In the context of reduced transmission of malaria, it is essential to re-evaluate and determine the level of transmission as it guides re-orientation of control measures which is appropriate to local disease epidemiology. However, little is known about level of malaria transmission in Ethiopia. The present study aimed to investigate the level of malaria transmission through combined application of classical methods and enzyme-linked immunosorbent assay (EIA) in low transmission settings of Ethiopia.This study was conducted in June 2016 on 763 apparently healthy children 2–9 years of age. Children were recruited from ten sites representing different malaria transmission settings in Ethiopia. Splenomegaly rate, infection rate and EIA antibody test were used to determine endemicity. The data were analysed using SPSS 21.0 and Stata 12.0.Plasmodium falciparum accounted for 62.63% of the infections. The prevalence of parasitaemia significantly varied by altitude and localities; the highest (5.8%) in areas below 1500 m above sea level. Overall, splenomegaly rate was 1.70% (95% CI 0.78–0.2.66%), making the overall malaria transmission hypoendemic. Infection rate was higher among males (2.7%), but rate of splenomegaly was higher in females. Incongruent with spleen rate and parasitaemia, EIA showed a higher level of cumulative exposure to malaria with spatially localized and highly heterogeneous transmission. Overall, 126  of the children were positive for total malaria antibodies with significant variations with altitude, age and sex; the higher in areas of < 1500 m asl (25.8%), children ≥ 5 years (22.1%) and among males (20.9%).The overall prevalence of malaria parasitaemia was 2.49% (95% CI 1.38–3.59) and 2.36% (95% CI 1.28–3.44) as detected using rapid diagnostic test and microscopy, respectively. Splenomegaly and parasitaemia are not good measures to show variations in the levels of malaria transmission in reduced and/or low endemic settings. The malaria antibody  test seems to be a good measure of malaria endemicity showing greater degree of heterogeneity and localized risk of transmission. Thus, malaria elimination efforts need to be supported with serological indicators to identify patterns of foci of transmission to set priorities for interventions. Despite remarkable achievements, the human toll of malaria and the global risk it still poses remains unacceptably high , 2. In 2In low transmission areas, many people who are infected with malaria parasites remain asymptomatic and, therefore, undetected by the health system. Such individuals make a significant contribution to sustain malaria transmission in the community , 5–11. IPlasmodium falciparum accounted for 63.7%, and the remaining were due to Plasmodium vivax [Despite promising progresses made to date –5, malarum vivax . In Ethium vivax , 15. In um vivax , 15. In um vivax , 15. In um vivax , 15.In Ethiopia, malaria prevention and control strategies have substantially remained unchanged over the past decade , 15. ThiA community cross sectional study was conducted, in June 2016, mainly in sentinel sites established by the National Malaria Control Programme for malaria surveillance in Ethiopia. Figure 1−α/2)2 p (1 − p)/d2) based on the following assumptions: Prevalence of malaria (parasite rate) among children (p = 10.5%) [The study participants were apparently healthy children 2–9 years of age. Children in this specific age group were chosen considering recommendations by the World Health Organization (WHO) for malaria endemicity study , 20. The= 10.5%) , 2% margChildren were sampled from ten PHCUs  was equally allocated to each PHCU. Second, two satellite health posts/villages, which are parts of the PHCU were selected randomly. Equal numbers of children were considered from each of the selected health posts. Within the selected health posts, list of households with eligible children (age 2–9 years) was obtained from family register from health posts. Simple random sampling was employed to select eligible households using the list obtained from health post level. Then, parents were invited to give consent for participation of their children, in the study through local administrators, community volunteers and community-based health workers. Parents were invited to the nearest health facility, . Up on arrival, parents/caretakers were given detail information about the study purpose, process and signed written consent for children that participated in the study.Three basic measures of malariometric indices were used to determine endemicity or malaria transmission which included: spleen rate (SR), parasite rate (PR), and enzyme immunoassay (EIA) malaria antibody test.This is the proportion of children with palpable enlargement of the spleen. Spleen palpation was conducted by clinicians at the ‘standing position’ technique as suggested by Dempster , 22. TheFor detection of malaria parasitaemia and antibody, 3–5 ml venous blood was collected from each child participated in the study. However, for those children who refused venous blood collection or venous blood unavailable, finger prick used to get the specimen for RDT and blood film examinations. For rapid qualitative identification of malaria infections, CareStart malaria Pf/Pv (HRP2/pLDH) Ag Combo RDT  was used. From the collected venous blood, 5 µl whole blood was used for RDT. The RDT test was performed as per the recommendation of the manufacturer .For microscopic examination, thick and thin films were prepared and air dried in the field, and the thin film was fixed with methanol. Both films were stained with 10% Giemsa. Each slide was read by experienced laboratory technologist at Jimma University Specialized Hospital. For positive slides, type of species was reported. In order to ensure the quality of reading, all positives slides and 15% of negatives slides were re-read by another microscopist who was blinded to the initial reading, which resulted in 16.0 and 0.02% discordant readings, respectively. Slides with discordant results were re-read by a third microscopist who was blinded to the previous results.Absence of malaria parasite in 1000× oil immersion objective in thin blood film was declared negative. A slide was considered as negative after 100 fields were examined and no parasite seen . Both foPlasmodium falciparum cultivation enriched with recombinant antigens from Plasmodium vivax . The antigens detect specific antibodies to the four species of Plasmodium parasite species; P. falciparum, P. vivax, Plasmodium ovale and Plasmodium malariae. Plasma obtained from whole blood collected, from venous of child, into ethylenediaminetetraacetic acid (EDTA) treated was used as specimen for antibody detection. The specimen was stored in − 20 °C freezer, but thawed and well mixed before testing. The test was based on the binding of specific antibodies present in the specimen to antigens immobilized on a 96-well EIA plate. The test was performed as recommended by the manufacturer [Plasmodium species) by the malaria EIA antibody test. The test does not distinguish between Plasmodium species, nor IgG and IgM and between an acute and chronic infection. The assay carried out at Jimma University Tropical and Infectious Disease Research Centre (TIDRC), using an automated procedure both for washing wells and reading EIA plates.Malaria EIA test uses four recombinant antigens in a sandwich form and detects antibodies to malaria during all stages of infections. The antigen used was a total extract from facturer  as folloBased on SR, malaria endemicity is defined as holoendemic (SR > 75%), hyperendemic (51–75%), mesoendemic (11–50%) and hypoendemic (SR < 10%) , 22. TheIn addition to blood sample collections, the background information of each child such as age, sex, body temperature was recorded.The data were analysed using SPSS version 21.0 and STATA version 12.0. The analysis was done by study sites, altitude and demographic factors. One-way ANOVA and t-test were used to compare mean optical density. Logistic regression was used to assess the association of age, sex and altitude with reactive malaria antibody. Spearman’s correlation was used to assess the correlation among malariometric indices. A 95% confidence interval and p < 0.05 level of significance were used to declare statistically significant association.Overall, 763 children participated in this study with response rate of 73.1%. Analysis of splenomegaly rate and parasitaemia was done on all 763 samples. Nevertheless, due to insufficient blood sample, malaria EIA antibody test was done only for 672 samples. More than half (53.1%) the included children were males and the remaining were females. Four hundred forty-one (57.8%) were between 5 and 9 years old and the remaining were between 2 and 4 years of age. In terms of place of residence, 555 (72.7%) were recruited from rural areas.Plasmodium falciparum was the predominant malaria parasite species, accounting for 62.63% (10/19) of the infections. In microscopy, 11 (57.89%) of the malaria cases were due to P. falciparum. No mixed infection was detected by microscopy.Table 2 = 23.255, p = 0.001): higher prevalence was found in areas below 1500 m asl (5.8%) and in areas with mid-altitude (1500–2000 m asl) which was 0.3%. No malaria parasite was detected at an altitude > 2000 m asl. When the analysis included only areas below 2000 m asl, prevalence was 2.70%, by RDT.Malaria parasitaemia was detected only in three study sites namely Dhera, Metahara and Goda Dhera, in which prevalence of 14.7, 3.6 and 2.3% was documented, respectively. In one of the villages in Dhera catchment (Dirre Kiltu), 33.3% of the sampled children had infection with detectable parasitaemia. Moreover, the prevalence of malaria parasitaemia significantly varied by altitude (xOnly 18 (2.4%) of the surveyed children had fever (axillary body temperature ≥ 37.5 °C) at the time of the study. Fever was less correlated with detectable parasitaemia . Of those positive children by RDT, only 4 (21.1%) were febrile. The difference in prevalence of malaria parasitaemia between males (2.7%) and females (2.2%) and children ≥ 5 and those below 5 years of age (2.9% vs 1.9%) was not statistically significant (p > 0.05).The prevalence of enlarged spleen was 1.70% (95% CI 0.78–0.2.66%), with mean enlarged spleen 1.85 (SD = 0.38). Enlarged spleen was detected in only five of the study sites. Relatively higher rates of splenomegaly were recorded in Dhera (5.3%), Goda Dhera (4.6%) and Goro (3.3%) sites. Rate of splenomegaly was higher among children living in areas < 1500 m asl (2.9%) and among children who resided in areas > 2000 m asl (1.4%). In areas between 1500 and 2000 m asl, splenomegaly was seen in only 0.8% of the children. Furthermore, splenomegaly was higher among children greater ≥ 5 years of age (2.3%) as compared to children < 5 years of age (0.9%). Similarly, the rate was slightly higher among females . Yet, none of these differences was statistically significant (p > 0.05).Figure In this study, ownership, access to and use of LLIN did not have significant association with exposure to malaria (p > 0.05). The prevalence of reactive antibody was 19.8 and 19.5% among households who own any nets and those without nets, respectively. Similarly, the prevalence of reactive antibody was 21.3 and 16.4% among children who slept under LLIN and those who did not, respectively. Likewise, rate of exposure to malaria did not vary by extent of access to LLINs: seropositive rate was 19.5, 17.9 and 21.3% among households with no access, sufficient access (i.e. 2 nets for every two people in the household) and insufficient access, respectively.The mean OD among malaria antibody positive samples was 2.01 (SD = 1.29). Figure Of the samples tested, reactive by malaria EIA (n = 126), 14.3% (18/126) and 13.5% (17/126) had detectable parasitaemia by RDT and microscopy, respectively. Table ‘Hypoendemic’. However, malaria antibody assay showed a higher level of cumulative exposure (18.75%) to malaria of the target communities with greater degree of heterogeneous and localized transmissions.The association between different measures of malariometric indices was weak Table . SplenomPlasmodium species. The overall prevalence of malaria parasitaemia was 2.5% (using RDT) although higher heterogeneity was observed among localities and with different altitude. The finding of this study was higher than many community-based reports in Ethiopia, including the 2007, 2011 and 2015 malaria indicator surveys (MIS) [P. falciparum was the predominant malaria parasite species in the study population. Moreover, the declining trend of malaria burden was less prominent in P. falciparum and there was an indication of resurgence of P. falciparum in 2010 and 2013 in the country [P. falciparum and P. vivax varies according to locality and seasons, in Ethiopia [P. falciparum conveys important messages as it is the primary target for malaria control and elimination programme [This study investigated the level of malaria transmission in Ethiopia based on classical malariometric indices (parasitaemia and spleen rate) and EIA for the detection of specific antibodies to ys (MIS) –30, but ys (MIS) , 32. Conys (MIS) –28, highys (MIS) , 34, P.  country . GeneralEthiopia –30, 33. rogramme .Splenomegaly has been used as stable indicator of malaria endemicity for many decades . In thisPlasmodium species following infection [Essentially, each individual develops antibodies to nfection , 38. Thunfection , there infection –11. The nfection . Therefomesoendemic considering partial immunity as manifestation of chronic malaria. According to this class of endemicity, malaria is typically characterized by regular seasonal transmission under normal rainfall conditions [Even though stratification of malaria endemicity on the basis of serological tests not common; generally, malaria in this study appeared to be nditions , 20, 22.nditions .Although chronic exposure to malaria was higher among children over the age of 5 years and among males, a high level of circulating antibodies was detected among younger children and females. It is not quite clear why younger children and females demonstrated stronger anti-malarial antibodies. Maybe, the intensity of antibodies varies with durations of exposure, and clinical and immunological response may also vary between males and females. This needs further investigation.Information on the level of herd immunity to malaria has several practical implications. High levels of immunity in the population could help to accelerate elimination success by reducing transmission rate . The levIn this study, no parasitaemia was detected among the majority of the febrile children; children with high intensity antibodies. This creates a critical challenge to malaria elimination programme since many people who are infected with malaria parasites remain undetected by the healthcare system , 6, 9–11The major strength of this study was the use of a combination of classical and immunological technique through community based approach; involving multiple sites with relatively large sample size. The finding of the study can be generalized to other similar malaria endemic settings. This is the first report from Ethiopia demonstrating serological tests to measure levels of malaria transmission and distribution of partially protective malaria anybody in the population. Thus, the finding can serve as first-hand information in planning malaria elimination. However, lower response rate might have affected reliability of estimates.P. falciparum. The classical methods used in measuring malaria endemicity, namely, rate of splenomegaly and parasite rate failed to reflect the level and heterogeneity of malaria risk in such low transmission settings. In contrast, EIA malaria antibody test was able to detect heterogeneity of levels of malaria transmission and provided more precise and clear picture of variations in levels of transmission in the Ethiopian context. The levels of transmission were quite local and heterogeneous by altitude and local ecology, manifesting multiple endemicity levels. Areas with low altitude carry the highest burden of malaria transmission whereas malaria transmission in high attitude settings was minimal. Therefore, in-depth understanding of the local level ongoing risk of transmission should be an initial step for countries with low transmission and different disease eco-epidemiology initiating elimination programme. The study also suggests the need to incorporate serological test into large scale periodic malaria surveys such as malaria indicators surveys, particularly in areas with low transmission and where elimination programmes are initiated. Serological test need to be considered in addition to data which is often extracted from existing health facilities, records of malaria control programmes and a point in time data from malaria indicator survey (MIS). Malaria elimination initiatives need to adopt location-specific strategies and tailor its approach to ongoing and local specific malaria transmission.The prevalence of malaria parasitaemia was somewhat high compared to many earlier studies, with high prevalence of


Although malaria is endemic in India, congenital malaria is not very common. Congenital malaria is a very rare condition in both endemic and nonendemic areas. We report a case of congenital malaria in a six-day-old neonate with fever and splenomegaly. The diagnosis was picked up accidentally on a peripheral smear examination. Congenital malaria should be kept as differential diagnosis of neonatal sepsis. Timely detection of this condition could lead to early diagnosis and treatment, thereby preventing neonatal mortality.  Congenital malaria is defined as malarial parasite demonstrated in the peripheral smear of a newborn from 24 hours to seven days of life. Clinically apparent congenital malaria is rare in countries where malaria is endemic and levels of maternal antibodies are high. With the symptoms of congenital malaria being nonspecific, it is often confused with neonatal sepsis.n = 0–6 mg/dL). Blood, urine, and cerebrospinal fluid cultures were sterile. Chest X-ray was unremarkable. The peripheral blood film revealed trophozoites and schizonts of P. vivax, with a parasite index of 2%. The baby was given chloroquine base at dose of 10 mg/kg stat followed by 5 mg/kg at 6, 24, and 48 hrs. There was prompt relief in fever and the spleen gradually reduced in size over a week. Five days after treatment, the patient's parasitemia completely cleared, TLC increased to 12000 cells/cu mm, platelet count was 150,000 cells/cu mm, and CRP fell to 5.5 mg/dL. The infant was discharged on day 5 and is doing well on follow-up.A six-day-old male neonate was brought to our outpatient department with complaints of not accepting feed, fever, and loose stools since two days. On examination the baby was pale, icteric, and lethargic, axillary temp was 100°F, and liver and spleen were palpable 3 and 6 cm below the right and left subcostal margins, respectively. Other systems examinations were normal. A provisional diagnosis of neonatal sepsis was made and the patient was started on intravenous ampicillin and gentamycin. Complete blood count (CBC) at the time of admission revealed a Hb of 9.0 gm/dL, total leucocyte count of 10,000 cells/cu mm, differential count of 50% polymorphs, 47% leucocytes, 3% monocytes, and a platelet count of 50,000 cells/cu mm. Other relevant results included a total bilirubin of 10 mg/dL, indirect fraction of 9 mg/dL, Serum Glutamate Pyruvate Transaminase (SGPT) of 485 IU/L, and C-reactive protein (CRP) of 23 mg/dL  antigenemia suggests that the infection was localized to placenta and had cleared. Discordance between maternal peripheral blood examination/antigen testing and placental parasitization is well described. The time of onset of symptoms in congenital malaria can vary from immediately after birth to few weeks though the median age of manifestation has been described as 21 days.More than 150 cases of congenital malaria have been reported in world literature –10. CongThe drug of choice for congenital malaria remains chloroquine. The therapy for the infecting plasmodium species is curative. As the infection is produced by transmission of infected erythrocytes rather than forms that invade the liver, the neonate does not require the treatment for exoerythrocytic stages of the parasite.Our case was accidentally picked up on peripheral blood film examination. This stresses the importance of a good peripheral blood film as a part of all suspected cases of neonatal sepsis. To conclude, congenital malaria is not rare as it was thought to be; in endemic zones malaria should be suspected in all neonates who present with fever and splenomegaly. Early diagnosis could prevent unnecessary antibiotics usage and could prevent neonatal mortality.


A wide array of vertebrates can serve as the intermediate hosts to malaria parasites (Apicomplexa: Haemosporida), such as birds, lizards, and several groups of mammals, including primates, bats, rodents, and ungulates. The latter group of hosts has not been intensively studied since early descriptions of a small set of taxa were published, but new reports of these parasites in both expected and new hosts have recently been published. Plasmodium odocoilei in farmed white-tailed deer in Florida, particularly in animals less than 1 year old, and provides evidence that the parasites may contribute to mortality in fawns. That paper opens new opportunities to study the malaria parasite-mammal interface in North America.A wide array of vertebrates can serve as the intermediate hosts to malaria parasites (Apicomplexa: Haemosporida), such as birds, lizards, and several groups of mammals, including primates, bats, rodents, and ungulates. The latter group of hosts has not been intensively studied since early descriptions of a small set of taxa were published, but new reports of these parasites in both expected and new hosts have recently been published. A new paper reports the presence of  Plasmodium odocoilei, a species described by Garnham and Kuttler in 1980  level of genetic diversity present in the Florida populations. Interestingly, the authors also showed a correlation between infection with the hemosporidians and mortality of the deer, providing evidence for virulence, particularly with respect to young animals, similar to the patterns observed in the human parasite Plasmodium falciparum, with which most mortality occurs in infants. The authors also found a correspondence of hemosporidian infection with the presence of a viral pathogen; thus, comorbidity may be a partial explanation for the higher mortality in malaria-positive animals. Although the deer parasites are quite distantly related to P. falciparum, this convergence of levels of virulence in young animals may offer an opportunity to better understand factors underlying these specific host-parasite dynamics. For instance, studies have shown that changes from the fetal to the adult microbiome in mice and humans are linked to susceptibility to malaria infections (A new paper by Guggisberg et al.  now expafections  and thatfections , akin to


P. vivax, 30 patients with uncomplicated P. falciparum, and 13 patients with complicated P. falciparum malaria on admission and on day 7, compared with healthy subjects (n = 18) as control group. The lowest level of serum S1P concentration was found in the complicated P. falciparum malaria group, compared with P. vivax, uncomplicated P. falciparum patients and healthy controls . In addition, serum S1P level was positively correlated with platelet count, hemoglobin and hematocrit levels in malaria patients. In conclusions, low levels of S1P are associated with the severity of malaria, and are correlated with thrombocytopenia and anemia. These findings highlight a role of S1P in the severity of malaria and support the use of S1P and its analogue as a novel adjuvant therapy for malaria complications.Sphingosine 1-phosphate (S1P) is a lipid mediator formed by the metabolism of sphingomyelin which is involved in the endothelial permeability and inflammation. Although the plasma S1P concentration is reportedly decreased in patients with cerebral malaria, the role of S1P in malaria is still unclear. The purpose of this study was to examine the impact of malaria on circulating S1P concentration and its relationship with clinical data in malaria patients. Serum S1P levels were measured in 29 patients with  Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Most of the fatal cases, which predominantly occur in P. falciparum infection, are due to cerebral malaria or severe anemia, but different clinical complications such as acute kidney injury, pulmonary edema, metabolic acidosis and thrombocytopenia also exist and vary in severity and outcome, depending on the parasite species, the organ involved and the access to care [It was estimated that 3.2 billion people still remain at risk of malaria in 2015. According to the latest reports, 214 million cases of malaria occurred globally with estimated mortality of 438,000 deaths [P. falciparum malaria [The pathogenicity of severe malaria infection is complex and it is regulated by both parasite and host factors. The process of cytoadhesion between infected red blood cells (RBCs) and vascular endothelium, as well as the production of inflammatory cytokines (ie. tumor necrosis factor (TNF)-α, interleukin (IL) 6, IL1β, or IL-10) are believed to contribute to the pathogenesis of the severe form of  malaria , 4–6. Af malaria , 8, dire malaria –11. The  malaria . Accumul malaria –16P. falciparum malaria, thrombocytopenia and anemia are common. Therefore, it is hypothesized that thrombocytopenia and anemia may lead to a reduction in S1P level in malaria. This study was designed to determine whether differences exist in the serum S1P concentrations in malaria patients and to investigate whether they are correlated with the clinical parameters, specifically parasite count, platelet count, hemoglobin (Hb) and hematocrit (Hct) levels.The major sources of circulating S1P are platelets , 18 and Plasmodium vivax malaria (n = 29), (2) uncomplicated Plasmodium falciparum malaria (n = 30), and (3) complicated P. falciparum malaria (n = 13). The diagnosis of malaria species was based on microscopic examination of blood smear. Blood films collected from all patients were examined by expert medical technologist and clinical diagnosis was confirmed by specialist doctor. Mixed infections  were not detected and tested by molecular methods. Severity of P. falciparum was defined by the World Health Organization (WHO) criteria [P. falciparum in blood smears, with other causes of coma excluded. Eighteen healthy volunteers with no history of malaria infection were recruited as control group. Written informed consent was obtained from all patients or their close relatives before enrollment in the study, and kept in the locked file cabinet accessible only by investigators. The study protocol was approved by the Ethics Committee, Faculty of Tropical Medicine, Mahidol University Malaria patients admitted to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand, were invited to participate in this study. Patients were divided into three groups: (1) criteria . ComplicWhole blood specimens were obtained from malaria patients on day 0  and day 7 (post-treatment) and directly collected into plastic tubes. The collected blood samples were left to stand for 15 min at room temperature (RT) to allow for clot formation, then centrifuged at 1500 g for 5 min. Subsequently, the supernatant as the serum was harvested and stored in an aliquot state at -80°C until analysis.The serum S1P concentrations were measured by S1P competitive ELISA kit according to the manufacturer’s protocol . Briefly, the serum was diluted (1: 10) in delipidized human sera. The diluted serum was incubated with the anti-S1P antibody in the mixing plate and incubated at RT for 60 min. After incubation, 100 μl of the reaction sample were transferred into wells of S1P coated detection plate and incubated for 60 min at RT, then washed with PBS for three times. After washing, 100 μl of streptavidin-horseradish peroxidase as the secondary detector was added in each well and incubated at RT for 30 min. The colour reaction was developed by incubation with 3,3′,5,5′ tetramethylbenzidine (TMB) substrate. Finally, the reaction was stopped with 100 μl of 1N sulfuric acid and read at OD 450 nm. S1P concentration level was expressed as μM. Each value from these indicated assays represents the mean ± SEM. All assays were carried out in triplicate.U test. Differences in levels of serum S1P within groups between day 0 and day 7 were tested by Wilcoxon signed-rank test. In addition, Spearman’s rank correlation (rs) was used to test the relationship between variables. Probability values (p) less than 0.05 were considered statistically significant.Statistical analysis was performed using SPSS version 17.0 software  and graphs were created using Graph Pad Prism version . Clinical variables are presented as mean and interquatile range (IQR). S1P levels are expressed as mean ± standard error of the mean (SEM). The normality of distribution was determined by the Kolmogorov-Smirnov test. Differences in levels of serum S1P between groups were compared by Mann-Whitney P. falciparum malaria patients. On admission, the mean malaria parasite count was significantly higher in the group of complicated P. falciparum malaria patients  compared to uncomplicated P. falciparum  and P. vivax malaria patients  . At day 7 post-treatment, malaria parasite was absent in the peripheral blood of all malaria patients. According to the hematological parameters, hemoglobin (Hb) and hematocrit (Hct) were significantly decreased in patients with complicated P. falciparuum, uncomplicated P. falciparum and P. vivax compared to healthy controls on admission and at day 7 post-treatment . On admission, the mean platelet count was significantly decreased in all malaria groups compared to the healthy controls . However, the platelet count in all malaria groups had increased significantly to normal value at day 7 post-treatment.Clinical data of malaria patients and healthy controls are shown in P. falciparum malaria patients (0.26 ± 0.04 μM), which showed significant differences when compared to P. vivax malaria patients (0.87± 0.10 μM), uncomplicated P. falciparum malaria patients (1.00 ± 0.07 μM), and normal controls (1.47 ± 0.09 μM) .Serum S1P concentrations of malaria patients and healthy controls are shown in P.vivax patients had increased significantly to normal level . However, S1P levels remained significantly decreased in patients with uncomplicated P. falciparum malaria (1.17 ± 0.04 μM) and complicated P. falciparum malaria (0.53 ± 0.07 μM), compared to the healthy controls .Serum S1P concentrations of all malaria groups were significantly increased on day 7 when compared with S1P levels on admission. On day 7 post-treatment, serum S1P level in rs = -0.623; p < 0.001)     and Hct levels . When segregated into groups, correlations were apparent that in P. vivax group, there was no association between S1P levels and laboratory parameters  at day 0  . However, in uncomplicated P. falciparum, positive correlations were obtained between S1P levels and parasitemia , Hb  and Hct   and Hct levels . While in complicated P. falciparum, on the day of admission, there were strong positive correlations between serum S1P levels and Hb , Hct , and platelet count   and Hct levels . S1P level was not correlated with white blood cells in patients with severe malaria .The number of parasite count, platelet count and the levels of Hb and Hct were important clinical parameters used to determine correlations with S1P concentration. Using pooled data, on admission, significant negative correlations was found between serum S1P concentration and parasitemia (< 0.001) . Signifi= 0.005) , Hct (rs< 0.001)  and plat= 0.010) . On day ctively)  and 7 (r= 0.028) . On day < 0.001) . On day P. falciparum malaria had reduced levels of serum S1P on admission and day 7 post-treatment. A decrease in S1P level had previously been documented in acute dengue infection [P. falciparum malaria. Nevertheless, limitations in this study include (1) lack of confirmation for mixed infection of P. falciparum and P. vivax, (2) lack of kinetics of S1P levels in all patient groups, and (3) only pertinent clinical and laboratory findings were used to find correlation with S1P, other malaria severity parameters such as cerebral malaria, pulmonary edema, acidosis, hyperbilirubinemia were not available for analysis.The main findings of our study indicated that patients with complicated nfection , sepsis nfection , chronicnfection  and obesnfection . In contnfection  and systnfection . In humanfection . The curThe preference for serum in S1P detection is widely discussed. During the coagulation process, platelets are activated by the clotting factors to release S1P in the serum . NormallP. falciparum malaria as SphK catalyzes sphingosine to S1P. Decreased in SphK activity has been reported in tuberculosis [P. falciparum had lower platelet counts than patients infected with P. vivax malaria. Previous findings reported that S1P receptor 1 on megakaryocytes is required for the formation of proplatelet-containing cytoplasmic protrusions and releases of platelet fragments [P. falciparum malaria  after 7 days compared to P. vivax malaria suggesting a lagging time in the synthesis and released of S1P from platelets in malaria.Platelets and RBCs are known to play a pathological role as main sources of circulating S1P. Platelets can accumulate and release S1P after local blood coagulation . Our datrculosis , bovine rculosis . Patientragments , 30, sugP. falciparum which indicates the decreased in RBC volume or anemia. It is well known that both P. falciparum and P vivax can induce anemia during blood stages of malaria infection. Severe malarial anemia caused by P. falciparum is responsible for approximately a third of the deaths associated with the disease [P. falciparum patients on day 0 and day 7. It is possible that reduction of RBCs or anemia may contribute to low S1P levels in malaria patients. Failure of Hb and Hct to normalize after 7 days of treatment suggest a lagging time of erythropoiesis. However, the regulatory mechanism that controls blood S1P levels is not well understood. Further investigation is required to study the receptors and signaling process of S1P on RBCs in malaria patients.A reduction in Hb and Hct levels was found particularly in complicated  disease . Anemia  disease , 32. In  disease . The supP. falciparum malaria than Hb and Hct, suggesting that serum S1P might derive largely from activated platelets in prepared serum samples in malaria. The finding agrees with previous study which reported that serum S1P concentration was closely correlated with the platelet count but was very weakly correlated with the RBC count [Platelet level was more positively correlated with S1P concentrations in complicated BC count . These rP. falciparum in this study could contribute to endothelial cell damage and affect the endothelial cell junction permeability. An experimental cerebral malaria reported that FTY720 (S1P agonist) administration improved vascular integrity in the brain, as well as reduced inflammation and reduced T-cell infiltration in the brain; and improved animal survival when used as adjuvant therapy with antimalarial treatment [S1P plays an important function is regulating pathophysiological processes involved in endothelial permeability and cytokine releases –35. The reatment . It was reatment . WhetherP. vivax and P. falciparum, suggesting that S1P signaling cascade is implicated in the severity of malaria. Decreased serum S1P concentration is associated with thrombocytopenia and anemia in P. falciparum malaria as well as malaria severity. These findings can be a proof of concept to support that S1P may represent a novel drug target for adjuvant treatment for severe malaria.Serum S1P concentration is decreased in malaria patient infected with


Serological data indicating the presence and level of antibodies against infectious disease antigens provides indicators of exposure and transmission patterns in a population. Laboratory testing for large-scale serosurveys is often hindered by time-consuming immunoassays that employ multiple tandem steps. Some nations have recently begun using malaria serosurveillance data to make inferences about the malaria exposure in their populations, and serosurveys have grown increasingly larger as more accurate estimates are desired. Presented here is a novel approach of antibody detection using bead-based immunoassay that involves incubating all assay reagents concurrently overnight.® multiplex bead-based assay (MBA) was used to detect total IgG against 8 malaria antigens: PfMSP1, PvMSP1, PmMSP1, PfCSP, PfAMA1, PfLSA1, PfGLURP-R0, PfHRP2. All sera and DBS samples were assayed by MBA using a standard immunoassay protocol with multiple steps, as well a protocol where sample and all reagents were incubated together overnight—termed here the OneStep assay. When compared to a standard multi-step assay, this OneStep assay amplified the assay signal for IgG detection for all 8 malaria antigens. The greatest increases in assay signal were seen at the low- and mid-range IgG titers and were indicative of an enhancement in the analyte detection, not simply an increase in the background signal of the assay. Seroprevalence estimates were generally similar for this sample Haitian population for all antigens regardless of serum or DBS sample type or assay protocol used.A serosurvey in was performed in Haiti in early 2017 with both sera (n = 712) and dried blood spots  collected for the same participants. The LuminexWhen using the MBA for IgG detection, overnight incubation for the test sample and all assay reagents greatly minimized hands-on time for laboratory staff. Enhanced IgG signal was observed with the OneStep assay for all 8 malaria antigens employed in this study, and seroprevalence estimates for this sample population were similar regardless of assay protocol used. This overnight incubation protocol has the potential to be deployed for large-scale malaria serosurveys for the high-throughput and timely collection of antibody data, particularly for malaria seroprevalence estimates. Plasmodium spp. parasites, the presence of IgG antibodies against malaria antigens has been used as an indicator to generate point estimates for malaria transmission as well as to monitor changes in malaria burden in a population over time carbodiimide hydrochloride (EDC) and 50 mg/mL sulfo-NHS (sulfo N-hydroxysulfosuccinimide) and incubating with rotation for 20 min at room temperature (RT) protected from light. After wash with antigen-coupling buffer , beads were suspended in antigen coupling buffer with the appropriate concentration of antigen  and 50 µL bead mastermix added to each well. Plates were washed two times with PBST and 50 µL of diluted sample (as described above) was added to each well and incubated with shaking at room temperature for 90 min. After 3 washes with PBST, beads were incubated with 50 µL biotinylated detection antibody mix consisting of: anti-human IgG  and anti-human IgG4 . Plates were incubated for 45 min and washed three times with PBST. 50 µL of streptavidin conjugated to phycoerythrin (PE)  was added to detect bound secondary antibody. After a 30-min incubation, wells were washed three times with PBST and incubated in Buffer A for 30 min under light shaking to remove any loosely bound antibodies. Samples were resuspended in 100 µL PBS and shaken for 30 min to resuspend beads; fluorescence data were collected immediately on the MAGPIX with Bio-Plex Manager™ MP software (BioRad) with a target of 50 beads per region per well. Median fluorescence intensity (MFI) signal was generated for a minimum of 50 beads/region, and background MFI from wells incubated with Buffer B was subtracted from each sample to give a final value of MFI minus background (MFI-bg) for analysis.The standard MBA protocol was performed as described previously , and ill4, 1:200 streptavidin-PE) was added to all wells, then 50 µL samples (or controls) were added to the reagent mix in the appropriate wells. Plates were incubated overnight with gentle shaking at room temperature and protected from light. The next morning , plates were washed 3×, and beads resuspended with 100 µL PBS and read on the MAGPIX machine. MFI signal was generated for a target of 50 beads/region, and background MFI from wells incubated with Buffer B was subtracted from each sample to give a final value of MFI-bg. Comparison in incubation times was accomplished by using a dilution curve of hyperimmune sera pooled from different areas of the world endemic for P. falciparum malaria.The OneStep assay was performed  curves created through the SGPLOT procedure with cubic interpolation and a degree of 2, and 95% confidence limits. Log-transformed MFI-bg values were fit to a two-component finite mixture model by the FMM procedure with normal distribution and maximum likelihood estimation outputs for component means and variances.Statistical analyses were performed in SASv9.4 . Direct comparisons between MFI-bg values using the two protocols were represented by Scatterplots comparing the MFI-bg values for the same person’s serum and DBS sampled assayed with both protocols are shown for all antigens in Fig. For the Haiti data, the 2-component finite mixture model (FMM) was used to estimate the antibody distributions in two putative subpopulations for each antigen: seronegative , and seropositive  , 23. ThiAs malaria serological data is typically presented in a binary fashion [an individual is +/− for antibodies against particular malaria antigen(s)], Table Plasmodium antigens when compared to a standard step-wise MBA protocol. This was found to hold true for both sera and whole blood that had been eluted from filter paper . Serological estimates from a serosurvey in Haiti of IgG prevalence in the study population were similar regardless of protocol or sample type, indicating that the novel OneStep protocol could generate malaria exposure prevalence estimates comparable to those generated using a standard protocol employing multiple incubation steps.Serosurveillance is increasingly becoming an attractive and economical strategy for multiple disease-specific public health programs hoping to gather information within a population regarding pathogen exposure or vaccination coverage , 13, 23.When comparing the OneStep to standard immunoassays, we found the greatest increases in signal detection capacity to be in the lower- to mid-range IgG levels. Augmented assay signal may be due to the overall increased incubation time in the OneStep protocol (~ 16 h) that extends the period for specific antigen–antibody binding. Signal detection may also be boosted by the formation of IgG-reagent complexes when all immunoassay reagents are present simultaneously, and reagent association is based on binding affinity and not tandem immunoassay steps. At the highest IgG concentrations, the data suggest that beads likely reach saturation of IgG-antigen binding regardless of incubation time, and this may explain why MFI-bg signals for the PfMSP1-19, PfAMA1, and PfGLURP-R0 antigens generated by the two protocols tended to be more similar at the highest IgG concentrations. However, for the PvMSP1-19, PmMSP1-19, PfCSP, PfLSA1, and PfHRP2 antigens, the y-axis for maximum MFI signals was actually extended when using the OneStep protocol. Some stark examples of this were for the DBS data for PmMSP1-19 where max OneStep MFI-bg was 18,745 (compared to standard protocol max of 3842) and DBS data for PfHRP2 where max OneStep MFI-bg was 72,370 . For IgG responses against many of the antigens, a noted limitation to the OneStep protocol was the loss of signal resolution at the higher IgG concentrations. When observing this shape of the non-parametric LOESS curves applied to scatterplots, many of the curves mirrored an exponential CDF curve which rapidly rises and eventually plateaus. If the OneStep protocol  is being used, and quantitative data is desired for analytical purposes, it is important to consider the range(s) of antibody concentration that are expected, as different protocols will provide clearer resolution to specific ranges of antibody concentrations. Additionally, no “correct” or “incorrect” total time of incubation is required by this simultaneous incubation protocol, and different laboratories can adjust according to the type of information they wish to gather.The increase in MBA signals from the OneStep protocol were indicative of improved detection of malaria antigen-specific IgG. In applying the two-component FMM approach, the MLE estimates for the mean and variance for the first component  were geRegardless of how many true components exist, having a separation of other components away from the first (putative seronegative) component with reduced overlap in distributions allows MLE statistics to more efficiently provide estimates for the mean and standard deviation of estimate for the first component, and provides higher confidence in determining the true assay signal.Malaria seroprevalence estimates for the Haiti serosurvey were similar if using different sample types (serum or DBS elution) or serology assay protocols (standard or OneStep). However, the estimate for PfAMA1 seroprevalence as generated by the OneStep protocol with serum samples (and the FMM approach) was notably lower than the three other experimental conditions. As DBS and serum samples were run in parallel, and the same bead couplings used throughout the study, the simple explanation for the dramatic difference in these estimates is that the modeled mean and variance of the first component of the OneStep sera data are markedly higher when compared to the other three FMM plots for PfAMA1. Due to these higher MLE outputs, the MFI-bg seropositivity threshold (mean + 3SD) was calculated to be much higher, and thus, some samples even with a high assay signal were not considered to have a MFI-bg signal “positive” for IgG against PfAMA1. This shows the susceptibility of forcing the mixture model to define two components, and comparison of seroestimates and cutoff determination schemes with various sample types and protocols should be investigated further. Overall, consistency among malaria seroprevalence estimates under different MBA conditions would indicate these estimates are accurate approximations of the true point prevalence.When compared to a standard MBA protocol, the OneStep serology protocol with samples and reagents incubated simultaneously overnight generated comparable estimates of overall seroprevalence for most antigens tested in a Haitian malaria survey, and may offer an efficient and flexible approach to large-scale serosurveillance studies. As malaria serosurveys become larger and more widely employed, options for the timely collection of laboratory data will be needed to allow a fast transition from sample collection to analyses.Additional file 1. Fittings to Two-Component Finite Mixture Models for Antigen Data Collected by both Assay Protocols and Sample Types for Low Seroprevalence Antigens. Histograms are displayed for log-transformed MFI-bg values for the four least immunogenic malaria antigens as fit to a two-component finite mixture model. On each panel, estimates for lognormal mean and variance are displayed for the two components.Additional file 2. Maximum Likelihood Estimates from Finite Mixture Model Comparing Standard to OneStep Protocols and Serum to Blood Elutions from Haiti Samples.Additional file 3. Scatterplots for assay signal for generic GST antigen for sera and dried blood spot samples when comparing standard and OneStep assay protocols for malaria antigens.Additional file 4. Increasing Incubation Times for the OneStep Assay Increases MFI-bg Assay Signal for Selected Malaria Antigens. Hyperimmune serum for malaria antigens was serially-diluted and incubated for 15, 30, 60, or 90 min with OneStep protocol, or assayed with standard protocol.


Plasmodium falciparum malaria is a major threat to global health. In response, countries in the Greater Mekong Sub-region, including Cambodia, have declared ambitious goals to eliminate malaria. Major challenges include the lack of information on the at-risk population-individuals who live or work in or near the forest where the malaria vectors are found, including plantation workers. This study aimed to address this knowledge gap through a cross-sectional survey conducted in rubber plantations in Cambodia in 2014.The ongoing spread of artemisinin resistant The survey was conducted in two rounds in four provinces and included a malaria prevalence survey, analysis for the K13 genetic mutation, and a comprehensive behavioural questionnaire. Forty plantations were included in each round, and 4201 interviews were conducted. An additional 701 blood samples were collected from family members of plantation workers.P. falciparum and 0.3% for Plasmodium vivax, and was very heterogenous between plantations. There was little difference in risk between permanent residents and temporary workers, and between the two rounds. The main risk factors for P. falciparum infection were smaller plantations, age under 30 years, lack of self-reported use of a treated net and recent travel, especially to the Northeastern provinces. Proximity of plantations to the forest was also a risk factor for malaria in round one, while male gender was also a risk factor for malaria by either species.Overall malaria prevalence was relatively low with adjusted PCR prevalence rate of 0.6% for P. falciparum elimination target on the horizon, identifying every single malaria case will become increasingly important. Plantations workers are relatively accessible compared to some other at-risk groups and will likely remain a high priority. Ongoing surveillance and adaptive strategies will be critical if malaria elimination is to be achieved in this setting.With Cambodia’s  Plasmodium falciparum, which was first detected along the Cambodia–Thailand border in the late 2000s [Malaria control efforts in Cambodia have led to a substantial decrease in cases over the past 15 years [te 2000s –5. Shoulte 2000s , much ofte 2000s , 7, puttte 2000s .In response, the World Health Organization and other partners have launched several initiatives to tackle artemisinin resistance , and in Approximately 27% of the population, or about 3.5 million people, in Cambodia are estimated to be migrant, defined as ‘a person who has moved to their enumeration area from another village or another country, which was the person’s last residence’ , 15. TheAnopheles vectors and that working conditions are often poor. Rubber tapping is carried out at night, exposing workers to nocturnally active exophilic mosquito vectors [Rubber plantation workers account for a significant proportion of MMPs working in forested areas in Cambodia . Access  vectors  and limi vectors . They ar vectors , 26.Rubber plantations have been shown to create situations of emergence and re-emergence of malaria in the region, with transient workers being an integral part of the malaria transmission cycle . BiologiOne approach that has been tried in Cambodia is a Plantation Malaria Worker (PMW) model. This is based on the village malaria worker (VMW) programme which has been implemented by the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM) since 2001 , with anThe VMW and PMW programmes have proven successful in providing access to quality assured diagnosis and treatment of symptomatic malaria. However, in the context of malaria elimination, it is felt that interventions such as mass drug administration or active screening and treatment may be needed in order to substantially reduce the reservoir of infection in asymptomatic malaria carriers , 29. TheIn order to address this knowledge gap, an epidemiological survey was conducted in 42 plantations involved in PSK’s PMW programme. The study aimed to document the prevalence of malaria and artemisinin resistance on plantations and to examine the potential risk factors for infection in order to inform the design of feasible and appropriate interventions to targeting plantation workers. A secondary objective was to describe the prevalence of malaria in non-plantation-working household members of plantation workers in order to inform how actively they should be included in future interventions.This was a cross-sectional survey of malaria prevalence, behaviour and knowledge in plantation workers. The survey was carried out in two rounds in recognition of the seasonal nature of plantation work and malaria transmission. Round 1 was conducted from June 1 to July 3, 2014 at the beginning of the wet season and Round 2 was conducted from October 1 to October 27, 2014, in the middle of the wet season.Forty plantations in four provinces were visited in each round Fig. . In the versus temporary workers. It was estimated that the prevalence in permanent workers would be around 1.0% and that there would be a minimum difference of 2.1% with temporary workers . A minimum sample size of 1273 was required for each study strata, or 32 of each type of worker from each of the 40 plantations. In order to estimate the prevalence of malaria among household members of plantation workers, a sample of 560 households members (14 per plantation) were required, to provide estimates of malaria prevalence accurate to within ± 1.5% points, assuming a prevalence point estimate of 2%. Family members had to be 5 years or above and not working in the plantation.All of the plantations involved in PSK’s PMW programme were eligible for inclusion. At the initiation of the study there were 42 plantations of which 40 agreed to participate. The PMW programme was expanding during the time of the survey, and therefore two plantations which had recently joined the PSK’s PMW programme were included as replacements during the 2nd round. The original sample size was based on comparing malaria in permanent Analysis of data from the first round showed that there was no statistical difference in malaria prevalence between permanent and temporary workers. The only statistically significant factor associated with malaria prevalence was the size of the plantation with smaller plantations (less than 6200 ha) being associated with a higher prevalence of malaria. For the second round, the sampling was adjusted to ensure that a comparison could also be made between smaller and larger plantations, as well as between permanent and temporary workers. This resulted in a required sample size of 1504, based on the survey including 25 small plantations and 15 big plantations, alpha error level of 95%, beta error level of 80% and an assumed dropout rate of 10%. From small plantations, the target sample size of 32 workers were sampled (15 temporary and 17 permanent) and on large plantations 48 workers (15 temporary and 33 permanent workers) ; had travelled to another commune in the previous 6 months (“mobility module”); or who had been into the forest in the previous month (“forest module”). The questionnaire was designed in English, translated into Khmer, back translated and piloted several times before being used.Prior to field the survey implementation, a team from PSK visited each plantation to inform the plantation manager or owner about the upcoming study and ask for approval to conduct the survey.The survey was conducted by four data collection teams consisting of eight or nine members including two supervisors, and four ‘advance teams’ consisting of four of five members. The survey team received 1 week of training and participated in a fieldwork practice.The advance teams visited plantations a few days before the arrival of the survey team to select, consent and enrol participants. Where the plantation manager had an up-to-date list of both permanent and temporary workers, this was used. Where no such list existed, a full census was undertaken with the help of the plantation managers who listed the workers and classified them as temporary (< 6 months) or permanent (> 6 months) according to their time working on site. The required sample of workers was then randomly selected from each group using simple random selection. On larger plantations this listing was facilitated with input from work team supervisors in the plantation, who have a closer day-to-day relationship with workers in the team than the plantation managers. The selected workers were visited and provided with verbal and written information about the survey and asked to give their written (or thumbprint) consent if they agreed to participate and were asked further information in order to confirm correct classification of their residency status. Participants were then informed of the time, date and location at which the survey would take place on the plantation.In five plantations access to the workers by the survey team was restricted by the plantation management because of the sensitive nature of some of the plantation’s activities. This is explained further in “On the day of the survey, participants arriving at the survey site were registered and cross-checked against the list drawn up by the advance team. Participants then had their temperature taken and finger prick blood sample taken for malaria RDT and one dried blood spot collected on filter paper for later analysis by polymerase chain reaction (PCR).All participants testing positive for malaria by RDT were offered immediate treatment with dihydroartemisinin–piperaquine, the national first-line treatment at the time of the study. The structured questionnaire was then administered to each worker by a data collector. The first 14 accompanying adult family members (one per worker) were provided with information about the survey and, if they consented, had blood taken for malaria, but did not undertake the survey questionnaire.Plasmodium, species identification and presence of resistance marker mutations using methods previously described by Canier et al. [P. falciparum positive samples were then processed to look for K13 mutations [All blood samples for PCR analysis were transported to Institut Pasteur du Cambodge in Phnom Penh twice per week and analysed for confirmation of r et al. . All P. utations . DiscordData were double entered using EpiData with in-built checks for consistency and range values. Each survey dataset was validated and all inconsistencies were checked against the original questionnaires and amended. Data were linked to the blood results using a unique identifier code and analysed in Stata version 12 . Stata survey design commands were used to account for clustering, stratification and weighting. Each participant was given a weight based on their probability of being selected into the study, calculated separately for each classification of residence status and the size of that worker population on the plantation. Differences in malaria prevalence by risk factor were calculated using the design based F-test. Results presented on plantation level characteristics .A total of 4201 workers were interviewed: 2664 in the first round and 1537 in the second round . The size of the selected plantations ranged from 80 to 40,000 hectares, with a median of 5214 hectares  workers were classified as temporary and 1457 as permanent in round one, and 476  and 1060, respectively, in round two. Residence status could not be determined for one worker in round two due to missing information. Overall just over half the plantation workers were male, and half were aged between 15 and 30 years . Of note PSK distributed long-lasting ITNs between the two survey rounds and in the 2nd round survey over 80% of people reported having received a net, the majority of which were reported to be ITNs received within the previous 2 months. However less than 60% of the temporary workers reported having received a net compared to 90% of permanent staff (p < 0.001).Although one fifth of workers reported having visited the forest in the previous 1 month, less than 2% had stayed overnight. About one fifth had travelled outside the commune within the previous 1 month of the survey with significantly more (35%) in the 2nd round than the 1st round (9%) (p < 0.001). This is most probably because the 2nd round was conducted just after the Pchum Ben holidays when most Cambodians try to travel back to their home villages. About one fifth of workers had travelled abroad, but less than 1% had done so in the previous 1 month.Plasmodium falciparum and 14 Plasmodium vivax infections, giving adjusted prevalence rates of 0.6% and 0.3% respectively. In the 2nd round, PCR positivity was 37 , which included 20 P. falciparum, 16 P. vivax and 1 mixed infection, giving adjusted prevalence rates of 0.6% and 0.5% and 0.01%, respectively , which included 25 P. falciparum and three for P. vivax) and in the second round 16 individuals tested positive . No family members tested positive by RDT in the first round while two tested positive in round two . Further analysis below is based on PCR results.By RDT among plantation workers, there were four positive tests in the first round  than among large plantation (above 6200 ha), which had a prevalence of 0.4% (p = 0.03), leading to a change in sampling design for the second round of data collection. A higher prevalence was also found in the second round , but this was not significant.P. falciparum, 12% P. vivax and 4% mixed infection), and another had prevalence of 13.3%, but neither of these plantations had been included in the 1st round. The plantation with a prevalence of 24% was relatively small (500 ha), with only 27 workers and was located within the forest, by a river, and was long in shape. All of the PCR positive individuals were permanent plantation workers or their family members. The small size of this plantation, and its elongated shape meant that the workers were in close proximity to the forest. There was no obvious pattern among the parasite positive workers in terms of individual characteristics.In the 2nd round, one plantation had a prevalence of 24% . Workers under 30 years had five times the odds of being infected than those over 30 (p < 0.01). Workers who did not regularly use a treated net had 2.9 times the odds of infection (p = 0.01) and people who had travelled had 2.5 times the odds of being infected (p = 0.01).Risk factors appeared to differ by round .Age, gender and reported net usage were also found to be risk factors for infection by Destination of recent travel was an important risk factor for parasitaemia Fig. . OverallThis study was designed to assess the prevalence of malaria infection in rubber plantation workers in Cambodia, and to describe the risk factors in order to inform the design of subsequent malaria interventions.There were several important findings. Overall malaria prevalence was lower than expected  and was very heterogenous, with smaller plantations and those that were surrounded by more forest tending towards have a higher prevalence. Unexpectedly, overall prevalence did not differ significantly between the two rounds, nor between temporary (“mobile and migrant”) workers compared to their more settled counterparts. One of the highest risk factors for malaria infection was reported recent travel to the Northeastern provinces. These and other findings and their implications are discussed in more detail below.P. vivax. It is not possible to differentiate between novel and relapsing infections. Similar to village based data, the survey showed that malaria in plantations was highly heterogenous [Previous studies have reported that people working in rubber plantations are at greater risk of getting malaria , with onrogenous . Howeverrogenous , 34, whirogenous . TherefoWorkers involved in clearing primary forest in preparation for planting, and those who venture into the forest for non-plantation related work, for example hunting, logging, and collecting forest products, would also be expected to be at high risk. In this study, most of the plantations were quite established (median age of 7 years) and the majority of workers were engaged in rubber tapping, with very few engaged in clearing forest, therefore the sample size may have been too small to show an association between age of plantation or the main type of work with risk of infection. However, travel outside the commune was associated with highest risk (adjusted OR of 2.5) in multivariable analysis, with travel to the Northeastern provinces, where many workers travel to engage in logging and other forest work, having the highest risk of malaria. This suggests that on mature plantations, plantation work itself (i.e. rubber tapping) may not be the major risk factor for malaria in this population.There was little difference between the two survey rounds which had originally been intended to measure prevalence during the dry, low transmission season and the wet, higher transmission season. The lack of difference may have been due to a number of reasons. Firstly, due to unavoidable delays the first round was carried out in June, during the transition from dry to wet season, therefore, malaria transmission may have already been on the increase. Secondly, ITNs were distributed between the two rounds and this was associated with a significant increase in their reported use from 33 to 56%, and would be expected to decrease the rate of infection. Indeed workers who reported regularly using an ITN were nearly three times less likely to have malaria, suggesting that despite reservations about their utility in this setting, they have an important role. Of note, the two plantations that were surveyed in the 2nd round that were included as replacements, were also the two with the highest prevalence. Had there been no change in the plantations between rounds, it could be expected that the prevalence would have been even lower in the 2nd round.It was also expected that a higher proportion of the plantation workers would be temporary mobile and migrant workers and that they would have a higher risk of being malaria infected than permanent residents. Other studies have found that MMPs are at higher risk of malaria than permanent more settled communities . HoweverInterventions that have been discussed in the context of malaria elimination include mass drug administration and mass or focal screening and treatment, however given the heterogeneity of malaria risk between plantations, it would be important to target such interventions appropriately. One of the highest risk factors for malaria was recent travel to the Northeastern provinces, presumably in order to work in the forest. Village-based pro-active case screening of individuals who have recently returned from the forest has been shown to be successful at identifying asymptomatic infected individuals in Cambodia , so expaThe following study limitations are identified. First, plantations visited during this study had a working relationship with PSK, however gaining entry remained challenging, and two plantations had to be replaced in the second round for this reason. Second, due to the quasi-legal nature of work on some sites, land concession disputes and issues related to labour regulations, the management on five sites only permitted the survey teams access to certain subgroups of workers, which meant that the sampling method according to the study protocol was not possible. Third, the most transient workers  employed on an ad hoc basis may have been missed from the census worker lists resulting in an underestimate of malaria prevalence. Other approaches such as ethnographic studies might be necessary to get a better understanding of this population. The data reported by the workers may have been subject to recall bias. Finally, there were challenges in obtaining data on plantations and defining their characteristics. There is no universally accepted definition of “forest” on which to base the “forest proximity” variable. Additionally, five plantations were not accounted for on the available maps and therefore forest proximity could not be calculated. The reported size of the plantations was based on economic land concession data, which did not necessarily represent the actual area under plantation, and may have reduced the relationship between size of plantation and malaria prevalence. Finally, defining the age of plantation was sometimes challenging as they sometimes underwent changes in management or different stages of clearing and planting and the age reported did not always reflect the same thing.P. falciparum elimination target on the horizon, identifying every single malaria case will become increasingly important. Real time surveillance at every point where fever cases present are critical. Plantation workers are relatively accessible compared to some other at-risk groups and will likely remain a high priority. Expanding the PMW programme, possibly by including active-case detection, and continuous surveillance of plantation level data should enable swift action to be taken, potentially focusing on the smaller plantations and those in close proximity to primary forest. Finally, as widely documented elsewhere, as malaria transmission falls, P. vivax will become increasingly dominant and without implementing radical cure it is difficult to see how the ambitious targets for malaria elimination will be reached.With Cambodia’s Additional file 1. Malaria prevalence by PCR among plantation workers (adjusted for survey design) and odds of being infected, by risk factor, by round.Additional file 2.P. falciparum prevalence by PCR among plantation workers (adjusted for survey design) and odds of being infected, by risk factor.Additional file 3.P. vivax prevalence by PCR among plantation workers (adjusted for survey design) and odds of being infected, by risk factor.


Plasmodium genetic material in the blood present in the gut of these mosquitoes may be possible. The process of assessing the blood meal for the presence of pathogens is termed ‘xenosurveillance’. In view of this, the present work investigated the relationship between the frequency with which Plasmodium DNA is found in culicids and the frequency with which individuals are found to be carrying malaria parasites. A cross-sectional study was performed in a peri-urban area of Manaus, in the Western Brazilian Amazon, by simultaneously collecting human blood samples and trapping culicids from households. A total of 875 individuals were included in the study and a total of 13,374mosquito specimens were captured. Malaria prevalence in the study area was 7.7%. The frequency of households with at least one culicid specimen carrying Plasmodium DNA was 6.4%. Plasmodium infection incidence was significantly related to whether any Plasmodium positive blood-fed culicid was found in the same household  and for indoor-collected culicids . Furthermore, the number of infected people in the house at the time of mosquito collection was related to whether there were any positive blood-fed culicid mosquitoes in that household for collection methods combined  or only for indoor-collected culicids . Our results suggest that xenosurveillance can be used in endemic tropical regions in order to estimate the malaria burden and identify transmission foci in areas where Plasmodium vivax is predominant.Vector-borne diseases account for more than 17% of all infectious diseases, causing more than one million deaths annually. Malaria remains one of the most important public health problems worldwide. These vectors are bloodsucking insects, which can transmit disease-producing microorganisms during a blood meal. The contact of culicids with human populations living in malaria-endemic areas suggests that the identification of  Culex mosquitoes do not transmit malaria, but due to their wide dispersion and anthropophilic behavior, may be used for the monitoring of certain vector-borne diseases, such as malaria, whose agent can be easily identified through molecular techniques in the ingested blood. Here, we evaluated a Plasmodium xenosurveillance approach to determine human malaria prevalence in three distinct peri-urban areas of Manaus. For this purpose, we evaluated the presence of Plasmodium DNA in engorged culicids sampled indoors regardless of their role in malaria transmission and compared them with the human infection status in the same households. The spatial correlation between the presence of Plasmodium DNA in mosquitoes was similar to the infection rate in humans which clearly indicates that xenosurveillance can be a complimentary strategy in endemic tropical regions in order to estimate the malaria burden.In the era of malaria elimination, novel surveillance strategies are needed in order to achieve and sustain malaria free status in the region. New malaria infection burden surveillance strategies should be simple to implement, technologically uncomplicated, cost-effective and applicable to areas where malaria cases are known to occur. In this context, the xenosurveillance is an important tool, especially when using widely distributed and anthropophilic mosquitoes.  Aedes: vector of Chikungunya, dengue, Zika, Rift Valley and yellow fever; 2) Anopheles: vector of malaria, Saint Louis encephalitis, West Nile fever and several Anopheles A and B orthobunyaviruses and 3) Culex: Japanese encephalitis, lymphatic filariasis, Saint Louis encephalitis, Oropouche and West Nile fever  and for indoor-collected culicids  ; p<0.001] or only for indoor-collected culicids .Plasmodium incidence ; p = 0.006].The total number of blood-fed culicids was also significantly associated with Plasmodium genetic material in the blood present in the gut of mosquitoes may be possible and correlated to infection prevalence in the human population. In view of this, the present study investigated the relationship between Plasmodium DNA presence in engorged culicids and the frequency of individuals carrying malaria parasites in the same environment and the suitability of this novel xenosurveillancetool to support malaria transmission surveillance.A large majority of mosquito species are hematophagous and rely on vertebrate blood for nourishment and reproduction. The contact of several anthropophilic and endophagic culicids with human populations living in malaria endemic areas suggests that the identification of Cx. quinquefasciatus and other less frequent Culex species predominated in the mosquito collections. Some mosquito species, such as Cx. quinquefasciatus, have a spatial distribution and abundance, which is strongly dependent on human presence [Cx. quinquefasciatus, for example, are inclined to blood feed in the evening and evening twilight and they use human dwellings as shelter during the day and at night [Cx. quinquefasciatus, make this species very susceptible to pathogens present in the human host blood [Plasmodium DNA. However, the identification is labour-intensive, though this may not be required when the proposed xenosurveillance method is used in operational settings.In this study, presence ,40–44. Tpresence –47. Cx. at night ,48. The st blood . After tst blood ,48–52. Ast blood –12,53,54Plasmodium detection in field-collected culicids. Through laser confocal microscopy, the development of P. falciparum in Cx. quinquefasciatus up to 30 hours after experimental infection was observed [Cx. quinquefasciatus were alive during this period of 30 hours. The results obtained through the follow-up of P. vivax experimental feeding in Cx. quinquefasciatus demonstrated the decay of the DNA detection rates. The detection was only possible up to 48 hours after feeding. Since the mechanism of parasite killing must be sufficiently powerful that Plasmodium is not able to overcome it, working only with visibly engorged mosquitoes is a feasible, less costly and less laborious option, and the limit of five mosquito abdomens per pool demonstrates the operational viability of the technique. The simple cut of the abdomen of the mosquito does not require more time-consuming procedures and provides resource saving of DNA extraction and qPCR consumables, fundamental characteristics for the feasibility of any surveillance tool.A variable that needs to be taken into account in this xenosurveillance method is the sensitivity of observed . HoweverPlasmodium infection prevalence in humans. This correlation is important for combined collection methods and for indoor-collected culicids. In terms of logistics, using only indoor aspiration collection methods appeared to be more reliable. Although BG Sentinel traps are more productive, culicids collected by this method are mostly not engorged. BG Sentinel traps are large and difficult to carry to the field. The transportation requires vehicles with higher freight capacity and a second home visit for trap removal. Differently, the electric aspirators collect a greater proportion of engorged mosquitoes. This technique permits speed and practicality, allowing only one technician to aspire ~15 houses in one morning, in a single visit for culicid capture. Furthermore, the portability of the equipment with low requirement of logistics, allows the accomplishment of the visits even by motorcycle, which is deemed favourable in operational settings. However, in unfurnished and single-room homes, capture was usually poorly productive or even negative, which is probably due to the absence of mosquito resting sites. Considering that indoor aspiration may be considered by some as invasive, there was an expectation of rejection, which was not confirmed. Participants exhibited a good receptivity, even at times when some members of a family were still asleep.The presence of positive blood-fed culicids was significantly correlated to the force of infection and Culex mosquitoes in parallel with higher odds to inhabitants who also have malaria. Unlike other studies, the samples of the present study were obtained in a seasonal period in which there was a lower prevalence of malaria, and in a single round of collections. Thus, the information generated may be important for estimating the circulation of the pathogen in the area, even in periods of low transmission, corroborating the possibility of using this tool as a new malaria indicator.In the force of infection analysis, the total number of blood-fed culicids was significantly associated with malaria infections. One explanation might be that some houses in poorer conditions are more permissive to Plasmodium DNA positive mosquitoes and infected humans was observed in the study area, where asymptomatic infection predominates. The existence of asymptomatic individuals who are carriers of Plasmodium sp. in significant proportions, even in a hypoendemic area, shows that the prevalence of infection, when based on the thick smear drops analyzed by microscopy, is underestimated. This indicates the importance of these reservoirs in the dynamics of malaria transmission, and suggests that the submicroscopic parasite may be important for the transmission between the high and low transmission seasonal periods [A good spatial correlation between  periods ,57. ThusPlasmodium DNA positive culicids and malaria prevalence. Our results suggest that xenosurveillance can be used in endemic tropical regions in order to estimate malaria burden and identify transmission foci in areas of a P. vivax asymptomatic carriers. As a perspective, this tool can be applied for the simultaneous surveillance of human pathogens, such as vector-borne, transmission areas overlapping with malaria, while taking advantage of the same collection efforts.New malaria infection burden surveillance strategies should be simple to implement, technologically uncomplicated, cost-effective and applicable to areas where malaria cases are known to occur. In this study, we found a significant association between blood-fed and S1 Checklist(DOC)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.


Identifying efficient and effective strategies to reach and monitor populations at greatest risk of malaria in low-transmission settings is a key challenge for malaria elimination. In Namibia’s Zambezi Region, transmission is ongoing yet its drivers remain poorly understood. A growing literature suggests that night-time social activities may lead to malaria exposure that is beyond the reach of conventional preventive interventions, such as insecticide treated bed nets and indoor residual spraying.Formative research was conducted with community members in March, 2015 in the catchment areas of six randomly selected health facilities in the western Zambezi Region to identify night-time locations where large numbers of individuals regularly congregate. Using time-location sampling, a survey was conducted between March and May, 2015 at community-identified venues (bars and evening church services) to develop representative estimates of the prevalence of parasite infection and risk factors among venue-goers.When compared to a contemporaneous household survey of the general population aged 15 and older (N = 1160), venue-goers (N = 480) were more likely to have spent the night away from their home recently , report recent fever , and were less likely to have sought care for fever . Venue-goers had higher, but not significantly different, rates of malaria infection . Risk factors for malaria infection among venue-goers could not be determined due to the small number of infections identified, however self-reported fever was positively associated with outdoor livelihood activities , not wearing protective measures at the time of the survey  and having been bothered by mosquitos at the venue .Prevention measures and continued surveillance at night-time venues may be a useful complement to existing malaria elimination efforts. Namibia is a country with low malaria transmission that has successfully reduced the burden of malaria over the past 15 years through the large-scale deployment of long-lasting insecticide-treated nets (LLINs), indoor residual spraying (IRS), artemisinin-based combination therapy (ACT), and improved availability of rapid diagnostic tests (RDTs) . MalariaAlthough Namibia aims to eliminate malaria by 2022, recent successes have plateaued, with outbreaks commonplace over the past 2 years. Transmission is known to be clustered in the northern regions of the country , 3, but Identifying the circumstances and behaviours that lead to increased risk of infection, as well as how to effectively reach subgroups at increased risk, is essential to reduce malaria transmission. Using time-location sampling (TLS), this study investigated whether locations where individuals tend to gather away from their homes during mosquito-biting hours could prove useful sites to conduct active surveillance and prevention activities in the Zambezi Region. TLS is a multi-stage cluster sampling method in which individuals are recruited from randomly selected venues during randomly selected venue-day-time intervals (VDTs), in order to develop representative estimates of the prevalence of malaria, risk behaviours and prevention uptake among venue-goers , 7. TLS This study was conducted in six randomly selected health facility catchment areas (HFCAs), out of 13 total HFCAs in the western Zambezi Region in northeastern Namibia. The Zambezi Region is arid and sparsely populated and borders Zambia and Angola to the north and Botswana to the south. Malaria transmission is seasonal, peaks between December and April and is highly dependent on rainfall patterns. This study was conducted in the western Zambezi Region, where prevalence is highest and access to services lowest. The population was 35,381 based on 2011 census data  and the The study was conducted simultaneously with a representative household survey in the same catchment areas , a facilA formative assessment was conducted in March, 2015 to identify types of venues that were active between dusk and dawn, to identify their peak attendance times, and to engage with the community and venue owners regarding the potential acceptability and feasibility of the venue-based study design.In each HFCA, one focus group discussion (FGD) was held with community leaders (“headmen”). Additionally, one FGD was held with community health volunteers in the combined study area. All FGDs were conducted in the Siliozi language and followed a semi-structured guide. Participants were asked to discuss the types of venues that would be likely to meet a set of pre-defined venue eligibility criteria, to identify potential challenges to recruitment, and to develop a map of such venues and a list of peak attendance times. The venue eligibility criteria included that the venues be open at least once weekly between sundown and sunrise, have clientele mostly older than age 14 years, have a peak attendance of at least 10 people, have clientele deemed likely to be willing to participate, and that carrying out the study would not be expected to pose a risk to survey staff. Based on findings from the FGDs, a limited number of types of venues that appeared to have the largest number of venue-goers, greatest duration of time outdoors during mosquito-biting hours, and accessibility, were selected for inclusion in the survey. Formative study staff then visited the venues identified to confirm peak attendance times, request permission to conduct the study, and identify locations within or outside of each venue to conduct malaria testing and confidential interviews.TLS was used to select venues and times to recruit survey participants. A sampling frame of VDTs was first constructed. VDTs were defined as 2- to 4-h intervals of peak attendance at a specific venue . At the start of each month, VDTs were randomly selected from the sampling frame and scheduled onto a calendar of recruitment events. As possible, four events (two per each of two field teams) were scheduled each Wednesday, Friday and Saturday (days when peak venue attendance was identified) during the approximately 10-week data collection period. VDTs were selected by simple random sampling subject to logistical constraints: to limit travel time, a field team could travel to only one HFCA per evening; to limit burden on venue-goers, a venue could be visited no more than once weekly. An alternate VDT beginning on or after the start of each of these primary VDTs was also randomly selected, in case the primary venue was found to be closed, inaccessible, or had low attendance. Sampling quota were a minimum of 11 and a maximum of 25 participants per event, to meet a planned total sample size of 800 participants. The sample size was chosen to provide 80% power to detect a difference in polymerase chain reaction (PCR) parasite prevalence of 4 percentage points between households (anticipated to be 2%) and venues (anticipated to be 6%), assuming a design effect of 2 due to clustering by sampling event. Each month, the sampling frame was updated to reflect new venues or venue closures.Field teams included an enumerator, a nurse and two interviewers. Enumerators designated an intercept line to count venue-goers present at any time during the event. When not conducting interviews, interviewers approached all venue-goers who crossed the line and verbally assessed eligibility.Males and females ages 15 years and older who lived or worked in the Zambezi Region, had not participated previously and did not appear too intoxicated to provide informed consent were assessed for eligibility using a screening form and, if deemed eligible, invited to a confidential area outside and nearby the venue entrance. Those willing to provide written informed consent then completed a 15- to 30-minute interviewer-administered questionnaire via electronic tablet, which included demographics, outdoor activities between dusk and dawn, protective measures against mosquitos at the venue and place of sleeping, fever and malaria history, treatment-seeking for fever, and venue attendance patterns. The nurse then collected a finger-prick blood sample, conducted a malaria RDT and prepared a dried blood spot for molecular testing.Participants with positive RDT results were provided free treatment on-site. Blood samples were frozen before being sent to a laboratory in Windhoek for loop-mediated isothermal amplification (LAMP), and follow-up PCR following all positive LAMP results, and on 10% of negatives. No monetary or other incentives were provided.Participant ID codes linked questionnaires, specimens and test results. Names were not recorded. The study protocol was approved by The University of California, San Francisco Committee on Human Research, Institutional Review Board, the University of Namibia Research Ethics Committee, and the Research Management Committee of the Ministry of Health and Social Services of Namibia.Estimates of characteristics of the population reached by TLS were compared to results from a cross-sectional household survey covering the same geographic region and conducted between April and June, 2015. In this survey, 684 households were randomly sampled, 529 households enrolled, and 1918 household members interviewed and tested for malaria . SamplinWhat are the occupations or activities that you do to support yourself and/or your family with income or food?”. Outdoor activities were defined as agriculture or fishing  and construction or security guard. Indoor activities were defined as professional, student or merchant , domestic work (“homemaker”) and “other” responses.Responses to the following question were grouped to differentiate indoor and outdoor livelihood activities: “Respondents were asked about protective measures against malaria in their home including IRS and LLIN ownership, as well as whether they had been ill with fever anytime in the past 3 months, the last 2 weeks, and whether they sought treatment.For categorical measures, proportions and 95% confidence intervals (CIs) were estimated; medians and interquartile ranges were estimated for continuous measures. Differences by venue type were assessed using Wald tests. Differences between the TLS and household surveys were assessed using two-sample Z-tests.Risk factors for malaria infection by RDT, PCR or LAMP could not be identified due to the small number of positives detected. Associations were identified between self-reported fever in the past 3 months using bivariate and multivariable logistic regression, examining variables related to demographics, potential exposure at the venue and at the place of sleeping. Response categories were combined in some cases where bivariate associations were similar. The multivariable model included all variables with bivariate associations at the 10% level. Analysis was conducted in Stata version 12.0 using procedures for complex surveys.Fe was the sampling fraction from respondent (i)’s sampling event and Ai was the frequency of attendance, defined as the number of days the respondent went to the same type of venue  in the past 30 days between sundown and sunrise [Estimates of prevalence and tests for associations from the TLS sample were weighted and standard errors were linearized to account for clustering by recruitment event –15. Samp sunrise . FrequenSeveral types of locations where individuals gathered between sundown and sunrise were identified during FGDs, including bars, churches, construction work sites, and cattle, fishing and police camps. The TLS study was limited to bars and churches as they were considered the most well-attended and accessible locations, often have windows or doors that are left open, and in some cases incomplete walls or open eaves. Twenty-one bars and four churches with evening hours were identified within the 6 catchment areas. VDTs, defined based on reported peak times, were generally between 3 and 8 pm at churches and 6 pm and 12am at bars. Selected HFCAs, venues, and households in the study area are shown in Fig. From March 27 to May 30, 2015, 480 study participants were recruited from 67 events held at 43 randomly selected VDTs, including 390 (81%) participants at bars and 90 (19%) at churches. Between 1 to 5 (median 3) sampling events were held per venue. Each venue contributed from 2 (0.4%) to 45 (9.4%) participants. Six additional events during the final month of data collection produced no new participants despite venue-goers in attendance. Of venue-goers approached, 80% at churches and 67% at bars were eligible and agreed to participate; this declined from 76% overall during the first 30 days to 51% during the latter half of the study period. The main reasons cited by field teams for non-participation were previous participation and lack of incentives.P = 0.001), male , and less likely to work in agriculture or fishing  than church-goers.Based on weighted analysis, an estimated 62.8% of individuals frequenting bars and evening church services were male and 37.2% were female Table . The medNearly a third (28.8%) of participants frequented bars or churches between sundown and sunrise at least weekly. Most (79.3%) spent more than 1 h during a typical visit to the venue where they were surveyed and 44.5% spent 3 h or more. About half (53.8%) had been bothered by mosquitoes at the venue previously; 2.5% were using mosquito repellent at the time of the interview. These estimates of potential exposure to mosquito bites were similar at bars and churches.About half (50.8%) of venue-goers slept in structures the previous night where IRS had been conducted in the past year, 43.7% owned a mosquito net and 26.9% had slept under a net the previous night. Fifty participants (10.5%) had slept outdoors in the past 2 weeks and 82 (17.3%) had spent the night outside of their home village in the past 4 weeks. Of the 82 participants reporting overnight travel, 78 travelled within Namibia, 2 to Zambia and 2 did not report their destination. These estimates were also similar at bars and churches.The estimated prevalence of malaria infection was 4.7% by LAMP and 0.5% by RDT, with no differences by type of venue. Of the 17 LAMP-positive participants, 13 were recruited from bars  and 4 from churches ; LAMP-positive participants’ ages ranged from 17 to 42 years (median 24 years) and were similar by sex and venue type. Only 2 of the 17 LAMP-positive participants tested positive by RDT. Two of the three RDT-positive participants tested positive by LAMP.An estimated 52.1% of venue-goers had self-reported fever in the 2 weeks prior to the survey and 64.2% in the prior 3 months. Of the latter, 37.9% (105) sought treatment or advice for the most recent fever episode. None of those who reported fever in the past 3 months reported receiving chloroquine or quinine: five received Coartem®; one received SP/Fansidar®; 61 received Panadol®; and 26 received other unspecified treatments. Of the six participants with fever in the past 3 months who took Coartem or SP/Fansidar anti-malarials at last fever, none tested positive for malaria by RDT, PCR or LAMP. Of the 88 who received other or no treatments, five (5.7%) tested positive for malaria: two by RDT, one by PCR and five by LAMP; all five reported fever in the past 2 weeks.P = 0.030), ever being bothered by mosquitos at the venue , not wearing personal protective measures when interviewed , and Zambian citizenship  , aged 15 to 24 years , and less likely to be age 45 years and older  (Table P = 0.008), to have experienced fever in the past 2 weeks  and 3 months , less likely to have sought treatment at last fever , and moderately less likely to reside in a structure where IRS had been conducted in the past year . Prevalence of malaria infection by LAMP between the venue-going population and the general population was not significantly different . Bar and church sub-samples both differed from the general population with respect to prevalence of fever and spending the night away from home; bar-goers differed from the general population with respect to age (P < 0.001), sex (P < 0.001), IRS in past year (P = 0.031), and treatment-seeking for fever (P = 0.011) , suggesting that youth may not be able to provide information needed to characterize malaria risk at their residence. It was not possible to assess the degree of bias in prevalence estimates due to potentially greater participation by individuals who may have been motivated to obtain free malaria testing because they were ill; future venue-based studies should record reasons for participation and refusal.The findings have other important limitations. Other types of venues, such as construction and agricultural work sites (identified in the formative research but not included in this study), could be associated with malaria risk and should be examined by future research. Due to time constraints, rates of venue attendance were not assessed by direct observation ahead of the study in order to inform the sampling plan; this may have contributed in part to failure to meet sample size targets . There wTLS was developed in the context of HIV in urban settings dense with venues and venue-goers , 7, wherIn the global push to eliminate malaria, many countries have identified high-risk populations that play an important role in sustaining transmission yet are not easily reached by conventional surveillance and control strategies . The preStrategies that should be considered at these sites include providing information on malaria prevention and testing, providing on-site testing and treatment, and distributing LLINs. Additional research is needed to clarify which venues actually play a role in transmission, in order to prioritize venue-focused interventions such as IRS. Such research should include gathering vector data around suspected venues, conducting TLS during a greater portion of malaria season in order to increase power to detect elevated risk, and including questions about venue attendance in malaria indicator surveys.While individuals who engage in outdoor social and occupational activities are increasingly seen as a target for onsite testing and treatment , 4, 20,