The objective of this study was to compare cases of imported malaria originating from the Spanish ex-colony of Equatorial Guinea (EG) with those originating from the rest of Africa (RA).visiting friends and relatives (VFR), species, hospital admission and chemo-prophylaxis were compared. Data were analysed by logistic regression, calculating the Odds Ratio (OR) and 95% Confidence Intervals (95% CI).All the African cases detected in Barcelona between 1989 and 2007 were investigated in a retrospective analysis. Clinical-epidemiological variables such as sex, age, visiting friends and relatives (VFR) category, and more individuals younger than 15 years or older than 37 years, and more women. They also visited a traveller's health centre more often, had fewer hospital admissions and were less likely to reside in the inner city.Of the 489 African patients, 279  had been born in EG and 210  in the rest of Africa. The cumulative incidence of imported malaria among those from EG was 179.6 per thousand inhabitants, while in those from the RA it was 33.7 per thousand (p < 0.001). Compliance with chemoprophylaxis (CP) was very low, but there were no differences between the two groups. Comparing those from EG to those from RA, the former were characterized by having more patients in the Cases of imported malaria originating in Africa, are more likely to come from the Spanish ex-colony of EG, and VFR are more likely to be affected. It is recommended that developed countries promote prevention programmes, such as CP advice directed at African immigrants, and develop programmes of cooperation against malaria in their ex-colonies. The population increase in endemic countries together with the increase in international travel and of migratory movements have made the worldwide risk of malaria higher now than it ever was ,2. Therevisit friends and relatives (VFR). Despite measures to prevent malaria, this creates an appreciable morbidity and mortality [Plasmodium falciparum [In developed countries, imported malaria predominates in tourists and in immigrants who travel to their country to ortality -6. As isortality ,8 and otortality ,9,10, imortality ,12, and lciparum -15. In Blciparum , are thelciparum ,18. Morelciparum , few of The objective of this study was to characterize the group of immigrants that contributes most cases of imported malaria, specifically comparing those from EG, an ex-colony of Spain, and to cases originating from other African countries, in a cosmopolitan city such as Barcelona.st 1989 and December 31st 2007 were studied in a retrospective analysis of prospectively collected data. Cases that were not laboratory-confirmed, those residing outside the city, those without a contact address in the city and cases of relapse from the same infection were excluded.The city of Barcelona, with an area of 100.4 square kilometers, has a population of 1,595,110 inhabitants, 15.6% from other countries January 2007 census) [07 censusPlasmodium vivax/Plasmodium ovale), the reason for travel and in the case of being an immigrant, if the reason for travel was to visit family and friends (VFR), work or volunteer work, or, if they had just arrived in Spain (recently-arrived immigrant), if this was verified by the municipal census. Immigrant resident is defined as an immigrant that lives in the city. Information about completion of the chemoprophylaxis treatment, and the area visited according to the risk level for multidrug-resistant P. falciparum transmission, as defined by the WHO (Types I to IV: minimum to maximum level of recommendation for prevention), was also noted [Reporting of cases malaria to the public healthcare system is mandatory throughout Spain. In Barcelona, active epidemiological surveillance is carried out, in which hospital admissions and microbiology listings are reviewed. Almost all cases are visited in two specialized units, regularly contacted by a public health nurse. After notification, an epidemiological survey is carried out, in which socio-demographic , diagnostic , chemoprophylaxis and treatment variables are collected, as well as the dates of onset of symptoms, diagnosis, and hospital admission and registration. In addition, the following information was collected: the endemic geographic areas visited in the 30 days prior to the onset of symptoms, or the last country visited if the patient returned from travel more than 30 days previously . Recently arrived patients were compared to those resident in the city, and also those born in EG were compared to other African citizens. For the purpose of this analysis, the immigrants' children were also considered immigrants, even if they were born in Barcelona.A descriptive analysis of the data was carried out. For the quantitative variables, the median and interquartile range (IR) was calculated for those that did not follow a normal distribution, and for the categorical variables the percentages were calculated. At the bivariate level the chi-test was used, and the corresponding non-parametric tests, where appropriate. The cumulative incidence rates per 1,000 inhabitants during the study period were calculated for patients from EG and for those from the rest of sub-Saharan Africa living in Barcelona using the average population in the city (January 2007 census) according to place of birth . At the multivariate level, logistic regression was used, comparing immigrants born in EG to those born in the RA. The 95% Confidence Interval (95% CI) was calculated and a significance level of 5% (p < 0.05) was accepted as statistically significant. Statistical analyses were performed using SPSS version 13.0 and EpiInfo version 6.During the study period, a total of 1,663 cases of malaria were detected, 1,072  of which were residents in Barcelona. All of these cases were imported except two, one of which was induced by transfusion and the other was contracted from another affected individual through the sharing of venipuncture materials. Figure Plasmodium sp.  were residents in the city, 89 18.2%) had recently arrived and in 32 cases (6.5%) it was not possible to determine if they were residents in Barcelona or not. Table 8.2% had P. falciparum with 373 cases (76.3%), followed by Plasmodium malariae with 20 (4.1%), P. vivax with 19 cases (3.9%) and P. ovale with 14 (2.9%). In 63 cases (12.9%), the diagnosis was of Plasmodium sp. A total of 108 cases (22.1%) required hospital admission. In 359 cases (73.4%) the reason for travel was VFR. Among these, only a single case of 359 (0.3%) completed the CP, 18 (5%) did not complete it and 239 (66.6%) did not undergo CP. In 101 cases (28.1%), it was not known if CP had been carried out . The two cases (0.4%) that came from the region of Magreb (Morocco and Algeria) were Plasmodium sp. The median time period, from the commencement of symptoms until the diagnosis, was 6 days (IR 3–13), 5 days (2–14) for those from EG and 6 days (IR 4–10) for the RA . During the period of the study no deaths among these immigrants were observed.The median age of all of the 489 Africans was 29 years (IR 20–39), 27 years (IR 16–41) among those from EG and 30 years (24–37) for the RA , with a slight predominance of women 249 (50.9%). A total of 68 patients (13.9%) lived in the inner city and 82 (16.8%) were children younger than 15 years. The most frequently isolated species was Comparing the cases from EG to those from RA, at the bivariate level, the variables that were associated with the group from EG were age, sex, being VFR, residing outside the Inner City, visiting the Tropical Medicine and International Health Unit, Drassanes (UMTSID), and requiring fewer hospital admissions Table . At the Plasmodium sp., and have some particular characteristics. Compared to other Africans, affected individuals from EG are predominantly women, children, persons older than 37 years and VFR immigrants. They are also less frequently admitted to hospital than other Africans and are less likely to live in the more disadvantaged districts.This study demonstrates that resident immigrants diagnosed with malaria in Barcelona are more frequently VFR, come from the Spanish ex-colony of EG, are infected by The frequent observation of malaria in individuals of African origin is consistent with that observed in other European countries, like Great Britain and France ,22,23, aAfter comparing the incidence of imported malaria in the group from EG to those from the RA, the study shows that the disease is diagnosed more frequently among the EG patients. It may be that coming from an ex-colony, and therefore having generally lived longer in the receiving country, may carry particular advantages, such as a better understanding of the language, a greater facility for obtaining nationalization and better knowledge of the healthcare system. These individuals may have greater possibilities to travel under more legally and economically stable conditions. Despite having the same risk of becoming ill and the fact that preventative measures should be similar to other immigrants, the results could suggest the use of different intervention strategies at the community level. Moreover, this difference may also be due to the fact that health checks are occasionally carried out upon returning to one's own country and that these individuals probably receive visits from family members, who may occasionally avail of the visit to have a health-check carried out.It was clear that the group of VFR immigrants carried the largest number of cases, perhaps because they travel more and, compared to the tourists, they take CP less often . The lowThe distinguishing features observed in the EG patients with imported malaria are likely to reflect, not so much the characteristics of the country of origin but rather of the greater number of years settled in the country, given it's relationship as an ex-colony. Established African immigrants in Barcelona are predominantly women, as is perhaps the case in other cities and countries . Having P. falciparum in Barcelona city during 1989–2007 occurred among immigrants  and thatourists ,32 suggetourists . This po prudent ,35, beca prudent ,28 born  prudent ,35,36.P. falciparum, might be explained by the fact that many cases are detected by screening during the visit to the UMTSID [With regard to admissions, if the same level of natural acquired semi-immunity among sub-Saharan immigrants because of coming from hyper-endemic countries is assumed, the Guineans are probably admitted less often because the disease is detected earlier as these individuals have spent more time in Spain, and are more familiar with the language and the functioning of the health system. Also, the low number of hospital admissions among the EG population despite the high percentage of cases with e UMTSID  after ree UMTSID . MoreoveIn order to control malaria, it is essential to combine different strategies against both the parasite and also the vector to reduce human-vector contact ,19,37-41To make the various preventative measures mentioned more effective, it is important that developed countries identify the most abundant immigrant groups, and those with the highest probability of importing malaria. In the context of the increase in international travel and of migratory movements, it is recommended that countries encourage specific prevention programmes for CP treatment and other preventative measures among all immigrants, which could potentially be VFR and their children.The authors declare that they have no competing interests.JPM, PG and JC designed the study, collected the data, analyzed and prepared the first draft. All authors put forward different ideas, contributed to the interpretation of the data, early drafts and agreed the final draft.


Plasmodium falciparum malaria by Freya Fowkes and colleagues aids identification of antigens that confer protection from malaria.A systematic review and meta-analysis examining the association between anti-merozoite antibody responses and incidence of  One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting.Plasmodium falciparum malaria. Two independent researchers searched six databases and identified 33 studies that met predefined inclusion and quality criteria, including a rigorous definition of symptomatic malaria. We found that only five studies were performed outside sub-Saharan Africa and that there was a deficiency in studies investigating antibodies to leading vaccine candidates merozoite surface protein (MSP)-142 and erythrocyte binding antigen (EBA)-175. Meta-analyses of most-studied antigens were conducted to obtain summary estimates of the association between antibodies and incidence of P. falciparum malaria. The largest effect was observed with IgG to MSP-3 C terminus and MSP-119  and there was evidence of a dose-response relationship. A tendency towards protective risk ratios (RR<1) was also observed for individual study estimates for apical membrane antigen (AMA)-1 and glutamate-rich protein (GLURP)-R0. Pooled estimates showed limited evidence of a protective effect for antibodies to MSP-1 N-terminal regions or MSP-1-EGF . There was no significant evidence for the protective effect for MSP-2 . Heterogeneity, in terms of clinical and methodological diversity between studies, was an important issue in the meta-analysis of IgG responses to merozoite antigens.We performed a systematic review with meta-analysis of prospective cohort studies examining the association between anti-merozoite immunoglobin (Ig) G responses and incidence of These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans, and to help identify antigens that confer protection from malaria. Further prospective cohort studies that include a larger number of lead antigens and populations outside Africa are greatly needed to ensure generalizability of results. The reporting of results needs to be standardized to maximize comparability of studies. We therefore propose a set of guidelines to facilitate the uniform reporting of malaria immuno-epidemiology observational studies.Please see later in the article for the Editors' Summary Plasmodium falciparum malaria, a mosquito-borne parasitic infection, kills about one million people every year. Around a week after an infected mosquito has bitten a person, “merozoites” (one of the life-stages of the parasite) infect the person's red blood cells where they replicate and then burst out and infect more red blood cells. Rapid replication of parasites can occur in the bloodstream, leading to massive numbers of parasites that can damage vital organs. Although individuals can lower their risk of becoming infected with malaria parasites by avoiding mosquito bites, a vaccine is urgently needed to reduce the global burden of malaria. When malaria parasites infect a person for the first time, the human immune system begins to produce antibodies, proteins that recognize molecules (antigens) on the parasite's surface and that act directly or cooperate with other parts of the immune system to kill malaria parasites. The production of these “naturally acquired” antibodies is initially slow so the individual can become ill when infected. However, because the immune system “remembers” how to make the antibodies, its response to subsequent infections is quicker. The levels of these antibodies also build up with each infection and become more effective at killing parasites. Vaccines, which contain malaria antigens, “prime” the immune system to respond rapidly to malaria infections and produce high concentrations of antibodies to prevent the infection from causing serious illness.P. falciparum malaria in naturally exposed populations in different regions of the world.A malaria vaccine that stimulates an efficient immune response against merozoites would limit the severity of malarial infections and prevent many deaths but no one knows which (if any) of the antigens on merozoites stimulate a protective immune response. Although many different types of antibodies are produced by the immune system, only some of these are effective in protecting against malaria. By investigating whether there is an association between naturally acquired antibodies, which recognize specific candidate antigens, and protection from malaria in populations living in areas where malaria is endemic , vaccine developers can get an idea about which antigens to include in their vaccines. Although many of these “malaria immuno-epidemiological studies” have been undertaken, their results are somewhat conflicting. In this study, the researchers reanalyze these results by doing a systematic review  and a meta-analysis . The researchers evaluated studies of the relationship between anti-merozoite antibodies and the incidence (the number of new cases of a disease in a population per year) of 42, a leading vaccine candidate). Conversely, the association between malaria incidence and antibodies to the antigen MSP-119, which has been included in only one candidate vaccine, was frequently studied. In their meta-analyses, the researchers found that among people with antibodies to the merozoite antigens MSP-3  and MSP-119, the risk of developing P. falciparum malaria was reduced by 54% and 18%, respectively, compared to people without antibodies to these antigens. There was also some evidence of a reduced risk of malaria for people with antibodies to AMA1 and GLURP. For other merozoite antigens, MSP1  and MSP2, there was either weak or no evidence for a protective effect of naturally acquired antibodies.The researchers' search of the published literature yielded 33 studies in which the incidence of malaria had been recorded over time in groups of people in whom levels of antibodies to specific merozoite antigens had been measured. These studies measured antibodies at the start of the study and examined the subsequent risk of malaria over several months of follow-up (these are known as prospective cohort studies). All but five of the studies were performed in Africa, and very few merozoite antigens had been well-studied in different populations, or studied at all. Of note, very few studies had examined naturally acquired antibodies to some leading vaccine candidates  and on the development of malaria vaccinesInformation is available from the malaria (in English and Spanish)The US Centers for Disease Control and Prevention provides information on Wellcome Trust on all aspects of malaria, including vaccine developmentInformation is available from the Malaria Vaccine Initiative provides information on the development of malaria vaccines and on ongoing trialsThe malaria (in English and Spanish)MedlinePlus provides links to additional information on  Plasmodium falciparum is a leading cause of mortality and morbidity globally, particularly among young children. After repeated exposure, individuals develop effective immunity that controls blood-stage parasitaemia, thereby reducing clinical symptoms and life-threatening complications  guidelines for the conduct of meta-analyses of observational studies P. falciparum malaria. Key words included: MSP, merozoite surface protein, MSA, merozoite surface antigen, GLURP, glutamate-rich protein, serine repeat antigen, SERA, S-Antigen, ABRA, AMA, apical membrane antigen, EBA, erythrocyte binding antigen, rhoptry, malaria, P. falciparum, immunity, antibodies, IgG, cohort, longitudinal, incidence, risk, epidemiology, vaccine. The key words variant surface antigen (VSA) were also used because merozoite antigens are sometimes used as comparative antigens in studies investigating VSAs. The reference lists of obtained papers were searched for further studies. Studies reported in languages other than English were included.PubMed, Web of Science, Scopus, Google Scholar, African Index Medicus, and LILACS   were searched for studies examining the association of antibody responses to merozoite antigens with The criteria for inclusion of studies were population-based prospective studies and population-based treatment to reinfection studies. Population-based cross-sectional studies to determine prevalence were excluded because causality cannot be established. Case-control studies, hospital-based studies, and vaccine efficacy trials of blood-stage vaccines were also excluded because of the rigorous inclusion and exclusion criteria applied during these studies, such that the participants would not be representative of the general population.P. falciparum status and studies investigating returned travellers or transmigrants were also excluded as they would not be representative of the general population.The criterion for inclusion of participants was individuals living in malaria endemic areas. Studies restricted to pregnant women and/or children <1 y  were excluded to remove the confounding effect of maternal transferred immunity. Studies where individuals were selected according to their Total immunoglobulin G (IgG) responses to recombinant or synthetic defined merozoite antigens measured at baseline  were considered. IgG responses to full length proteins, processing products, and defined regions of merozoite antigens were included, but IgG responses to peptides that represent undefined regions or incomplete domains or subdomains of antigens were excluded.P. falciparum infection , symptomatic P. falciparum malaria, severe P. falciparum malaria, and P. falciparum malaria-associated mortality. In treatment-to-reinfection studies P. falciparum reinfection was also included as an outcome. Newly established blood-stage infection must have been differentiated from treatment failure by either PCR or documented clearance of infection within a specified time frame appropriate for the chosen antimalarial.The following malaria outcome measures during follow-up were included: high density The minimum quality criteria for inclusion in the review were that: detection of malaria was by active case detection (ACD) and/or passive case detection (PCD); parasitaemia was confirmed by slide microscopy, rapid detection kit, or PCR; symptomatic malaria was defined as fever and/or history of fever (within the past 72 h) plus a high density parasitaemia threshold (to increase specificity because low-grade parasitaemia is common in most settings); severe malaria was defined by the World Health Organization criteria and other causes of morbidity excluded; and other common causes of mortality excluded before a diagnosis of malaria-associated mortality Review authors  identified possible studies, FJIF and JSR assessed the methodological quality of included studies independently, with discrepancies resolved by discussion with JGB.P. falciparum parasitaemia of any density  were invited to provide estimates or data meeting the quality criteria. Some studies had analysed antibody levels at baseline as the outcome variable, comparing baseline levels in those who had or did not have P. falciparum malaria during follow-up. For these studies, data was extracted and reanalysed so that malaria was the outcome variable and related to antibodies at baseline. If the raw data were not presented, authors of the study were invited to reanalyse or provide data for the inclusion of their study in the systematic review. In addition, we contacted several authors whose studies did not meet the inclusion criteria yet contained data that were eligible for the systematic review. These were the authors of studies that had measured antibody responses after baseline  and invited them to provide estimates or data concerning prospective P. falciparum incidence only. We also contacted authors who had restricted analysis to individuals who were parasite positive at baseline and invited them to provide estimates or data on the whole cohort where possible. If authors were unable to provide estimates or data, the study was classified as not meeting inclusion and/or quality criteria and excluded from the systematic review.Authors of studies that had defined a case of symptomatic malaria as fever and/or history of fever plus a Measures of association  and their 95% confidence intervals (CIs) were extracted or derived using data reported in the publications. Data extraction was performed independently by FJIF and JSR, using proforma designed by FJIF, JAS, and JGB. The investigators of the original studies were contacted if relevant information on eligibility or key study data were not available in the published report. An email was sent to authors explaining the nature of the systematic review and the information required together with proforma. If the author did not respond within three email attempts then no further action was taken. Where a study does not provide measures of association , the study results will be described only in narrative terms.A major issue in reviewing the published results of different epidemiologic studies examining the relation between an exposure variable and risk of the outcome is that the results are presented in many different ways. Determining antibody levels by enzyme-linked immunosorbent assay (ELISA) does not produce a common metric measurement among studies. Individuals can be classified as “responders” or “nonresponders” relative to a negative control (unexposed sera) within each study. Study-specific comparisons of these exposure variables can then be pooled. However, categories based on arbitrary cut-offs  cannot be pooled across studies.For studies where the antibody measures were analysed as continuous exposure variables we either asked the authors to reanalyse their data by collapsing the antibody data into categories or asked them to provide the standard deviation of the data so we could calibrate the estimate to represent the relative change in the risk of malaria associated with a change of one standard deviation of the antibody level. For log transformed antibody data we used log base 2 so that the relative change in malaria risk corresponds to a doubling of antibody level.P. falciparum parasitaemia (at baseline or preseason), and/or bed net use are reported. Estimates adjusted for other anti-merozoite antibodies (including antibodies to schizont protein extract) are not reported because antibody responses are typically highly correlated making it difficult to estimate their individual regression coefficients reliably; in these cases unadjusted estimates are reported. For all malaria outcomes the study-assigned P. falciparum definitions were used.ORs considerably overestimate the RR, if the incidence risk is >20%, which is often the case in highly malaria endemic areas 42, MSP-119 ). Estimates from the above-mentioned regions/alleles were used to ensure maximum comparability between studies. Separate analyses were not done for MSP-3-Ct or MSP-119 alleles because of the conserved nature of MSP-3-Ct and MSP-119 (similarly EGF domains). For these antigens, if responses to multiple alleles were investigated in the same study, the most common circulating allele in the population was included in the meta-analysis.Our aim was to obtain a single RR estimate for each study. If antibody responses to the same antigen, in the same population-based study, were reported in several publications, results from the largest sample size were used. Separate estimates were obtained for the RR associated with AMA-1 (full-length ectodomains of FVO [pro-DI-DII-DIII] and 3D7 [DI-DII-DIII]), EBA-175 , GLURP , MSP-2 , MSP-3 . For MSP-1, separate analyses were done for each region and allelic type , MAD20-like [MAD20], and RO33-like [RO33]), and processing fragments  of the RR was calculated using the formula (ln[upper limit of CI]−ln[RR])/1.96. Heterogeneity between studies was tested with the Clinical and methodological heterogeneity was explored using prespecified variables to minimize spurious findings. Variables evaluated included study design , length of follow-up, age of study participants , malaria endemicity , source of malaria cases , definition of symptomatic malaria, preparation of antigen , and method of antibody determination . Influence analysis was also performed whereby pooled estimates were calculated omitting one study at a time. Where possible, publication bias was assessed visually by plotting a funnel plot P. falciparum malaria was examined in 19, eight, seven, five, three, and six studies, respectively. Details of recombinant proteins and sero-prevalences can be found in P. falciparum malaria during follow-up was the most common outcome, examined in 29 studies; with reinfection and high density infection during follow-up examined in five and three studies, respectively. No study examined the association of anti-merozoite responses with incidence risk of severe P. falciparum malaria or P. falciparum malaria-associated mortality.The 33 studies reported data obtained from 14 separate prospective cohort studies and six separate treatment-to-reinfection population studies (42) fragments 42 is further processed into MSP-119 and MSP-133 fragments. Both MSP-119 and MSP-142 are regarded as potential vaccine candidates and have been shown to be protective in animal models 19 IgG responders had an 18% reduction in the risk of symptomatic P. falciparum malaria compared to nonresponders  with the greatest magnitude of effect seen with MAD20 and Palo Alto alleles  that is proteolytically processed into 83 kDa, 30 kDa, 38 kDa, and C-terminal 42 kDa (MSP-119 levels (log base 2) and the risk of malaria P. falciparum per doubling of antibody levels was observed  19 responses with P. falciparum high density infection or reinfection . Meta-analyses showed no association between the presence of responses to either MSP-1-EGF1 or MSP-1-EGF2 with protection against symptomatic P. falciparum  MSP-142 levels (log base 2) with incidence risk of symptomatic P. falciparum and found a reduced risk  Only one study examined the association of MSP-1P. falciparum with allelic specific MSP-1 block 2 responders compared to nonresponders was examined in four studies p = 0.39) or RO33-like allele   . There wP. falciparump = 0.031) but not MSP-1 block 2 MAD20-like repeats   and incidence risk of symptomatic  = 0.35) . There wp = 0.88; Combined results from two studies showed no evidence of an association of MSP-1 block 1 responses with risk of symptomatic falciparum malaria  and risk of symptomatic P. falciparum  but not MSP-2FC27  P. falciparum  The single P. falciparum in responders versus nonresponders  is highly conserved whereas the remainder of the sequence is defined by two major allelic types, 3D7 and K1 3D7 and MSP-3K1 responses (responders versus nonresponders) with risk of symptomatic P. falciparum3D7 responses . A high degree of heterogeneity was also seen for MSP-3K1 associations  so results were not combined , and there was also a tendency towards a protective effect in the study that examined tertiles , but combined results of two studies showed no association of anti-AMA-1FVO levels (log base 2) with incidence risk of symptomatic P. falciparum  tertiles  [40],,[74Text S1PRISMA checklist.(0.07 MB DOC)Click here for additional data file.Text S2Excluded studies, supplementary tables, and analyses.(0.54 MB DOC)Click here for additional data file.


Plasmodium infection rate and on clinically-confirmed malaria cases in sub-Saharan Africa. We carried out an epidemiological study to investigate whether LLINs impact on Plasmodium prevalence rate and the proportion of clinically-confirmed malaria cases, in five villages in the district of Toumodi, central Côte d'Ivoire.Long-lasting insecticidal nets (LLINs) are an important tool for controlling malaria. Much attention has been devoted to determine both the effect of LLINs on the reduction of Plasmodium prevalence rate, clinically-confirmed malaria cases and proportion of high parasitaemia rates in children aged 6-59 months through a series of cross-sectional surveys starting in April 2007 and repeated once every 6 months.From April 2007 to November 2008, a community-based malaria control programme was implemented in the study villages, which involved large-scale distribution of LLINs, and training and sensitization activities within the community. We determined the effect of this programme on P. falciparum prevalence rate from April 2007 to April 2008 (p = 0.029). An opposite trend was observed from November 2007 to November 2008 when P. falciparum prevalence rate increased significantly (p = 0.003). Highly significant decreases in the proportions of clinical malaria cases were observed between April 2007 and April 2008 (p < 0.001), and between November 2007 and November 2008 (p = 0.001).We observed a significant decrease in the mean Plasmodium prevalence rates among young children in the first year of the project, whereas overall clinical malaria rates dropped over the entire 18-month project period. A decrease in community motivation to sleep under bed nets, perhaps along with changing patterns of malaria transmission, might explain the observed increase in the Plasmodium prevalence rate between November 2007 and November 2008.Large-scale distribution of LLINs, accompanied by training and sensitization activities, significantly reduced  In Côte d'Ivoire, malaria remains the primary cause of health seeking at dispensaries, particularly during and shortly after the rainy season (from approximately mid-August to mid-October), accounting for 42% of all visits . MoreoveWith regard to malaria prevention, insecticide-treated nets (ITNs) have a track record of reducing malaria-related morbidity and mortality , and henResearch carried out in rural Tanzania showed that a social marketing campaign with key messages and products readily adapted to the local settings can boost the coverage rates of ITNs, result in significant health gains and represents a cost-effective intervention to prevent malaria-related death -10. In vPlasmodium prevalence rate and the number of clinical malaria cases, determined by repeated cross-sectional surveys. Plasmodium transmission by malaria vectors was investigated through entomological surveys in parallel to parasitological surveys with details presented elsewhere [The goal of the project reported here was to reduce malaria morbidity in young children by promoting the use of LLIN within households, facilitated by CHWs. The study was implemented in five villages located in central Côte d'Ivoire that are characterized by the same climate and agricultural activities. We report a descriptive study with no defined control groups, using a 'before-after' evaluation design. Particular emphasis was placed on lsewhere .The study was conducted in five villages in the health district of Toumodi, in the commune of Djekanou in central Côte d'Ivoire: Tafissou-Bringakro (Taf-Bri), Gbohua-Angbavia (Gbo-Ang), Alluminankro-N'Kloidjo (All-N'Kl), N'Da Dibykro-N'Da Kouassikro (N'Da-N'Da) and Gbakokro-Abokro (Gba-Abo) Figure . AccordiThe mean annual precipitation in this area is slightly above 1150 mm, and the mean annual temperature is 26.5°C. There are two seasons; a rainy season between April and October, and a dry season between November and March. The main activity in these communities is subsistence agriculture, supplemented with some cash crop, mainly consisting of cocoa, coffee and rubber production. The nearest health infrastructure is located in Taf-Bri, which is run by a nurse and three assistant nurses. Four of the villages have a primary school each . At the time of our study, access to the power grid was available in two villages only (Taf-Bri and Gbo-Ang). Four villages had access to running water .Anopheles gambiae s.s., the main malaria vector in central Côte d'Ivoire [An. funestus and An. pharoensis were found to be infected .Two entomological surveys were carried out in April and July 2008, after net distribution to householders. We recorded biting rates and entomological inoculation rates (EIRs) for d'Ivoire -16. The An. gambiae collected in this study area showed very high efficacy of these compounds. For example, we recorded a high KD rate with low fluctuations (range between 96.7% and 100%) and also a high mortality rate (range between 91.6% and 100%). Hence, there is no indication of resistance among adult An. gambiae s.s mosquitoes in the study area.The mean knock down (KD) and mortality rates recorded after performing standard World Health Organization (WHO) Pesticide Evaluation Scheme (WHOPES) tube bioassay tests in October 2008 with DDT, deltamethrin and permethrin on Our study was approved by the Ivorian Ministry of Health through the national malaria control programme (NMCP). The heads of households in the study villages were informed about the objectives and procedures of the study and the parents or legal guardians of participating children gave written informed consent. Patients with malaria-related symptoms were treated free of charge according to the national malaria policy (i.e. artesunate plus amodiaquine) regardless of whether or not they participated in the cross-sectional surveys.In April 2007, in parallel with a cross-sectional clinical survey, a questionnaire was administered to the heads of households in the five villages. We employed a pre-tested questionnaire to obtain data on demographics , professional and economic activities of the adult population, assessment of construction material for walls and roofing of houses, connection to the power grid, and appraisal of wealth indicators based on the presence of various assets . Householders were interviewed about major health problems, preventive measures against mosquito bites , the availability of latrines, and disposal of wastewater and solid waste. Additionally, in May 2007, a total of 16 focus group discussions (FGDs) were conducted with young men and women, adults and older individuals in three of the study villages . Participants in the FGDs were recruited randomly with the help of CHWs. These FGDs centred on malaria causes and symptoms, and preventive measures employed against mosquito bites and commonly used antimalarial treatments.Malariometric measurements were carried out in April 2007 before net distribution in the study villages to assess malaria parasitaemia and clinical cases in children aged 6-59 months. A finger prick blood sample was taken, thick and thin blood films were prepared on microscope slides and they were air-dried on the spot. The slides were transferred to a nearby laboratory and processed as detailed below.In July 2007, we received 3600 LLINs (PermaNet 2.0) from Vestergaard-Frandsen, of which approximately 2800 nets were distributed in the study villages. The remaining 800 nets were distributed in small hamlets ("campements") that surround the study villages. LLIN distribution started in early August 2007 and was continued until October 2008. Heads of households were encouraged to use the nets for children younger than 5 years of age in order to protect them from mosquito bites. In July 2007, in collaboration with the non-governmental organization Stop Malaria International (SMI), all nets previously in the area and used for more than 30 months were treated free of charge with a long-lasting insecticide treatment . Heads of households were invited to send nets to a designated community location and the CHWs were trained by SMI to treat the untreated nets. Overall, 90 nets were treated according to the manufacturer's procedures.Subsequent to the initial evaluation, a series of sensitization campaigns  were conducted in each village. The aim of these campaigns was to improve knowledge on malaria transmission and preventive measures, and to increase acceptance of LLINs by the beneficiary communities. The activities started with a mass sensitization campaign that focused on the promotion of LLINs by the CHWs. In each village, CHWs held two meetings with the communities, during which everyone was invited to a designated location where the advantages of sleeping under a LLIN were explained and each householder received additional information to ensure that LLINs are used properly. Regular follow-ups were held in the intervention zones after net distribution. Communities were also informed about the importance of re-treating old nets. The sensitization campaign started in July 2007 and ended after reaching a high coverage level in March 2008 .LLINs were ordered and packaged according to community preferences determined during an initial social survey carried out in April 2007. In agreement with village authorities, the CHWs were identified as net sellers. LLINs were sold at a low price .Two training activities were carried out in June and August 2007. First, with the support of the NMCP and the regional health district, CHWs were trained on the causes of malaria transmission, preventive measures against mosquito bites and the management of uncomplicated malaria cases among children under the age of 5 years. Training focussed on the recognition of typical symptoms of uncomplicated malaria, as well as severe symptoms warranting referral to a nearby health centre. Secondly, the CHWs were also trained to perform a follow-up of severe malaria cases, to act as intermediaries between the health services and the community, and to provide advice to mothers/caregivers of under-5 year-old children on how to manage uncomplicated malaria cases. The activities related to mothers' and caregivers' practices and behaviours in response to malaria attacks were assessed by an MSc student specializing in socio-anthropology who was in charge of this appraisal.P. falciparum were treated free of charge, and ACTs were given to CHWs to treat new uncomplicated malaria cases detected in agreement with the local nurse after the departure of our research team. CHWs kept drugs and supplied them to mothers/caregivers on demand for treating new uncomplicated malaria cases. CHWs supervised and monitored mothers' and caregivers' practices and behaviours directly by following them while they treated malaria cases that occurred in the household. A part of the money collected when nets were sold was used to assure that ACTs were available during the length of the study.Antimalarial drugs (i.e. artemisinin-based combination therapy (ACT)) were kindly provided by a pharmaceutical company  and administered free of charge. Drugs were packaged in plastic bags labelled for one age group (0-59 months) according to standard recommendations by WHO. During the survey, children with measured fever (axillary temperature > 37.7°C) and infected with Four repeated cross-sectional surveys were carried out in the study villages to assess malaria parasitaemia and clinical malaria in children aged 6-59 months. The surveys were done always in April and November, both in 2007 and 2008, i.e. once before the distribution of LLINs in August 2007 and three times afterwards. During the surveys, mothers and caregivers of under 5-year-old children were invited to accompany their children to a designated community location where a finger prick blood sample was taken from each child. Previously, all mothers who had one or several children below the age of 5 years were listed by the CHWs. During each cross-sectional parasitological survey, previously selected children aged 6-59 months came to a designated place with their mothers or caregivers and the children were examined, adhering to a standard protocol.Plasmodium species and gametocytes were identified and counted against 200 leucocytes. In case fewer than 10 parasites were found, the reading was continued for a total of 500 leucocytes. Parasitaemia was expressed by the number of parasites per μl of blood, assuming a standard count of 8000 leucocytes/μl blood. For quality control, 10% of the slides were randomly selected and re-examined by a senior laboratory technician.In the laboratory, air-dried thick and thin blood smears were stained with Giemsa for 45 min. Slides were examined by the same experienced laboratory technician throughout the study under a microscope at high magnification (x 100). In our study, all children having an axillary temperature > 37.7°C were considered as fever cases. Clinical malaria was defined as fever plus parasitaemia . In all Every 2 months, the coordinator of the project had meetings with the CHWs in order (i) to assess the number of new LLINs sold and (ii) to evaluate the number of new LLINs installed in household sleeping rooms. The goal of these meetings was to assess the new bi-monthly coverage rate of LLINs in each village, among children aged 6-59 months, and at the household level in general.The monthly number of new LLINs sold was used to assess the overall number of LLINs sold. Additionally, every two months, CHWs assessed the number of LLINs installed and properly used in households. During the net promotion campaign, the number of LLINs sold per household was known because CHWs sold the LLINs only to heads of households. As the number of persons living in each household was determined beforehand, CHWs sold exactly the number of nets that were needed per household. Net coverage rates recorded in CHWs log-books were checked during two monitoring and evaluation visits in the study sites (April and October 2008).village code)" subcommand in the "logistic" subroutine in Stata 9.0). To allow for known seasonal variation in malaria rates in the study area [vs. April 2008 and November 2007 vs. November 2008. No formal comparisons were made within individual villages as this was outside the remit of the study; differences between villages were adjusted for by treating these as clusters. Statistical significance was set at the conventional alpha = 0.05 (5%) level.Data were analysed in STATA version 9.0 . Monthly malaria prevalence rates were computed for April 2007, November 2007, April 2008 and November 2008 for each village separately and then for all villages combined. These rates are reported with their 95% confidence intervals (CIs). As it was only possible to evaluate random samples of children in each village at each of the four assessment times, the CIs are finite population corrected (i.e. adjusted for the known population of each village). Overall monthly prevalence rates were compared before and after the distribution of the LLINs, using logistic regression models that were adjusted for the clustering of individuals within villages  possessed a net, only 3.2% of householder reported that they actually used these nets (corresponding to 237 persons). A common reason stated by adults for not using a net was that the nets were not felt appropriate. The proportion of children below the age of 5 years who slept under a net was low (14.2%). With regard to personal protective measures against mosquito bites, fumigating coils was the most widely used tool (28.0%). Approximately one out of seven households used insecticide sprays, mainly applied on walls and ceilings (15.2%).The preferred colour for the use of LLINs was blue; almost half of the interviewed households mentioned this colour (45.3%), followed by green (22.8%) and white (12.1%). The preferred shape of LLINs mentioned by the households is rectangular (64.1%). More than three-quarter of the householders interviewed (77.1%) agreed to pay F CFA 1000 (approximately US$ 2), corresponding to one third of the LLIN price at the national public health pharmacy at the time of our study.According to the FGDs, the main causes of malaria were the sun, tiredness due to field work and household work, inadequate nutrition, cold and constipation. Concerning the use of preventive measures against mosquito bites, the majority of respondents mentioned anti-mosquito measures such as ITNs, fumigating coils, modern and traditional insecticides.With regard to malaria symptoms, fever was clearly the most important one, followed by loss of appetite, tiredness, abdominal discomfort and diarrhoea. Additionally, yellow eyes and yellow urine were frequently mentioned. Modern health care providers interviewed moreover indicated constipation, diarrhoea, nausea, body aches and pains.Traditional medicine is primarily sought when a malaria episode occurs. Modern drugs were used to a lesser extent, often simultaneously with traditional medicine.A total of 11 CHWs in the five study villages were trained on malaria transmission, preventives measures against mosquito bites and the management of uncomplicated malaria cases among children aged 6-59 months.With the support of SMI, CHWs were trained in the treatment of old nets. CHWs also served as intermediates between the health services and the community, and they were responsible for providing advice to mothers and caregivers of under-5 year-old children on how to manage uncomplicated malaria cases.Following the sale of LLINs to household members, CHWs assisted in the installation of the LLINs. Fourteen months after net distribution, the proportions of children below the age of 5 years who slept under a LLIN were 47.3%, 56.4%, 51.1%, 55.4% and 50.2% in the villages of All-N'Kl, Gba-Abo, N'Da-N'Da, Taf-Bri and Gbo-Ang, respectively. Table P. falciparum was clearly the predominant malaria parasite in the study villages. Its frequency among positive blood films obtained during the repeated cross-sectional surveys was above 87%. Single species infections with P. malariae were found in 3.7-6.2% of the positive slides. Mixed species infections with P. falciparum and P. malariae ranged between 1.2% and 3.8%. Neither P. ovale nor P. vivax single infections were diagnosed throughout the study. The annual gametocyte rates ranged between 2.4% and 3.7%.P. falciparum prevalence (number of positive children/number of children examined) obtained during repeated cross-sectional surveys carried out between April 2007 and November 2008, in the five study villages among children aged 6-59 months. The data revealed that the prevalence of P. falciparum was high in November 2007 and 2008 (during the short rainy season) and low in April 2007 and 2008 (beginning of the long rainy season).Table P. falciparum prevalence occurred from April 2007 to April 2008 (p = 0.029), although it was noted that the rate increased in this period in one of the study villages (Gbo-Ang). An opposite trend was observed from November 2007 to November 2008; P. falciparum prevalence rate increased significantly (p = 0.003), with consistent increases observed in each of the five villages.An overall significant decrease in the Table Sleeping under a LLIN did not have any effect on the proportion of high parasitaemia in the study villages. Indeed, the differences between the proportions of high parasitaemia recorded among children who slept under a LLIN and those who did not in the night before the survey showed no statistical significance: All-N'Kl (p = 0.246), Gba-Abo (p = 0.084), N'Da-N'Da (p = 0.365), Taf-Bri (p = 0.953) and Gbo-Ang (p = 0.111).P. falciparum after blood examination . A highly significant fall in clinical malaria rates was observed both between April 2007 and April 2008 (p < 0.001), and between November 2007 and November 2008 (p = 0.001). These trends were reflected in all five villages.Table Plasmodium prevalence rate from November 2007 to November 2008 could be explained by the fact that during the latter period, conditions such as rainfall and humidity were more conducive to mosquito development. Generally, when environmental conditions are favourable , more productive An. gambiae breeding sites are formed, contributing to the increase of Plasmodium infection [Plasmodium prevalence rate (13% reduction when the control group did not have any nets and 10% reduction when the control group had untreated nets) [Plasmodium prevalence rate could also be due to the fact that LLINs were not properly used by householders, specifically because we recorded a reduction in malaria transmission in April and July 2008, 9 months after net distribution (unpublished data). During this low transmission and biting period, the perceived benefits of reduction in mosquito bites and of malaria were considered not to be important by household members. Recent studies carried out in Burkina Faso [The increase recorded in nfection . Previoued nets) -21. Incrina Faso  showed tina Faso .Plasmodium infection rates recorded among children aged between 6 and 59 months. Indeed, activities carried out few months after net distribution by CHWs and mass sensitization campaigns undertaken in the study villages contributed to reduce drastically Plasmodium prevalence rate. It is conceivable that the motivation for the use of LLINs is high shortly after mass and household-to-household sensitization campaigns. This argument is confirmed by a recent study carried out in Burkina Faso showing that the motivation for the use of LLINs decreased after less than a year [An. gambiae biting rates are supposed to be high. During this study period, particularly in April, during the rainy season, previous studies carried out in three urban cities of West Africa  showed that sleeping under an ITN the night before the survey was protective against Plasmodium infection [Plasmodium prevalence rate was strongly influenced by socioeconomic status, and the methods used for prevention depended on their perceived cost [Plasmodium infection rates, perhaps because in villages where we recorded the highest Plasmodium infection rates, the use of fumigating coils and insecticides was high.From April 2007 to April 2008, a statistically significant reduction was observed in the overall n a year . The incnfection -25. Addived cost . In our An. gambiae s.s., is resistant to permethrin and other pyrethroids [Another important finding of our study is that we observed a significant decrease in the overall proportions of clinical malaria cases among children less than 5 years old during both study periods, from April 2007 to April 2008 and from November 2007 to November 2008. This observation might be explained by the considerable number of household members sleeping under LLINs instigated by the household-to-household and mass sensitization campaigns undertaken before and after net distribution. Of note, in this project, although the price of nets was relatively low, we did not carry out free distribution of nets. Similarly, in Tanzania, 10 years ago, social marketing of treated nets was identified as an effective means for malaria control in rural settings. Eighteen months after launching promotion of treated nets, 46% of 312 families with children aged less than 5 years reported that their children were sleeping under treated nets . A reducethroids ,28.Finally, in our study, no significant statistical difference was found between users and non-users of LLINs concerning the prevalence of high parasitaemia levels (> 5000 parasites/μl blood). This observation is in agreement with previous studies carried out in the savannah village of Kafine, northern Côte d'Ivoire, where no difference was found between user and non-user groups of mosquito nets with regard to the prevalence of high parasitaemia, or gametocytes or to the mean parasite load . GeneralPlasmodium prevalence from April 2007 to April 2008 and to significantly reducing the number of clinical malaria cases. However, 8-9 months after net distribution, a decrease in the motivation for the use of nets probably favoured a significant increase in the overall Plasmodium prevalence rate from November 2007 to November 2008. Thus, continued long-term social marketing for promoting LLINs holds promise for effective malaria control in rural and urban malaria endemic countries.To conclude, training and sensitization activities undertaken before and after net distribution contributed to reducing The authors declare that they have no competing interests.BGK: designed experiments, coordinated field activities, collected and analyzed data, wrote and revised the paper; HG: designed the study and participated in the coordination of the field activities; CE: designed experiments, collected data and analysed socio-anthropological data; CN: contributed to the study design and revised the paper; FR: designed the study, participated in the coordination of field activities and revised the paper; JU: participated in the design of the study and revised the paper; BEF: assisted with the statistical analysis and revised the paper; ABT: designed the study, participated in the coordination of field activities and revised the paper. All authors have read and agreed with the content of the submitted manuscript.


Plasmodium falciparum. Polymorphisms at the human IRF1 locus have been previously found to be associated with the ability to control P. falciparum infection in populations naturally exposed to malaria. In order to test whether genetic variation at the IRF1 locus also affects the risk of developing severe malaria, we performed a family-based test of association for 18 Single Nucleotide Polymorphisms (SNPs) across the gene in three African populations, using genotype data from 961 trios consisting of one affected child and his/her two parents . No significant association with severe malaria or severe malaria subphenotypes  was observed for any of the SNPs/haplotypes tested in any of the study populations. Our results offer no evidence that the molecular pathways regulated by the transcription factor IRF-1 are involved in the immune-based pathogenesis of severe malaria.Interferon Regulatory Factor 1 (IRF-1) is a member of the IRF family of transcription factors, which have key and diverse roles in the gene-regulatory networks of the immune system. IRF-1 has been described as a critical mediator of IFN-gamma signalling and as the major player in driving TH1 type responses. It is therefore likely to be crucial in both innate and adaptive responses against intracellular pathogens such as  Plasmodium falciparum malaria during childhood, but a small proportion of children experience severe complications, the clinical outcome depending on many factors, including the genetic make-up of the human host. Malaria is recognised as a very strong selective force in the recent evolutionary history of the human genome Severe malaria is a life-threatening disease that kills over a million individuals every year, with 90% of the deaths occurring in Sub-Sahara African children below the age of five P. falciparum.Members of the Interferon Regulatory Factor (IRF) family of transcription factors have key and diverse roles in the gene-regulatory networks of the immune system IRF1 polymorphisms were associated with the ability to control P. falciparum infection: in adult asymptomatic individuals living in rural villages IRF-1 alleles were associated with a higher prevalence of P. falciparum infection; the same alleles were associated with a higher parasite density in children with uncomplicated and severe malaria In two recent complementary association studies conducted in rural communities and malaria patients of Burkina Faso, we found that IRF1 locus contributes to a child's risk of developing severe malaria, we conducted a large multi-centre association study. As human populations in Africa show a great genetic diversity P. falciparum transmission intensity In order to investigate whether genetic variation at the IRF1 genetic variation in severe malaria, and by considering multiple populations, we able to investigate regional differences, whilst standardizing phenotype definition and study design.Here we assess the role of IRF1 genetic region (Nucl3ar software package We have genotyped 18 Single Nucleotide Polymorphisms (SNPs) in the c region  in 961 nThe allele frequencies calculated among the trio founders  differ fG = 40.8%; Kenya, HK = 38.7%; Malawi, HM = 38.0%. For comparison, in the HapMap populations Yoruba  and CEPH (Utah residents with ancestry from northern and western Europe) the heterozygosity of the IRF1 gene is the highest among the Th2 cluster (HY = 38% and HC = 45%) while the heterozygosity of the neighbouring IL5 gene is the lowest among the Th2 cluster (HY = 25% and HC = 21%) (HapMap data release 3.1).The power of a TDT analysis to detect transmission distortion is a function of the number of heterozygous parents in the sample and the level of distortion at the locus We conducted single marker and haplotype TDT association analysis using a custom script implemented in R statistical software and based on Transmit IRF1 SNPs revealed no evidence of association  across all populations, neither with severe malaria or with the sub-phenotypes of cerebral malaria (CM) and severe malaria anaemia (SA) under any of the models tested . The results for the test of association with severe malaria under the additive model are shown in The application of the single locus TDT to the 18 −19) and Kenyan (P = 9×10−5) populations, respectively. The effect of the HbS allele could not be tested in the trios from Malawi, due to its low allele frequency in this population and therefore the number of informative trios is not sufficient to compute the statistic.In comparison, the HbS allele shows, as expected, a very strong protective effect against severe malaria, with a reduction in risk of 81% and 87% in the Gambian (P = 6×10Linkage Disequilibrium (LD) analysis was performed using the HAPLOVIEW application We tested the common haplotypes (frequency higher than 5%) within each LD block for association with severe malaria phenotypes. Similarly to the single locus TDT, no significant association was observed  in any of the study populations. Rodent malaria studies have shown that IFN-gamma is a key inducer of the immune effector mechanisms that are responsible for the control of both pre-erythrocytic and blood-stage malaria infection IRF1 polymorphisms are associated with the control of P. falciparum infection , both in healthy adult subjects and in children with uncomplicated and severe malaria. In those studies we did not find conclusive evidence for the involvement of this locus in the severe manifestations of malaria disease. Indeed, only a trend of association with small differential risk of severe disease was suggested We have previously investigated the role of the transcription factor IRF-1, a critical effector molecule in IFN-gamma signalling, in susceptibility to malaria by conducting genetic association studies in Burkina Faso based on cross-sectional parasitological surveys and case-control clinical studies. We have shown that IRF1 locus affects susceptibility to severe malaria by performing a family based association test in nuclear trios consisting of an affected child and his/her two parents from The Gambia, Kenya and Malawi. The trio design is robust to population stratification. We did not observe any significant associations between the SNPs or haplotypes analysed in severe malaria in any of the three study populations.In the present study we investigated whether genetic variation at the IRF1 polymorphisms on severe malaria may be too small to be detected with this sample size, or may affect only a sub-phenotype of severe malaria. It is likely that the genetic basis of susceptibility to malaria is built upon many different protective genes, each individually resulting in small effects 2 = 1) with the causal polymorphism. Overall, we find no evidence that IRF1 is a strong determinant of susceptibility to severe disease in our study populations.There are various possible interpretations of these findings. The effects of IRF1 functional variants with an effect on malaria susceptibility/resistance exist in the Fula and Mossi populations of Burkina Faso but not in the study populations presented here. Moreover, we cannot rule out that a different pattern of association could be observed in other African populations as a consequence of a different genetic background. Further disease-association studies across Africa and including Burkina Faso will be needed to answer this question.When considering the exceptionally high diversity of the human genome in African populations IRF1 polymorphisms with the ability to control P. falciparum infection and the lack of association with susceptibility to severe disease actually reflect differences in the molecular mechanisms underlying protective/pathological immune responses at different stages of infection. The gene regulatory networks of the immune system that are activated during malaria infection and that are responsible for parasite killing may differ from those leading to an over-reaction to the parasite that is harmful to the host, depending for example on the molecular triggers from the parasite or on the tissues involved. That this could be the case has been recently suggested by a genome-wide linkage analysis of parasite density and mild malaria in two Senegalese villages. Regions of linkage showed little if any overlap with genes previously described to be associated with severe malaria, while showing an overlap with genes involved in asthma and atopy related traits Finally, it might be possible that the association of Severe malaria patient samples were collected as part of ongoing epidemiological studies at the Royal Victoria Hospital, Banjul, The Gambia (555 trios); the Kilifi District Hospital, Kilifi, Kenya (204 trios); and the Queen Elizabeth Central Hospital, Blantyre, Malawi (202 trios). Nuclear family trios comprised one affected child and his two parents.The research protocols used in this study have obtained approval from the relevant ethics committees: the United Kingdom approval was given by the Oxfordshire Clinical Research Ethics Committee (OxREC); the Gambian approval was given by the Gambia Government/Medical Research Council (MRC) Joint Ethics Committee; the Kenyan approval was given by Kenya Medical Research Institute (KEMRI)/National Ethical Review Committee; the Malawi approval was given by the Health Sciences Research Committee and College of Medicine Research Committee (COMRC).All DNA samples were collected and genotyped following informed consent from participants. Some of the samples for this study were collected with written consent, others with verbal consent. In all cases the advice of the local ethics committee about the appropriate ways of recording consent in the local research context was followed.Nucl3ar software package The family trios were assessed for pedigree misspecification using a panel of 48 additional markers spread across the genome and the P. falciparum on blood film and clinical features of severe malaria All cases were children admitted to hospital with evidence of The sub-clinical phenotype frequencies in the probands were: (i) The Gambia , (ii) Kenya  and (iii) Malawi .The median (range) ages of the Gambian, Kenyan and Malawian cases expressed in months were 41 (0-156), 26 (0-121) and 31.5 (2-156), respectively.IRF1 gene were identified from public databases  and the literature.Single Nucleotide Polymorphisms (SNPs) in the region 10 kb upstream to 10 kb downstream of the IRF1 gene of the 18 SNPs are displayed in 18 polymorphisms were selected on the basis of the following criteria: validated status by frequency, haplotype tagging SNP (htSNP) in the HapMap Yoruba population, overall coverage of the locus with regular spread (density of 1 SNP every 2 Kb on average), encompassing exonic, intronic, regulatory regions of the gene. Database identifiers and location with respect to the Genomic DNA samples underwent whole genome amplification through either Primer Extension Pre-amplification (PEP) The genotypes for the selected polymorphisms were determined through the SEQUENOM© MassARRAY™ System Haplotypes have been constructed using family data and the Stephens-Donnelly method http://www.gmap.net/perl/marker/marker_entry) application. Typed single nucleotide polymorphisms (SNPs) are represented on the vertical axis and ordered by chromosome position as in LD metrics and graphs showing LD patterns have been generated using the MARKER  has been used for comparison of the results.Trio association analysis was performed using the transmission disequilibrium test (TDT). This tests for family-based association in the presence of genetic linkage between a SNP and severe malaria Haemoglobin genotyping was previously performed on all samples included in the study A P-value of 0.05 has been considered as the threshold for statistical significance.


Anders Bjorkman and colleagues report results from a cross-over trial evaluating rapid diagnostic testing for malaria diagnosis in Zanzibar. Plasmodium falciparum malaria is being suggested to improve diagnostic efficiency in peripheral health care settings in Africa. Such improved diagnostics are critical to minimize overuse and thereby delay development of resistance to artemisinin-based combination therapies (ACTs). Our objective was to study the influence of RDT-aided malaria diagnosis on drug prescriptions, health outcomes, and costs in primary health care settings.The use of rapid diagnostic tests (RDTs) for p<0.001). Prescriptions of antibiotics were higher after RDT than CD alone, i.e., 372/1005 (37%) and 235/882 (27%) , respectively. Reattendance due to perceived unsuccessful clinical cure was lower after RDT 25/1005 (2.5%), than CD alone 43/882 (4.9%) . Total average cost per patient was similar: USD 2.47 and 2.37 after RDT and CD alone, respectively.We conducted a cross-over validation clinical trial in four primary health care units in Zanzibar. Patients of all ages with reported fever in the previous 48 hours were eligible and allocated alternate weeks to RDT-aided malaria diagnosis or symptom-based clinical diagnosis (CD) alone. Follow-up was 14 days. ACT was to be prescribed to patients diagnosed with malaria in both groups. Statistical analyses with multilevel modelling were performed. A total of 1,887 patients were enrolled February through August 2005. RDT was associated with lower prescription rates of antimalarial treatment than CD alone, 361/1005 (36%) compared with 752/882 (85%)  for P. falciparum malaria in all regions with drug-resistant malaria. In ACT, artemisinin derivatives  are used in combination with another antimalarial to reduce the chances of P. falciparum becoming resistant to either drug.Every year, nearly one million people (mainly children living in sub-Saharan Africa) die because of malaria, a subtropical and tropical parasitic disease. Although several parasites cause malaria, P. falciparum becoming resistant to ACT should also be reduced by giving ACT only to people who definitely have malaria. Unfortunately, many people who do not have malaria are given ACT because symptom-based  diagnosis cannot always distinguish between patients whose fever is caused by malaria and those who have a different infection and who would, therefore, gain more benefit from other treatments. Microscopic detection of parasites in blood smears would greatly improve the accuracy of malaria diagnosis, but this test is rarely available in rural clinics in developing countries. Might the recently developed “rapid diagnostic tests” (RDTs) for P. falciparum provide an alternative way to improve malaria diagnosis and thus reduce the overuse of ACT? In this “cross-over trial,” the researchers investigate the effect of the routine use of an RDT for the diagnosis of malaria on ACT prescribing, health outcomes, and costs in four primary health-care clinics in Zanzibar (part of the United Republic of Tanzania), one of the first regions in sub-Saharan Africa to introduce ACT.The chances of Each clinic used RDT-aided symptom-based clinical diagnosis of malaria  and symptom-based clinical diagnosis (the CD arm) in alternate weeks to decide whether patients attending with fever had malaria. ACT was prescribed to everyone diagnosed with malaria; during RDT weeks only patients with positive RDT results were prescribed ACT. During the trial, 36% of the 1,005 patients in the RDT arm were prescribed ACT compared to 85% of the 882 patients in the CD arm. 37% and 27% of the RDT and CD arm patients, respectively, were prescribed antibiotics and fewer RDT-arm patients than CD-arm patients returned to the clinic because they still felt ill. The overall cost per patient was similar in both arms. The researchers also report that 23% of the antimalarial treatments given to patients in the RDT arm and 80% of those given to patients in the CD arm were given to people with no microscopically detectable parasites in their blood. Importantly, none of the 26 patients in the RDT group who had positive smears but who were not treated with antimalarial drugs because of a negative RDT result developed severe malaria.These findings suggest that the replacement of clinical diagnosis alone with RDT-aided diagnosis may reduce the number of people prescribed ACT who do not have malaria and may increase the number of patients given antibiotics for nonmalarial illnesses without increasing costs. However, while the health-care workers involved in this study only prescribed ACT to those patients in the RDT arm who had a positive RDT result , evidence from other studies suggests that health-care workers often give antimalarials to patients with negative RDT results. Consequently, these findings may not be generalizable to other clinics. Nevertheless, it is reassuring that none of the patients who had malaria that was detected by blood smear but that was missed by RDT subsequently developed severe malaria. This finding, if replicated, might persuade health-care workers to trust RDT results rather than prescribing ACT to everyone with a fever “just in case.”http://dx.doi.org/10.1371/journal.pmed.1000070.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Zeno Bisoffi and colleaguesThis study is further discussed in a malaria (in English and Spanish)The MedlinePlus encyclopedia contains a page on malaria  and on rapid diagnostic tests for malaria. Their 2008 World Malaria Report includes information about global efforts to control malaria and the latest information on malaria in the United Republic of TanzaniaInformation is available from the World Health Organization on malaria (in English and Spanish)The US Centers for Disease Control and Prevention provide information on Roll Back Malaria Partnership on its approach to the global control of malaria and on artemisinin-based combination therapiesInformation is available from the  Plasmodium falciparum malaria have been increasing in sub-Saharan Africa since the early 1990s, concomitantly with spread of resistance to commonly used monotherapies, i.e., chloroquine and sulfadoxine-pyrimethamine Morbidity and mortality due to P. falciparum malaria offers a potential alternative in remote and poorly resourced health facilities that are beyond the reach of high-quality microscopy services Symptom-based or clinical malaria diagnosis has proven to be quite unspecific Zanzibar was among the first regions in sub-Saharan Africa to introduce ACT, free of charge through public health care, as both as first- and second- line treatment for uncomplicated malaria, which are provided free of charge through public health care. In view of the fact that many patients with fever are prescribed ACT without being malaria infected, the present study was undertaken to assess, on a wide scale, the added value of RDT to clinical diagnosis (CD) alone for management of patients of all ages presenting with fever at primary health care facilities. The hypothesis was that RDT-aided diagnosis of fever patients would improve rational use of ACTs and possibly other necessary treatments, such as antibiotics to non-malaria patients, with an overall improved health impact.The trial was conducted in four Primary Health Care Units (PHCUs) in Zanzibar, namely, Muyuni and Uzini on Unguja Island, and Kinyasini and Mzambarauni on Pemba Island. The selection of the four study sites aimed to provide a representative picture of Zanzibar with regard to malaria epidemiology as well as previous use of RDT in Zanzibar. By the time of the trial, malaria transmission in Zanzibar was generally considered endemic As in other PHCUs in Zanzibar, registered nurses, with 3 to 4 y of formal training, were responsible for providing out-patients care in all study sites. All nurses involved in the study had previously been trained in malaria case management, i.e., diagnosis and treatment including the IMCI algorithm, by Malaria Control Programme and District Health Management Teams of Ministry of Health. In addition and prior to the study start the staff members of all four health facilities received a one-day training on the use of RDTs according to the manufacturer's instructions. This training covered performance of the test and interpretation of the result. Specific instructions were also given to the nurses to consider other treatments and referral especially in children with RDT-negative results and to encourage their guardians or mothers to come back with their children if fever persisted or condition deteriorated. The study design was described in detail to the staff. The nurses were responsible for study implementation. The nurses received a salary supplementation of Tanzanian Shillings (TSh) 25,000  per study week. Their ordinary salary was equivalent to about USD 136 at the time of the trial.This was a nonrandomized four-centre clinical trial but with weekly cross-over validation comparing CD plus RDT (CD+RDT) versus CD alone. Each health facility alternately used CD+RDT or CD alone on a weekly basis. Two sites were allocated to CD+RDT the first week of the trial and the other two to CD alone. During the CD-alone weeks RDTs were not to be used. During RDT weeks the study nurses were encouraged to give antimalarial treatment based on the RDT results, i.e., to prescribe ACT only to RDT-positive patients. They were visited by a member of the research team at least once weekly.http://www.clinical.trials.gov with study identification NCT00549003.The study was conducted in accordance with Good Clinical Practice The study was conducted during two periods in 2005: 15 February to 15 April, and 30 May to 10 August. These periods thus included both low and peak seasons for malaria transmission in Zanzibar. All patients of all ages with a past history of fever within last 48 h and symptoms compatible with uncomplicated malaria were eligible and screened for the study. Other inclusion criteria included provision of informed consent and living within the catchments area of the health facility. Patients were not enrolled if previously included in the study or if the study nurse was not present at the health facility at the time of attendance.Treatment of uncomplicated malaria was prescribed according to national policy in Zanzibar: artesunate with amodiaquine as first-line treatment and artemether with lumefantrine as second-line treatment. Drug intake was unsupervised. Patients with severe manifestations of malaria or any other danger signs were required to be referred to the next level of care for further treatment. Study nurses were required to prescribe other treatments in accordance with the IMCI guidelines and their general clinical judgement.All enrolled patients were issued a prescription book in which clinical findings and prescribed medications were to be recorded during a 14-d follow-up.Patients were instructed to return for assessment at any time if symptoms persisted or deteriorated, but also if symptoms recurred during the 14-d follow-up period. All patients were also asked to report back to the health facility routinely 14 d after enrolment. Patients were to be actively followed up if they did not report back within 14+2 d. Upon the study completion, each participant then received one insecticide-treated net free of charge.If a patient made an unscheduled visit between days 1 through 13 due to fever, the study nurse was to prescribe new treatment(s) based on the diagnostic tool available  at time of reattendance or to refer the patient. Patients developing symptoms of severe malaria were to be referred to the next level of health care. On the scheduled day 14 follow-up visit, patients were asked to report their perceived health status and intake of any concomitant medicines during follow-up. Concomitant medications were to be recorded in the patient case record form, regardless of whether the patient returned to the health facility of enrolment or attended any other health facility.CD of uncomplicated malaria was generally based on presence or history of fever and absence of clear symptoms indicating alternative causes of fever, e.g., otitis, acute respiratory tract infection, etc. For children below age 5 y, CD was based on the IMCI algorithm. We used a histidine-rich protein (HRP) 2 based test, Paracheck Pf , as RDT. The RDT result was provided to the patient in support of the treatment prescribed.Thick blood smears (BSs) were collected from all patients at enrolment, on the scheduled day 14 follow-up visit, and at any unscheduled reattendance at the health facility during follow-up days 1 through 13. All BSs were brought to a central laboratory and independently examined by two qualified microscopists, reader 1 (R1) and reader 2 (R2), after staining with 3% Giemsa according to standard procedures According to initial sample size calculations, a total of 850 participants were needed to identify an assumed reduction in malaria diagnosis of 50% with the addition of RDT to clinical diagnosis, at a 5% significance level and a power of 80%, without controlling for design effect of clustering within PHCUs. However, during the initiation of the trial the Ministry of Health and Social Welfare requested that the study be powered also for secondary outcomes. A higher number of participants were thus included in the trial, i.e., up to 1,887. The design effect was not initially accounted for in the power calculation but was accounted for in the final statistical analyses by multilevel modelling. Multilevel analysis with regard to weeks was not applied since observed slight seasonal variation is not based on weekly differences.p-values (two-sided) were calculated, where applicable. Statistical significance was defined as p<0.05.Patient data were recorded in case report forms. The final BS, see above, was regarded as the gold standard in the analyses of sensitivity and specificity for the respective diagnostic procedures. Data were entered in Microsoft Excel and analyzed in Epi Info version 3.3.2 and Stata 10. Two-level models with sites (PHCUs) as second level clustering were applied. Odds ratios (ORs) with 95% CIs and associated Chi-square tests and A total of 9,346 patients attended the four health facilities during the study. There was no significant difference in rate of fever among patients attending during the first and second substudy period, i.e. 1,587/4,177 (38%) and 2,108/5,169 (41%), respectively. Of these 3,695 fever patients a total of 1,887 met the inclusion criteria , of whomA total of 1,005 and 882 patients were allocated to the CD+RDT and CD-alone group, respectively. There was a balanced enrolment rate among the four health facilities (range 424 to 528 patients per site). There was a tendency for the number of patients enrolled per week to increase with time, i.e., in parallel with the seasonal increase of malaria transmission during the second study period. However, no systematic difference in patient attendance or enrolment was found between CD+RDT and CD-alone weeks. The study period included an additional CD+RDT week in two health facilities, explaining the higher overall number of patients enrolled in the CD+RDT group. Baseline characteristics  were similar in the two study arms (unpublished data). Parasite prevalences were also similar—305/1005 (30%) and 247/882 (28%)—as were mean parasite densities (unpublished data) in the CD+RDT and CD alone groups, respectively.p<0.001). The rate of antimalarial prescriptions in the CD+RDT group was particularly low (16%) in patients above age15 y, whereas the prescriptions rates were similar in all age categories of CD-alone patients, ranging from 84% to 87%  in CD+RDT group and 423/752 (56%) in CD alone group. Prescription in relation to microscopy results are presented in p<0.001), respectively (p<0.001). In the CD+RDT group 343/1,005 (34%) received neither antimalarial nor antibiotic treatment, whereas this occurred in only 24/882 (3%) in the CD-alone group .A total of 607/1,887 (32%) patients were prescribed antibiotics, including mainly cotrimoxazole, but also ampicillin, amoxicillin, and erythromycin. Prescription of antibiotics was significantly higher in the CD+RDT than CD-alone group, 372/1,005 (37%) and 235/882 (27%)  of patients in the CD+RDT and CD-alone groups, respectively, but in similar proportions to all age groups .A total of 1,601/1,887 (85%) patients were prescribed antipyretics, mainly paracetamol but also acetyl salicylic acid. Antipyretics were prescribed to 87.0% versus 82.4%   .p = 0.005).The majority  of patients returned for the scheduled follow-up visit between 14 and 16 d after enrolment. The remaining 74 patients were actively followed up by the research team within three weeks. No deaths occurred during the study. A total of 68 patients returned to the health facility because of ill health during the 2-wk follow-up, mostly (51/68 [75%]) within the first week. Of these 68 patients, 52 were children below age 5 y, nine were 5–15 y old, and seven were adults. The rate of reattendance due to perceived unsuccessful clinical cure was significantly lower in the CD+RDT (25/1005 [2.5%]) than in the CD-alone group (43/882 [4.9%])  of these patients. The three positive slides, all from the CD-alone group, had low densities: 64, 80, and 1,866 parasites/µl of blood. All three patients had initially been prescribed ACT.The health outcomes in patients with incorrect malaria diagnoses, both false negative and false positive, are of particular importance. Out of 28 patients  with positive BS and not treated with antimalarial drugs, only one returned because of any illness during the two-week follow-up, carrying a low density of 32 parasites/µl of blood. On day 14, an additional four of 28 patients were found to be still BS positive . Among 549 patients having negative BS but clinically diagnosed and treated for malaria, only 20 (3.6%) returned to the health facility with persistent symptoms.Of 1,113 patients treated for malaria in both groups, 42 (3.8%) returned with illness, compared with 26/773 (3.4%) who did not receive antimalarial treatment. Among patients with positive BS on day 0 and treated for malaria, the frequencies of positive slides on routine follow-up between days 14 and 16 were similar in the two groups, 39/279 (14%) in the CD+RDT and 38/245 (16%) in the CD-alone group. The overall malaria treatment failure after nonsupervised drug intake was thus 77/524 (15%) on day 14, with a GM of 611  parasites/µl.The mean total costs per patient by age group are presented in We found an overall 2-fold reduction in prescription of antimalarial drugs and reattendance of patients due to illness during the two-week follow-up period in the CD+RDT group compared with CD-alone group. Overall costs were, however, similar in the two groups despite a significant reduction of cost among the adult patients after RDT-aided diagnosis.Almost all enrolled fever patients in the CD-alone arm were considered and treated as malaria patients, resulting in high diagnostic sensitivity (99%) but low specificity (20%). This result follows the suggestion that fever alone may be a better criterion for malaria treatment than more complicated algorithms P. falciparum–endemic areas are normally high with a mean sensitivity of 93% Health workers learnt to use RDTs correctly with relative ease, confirming that the tests are simple to perform and interpret The use of confirmatory malaria diagnosis with RDT is expected to reduce the overuse of antimalarial drugs by ensuring that treatment is targeted to patients suffering from malaria infections as opposed to treating all patients with fever. Our findings confirm this expectation, although the impact of RDT-aided diagnosis will obviously be highly dependent on the malaria incidence  in a given situation.Importantly, in our study, the study nurses showed great confidence in the RDT results as a guide to choice of treatment, as did the patients. This is in contrast to the assumption that care providers, although willing to perform diagnostic tests, do not always comply with the results, especially when the result is negative Fearing false negative test results and being aware that delays in providing effective treatment can be fatal for malaria patients is reported to be the main reason to prescribe antimalarial drugs despite a negative RDT result. Importantly, in our study, the patients with malaria detected by BS but non-detectable by RDT and therefore not treated with antimalarial drugs had relatively low parasite densities and no patients developed any severe malaria manifestations during the two week follow-up. This supports a general recommendation of consistence in not treating RDT negative patients. Re-testing will, however, obviously be required if the illness remains or aggravates.Our finding of a reduction in perceived illness during a two-week follow-up in the CD+RDT group of patients is critical. This was probably attributed to improved treatment of patients with fever not associated with malaria. More antibiotics were prescribed to the RDT-negative patients. The introduction of RDT and ACT thus provides an opportunity to improve the treatment of both malaria and bacterial diseases.We did consider the potential selection bias of the four health facilities; indeed, significant heterogeneity was observed with regard to the primary effect parameter. We do, however, assume this heterogeneity was at least partly accounted for by multilevel analysis and, since the RDT effect on drug prescriptions was quite large in each PHCU, it seems unlikely to be due to selection bias.The selection of the four study sites was done to provide a relatively representative picture of both malaria epidemiology and previous use of RDT in Zanzibar. Since RDTs had already been introduced by MSF in some parts of Zanzibar we opted for including PHCUs both with previous experience (two sites) and without previous RDT use (two sites). Beside the previous RDT exposure, the selection of the four study sites was based on representing a common rural situation and representing both of the two major islands in Zanzibar, i.e. Unguja and Pemba (two PHCUs on each island). However, we do of course acknowledge that the choice of the four sites remains arbitrary and of low number and thus cannot be fully representative of an overall Zanzibar situation and even less so of an overall situation in sub-Saharan Africa, which indeed is very diverse itself with regard to epidemiology of malaria, cultural and behavioural aspects, health care structure, etc.With the understanding that four PHCUs is a very low number, we used a cross-over design of RDT versus non-RDT weeks within sites. The choice of RDT or non-RDT the first week was based on an allocation with one previous MSF/RDT site being in either arm and one non previous MSF/RDT similarly being in either arm. Still, we acknowledge that there may still be confounding effects with regard to health-seeking behaviour or even selection bias by study nurses on respective weeks by (a) patients/caretakers postponing health care attendance to a week with RDT or staff applying exclusion criteria on a CD week and instead request the patient to return on a RDT week, and/or (b) attending alternate PHCUs where RDT is performed. However, we assume that (a) is less realistic, considering that uncomplicated malaria requires urgent treatment and patients or their caretakers as well as health care workers are therefore not likely to wait and postpone treatment. We also do not believe (b) is realistic because systematic RDT use was not implemented outside study PHCUs at the time of the trial and the study sites were located far from each other. And indeed statistical analysis showed no significant difference between frequencies of fever patient attendance on RDT and non-RDT weeks. The only trend observed with regard to frequency of attendance was a tendency to a relative increase toward the later period of the study, compatible with increased malaria transmission.In summary, RDTs were well performed in peripheral health facilities with acceptable sensitivity and specificity for identifying malaria-attributable fever episodes. The RDT results were adhered to and did provide consistent and significant reduction in antimalarial treatment in parallel with an increase in prescribed antibiotics. This probably contributed to the significant reduction in reattendance due to illness during the two-week follow-up. Our results indicate that RDTs may represent an important tool for improved management of fever patients in peripheral health care settings in malaria-endemic areas, especially where ACT has been introduced for treatment of uncomplicated malaria.Text S1Trial protocol.(0.15 MB DOC)Click here for additional data file.Text S2CONSORT checklist.(0.06 MB DOC)Click here for additional data file.