In 2011, Uganda’s Ministry of Health switched policy from presumptive treatment of malaria to recommending parasitological diagnosis prior to treatment, resulting in an expansion of diagnostic services at all levels of public health facilities including hospitals. Despite this change, anti-malarial drugs are often prescribed even when test results are negative. Presented is data on anti-malarial prescription practices among hospitalized children who underwent diagnostic testing after adoption of new treatment guidelines.Anti-malarial prescription practices were collected as part of an inpatient malaria surveillance program generating high quality data among children admitted for any reason at government hospitals in six districts. A standardized medical record form was used to collect detailed patient information including presenting symptoms and signs, laboratory test results, admission and final diagnoses, treatments administered, and final outcome upon discharge.Between July 2011 and December 2013, 58,095 children were admitted to the six hospitals .A total of 56,282 (96.9 %) patients were tested for malaria, of which 26,072 (46.3 %) tested positive . Among those testing positive, only 84 (0.3 %) were first tested after admission and 295 of 30,389 (1.0 %) patients who tested negative at admission later tested positive. Of 30,210 children with only negative test results, 11,977 (39.6 %) were prescribed an anti-malarial . The proportion of children with a negative test result who were prescribed an anti-malarial fluctuated over time and did not show a significant trend at any site with the exception of one hospital where a steady decline was observed. Among those with only negative test results, children 6–12 months of age  and those greater than 12 months of age  were more likely to be prescribed an anti-malarial compared to children less than 6 months of age. Children with findings suggestive of severe malaria were also more likely to be prescribed an anti-malarial after a negative test result .Despite high testing rates for malaria at all sites, prescription of anti-malarials to patients with negative test results remained high, with the exception of one site where a steady decline occurred. Malaria is reported by the Ministry of Health’s Health Management Information System to be the leading cause of morbidity and mortality in Uganda, accounting for 30–50 % of outpatient visits, 35 % of hospital admissions, and 9–14 % of hospital deaths . For chiIn 2010, WHO introduced two major changes to treatment guidelines for malaria case management. First was the replacement of presumptive treatment with parasitological diagnosis whenever possible, and second was the replacement of intravenous quinine with intravenous artesunate as first-line treatment for severe malaria . The UgaDespite changes in policy recommendations and reported benefits, reports indicate that anti-malarial therapy continues to be prescribed to children even following a negative malaria test result . UtiliziBetween April 2010 and June 2011, the Uganda Malaria Surveillance Project (UMSP) in collaboration with the National Malaria Control Programme (NMCP) established an inpatient malaria surveillance system at public hospitals in six districts—Tororo, Jinja, Kanungu, Mubende, Apac and Kabale—representing distinct geography and malaria transmission intensity  was developed by UMSP in consultation with the NMCP. The MRF collects detailed patient information including presenting symptoms and signs, laboratory test results, admission and final diagnoses, treatments administered, and final outcome upon discharge. The MRF uses check boxes to minimize transcription errors, and incorporates Integrated Management of Childhood Illnesses (IMCI) terminology for signs and symptoms, a standard framework familiar to most clinicians in Uganda. UMSP was responsible for ensuring that MRFs were available at all study sites.Upon initiation of the inpatient malaria surveillance system, training was provided to the clinical staff on the use of the new MRF, the importance of good medical record keeping, the use of data to improve the quality of services, and proper case management of uncomplicated and severe malaria. Case management training was conducted with all clinicians during a 1-day session that was repeated on a second day to maximize the proportion of staff who received training. Through lectures and the distribution of job aides, case management training focused on the importance of malaria diagnosis based on parasitological confirmation, the use of quinine for severe malaria (prior to the policy change recommending intravenous artesunate), and strategies aimed at streamlining patient flow within the hospital with the goal of reducing wait time. Hospital staff were also trained to test all paediatric admissions for malaria as a component of the programme’s goal of measuring the test positivity rate of all inpatients. This training included political and administrative district leadership, hospital administration, and hospital staff.Prior to start of the surveillance programme a laboratory staff at respective hospitals underwent a 3-day refresher course in malaria microscopy. The curriculum and training materials were developed by a team of laboratory experts from UMSP, Ministry of Health, and Central Public Health Laboratories as well as district laboratory focal persons, the details of which have been previously published . TraininPatient care in all hospitals except Jinja was provided by clinical officers (diploma level training in medicine) and nurses. A clinical officer made the decision for admission after the child was initially seen in the outpatient department. All admitted children had an MRF completed by the clinical officer, documenting the child’s presenting symptoms and signs, initial diagnoses, and treatments prescribed. For most hospitalized children, nurses provided routine care. For critically ill children, a clinical officer or a medical doctor was often consulted. Mubende Hospital also had a consultant paediatrician who provided care in the paediatric ward.Jinja Hospital staffing capacity was different in comparison to the other five hospitals. In addition to clinical officers, intern doctors were responsible for making admission decisions and reviewing admitted children on a daily basis. A team of three consultant paediatricians supervised the interns, reviewed children in the unit, and provided daily support including weekends. In all hospitals, nurses made the admission decision if a clinician was not available.Most children admitted at all six hospitals were severely ill. Occasionally children with uncomplicated disease were admitted. Possible reasons for admitting children with uncomplicated disease states include, existence of co morbid states warranting admission, self-request for hospitalization, and lack of oral medicines required to treat uncomplicated illness warranting admission for treatment with intravenous medications.Following the initiation of surveillance at the six public hospitals, supportive supervision visits were conducted by the UMSP surveillance team consisting of a physician, laboratory supervisor, and data supervisor. The visits were initially conducted every 2–3 months and were later spaced out to every 4–6 months by 2013. During each visit data quality was assessed and feedback provided. Additional data dissemination workshops were held biannually at each site, and included presentations on data quality, key malaria indicators, morbidity and mortality trends, and treatment practices. Administrative issues, potentially affecting performance at each site, were also discussed at the workshops. Continued support to laboratories, including external quality control, was provided by UMSP and the district health office to ensure adequate staffing, supply of reagents and slides, and adherence to high standards of malaria microscopy. The quality control system used expert microscopists to re-read 50 randomly selected slides and any hospital microscopist with less than 90 % concordance was re-trained. Quality control was carried out monthly at the beginning of the project, but changed to every 6 months after the UMSP team felt that quality was being maintained.MRFs of discharged patients were collected on a daily basis and entered into an Access database  and transmitted to a central database located in Kampala, Uganda on a monthly basis. Prior to being uploaded to a secure server, data were checked for errors. Stata version 12 was used for data analysis .All children (age <14 years) admitted to the six hospitals from July 2011 through December 2013 were included in the analysis. The following malaria case management indicators were examined: (1) proportion of admitted children with any malaria test result performed; positive or negative, (2) proportion of children prescribed anti-malarial drugs, stratified by positive or negative test result, and (3) type of anti-malarial drug prescribed, stratified by positive or negative result. A patient was reported to have a positive malaria test if any test result [presence of asexual parasitaemia by light microscopy or positive rapid diagnostic test (RDT)] was positive during hospitalization. A patient was reported to have only negative malaria test results if all test results were negative during hospitalization. A patient was defined as having complicated malaria, if had a positive malaria test result and presented with symptoms or signs consistent with WHO definition of severe malaria or had danger signs warranting intravenous treatment [A total of 58,095 children were admitted to the six hospitals over the 30-month period. Total number of hospital admissions during this period was highest in Jinja  and lowest in Kambuga . Fever was reported or documented in 91.6 % of patients and 49.0 % of patients had signs or symptoms suggestive of complicated malaria. The proportion of children tested for malaria was greater than 95 % at all hospitals and testing rates were consistently high throughout the study period. Among 56,282 patients tested for malaria, 26,072 (46.3 %) tested positive, ranging from 5.9 % in Kabale to 57.3 % in Apac , the site with the lowest malaria test positivity rate. In Kabale, the practice of prescribing anti-malarials to patients with any positive malaria test decreased significantly in 2013 compared to the previous 18 months . In all 22 cases where no anti-malarial was prescribed despite a positive test in Kabale, the positive test was performed at the time of admission.Among children with a positive malaria test result who were prescribed an anti-malarial, intravenous quinine was prescribed most frequently (81.9 %), ranging from 52.5 % in Kabale to 87.8 % in Tororo Table . The incAmong the 30,210 children with only negative malaria test results, 11,977 (39.6 %) were prescribed any anti-malarial drugs, ranging from 14.5 % in Kabale to 53.6 % in Jinja and Mubende  children were prescribed any anti-malarial drug during the first 3 months of the study, decreasing to only 4 of 337 (1.2 %) children during the last 3 months of the study. However, the other five hospitals showed greater fluctuation and no significant trend in the practice of prescribing any anti-malarial drug to children with only negative malaria test results (Fig. Associations between covariates of interest including the hospital, age, the presence of fever, and clinical findings suggestive of complicated malaria with the odds prescribing any anti-malarial drugs to children with only negative malaria test results are presented in Table Demonstration of the presence of malaria parasites prior to treatment with appropriate anti-malarial medicine has recently been adopted as a global standard of care for malaria case management . Proper Through sustained supervision, adopting a goal of universal testing, and buffering of the supply chain for essential materials required for malaria diagnostic testing, testing rates over 95 % among children admitted to the six public hospitals were achieved. Among children with positive tests results, intravenous quinine was the most commonly prescribed anti-malarial. At the start of the programme, intravenous artesunate, recommended by WHO and the Ministry of Health as first line treatment for complicated malaria in children, was rarely prescribed. However, in the latter half of 2013, a dramatic increase in prescription rates of intravenous artesunate was noted across all sites, though this was not sustained at some sites in subsequent months. The rise in prescription rates of intravenous artesunate corresponded to the actual implementation of the policy change adopted 2 years earlier in Uganda. The rapid decline in prescription rates observed at two sites following the initial surge was attributed to failed reacquisition of intravenous artesunate. These difficulties highlight the challenges of implementing and sustaining new policies in resource-limited settlings like Uganda.Among children with only negative malaria test results, data showed high rates of prescription of anti-malarials, results comparable to those reported in a similar setting in Tanzania prior to the global recommendation for universal diagnostic testing for malaria . DespiteKey to lowering the rate of prescription of anti-malarials to children with negative test results will be a better understanding of factors associated with this case management decision, and the results suggest possible reasons for this practice. With limited clinical skills and diagnostic capacity to evaluate patients, clinicians are unable to confirm alternate diagnoses and often disregard negative test results. Presented data support this hypothesis—children presenting with signs and symptoms similar to those of complicated malaria, such as pallor, and histories of fever, convulsions, and passing tea coloured urine were more likely to be prescribed anti-malarials after a negative test than children who did not have these findings. Additional evidence in support of this hypothesis is that the magnitude of association between study sites and anti-malarial prescription among children with negative test results doubled upon regression analysis limited to children with clinical findings suggestive of complicated malaria, as compared to analysis limited to those without clinical findings suggestive of complicated malaria. In addition, qualitative research work has shown that health worker beliefs towards perceived benefits of anti-malarial treatment are often overrated superseding rational clinical judgment. This behaviour is likely influenced by promotion of malaria as an important disease , peer prThis study had some limitations. First, it is possible that some treatments documented on the MRF were never administered and others were administered without documentation. Additionally, study data came from a malaria sentinel surveillance site programme and may not reflect what is happening at other public hospitals in Uganda. Furthermore, classification of those with malaria was limited to diagnostic testing results and it was not possible to ascertain where patients fell on the spectrum of disease from asymptomatic parasitaemia to complicated malaria. Lastly, data on treatment prior to hospitalization was not captured, which may have influenced some clinicians to prescribe anti-malarials even when test results were negative.Demonstrated is that the practice of irrational prescription of anti-malarials remained high at six hospitals serving as malaria sentinel sites. These findings are of concern considering that Uganda changed policy to laboratory based confirmation, and that testing rates were high in the hospitals where the study was done, implying that the situation could be more serious in government hospitals where opportunities for testing, training and supervision are not comparable. Given this, it will continue to be extremely challenging to implement the WHO test and treat policy in Uganda and similar settings . Sustain


Plasmodium falciparum predominates. The nature and impact of malaria, however, is not well understood in pregnant women residing in areas of low, unstable malaria transmission where P. falciparum and P. vivax co-exist.Pregnancy is a known risk factor for malaria which is associated with increased maternal and infant mortality and morbidity in areas of moderate-high malaria transmission intensity where P. falciparum infections was assessed by rapid diagnostic test and blood smear and compared among men, non-pregnant women and pregnant women. A subset of samples was tested for infection by PCR. Hemoglobin was assessed. Independent risk factors for malaria infection were determined using a multivariate logistic regression model.A large longitudinal active surveillance study of malaria was conducted in the Chittagong Hill Districts of Bangladesh. Over 32 months in 2010–2013, the period prevalence of asymptomatic P. falciparum infections were detected by RDT/smear from 3,110 tests. The period prevalence of asymptomatic P. falciparum infection in pregnant women was 2.3%, compared to 0.5% in non-pregnant women and 0.9% in men. All RDT/smear positive samples that were tested by PCR were PCR-positive, and PCR detected additional 35 infections that were RDT/smear negative. In a multivariate logistic regression analysis, pregnant women had 5.4-fold higher odds of infection as compared to non-pregnant women. Malaria-positive pregnant women, though asymptomatic, had statistically lower hemoglobin than those without malaria or pregnancy. Asymptomatic malaria was found to be evenly distributed across space and time, in contrast to symptomatic infections which tend to cluster.Total of 34 asymptomatic P. falciparum infection in the Chittagong Hill Districts of Bangladesh, and pregnancy and malaria interact to heighten the effect of each on hemoglobin. The even distribution of asymptomatic malaria, without temporal and spatial clustering, may have critical implications for malaria elimination strategies.Pregnancy is a risk factor for asymptomatic  Plasmodium falciparum parasites in the placenta, with or without detectable parasites in the peripheral circulation, is a distinct feature of pregnancy-associated malaria Pregnant women are at greater risk of acquiring malaria infection and developing symptomatic and complicated malaria disease than their non-pregnant counterparts The major risk factors associated with malaria during pregnancy include young maternal age, a low number of previous pregnancies (primi- or secundigravidae), and gestational age in pregnancy P. falciparum dominates. Data on the nature and magnitude of malaria on pregnant women and their pregnancy outcomes is relatively sparse in regions where malaria transmission intensity is low or unstable and mixed infections with P. falciparum and P. vivax are common Much of the knowledge about pregnancy-associated malaria is primarily based on data from sub-Saharan Africa where malaria transmission intensity is moderate to high and P. falciparum malaria in the Chittagong Hill Districts (CHD) of Bangladesh. In Bangladesh, 26.9 million people residing in 13 of its 64 districts are at risk of malaria and approximately 50,000 clinical malaria cases and 100–500 malaria-related deaths were reported annually . The highest incidence of malaria is found in the CHD P. falciparum malaria transmission is considered hypoendemic in the region, since the incidence rates were estimated as 1.48 and 2.75 per 1,000 person-months in 6–59 months and 5–14 years old, respectively We conducted a field clinical longitudinal study to assess whether pregnancy is a risk factor for asymptomatic P. falciparum malaria. The study was conducted as part of a large passive and active malaria surveillance system established in the CHD of Bangladesh to study the epidemiology of malaria in the region, and to link demographic, clinical, and entomologic factors in the determination of important risk factors for malaria. The details of this study system and its methods of data collection have been published elsewhere 2  were included in this analysis. Active surveillance was conducted as cross-sectional, random screening of the population using a two-stage cluster sampling design, as previously described P. falciparum and P. vivax malaria, and microscopic examination of thick and thin blood smears. The reported sensitivity and specificity of FalciVax RDT were relatively high  in febrile patients who typically present with a medium-high parasite density P. falciparum in the absence of any clinical signs or symptoms suggestive of malaria, and only asymptomatic infections were included in this analysis. P. vivax infections were not included in this analysis because only one asymptomatic P. vivax infection was detected in the study population during the time period of the analysis. The presence of symptoms was subjectively investigated by general open-ended and specific questions, and body temperature was measured. Finger prick blood was collected for RDT and smear reading, and for retrospective PCR analysis. A positive infection was defined if either or both an RDT or blood smear was positive. A standard protocol for malaria microscopy quality management established at the International Center for Diarrhea Disease Research, Bangladesh  was followed, where a random sample of 100 positive and 100 negative samples were examined and independently validated by two microscopists not associated with the study. Positive RDT or blood smear, regardless of clinical symptoms, prompted a treatment with a standard three-day course of artemether-lumefantrine. Pregnant women positive for malaria during the first trimester were treated with quinine as per national treatment guidelines Individuals were tested for asymptomatic malaria infection by a rapid diagnostic test (RDT) using FalciVax test strip, which detects both P. falciparum ribosomal gene with a Cy5 labeled probe. Samples were run in duplicate on manually loaded 96-well plates. A 40 cycle standard PCR protocol was run on a BioRad CFX96 PCR Detection System . Baseline relative fluorescence unit (RFU) values were readjusted within the Bio-Rad CFX manager software. The rt-PCR method demonstrated the highest sensitivity in the latent class analysis in febrile patients, and was able to detect 1–100 parasites per microliter. PCR testing was conducted for study samples collected between May 2010 and March 2012. Blood samples from non-endemic patients at Johns Hopkins University were used as negative control. Blood was also collected from the enrolled pregnant and non-pregnant women in the longitudinal surveillance, for the measurement of hemoglobin concentration using the hemocue system . Testing was performed in all women who were enrolled in the study from July 2011 to the end of the study.Detailed description of DNA extraction and real-time PCR (rt-PCR) analysis is available elsewhere P. falciparum incidence rates determined in a previous analysis Household locations for all individuals tested for malaria infection were mapped to show the spatial distribution of malaria tests in the study area. GPS coordinates for households were collected by local surveillance workers in March-April 2009, and mapped in ArcGIS . Using these GPS coordinates and passive malaria surveillance data, incidence clusters were defined based on the symptomatic P. falciparum infection among pregnant women, non-pregnant women and adult men from May 2010 to January 2013, and investigated the relationship between asymptomatic malaria infection and the following risk factors over the same time period: pregnancy status, study union, the symptomatic malaria incidence of the study cluster, age, ethnicity, occupation, bed net use, and transmission season. Asymptomatic malaria infection was defined as a positive blood smear or RDT for P. falciparum malaria without any clinical signs or symptoms suggestive of malaria. The analysis was restricted to asymptomatic individuals 16–44 years old who were tested at least once by active surveillance between May 2010 and January 2013 and had complete lab data. Only day 0 tests were included in this analysis, and tests on follow-up days 2, 7 and 28 were excluded.This study was designed to estimate the period prevalence of asymptomatic P. falciparum infection in this study area P. falciparum incidence rates as described in the Age was stratified into two groups, those at or above the median age (25 years) and those below the median age. Anemia was defined as hemoglobin concentration less than 11.0 g/dL. Mild, moderate or severe anemia was defined as hemoglobin concentrations 10–10.9 g/dL, 7–9.9 g/dL, or less than 7 g/dL, respectively. Occupations of interest included housewives, agriculture, daily labor, jhum cultivation, and others (mostly students and unemployed). Jhum cultivation is a form of migrant agriculture in the hilly CHD region and has been implicated as a risk factor for symptomatic 2 test. The period prevalence of asymptomatic malaria infection between pregnant women, non-pregnant women and men as well as within each of these risk categories were calculated and assessed for statistically significant differences using a Chi2 test. The period prevalence was calculated for both postpartum non-pregnant women and non-postpartum, non-pregnant women and compared using a Chi2 test.The sociodemographic and temporal factors were compared between pregnant women, non-pregnant women and men ages 16–44 years using a ChiP. falciparum infection was determined using multivariate logistic regression analysis. Dummy variables were created for demographic and temporal factors of interest and used to calculate the univariate and multivariate associations between these factors and asymptomatic malaria infection. Because very few asymptomatic P. falciparum cases occurred in the eight low incidence clusters, only data from the sixteen medium and high incidence clusters were used to maximize statistical power. The final multivariate logistic regression model was selected based using forward and backward stepwise regression  after assessing the interactions between covariates. Models were assessed for fit using Hosmer-Lemeshow and Pearson's Chi2 goodness of fit tests and checked for collinearity by calculating variance inflation factors.The independent effect of pregnancy and other factors on asymptomatic 2 test. All statistical analyses were conducted in Stata .The effect of asymptomatic malaria infection and pregnancy status on blood hemoglobin level was assessed. The median and inter-quartile range of blood hemoglobin concentration (in g/dL) were calculated and presented as box plots for asymptomatic malaria cases and non-cases, pregnant women and non-pregnant women, and the interaction of the two. Statistical significance was determined using a non-parametric Kruskall-Wallis equality of populations test. The percent of each group that was moderately or severely anemic (<10 g/dL) was calculated for each group and assess for statistical significance using a ChiThe overall period prevalence of asymptomatic malaria infection detected by PCR was calculated for pregnant women, non-pregnant women and men, and stratified into RDT- or smear-positive and PCR-positive infections that were negative by RDT or smear.A written informed consent or assent was obtained prior to enrollment from all adult or children (as defined as less than 16 years of age) participants, respectively. Permission from a guardian was also obtained for children. Additional consent was obtained at the time of enrollment into the active surveillance cohort. The study protocol and informed consent form were approved by the icddr,b Ethical Review Committee (PR#09021) and the Johns Hopkins Bloomberg School of Public Health Institutional Review Board (IRB#1965). The study was conducted in accordance with the principles of research ethics stated in the Declaration of Helsinki and the local and international regulatory guidelines.A total of 3,110 tests of malaria RDT and blood smear from individuals ages 16–44 years old were performed as part of active surveillance from May 2010 to January 2013. Among these, 909 tests were taken from 526 pregnant women, 1,753 tests from 911 non-pregnant women, and 448 tests from 316 men. Tests gathered from active surveillance were distributed evenly across the study area for men, pregnant women and non-pregnant women .P. falciparum incidence rates. There were no significant differences observed in ethnicity and bed net use among the three groups.The demographics of these different groups are described in P. falciparum infection was assessed by calculating the percentage of tests that were positive for P. falciparum malaria infection by a RDT or blood smear microscopy from May 2010 to January 2013  compared to non-pregnant women (0.5%) and men (0.9%) . Statistically significant risk factors for asymptomatic P. falciparum infection included residence in Rajbila union , and younger age (1.7% in those 16–24 years old compared to 0.6% in those 25–44 years). Individuals living in study clusters of high symptomatic P. falciparum incidence had the highest period prevalence of asymptomatic infection (1.8%) compared to study clusters of medium (1.1%) and low incidence (0.5%). There were no significant differences in the period prevalence of different occupation groups, among those reporting bed net use the previous night compared to those who did not, and across tests taken in the high and low transmission seasons. Among non-pregnant women, there was no statistically significant difference between the period prevalence of asymptomatic malaria among those tests taken ≤6 months postpartum  and those taken from women who have never been pregnant or >6 months postpartum .The period prevalence of asymptomatic ary 2013 . There wResults of the PCR analysis were available for the first 2,362  blood samples, collected from May 2010 to March 2012. Of these, a total of 67 samples (2.8%) were PCR-positive: 33 (4.6%), 23 (1.9%) and 11 (2.7%) in pregnant women, non-pregnant women, and men, respectively. Of these PCR-positive samples, 32 were also positive by RDT/smear. Therefore the PCR detected additional 35 infections that were negative by RDT or smear: 14 (2.0%), 14 (1.1%) and 7 (1.7%) samples in pregnant women, non-pregnant women, and men, respectively.P. falciparum infection were calculated using univariate and multivariate logistic regression modeling . Men had 2.5-fold higher odds of infection than non-pregnant women, however this relationship was not statistically significant (p = 0.214). Other factors selected into the final model included living in Rajbila union as compared to Kuhalong union  and age, with the odds of infection decreasing by 15% per year of age . The final model was assessed for goodness-of-fit and found to adequately fit the data .The unadjusted and adjusted odds of asymptomatic modeling . Only daanalysis . After uvs. 10.7 g/dL), and in women with than without asymptomatic falciparum infection (8.3 g/dL vs. 10.4 g/dL). The presence of both pregnancy and malaria appeared to interact and magnify the negative impact, as pregnant women with asymptomatic P. falciparum malaria had the lowest median blood hemoglobin (8.0 g/dL) compared to women with non-pregnant, malaria negative women (10.7 g/dL), non-pregnant, malaria positive women (10.8 g/dL), and pregnant, malaria-negative women (10.0 g/dL). A greater proportion of pregnant than non-pregnant women  and malaria positive than malaria negative women  had moderate or severe anemia (hemoglobin less than 10 g/dL). Of 21 malaria positive pregnant women, 10 were tested for hemoglobin, and all were moderately to severely anemic. Of 9 malaria-positive non-pregnant women, 3 were tested for hemoglobin and 1 was moderately to severely anemic.Hemoglobin concentrations were available in 495 (54.5%) and 444 (25.3%) pregnant and non-pregnant women, respectively. The median and interquartile range of blood hemoglobin concentration was calculated and stratified by pregnancy status and asymptomatic malaria infection status . OverallP. falciparum malaria in the Chittagong Hill Districts of Bangladesh, pregnant women had significantly higher period prevalence of asymptomatic P. falciparum infection compared to men and non-pregnant women over 32 months of active and longitudinal malaria surveillance. Using a real time PCR analysis we detected a similar ratio of malaria prevalence between pregnant women, non-pregnant women, and men, as compared to RDT or blood smear. RDT or smear detected about one-half of infections detected by rt-PCR. None of the submicroscopic infections detected by PCR progressed to clinical malaria.In this study of asymptomatic P. falciparum infection.This association held true in a multivariate logistic regression model controlling for maternal age and study union in the 16 study clusters with the highest malaria incidence. These results are consistent with other studies from high-transmission settings in sub-Saharan Africa P. falciparum infection in all age groups was 6-fold higher in the high transmission season (May-October) than the low transmission season (November-April), and 18.3-fold higher in the high incidence cluster than in the low incidence clusters P. falciparum infection between the two transmission seasons. While a subsequent analysis will investigate the epidemiology of asymptomatic infection in all age groups in this study area, our study results suggest that asymptomatic malaria infection is more evenly distributed across time and space than symptomatic infections. This observation will have a critical implication in the development of strategies for malaria control, particularly in the era of malaria elimination in regions such as Bangladesh where low transmission intensity justifies such an attempt. Further research is urgently needed to understand the nature and distribution of asymptomatic malaria infection serving as an important infected reservoir to continue malaria transmission.This study shows two important findings. First, the temporal and spatial distributions of asymptomatic infections differ from that of symptomatic disease. These observations were made over a similar time period, excluding the possibility of changes in transmission over time potentially confounding the observed infections. We previously found that the incidence of symptomatic P. falciparum infection and pregnancy synergistically contribute to maternal anemia in a hypoendemic malaria setting. Either pregnancy or malaria infection was associated with low hemoglobin concentration, and the women who were both pregnant and malaria positive had the lowest hemoglobin concentration. While hemoglobin values were available only in a portion of women, and the number of malaria-positive was too low to draw a definitive conclusion, the results are in agreement with evidence from high transmission settings Second, our observation in this study also suggested that While this study was grossly underpowered to detect the negative health consequences related to malaria during pregnancy, our findings echoed previous observations of detrimental effects of malaria in pregnant women and the fetus, and highlighted an urgent need to optimize the management of asymptomatic malaria infection in pregnant women residing in hypoendemic settings. While the risk-benefit balance may not favor of the use of IPTp, a mass preventive malaria treatment to minimize negative consequences of malaria in pregnant women widely used in regions of sub-Saharan Africa with moderate to high malaria transmission, screening of all pregnant women using RDT at prenatal visits may be effective. Further research is warranted to assess the effects of malaria detection and treatment targeting among this complex and vulnerable group of women.The reported rate of the use of bed nets (ITN) was high in our study, but we did not systematically measure the effectiveness of, or the lack thereof, ITNs in our study populations. The use of ITNs is one of the strategies that has been shown in other studies to improve malaria control and childhood mortality and morbidity P. falciparum infections, including only two infections in non-tribal Bengalis. These low numbers could not accommodate a multivariate model to assess potentially important factors risking over-stratification of the data and leading the large confidence intervals. Therefore, important confounders and effect modifiers of the effect of pregnancy on malaria infection may be missing from the model. Despite this limitation, this study was still powered enough to detect a clinically relevant 2–4 fold increase in the period prevalence of P. falciparum infection in pregnant women compared to non-pregnant women, adjusted for age and study location. Third, there is potential discrepancy between the estimated date of delivery and actual date of pregnancy termination, raising a risk of incorrect assignment of some women who had miscarriage or premature delivery. However we were able to confirm the date of pregnancy termination in 28 of 30 women, using information on the delivery outcome in the demographic surveillance system, and their status of pregnancy at the time of the detection of asymptomatic malaria infection. Moreover we were able to measure hemoglobin only in a portion of study women, and did not examine the association between asymptomatic malaria risk and gestational age of pregnancy, gravidity or parity, or pregnancy outcomes, for which a larger stratified study will be needed. Similarly, we did not address the impact of pregnancy on P. vivax infection, an important known cause of pregnancy anemia and maternal and infant morbidity P. vivax infection and pregnancy status in Bangladesh, especially in areas where P. vivax is more common.There are a number of important limitations to this study. First, we performed repeated testing in the same individual and the effect of non-independent measurement may have systematically biased our results. However, for this analysis, we assume that data points are independent since all infections originated from independent participants and the majority of positive infections were positive on the first testing. It is, therefore, unlikely that our study finding can be explained by a systematic error of repeated testing, or by the disproportionate testing frequency in one study population over the other. Similarly, this assumption of independence among repeated testing in the same individual may not be true since previous malaria infection may provide some immunity to subsequent infections. However, the likelihood that this assumption biased the results in this hypoendemic setting is low. Second, the study is underpowered with only 34 asymptomatic P. falciparum infection is significantly higher in pregnant women than in non-pregnant adult women or men in the Chittagong Hills Districts of Bangladesh, and suggests that the presence of P. falciparum infection in pregnant women may heighten the risk of anemia. These results underscore the need to further evaluate ways to optimize management of malaria infection in pregnant women living in areas where malaria transmission intensity is low. Management of asymptomatic infections at a population level, particularly in a low transmission setting, is complex owing to the risk-benefit ratio as well as to its social acceptance. Pregnant women present a different and important argument due to the known risk of malaria, regardless of symptomatic illness, to the mother and fetus. While argument for mass treatment such as IPTp widely used in high-transmission settings in sub-Saharan Africa may not be favorable in areas of low malaria transmission intensity such as Bangladesh, active screening and prompt treatment, with or without clinical symptoms, should be considered for high-risk populations. Our study also shows that asymptomatic P. falciparum infections, the most critical and problematic parasite population in breaking the transmission cycle, are evenly distributed throughout time and space, differing from the observed pattern of symptomatic infections which tend to cluster geographically and with time. These findings have critical implications for malaria control or elimination strategies. Further research is warranted to understand the distribution of asymptomatic infected reservoir, and to assess ways to eradicate it at the population level.In summary, this study shows that the risk of asymptomatic


Plasmodium species, the causative agents of malaria, are intracellular parasites that remodel host erythrocytes by expressing their own proteins on erythrocyte membranes. Although various aspects of the modifications made to the host erythrocyte membrane have been extensively studied in some human Plasmodium species , details of the structural and molecular biological modifications made to host erythrocytes by nonhuman Plasmodium parasites have not been studied. We employed zeta potential analysis of erythrocytes parasitized by P. chabaudi, a nonhuman Plasmodium parasite. From these measurements, we found that the surface potential shift was more negative for P. chabaudi-infected erythrocytes than for P. falciparum-infected erythrocytes. However, electron microscopic analysis of the surface of P. chabaudi-infected erythrocytes did not reveal any modifications as compared with nonparasitized erythrocytes. These results suggest that differences in the membrane modifications found herein represent unique attributes related to the pathogenesis profiles of the two different malaria parasite species in different host animals and that these features have been acquired through parasite adaptations acquired over long evolutionary time periods.Membrane electrochemical potential is a feature of the molecular profile of the cell membrane and the two-dimensional arrangement of its charge-bearing molecules.  Plasmodium. The parasite is widely distributed across tropical regions and affects large numbers of people living in Africa, South America, and Southeast Asia. Malaria infections are established by parasites released into the blood stream when parasite-infected female mosquitoes bite a vertebrate host; the consequent infection that follows occurs first in the liver followed by the erythrocytes. The clinical signs and symptoms of malaria infections in humans include chills, fever, body aches, headache, anemia, and spleen enlargement, while infections with Plasmodium falciparum, the most virulent, cerebral malaria-causing species [ Plasmodium also induce cellular and molecular alterations to erythrocytes, such as cell adhesion [Malaria, a serious infectious disease in humans, is caused by protozoan parasites of the genus species , can be adhesion –5, Band adhesion , erythroadhesion , increasadhesion , increasadhesion , changesadhesion –11, and,adhesion –16. ThesP. falciparum is known to express a variety of proteins after it invades human erythrocytes. These proteins include the P. falciparum erythrocyte membrane protein-1 (PfEMP-1), knob-associated histidine-rich protein (KAHRP), and RIFIN, STEVOR, and SURFIN proteins amongst others, and these proteins are transported to the erythrocyte membrane. PfEMP-1, PfEMP-3, KAHRP are main components of the interconnect-protein complex known as “knobs” [ P. falciparum have a characteristic morphology and uniform size , their function has not been extensively studied [ “knobs” ; these p “knobs” –19. Knob–25.3 nm ; diamete–25.3 nm ). Also, –25.3 nm –16. Furt studied . Express studied –25. Addi studied , as well P. berghei-, P. yoelii-, and P. chabaudi-infected erythrocytes is their ability to sequestrate in various organs of the body [ P. falciparum-infected erythrocytes [ Plasmodium in the same host and in different host animals. Hence, investigating the types of modification made to erythrocytes by different malaria parasite species in different host animals should further our understanding of malaria parasite biology. In this study, we investigated the physicochemical aspects of the erythrocyte membrane in terms of the structural modifications induced by infection with the rodent malaria parasite P. chabaudi and we describe here the host cell modifications that are characteristic of this parasite species.Different species of malaria parasites infect humans, reptiles, birds, and rodents. One study has reported that chickens experience fevers, enlarged spleens, hemolysis, and other clinical signs when they become infected by avian malaria parasites . In the the body –33; howehrocytes , 35. Des P. chabaudi AS strain chloroquine sensitive was used. Four- to five-week-old female mice  were injected intravenously with 5 × 106 parasitized erythrocytes. Mouse blood was collected when the parasitemia was ≈30–48% in ~1 mL of blood. The heparinized mouse blood was centrifuged at 300 ×g for 5 min to remove the buffy coat and washed twice with RPMI . Mature parasitized erythrocytes were separated using the MACS-LS Column system  [ P. chabaudi-infected erythrocytes were not separated from the uninfected erythrocytes because ~1 mL is the maximum volume that can be collected from a mouse heart after isoflurane  treatment and this amount is too small for measurement when only the parasitized erythrocytes were separated and used for the measurements).Animal infections were approved by “The Keio University Institutional Animal Care and Use Committee” and followed the “Institutional Guidelines on Animal Experimentation at Keio University.” Infection and erythrocyte collections were conducted as described in previous studies , 37. BriGermany)  for dete P. falciparum was approved by the Bioethics Committee for Epidemiologic Research, Jichi Medical University (authorization number: 12–20), for clinical research with human blood samples. O+ human erythrocytes were purchased from the Japanese Red Cross Society (authorization number: 25J-0045). P. falciparum was cultured as described previously [ P. falciparum-infected erythrocytes were separated by MACS Separators LS columns (Miltenyi Biotec K.K.) and then used for electron microscopy and zeta potential measurements or for obtaining the detergent-resistant membrane (DRM) fractions.Cultivation ofeviously . P. falcFor TEM analysis, nonparasitized and parasitized erythrocytes were fixed for 60 min on wet ice in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in HEPES buffer solution (pH 7.05)  and then fixed for a further 60 min in 1% osmium oxide in the same buffer. The fixed specimens were dehydrated with stepwise concentrations of ethanol and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Samples were examined with a JEM-1230 transmission electron microscope . For the SEM analysis, erythrocytes were fixed for 2 hours on wet ice in 2% paraformaldehyde in 0.1 mol/L HEPES buffer (pH 7.4) The erythrocytes were then spread on a slide glass covered with MAS coating . The samples were further fixed with 1% osmium oxide in phosphate-buffered saline  for ~1 hour at room temperature and then dehydrated with ethanol. The samples were treated with isoamyl acetate  and 100% ethanol (1 : 1 vol/vol) (Wako), dried using a critical point Dryer HCP-2  for 5 min at 40°C, and then sputtered by an Ion Sputter  with Pt and Pd. Sample observations were performed using S-4300 scanning electron microscopy .The surface potentials of erythrocyte membranes were measured by a zeta potential analyzer  as described according to a previous study . MeasureμL volume of the packed erythrocytes was thoroughly mixed with 800 μL of ice cold 1% Triton X-100 (Sigma-Aldrich Co. LLC) in TBS buffer solution (Wako) and a proteinase inhibitor  was included in each tube . The samples were kept on ice for 20 min, after which they were each mixed with the same volume of 0.2 mol/L-Na2CO3 in 80% sucrose in TBS . The 2 mL sample was transferred to a centrifugation tube . Then, 6 mL of 30% sucrose in TBS was overlaid at 4°C, followed by an additional 3 mL of 10% sucrose in TBS as the upper layer. Ultracentrifugation was performed in a SW-41Ti rotor (Beckman Coulter) at 200,000 ×g for 18 h at 2°C and 300 μL of each fraction was collected at 4°C and kept at −20°C.The effect of parasite infection on the lipid domain fraction of host erythrocytes was assessed by preparing lipid domain fractions according to an established method , 41. BriTo detect the DRM fraction and protein distribution, a NuPAGE 4–12% Bis-Tris Gel (Invitrogen) in MOPS SDS running buffer  was used for SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a PVDF membrane using the iBlot Gel Transfer system (Invitrogen) and NuPAGE transfer buffer (Novex). Flotillin-1 was used as a reference raft marker and mouse anti-flotillin-1 was used as the monoclonal antibody against it . Next, the membrane was washed with 0.2% Tween 20 in PBS for 10 min (×3). The second antibody reaction was performed using peroxidase-conjugated AffiniPure Donkey Anti-Mouse IgG (H + L) . ECL Advance Western Blotting Detection Reagents  were used as the developing reagents. P. chabaudi-infected mouse erythrocytes  in comparison with the nonparasitized erythrocyte controls .The zeta potential is an important index for evaluation of a material's surface in terms of its reflecting interfacial properties, dispersion, aggregation, and interaction with other materials. To investigate the properties of the erythrocyte membrane further, despite there being no obvious surface alterations to Figures . This te P. chabaudi altered the distribution of the lipid domain and the interaction between lipids and proteins in the erythrocyte membrane, even though the surface structure of the erythrocyte was not altered by the presence of the parasite.To further characterize how parasite infection affected the properties of the erythrocytes, we examined the lipid domain fraction after sucrose gradient density centrifugation separation. This fraction influences the raft domain in the nonparasitized and parasitized erythrocyte membrane. Anti-flotillin-1 was used as a marker for raft/DRM. With the nonparasitized mouse erythrocytes, the lipid domain was present in the upper-to-middle and lower fractions, and most fractions existed in the upper fraction . In cont P. falciparum results in significant alteration of the erythrocyte surface topography, with alteration of the inner structure of the parasitized erythrocyte [ P. chabaudi-infected erythrocytes . We found no direct evidence of an alteration in erythrocyte topology or a change in the erythrocyte surface under the influence of P. chabaudi infections. However, our data suggest that parasite-encoded proteins, such as knob-like structures on erythrocytes, possibly play a role in altering the surface potential charge on the erythrocyte by altering the distribution of cytoskeleton proteins and/or lipid-protein interactions.Generally, lipids are asymmetrically distributed between the inner and outer bilayer membranes of the erythrocyte with phosphatidylserine and phosphatidylethanolamine existing in the inner membrane and phosphatidylcholine in the outer membrane. Various studies have shown that not only malaria parasites modify the erythrocyte membrane surface structure, but also the internal structure and properties of the infected erythrocytes are modulated after invasion. In particular, infection withthrocyte , alteratthrocyte , 48, modthrocyte , and chathrocyte  and the throcyte , 49. In  Figures , even thpography . The sur Figures . The dir P. falciparum erythrocyte adhesion . Our data showed a shift away from a zero point for P. chabaudi-infected erythrocytes; however, the absolute shift values for P. chabaudi- and P. falciparum-infected erythrocytes were in a similar range and the shift range did not depart far from the zero point. Parasitized erythrocytes exhibit similar phenomena to aging erythrocytes compared with normal erythrocytes, such as increased levels of hemichrome, higher levels of oxidization, alterations in Band 3 aggregation and the cytoskeleton, and changes in protein expression related to immune function, adhesion, and permeability of the erythrocyte [Zeta potential is an electrochemical aspect of a particle's surface, and information about a particle's dispersibility, aggregability, and adhesion ability can be obtained from this measurement. If zeta potential takes a cross value around zero, the repulsive force between particles becomes weak and the particles will eventually aggregate. This physicochemical aspect corresponds to the phenomenon ofthrocyte , 50.P. chabaudi- and P. falciparum-infected erythrocytes are reported to have properties that promote different patterns of sequestration [ P. chabaudi-infected erythrocytes are reported to sequester in the lungs and liver of mice but do not adhere to the mouse brain, which is the cause of cerebral malaria in P. falciparum infections in humans. These differences in the behavior of P. chabaudi-infected mouse erythrocytes and P. falciparum-infected human erythrocytes are consistent with our zeta potential data. One possible explanation for the different pattern of sequestration observed between P. falciparum- and P. chabaudi-infected erythrocytes is that the var multigene family of P. falciparum, which is a component of knobs and is necessary for sequestration of P. falciparum-infected erythrocytes, does not exist in P. chabaudi [stration , 51, 52 chabaudi . P. falciparum-infected erythrocytes. However, knob-associated proteins are one of the candidates for the difference observed in the zeta potential data between P. falciparum- and P. chabaudi-infected erythrocytes. Taken together, our data are consistent with previous reports and physiological evaluation of parasite-infected erythrocytes.Zeta potential provides information on the “total surface charge” of an erythrocyte, and this parameter is modulated by complexes formed between different proteins, the lipid bilayer, and the erythrocyte cytoskeleton. We do not know which proteins or protein complexes reduce the absolute value of the surface charge of P. falciparum malaria infections for over 4,000 years [ Plasmodium spp. Furthermore, the role of the furrow-like structure observed in P. gallinaceum-infected erythrocytes from chicken [ P. chabaudi rodent malaria parasites do not modulate the surface of the parasitized erythrocyte  that encodes P. falciparum PfEMP-1; thus, P. chabaudi-infected erythrocytes probably do not express PfEMP-1, neither do they express KAHRP, which together form the components of knobs. Hence, rodent parasite-infected erythrocytes do not become trapped in the capillary blood vessels of the brain and rodents infected with this parasites species are not subject to the lethal effects of cerebral malaria. This feature of infection with P. chabaudi parasites allows them to survive for longer time periods in their hosts and was possibly selected for over the course of parasite evolution.Humans have been exposed to00 years , but dif00 years –57. Mala chicken  is also throcyte , Table 1hrocytes . One pos P. chabaudi- and P. falciparum-infected erythrocytes that we observed in this study may be one of the ways in which these parasites have adapted to their respective host animals (rodents and humans). Parasites eventually evolve to not kill their hosts but coexist with them.Coevolution between some malaria parasites and their hosts has occurred over very long evolutionary time periods, during which time these parasites have developed strategies that enable them to survive for long time periods in their respective host animals. The differences in theSupplemental data 1 Z-potential of (a) Non-parasitized human erythrocytes and (b) P. falciparum-infected erythrocytes. The absolute value of the membrane potential is reduced in the parasitized erythrocytes.


The World Health Organization recommends malaria be diagnosed by standard microscopy or rapid diagnostic test (RDT) before treatment. RDTs have been used with greater frequency in the absence of matching blood slide confirmation in the majority of RDT reported cases in Mimika Regency, Papua Province, Indonesia. Given the importance of RDT in current health system as point-of-care tool, careful validation of RDT product performance for providing accurate malaria diagnosis is critical.Plasmodium falciparum and Plasmodium vivax infections.Plasmotec Malaria-3 (XW-P07) performance was evaluated by comparing it with paired blood film microscopy and quantitative real-time PCR (qPCR). Consecutive whole blood samples were derived from one clinic in Mimika as part of routine passive malaria case detection. RDT results were read by two trained technicians and interpreted by consensus. Expert microscopic examination of blood slides was cross-checked by observer-blinded second reader and a third examiner if discordant between examinations. qPCR was used as the ‘gold standard’, followed by microscopy for the outcome/disease variable. Comparison analysis included sensitivity (Sn), specificity (Sp), positive and negative predictive values (PPV & NPV), and other diagnostic screening performance measures for detecting P. falciparum and 16.5% of P. vivax samples examined. Overall RDT performance when compared with microscopy for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:72.9%, Sp:99.1%, PPV:95.4%, NPV:93.4%, Kappa:0.79. Overall RDT performance when compared with qPCR for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:66%, Sp:99.1%, PPV:95.4%, NPV:90.9%, Kappa:0.73.Overall malaria positive samples from qPCR was 42.2% (175/415 samples); and from matching blood slides 40.5% (168/415) of which those infections with relatively low parasite densities ≤100/μl blood was 5.7% of P. falciparum, but lower values regarding sensitivity and negative likelihood ratio for detecting P. vivax, a finding partly associated with greater frequency of lower density P. vivax infections compared to P. falciparum in this study. In particular, the negative likelihood ratio (>0.1) for P. vivax detection indicates RDT lacked sufficient discriminating exclusion power falling below general acceptance criteria.Plasmotec Malaria-3 test showed good overall performance scores in precision for detecting  Malaria remains a serious public health problem in Indonesia. Approximately 45% of the populations across the archipelago are at risk for malaria infection , with 41Plasmodium parasites are present in Papua, with Plasmodium falciparum and Plasmodium vivax as the most common infections, followed with far less frequency by Plasmodium ovale and Plasmodium malariae. In high transmission areas, mixed species infections are not uncommon. Mimika Regency, covering a vast area of the southern part of Papua Province, had an API of 531.3‰ in 2012 with an overall P. falciparum/P. vivax case infection ratio of 1.3:1 [While several areas have witnessed significant reductions in malaria prevalence, other regions  have remained problematic for performing effective control strategies because of remoteness, lack of adequate resources and sufficient budgets to combat both vectors and parasites. Papua and West Papua provinces of Indonesia, located on the western half of the island of New Guinea, have the highest malaria burdens in the country, with recent province-wide APIs of 60.6‰ and 52.3‰, respectively [of 1.3:1 . Other rThe World Health Organization (WHO) recommends all clinically suspected malaria cases have parasitological confirmed diagnosis, using either a malaria-specific rapid diagnostic test (RDT) or direct visualization of parasites using microscopy, before treatment . For morAmong other criteria, a useful and effective RDT must have sufficiently high sensitivity to be able to accurately identify as many ‘true’ malaria cases as possible, especially in areas where reliable microscopy is not available or used infrequently. This is particularly important so that infections can be effectively and specifically treated based on parasite species. The screening sensitivity of an RDT can be influenced by the epidemiological characteristics and infection dynamics in the target population. As parasite antigen concentrations in the blood and parasitaemia levels can vary due to multiple host and parasite factors, the performance level of an RDT can be similarly affected depending on the malaria-endemic population involved in the product assessment .Establishing test performance accuracy for disease screening should be considered before investing and committing to a specific product. Although certain tests may be relatively inexpensive and easy to use, they must be valid and provide consistent reproducible results. Test accuracy describes the diagnostic strength of the association between the predictor variable (RDT result) and outcome variable (disease) as measured against a ‘gold standard’ test. The most common and useful complementary measures for evaluating a test are sensitivity (the proportion of true diseased persons in a population who are test positive – the true positive rate), specificity (the proportion of truly non-diseased persons who are so identified by the test – the true negative rate), and the positive and negative predictive values . However, unlike the previous measures, another set of complementary statistics, the positive and negative likelihood ratios, are less likely to be affected by background disease prevalence , and areThe XW-P07 has not been listed, or data presented, as formally evaluated by the World Health Organization’s Malaria RDT Product Testing Program in Rounds 1 through 5 (2008–2013) ,16; therP. falciparum/P. vivax ratio = 1.3:1) and 38%  respectively, indicating no significant changes in SPR while having a notable shift between the two years in the proportion of parasite species in favor of P. vivax. The clinic is equipped with a modern laboratory and full-time expert laboratory staff and malaria microscopists with access to detailed medical records on all malaria cases reported. The inclusion criteria for study samples included one or more of the following: 1) passive patient visit to clinic through either the emergency or outpatient department; 2) patient presenting with suspected malaria infection that included one or more of these following symptoms: fever, chills, headache, nausea, vomiting, abdominal pain and/or diarrhea, myalgia; 3) patient having lived in a malaria-endemic area or visiting one in the past four weeks; and 4) the patient may have experienced malaria before [Data collection was conducted in Kuala Kencana, a township near Timika, in April and May 2014. The health facility was selected as the XW-P07 was in current use for routine malaria diagnosis with microscopy. The annual slide positive rate (SPR) at the Kuala Kencana Clinic for 2012 and 2013 was 40% (a before .2) EDTA  from each suspected malaria case. Blood was used to prepare an RDT with a matched thick and thin blood slide for microscopy, and qPCR testing. Approximately 1 ml of blood was transferred into a PCR sample tube and immediately stored at −20°C. The RDT and first blood slide reading were recorded immediately. A second slide reading and PCR test were conducted within a week of the initial blood draw. Results based on the RDT and first blood slide reading were provided to the patient within an hour of sampling and if found infected, malaria treatment provided (artemisinin-based combination drugs and primaquine as appropriate to specific infection).Following informed consent by patient adults or minors , using sterile procedures, a single 3 ml venous blood sample was collected in a glass Vacutainer® tube containing 5.4 mg dipotassium (KP. vivax), the Mimika 2012 estimated malaria incidence of 0.53 infections per person-year in the resident population, and an absolute precision value of 0.05. Those cases with incomplete information regarding symptom presentations or laboratory findings, and infections with only P. ovale or P. malariae parasites based on qPCR were excluded from the final analysis.The study sampling continued in a consecutive manner until the target sample size was obtained. The minimum sample size (n = 400) for accurately estimating sensitivity and specificity  was baseP. falciparum-specific histidine rich protein-2 , P. vivax-specific Plasmodium lactate dehydrogenase (P. vivax-pLDH), and pan-specific pLDH for all Plasmodium species .The XW-P07 is a rapid, qualitative immunoassay, lateral flow cassette device that uses 5 μl whole blood for the detection of P. vivax, while the RDT showed all four bands reactive, the interpretive result of the RDT would be P. falciparum false positive, P. vivax true positive, P. ovale and/or P. malariae as equivocal and undetermined. Given the low prevalence of the latter two species, this study restricted performance analysis on P. falciparum and P. vivax infections only.The RDT  was used according to the manufacturer’s instructions provided in the product insert. Reading and interpretation of test results were done by two trained technicians and interpreted by consensus within the specified 15–30 min test window. No test was scored beyond the 30 min limit. Any test that failed to produce a control band was considered invalid and the test repeated. Test interpretation criteria are provided in Figure Microscopy and qPCR were used as the reference standards in this study. With each RDT, a matching thick and thin blood film (one slide per sample) was prepared and stained with Giemsa solution (1:10 dilution for approximately 20 min). Slides were examined using a compound light microscope under x1,000 oil-immersion magnification by a qualified laboratory technician in the clinic. All blood films were examined for a minimum of 100 high-magnification fields before being recorded as either negative for malaria parasites and for the detection of low density mixed species infections. The parasite densities were estimated for each sample counted separately by parasite species. Parasite numbers were reported per 200 white blood cells (WBC) to estimate parasite density per μl of blood, assuming a standard mean WBC count of 8,000/μl blood. Samples were also categorized into one of four groups based on overall parasite density .All slides were subsequently examined by an independent expert malaria microscopist as an observer-blinded cross-check and confirmation of first examination. A third, observer-blinded expert microscopist was used for slides where there was either discordant or significant discrepancies in findings (infection density or parasite species discrepancies) between the first and second examiners. In such cases, the results for the third examination were regarded as final. All microscopists in this study were experienced in malaria slide preparation and diagnosis, and certified through quality assurance procedures.Plasmodium species.Quantitative real-time PCR (qPCR) amplification was performed using a Rotor-Gene Q . Parasite DNA (if present) was extracted from whole blood using Qiagen DNeasy Blood and Tissue Kit , according to the manufacturer’s instructions. Extracted DNA was eluted in a final volume of 100 μl for the first elution and 50 μl for the second. DNA concentration and purity were measured using a nanophotometer  and then immediately stored at −20°C. Amplification was carried out in a 20 μl reaction volume, containing 10 μl SYBR Green , 0.3 mM of each primer, 30 ng DNA template, and nuclease-free water . A pair of primers was used to amplify the 18S rRNA gene sequences: PL1473F18 (5′TA CGA ACG AGA TCT TAA-3′) and PL1679R18 (5′GTT CCT CTA AGA AGC TTT-3′) for the four Plasmodium species differentiation was achieved using melting curve analysis. Above steps and subsequent data interpretation for qPCR followed Mangold et al. [P. falciparum 75.5-77.5°C, P. vivax 79.0-81.0°C, P. ovale 77.5-79.0°C, and P. malariae 73.5-75.5°C [The conditions for the qPCR consisted of initial denaturation at 95°C for 10 minutes, 40 cycles amplification at 95°C for 10 seconds, 50°C for 5 seconds, and 72°C for 20 seconds each, with fluorescence acquisition at the end of each extension step. The melt program consisted of 2 minutes at 95°C and 68°C each, followed by a stepwise temperature increase of 0.2°C/s until 90°C, with fluorescence acquisition preformed at each temperature transition. d et al.  for dete5-75.5°C .p-value of <0.05.Data was analyzed using Stata 12.0 software . RDT performance was calculated compared with matched microscopy and qPCR results with 95% confidence intervals (CI) for the following values: sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and Kappa score. RDT sensitivity was also calculated based on parasite density. Odds ratios and confidence intervals were calculated for correlation between low parasite counts (≤100/μl blood) and RDT false negative results based on matching microscopy and qPCR results; as well as between low parasite counts and body temperature at the time of blood sampling. For statistical inference, the Fisher’s exact test was used for interpretation of false negative RDT results between malaria infections with densities above and below 100 parasites/μl blood with significance set at All observations and reporting contained herein are based on informed consent by volunteers  prior to physical and laboratory examinations. A unique medical record number was used as an identifier of each sample and all information was kept confidential throughout the study. Ethical review and clearance was obtained from the Medical and Health Research Ethics Committee (Reference no. KE/FK/320/EC), Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia.P. malariae infection, and four lacked matching qPCR testing; therefore, only 415 samples were included in the study and analysis. No adverse events were recorded with any patient during the blood draw process or thereafter. In this study, 80% (n = 331) of patients reported ‘fever’, while only 47.5% (n = 197) actually had a body temperature ≥37.5°C at the time of blood sampling. Baseline characteristics of study population are shown in Tables Between 10 April and 14 May 2014, 428 suspected malaria patients attending a local clinic in Kuala Kencana, Mimika, voluntarily provided blood samples  for P. vivax, and 1.2% (5/415) with mixed P. falciparum/P. vivax infections. Microscopy produced a total SPR of 40.5% (168/415); with P. falciparum slightly more common than P. vivax. To determine RDT performance using matching microscopy, positive slides were divided between P. falciparum and P. vivax; and those with mixed species infections were counted twice, separately as P. falciparum and P. vivax. Composite determinations from first, second, and third slide (if required) examinations were used to calculate final RDT performance. A total of 64 blood slides (15.4%) out of 415 observations required a third reading for arbitration over any discrepant results from the initial first and second examinations. When compared with matched qPCR, the microscopy error rates for the first and second examinations were 12% and 8%, respectively; therefore, the parasite density determinations from the second reading were used for the final analysis. Overall, compared to the RDT, microscopy resulted in an increased detection of infection, with Kappa scores (inter-procedure agreement) between composite microscopy and qPCR of 0.97 for P. falciparum and 0.92 for P. vivax.The RDT cassettes used in this study showed clear line intensities and had good background clearing after addition of the buffer solution; however, 3 out 415 tests failed to produce a ‘control’ line and thus had to be repeated with a new cassette using the same blood sample. Laboratory test findings are shown in Table P. falciparum was Sn 92%, Sp 96.6%, PPV 88%, NPV 97.8%, with Kappa score 0.87; and for P. vivax: Sn 72.9%, Sp 99.1%, PPV 95.4%, NPV 93.4%, with Kappa score 0.79. Final RDT performance compared with qPCR for detecting P. falciparum was Sn 92%, Sp 96.6%, PPV 88%, NPV 97.8%, with Kappa score 0.87; and for P. vivax: Sn 66%, Sp 99.1%, PPV 95.4%, NPV 90.9%, with Kappa score 0.73.Table P. falciparum or P. vivax was different depending on parasite density as measured from peripheral blood  and P. vivax (p <0.001). The RDT achieved 100% sensitivity at high parasite densities ≥4,800 parasites/μl blood for P. falciparum and at a lower threshold of ≥640 parasites/μl for P. vivax. Parasite densities at or below 100/μl increased the probability of the RDT producing a false negative finding; thus, impacting the overall malaria detection performance of the test. When compared with microscopy, the odds ratio with low parasite densities resulting in RDT false negative findings compared to densities >100/μl was 10.4 (p = 0.05) for P. falciparum, and 18 (p <0.001) for P. vivax. Similarly, when compared with qPCR, the odds ratio for false negative results at low parasite densities was 6.5 (p = 0.2) for P. falciparum and 16.4 (p <0.001) for P. vivax  to 72.8% (61.8-82.1%), along with adjusted Sp 99.1% (97.3-99.8%), PPV 95.2% (86.5-99%), NPV 93.5% (90.3-95.9%), PLR 77.7 (25–242), NLR 0.3 (0.2-0.4), and Kappa score 0.79. Similarly, when removing the lower density P. falciparum infections, the RDT performance compared with qPCR remained nearly the same, increasing sensitivity only slightly from 92% (84.3-96.7%) to 92.9% (85.1-97.3%), followed by Sp 96.6% (94–98.3%), PPV 87.6% (79–93.7%), NPV 98.1% (96–99.3%), PLR 27.5 (15.3-49.3), NLR 0.07 (0.03-0.16), and Kappa score 0.88.RDT sensitivity for detecting od Table . This diP. falciparum and P. vivax infections. The odds ratio of having a normal body temperature (<37.5°C) and lower parasite densities (≤100/μl) was 12  for P. falciparum, and 5.5  for P. vivax.Normal body temperature recorded at the time of blood sampling was significantly associated with lower parasite density in both Definitive diagnosis and confirmation of disease status is the cornerstone of evidence-based medicine. Many malaria-endemic areas of the world lack sufficient capacity and resources to accurately diagnose the infection and where reliance on the presentation of clinical signs and symptoms alone are inadequate and imprecise indicators of specific disease. The WHO recommendations for procurement of malaria RDTs are currently based on the attainment of a set of minimum performance criteria  in the WHO Malaria RDT Product Testing Program  and recoP. falciparum infections when compared with microscopy and qPCR. On the other hand, for P. vivax, the RDT showed the same specificity >90% and PLR >10; while the overall sensitivity was much lower when compared with microscopy and qPCR (73% and 66% respectively), with NLR >0.1 and Kappa score slightly <0.8. The test specificity for both parasite species easily met the WHO recommended minimum performance criteria of >90%  detection at 200 parasite/μl [The RDT cassettes were easy to use and provided distinct, easy to interpret test lines. In only 3 tests did the RDT fail to show a control line, producing an ‘invalid rate’ of 0.72%, well within the acceptable limit (<5%) established by WHO . The RDTP. falciparum and P. vivax [P. falciparum; and Sn 84% (75-92%), Sp 100%, PPV 100%, NPV 96% (94-98%) for P. vivax based on comparisons with matched microscopy [An unpublished product evaluation by the XW-P07 manufacturer reported findings from 251 samples compared to microscopy SPR 16.3%) showing Sn and Sp of 100% for both P. vivax . A studyn 84% 75-2%, Sp 10croscopy . Any dir.3% showicroscopy ,23. In tcroscopy ,25.P. ovale and P. malariae were not specifically included in the panel assay ; however, the majority of these infections in Papua are often coincident (mixed) with other plasmodial species. In this study, three P. malariae infections were either mixed with P. falciparum (two cases) or P. vivax (one case). Lastly, a set minimum of 100 high magnification fields were used for blood examination of films which possibly contributed to the relatively high (15.4%) discordant results between the first 2 microscopists. The detection accuracy would have likely been enhanced had each reader examined a minimum of 200 fields.This product evaluation was not without some underlying limitations. Firstly, this study only used RDT cassettes from a single lot number; thus, possible inter-lot variability in performance between product production periods was not assessed. This study was conducted in a reasonably controlled setting with trained laboratory technicians; therefore, extrapolation of findings to areas under more demanding environmental conditions and clinical expertise  should be made with caution. Albeit relatively uncommon infections, P. falciparum infections have been regarded as the only human plasmodial species responsible for common causes of severe morbidity and mortality; however, that general perception has changed and been challenged by a number of recent studies showing that P. vivax can produce substantially greater morbidity manifesting as severe infections, causing acute and chronic anaemia, and ultimately resulting in death [P. vivax infections, that accurate diagnosis of this species becomes an even greater priority to ensure early and effective treatment. An RDT that lacks the necessary sensitivity for detecting P. vivax (>90% preferred) and poor exclusion power (a negative diagnostic likelihood ratio of >0.1) to adequately rule out infection presents a distinct disadvantage to both patient and health care provider in areas where the parasite is common.Various host and parasite factors are possible reasons for varying RDT performance values between different malaria endemic populations ,26-31. Gin death -36. It iP. vivax than for P. falciparum, even when excluding low density infections below 100 parasites per μl/peripheral blood. Published WHO product evaluation on different malaria RDT products submitted for testing has shown that targeting P. falciparum HRP2 has the highest and most consistent detection rate [P. falciparum, as the pLDH capture system is not affected by a possible ‘prozone’ effect, parasite antigen polymorphisms or gene deletions [P. vivax and P. malariae were detected to a far lesser extent  than P. falciparum [P. vivax detection, separate aldolase and pLDH targeting RDTs have been shown to perform differently depending on the samples tested, increasing the risk of misdiagnosis and therefore suggesting that test sensitivity for P. vivax can be improved by using a combination of both aldolase and pLDH in a single RDT [P. vivax, thus one possible explanation for the inferior sensitivity seen. The XW-P07 showed considerably lower sensitivity and a poor negative likelihood ratio, both measures falling below general acceptance criteria for detecting P. vivax infections. This is especially problematic for infections presenting with lower parasitaemia; thus, RDT results with such limitations must be interpreted with caution if the test is the sole method of diagnosis, particularly given the importance and high prevalence of vivax malaria in the Mimika area.This study showed that XW-P07 has a significantly lower detection rate for ion rate . Howevereletions -41. In Mlciparum . For P. ngle RDT . The RDTP. ovale and P. malariae is relatively low, a three-band RDT with better overall performance and able to differentiate P. falciparum and P.vivax, or possibly a P. falciparum/Pan-malaria test, may be a better format. Whenever possible, it would also be prudent and strongly advised to back-up all RDT diagnosis, regardless of RDT performance rating, with matched blood films and proficient microscopic examination.Furthermore, as a four-band RDT with one control line and three test lines Figure , the XW-Plasmodium infections when compared with microscopy, taking into account the high number of sub-microscopic infections in a relatively low transmission area [A recent study in Flores, Indonesia, found that qPCR revealed almost eight times more ion area . Moleculion area -48 that P. vivax infection, from a cost-effectiveness point of view, standard expert microscopy should continue to be used as the reference gold standard for malaria diagnosis despite the likelihood of missing some low density parasitemia and sub-microscopic infections. With skilled technicians and experienced health care providers, microscopy has more than sufficient, if not excellent, diagnostic capacity in most instances. All public-funded health facilities and private clinics in the Mimika Regency must either begin, or ensure the continuation of microscopy, as their primary means of malaria diagnosis. Microscopy should be used for routine confirmation of all RDTs performed in clinical settings whenever possible. Lastly, in many circumstances without external funding to support procurement and routine access to RDTs, the sustainability for maintaining these devices in all clinics is vulnerable to supply disruptions without adequate safeguards and reliable logistical support in place. The use of microscopy, even absent the aid of electricity in the most remote areas, is a sustainable approach and within the supportive framework of the Indonesian health care system. Moreover, providing basic electrical power using efficient solar capture devices and battery storage for a microscope and basic clinical equipment is well within the means of most local health budgets. The availability and routine use of microscopy also enables a facility to diagnose other important endemic diseases  and hematological conditions and indicators without the need of more sophisticated and costly techniques and medical instrumentation.In areas with high prevalence of As malaria represents one of the leading and arguably most important health concern in the Mimika area, various health program stakeholders should continue or adopt a policy of investment in the procurement and maintenance of quality microscopes, the recruitment of additional trained laboratory technicians, and organize regular refresher training on microscopy and RDT proficiency. Nevertheless, for logistical and operational rationale, the use of RDTs will continue to play a valuable and important role in remote areas for first line diagnosis of malaria. Health care facilities in remote locations with limited laboratory capacity should continue to use high quality malaria RDTs combined with evidence from good clinical observations until microscopy can be introduced.P. falciparum infections. For P. vivax infections, the test provided acceptable specificity and positive likelihood ratio, but with lower sensitivity, negative likelihood ratio (below test acceptance criteria), and inter-procedure agreement comparison. Low parasite blood densities (≤100 parasites/μl), especially with P. vivax, increased the probability of false negative test results. Normal body temperature was strongly associated with the incident of lower parasitaemia, further complicating diagnosis. The RDT meets WHO minimum performance criteria for test specificity (>90%) and invalid rate (<5%). As vivax malaria is a very common and important parasitic infection in the population studied, all primary health centers in the Mimika Regency should begin, or continue using, expert-level and quality assured standard microscopy in a sustainable manner for greater accuracy in malaria detection.As a point-of-care device, the XW-P07 provided good test sensitivity, specificity, positive and negative predictive values and likelihood ratios, as well as inter-procedure agreement for detecting


Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life.Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia.An assay based on 14 A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults.The multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection.The online version of this article (doi:10.1186/s12936-015-0868-z) contains supplementary material, which is available to authorized users. Globally an estimated 3.4 billion people in 107 malaria-endemic countries are at risk of malaria, of which 1.2 billion at high risk [Plasmodium Abs can persist for months after infection. Therefore, these Abs have been suggested as indicators of malaria transmission [Detection of malaria-infected persons by microscopy, rapid diagnostic tests (RDTs) and even PCR lacks sensitivity because of low numbers of positive samples , represesmission –8, and asmission . Previousmission , 10–12.Since the 1960s, serological markers detected by indirect immunofluorescence antibody tests (IFAT) were used to assess malaria transmission intensity and reductions in transmission . This haIn this context, the general objective of this study was to implement an existing assay based on the Luminex technology for detection of Abs against malaria parasites in blood samples from Ratanakiri Province, Cambodia. This is the first and most extensive multiplex assay in malaria serology executed in the Southeast Asian region, including 20 Ags (recombinant proteins and peptides) directed against different specific malaria parasites. Furthermore, this study includes a detailed analysis on the stability of coated beads over time and the reproducibility of the beads coupling and immunoassay.Plasmodium falciparum- and two Plasmodium vivax-infected patients from Ratanakiri Province in Cambodia. Dilutions of this positive control pool were prepared at 1:100, 1:400 and 1:1600 in PBS-CR . These dilutions were used to assess the performance of the assay and as positive control samples in the immunoassay applied on field blood samples. The latter were collected in Ratanakiri Province, Cambodia, on filter papers through finger prick, during April–May 2012 . OuP. falciparum representing different life stages of the parasite was based on the work of Ambrosino et al. [Anopheles gambiae saliva protein [P. vivax and Plasmodium malariae were included in the assay, as well as specific recombinant proteins for P. falciparum and P. vivax  was coupled to an additional set of beads to serve as a background control [Covalent coupling of paramagnetic beads  was carried out as described by Ambrosino et al.  and the  control , 23.+-conjugated AffiniPure F(ab′)2 fragment of goat anti-human IgG, Jackson Immuno Research Laboratories) at a dilution of 1:500 was added [® system. A minimum of 400 beads per spectral address were analysed and results were expressed as the median fluorescence intensity (MFI) [The immunoassay was carried out as described previously , 24, 25,ty (MFI) .For performance assessment, two bead concentrations of 1000 and 2000 beads/Ag/well were compared. The assays performed in monoplex (each Ag separately) and multiplex  were compared. Both assays were carried out in triplicate on the positive control pool dilutions  as previously described , 25.Stability of the Ag coupled beads was assessed by performing a multiplex assay directly after coupling the beads and after storage at 4 °C during two, three, four, eight, and 16 weeks.Reproducibility of the beads coupling was tested by coupling all Ags at three different time points, followed by an immunoassay on positive control pool dilutions (inter-coupling variability) . Inter- ® system.The immunoassay was applied on 2000 field blood samples. Therefore, the microsphere working mixture was prepared at a final concentration of 1000 beads/Ag/well. All field blood spots were analysed in duplicate on separate plates. In each plate, the positive control pool dilutions , negative control serum (1:200) and blanco (PBS-CR) were analysed in duplicate. In total, 50 plates were analysed and read by the MAGPIXBSA) to the median value of the Ag-coupled beads (MFIAg), defined by: ΔMFI = MFIAg − MFIBSA [All data were incorporated and analysed with R software package version 3.1.0. . Results− MFIBSA .Ags of which the positive control sera did not give a clear signal (ΔMFI < 1500) were excluded from the data-analysis for performance assessment. Non-parametric Spearman’s rank correlation tests were used to analyse the relation between the ΔMFIs for comparing 2000 vs 1000 beads/Ag/well, as well as the monoplex versus the multiplex assay. Correlations were considered significant at p <0.05. Segmented regression models were used for assessing the stability of the beads over time (R package ‘segmented’ ). First To assure the validity of the plates for screening the field blood samples, a quality control was performed on the ΔMFI values of the high positive control pool samples and on the percentage positivity (PP) calculated from the low positive control pool samples. Results were plotted in Levey Jenning Charts  . The meaThe study protocol was reviewed and approved by the Cambodian National Ethics Committee on Health Research , the Institutional Review Board of the Institute of Tropical Medicine Antwerp  and the Ethics Committee of the University of Antwerp . Gatekeepers provided informed written consent for the participation of their village. The survey participant or his/her parents or guardian provided informed written consent for individual participation.No clear ΔMFI signal was found for the positive control sera for Ags SR11.1, PvlikeCSP, PvVK210CSP, PvVK247CSP, PmCSP, SALIV1 and SALIV2 . These antigens were therefore excluded from further analysis in the performance assessment . This stability is lower compared to previous studies, stating that beads are stable for up to 3 or 7 months , 33, excGood reproducibility is an important aspect of an assay as it guarantees that results obtained with the assay performed on different plates and at different time points are comparable to each other. In the experimental set-up used in this study, there is no big difference in reproducibility between and within different plates as shown by similar RSDs. This is in line with other studies that showed accurate and repeatable results according to the interplate, intraplate , 27, 31,Plasmodium-specific peptides , one peptide specific for the An. gambiae saliva protein (SALIV2), and five Plasmodium-specific recombinant proteins  were included in the immunoassay.In the present assay the use of peptides and recombinant proteins was combined. Fourteen After assessment of its performance, the multiplex assay was applied to blood spot samples collected in Ratanakiri Province in Cambodia to detect Ab responses against 20 different Ags. The seroconversion rate (SCR) was then estimated per Ag by constructing age-seroprevalence curves based on a threshold approach. This is currently the most frequently used way of analysing serological data in malaria epidemiology , 7, but Plasmodium Abs.Until the present very limited information was available on the half-life of the Ab responses to the Ags used and on the individual variation in those responses. This is crucial for interpreting the results obtained with this assay. Serological markers with a long half-life can be used for assessing past exposure, while a short half-life will be useful for assessing recent or even present exposure. The present study has obtained information on the Ab half-life estimated from the SRR as modelled in the reverse catalytic model . Based oPlasmodium Ags of three different Plasmodium parasites, which has never been done before. The assay was used to screen almost 2000 field blood spot samples in duplicate and proved to be very time efficient and informative. It was also possible to estimate differences in the force of malaria infection, by looking at the differences in SCR over time between the different Ags. For a better estimation of changes in the force of infection on long-term as well as short-term in areas with low malaria endemicity, more information on the longevity of the Ab responses is needed. The simultaneous detection of several Ags of different parasites offers an opportunity to look for a potential serological marker of very recent malaria transmission, which is the next step of this research. This will prove to be key knowledge for malaria elimination.As many countries engage in malaria elimination it is crucial to improve the understanding of the effectiveness of additional malaria control tools and to be able to identify malaria hotspots for targeted and effective interventions in countries in an advanced elimination phase. With the current malariometric and entomological tools it is challenging to determine the malaria transmission rate in areas where prevalence of infection and incidence is decreasing. The multiplex assay measuring serological markers for malaria transmission was successfully implemented as the first in the Southeast Asian context. Moreover, it proved to be a reliable, fast and useful method for simultaneous detection of Abs against