Plasmodium falciparum endemic areas need an effective chemoprophylaxis.Malaria chemoprophylaxis prevents the occurrence of the symptoms of malaria. Travellers to high-risk A literature search to update the status of mefloquine as a malaria chemoprophylaxis.Plasmodium knowlesi. New data were found in the literature on the tolerability of mefloquine and the use of this medication by groups at high risk of malaria.Except for clearly defined regions with multi-drug resistance, mefloquine is effective against the blood stages of all human malaria species, including the recently recognized fifth species, Use of mefloquine for pregnant women in the second and third trimester is sanctioned by the WHO and some authorities (CDC) allow the use of mefloquine even in the first trimester. Inadvertent pregnancy while using mefloquine is not considered grounds for pregnancy termination. Mefloquine chemoprophylaxis is allowed during breast-feeding. Studies show that mefloquine is a good option for other high-risk groups, such as long-term travellers, VFR travellers and families with small children. Despite a negative media perception, large pharmaco-epidemiological studies have shown that serious adverse events are rare. A recent US evaluation of serious events  found no association between mefloquine prescriptions and serious adverse events across a wide range of outcomes including mental disorders and diseases of the nervous system. As part of an in-depth analysis of mefloquine tolerability, a potential trend for increased propensity for neuropsychiatric adverse events in women was identified in a number of published clinical studies. This trend is corroborated by several cohort studies that identified female sex and low body weight as risk factors.The choice of anti-malarial drug should be an evidence-based decision that considers the profile of the individual traveller and the risk of malaria. Mefloquine is an important, first-line anti-malarial drug but it is crucial for prescribers to screen medical histories and inform mefloquine users of potential adverse events. Careful prescribing and observance of contraindications are essential. For some indications, there is currently no replacement for mefloquine available or in the pipeline. Plasmodium falciparum malaria will die [P. falciparum. Moreover, the incidence of malaria in travellers is likely to be an under-estimate as it does not include those diagnosed and treated abroad and because it is estimated that 40-70% of imported malaria cases are not reported to health authorities [Malaria is often imported into industrialized areas classified "malaria free" due to migration and tourist travel to malaria endemic areas. Approximately 80-90 million travellers will visit malaria endemic areas annually. In particular, travel to Africa has increased by 10% and sub-Saharan Africa has seen a recent 13% growth in international tourist arrivals [will die . The trehorities .Travellers to sub-Saharan Africa are most at risk of contracting malaria. Recent estimates suggest an attack rate of 302 in 100,000 travellers to West Africa compared to lower rates in Southern Africa 49/100,000 and much lower rates in Eastern Asia 5.4/100,000 and the Americas 1/100,000 [Travellers who return to their country of origin to visit friends and relatives (VFR) have been shown to have a higher risk of acquiring malaria than regular tourists . This isP. falciparum malaria varies from 0.6 to 3.8% [The overall case fatality rate of imported  to 3.8%  but may  to 3.8% . HoweverAnopheles mosquito), which remain only briefly in the bloodstream before entering the liver. Drugs which act on the parasite in the liver tissue are termed "causal prophylactics", for example, atovaquone and proguanil. Doxycycline has only a weak causal effect. "Suppressive prophylactics" or blood schizontocidal drugs act in the bloodstream when parasites invade the erythrocyctes. Most anti-malarial drugs fall into this category, for example, mefloquine and doxycycline.Malaria chemoprophylaxis can be defined as the use of anti-malarial medication to prevent the occurrence of the symptoms of malaria. No available drug can destroy the sporozoites  [There is a lack of international harmony in guidelines for malaria prevention Table , but almm (CRPF) -17. Othem (CRPF) ,12  has an overall anti-malarial efficacy of circa 95% [P. falciparum allowing discontinuation of the regimen 1 week after leaving the malarious area. The combination is not effective against the latent stages of relapsing malaria, such as Plasmodium vivax. Disadvantages are the high cost of the regimen and the limited experience and applicability in risk groups such as pregnant women, small children  and long-term travellers.The fixed dose combination Malaroneirca 95%  and is wirca 95%  with theDoxycycline is third priority anti-malarial and an adult dosage of 100 mg daily is >95% effective in malaria chemoprophylaxis and has a weak activity against malaria liver stages. Doxycycline monohydrate is better tolerated than the older form doxycycline hyclate , which iDifferences exist among the chemoprophylaxis regimens that are licensed, recommended, and distributed in each country. Table The ideal chemoprophylactic medication should be highly effective, cause few or no adverse events, be indicated for all travellers including pregnant women, nursing women, small children, long-term travellers, should be cheap and easy to use and should be registered globally for this indication. Currently no anti-malarial satisfies all the criteria for a "perfect" chemoprophylaxis regimen. Table Plasmodium knowlesi [Mefloquine is recognized as an effective malaria chemoprophylaxis for travellers to high risk CRPF areas . The druknowlesi ,20. The knowlesi .P. falciparum infection in persons with mefloquine blood levels in excess of this protective level. Using this definition, an analysis of 44 confirmed P. falciparum cases, acquired in sub-Saharan Africa showed five volunteers with mefloquine-resistant P. falciparum malaria [in vitro studies have documented an inverse relationship between chloroquine and mefloquine resistance. Mefloquine resistance is associated with halofantrine resistance [P. falciparum in vitro [In many geographic regions, mapping of prophylactic failures, mainly in non-immune individuals has been used to detect early resistance development although it should be emphasized that prophylactic failures do not prove resistance. Mefloquine blood concentrations of 620 ng/ml are generally considered necessary to achieve 95% prophylactic efficacy . As defi malaria . Other c malaria , a drug sistance  and quinsistance . Penflurin vitro .Pgh1, an energy-dependent transporter encoded by the mdr (multi-drug resistant) homolog Pfmdr1. Later studies demonstrate that mutations in Pfmdr1 may confer mefloquine resistance to sensitive parasites [Innate resistance is still controversial and may to some extent be explained by cross resistance to other drugs . The molarasites .Plasmodium falciparum with the exception of clearly defined areas of multi-drug resistance mainly limited to Thai border areas.In summary mefloquine is recognized as a highly effective malaria chemoprophylaxis for non-immune travellers to high risk areas of chloroquine-resistant P. falciparum (CRPF) malaria is transmitted should be avoided or deferred when possible.Falciparum malaria in a pregnant woman poses significant risks for the mother, foetus and the neonate. A study of imported malaria cases from 2002 showed that 7% of imported malaria cases in the USA occurred in pregnant women of whom only 28% reported taking a recommended malaria chemoprophylaxis . In FranP. falciparum need effective strategies to prevent malaria. Protection against mosquito bites is essential and a combination of protective measures including insecticide treated bed nets and DEET-, containing repellents are considered safe in pregnancy and are recommended although the efficacy of these measures for pregnant women has not been unequivocally proven. Chloroquine and proguanil can be used by pregnant women but because chloroquine resistance is widespread, this combination has limited applicability. For high risk areas of chloroquine resistant P. falciparum, an effective chemoprophylaxis is imperative and the choice is a difficult one. Doxycycline is not recommended in pregnancy mainly based on the experience that tetracyclines cross the placenta and can lead to disturbances of skeletal growth, permanent discoloration of teeth, corneas and lenses [®) is not recommended in pregnancy although proguanil is widely used and considered safe in pregnancy and no teratogenicity has been observed in animal studies using atovaquone. Because of ethical and safety restrictions, few anti-malarials have been evaluated for pregnant, non-immune travellers [Travellers to areas of chloroquine resistant d lenses . Due to avellers  and a Coavellers .®) during pregnancy. This level is within the background population level of congenital malformations estimated at between 5-6% [Currently, therefore, mefloquine is the only option for pregnant women who cannot defer travel and who need an effective chemoprophylaxis when visiting chloroquine resistant malaria endemic areas. Literature reviews and post-marketing surveillance studies of the Roche Drug Safety Database ,37 have een 5-6% .These data have allowed expert groups to recommend mefloquine during pregnancy although a survey of the recommendations made by expert groups shows a variation in the advice given Table . The WHOYoung children are at special risk for malaria because of their inability to protect themselves from mosquitoes, the difficulty in administering anti-malarial drugs and the rapidity at which they become severely ill. Parents and guardians must pay particular attention to insect protection measures including repellents and treated bed nets.Malaria chemoprophylaxis in the very young infant is a challenge. Although most anti-malarials taken by the mother will be present in breast milk, the drug concentrations are not considered high enough to provide an adequate protective dose for the infant.With regard to breastfeeding, chloroquine, atovaquone/proguanil and mefloquine are considered compatible with breastfeeding. Proguanil is excreted into human milk in small quantities and in a rat study, atovaquone concentrations in milk were 30% of the concurrent atovaquone concentrations in the maternal plasma. Doxycycline is not routinely recommended.The use of chloroquine prophylaxis is allowed in very small children but use is limited by widespread resistance to the drug. Atovaquone/proguanil prophylaxis (as paediatric tablets) can be used for children weighing more than 11 kg in Europe and in the US for children >5 kg according to new CDC guidelines . DoxycycA review of the literature shows that 15-82% of long-term travellers, who spend lengthy periods in Africa, will report malaria . In highThe terminology "long-term" usually refers to non-immune travellers who visit malaria endemic areas for a period of six months or longer. Typical long-term travellers include urban expatriates , rural expatriates , military groups, Peace Corps volunteers, students, backpackers and occupational travellers . MalariaBecause many reports highlighted neuropsychological mefloquine events, concern emerged that the use of mefloquine prophylaxis may impair performance and precision while driving, operating machinery or for soldiers in combat situations. For travellers, the impact of mefloquine on driving performance, in particular, required clarification. A review of mefloquine clinical toxicity studies including a study on driving performance with alcohol challenge showed that mefloquine, with or without small quantities of alcohol, does not impair driving . AdditioOlder travellers are less likely to report adverse events with mefloquine than their younger counterparts . One stuTravellers visiting friends and relatives (VFR), mainly immigrants and their children returning to their home country for vacations, are at particularly high risk for largely preventable infectious diseases such as malaria. In Western Europe, there are currently 20 million foreigners of which the majority are settled immigrants. One third come from a country outside of Europe. Increased global mobility and eased travel as well as the growing number of immigrants from malaria endemic countries have contributed to increased numbers of imported malaria cases in Europe. The immigrants from malaria endemic countries constitute a special risk group with high levels of malaria importation because they visit high risk areas for prolonged periods, are less likely to stay in air-conditioned hotels and often their status and financial circumstances hinder access to malaria chemoprophylaxis . MefloquMefloquine should not be used concomitantly with quinine or halofantrine and other structurally related anti-malarials or with medications that have central or peripheral nervous system activity. These interactions are well described in the labelling. A retrospective analysis of a database of anti-malarial tolerability data showed that other co-medications commonly used by travellers (such as anti-diarrhoea medication) have had no significant clinical impact on the safety of prophylaxis with mefloquine . MefloquMefloquine and its metabolite are not appreciably removed by haemodialysis . No specThere is considerable controversy among international experts regarding the tolerability of mefloquine prophylaxis versus alternative regimens, such as doxycycline, chloroquine/proguanil and, the combination atovaquone/proguanil.Adverse events can be divided into common events, usually mild and affecting large percentages of users and rare events, which are seen much less frequently and often recognized only after millions of users have used the drug. Rare events are usually not discovered in phase III trials and rely on post-marketing surveillance, which make the true incidence very uncertain as unreported events are likely.®), mefloquine (Lariam®), doxycycline (Vibramycin®) and chloroquine plus proguanil (Savarin®). Forty five percent of chloroquine and proguanil users reported mild to moderate adverse events, 42% of mefloquine users, 33% of doxycycline users and 32% of atovaquone/proguanil users. Significant adverse events (that interfered with daily activity) were reported in 11% of mefloquine users, 12% in chloroquine plus proguanil users, 6% in doxycycline users and 7% in atovaquone and proguanil users. Thus all malaria chemoprophylaxis regimens are associated with adverse events and many of the differences between the arms were not significant. It should be emphasized that an adverse event is not necessarily attributable to the anti-malarial drug, but reflects all intercurrent events experienced during the use of the drug.There is only one double blind, randomized controlled trial  which coAn overview of the studies and databases comparing use of malaria chemoprophylactic agents in travellers Tables  and 5 shAlthough often a subjective report by the traveller, when some measure of severity is applied to AE reporting, it appears that between 11-17% of travellers using mefloquine and other anti-malarials are, to some extent, incapacitated by adverse events. The extent of this incapacitation is often difficult to quantify and a good measure of the impact of adverse events is the extent of chemoprophylaxis curtailment or withdrawal from chemoprophylaxis. In a study of 5,120 Italian soldiers, deployed in Somalia and Mozambique in 1992-1994, the rate of prophylaxis discontinuation in chloroquine/proguanil users was 1.5% compared with a significantly lower rate of discontinuation in mefloquine users (0.9%) . A contrThe latest Cochrane report  used eigThese are adverse events that constitute an apparent threat to life, which require or prolong hospitalization or which result in severe disability. Steffen's large cohort study in travellers to East Africa estimated that one in 10,600 mefloquine users to have a serious adverse event (53). The study observed five probably mefloquine associated hospitalizations: two cases of seizures, 2 psychotic episodes and one case of vertigo. The rate of such events for chloroquine/proguanil users was 1/5,100 .Data on serious adverse events only become available after widespread use of the anti-malarial and data is available for older drugs, such as mefloquine and chloroquine.In one retrospective cohort analysis, serious neuropsychiatric AEs involving hospitalization were noted for 1:607 mefloquine users versus 1:1,181 chloroquine-proguanil users .The British army's experience with mefloquine prophylaxis found the incidence of severe neuropsychiatric reactions to be ≤1:6,000 . The moset al [et al [This is the main area of controversy in the literature regarding the tolerability of mefloquine. Neuropsychiatric disorders include two broad categories of symptoms namely central and peripheral nervous system disorders  and psychiatric disorders . Lobel et al  found anl [et al . reportel [et al  significl [et al ,59.More recent studies have used databases of electronically recorded prescriptions and diagnoses to define mefloquine exposure and outcome. The analysis of the UK-based General Practice Research Database  comparedThe precise role of anti-malarial drugs in neuropsychiatric adverse events is difficult to define and the role of travel as a catalyst for such events should be considered together with other confounding factors such as gender predisposition and the use of recreational drugs. The WHO recommends that mefloquine be contraindicated for persons with a personal or family history of psychiatric disorders. In terms of all AE, studies have shown that women are significantly more likely to experience AE ,58,61-63connexin blockade" by mefloquine (and other substances) is receiving a lot of attention in the field of neuroscience as a possible mechanism for certain mefloquine associated adverse events. "Connexins" are neuronal gap junction proteins that occur in the brain, lens, retina, and elsewhere. These connexins are assigned numbers according to their molecular weight. The connexins Cx36 and Cx43 are widely distributed in neuronal tissue. With regard to its sensitivity to mefloquine, Cx36 is an important gap junction protein and is thought to be involved in synchronizing rhythmic activity of neurons in several brain regions. Experiments in a rat model show that mefloquine effectively blocks connexin 36 which may explain the biological basis of some of mefloquine's side-effects such as anxiety, confusion, dizziness and other neuropsychiatric effects [et al [Recent research in animal models proposes mechanisms that may explain the neuropsychiatric profile of adverse events associated with mefloquine. Some of the mechanisms that have been proposed to explain neuropsychiatric adverse events with mefloquine include disruption of calcium homeostasis of neuronal cells, inhibition of enzymes such as acetylcholinesterase or butylcholinesterase, inhibition of cellular transport systems , blockage of receptors  and blockage of intercellular channels (gap junctions) . The phe effects . A parads [et al .Most of the studies on possible mechanisms of mefloquine neurotoxicity were conducted at relatively high mefloquine concentrations in animal models such as knockout mice or in rat brain slices and the transfer of knowledge acquired in these studies to healthy humans using mefloquine at chemoprophylaxis dosage still requires the bridging of a large knowledge gap.The use of genetic analyses to explain a predisposition to mefloquine neuropsychiatric events including seizures is generating warranted interest but careful attention must be paid to confounding factors in order to minimize the chance of spurious associations.P. falciparum in vitro. A study in human volunteers documented that the pharmacokinetic of mefloquine is highly stereospecific. Higher plasma concentration, longer half-live and a higher systemic exposure have been shown for the (-)-enantiomer [Mefloquine crosses the blood brain barrier in a rat model, and human data shows that there is accumulation in brain tissue particularly of the (+) enantiomer . Mefloquantiomer .Another study investigated the enantioselective kinetics of mefloquine during long term prophylactic treatment . These aFurthermore, new research shows that genes encoding various receptors, transporters, ion channels and connexins differ between the sexes  so that A role has been suggested for the concomitant use of mefloquine and recreational drugs or an interaction between mefloquine and large quantities of alcohol , althougMefloquine is considered to be well tolerated in small children where the main adverse events are of gastrointestinal nature, probably associated with the bitter taste of the medication which should be disguised with chocolate or yoghurt.®) spontaneously reported adverse events reported to F. Hoffmann-La Roche are categorized by system organ class and age of the mefloquine user, the adverse event profile shows that the neuropsychiatric adverse events dominate across all age groups with the exception of children under two where gastrointestinal events predominate  that has been widely used by a broad spectrum of age groups. Based on the literature reports, studies, national and international guidelines mefloquine can be particularly useful in special populations such as pregnant women, infants and small children, as well as long-term travellers and VFRs, in which efficacy, safety or cost-benefit profile have been well-documented. Careful prescribing of mefloquine with attention to contraindications is essential, as is a clear warning about the potential side effects.Because many mefloquine-associated adverse events occur early in dosing, during the intake of the initial three tablets, starting mefloquine prophylaxis 2-3 weeks before departure may allow for evaluation of tolerability to the regimen. Stopping the drug with early signs of such events should minimize the severity and duration of adverse events and women may consider the use of the split dose.Mefloquine has a pivotal position as an important first-line anti-malarial drug and needs to be preserved. For many of its indications, there is currently no replacement available or in the pipeline.This paper is based on data collated for an F. Hoffmann-La Roche regulatory update. PS was the external consultant who received consultancy fees for preparing the regulatory update. PS has also received research funding and/or speakers' honoraria from GlaxoSmithKlein, F. Hoffmann-La Roche and Pfizer.MA, LR, MTS, HGR are employees of F. Hoffmann-La Roche, Basel, SwitzerlandPS was responsible for the concept and design, acquisition of data, analysis and interpretation of data and the writing of the final manuscript. MA, LR were responsible for certain portions of data acquisition and MA, LR, MTS, HGR critically revised the paper.All authors have seen and approved this final version.


We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from  P. falciparum malaria. Erythrocytes containing mature malaria parasites adhere to the endothelium via a range of endothelial receptors in order to escape removal by the spleen. Endothelial activation is an early feature of malaria, which is likely to favor sequestration of parasitized erythrocytes The endothelium plays a central role in the pathophysiology of Plasmodium falciparum malaria from whom platelet poor plasma was obtained under special conditions to prevent ex vivo platelet activation. A second aim was to correlate plasma levels of these angiogenic proteins with circulating platelet numbers and markers of platelet activation.Both Ang-1 and VEGF are both stored in high quantities in alpha granules from platelets This study received ethical clearance for the use of human subject from the Eijkman Institute Research Ethics Committee, Jakarta, Indonesia and all enrolled patients gave written informed consent.P. falciparum and P.vivax malaria transmission. Consecutive patients presenting to hospital with recent or current fever, clinical symptoms of malaria and a positive blood slide for P. falciparum were enrolled in this study following informed consent. All patients were assessed according to a predefined protocol which included a standardized history and physical examination performed by an experienced internist-infectious diseases specialist. All patients were treated with intravenous quinine and an antipyretic (paracetamol). Concurrent administration of antibiotics for severely ill patients was on the discretion of the treating physician. Venous blood for this study was collected at enrollment and on day two and five after enrollment. A group of 25 healthy young adults were recruited among local hospital staff as controls. All controls had no signs or symptoms of any illness and a negative malaria blood slide. Treatment for malaria in the past two months was an exclusion criteria for both malaria patients and controls.This study was conducted in the Rumah Sakit Karitas Hospital in Waitabula, West Sumba, East Nusa Tenggara Province, Indonesia, an area of hypo- to meso-endemic In vitro platelet activation is almost completely absent in whole blood anticoagulated with CTAD Five ml of venous blood was collected in EDTA tubes for a full blood count and malaria slides and in CTAD tubes  for measurement of angiogenic and platelet activation marker proteins. Data are presented as median followed by interquartile range in parentheses unless otherwise stated. Within the group of malaria patients, the Freidman test with post-tests was used to compare laboratory parameters on the three time points.Mann-Whitney U test was used for comparisons with the controls. Relationships between laboratory parameters were assessed using Spearman correlation coefficient. All analyses were performed with GraphPad version 5.0.P. falciparum malaria and 25 controls were included. Demographic and clinical characteristics are shown in A total number of 27 patients with The time course of Ang-1 and VEGF resembled the course of the platelet activation markers P-selectin and CXCL7. Correlation analysis, presented in in vitro platelet activation ex vivo platelet activation, CTAD tubes almost completely prevent in vitro platelet activation and several authors have already recommended using CTAD when platelet-stored angiogenesis proteins are being measured ex vivo platelet activation was responsible for the lower Ang-1 and VEGF concentrations in previous studies. Serum concentrations of platelet-stored angiogenic proteins may also have prognostic value. Generation of serum results in complete in vitro platelet activation and serum concentrations will largely reflect the total platelet content of angiogenic proteins. Possible drawbacks of using serum for this objective is that clotting processes may not release all platelet-stored angiogenic growth factors into the serum These findings are in contrast to previous studies which showed that patients with malaria had lower Ang-1 levels than healthy controls 9/L) in the remaining volunteer.We only used a single centrifugation step to generate platelet poor plasma in our study, while a double centrifugation step is often advised. We assumed that only under special circumstances single centrifugation does not result in near complete removal of platelets from the top layer of the plasma. We tested this assumption by centrifuging venous blood in CTAD tubes from five volunteers using the same procedures as in our study and we found that a singly centrifugation step resulted in no detectable platelets in the plasma in four volunteers and a negligible platelet number (2×10Several groups of researchers have investigated the utility of angiopoietins and VEGF as biomarkers to identify patients with severe malaria. Due to its limited sample size, our study did not allow to explore the role of these angiogenic proteins in malaria. In general, we strongly recommend that a similar protocol for measurement of platelet-stored angiogenic proteins is used in future biomarker studies, including use of CTAD plasma and consideration of circulating platelet numbers. These precautions do not apply to Ang-2, which is derived from endothelial cells and of which plasma levels are not affected by platelet activation.What are the implications of these findings for the pathophysiology of malaria? Increasing evidence supports a central role for platelets in protecting the vasculature during inflammation In conclusion, we show that platelet activation is an important determinant of circulating Ang-1 and VEGF levels in malaria. Optimal biomarkers for malaria severity would rely on inexpensive point-of-care devices using whole blood. The fact that assays measuring these platelet-stored angiogenic proteins require special precautions to avoid inadvertent platelet activation during blood processing and are prone to artifacts limits widespread use of these proteins as possible biomarkers.


P. falciparum-infected erythrocytes and bind to multiple host receptors. Using a mammalian expression system and BioPlex technology, we developed an array of 24 protein constructs representing 38 PfEMP1 domains for high throughput analyses of receptor binding as well as total and functional antibody responses. We analyzed the reactivity of 561 plasma samples from 378 young Tanzanian children followed up to maximum 192 weeks of life in a longitudinal birth cohort. Surprisingly, reactivity to the DBL5 domain of VAR2CSA, a pregnancy malaria vaccine candidate, was most common, and the prevalence of reactivity was stable throughout early childhood. Reactivity to all other PfEMP1 constructs increased with age. Antibodies to the DBL2βC2PF11_0521 domain, measured as plasma reactivity or plasma inhibition of ICAM1 binding, predicted reduced risk of hospitalization for severe or moderately severe malaria. These data suggest a role for VAR2CSA in childhood malaria and implicate DBL2βC2PF11_0521 in protective immunity.PfEMP1 proteins comprise a family of variant antigens that appear on the surface of  Plasmodium falciparum kill over 1 million African children each year. P. falciparum-infected erythrocytes (IE) adhere to host endothelium and to red blood cells, allowing IE to sequester in deep vascular beds of various organs Severe malaria syndromes caused by The role of specific PfEMP1 proteins in severe malaria syndromes remains unclear. A single member of the PfEMP1 family, VAR2CSA, is upregulated by IE that sequester in the placenta during pregnancy malaria (PM)  and functional (inhibition of receptor binding) antibody levels In their native state, proteins exist in a folded form, which can create conformationally dependent epitopes and also mask cryptic epitopes. For this reason, correctly folded recombinant proteins may have lower seroreactivity than unfolded proteins We previously expressed all DBLβC2 domains encoded in the genome of parasite clone 3D7, and surveyed their binding properties The CD36 binding results obtained with multidomain constructs that contain CIDR1  correspoIn an earlier study, seroreactivity in African children was acquired most rapidly against group A or A/B DBL1 domains, but conversely was acquired most rapidly against group B, B/C, or C CIDR1 domains var gene classification var gene expression has been associated with severe malaria in some studies var genes.Eleven out of 24 domain constructs were recognized by >10% of serum samples from children above 2 years of age . These iPF11_0521, which binds strongly to ICAM1, was the second most commonly recognized PfEMP1 domain after VAR2CSA. In general, antibodies against ICAM1-binding domain dominate at early ages, while antibodies against CD36-binding domains increase at later ages  children's parasites The frequency of plasma reactivity to VAR2CSA DBL5 among children is similar to that among adult males in our East African cohort At the population level, the number of reactive PfEMP1 domains  and the sum of anti-PfEMP1 IgG reactivities  positively correlate with the number of preceding parasitemia episodes . The relPF11_0521 and VAR2CSA DBL5) that were recognized most commonly. In this cohort, eighteen children were hospitalized after age 76 weeks with severe or moderately severe malaria, and these included children who suffered severe anemia (n = 1), respiratory distress (n = 7), prostration (n = 4), convulsions (n = 2), hyperpyrexia (n = 9), or hypoglycemia (n = 1). Hyperparasitemia without other features of serious disease did not meet our criteria for severe or moderately severe malaria.Infants rarely develop functional IgG PF11_0521 domain PF11_0521-reactive IgG, and none of the children found to have functional antibody by binding-inhibition assay, subsequently suffered severe or moderately severe malaria. Because both assays have incomplete sensitivity, and because plasma IgG reactivity and inhibition of ICAM1 binding represent two facets of the specific anti-domain immune response that can contribute to protection, we analyzed whether responses detected by either assay would predict reduced risk of disease. Children with antibody measured in either format demonstrated a reduced risk of Severe/Moderately severe malaria hospitalizations  time from the most recent parasitemia ; 2) duration of follow-up ; 3) number of previous parasitemia episodes . Time from the most recent parasitemia (p = 0.36), duration of follow-up (p = 0.76), and the number of previous parasitemia episodes (p = 0.68) did not differ significantly (Mann-Whitney test) between children with versus those without DBL2C2er assay . Furtherizations , suggestIn an earlier study, IgG reactivity against the PF11_0008 CIDR2 domain was associated with protection against clinical malaria among children 4–9 years of age, but not among younger children, and plasma reactivity to the DBL1 and DBL2 domains of the same protein were not related to risk. PF11_0521 domain by children older than 2.5 years of age ; two or more observed convulsions in the past 24 hours; glucose <2.2 mmol/L; prostration; hemoglobin <5.0 g/dL. Moderately severe malaria was defined as parasitemia with symptoms that did not meet WHO criteria for severe malaria but that met one or more of the following criteria: a respiratory rate ≥40; observed convulsions in the past 24 hours; hemoglobin <6.0 g/dL; temperature ≥40°C. Children with these presentations were commonly hospitalized for parenteral therapy Construct composition and corresponding PCR primers for 26 PfEMP1 constructs encompassing 39 DBL and CIDR domains are shown in Binding of CD36 at increasing concentrations to various N-terminal head structures  was measThis reactivity was measured as previously described To confirm by standard ELISA method that anti-human IgG does not recognize human IgM we incubated 0.1 µg of human IgG and IgM in carbonate/bicarbonate buffer in duplicates using 96-well plates. After overnight incubation at 4°C, washing, and blocking in 4% milk wells were probed with various dilutions of FITC-conjugated anti-human IgG (see above). After washing, fluorescence  was measured using Fluoroscan Ascent FL fluorometer/luminometer (ThermoLab systems). Results are shown in These measurements (including controls) were performed as described earlier Analyses of proportions (contingency tables), correlation, and Mann-Whitney tests were performed using GraphPad Prizm software . Multiple regression analyses were performed using StatView for Windows Figure S1A. Age-dependent prevalence of plasma reactivity to PfEMP1 domains ordered by decreasing reactivity measured at week 76 of life. Note that all constructs with DBL1 domain also contain NTS domain but it is not included in the names of constructs in this Figure. B. Percent of children responding to any construct in each particular PfEMP1 group, stratified by age. The total number in each age group exceeds 100% because individual children may respond to more than one antigen.(PPT)Click here for additional data file.Figure S2A. IgG reactivity of VAR2CSA DBL5 with plasma from children of different age after subtraction of background reactivity (mean plus 2SD threshold). AU, arbitrary units. Reactivity of pooled multigravida plasma (positive control) was 11093 AU. Highest reactivity of child plasma was 6674. Red lines indicate mean values . Pink and blue lines indicate 2 SD and 4 SD threshold used for analysis in (PPT)Click here for additional data file.Figure S3Number of reactive PfEMP1 domains (A) and the sum of anti-PfEMP1 IgG reactivities (B) positively correlate with the number of preceding parasitemia episode. Corresponding Spearman correlation coefficients (r) and P-values are indicated. Blue line demonstrates linear regression.(PPT)Click here for additional data file.Figure S4Opposite trends in the percentage of children with subsequent hospitalizations for severe/moderately severe malaria (A) vs. percentage of children with total or functional seroreactivity against DBL2C2PF11_0521 domain (B) with age. Total/functional reactivity is Positive if IgG or binding inhibition activity of serum against this domain is above 0. “n” indicate actual numbers of hospitalized children (A) and children with Positive reactivity (B) in the cohort.(PPT)Click here for additional data file.Figure S5Analysis of potential confounding factors for groups of children with Positive (IgG>0 or BI>0) and No reactivity against DBL2C2PF11_0521 domain at week 76 of age. Red lines indicate medians. P values calculated using Mann-Whitney test.(PPT)Click here for additional data file.Figure S6Number of children with subsequent severe or moderately severe malaria, stratified by positive and no plasma IgG reactivity against 3 most reactive constructs measured at 76 weeks of age, and P values against all constructs obtained in similar analyses.(DOC)Click here for additional data file.Figure S7Reactivity of anti-human IgG with human IgG and human IgM. PBST, Phosphate-buffered saline buffer containing 0.05% Tween-20 (negative control). The preparation of anti-human IgG does not recognize human IgM.(PPT)Click here for additional data file.Table S1Construct composition and corresponding PCR primers for 26 PfEMP1 constructs encompassing 39 DBL and CIDR domains. Amino acid residue numbers are shown according to PlasmoDB database.(XLS)Click here for additional data file.


Although global malaria mortality is declining, estimates may not reflect better inpatient management of severe malaria (SM) where reported case fatality rates (CFRs) vary from 1–25%.A meta-analysis of prospective studies of SM was conducted to examine i) whether hypothesized differences between clinical features and outcome in Melanesian compared with African or Asian children really exist, and ii) to explore temporal changes in overall and complication-specific CFRs. The proportions of different SM complications and, overall and complication-specific CFRs were incorporated into the meta-analysis. Adjustments were made for study-level covariates including geographic region, SM definition, artemisinin treatment, median age of participants and time period.I2>80%) was demonstrated for most outcomes. SM definition contributed to between-study heterogeneity in proportions of cerebral malaria (CM), metabolic acidosis (MA), severe anemia and overall CFR, whilst geographic region was a significant moderator in for CM and hypoglycemia (HG) rates. Compared with their African counterparts, Melanesian children had lower rates of HG , lower overall CFR  and lower CM-specific CFR . There was no temporal trend for overall CFR and CM-specific CFR but declining HG- and MA- specific CFRs were observed.Sixty-five studies were included. Substantial heterogeneity (These data highlight that recent estimates of declining global malaria mortality are not replicated by improved outcomes for children hospitalized with SM. Significant geographic differences in the complication rates and subsequent CFRs exist and provide the first robust confirmation of lower CFRs in Melanesian children, perhaps due to less frequent HG. Plasmodium falciparum estimated to occur globally are uncomplicated P. falciparum are detected in the peripheral blood and there is impaired consciousness or coma, prostration, multiple seizures, hyperlactatemia or metabolic acidosis, severe anemia, dark urine, hypoglycemia, jaundice, respiratory distress, persistent vomiting, abnormal bleeding, shock, and/or renal failure P. falciparum infections. It has evolved from previous iterations, published in 1986 Although most of the 500 million clinical cases of malaria due Both the proportions of children with different clinical features defining severity and their subsequent case fatality rates (CFRs) will depend on selection/recruitment strategies, the sensitivity of malaria diagnosis/speciation and the definitions of the features themselves, as well as inpatient management including identification of co-incident disease, and social, cultural and genetic factors that could influence presentation and clinical course. Apparent inconsistencies between published studies may reflect differences in one or more such factors. Indeed, reported CFRs in severe childhood malaria studies vary from 1–25% In the light of this background, we performed a standardized review and meta-analysis to examine whether i) there are significant differences between clinical features and outcome in Melanesian children with severe malaria and those in cases from Africa or Asia, and ii) there have been changes in overall and complication-specific CFRs over time. We incorporated geographic region, the year in which the study was performed, the case definition used, patient age and antimalarial therapy as potential moderators of outcome.The supporting PRISMA checklist is available as P. vivax or mixed P. vivax/P. falciparum malaria were not.A search of all descriptive studies of severe childhood falciparum malaria published before the end of 2012 was conducted using Medline. The following MeSH terms were employed: “child*” OR “pediatric” OR “paediatric” AND “severe malaria” OR “cerebral malaria” OR “severe anemia” OR “severe anaemia” OR “severe malarial anemia” OR “severe malarial anaemia” OR “metabolic acidosis” OR “lactic acidosis”. The bibliographies of key papers were reviewed and additional studies obtained. Publications in French were included but those describing children with severe The definition of severe malaria was categorized as ‘all malaria requiring admission’, ‘clinician-determined severe malaria’, WHO 1990 Studies focusing on children with only severe malarial anemia, cerebral malaria, hypoglycemia and metabolic acidosis were included only in CFR analyses of the relevant specific clinical feature. The term ‘deep coma’, ‘unrousable coma’, ‘comatose not responding to painful stimuli’ were considered synonymous. This equated to a Blantyre Coma Score (BCS) Definitions of severe malarial anemia (hemoglobin <50 g/L) and hypoglycemia (blood/plasma glucose <2.2 mmol/L) were uniform across time, geographic region and WHO definition. Either hyperlactatemia (blood/plasma lactate ≥5.0 mmol/L), or serum bicarbonate ≤15 mmol/L or ≤12.2 mmol/L The R statistical package ‘metafor’ was used for meta-analyses I2 statistic, a measure of the percentage of total variability due to between-study heterogeneity. Values <50% suggest a lack of significant heterogeneity. The τ2 statistic is a measure of total heterogeneity before and after incorporation of moderator variables, allowing the contribution of moderator variables in mixed effects models to be quantified as a relative proportion of the overall between-study heterogeneity. The mixed effects model with the best fit for each outcome of interest was determined by comparison between derived models using backward stepwise testing, incorporating moderators with a statistically significant effect on heterogeneity, and by using Aikake's Information Criterion and analysis of variance testing at each step to identify the most parsimonious model.Heterogeneity was determined using the The results of random effect models are depicted using forest plots. In each such plot, the study is denoted by author and year of publication with the exception of the Severe Malaria in African Children (SMAC) studies This study is registered with PROSPERO (CRD42013005510).The initial Medline search yielded 4,629 citations. A PRISMA Plasmodium species, making it difficult to identify children with P. falciparum. The third study reported clinical data collected from children recruited to the SEQUAMAT intervention trial The meta-analysis included studies performed from 1987 to 2009 (median 1999) with average patient age ranging from 1 to 8 (median 3) years. Three highly cited articles, all of which reported studies of Melanesian patients, were captured by the search strategy but excluded from analysis. The first, a retrospective study of morbidity surveillance in a cohort of PNG children, did not have detailed data available for ascertainment of clinical manifestations and did not report overall or phenotype-specific CFRs Of the studies included, only one systematically reported HIV status in children with severe malaria. Blood cultures were taken from at least some of the children with severe malaria in 7 studies I2>80%) for all but three outcomes of interest. The exceptions were the proportions and CFR of children presenting with the overlap syndrome  and CFR for children presenting with a BCS of 0 (I2 0%). The overall heterogeneity (I2), total estimated heterogeneity (τ2) and the effect of moderators on τ2 for the proportions of clinical features in descriptive studies are shown in There was substantial heterogeneity , while Asian children also had a higher mortality due to severe anemia than African children.Forty-five studies reported overall CFRs. A forest plot showing random effects models for overall mortality according to region is shown in The average patient age in studies included in the meta-analysis was a significant moderator and was incorporated into the final mixed effects model for proportion of deep coma in severe malaria  and mortality due hypoglycemia . Artemisinin use was not a variable in any final mixed effect model for any outcome of interest.P<0.05).For children with deep coma, the proportions in BCS categories were highly heterogeneous, with at least some of the heterogeneity accounted for by the year the study was performed. There were no apparent regional differences in the proportions of children with BCS of 0, 1 or 2, although no Asian studies were represented. The CFRs for children with a BCS of 0 had no heterogeneity  Regional differences accounted for at least some of the overall heterogeneity between studies. When compared with their African counterparts, PNG children (in the only representative studies from Melanesia or Oceania) had lower overall and cerebral malaria-specific CFRs. The proportions of children from PNG with deep coma who had a BCS of 0, 1 or 2 were comparable to those in African studies. The CFRs for a BCS of 0 were extremely high but without heterogeneity. For children with BCS of 2, the CFR was lower in PNG children. These findings reflect epidemiological studies, historical information P. vivax rather than a survival benefit mediated via protection against cerebral malaria due to P. falciparumLocally prevalent protective genetic polymorphisms are, however, unlikely to account for the observed differences in CFRs. First, SAO prevalence is highly heterogeneous and been identified in coastal PNG peoples with Austronesian language lineage, but not elsewhere in Melanesia Plasmodium species P. falciparum when there is a mixed infection with P. vivaxP. vivax co-infection appears to abrogate the nadir in hemoglobin after treatment for falciparum malaria P. vivax protects against P. falciparum clinical disease P. falciparum/P.vivax infections in PNG is inconsistent with this concept Cross-sectional and longitudinal surveys in Melanesia have shown negative correlations between the prevalence of different Two alternative explanations for the low CFRs in Melanesian children with severe malaria include the low rates of hypoglycemia in this setting, as demonstrated in the present meta-analysis, and a more recent observation that concomitant bacteremia, particularly non-typhoidal salmonellae (NTS), occurs much less frequently than in African children with severe malaria. A very low prevalence of hypoglycemia in Melanesian children with severe malaria (1%) might account for lower CFRs observed in comatose children as well as overall CFR. Our analyses suggest that an absolute difference in hypoglycemia between PNG and other children of 8% might account for only 2% of the differences in overall CFR. Differences in nutritional status and hepatic glycogen reserves, pre-referral treatment with quinine with attendant hyperinsulinemia and as yet undiscovered genetic polymorphisms in glucose metabolism are possible explanations for geographical variability in hypoglycemia rates.S. aureus nasal carriage rates or circulating S. pneumoniae serotypes), HIV seroprevalence and sickle cell disease rates, pre-treatment with antibiotics, and nutrition status Concomitant bacteremia in severe childhood malaria in African children is well described, occurring in 5–16% of children There were no significant temporal trends for overall CFR or for CFRs due to deep coma and severe anemia in the present study. In addition, artemisinin use was not a significant moderator of any outcome of interest. These observations appear surprising in the light of recent models demonstrating that estimates of global malaria-attributable mortality have declined since 2004 The lack of change in CFR due to cerebral malaria over recent decades could reflect diagnostic uncertainty, with a true decline masked by misdiagnosis of other causes of encephalopathic illness as cerebral malaria Artemisinin use was not a significant moderator of any outcome of interest in the present analyses. Only one third of the studies involved an artemisinin derivative, nearly all of which were comparative intervention trials. The artemisinin formulations used varied widely in terms of derivative  and route of administration . Due to the limited number of studies and the different treatment regimens, we pooled the data for all preparations and routes of administration. This may have masked a beneficial effect of intravenous artesunate that was clearly demonstrated in AQUAMAT et al.et al. study et al.Our study had other limitations. First, the four Melanesian studies eligible for inclusion were from the same regional hospital in PNG. Each study was independent and conducted at different times, but the applicability of the present findings to other Melanesian geographical areas is unknown. Second, because of methodological issues described in the In conclusion, the present meta-analysis supports the contention from individual studies and observations that there is a lower CFR for PNG children when compared with African children presenting with severe malaria. Although this may be mediated in part by low rates of hypoglycemia and concomitant bacteremia, there is still substantial heterogeneity across all studies. Although there is good evidence that CFRs due to hypoglycemia and metabolic acidosis have been declining, the lack of temporal change in overall and cerebral malaria CFRs in an era of improving access to parenteral artesunate should be a catalyst for renewed efforts to refine the diagnosis of severe malaria, improve pre-referral practices, optimise inpatient management and continue the search for effective adjunctive therapies.Checklist S1The supporting PRISMA checklist.(DOC)Click here for additional data file.


Plasmodium falciparum malaria.Considering the natural history of malaria of continued susceptibility to infection and episodes of illness that decline in frequency and severity over time, studies which attempt to relate immune response to protection must be longitudinal and have clearly specified definitions of immune status. Putative vaccines are expected to protect against infection, mild or severe disease or reduce transmission, but so far it has not been easy to clearly establish what constitutes protective immunity or how this develops naturally, especially among the affected target groups. The present study was done in under six year old children to identify malaria antigens which induce antibodies that correlate with protection from In this longitudinal study, the multiplex assay was used to measure IgG antibody levels to 10 malaria antigens  in 325 children aged 1 to 6 years in the Kassena Nankana district of northern Ghana. The antigen specific antibody levels were then related to the risk of clinical malaria over the ensuing year using a negative binomial regression model.IgG levels generally increased with age. The risk of clinical malaria decreased with increasing antibody levels. Except for FMPOII-LSA, (p = 0.05), higher IgG levels were associated with reduced risk of clinical malaria (defined as axillary temperature ≥37.5°C and parasitaemia of ≥5000 parasites/ul blood) in a univariate analysis, upon correcting for the confounding effect of age. However, in a combined multiple regression analysis, only IgG levels to MSP1-3D7  and AMA1 3D7  were associated with a reduced risk of clinical malaria over one year of morbidity surveillance.The data from this study support the view that a multivalent vaccine involving different antigens is most likely to be more effective than a monovalent one. Functional assays, like the parasite growth inhibition assay will be necessary to confirm if these associations reflect functional roles of antibodies to MSP1-3D7 and AMA1-3D7 in this population. In malaria endemic regions, clinical malaria is responsible for high morbidity and mortality in less than five year old children and pregnant women. In these regions, individuals develop a partial 'non-sterile' immunity against erythrocytic stage disease in an age and exposure dependent manner and, therefore, older individuals suffer less clinical symptoms and disease complications. Sero-epidemiological studies show three sequential phases of development of acquired immunity to malaria: first, immunity to life-threatening disease; second, immunity to symptomatic infection; and only then, can the third phase, partial immunity to parasitization be achieved ,2. PassiSeveral studies have reported associations between levels of antibody to various malaria parasite specific antigens and reduced risk of infection -9. Howevin vivo remains unclear  and re-suspended in 100 ul of coupling buffer. To determine the optimal concentration of protein for coupling, 1.6, 4.0, 10, 25, and 62.5 μg/ml of recombinant proteins and BSA were assessed. Various concentrations of the recombinant malarial antigens such as GLURP R2 at (1.6 μg/ml); GLURP-R0, AMA1-FVO, MSP1-FVO, MSP1-3D7, LSA-1, MSP3 FVO, AMA1 LR32, and EBA175RII ; AMA1 3D7 at 62.5 μg/ml were added to the microspheres. They were then mixed gently by vortexing, followed by 20 seconds ultrasonic sonication. The total volume was adjusted to 500 μl by adding the coupling buffer. The microspheres were incubated at RT in the dark for 2 hours with rotation, and then washed twice with 500 μl of storage buffer . The coupled microspheres were re-suspended in 500 μl of blocking/storage buffer and stored at 4°C in the dark until ready for use.Antigen specific IgG levels were meseasured against these antigens using the multiplex assay, a suspension array technique as described in a previous publication  with modStock suspensions of antigen-coated microspheres were thoroughly re-suspended by vortexing and sonication before use. Filter plates  were washed with 200 μl of washing buffer (PBS plus 0.05% Tween 20) and then washed with 200 μl of dilution buffer (PBS plus 1% BSA) using a vacuum manifold  to pre-wet the plates. Then, 25 μl of mixed microspheres of the antigens were distributed to microtiter wells  and 50 μl of diluted plasma (1:50 and 1:2000 dilutions) was added. The plates were placed on a Microplate Shaker  and incubated at RT in the dark for 1 hr at 500 rpm. The plates were washed five times with 200 ul washing buffer. A total of 25 ul of 5 ug/ml (1:200) of goat anti-human immunoglobulin G  was added to each well, and plates were incubated for 1 hr in the dark at RT on a microplate shaker. After the incubation period, plates were washed five times with washing buffer, and then 50 ul of strepavidin phycoerythrin (1:800) was added and incubated for 30 mins at RT in the dark on a shaker. The reader  was programmed to read a minimum of 100 microspheres per bead region, and results were expressed as mean fluorescent intensity (MFI), which is directly proportional to antibody concentration. For quaThe study was reviewed and approved by the IRBs of both Noguchi Memorial Institute for Medical Research and the Navrongo Health Research Centre.P. falciparum infection was prevalent all year round; with about 60-80% study subjects carrying parasites throughout the study period ] and AMA1-3D7  were shown to be independently associated with reduced risk of clinical malaria  or the antibody-dependent cellular inhibition (ADCI) assay. Unlike other immuno-epidemiological studies which are usually conducted in older individuals whose immune response mechanisms may not be similar to that of children, the focus of this study was on young children whose immune systems may be in a state of flux as they develop partial immunity to malaria. This, therefore, gives us the opportunity to study more clearly which antibodies may be associated with a reduced risk to malaria.In a final model involving all the immunological variables and age, IgG to MSP1-3D7 and AMA1-3D7 were found to be independently associated with reduced risk of clinical malaria indicating their potential as candidate antigens for vaccine development. However, these findings are only associations and the antibody responses to these antigens may be surrogate markers of other malaria antigen specific immunity that were not assessed in this study. The functionality of these antibodies that correlated with protection from clinical malaria in the present study can, however, be evaluated in a functional assay such as the The results from this study also supports the view that a multivalent vaccine involving different antigens, such as MSP1-3D7 and AMA1-3D7, is most likely to be more effective than a monovalent one, at least in this area. These antigens can, therefore, be tested in the study site, which is under demographic surveillance and has high malaria transmission. This study also confirms the feasibility of using the multiplex assay to assess a panel of antigens with small volume of sample.The authors declare that they have no competing interests.DD designed the experiments and wrote the manuscript, FA performed and supervised field work, SB analysed the data, NAA supervised the field work, PA supervised the field work, HN and BE performed the experiments, ARO conducted and supervised the field work, BG designed the experiments, AH conceived the study, and KAK conceived the study and wrote manuscript. All authors read and approved the final manuscript.


The objective of this study was to measure the rate of asymptomatic carriage of plasmodium in the Dakar region two years after the implementation of new strategies in clinical malaria management.Between October and December 2008, 2952 households selected in 50 sites of Dakar area, were visited for interviews and blood sampling. Giemsa-stained thick blood smears (TBS) were performed for microscopy in asymptomatic adult women and children aged 2 to 10 years. To ensure the quality of the microscopy, we performed a polymerase chain reaction (PCR) with real time qPCR in all positive TBS by microscopy and in a sample of negative TBS and filter paper blood spots.The analysis has concerned 2427 women and 2231 children. The mean age of the women was 35.6 years. The mean age of the children was 5.4 years. The parasite prevalence was 2.01% (49/2427) in women and 2.15% (48/2231) in children. Parasite prevalence varied from one study site to another, ranging from 0 to 7.41%. In multivariate analysis, reporting a malaria episode in 2008 was associated with plasmodium carriage  in women; in children, a malaria episode  and a travel out of Dakar during last 3 months  were associated with plasmodium carriage.Among the positive TBS, 95.8% (93/97) were positive by plasmodium PCR. Among the negative TBS, 13.9% (41/293) were positive by PCR. In blood spots, 15.2% (76/500) were positive by PCR. We estimated at 16.5% the parasite prevalence if PCR were performed in 4658 TBS.Parasite prevalence in Dakar area seemed to be higher than the rate found by microscopy. PCR may be the best tool for measuring plasmodium prevalence in the context of low transmission. Environmental conditions play a major role in the heterogeneity of parasite prevalence within sites. Malaria is reported to be more prevalent in rural areas compared to urban settings However, despite low transmission, malaria is still a major public health problem in urban areas, as it currently concerns more than 50% of African population and is expected to concern at least 60% in 2050 In Senegal in 2005, the Health Ministry reported high levels of malarial diagnoses and anti-malarial prescriptions Anopheles arabiensis aggressiveness was low, and no infected Anopheles was collected With regard to entomological data, in the south and central sanitary districts of Dakar in 1994–95 and 1996–97 respectively, Available prevalence data for the parasite are out-dated. The city has changed considerably since the last studies on the prevalence of malaria, both demographically and in terms of urbanisation, and it is necessary to obtain useful information to demonstrate the burden of malaria in Dakar.In 2008, a three-year multi-disciplinary research program was undertaken in Dakar to identify determinant factors of health care access and the success of new strategies in malaria control at the population level.As a part of this program, the aim of the present work is to update the prevalence of asymptomatic carriage of plasmodium in the Dakar region two years after the implementation of new strategies in clinical malaria management (i.e use of artemisinin-based combination therapy since 2006 and rapid diagnostic tests since 2007), and obtain reliable and representative data.districts of Dakar, Pikine, Guediawaye and Rufisque.The study took place in the region of Dakar, Senegal, which comprises the districts are sub-divided into 14 boroughs and 42 neighbourhoods (akin to boroughs).The districts had a population of 2,168,314 inhabitants and 302,551 households In 2002, these 4 urban The region of Dakar is in a Sudano-Sahelian climate with a long dry season (October to June) and a short rainy season (July to September). The level of recorded rainfall rarely exceeds 500 mm per annum. However, it has been increasing since 2005 Due to the proximity of the sea, this coastal area benefits from particularly mild conditions, with a maximum temperature of 27–28°C in September and a minimum of 21–22°C in February.We performed a cross-sectional survey from October to December 2008.Following the model of household surveys using the IRIS (Grouped Islets for Statistic Indicators) carried out within the SIRS research program  The eligible households must contain at least one child younger than ten.The urban environment is known to be a heterogeneous space from both a socio-economic and an environmental perspective. The aim of the choice of study sites was therefore to highlight the diversity of the urban zone. It was necessary to have homogeneous zones and the most heterogeneous zones between them.district de recensement” (DR - or census area) given by the national agency of statistics and demography collection. The population average in each DR was 1037 inhabitants, 141 households and 86 concessions. One DR constituted a homogeneous entity from a socio-economic, demographic and environmental point of view. Dakar comprises approximately 2000 DR.The “The selection of study sites followed a 3-step procedure, described below.Census data are too bulky to be processed directly. Data synthesis is thus necessary. We first regrouped various clusters of variables belonging to the same topic . For each group, a principal component analysis (PCA) was performed to extract the first two factorisation axes summarising the data for each group.Finally, a principal component analysis was carried out across all factors to perform a factorial analysis.A classification by dynamic clusters (K-means method) using variables selected from the PCA enabled us to identify 5 types of DR.Type 1: 307 DR, located mostly in the district of Dakar. This type mainly consists of the high-income population, which enjoys good housing and living conditions.Type 2: 274 DR, located mostly in the district of Dakar. This type mainly consists of the middle-income population.Type 3: 335 DR can be found in clusters in various areas. This type contains the low-income populations, mainly in the district of Dakar.Type 4: 528 DR can be found in clusters across various built-up areas, especially to the west of Pikine. This type mainly contains low-income populations from the old Pikine.Type 5: 526 DR found in clusters across various built-up areas, especially in the east Dakar area. It is mostly composed of the low-income populations from recently occupied zones found mainly to the east of Pikine.neighbourhood were chosen. Then, 42 DR were randomly chosen. To reach the number of 50 sites planed for the study, 8 other DR were then added according their socio-demographic and economic characteristics as well as to their proximity to the swamps. To avoid the risk of insufficient numbers of households in a DR, we have paired each of the chosen DR with the nearest same type of DR, thereby constituting one study site.The most representative types in each Selected DR were visited according to itineraries pre-established by the head supervisor; the field worker picked up a concession within the study zone and then moved from one household to the next. If a concession encompassed several households, the field worker chose the head of the household using the first name ranked alphabetically.Sixty households in each site  should be selected. The first criterion for household selection was the presence of at least one 2- to 10-year-old child. After collecting a family agreement, socio-demographic investigators completed a questionnaire about the household lifestyle, education level, income, and the access mode to healthcare facilities. The adult woman  who answered the questionnaire and a child aged between 2 and 10 years old were selected by the nurses for blood sampling. Asymptomatic individuals were selected on the basis of their own declarations and interviews by nurses. Pregnant women and individuals who were sick and/or had taken anti-malarial drugs during the 15 days preceding the blood sampling were not included in the study. Then a second questionnaire was completed by a team of nurses, exploring events linked to a reported malarial attack, including the history of a malarial episode during 2008, the use of a bed net, any overnight stays outside the region of Dakar (for women), or any travel (children).Reporting a malaria episode in 2008: we asked individuals (women and caretakers for children) if they have experienced a malaria attack during the year 2008.This protocol was approved by the ethics committee of Senegal's Department for Health (SEN 12/08). Information about the objective of the study was given to the head of the household, and verbal consent was requested. For minors, the consent of their legal guardians was requested. This procedure was used because most of the head of household could not write. The nurse explained the aims and interest of the study, and then he signed a document proving that he has obtained the consent. The ethics committee agreed on this procedure.All participants were informed of the results of the thick blood smear examination by telephone or home visits. Those who tested positive were treated through the local health facility.A thick blood smear for malaria parasite research and a dried blood filter paper spot were collected from each volunteer. The staining and the reading of the thick smears were carried out at the laboratory of the research institute for development (IRD) in Dakar.To declare that a thick smear is negative, the microscopist examined at least 200 oil immersion fields. A control examination of all thick smears that were found to be positive was performed. Ten percent of the negative thick smears have been examined a second time to guarantee the quality of the reading. All discrepancies were discussed, and if necessary, the thick smears were read by a third microscopist. The plasmodium species and the presence or absence of gametocytes was noted. We counted the number of trophozoites per 200 leucocytes. If the number of trophozoites counted is less than 10 per 200 leucocytes, the count is made from 500 leucocytes. We calculated the parasite density per microliter of blood using the following formula: (Number of trophozoites ×8000)/Number of leucocytesDNA was extracted from filter spots by the use of chelex as previously described GTT AAG GGA GTG AAG ACG ATC AGA) and Plasmo2 (AAC CCA AAG ACT TTG ATT TCT CAT AA) and the Plasmodium falciparum-specific forward primer Fal-F (CCG ACT AGG TGT TGG ATG AAA GTG TTA A) designed from sequences of the small subunit of 18S rRNA and reported by Shokoples et al. TCGTAATCTTAACCATAAAC and Falciprobe: Fam - TCTAAAAGTCACCTCGAAAGA) to increase specificity and limit cross-hybridisation of primers and probes in duplexes. Forty cycles of PCR were carried out and included the following controls: extractions of PBS and/or chelex as a negative control, a human host gene ) as a positive control for DNA extraction using primers and probe described in Duplex real-time PCR was performed using the conditions 95°C for 5 s and 60°C for 45 seconds with the Rotor Gene 6000 analytical PCR system. The PCR reaction was performed in a single reaction tube with a final volume of 20 µl, containing 5 µl of DNA, 10 µl of PerfeCTa qPCR FastMix, UNG (Quanta Biosciences), 300 nM of each primer, and 100 nM of each probe. We used the genus-specific primers Plasmo1 (www.stata.com). The socio-demographic characteristics  were described.Statistical analysis was performed using Stata version 11 :Parasite prevalence: the percentage of thick blood smears carrying a plasmodium.Gametocyte index: the percentage of positive thick blood smears carrying a gametocyte.The parasite density (DP): e.g., parasitaemia. The parasite density was estimated in relation to leucocytes, on the basis of 8,000 leucocytes per µl of blood, as explained above.The parasite prevalence was calculated for two groups, women and children, in each study site.The χ2 test was used to compare the plasmodium carriage rate in women and children.To estimate the association between the presence of plasmodium and the variables mentioned above, a univariate analysis by logistic regression was performed first. The ages in the women's group were divided into 4 classes based on quartiles. In children, two age classes were created (<5 years and ≥5 years). Finally, a multivariate logistic regression was performed to test the association between plasmodium carriage and independent covariates.A test was considered statistically significant if the p-value was <0.05.Two thousand nine hundred and fifty-two households in 50 study sites were visited for both socio-demographic and biological investigations. Of these, 328 households did not give consent for biological measures, and 63 households were missing or unavailable at the time of the home visit. Other subjects were not selected due to protocol requirements . The analysis of biological data has finally concerned 2427 women and 2231 children.The mean age of the women was 35.6 years (range = 17–81). The mean age of the children was 5.4 years (range = 2–10). Forty-nine percent of the children were male.The parasite prevalence was 2.01% (49/2427) in women and 2.15% (48/2231) in children, while the overall parasite prevalence was 2.08% (97/4658).Plasmodium falciparum; two thick smears (2.06%) harboured Plasmodium malariae, while one individual had a mixed infection .A total of 96.9% of infections were caused by Gametocytes were found in 33 individuals among the total of 97 infected by plasmodium (34.02%): 34.7% (17) in women and 33.3% (16) in children. Gametocyte carriage was higher among children younger than 5 .The parasite density (DP) for asexual forms ranged from 1.1 to 41963 parasites per microliter, with a median value of 190. Among positive thick blood smears, 25% had parasitaemia of less than 12 parasites per microliter of blood.The overall parasite prevalence did not show monthly variations. It was at 2.10%, 2.15% and 1.95% in October, November and December, respectively.In women, the plasmodium prevalence was 2.93% in the population younger than 28 years old, compared to 1.08%, 1.96% and 1.91% for the age classes of 28–34, 34–42 and more than 42 years, respectively. The difference was not significant.In children, there was no difference between the group younger than 5 (2.03%) and the group 5 and older (2.29%).Parasite prevalence varied from one study site to another, ranging from 0 to 7.41%. No plasmodium carriers were found in fifteen sites .In the women, the parasite prevalence ranged from 0 to 10.71%, whereas in children, it ranged from 0 to 8.33%. Differences of parasite prevalence are found throughout the area of Dakar. Differences are more pronounced in peripheral sites located nearest the swamps. A chi-square fisher exact test, have been performed to compare prevalence between sites. For example, prevalence in Darou missette is 7.41 whereas it is 1% in Darou salam 2 (p = 0.036). Similarly, prevalence is 0% for Diokoul and 3.88% for Merina (p = 0.053).Three sites closest to the downtown of Dakar  had parasite prevalences as high as those found in peripheral areas.In the univariate analysis, only “reporting a malaria episode in 2008” was significantly associated with parasite carriage. Among women, 45% declared using a bed net; we have found no differences in parasite prevalence between women who reported using a bed net and those who did not.In the multivariate analysis, only age and “reporting a malaria episode in 2008” were significantly associated with parasite carriage. Women between 28 and 34 years were less likely to be infected compared to those aged younger than 28 .2 = 4.28, p = 0.74) confirms the good fit of the model.A Hosmer-Lemeshow test was performed to verify the quality of model fit. The results of the test  and “a trip out of Dakar in last three months “  were significantly associated with parasite carriage. We did not find any differences in risk according to age group or sex.In children, bed net use as reported by mothers was 43%; no difference in prevalence was found according to bed net use.In the multivariate analysis, “a malaria episode in 2008” and “a trip out of Dakar in 2008 in last three months” were associated with parasite carriage, with an OR of 6.19 (P<0.001) and 2.27 (p = 0.023), respectively.2 = 6.62, p = 0.46) and confirmed the hypothesis of equality between the observed and the predicted number.A Hosmer-Lemeshow test was performed  were positive by plasmodium PCR. Among the negative thick smears, 13.9% (41/293) were positive by PCR. In the dried blood spots, 15.2% (76/500) were positive by PCR.To calculate the overall parasite prevalence, we first calculated the total percentage of positive PCR results in a selected sub-sample; 41 of 293 thick smears and 76 of 500 blood spots were positive, giving an overall rate of 14.75%.Second, we estimated 673 positive thick smears if PCR was performed on 4561 negative thick smears (97 positive thick smears removed) using the rate of 14.75% found above in the sub-sample.Based on these results, the estimated parasite prevalence using PCR and microscopy is approximately 16.5% in our sample .The aim of this study was to measure parasite prevalence in a sample of asymptomatic individuals in the region of Dakar. The selection of study sites and households was performed by following a methodology that takes in account the diversity of the urban environment. The representativity of the sample was also established with regard to population density. Data were obtained from the 2002 population census provided by the national agency for statistics and demography of Senegal. During the period 2002–2008, there was a small increase in population density; indeed, Dakar was urbanised at 97.2% in 2002. Moreover, Dakar is built on a peninsula, which allows possible extensions only on the east side of the area. Changes have mostly concerned the newly occupied areas in the east of Dakar, where only 3 study sites were selected in our sample.Our study was mainly focused on women and children aged 2 to 10 years old. It was easier for us to interview women, who are responsible for the daily activities of the home. Men are often missing due to work or travel. Children aged 2 to 10 constitute a standard group for studies of malaria endemicity, but measures in women could present a bias in the estimation of parasite prevalence in the adult population. No study has demonstrated that women have a higher risk of carrying the parasite than male adults in Dakar. Nevertheless, to obtain an indication of the prevalence in men, 224 thick smears were collected in volunteer men present at home during our visit. Five of them were found to be positive (2.23%). This prevalence is similar to that found in women.Several population studies have been conducted in Dakar area since 1979. Four (4) main studies were still references for malaria transmission in Dakar. Two studies conducted in 1979 and 1987 in Pikine located at 15 kilometers away of downtown of Dakar; the target population were children aged 6 months to 6 years in the first study Between 1995 and 1997, Diallo et al. In Pikine parasite prevalence were 8.8% in 1980 and 3.8% in 1988 respectively, while in our survey, parasite prevalence varied from 0.88 to 3.88% in the sites located near these areas. Thus, there is variation in parasite prevalence, mostly explained by increasing urbanisation, since Pikine was not highly urbanised in the early 80 s.Parasite prevalence in Diallo studies, were 0.9% (October–November 1994) and 1.5% (October–November 1996) in downtown Dakar and the nearby respectively, whereas in our study, it was 2.08%, 4.12% and 6.1% for the sites located at nearby. Accordingly, prevalence is higher in our study.The methodologies in all these studies are different in terms of area and target population.In our sample, children under 5 years old have a carriage rate of 2.03%. This rate is higher than the 0.8% found recently by the ENPS  in infants aged 6 months to 59 months in 2008 in Dakar Thus, the variability of parasite prevalence across time and space is evident in Dakar.The same technique was used in all studies mentioned above, i.e., thick blood smears (BS). We used real-time qPCR to confirm our microscopy results. Unfortunately, we were unable to carry out PCR on all the blood samples, given the high cost of the technique. Several studies have shown the superiority of real-time qPCR over microscopy Nevertheless, in our study, 4 positive BS were negative by PCR; parasitaemia was 19.4, 12.9, 4.8 and 137.1 parasites per microliter. For these BS positive samples that were not detected by PCR, several reasons could explain this: artefacts in thick smear reading due to red cell fragments, other blood elements or environmental contaminant, which might produce false-positive in microscopy. This has been discussed in other studies Most clinical and field studies on malaria rely on BS and PCR has not been widely used principally because of poor resources and infrastructures in study areas. The application of microscopy in areas with low parasite prevalence or parasitaemia can compromise its predictive value. The overall prevalence estimated by PCR in our study is approximately 16.5% which is 8-time higher than the microscopy.Plasmodium falciparum resistance to new anti-malarial drugs is quite plausible in this context, as artemisinin resistance has been reported recently in Cambodia If we conclude that malaria remains endemic in Dakar and that the prevalence can reach high levels in some areas (more than 10%), these results could explain the high consumption of anti-malarial drugs that has been found in Dakar, with high self-medication Anopheles development. In urban Dakar, malaria infections were imported from rural villages. However, recent data suggest that transmission occurs in Dakar. Indeed, a two-year study (2005/2006) in two sites near Dakar-centre (Ouakam and Bel-air) showed an annual EIR of 3 and 3 in Ouakam and 9.5 and 4 in Bel-air in 2005 and 2006, respectively These results could be related to new entomological data collected in the same areas. Entomological data collected in Dakar before 2000 suggested very low or no malaria transmission, with an EIR of less than 1 infected bite per person per year The hypothesis is that the mosquito responsible for the transmission of malaria has adapted to urban sites and now can develop in polluted waters, as reported by Awolola et al. in Lagos We found that approximately one-third (34%) of positive thick smears were gametocyte carriers. This rate rises to 50% in children under 5 compared to 17% for the other children. These findings are similar to those found in other studies In the low transmission context, such as urban areas, the constitution of immunity against malaria is delayed and often incomplete. Therefore, adults and children could face the risk of infection equally in areas with instable transmission. We did not find a significant difference between the prevalence in children (2.15%) and women (2.01%). Adults seem to be vulnerable in urban areas and can present with severe malaria Our study has demonstrated that the plasmodium is widely spread in the region of Dakar. Indeed, among the 50 sites surveyed, an absence of parasites was found by microscopy in only 15 areas.The built-up area of Dakar presents a diversified environment, with varying levels of sanitation from one zone to another. The city centre and surrounding districts are well urbanised. In contrast, the peripheral zones, which include the marshlands , are characterised by a mix of urbanised neighbourhoods and those occupied illegally.Depending on the study area, the prevalence varied from 0 to 10.8% in women and from 0 to 8% in children. Similarly, we observed huge variations in neighbouring sites. Three sites  located downtown Dakar presented a rather high prevalence. This could be explained by local characteristics that must be explored by geographical and entomological studies. The other sites located downtown Dakar presented rather low levels of parasite prevalence. In contrast, parasite prevalence is high at sites located in peripheral areas. Indeed, these areas presented 2 characteristics: first, urban farming is developing near the swamps (“Niayes”), and second, illegal dwelling has modified local conditions, causing major floods during rainfall.In the present study, we found differences in parasite prevalence between sites; but it was not possible to demonstrate the heterogeneity of prevalence. However the differences observed between sites suggest strongly the existence of the heterogeneity of malaria transmission, as it is showed in an entomological study conducted between 2007 and 2010 in Dakar Environmental factors play an important role in relation to the risk of infection. People living near a potential larval habitat are more at risk of infection than others The use of bed nets is known to reduce the risk of malarial infection. In this study, self-reported bed net use was 45% for women and 43% for children. These rates do not differ from those found in the National Survey for Malaria in Senegal Surprisingly, we have found no significant difference in the prevalence of malaria between those who used a bed net and those who did not. Data were collected by interview. These results show the limitation of self-reported information. Indeed, if individuals have a bed net, this does not mean that they use it correctly and consistently. To establish any link between malaria infection and the use of bed nets in the general population, it would be necessary to perform a questionnaire on habits and day-to-day usage.The period of the survey coincided with the maximum period of malaria transmission in Dakar We have found that most children carrying plasmodium have had a clinical attack of malaria . The same situation was found in women.2 = 12, p = 0.001). Similarly, by regrouping all sites with a lower prevalence, the carriage rate was 1.85% in individuals who have a history of malaria and 1.15% for others (the difference is not significant).We suggest that the majority of infections occurred recently, since the beginning of rainy season. Most treatments were self-medication, with problems of compliance and adequate treatment, which can explain the lack of parasite clearance. The other hypothesis is that the sites with a high risk of infection were most concerned in our study. This could explain the correlation between a history of malaria and plasmodium carriage. When regrouping the sites with higher prevalence, we found a carriage rate of 7.43% in individuals with a history of malaria and 2.23% for others  during holidays corresponding with the rainy season. The risk of infection is more important in rural areas, especially if the stay is long.The movement of this population affects malaria transmission Our results outline a new urban malaria epidemiology. Contrary to what is commonly accepted, malaria is not uncommon in urban Dakar, and malaria is not unstable. Data suggest that malaria may still be a threat to the urban population. As a confirmation, three facts must be considered. First, entomological data clearly show an increased risk of transmission. Second, our data show a parasitological prevalence that is variable but very significant in certain areas, especially in the most popular part of the town. Third, self-medication in drugstores is frequent and suggests a significant rate of malaria attack in population.The weight of malaria in common diseases is certainly underestimated due to the huge distribution, mostly un-regulated, of new treatments (ACT), reduced access to care and high rate of self-medication.Our results and entomological findings should help to initiate further studies in populations and to alert health authorities to take into account environmental conditions. According to these findings, it should be important to update the burden of clinical malaria in the Dakar area, including severe malaria, to set up a monitoring system for severe cases and for screening of the emergence of drug resistance.Our study also suggests that PCR may be the best tool for the estimation of parasite prevalence in the general population in urban Dakar due to the low rate of parasitaemia occurring in this context.