmed malaria diagnoses are a more reliable predictor of persistent vivax parasitaemia .P. falciparum prevalence in these villages was very low, six of seven uPCR-positive P. falciparum infections did not have recent history of travel to another province, suggesting some ongoing local transmission. Without the contribution of migrants to the clinical malaria records Samlout District may be close to interruption of P. falciparum transmission, but imported cases may undermine elimination. In a recent study in neighbouring Pailin Province, a high proportion of P. falciparum clinical cases were found to have C580Y Kelch mutations, and in 2013-14 prevalence surveys in Pailin, approximately half of all asymptomatic P. falciparum infections had markers associated with artemisinin resistance [To accelerate malaria elimination, malaria control programmes in Cambodia and other countries are evaluating various strategies for mass drug administration (MDA) , 23–25. sistance , 14. In sistance .P. falciparum prevalence over one year; therefore, it is not possible to estimate the overall contribution of these infections to transmission in these communities. It was also not possible to determine the Plasmodium species of some parasitaemias. Generally these were the participants with the lowest quantity of parasite DNA. The incidence data could be incomplete as VMW data from 2014 may have missed some people who went to other places for diagnosis and treatment.The cross-sectional survey design was unable to resolve whether past clinical malaria left persistent parasites or whether these individuals were parasitaemic because they were generally at a higher risk. This study was conducted during the dry season and studies from Pailin in 2013–2014 reported a several-fold variation in P. vivax infections with very low P. falciparum prevalence. Plasmodium vivax prevalence correlated with incidence of clinical malaria episodes while P. falciparum did not. Knowledge of the crude P. falciparum incidence alone was not a reliable tool to predict the hidden parasite reservoir during the dry season in this instance. More detailed knowledge of the characteristics of the P. falciparum cases, such as occupation and travel history could potentially distinguish indigenous and imported malaria and thereby improve the use of local treatment records to predict the sub-clinical P. falciparum reservoir. This can be a particularly useful tool to target malaria elimination activities such as mass screening and treatment or mass anti-malarial drug administration.The low season reservoir consisted chiefly of P. falciparum was among migrants, forest workers or those recently returned from other provinces, suggesting transmission inside the villages themselves is now very low. Young men were more likely to have sub-microscopic malaria than older men or females of any age and to exhibit behaviour that exposes them to the risk of infection (and transmission). Imported malaria has the potential to undermine provincial P. falciparum elimination efforts as clinical and sub-clinical infections are brought into the district by migrant workers and by local people returning from other provinces.Much clinical Plasmodium prevalence between neighbouring villages shows that there is important variability in malaria epidemiology at a local level. Knowledge of the recent, detailed travel history of clinical malaria cases could provide crucial information about the probable source of infection and could help determine the risk of infection within individual villages. Even in villages with indigenous transmission, strategies to screen or treat sub-microscopic malaria in the dry season may be undermined by subsequent immigration of parasitaemic individuals. Therefore, targeting high-risk groups, e.g., migrant labourers, forest goers, by offering free screening and treatment, may prevent re-importation of malaria parasites from areas of higher transmission to areas in pre-elimination state and this could be an effective tool to eliminate falciparum malaria. Simultaneously, mass treatment in high transmission areas around the start of the malaria transmission season at community level may reduce the incidence of falciparum malaria significantly. It could have a collateral effect on a whole geographical area moving towards malaria elimination.The spatial heterogeneity of