To design and implement surveys of malaria infection and coverage of malaria control interventions among school children in Kenya in order to contribute towards a nationwide assessment of malaria.Plasmodium infection using rapid diagnostic tests, assessed for anaemia and were interviewed about mosquito net usage, recent history of illness, and socio-economic and household indicators. Children's responses were entered electronically in the school and data transmitted nightly to Nairobi using a mobile phone modem connection. RDT positive results were corrected by microscopy and all results were adjusted for clustering using random effect regression modelling.The country was stratified into distinct malaria transmission zones based on a malaria risk map and 480 schools were visited between October 2008 and March 2010. Surveys were conducted in two phases: an initial opportunistic phase whereby schools were selected for other research purposes; and a second phase whereby schools were purposively selected to provide adequate spatial representation across the country. Consent for participation was based on passive, opt-out consent rather than written, opt-in consent because of the routine, low-risk nature of the survey. All children were diagnosed for 49,975 children in 480 schools were sampled, at an estimated cost of US$ 1,116 per school. The overall prevalence of malaria and anaemia was 4.3% and 14.1%, respectively, and 19.0% of children reported using an insecticide-treated net (ITN). The prevalence of infection showed marked variation across the country, with prevalence being highest in Western and Nyanza provinces, and lowest in Central, North Eastern and Eastern provinces. Nationally, 2.3% of schools had reported ITN use >60%, and low reported ITN use was a particular problem in Western and Nyanza provinces. Few schools reported having malaria health education materials or ongoing malaria control activities.School malaria surveys provide a rapid, cheap and sustainable approach to malaria surveillance which can complement household surveys, and in Kenya, show that large areas of the country do not merit any direct school-based control, but school-based interventions, coupled with strengthened community-based strategies, are warranted in western and coastal Kenya. The results also provide detailed baseline data to inform evaluation of school-based malaria control in Kenya. Plasmodium infection collected among young children and pregnant may not be optimal due to the modifying presence of maternal antibodies and sequestered parasites [The epidemiology of malaria in sub-Saharan Africa (SSA) is in transition, with funding agencies dedicating substantial resources in tackling malaria and national governments making great efforts in increasing access to malaria control interventions. It is essential that this transition is accurately monitored in order to evaluate the impact of interventions but also to allow for better targeting of interventions. A number of studies provide evidence of declining malaria-related mortality and morbidity -6, but tarasites . A cheaparasites .Historically, such school surveys were routinely conducted as part of malaria surveillance in Africa  and todaThe epidemiology of malaria in Kenya has been changing with reported reductions in malaria associated hospital admissions and mortality in children under the age of five years -17. ThesIn 2009, the Government of Kenya launched its National Malaria Strategy (NMS), 2009-2017. This identified the need to tailor malaria control interventions to the local diversity of malaria risk, target specific population sub-groups to achieve effective and sustainable control, and strengthen the surveillance, monitoring and evaluation systems  thereby The Kenya NMS also included the proposal to undertake school malaria surveys to monitor trends of malaria transmission in the context of increasing intervention coverage. Such school surveys have a historical precedent in Kenya, dating back to the 1950 s, when the Division of Vector Borne Diseases (DVBD) was established and school surveys of malaria, helminths and other parasites were one of its core activities. Routine school survey stopped in the 1980 s due to financial constraints and deteriorating school enrolment rates .The renewed potential for school malaria surveys builds on the increased funding for malaria surveillance but also recent improvements in primary school enrolment in Kenya. There are a total of 19,177 government primary schools, the majority (98.5%) of which are day schools with pupils living at home. Primary education in Kenya begins at the age of 6 or 7 years old after completion of a year of nursery school and includes eight years of schooling. The Kenyan school year runs from January to December. In the 1980 s and 1990 s, there was a growth of privately owned schools while the government schools deteriorated. In 2003, the Government of Kenya re-introduced free primary education, resulting in a marked increase in school enrolment. However, parents must pay fees for uniforms and other items and some poorer children still do not attend primary school. The overall net enrolment rate  in Kenya was 91.6% in 2007, but this ranged from 27.5% in North Eastern Province to 97.8% in Nyanza Province .Plasmodium infection in a given district based on 95% confidence limits, 80% power, and a design effect of 2. Based on these assumptions, a minimum sample size of 16 schools per district was calculated as necessary to estimate prevalence of 5%, with 1% precision. An additional 54 schools were sampled as part of an evaluation of school net distribution programmes along the Tana River  per school would be optimal as this was the number of children, which could practically be sampled in a single day. In each school, 11 boys and 11 girls were selected from each of classes 2-6 using computer generated random table numbers. If there were insufficient pupils in these classes, additional pupils were sampled from class 1. Some of the schools visited were small, and this meant that in these schools all children were selected to achieve the target sample size and fewer than 110 children were present and, therefore, examined.Mobile survey teams consisted of a team leader, three laboratory technicians and three interviewers. Technicians were typically from the Division of Vector Borne and Neglected Tropical Diseases (DVBNTD) of Ministry of Public Health and Sanitation, holding diplomas or first degrees and who had extensive experience of conducting school surveys. Interviewers were either from the Ministry of Public Health and Sanitation or Ministry of Education, who had previous survey experience. Each team was supervised from an experienced researcher from the Kenyan Medical Research Institute (KEMRI) or KEMRI-Wellcome Trust Research Programme. These teams were accompanied by an education officer from the district education office who helped teams locate schools.All team members underwent training in all survey procedures and received a field manual outlining the survey purpose and methods  according to national guidelines.Selected children were asked to provide a finger-prick blood sample, which was used to assess dipstick , these ttiMAL-IT  able to Pv Combo  which caIn all 480 schools, thick and thin blood smears were also prepared for each child. Slides were labelled and air-dried horizontally in a carrying case in the field, and stained with 3% Giemsa for 45 minutes at the nearest health facility when the teams returned from the school. Due supply difficulties in securing Hemocue curvettes for all schools, haemoglobin concentration was assessed in 399 schools and estimated to an accuracy of 1 g/L using a portable haemoglobinometer . Children identified as severely anaemic (haemoglobin levels < 70 g/L) were referred to the nearest health facility for treatment according to national guidelines. Transportation costs were provided and an agreement was reached with facilities to waive drug costs.A questionnaire was administered to pupils to obtain data on mosquito net ownership and use and when treated, recent travel history, recent history of illness, key socio-economic variables such as household construction, education level of the child's guardian and ownership of household items such as mobile phones. An additional questionnaire was administered to the head teacher to collect information on ongoing school health activities, including malaria control, as well as information, education and communication (IEC) material on malaria. The pupil and school questionnaire data will be used in future analyses. The geographical location of each school was determined using a Garmin eTrex global positioning system .Blood smears of all RDT-positive children, where available, and an equivalent number of randomly selected blood slides from RDT-negative children were examined by expert microscopy either at the KEMRI-Wellcome Trust laboratory in Kilifi or the KEMRI laboratory in Nairobi. Parasite densities were determined from thick blood smears by counting the number of asexual parasites per 200 white blood cells (or per 500 if the count was less than 10 parasites/200 white cells), assuming a white blood cell count of 8,000/μl. A smear was considered negative after reviewing 100 high-powered fields. Thin blood smears were reviewed for species identification. Two independent microscopists read the slides, with a third microscopist resolving discrepant results . The locations of schools were linked with survey data and mapped using Arc GIS 9.2 .Anaemia was defined as a haemoglobin concentration <130 g/L for male children above 15 years, <120 g/L for children aged 12-14 years and female children above 15 years, <115 g/L for children aged 5-11 years and <110 g/L for children aged less than five years, with adjustment made for altitude of the school . Severe Plasmodium infection and corresponding 95% binomial confidence intervals (CI) were estimated using a zero inflated Poisson (ZIP) model to account for the excess of schools with zero prevalence. The ZIP model was favoured over a standard Poisson model on the basis of the Vuong test [Plasmodium infection except for Nairobi and Rift Valley provinces where a standard Poisson model was used. National and Province-level estimates of anaemia and net use were estimated using generalized linear and latent mixed models (GLLAMM) adjusted for clustering at the school level.Results were adjusted for clustering at the school-level using random effects regression modelling . Specifiong test . The ZIPThe overall financial cost of the survey was estimated from the project accounting system, with costs divided into staff, transport, operating costs and consumables.The study protocol received ethical approval from the Kenya Medical Research Institute and National Ethics Review Committee (numbers 1407 and 1596). Additional approval was provided by the Permanent Secretary's office of the Ministry of Education (MoE) and the Division of Malaria Control, Ministry of Public Health and Sanitation. All national, provincial and district-level health and education authorities were briefed about the survey purpose and selected schools. Official letters of support were prepared by Provincial MoE officers.Head teachers were briefed about the survey and were provided with an information sheet detailing the survey procedures and asking for their permission to have their school involved in the survey. The head teachers were also asked to inform the students, parents and the school committee members about the survey and obtain their approval for the study. Parents/guardians who did not want their children to participate in the study were free to refuse participation. If a parent or guardian chose not to allow their children to participate in the survey, the child's name was removed from the school rolls. On the survey day, the survey team leader informed all children in the school about the sampling and survey procedures, making it clear to their participation was voluntary and that they may opt out of the testing at any time if they choose to. After randomly sampling the students from the classrooms, individual assent was also obtained from the children before samples were collected. Very few children refused to participate in the survey and therefore replacement sampling was not required. Individual written parental consent was not sought since the survey was conducted under the auspices of the Division of Malaria Control, Ministry of Public Health and Sanitation, which has the legal mandate to conduct routine malaria surveillance, and because only routine diagnostic procedures were undertaken.The surveys were carried out in two main phases Figure : first, The average cost of surveying one school was estimated to be US$ 1,116. The largest cost component was staff (32.0%), following by transport (26.9%). Operating costs included laboratory consumables, courier services and hiring of mini-laptop computers and accounted for 24.7% of total costs. Other costs included slide reading (7.2%) and administration costs (9.2%).A total of 49,975 children in 480 schools across Kenya were included in the survey. Table P. falciparum, with the remainder being either P. ovale (0.1%) or P. malariae (0.6%) or mixed infections (2.6%); no P. vivax was detected. Prevalence was significantly higher in children aged 5-9 and 10-14 years old (4.4%) than children older than 15 years , but did not significantly differ between males and females . The prevalence of malaria infection by province is shown in Table The overall prevalence of infection, based on slide-corrected RDT positivity, was 4.3 . The vast majority (96.8%) of these infections were The overall prevalence of anaemia was 14.1% 95% CI: 13.0-15.3%) and the mean haemoglobin concentration was 128.8 g/L (95% CI: 127.9-129.7 g/L). Anaemia was more common among children aged 15 years and above  than 10-14 years  and 5-9 year olds . There was no difference in prevalence of anaemia among males and females (13.3% (95% CI: 12.1-14.7) vs 13.3 (95%CI: 12.0-14.8)). Anaemia varied markedly by school  of children reported having a bed net and 42.1% (95% CI: 40.9-42.8%) reported sleeping under a net the previous night. However, of the children asked about sleeping under an ITN, less than a quarter  reported sleeping under an ITN while 6.4% did not know whether their nets were ITNs or not. The majority  of nets, non-ITNs or ITNs, were reportedly obtained from the health facilities. Reported use of ITNs varied markedly across the country Figure , and wasOverall, 13.5% of children reported a fever on the day of the survey, but of the children that had their axillary temperature measured only 733 (2.4%) children had a temperature >37.5°C. Of these febrile children, only 55 (7.5%) children had a malaria infection. Of the children asked about their absenteeism history , 26.9% of children reported being absent from school due to illness for at least one day in the last two weeks, with the commonest cause of illness being headache (56.7%), whilst 17.0% reported malaria as the cause for absenteeism.A comprehensive school level questionnaire was administered in 344 schools, predominantly in Western, Central, Rift Valley and Nyanza provinces during the second phase of the survey. Of these schools, only 59 (17.2%) reported having had any malaria control activities, such as indoor residual spraying of the school buildings and draining of stagnant water, in the last 12 months and the majority (21) of these schools were located in the malaria high transmission zone. Only, seven schools had malaria IEC materials in at least 1 classroom, 8 had IEC materials in the head teacher's office while 17 schools had IEC booklets in the school library.Plasmodium infection was relatively low at 4.3%, there existed marked variation across the country. This finding is consistent with national level malaria prevalence estimate observed in the Kenya MIS in 2007, where the malaria prevalence by RDTs was 7.6% and 3.4% by microscopy [Reliable, contemporary data are essential prerequisites for the planning and implementation of effective malaria control. Each national programme needs to be tailored to its specific national context, based on a cartographic understanding of malaria transmission intensity and current intervention coverage. The data from the present study show that although the national prevalence of croscopy .The observed distribution of low transmission in most of Nairobi and Central provinces and some parts of the Eastern and Rift Valley provinces and high transmission along the shores of Lake Victoria and the south coast is consistent with a recent model of malaria risk in Kenya . The curPfPR than estimates among under fives and pregnant women [The current survey took advantage of the existing school infrastructure and historical experience in carrying out school surveys in Kenya to achieve a rapid, inexpensive approach to malaria surveillance. The adopted approach has a number of advantages over malaria indicator surveys (MIS) which provide nationally-representative household survey data on coverage of malaria interventions as well as malaria parasitaemia and anaemia among household members most at risk, namely children under five years and pregnant women . First, nt women . FinallySchool malaria surveys are not without their limitations, however (for a review see ), and a A further drawback of school surveys is that they cannot provide complete information on household ownership of insecticide-treated mosquito nets and their use by children under five years of age and pregnant women, or on the use of the intermittent preventive treatment during pregnancy and the type and timing of treatment of fever in children under five years of age. However, a study in Uganda found that reports by schoolchildren on household net ownership provide a rapid method to collect reliable coverage data at the community level .There are also several practical features of the present survey worth highlighting. First, the survey used modern technology to achieve a more cost efficient approach to data collection. In particular, data captured were achieved using netbook computers with customized data entry screens. Electronic data capture systems, mainly based on the use of personal digital assistants (PDAs), are shown to be acceptable and reduce data entry errors considerably -42. ExpeSecond, consent for the survey was based on a passive, opt-out method of parental permission. This approach is considered to be an ethical and practical way of informing participants in low-risk studies and interventions , and hasThird, malaria parasitaemia was ascertained using a two-stage approach of malaria RDTs and blood slides. Importantly, this approach enabled appropriate treatment of clinical malaria in schools on the day of the survey, but also allowed assessment of the reliability of RDTs at a later stage. A drawback of using RDTs is that they can result in false positives, especially those RDTs that detect the histidine-rich protein-2 (HRP-2) antigen -51, leadIn conclusion, the current study describes recent experiences of school malaria surveys in Kenya and highlights the potential of school surveys as a complementary approach for malaria surveillance to MIS. The adopted survey approaches evolved over time and important practical lessons were learnt which can inform the conduct of future school malaria surveys. Notwithstanding, there remain several issues requiring further investigation, including the representativeness of schools, the reliability of school children's reports of ITN ownership and use, the reliability of RDTs among school children. It is hope that addressing these issues will provide clearer direction on the role, in Kenya and elsewhere, of schools in an integrated national malaria surveillance system, which also includes household surveys and health facility reporting.The authors declare that they have no competing interests.CWG participated in the data collection, analysis and developed the draft manuscript. PNK, JK and MM were responsible for fieldwork supervision and contributed to the final manuscript. EJ, RWS and AN were responsible for the study design, interpretation and scientific guidance. SB was responsible for the overall project management, study design, scientific guidance and writing of the manuscript. All authors read and approved the final manuscript.Field manual for school malaria surveys, Kenya. The field manual developed for the national school malaria survey and used the field teams.Click here for fileMicroscopy results flowchart. A flow chart showing the numbers of slides examined and the microscopy results, including discrepant results, in the school malaria surveys in Kenya, 2008-2010.Click here for file


Diligent and correct laboratory diagnosis and up-front identification of risk factors for progression to severe disease are the basis for optimal management of malaria.® instrument.Febrile children presenting to the Medical Research Unit at the Albert Schweitzer Hospital (HAS) in Lambaréné, Gabon, were assessed for malaria. Giemsa-stained thick films for qualitative and quantitative diagnosis and enumeration of malaria pigment, or haemozoin (Hz)-containing leukocytes (PCL) were performed, and full blood counts (FBC) were generated with a Cell Dyn 3000® instrument detected significantly more patients with PCL (p < 0.01). Both PCM and PCN numbers were higher in severe versus non-severe malaria yet reached statistical significance only for PCN . Of note was the presence of another, so far ill-defined pigment-containing group of phagocytic cells, identified by laser-flow cytometry as lymphocyte-like gated events, and predominantly found in children with malaria-associated anaemia.Compared to standard light microscopy of Giemsa-stained thick films, diagnosis by platelet count only, by malaria pigment-containing monocytes (PCM) only, or by pigment-containing granulocytes (PCN) only yielded sensitivities/specificities of 92%/93%; 96%/96%; and 85%/96%, respectively. The platelet count was significantly lower in children with malaria compared to those without (p < 0.001), and values showed little overlap between groups. Compared to microscopy, scatter flow cytometry as applied in the Cell-Dyn 3000In the age group examined in the Lambaréné area, platelets are an excellent adjuvant tool to diagnose malaria. Pigment-containing leukocytes (PCL) are more readily detected by automated scatter flow cytometry than by microscopy. Automated Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria. However, this is possibly because of the instrument detection algorithm was not geared towards this task, and data lost during processing; and thus adjusting the instrument's algorithm may allow to establish a meaningful cut-off value. Malaria is known to cause several changes in full blood count (FBC) parameters, of which the most prominent are anaemia and thrombocytopaenia . However®, Abbott, Santa Clara, California) allows the automated detection of Hz-containing cells. The instrument has been shown to be useful in the diagnosis of malaria [®) has been installed in a remote malaria-endemic area in Central Africa . The objective of this study was to investigate the haematological parameters in children with malaria as well as the importance and potential usefulness of the automated detection of PCL.Haemozoin (Hz), the end product of the detoxification of haem, is phagozytosed by monocytes and granulocytes. Some studies have reported a link between Hz and dyserythopoiesis and anaemia -6. Using malaria -15. One ® (CD3000) instrument .The study took place at the HAS, Lambaréné, Gabon, in 2003 and 2004. The area is mainly tropical rainforest with holoendemic malaria ,17. Bloo® instruments generate a five-part differential white blood cell count, using scatter flow cytometric principles based on the manufacturer's patented multi-angle-polarized-scatter-separation (M.A.P.S.S.®) [® instruments and the transformation of the image, the maximum resolution of the analysed bitmap image was 140 × 140 pixels, rather than the 256 × 256 channels.The Cell-DynP.S.S.®)  as descrP.S.S.®) ,23. The P.S.S.®) . As the P.S.S.®) . Due to In the granularity/lobularity plot, two different areas were defined to classify monocytes as pigment-containing monocytes (PCM), shown in Figure A higher degree of depolarization  may be caused by either a higher amount of phagozytosed Hz or larger Hz crystals. Thus, Hz-containing monocytes and granulocytes with higher depolarization values contain possibly more Hz and thus could be correlated with severity. To test this hypothesis, two "severity indices" with the intention to weigh the degree of depolarization were constructed: (i) the sum of the y-axis values of Hz-containing monocytes and granulocytes and (ii) the sum of the y-axis values after mathematical transformations . These indices were then used to determine a meaningful cut-off value that would allow to distinguish between severe and non-severe malaria.During the analysis of the granularity/lobularity plot it was noted that blue coloured events, that usually represent lymphocytes, showed depolarization Figure . A horizData was analysed using SPSS 14.0 software. The student t-test for unpaired samples with unequal variance was used to test quantitative data and the chi-square test to test qualitative data.During the study period a total of 368 children  were included, of which 152 had falciparum malaria and 216 children were either healthy or had other diseases. More than 88% of malaria occurred in children older than one year, while 99.1% of all children without malaria were below one year of age. The children in the malaria group were significantly older than the children without malaria . Of the 152 children with malaria, 48 had severe malaria, classified as 15 cases with severe anaemia, 13 cases with hyperparasitaemia, three cases with hypoglycaemia and 17 children with cerebral malaria. In three cases, children had both cerebral malaria and severe anaemia, which were included in the cerebral malaria group. The mean age of children with severe malaria (3.8 years) and non-severe malaria (3.4 years) was not significantly different (p = 0.27).The FBC results are shown in Table Sixtyfive percent of children with malaria had < 150,000 thrombocytes/μL. Given the little overlap of the platelet counts between the malaria and non-malaria groups we computed a Receiver-Operator-Characteristic (ROC) curve Figure . The ROC® instrument. Concerning Hz-containing monocytes, no significant difference was observed between the two defining lines  the sensitivity was 97% and the specificity of 93%.The other parameters that were analysed for their usefulness to diagnose malaria were the presence of Hz-containing leukocytes  detected by the Cell-Dyn 3000s Figure  yielded s Figure . When th® instrument which showed that the analyser detected significantly more patients harbouring PCL (p < 0.01) (Table The detection of PCL by microscopy was compared with results from the Cell-Dyn1) Table . This wa1) Table . FurtherThe results for the automated detection of Hz-containing leukocytes in severe and non-severe malaria are shown in Figure The number of Hz-containing leukocytes showed a wide distribution with an overlap between the non-severe and severe malaria groups Figure . This diConcerning the presence of depolarizing blue coloured events (lymphocytes), they were found in 77 of 152 children with malaria (51%). The mean number of these events in the 77 children was 4.2 (range: 3 – 24). Although they appeared to be evenly distributed between the non-severe and severe malaria groups, they seemed to be more frequent in children with anaemia, with 11/14 FBC results yielding depolarizing blue coloured events.This study underpins the value of thrombocytopenia for malaria in the investigated age groups and in the setting of a Central African rainforest area and provides more insight into the usefulness of PCL analysis as a prognostic marker in malaria. It furthers the knowledge about the utilization of scatter flow cytometry for this purpose, which may be applied in future in low-cost, robust devices needed for other applications such as CD4+ cell identification.Concerning the FBC parameters, most of the values showed a significant difference between the malaria and non-malaria groups. As the children with malaria were significantly older (mean: 3.7 years) than the ones who had no malaria (mean: 0.6 years), these results may in great part simply reflect the different reference values in both age groups . This efA notable observation was not only the significantly different platelet count between the malaria and non-malaria groups, but even more so, that almost no overlap of values was observed. However, it could be argued again that this observation reflects rather the different age in both groups. Interestingly, most textbooks give only one set of reference values for all age groups, which implies the same or very similiar values for children of different ages . On the Given the frequently-used cut-off value of 150,000 platelets/μL, this cut-off would have identified 66% of malarious children, while almost none of the children without malaria had such a value (specificity 99%). These values are in keeping with other reports and confirm the usefulness of a platelet count as marker of acute malaria. In fact, ROC curve analysis indicates that this parameter showed a high degree of accuracy Figure ; and eve® instrument has a large potential for the rapid and accurate detection and enumeration of PCL as it identified more PCL-positive samples than microscopy , or were lost during the "screen shot" transformation that resulted in a decrease from 256 × 256 channels to a 140 × 140 pixel image. In fact, the number of coloured pixels that represent leukocytes were analysed and compared to the total number of leukocytes in a subset of FBC results. It was found that the granularity/lobularity plot  events that are highly depolarising. However, lymphocytes do not phagozytose and consequently, these cells cannot be lymphocytes, but must be phagocytic cells with cell characteristics that are similar to lymphocytes  are more readily detected by automated laser-flowcytometry than by microscopy. Mechanical Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria; possibly because of a detection algorithm not geared towards this task, and data lost during processing. Newly described 'lymphocyte-like' gated events warrant further examination and should be analysed by flow cytometry to establish their nature and role in the pathophysiology in malaria.FBC: full blood count; Hb: haemoglobin; Hz: haemozoin or malaria pigment; IPTi-SP: Intermittent Preventive Treatment in infants of malaria with sulfadoxine-pyrimethamine; PCL: pigment-containing leukocyte(s); PCM: pigment-containing monocyte(s); PCN: pigment-containing neutrophil(s) or granulocytes; WBC: white blood cell countTH and MPG designed the study, collected and analysed data and prepared the manuscript.ML helped designing the study, collected and analysed data and contributed to the manuscript's final version.MP and SO helped collecting the data and contributed to the manuscript's final version.BL contributed to study design, data analysis and the manuscript's final version.PGK contributed to the study design and to the manuscript's final version.


Haem oxygenase (HO) enzyme and the pro-inflammatory cytokine tumour necrosis factor (TNF) have been proposed as one of the factors that may play significant role in pathogenicity/severity of malaria infection. HO is the enzyme of the microsomal haem degradation pathway that yields biliverdin, carbon monoxide, and iron. In this study, the association between malaria disease pathogenicity/severity and (GT)Plasmodium falciparum malaria (UM) and 80 cases with Plasmodium vivax malaria (VM), and 77 cases with severe or cerebral malaria (SM) for analysis of genetic polymorphisms of HO-1 and TNF and cadmium levels. These patients consisted of 123 (25.3%) Thai, 243 (50.0%) Burmese and 120 (24.7%) Karen who were present at Mae Sot General Hospital, Mae Sot, Tak Province, Thailand.Blood samples were collected from 329 cases non-severe malaria with acute uncomplicated n repeats of the HO-1 gene in all patients varied between 16 and 39 and categorized to short (S), medium (M) and long (L) GTn repeats. The genotype of (GT)n repeat of HO-1 was found to be significantly different among the three ethnic groups of patients. Significantly higher frequency of S/L genotype was found in Burmese compared with Thai patients, while significantly lower frequencies of S/S and M/L but higher frequency of M/M genotype was observed in Burmese compared with Karen patients. No significant association between HO-1 and TNF polymorphisms including the inducing effect of cadmium and malaria pathogenicity/severity was observed.The number of (GT)n repeat as well as TNF observed in this study may not entirely exclude their possible link with malaria disease pathogenicity/severity. Further study in larger sample size is required.Difference in the expression of HO-1 genotype in different ethnic groups may contribute to different severity of malaria disease. With this limited sample size, the finding of the lack of association between malaria disease pathogenicity/severity genetic polymorphisms of HO-1 (GT) Plasmodium falciparum infection in humans, manifesting as coma that can lead to death. The pathogenesis of cerebral malaria remains controversial, but major factors involved, i.e., cytokines and adhesion molecules are well documented  compared with VM [0.10 (0.01-0.33) μg/L], was observed. In the group with cadmium levels < 0.5 mg/L, the proportions of patients with SM  and UM  were significantly lower than VM , and in addition, the proportion of Thai patients  was significantly lower than Burmese .in vitro including molecular epidemiological investigation from brain lesion [n repeat polymorphism of HO-1 and malaria pathogenicity/severity was reported in Karen ethnic minority group with cerebral malaria and acute uncomplicated falciparum malaria who resided in areas along the Thai-Myanmar border [n alleles was found to be significantly higher in cerebral malaria (SM) than UM. Short (GT)n alleles in the promoter region may therefore represent a genetic risk factor for cerebral malaria as in may directly enhance the transcription of HO-1 in malaria patients, and as a consequence, the products of haem degradation, i.e., CO, iron and bilirubin, may increase in the brain lesion. Furthermore, the influence of the (GT)n repeat polymorphisms in the HO-1 gene promoter was investigated in different cell lines where constructs with lengths of less than 25 repeats (short repeat) showed an increasing HO-1 basal promoter activity [The association between human haem oxygenase-1 (HO-1) and pathogenicity/severity of malaria infection was described in a few studies n lesion ,29,30 ann lesion . Howeverr border . In thatactivity  or transactivity . Increasn repeat polymorphism of HO-1 in different patient populations with different malaria severity, estimation of proper sample size was not possible. There appears to be a significant difference in HO-1 genotypes among the three ethnics. This may explain difference in pathogenicity/severity of malaria infection in various ethnics. The frequencies of short (GT)n alleles (S/S) genotype in the three groups of patients with different disease severity (combined ethnics) were found to be comparable. This lack of association could represent a genuine finding, or else, a low study power due to inadequate sample size (particularly in the SM group). In addition, the major confounding factor ethnics cannot be excluded. In a previous study, a significant association between the short (GT)n alleles (S/S) polymorphism of HO-1 gene promoter and increased risk of cerebral malaria  has been demonstrated in 150 Burmese and Karen patients [n repeat and malaria disease pathogenicity/severity found in this study may not entirely exclude the possible link between HO-1 enzyme activity and malaria disease pathogenicity/severity. Results from recent in vitro study provide insight into the molecular mechanism of induction of HO-1 activity and the link with malaria pathogenicity/severity [2 (PGD2) and 15d-PGJ2 (metabolite of PGD2) consistently induced the expression of HO-1 mRNA and protein [2 is a major prostanoid produced in the brain and is involved in the regulation of sleep and pain responses [The present study, due to limitation of available data on (GT)patients . Neverthseverity ,24. Trea protein ,33. PGD2esponses .P. falciparum but not with P. vivax by inducing HO-1 activity to produce iron. The mechanism(s) by which parasite transport or demolish cadmium are, therefore, important for their survival. It has been reported that P. falciparum efflux cadmium from cells through PFMDR2 protein [vs Burmese) and cadmium levels in group with level of ≤ 0.5 μg/L.Cadmium has been shown to be a potent inducer of HO-1 activity ,35, and  protein . Again,  protein ,43 and rvs 39% in Thai, 40 vs 38% in Burmese and 41 vs 40% in Karen patients, respectively. The association of single SNP of TNF was also found in other studies in African patients. Two studies were performed in Gambian children and results showed that the TNF -238 A allele was found to be associated with susceptibility to severe malarial anaemia but not to CM [P. falciparum parasitaemia. Among low birth weight children, the TNF -308 A allele was also found to be associated with severe anaemia with a trend toward a risk for severe malaria anaemia [Several articles have reported the involvement of cytokines in the histopathology of cerebral malaria ,45. Amonot to CM . On the ot to CM . Further anaemia . In cont anaemia . MoreoveDifference in the expression of HO-1 genotype in different ethnic groups may contribute to different severity of malaria disease. To definitely conclude on the association between malaria pathogenicity/severity and the involvement of genetic polymorphisms of HO-1 (including the inducing effect of cadmum) and TNF, further study with larger sample size estimation based on this preliminary data should be performed with focus on only one ethnic group .The authors declare that they have no competing interests.JK performed the molecular genetic studies, participated in the sequence alignment, interpretation of data, performed the statistical analysis and drafted the manuscript. KH participated in the design of the study. MK participated in the molecular genetic studies and sequence alignment. RR participated in sample collection and the study design and coordination. KN participated in the design of the study, performed the statistical analysis and finalised the manuscript. All authors read and approved the final manuscript.


Plasmodium species, and its association with malaria severity is unknown.Hyponatraemia (serum sodium < 135 mmol/L) has long been recognized as a complication of malaria. However, few studies have been done in non-immune adult populations. It has not been investigated previously how hyponatraemia is distributed among the various Plasmodium species.The aim of this retrospective cohort study was to determine the prevalence of hyponatraemia and its association with malaria severity in a large cohort of patients with imported malaria caused by various All patients that were diagnosed with malaria in the Harbour Hospital and Institute for Tropical Diseases in Rotterdam in the period 1999-2009 and who had available serum sodium on admission were included. Severe malaria was defined according to the modified WHO criteria. Prevalence of hyponatraemia and its association with malaria severity were investigated by univariate comparison, ROC analysis and multivariate logistic regression analysis.A total of 446 patients with malaria  was included. Hyponatraemia was present in 207 patients (46%). Prevalence and severity of hyponatraemia were greatest in severe falciparum malaria , followed by non-severe falciparum malaria , and non-falciparum malaria . Admission serum sodium < 133 mmol/L had a sensitivity of 0.69 and a specificity of 0.76 for predicting severe malaria. Multivariate logistic regression showed that serum sodium < 131 mmol/L was independently associated with severe falciparum malaria . In patients with hyponatraemia, hypovolaemia did not appear to play a significant role in the development of hyponatraemia when prerenal azotaemia and haematocrit were considered as surrogate markers for hypovolaemia.Hyponatraemia is common in imported malaria and is associated with severe falciparum malaria. From a clinical point of view, the predictive power of hyponatraemia for severe malaria is limited. The precise pathophysiological mechanisms of hyponatraemia in malaria require further study. Plasmodium falciparum malaria and was approximately 55% [Plasmodium species.Hyponatraemia has long been recognized as a complication of malaria . The inctely 55% -3. Two stely 55% ,5. HowevThe pathophysiology of hyponatraemia in malaria remains unclear but several studies have suggested that increased secretion of vasopressin, either appropriately or inappropriately, plays an important role ,6-10. AlPlasmodium species and its relationship with malaria severity.The aim of this retrospective cohort study was to investigate the prevalence and severity of hyponatraemia in a large cohort of predominantly adult non-immune travellers with imported malaria caused by various st 1999 and January 1st 2009 were included. Patients were identified by screening the malaria database of the Department of Parasitology. Of all patients thus identified, demographic, clinical and laboratory data were collected using a standardized form, and subsequently stored in an electronic database.The Harbour Hospital is a 161-bed general hospital located in Rotterdam, The Netherlands. It also harbours the Institute of Tropical Diseases, which serves as a national referral centre. All patients diagnosed with malaria in our hospital in the 10-year-period between January 1P. falciparum Histidine-Rich-Protein 2 screening  and conventional microscopy with subsequent specification of the Plasmodium species. Multiple malaria episodes in a single patient were only regarded as separate cases if caused by true re-infection; recrudescent infections of P. falciparum and Plasmodium malariae and relapses of Plasmodium vivax and Plasmodium. ovale were excluded. Patients with a mixed infection of P. falciparum with another Plasmodium species were considered as having P. falciparum malaria.Available laboratory examinations included red and white blood cells, haematocrit, platelets, serum electrolytes, C-reactive protein (CRP), total bilirubin, serum creatinine and urea, liver enzymes, lactate dehydrogenase (LDH), blood glucose and plasma lactate. Serum sodium concentration was measured using indirect potentiometry . Blood smears (thin and thick films) were obtained from finger pricks and stained with Giemsa for parasite counts. Malaria was diagnosed by Quantitative Buffy Coat analysis, Severe malaria: Patients were considered as having severe P. falciparum malaria if they met predefined modified World Health Organization (WHO) criteria for severe malaria on admission or during hospitalization [lization   score < 11  or• Anaemia (haematocrit < 0.20 L/L with parasite count > 100.000/μL) or• Jaundice (serum bilirubin > 50 μmol/L with parasite count > 100.000/μL) or• Renal impairment (urine output < 400 mL/24 h and serum creatinine > 250 μmol/L) or• Hypoglycaemia (blood glucose < 2.2 mmol/L) or• Hyperparasitaemia (> 10% parasitaemia) • Shock (systolic blood pressure < 80 mm Hg with cold extremities)Hyponatraemia: Hyponatraemia was defined as a serum sodium concentration of less than 135 mmol/L.s) and Wilcoxon signed rank test. The prognostic value of serum sodium for malaria severity was determined by a receiver operating characteristic (ROC) analysis. To analyse if hyponatraemia was also independently associated with malaria severity, a logistic regression analysis was performed using a backward stepwise conditional approach. In the latter analysis only patients with falciparum malaria were included  and the presence of severe malaria was defined as the outcome.All data were not Normally distributed (Kolmogorov-Smirnov test) and are, therefore, presented as medians and range. Univariate comparisons were performed using the Kruskall-Wallis test (three groups) with Dunn's post-hoc tests, or the Mann-Whitney test (two groups). Correlations were analysed using Spearman rho . P. falciparum infection was classified as severe (n = 35) or non-severe (n = 280). One hundred and thirty-one patients had non-falciparum malaria, which consisted of P. vivax , P. ovale  and P. malariae . True re-infection occurred in 13 patients, and four patients had a mixed infection of P. falciparum and another Plasmodium species. Table Serum sodium concentration on admission was available for 446 of the 477 malaria cases (93.5%), and they comprised the study population. Infection was most commonly acquired in Africa (75%) and Asia 14%). Infections with 4%. InfecThirty-two patients fulfilled one or more of the severity criteria at initial presentation . Three patients did not fulfil the criteria for severe disease on admission but their clinical course deteriorated shortly hereafter. Admission plasma lactate levels were significantly increased in patients with severe malaria as compared with patients with uncomplicated falciparum malaria and patients with non-falciparum malaria  had a serum sodium concentration exceeding 145 mmol/L. When only patients with hyponatraemia were considered, hyponatraemia was more severe in severe falciparum malaria (median serum sodium 129 mmol/L) compared with both non-severe falciparum and non-falciparum malaria . In the whole population, prerenal azotaemia (defined as a serum urea: creatinine ratio > 1: 10) was not more common  and haematocrit values  were not higher in patients with hyponatraemia and malaria. Other causes of hyponatraemia appeared rare. Only three patients with malaria and hyponatraemia also used drugs associated with hyponatraemia  [s = -0.36; p < 0.0001) but also for patients with infections solely caused by Plasmodium falciparum .Hyponatraemia was present in 207 patients (46%). The distribution of serum sodium in the whole population seemed to be shifted to lower values Figure . Figure P. falciparum infections and 0.74 for all patients with malaria .The median duration of hospital stay is shown in Table P. falciparum malaria. The incidence of hyponatraemia in this study  is comparable to that found in other studies. Holst et al [P. falciparum infection, whereas Kockaerts et al [et al [et al [The present study shows that hyponatraemia is common in patients with imported malaria, and is associated with severe disease in ts et al  reportedl [et al , 16/192 9/L, and leukocyte count > 6.5 × 109/L were all independently associated with severe falciparum malaria . Some ocriteria  have beecriteria . When thpatients . As suchpatients , performpatients  involvinpatients  and diffpatients . For exapatients , 68% of patients , elevatepatients ,26 or bapatients ) have beP. falciparum malaria [et al [Even though the predictive power of hyponatraemia for severe malaria may be limited in clinical practice, its pathophysiology is certainly puzzling. Suggested mechanisms are absolute sodium deficit due to cerebral salt wasting, excessive sweating or loss in the gastrointestinal or urinary tract , and the malaria . In the  malaria . Althouga [et al  presentea [et al ,29. The a [et al . Previoua [et al  but alsoIn conclusion, hyponatraemia is common in imported malaria and associated with severe falciparum malaria. From a clinical point of view, however, the predictive power of hyponatraemia for severe malaria is limited. The precise pathophysiological mechanisms of hyponatraemia in malaria require further study.The authors declare that they have no competing interests.MEvW, DAH and EJH performed the data analysis and wrote the first draft of the manuscript. PJJvG and MEvW contributed to the data acquisition. RZ and PJJvG contributed to the data analysis and made substantial changes to the manuscript. All authors have seen and approved the final version.


Plasmodium falciparum from different endemic regions, but few studies have examined the characteristics of isolates from patients with different clinical outcomes. The population genetic structures of P. falciparum isolates from patients with either severe or uncomplicated malaria were examined.There have been many reports on the population genetic structures of P. falciparum were used to assess the population genetic structures of 50 isolates  collected in the Thai-Myanmar border area between 2002 and 2005.Twelve microsatellite DNA loci from P < 0.05), and allele frequencies for 3 of the 12 microsatellite loci differed significantly between the two groups.Genetic diversity and effective population sizes were greater in the uncomplicated malaria group than in the severe malaria group. Evidence of genetic bottlenecks was not observed in either group. Strong linkage disequilibrium was observed in the uncomplicated malaria group. The groups demonstrated significant genetic differentiation (P. falciparum populations in patients with severe malaria differs from that in patients with uncomplicated malaria. The microsatellite loci used in this study were presumably unrelated to antigenic features of the parasites, but, these findings suggest that some loci may influence the clinical outcome of malaria.These findings suggest that the genetic structure of  Plasmodium falciparum is the most pathogenic of the protozoan parasites that cause human malaria. According to the World Health Organization, more than 1 million people die from malaria each year  [1 - ∑pi2], where n corresponds to the number of isolates examined and pi is the frequency of the ith allele.Expected heterozygosity (Ne) was estimated based on H and the microsatellite mutation rate  for P. falciparum .,15.Ne vaH excess and deficiency. Statistically high levels of H excess compared with H deficiency were not observed in either study population when IAM and SMM were applied to the analyses, indicating the absence of genetic bottleneck events. The mode-shift indicator test revealed a normal L-shaped allele frequency distribution in the two study populations  ,17. Tablns Table , furtherIAS values were calculated for 12 loci from 22 isolates in the severe malaria group and 23 isolates in the uncomplicated malaria group. IAS values were equal to 0.010 and 0.127 for the severe and uncomplicated groups, respectively (Table p < 0.0001).ly Table . SignifiFST value of 0.014. This value was found to be significantly different from 0 (p < 0.05). The distribution of alleles for each locus in the severe malaria group was compared with that in the uncomplicated group. Distribution patterns for 3  of the 12 loci were significantly different (p < 0.05) between the two populations  reported for the same region in 1997 and 1998 . The freP. falciparum population in the severe and uncomplicated malaria groups differed in regards to several genetic indexes. Specifically, H values in the severe population were lower than those in the uncomplicated population. H values determined for both the severe and uncomplicated groups were higher than previously reported values for the same area [Ne values for the severe population were also lower than those in the uncomplicated population, and our values were generally higher than previously reported values for this area [H and Ne values in the P. falciparum populations.The genetic structure of the ame area . Ne valuhis area . Discrepp < 0.05), even though the groups were collected from the same endemic area. This may reflect differences in transmission intensity between the two groups.Significant genetic differentiation was observed between the severe and uncomplicated malaria groups (P < 0.05). Pfg377 is located within the telomeric region of chromosome 12, while TA109 is located within a hypothetical protein on the centromeric region of chromosome 6, and TA42 is located within a hypothetical protein on the telomeric region of chromosome 5.Allele frequency distributions for 3  of 12 loci differed significantly between the uncomplicated and severe malaria groups  of loci within the two study populations.Click here for file


Plasmodium falciparum, are thought to play an important role in acquired immunity to malaria. Evaluating such responses in longitudinal sero-epidemiological field studies, allied to increasing knowledge of the immunological mechanisms associated with anti-malarial protection, will help in the development of malaria vaccines.Antibodies, particularly cytophilic IgG subclasses, with specificity for asexual blood stage antigens of P. falciparum. In age-adjusted analyses, children resistant to malaria as well as to high-density parasitemia, had significantly higher IgG1 responses to GLURP and IgG3 responses to MSP2 than their susceptible counterparts. Among those resistant to malaria, high anti-MSP1 IgG1 levels were associated with protection against high-density parasitemia. To assess functional attributes, we used an in vitro parasite growth inhibition assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1, MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked age-related decline was observed in these effects.We conducted a 1-year follow-up study of 305 Senegalese children and identified those resistant or susceptible to malaria. In retrospective analyses we then compared post-follow-up IgG responses to six asexual-stage candidate malaria vaccine antigens in groups of individuals with clearly defined clinical and parasitological histories of infection with P. falciparum malaria in children depends on acquisition of a constellation of appropriate, functionally active IgG subclass responses directed to multiple asexual stage antigens. Our results suggest at least two distinct mechanisms via which antibodies may exert protective effects. Although declining with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The latter could act on parasite growth via indirect parasiticidal pathways.Our data are consistent with the idea that protection against  P. falciparum, although the precise mechanism(s) involved remains unclear. Specific IgG are proposed to have either a direct IgG antibodies' pivotal role in anti-malarial protection was demonstrated by seminal studies involving the passive transfer of IgG, purified from sera of semi-immune adults, to non-immune patients resulting in clearance of parasitaemia P. falciparum, 2) to retrospectively assess the association between anti-malarial protection and IgG antibody isotype responses to a panel of asexual stage antigens representing the foremost vaccine candidates, and 3) to use functional assays of antibody activity in vitro to determine their predictive value for the results of the sero-epidemiological study.Defining immune surrogates or, even better, correlates of protection is considered an essential step in the rational development of malaria vaccines and sero-epidemiological studies are one of the valuable tools with which putatively protective anti-malarial antibody responses can be identified. Here, we present the results of a sero-epidemiological study in the Niakhar area in Senegal in which parasitological, clinical and epidemiological data were collected during one-year of close, active follow-up of a large cohort of children and adolescents. Plasma samples, used for antibody assessments, were collected from participants at the end of the study period. The objectives we set were 1) to identify groups of individuals based on clearly defined differences in their capacity to control infection and/or disease due to Anopheles gambiae s.l. complex The study took place during the whole of 2003 in Diohine and Toucar, two villages located 6 km apart in the Niakhar district situated 135 km south-east of the capital, Dakar. The study area, study design, and local epidemiology of malaria have been described in detail elsewhere The study was explained in detail to all participants and their parents, and either they or their parents gave their signed informed consent. The ethics committee of the Health Ministry of Senegal approved the study protocol (N°000526/MS/DERF/DER).We defined uncomplicated malaria attacks (UMA) as the association of an axillary temperature greater than 37.5°C with a PD equal to or higher than 2,500/µl and with no other apparent cause of fever, in order to avoid potential bias. Any individual identified as having had at least one UMA during the follow-up period was included in the UMA group.Individuals with no UMA during the follow-up and with at least one parasite-positive TBS were considered as asymptomatic carriers (AC). All TBS performed in the 15 day period immediately following anti-malarial treatment were excluded when assessing the study participants' parasitological phenotype. The AC group was further segregated according to their levels of parasitaemia (low vs high). As a UMA was defined as the association of fever with a parasite density equal to or higher than 2500/µl, we selected the same threshold to discriminate between two AC groups. The AC group with low parasitaemia (ACLP) group thus comprised individuals who had at least one parasite-positive TBS but with a PD below 2,500/µl on each occasion. The AC group with high parasitaemia (ACHP) comprised individuals who had at least one positive TBS during the follow-up but with a PD equal to or above 2,500/µl on at least one of the parasite-positive TBS. This sub-segregation was designed to allow evaluation of antibody response(s) potentially associated with anti-parasite (ACLP vs ACHP groups), anti-disease (ACHP vs UMA groups) and/or combined anti-parasitic/anti-disease (ACLP vs UMA groups).P. falciparum parasites on any of their six TBS could be considered to either be completely protected or simply not exposed to infection. To avoid possible confounding we excluded all such individuals (n = 33) from all analyses.Children with no UMA during the active follow-up and no P. falciparum:An Enzyme-Linked Immuno-Sorbent Assay (ELISA) following a standardized methodology described in the Afro-immunoassay network standard operating procedure (procedure number AIA-001-02) was used to assess the antibody response, at the end of the follow-up (December 2003), to the following panel of recombinant proteins derived from sequences of asexual stage antigens of 19 20–43, 1615–1723/Uganda-Palo-Alto strainMSP1MSP2/3D7 & MSP2/FC27161–276/FC27 strainMSP3 25–545/FVO strainAMA1 25–514/F32 strainGLURP 19  was expressed in a Baculovirus/insect cell system Pichia pastoriusEscherichia coli2SO4 (100 µl/well). Plates were extensively washed between each incubation period with PBS Tween-20 (0.1%) NaCl (0.5 M). The optical density (OD) was read at 450 nm (reference filter 620 nm). Positive-control plasma samples from Gabonese individuals and negative-control plasma samples from Dutch individuals were included in each plate and results were expressed in arbitrary units (AU) calculated from the formula: 100× [ln(OD test plasma)−ln(OD negative control plasma)]/[ln(OD positive control plasma)−ln(OD negative control plasma)] MSP1HBB AS genotype (n = 32) were excluded from analyses that compared antibody responses between groups. The determination of the different HBB genotypes was performed by PCR/RFLP as previously described Previous studies have shown that carriage of the sickle cell trait is associated with protection against uncomplicated malaria and with the modulation of anti-parasite antibody responses Thirty plasma samples, matched according to age, gender and area of residence, were randomly selected from each of the ACLP, ACHP and UMA groups. IgG was purified from 600 µl of each of these plasma samples using SpinTrap Protein G columns  using procedures recommended by the manufacturer. The purified IgG samples were concentrated using Vivaspin 20 ultrafiltration spin columns  according to the recommended protocol to a final concentration of at least 20 mg/ml and the concentrated IgG were then filter-sterilized using 0.22 µm centrifuge filters . The IgG were quantified using the NanoDrop® ND-1000 spectrophotometer and the required IgG concentration for the parasite growth assay (20 mg/ml) was obtained through appropriate dilution in RPMI 1640 medium before each functional assay.P. falciparum parasites maintained in culture in vitro were synchronized using alanine treatment and collected for use at the beginning of schizogony. Assays were initiated in flat-bottomed 96-well tissue culture plates with a starting parasitaemia of 0.3% in a final volume of 100 µl containing 10% normal human serum, 20 µg/ml of gentamicin, a final concentration of test IgG of 10 mg/ml (50 µl of the 20 mg/ml IgG preparation) at a haematocrit of 2% in RPMI 1640. The growth inhibitory capacity of each sample was assayed in triplicate and each assay plate included positive (purified rabbit anti-AMA1 IgG) and negative control samples . Assays were collected for analysis 42–44 h after initiation and the PGI in the presence of IgG was assessed by measuring the Plasmodium lactate dehydrogenase levels in parasite cultures Parasite Growth Inhibition (PGI) was performed as previously described with minor modification The association of specific IgG responses with anti-malarial protection was first assessed using a linear model (ANCOVA) taking into account the age effect on the IgG levels. The comparison of IgG levels between the UMA and AC groups was performed to investigate the potential anti-malarial protective responses and the comparison of the IgG levels between the UMA vs ACHP, ACHP vs ACLP, UMA vs ACLP groups aimed to identify more precisely anti-parasite and/or anti-malarial  protective responses.Logistic regression was applied for the analysis of associations with protection against malaria attacks (UMA vs AC groups) which was used as the dependent variable against the explanatory variables (IgG responses/age) shown to be associated by ANCOVA. This approach allowed for assessment of the independence of associations of IgG responses with protection in the linear model and to additionally check for the presence or absence of interactions between putatively protective IgG responses. The relationship between the number of antigens recognized at high titre by each individual and group  was also investigated using logistic regression. For this purpose, antibody titres  in the whole study population were re-coded into tertiles and values in the top tertile were considered a high response for each specific antigen. Next, the number of high responses was calculated per individual. Logistic regression was then performed with group  as outcome and age and number of antigens recognized at high titre as explanatory variables.The non-parametric Kruskal Wallis and Mann-Whitney tests were used to test for differences in levels of PGI and antibody levels between the different groups. All analyses were carried out in STATA version 8 .HBB AS carriers were significantly less frequent in the UMA compared to the AC group. After exclusion of those with HBB AS, the UMA group for analyses comprised 89 children among whom 64, 21 and 4, had had, respectively, 1, 2 and 3 malaria attacks during the follow-up period. The AC group for analyses comprised 157 children .Details of the study participants are presented in P. falciparum parasitemia at the time of the blood draw in December 2003  did not significantly influence the level of IgG responses (data not shown); (ii) the proximity of malaria episodes to blood draws in the UMA group  did not affect antibody levels (data not shown). Age had an effect on the levels of specific antibody responses, most of which increased with age. Particularly notable was IgG1 with specificity for GLURP and IgGt with specificity for MSP2 FC27  up to 100% (IgG3 to MSP2 3D7). IgG1 anSP2 FC27 . Given tP. falciparum malaria conferred by carriage of HBB AS The putative protection against uncomplicated The logistic regression model confirmed the apparent anti-malarial protective effects of the IgG isotype responses identified by ANCOVA. Thus, the levels of IgG3 with specificity for MSP2 3D7, and of IgG1 with specificity for GLURP were both shown to be significantly higher in the AC versus UMA groups, while a similar trend, but of borderline significance, was found for IgG3 directed to the FC27 allele of MSP2. Independently of the IgG activity, age  also differed significantly between the groups. Possible interactions between the three antibody responses were investigated to determine potential combination effects. No interaction was observed in the logistic regression model, indicating that the three responses act independently, and, possibly, additively.P. falciparum infection, thus aimed to determine whether an ‘anti-disease’ type of immunity was associated with any of the antibody activity measured here. By ANCOVA, the levels of IgGt to both MSP2  and to GLURP, of IgG1 to GLURP and of IgG3 to MSP2  were found to be higher in the ACHP compared to the UMA group (The sub-segregation of the AC group (ACHP and ACLP) was designed to allow for assessments of possible associations between specific IgG responses and different aspects of acquired immunity. The comparison of IgG levels between the ACHP and UMA groups, respectively resistant and susceptible to the clinical symptoms associated with MA group , but aftAssociations between antibody responses and protection against higher-grade parasitemia were assessed through comparison of IgG levels in the ACHP and ACLP. Here, the levels of IgGt directed to GLURP, of IgG1 to MSP1 and of IgG3 to both MSP2  and MSP3 were higher in the ACLP compared to the ACHP group . Again, The association between specific antibodies and the combined ability to protect against disease and high-grade parasitemia was assessed through comparison of the IgG levels in the ACLP and UMA groups. This analysis showed that the levels of IgGt directed to MSP1, to MSP2  and to GLURP, as well as of IgG1 to MSP1 and to GLURP and of IgG3 to both MSP2  and to GLURP were all higher in the ACLP compared with the UMA group. After multiple test adjustments, the higher levels of IgGt and of IgG1 to GLURP and of IgG3 to MSP2  remained statistically significantly different between the groups . In the P. falciparum. Here, logistic regression analyses revealed 3.6-fold greater odds (p = 0.0003) of being in the ACLP group versus the UMA group when responding strongly to 2 or more antigens, and 2.4-fold greater odds (p = 0.019) of an individual being in the ACLP group versus the ACHP group when responding strongly to 3 or more antigens.Study participants' cumulated antibody-mediated recognition of multiple antigens at high levels was also found, independently of age, to be associated with the outcome of infection with The PGI of the matched  samples selected from the UMA, ACHP and ACLP groups did not differ significantly , althougIn further analyses, ELISA-detectable antibody levels in the original plasma were compared in the same set of samples segregated according to their PGI activity: a ‘high’ group  with PGI>50%, and a ‘low’ group  with PGI≤50%. The group with high PGI activity had, after correction for multiple comparisons, significantly higher levels of IgGt directed to MSP1 and to MSP2  than those with low PGI activity. Among the cytophilic subclasses, elevated IgG1 responses to MSP1 and IgG3 responses to MSP2  were associated with a higher capacity to inhibit parasite growth . Anti-AMP. falciparum asexual stage antigens. A distinguishing feature of our study is that we combined quantification of IgG subclass responses to a panel of P. falciparum vaccine candidate antigens with determinations of functional antibody activity using purified IgG in a standardized in vitro assay of P. falciparum asexual stage growth inhibition. In addition, the continuous and close active surveillance of study participants we conducted over 1 year allowed for precise definitions of their clinical and parasitological phenotypes. The study therefore had sufficient power, using adjusted analyses where appropriate, to identify independent associations between anti-malarial protection and defined antibody responses. Of equal importance, we feel, is the fact that our findings emphasize the need to identify IgG subclass-specific responses in sero-epidemiological studies of this kind rather than simply total IgG.In this study we used retrospective analyses to assess the associations between parasite-specific antibody activity and protection from malaria and/or high grade parasitaemia. Although therefore differing in design from more commonly-used prospective studies, precluding direct comparison of results, our findings do support the continued development of vaccines based on specific P. falciparum malaria is associated specifically with (i) cytophilic IgG1 and IgG3 responses (the latter of borderline significance) to GLURP and (ii) IgG3 responses to MSP2, measured after the transmission season. We found, furthermore, an association of borderline significance between anti-parasite immunity and higher levels of IgG1 directed to MSP1. All of these associations, importantly, were independent both of each other and of age. The possible functional attributes in vivo of both the anti-MSP1 and -MSP2 responses were further emphasized by their associations with enhanced inhibition of parasite growth by purified IgG in vitro.Our data show that naturally-acquired clinical and parasitological immunity in children living in an area characterized by stable, highly seasonal transmission of P. falciparum and P. vivax.Despite their evident design differences, then, the results of our retrospective study are nevertheless consistent with those of other prospective sero-epidemiological studies that have shown associations between anti-malarial protection and IgG responses directed to either the GLURP R0 domain in vitro in samples containing the highest levels of anti-MSP1 IgG1 lend strong support to the notion that such antibodies indeed have a functional role in vivo. Although displaying only non-significant trends, differences in the levels of anti-MSP2 IgG3 suggested some degree of association with clinical immunity  able to control parasitemia above a defined threshold from those (the ACHP group) that could not. This finding is consistent with the results of other sero-epidemiological studies of this molecule, some of which also identified an association of anti-MSP1 IgG with control of high-grade parasitaemia in vitro but no such association was detectable for anti-GLURP IgG (We observed markedly contrasting associations for IgG responses directed to GLURP compared with those directed to AMA1. On the one hand, the sero-epidemiological analyses revealed that cytophilic anti-GLURP IgG activity was strongly associated with anti-malarial protection  & 3, butLURP IgG . It is kin vitro assay - we did not find significant differences in functional activity between the groups segregated on the basis of clinical and parasitological phenotypes. This is in line with the results of studies that used either dialysed or whole serum rather than purified IgG, but that nevertheless found no association between the amounts of growth-inhibitory antibodies detectable in vitro and reduced risk of malaria in vitro assay concerns the age-related decline in growth inhibitory activity in vitro assays that faithfully reflect in vivo antibody function.Finally, some aspects of the growth inhibition assay employed here are worthy of mention. Despite using purified IgG - effectively precluding any influence of anti-malarial metabolites or other potentially parasitistatic molecules in the in vivo to suppress parasitaemia to levels below those causing disease. We interpret the results as providing strong support for the development of a vaccine designed to elicit antibody responses simultaneously to multiple asexual stage antigens of P. falciparum.In summary, the results of this study clearly highlight the role of IgG antibody subclass responses in acquired anti-malarial immunity. On the basis of the data presented, we conclude that naturally-acquired immunity to malaria, in the setting described, relies on the presence of a constellation of cytophilic IgG antibodies, displaying differing specificities and probably having disparate functional attributes, but that may operate in concert


The time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured.Plasmodium falciparum every week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes.One hundred and ten individuals from the village of Ndiop , aged between one and 72 years, were cured of malaria by quinine . Thereafter, thick blood films were examined to detect the reappearance of P. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals  and in adults > 30 years than in younger subjects . In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age ≥ 30 years were independently predictive of a reduced risk of re-infection .Malaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals  had been re-infected with Results indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines. Plasmodium falciparum enables them to control parasite densities to low levels and, thereby, to reduce the incidence of clinical malaria episodes. This control is usually considered to be mainly induced by and effective against erythrocytic forms of P. falciparum  for age (age < 30 years as reference group) and 0.4 [95% CI: 0.2–0.6] for prior parasitaemia . Using different cut-of values than 30 years also led to conclude about the role of age, but in less significant manner.P. falciparum were 22, 39, and 53 days among age-groups 1–6, 7–14, and ≥ 15 years, respectively, i.e. confirming the strong effect of age, and showing long positivation delays in adults. After 98 days of follow up, corresponding to an average 35 infective bites, 12% of the population had not been yet become positive again, showing a stronger level of resistance to blood positivation than in Ndiop. In Diohine, the median time to reappearance of P. falciparum was 28 days in individuals < 40 years[In this medium-low transmission area, reinfection studies show a substantial resistance of individuals to sporozoite inoculations. Indeed it required an estimated average of more than six inoculations of sporozoites to positivate 25 percent of the population and more than 10 to positivate half of the cohort. However, after an average of 15 infective bites, all but seven individuals had been undergoing a successful liver schizogony, as shown by patent blood infection. Similar resistance to sporozoite inoculations has already been reported in individuals exposed to both high and low transmission conditions in Senegal, such as in Dielmo, with average 200 infective bites/person/year  and in D 40 years,22, wher 40 years, the fir 40 years. Results 40 years. The EIRThe reappearance time was longer in subjects with parasitaemia upon enrolment than in individuals without, independently of age or administration of antimalarial treatment over the year preceding the study. This result is in apparent contradiction with that recorded in the higher endemic area of Dielmo, where previous parasitaemia was associated with an increased risk of the patient becoming prositive again . HoweverAge was significantly associated with reappearance time in all studies. However, the age at which the risk of reappearance of parasitaemia decreased significantly varied according to the studies and was grossly in relation with transmission intensity, i.e. in relation with exposure to sporozoites and hence potentially to anti-sporozoite immunity. It was significant in individuals > 40 years in Diohine , where tSimilar findings have been reported for other stages such as gametocytes, with evidence for gamete-blocking antibodies. Thus exposure to various stages of the parasite life cycle, sporozoite, liver stages, asexual blood stages, gametocytes, would generate a parasite-dependent, stage-specific immunity that modulate the survival of the corresponding stage. The lack of significant effect of the sickle cell trait upon the time it takes for a patient to become positive again may be significant in that respect.As sickle cell trait is the strongest known factor of protection against erythrocytic stages, this observation brings support to the view that re-positivation studies measures mostly pre-erythrocytic immune defences. Indeed, in the same community, the well-documented strong effect of the sickle cell trait against clinical malaria, including uncomplicated clinical malaria, was confirmed , and thiResults obtained in this and in previous studies bring support to the existence of a resistance to sporozoite inoculation which increases, as a function of parasite transmission levels and age, i.e. depending on previous exposure to pre-erythrocytic stages. Further similar studies are required to better delineate the part related to pre-erythrocytic and to erythrocytic stages immune responses. Indeed, several malaria control measures are targeted against the pre-erythrocytic phase of the cycle, such as vector-control measures, i.e. intra-domiciliary spraying, insecticide-impregnated curtains or bed nets, repellents, and experimental vaccines directed against the sporozoite and/or liver stages, the impact of which would be best evaluated using stage-specific tools. The design and results of re-positivation studies indicate that they can contribute to monitor the efficacy of those interventions.The authors declare that they have no competing interests.AT responsible for field-implementation, supervision of the team in the field, data collection, analyses, writing of and finalising this paper. CS, DF contributed to entomological protocol design and data collection ABL, FDS, LM supervision of the team in the field, data collection, supported data analyses. AT supported supervision of laboratory team. RP, CR supported writing of this paper. CR established the longitudinal study of the population. PD, JFT, AS contributed to study design, overall supervision of the study, and supported data analysis and writing of this paper. All authors read and approved the final manuscript.This study was supported by a grant from the Ministère Français de la Coopération et du Développement


The malaria vaccine candidate GMZ2 is a recombinant fusion protein of Plasmodium falciparum Glutamate Rich Protein and Merozoite Surface Protein 3, adjuvanted with aluminium hydroxide. Trials in malaria naive adults in Germany and in Gabon in adults exposed to malaria and subsequently in children 1-5yrs showed that three doses of 100ug GMZ2 are well tolerated and immunogenic. The aim of this phase 2b trial is to determine whether the GZ2 vaccine can protect against clinical attacks of malaria in children aged 1-5yrs, to obtain more extensive data on safety, tolerability and immunogenicity, and to determine if the vaccine can prevent anaemia and reduce the parasite density and gametocyte carriage.The primary endpoint is the incidence of clinical malaria defined as fever or history of fever in the previous 24 hours with parasite density of 5000 asexual parasites per ul or more, detected by passive surveillance over a 6 month period from the third vaccination. All malaria episodes will be included in the calculation of vaccine efficacy. This endpoint is chosen in order to have the optimum power to detect a protective effect; efficacy will also be calculated using a range of different parasite density cutoff values. Children will be followed for a total of 22 months from the third vaccination. Immune responses to the vaccine antigens GMZ2, GLURP and MSP3 will be assessed by measuring antigen specific IgG by ELISA, and antigen specific memory B-cell responses by ELISPOT. Functionality of the immune response will be assessed by growth inhibition of P. falciparum in the presence or absence of monocytes. Cell mediated immunogenicity will be assessed by cytokine profiling and intracellular cell staining following stimulation with the vaccine antigen, and the quality of the antibody response by type and subclass-specific ELISA.The planning of the trial was complicated by the need to take account of the changing epidemiology of malaria. Baseline studies were conducted in five potential trial sites to determine the current incidence of malaria; in each site two cohorts of children were followed to estimate the incidence of malaria by active and passive surveillance and by passive surveillance alone. Four sites with higher malaria incidence were chosen for recruitment. A total sample size of 1840 will be enrolled in each of the four sites in 2010/11.


Prompt, accurate diagnosis and treatment with artemisinin combination therapy remains vital to current malaria control. Blood film microscopy the current standard test for diagnosis of malaria has several limitations that necessitate field evaluation of alternative diagnostic methods especially in low income countries of sub-Saharan Africa where malaria is endemic.We compared the accuracy of axillary temperature, health centre microscopy, expert microscopy and a HRP2-based rapid diagnostic test (Paracheck) in predicting malaria infection using polymerase chain reaction (PCR) as the gold standard. Three hundred patients with a clinical suspicion of malaria based on fever and or history of fever from a low and high transmission setting in Uganda were consecutively enrolled and provided blood samples for all tests. The endpoints were: sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). This study is registered with Clinicaltrials.gov, NCT00565071.Of the 300 patients, 88(29.3%) had fever, 56(18.7%) were positive by health centre microscopy, 47(15.7%) by expert microscopy, 110(36.7%) by Paracheck and 89(29.7%) by PCR. The overall sensitivity >90% was only shown by Paracheck 91.0% [95%CI: 83.1-96.0]. The sensitivity of expert microscopy was 46%, similar to health centre microscopy. The superior sensitivity of Paracheck compared to microscopy was maintained when data was stratified for transmission intensity as well as by age. The overall specificity rates were: Paracheck 86.3% [95%CI: 80.9-90.6], health centre microscopy 93.4% [95%CI: 89.1-96.3] and expert microscopy 97.2% [95%CI: 93.9-98.9]. The NPV >90% was shown by Paracheck 95.8% [95%CI: 91.9-98.2]. The overall PPV was <88% for all methods.High sensitivity of malaria diagnosis is essential because the infection produces an acute illness in vulnerable populations that can rapidly progress to death. The HRP2-based rapid diagnostic test has shown superior sensitivity compared to microscopy and may be more suitable for screening of malaria infection.


A case-control study was performed to determine the role of rural factors including occupation and previous malaria exposure in the development of classic Kaposi's sarcoma (CKS) in a high incidence area of Europe. The occurrence of CKS association with other malignancies was also examined. The results showed that the risk of having CKS was significantly increased in subjects farming cereals, while a previous history of malaria did not influence the risk of developing CKS. A near-significant increase in associated tumours was found.


Accurate and practical malaria diagnostics, such as immunochromatographic rapid diagnostic tests (RDTs), have the potential to avert unnecessary treatments and save lives. Volunteer community health workers (CHWs) represent a potentially valuable human resource for expanding this technology to where it is most needed, remote rural communities in sub-Saharan Africa with limited health facilities and personnel. This study reports on a training programme for CHWs to incorporate RDTs into their management strategy for febrile children in the Democratic Republic of Congo, a tropical African setting ravaged by human conflict.Prospective cohort study, satisfaction questionnaire and decision analysis.Twelve CHWs were trained to safely and accurately perform and interpret RDTs, then successfully implemented rapid diagnostic testing in their remote community in a cohort of 357 febrile children. CHWs were uniformly positive in evaluating RDTs for their utility and ease of use. However, high malaria prevalence in this cohort  limited the cost-effectiveness of RDTs compared to presumptive treatment of all febrile children, as evidenced by findings from a simplified decision analysis.CHWs can safely and effectively use RDTs in their management of febrile children; however, cost-effectiveness of RDTs is limited in zones of high malaria prevalence. Malaria remains the leading parasitic cause of morbidity and mortality worldwide, causing an estimated 515 million clinical cases and 2 million deaths annually,4.Accurate malaria diagnosis remains a cornerstone of global control efforts. However, several authors have noted that malaria misdiagnosis in endemic areas is common, resulting in harm to vulnerable populations. It is eIn this setting, community health workers may represent a valuable human resource for the case management of malaria. Previous reports from South America,10, AsiaAs a promising strategy for improving capacity for malaria management, the use of RDTs by CHWs in a rural setting in eastern Democratic Republic of Congo was investigated. This malaria holoendemic area has been crippled by civil and international war and currently has limited health personnel and facilities. This study reports the outcome of a training module for community health workers, and demonstrates the feasibility of RDT use by community health workers in a cohort of 357 febrile children in their remote village. However, the findings of this study, supported by a decision analysis using locally relevant cost parameters, call into question the cost-benefit of rapid diagnostic tests in a zone of malaria holoendemicity.Plasmodium falciparum accounts for more than 95% of clinical cases of malaria, and children may experience an estimated three to four clinical episodes of malaria annually.Over the past decade, the Democratic Republic of Congo has been the stage of the most deadly humanitarian crisis in recent history,4. DespiIn this underserved area, community health workers form an integral part of the health care system. They are volunteers that have chosen to devote some of their time to manage health problems in their community, acting as a bridge between the community and the government health care system. They are chosen by community members and contribute to the sustainable development of the health system at the village level. They have minimal  training in epidemic and endemic diseases , generally provided by non-governmental organizations. Although they do not receive a salary from the central government, CHWs may derive a profit  from their activities. The CHWs in this study had previous experience in the management of febrile children in their community. The goal of this study was to build on their existing capacities and extend their diagnostic armamentarium with RDTs.Ethics approval for this study was obtained by the local Congolese university institution .(R) kit . One instructor (JPK) taught and evaluated CHWs. Tools used for teaching included materials published by the WHO[Twelve experienced health care workers from Yakusu village were selected to attend a one-day training session on malaria, its clinical manifestations, treatment, and the use of RDTs for parasitological diagnosis. The RDT in this study was the commercially available Paracheck-Pfy the WHO, includiy the WHO. Teachiny the WHO. A 10-ity the WHO. ParticiFollowing training and verification of knowledge and skills in the safe and reliable use of RDTs, CHWs were distributed thermometers, RDTs and malaria treatment  for use upon return to their community. Interpretation of the malaria RDT was positive if both the control and test lines were present and negative if only the control line was present. In cases where the control line did not appear, the result was considered indeterminate. Children (0 to 14 years old) with fever (>38°C axillary temperature) were eligible to be tested with RDTs and were treated with ACT  dosed by weight, according to WHO guidelines. ParticiAfter the field trial of RDTs, CHWs were assembled and surveyed using a 16-item written survey questionnaire. Questions consisted of provocative statements with which participants ranked their agreement or disagreement on a 5-point Likert scale.To examine the cost-effectiveness of malaria RDTs in this setting, a decision analysis tool was used, modified from previous studies. The goa(R)). Median health worker age was 32 (range 23 to 47) years, and 4/12 were female. Participants had a median of 4.5 (range 1 to 10) years previous health care experience, all had previously managed cases of malaria, but only 2/12 had used a malaria RDT previously. Participants were highly satisfied with the training. Eleven of 12 agreed or strongly agreed that the information provided was useful; and although 7/12 strongly agreed that they already knew about malaria management beforehand, 11/12 agreed or strongly agreed that their knowledge had improved as a result of the training session. Qualitative feedback emphasized the comprehensiveness, clarity and quality of the teaching materials and trainer.Twelve community health workers were trained to perform and interpret malaria RDTs (Paracheck-PfParticipants demonstrated satisfactory knowledge and skills following the training session. Median (range) scores on a 10-item test of the causes and management of fever and malaria in children were 80% (60% to 90%). Questions pertaining to the causes of fever in children and the clinical signs of malaria were answered correctly by all participants. Poorer performance was observed on questions involving clinical scenarios: for example, 5/12 participants incorrectly answered that a febrile well-appearing 10 year old child with a negative RDT result should receive anti-malarial treatment. Median (range) scores on a standard WHO 16-item objective RDT skills assessment were 100The 12 trained health workers used the RDTs to manage 357 children with fever in their remote community over a one-week period in February, 2009 (during rainy season). Each health worker managed a median of 4 (range 0 to 9) febrile children each day, which included the administration of a fingerprick malaria RDT. The median age of the children was four years (range 1 month to 13 years), and 51% were less than five years old. The mean axillary temperature of children was 39.3 (standard deviation ± 0.8)°C. Associated signs and symptoms are shown in Table Given the high rate of RDT positivity among febrile children in this community, an alternative diagnostic modality (light microscopy) was used to verify the malaria prevalence. An experienced microscopist prospectively examined Giemsa-stained peripheral blood smears by light microscopy in an additional sample of 40 children with fever in the same community. The prevalence of parasitaemia among febrile children was confirmed to be high  using this gold-standard diagnostic modality.Following the trial period of RDTs, CHWs were surveyed to determine their views regarding their experience using RDTs in the field. As a rule, responses were uniform across the 12 health workers. Ten of 12 respondents agreed or strongly agreed that fever was an important clinical problem among children in their community, and all 12 agreed or strongly agreed that the febrile child poses a diagnostic dilemma for which RDTs are useful. All agreed or strongly agreed that RDTs were easy to learn, easy to perform, useful in the management of febrile children, and easy to interpret. All 12 agreed or strongly agreed that the initial training course had been useful. All noted that RDTs were not readily available in their community, 11/12 felt that RDTs would be affordable, and all 12 felt that the government should supply RDTs. Finally, all agreed or strongly agreed that they would continue to use RDTs after the study was finished.Next, a simplified decision analysis was employed to examine the cost-benefit of using RDT in a context of high malaria endemicity. Comparison was made between the scenario of universal treatment of all febrile children to the alternative scenario of treating according to RDT result, thereby withholding anti-malarial treatment from children five years and older with a negative RDT Figure . The cosThis study demonstrates the feasibility of training community health workers in the use of malaria RDTs in a setting of high malaria endemicity with scarce human and technological resources for health, exacerbated by violent conflict and human insecurity. In a single day, community health workers were trained to use and interpret a commercially available RDT, demonstrating appropriate knowledge and skills at the end of the training session. They subsequently demonstrated competence in point-of-care parasitological diagnosis of malaria in a group of 357 febrile children under field conditions. Following the field implementation of rapid diagnostic testing, CHWs were uniformly positive about the utility and ease of use of RDTs in their setting. On the other hand, cost remains a major barrier to the use of RDTs in areas of high malaria prevalence, as demonstrated by a simple decision analysis.This study is noteworthy for its setting in a zone of ongoing human insecurity. Over more than a decade, a civil and international conflagration has claimed over 3.9 million lives in the DRC, more than any other conflict since World War II. Despiteper capita public expenditure on health care ($15/yr)[The principal barrier to the utilization of RDTs in community-based malaria care is the sustainable purchase and supply of RDTs. In the decision analysis presented, health service payers would have to value the cost of saving one unnecessary treatment at $8.79, which represents eight times the cost of an ACT treatment course, and 60% of the annual  ($15/yr). This co ($15/yr). In one  ($15/yr). Likewis ($15/yr),20. Few As in this cohort of febrile children in tropical Congo, previous studies have documented a high prevalence of parasitaemia among children with fever in holoendemic zones. Test positivity among febrile children in excess of 80% has been documented following a flood disaster in Mozambique 81%) and in t% and in Although RDTs appear to be prohibitively expensive in this context, the mobilization of CHWs for malaria management and control remains a relevant strategy. In this study, 12 highly motivated CHWs who had previous experience managing children with malaria , demonstrated efficient integration of knowledge and skills in malaria diagnosis and treatment, and were rapidly mobilized to treat a large number of febrile children in a remote setting. Access to government sponsored health services may be as low as 50% in this and similar areas of sub-Saharan Africa. In the Limitations of this study include the relatively small sample size of CHWs from a single geographic area, and the short time span (one week) during which children were diagnosed and treated for malaria. Although a study involving multiple sites over a prolonged period might provide more generalizable information, these results nonetheless underline some important considerations for the roll-out of RDTs in other areas, particularly the prevalence of malaria among the target population for testing. During dry season, or in areas where malaria prevalence is lower, RDTs may be more cost-effective than determined in this study. With respect to survey questionnaires on the utility of the training session and the acceptability of RDTs, we cannot exclude the possibility of "social courtesy bias" influencing CHW to give positive feedback, although surveys were conducted anonymously to minimize this potential bias. Despite the lack of formal medical or nursing training, the CHWs in this study were relatively experienced in malaria management, which may explain their ability to rapidly incorporate RDTs into their clinical practice. Thus, successes in training CHWs in this context should not necessarily be extrapolated to less experienced personnel. RDT test performance characteristics (sensitivity and specificity) were not assessed against a gold standard, because this has been extensively documented in previous studies (reviewed in ).In summary, this study demonstrates the feasibility of training CHWs in the use of RDTs for malaria diagnosis and management among febrile children in a remote community affected by violent conflict. This study illustrates the importance of considering local factors  in assessing the appropriateness of RDTs for remote and vulnerable populations.The authors declare that they have no competing interestsMH conceived the study, performed the analysis, and wrote the manuscript. JPK trained the CHWs, supervised the field study of RDTs, and collected the data. CKM provided intellectual input into study design and implementation, and revised the manuscript. All authors have read and approved the final manuscript.