Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.Severe malaria has been attributed partly to the sequestration of P. falciparum isolates from Mozambican children under 5 years of age with severe malaria (cases) were examined and compared to 46 isolates from sex and age matched Mozambican children with uncomplicated malaria (controls). Cytoadherence properties such as platelet-mediated clumping, rosetting and adhesion to purified receptors , were compared between these matched pairs by non-parametric tests. The most common clinical presentation associated with severe malaria was prostration. Compared to matched controls, prevalence of platelet-mediated clumping was higher in cases (P = .019), in children presenting with prostration (P = .049) and in children with severe anaemia (P = .025). Prevalence of rosetting and gC1qR adhesion were also higher in isolates from cases with severe anemia and multiple seizures, respectively (P = .045 in both cases), than in controls.Forty-six These data indicate a role for platelet-mediated clumping, rosetting and adhesion to gC1qR in the pathogenesis of severe malaria. Inhibition of these cytoadherence phenotypes may reduce the occurrence or improve the prognosis of severe malaria outcomes. Plasmodium falciparum causes approximately 500 million clinical episodes of malaria per year P. falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs leading to mechanical obstruction of blood flow, hypoxia, tissue damage and, ultimately, organ failure P. falciparum IEs to host receptors expressed on the endothelium lining host capillaries, on uninfected erythrocytes to form ‘rosettes’ P. falciparum, which is responsible for most of the malaria related deaths, is known to sequester P. falciparum variant types suggests that IEs can exhibit preferential adhesion in the body The following two theories are proposed to explain the patho-physiological mechanisms leading to severe malaria. The cytokine-based hypothesis suggests that inflammatory mediators such as cytokines and nitric oxide released by host cells following exposure to malaria ‘toxins’ have a major role in the onset of the pathology of severe malaria In vitro analysis of the cytoadherence phenotypes of P. falciparum isolates derived from infected individuals has shown a wide range of binding affinities to numerous host receptors such as intercellular adhesion molecule ICAM-1 P. falciparum isolates, namely, adhesion to ICAM1, CD36 and gC1qR, rosetting and platelet-mediated clumping, and disease severity in children less than 5 years of age residing in a malaria-endemic area of Mozambique.Here we have examined the relationship between specific adhesion phenotypes of Among the 142 parasite isolates included in the study, 111 (78%) matured to trophozoite stages. The proportion of isolates that did not recover from cryopreservation was similar for parasite isolates collected from children with severe  or uncomplicated malaria . Parasitemia at thawing was higher for isolates from children with severe malaria  than in children with uncomplicated malaria . Of the 111 isolates that matured to trophozoite stage, 46 matched case-control pairs of parasites could be used for analysis. The characteristics of the 92 patients that provided the parasite isolates used are summarized in 2; standard deviation 8.78), we established the cut-off for positive binding to endothelial host receptors as 19.5 per mm2 (mean+ 2 standard deviations).Isolates were considered positive for rosetting if frequency of rosettes was higher than 2% P. falciparum isolates tested (65%) formed clumps in presence of platelets. Among those isolates showing platelet-mediated clumping, patient's platelet count decreased with increasing frequency of clumps . No correlation was found between frequency of clumps and parasitemia at thawing . Results from platelet-mediated clumping assays performed without adjusting parasitemia correlated well with results from the same assay performed with parasitemias adjusted to 1% . Both assays gave similar results in terms of prevalence of clumping , although medians for frequency of clumping were slightly higher in assays with unadjusted parasitemias  compared to assays performed at 1% parasitemia .I was doing it and I will do my best to finish Sixty out of 92 P. falciparum isolates , followed by platelet-mediated clumping , adhesion to ICAM1 , to gC1qR  and rosetting . Among the isolates showing cytoadherence, median adhesion (parasites per mm2) was highest for CD36 , followed by gC1qR  and ICAM1 . Median frequency of rosetting and platelet-mediated clumping was 3.0% (IQR [1.9–5.3]) and 7.2% (IQR [1.9–23.3]), respectively.Adhesion to CD36 was the most frequent cytoadherence phenotype among Cytoadherence phenotypes were compared between isolates from children presenting with severe malaria or specific severe malaria syndromes and their matched controls. For the rest of the cytoadhesion phenotypes, a uniform trend towards higher levels of cytoadherence among parasites isolated from children with severe malaria was observed, although these differences were not statistically significant. Platelet-mediated clumping assessed at unadjusted parasitemias was also higher for those parasites isolated from children with prostration and severe anemia, as compared to their matched controls  and 3. SThe results of this case-control study suggest that platelet-mediated clumping is associated with severe malaria. Moreover, to our knowledge, this study reports for the first time that platelet-mediated clumping is associated with prostration, and that adhesion to the recently identified cytoadherence receptor gC1qR The 46 patients with severe falciparum malaria participating in this study are representative of this condition in Manhiça (Mozambique) P. falciparum isolates infecting Mozambican children were characterized by their predominant adhesion to CD36 (87%), followed by platelet-mediated clumping (65%), adhesion to ICAM1 (45%), to gC1qR (42%) and rosetting (36%). It could be argued that cryopreservation of parasites may reduce the adhesive ability of IEs, although it has been reported that it does not alter the amount of parasite proteins on the surface of IEs as assessed by flow cytometry In accordance with previous studies from other sites Since pathogenesis may be quite different among the syndromes used to define severe malaria, we have also analyzed cytoadherence separately for each syndrome. Prostration, the most prevalent severe malaria presentation in this study population, was found to be associated with platelet-mediated clumping. Multiple seizures were observed to be associated with adhesion to gC1qR. Seizures are frequent among severe malaria cases. They may occur as part of CM or independently Severe anemia was associated with higher platelet-mediated clumping and rosetting, suggesting a role for these cytoadherence phenotypes in the etiology of malaria-related anemia. Destruction of uninfected erythrocytes attached to IEs in rosettes or down regulation of erythrocyte production by abnormal erythrocyte aggregates might also contribute to severe anemia. The role of platelet-mediated clumping in severe anemia is unclear and requires further investigation. If platelet-mediated clumping is found to have a causative role in severe anemia, novel anti-adhesion interventions that prevent platelet-mediated clumping might have an impact on reducing severe anemia in malaria endemic areas. Such interventions might be of public health relevance, as severe anemia is an important contributor to malaria-related morbidity and mortality in children residing in sub-Saharan Africa Role of adhesion to CD36 in the physiopathology of severe malaria is still a subject of debate, as some studies have found higher binding in isolates infecting children with severe malaria P. falciparum may be an important virulence factor. The potential for inhibiting or reversing platelet-mediated clumping and adhesion to gC1qR with antibodies or receptor analogs could improve severe malaria outcomes and should be actively explored.In summary, the results of this case-control study support the hypothesis that cytoadherence properties of The study protocol was approved by the National Ethics Review Committee of Mozambique and the Ethics Review Committee of the Hospital Clinic of Barcelona.P. falciparum infections with an average entomological inoculation rate of 38 infective bites per person per year in 2002 The study took place in Manhiça District, southern Mozambique. The study area, which is characterized by perennial malaria transmission with some seasonality, has been extensively described elsewhere P. falciparum malaria were recruited into the study after written informed consent was given by their parents or guardians. Blood smears were stained with Giemsa and examined by microscopy according to quality-controlled procedures P. falciparum≥500/µLon thin blood film examination. This definition has both a sensitivity and specificity of greater than 90% in children from Manhiça Between April and November 2006, a sex and age (+/−3 months) matched case-control study was conducted, the cases being children under five years of age with severe malaria (n = 71) and controls being outpatient children with non-severe malaria (n = 71). Children attending the Manhiça District Hospital with a primary clinical diagnosis of Before treatment, 10 mL of peripheral blood was collected by venipuncture into a tube containing lithium heparin. All blood samples were collected by trained persons in presence of a paediatrician. The amount of blood collected was less than the limit of 3 ml per kg of bodyweight as recommended by ethical guidelines msp-1 ormsp-2 alleles detected in the sample by PCR DNA was extracted from blood onto filter papers using the QIAGEN QIAamp DNA Mini Kit. The number of concurrent infections  was estimated as the highest number of P. falciparum isolates were thawed by serial dilutions in a gradient of salt solutions following standard methods. Erythrocytes were washed in RPMI 1640 medium containing 2 mM L-glutamine, 25 mM HEPES, 25 mM sodium bicarbonate and 10 µg/mL gentamicin (incomplete RPMI) and cultured at 37°C in complete RPMI (incomplete RPMI supplemented with 0.5% Albumax I and 50 mM hypoxanthine) as previously described Cryopreserved 2 was counted by observation of 10 fields (using 100× objective) under light microscopy. Adhesion to BSA was subtracted to yield specific binding to each receptor.Measurement of parasite adhesion to purified receptors was done as previously described An aliquot of culture suspension at pigmented trophozoite stage was stained with 20 µg/mL of ethidium bromide in rosetting media (incomplete RPMI supplemented with 10% AB+ human sera). Twenty µL of the suspension at 2% hematocrit was examined by fluorescence and direct light microscopy, and the proportion of IEs in rosettes was measured for each sample in triplicate. Two hundred IEs were scored in each experiment, with the adhesion of two or more uninfected erythrocytes to an IE constituting a rosette. The level of rosetting is reported as the percentage of IEs forming rosettes.g for 5 min. Platelet rich plasma was transferred to a separate tube, equal volume of fresh CCDAT buffer  was added drop wise and incubated for 15 min. Platelets were collected by centrifugation at 1000 g for 20 min and resuspended in CCDAT buffer. Homogenous platelet suspension was transferred to new tubes and centrifuged at 1000 g for 20 min. Platelets were finally resuspended in Hanks buffer at 108 platelets per mL, stored in sterile condition at room temperature and used within 4 days of collection. All steps were performed at room temperature. P-selectin (CD62) was not detected on the surface of platelets by flow cytometry Blood was collected from donors by venipuncture in citrate-phosphate dextrose solution (Sigma Aldrich), and centrifuged at 250 Parasites were thawed and used for clumping assays as described below. The assays were performed at thawing parasitemias and 2% hematocrit, as well as at 1% adjusted parasitemias  and was analysed using the Stata version 9.0 (Stata Corporation). A Table S1Platelet-mediated clumping results in assays conducted at 1% parasitemia and 5% hematocrit.(DOCX)Click here for additional data file.


In the fight against malaria new medicines are an essential weapon. For the parts of the world where the current gold standard artemisinin combination therapies are active, significant improvements can still be made: for example combination medicines which allow for single dose regimens, cheaper, safer and more effective medicines, or improved stability under field conditions. For those parts of the world where the existing combinations show less than optimal activity, the priority is to have activity against emerging resistant strains, and other criteria take a secondary role. For new medicines to be optimal in malaria control they must also be able to reduce transmission and prevent relapse of dormant forms: additional constraints on a combination medicine. In the absence of a highly effective vaccine, new medicines are also needed to protect patient populations. In this paper, an outline definition of the ideal and minimally acceptable characteristics of the types of clinical candidate molecule which are needed (target candidate profiles) is suggested. In addition, the optimal and minimally acceptable characteristics of combination medicines are outlined (target product profiles). MMV presents now a suggested framework for combining the new candidates to produce the new medicines. Sustained investment over the next decade in discovery and development of new molecules is essential to enable the long-term delivery of the medicines needed to combat malaria. Malaria is a critical public health challenge, historically being responsible for the deaths of millions, particularly young children and expectant mothers. The past decade has seen significant progress in the control of malaria, with a reduction in reported cases . There wAnopheles) is developing behavioural strategies to evade ITNs [Two major types of challenge for the development of new medicines against malaria remain: those external to the drug discovery community, and those internal. The external challenge is the changing malaria landscape. Emergence and spread of resistance are always major concerns in infectious disease, and recent reports in the literature confirm decreased patient responses to artemisinin derivatives in South-East Asia ,10 combiade ITNs , and resOther public health factors will influence the type of medicines needed in the future, such as the need for anti-malarial treatments for patients who are already receiving treatment for Human Immunodeficiency Virus (HIV), tuberculosis (TB) or other co-infections. Co-treatment for HIV infection is especially relevant due to risks for interactions between the medicines used to treat HIV and those for malaria, through interference with metabolic pathways involving cytochromes, especially P450 3A4. This increases the risks for altered pharmacokinetics leading to either reduced efficacy or enhanced drug exposure and side effects. In addition the pathology of co-infection means that such patients are especially vulnerable, and this may present additional constraints for the safety of new medicines. Finally, the chemical stability of new medicines is another key challenge. Fixed-dose artemisinin combination therapies are stable in Zone IV conditions  for 2–3 years. Given the difficulties of distribution, then any improvement over this level of stability would be of considerable advantage in the future.0 < <1). Second, they need to safely prevent the relapses with Plasmodium vivax and Plasmodium ovale. Third, significant post-treatment prophylaxis  may help to reduce the clinical burden of malaria especially in high-transmission areas. Fourth, new medicines will be needed for chemoprotection  that are necessary to protect vulnerable populations such as infants and expectant mothers. All of these medicines must be safe enough for use in sensitive patient groups, including pregnant women [In addition, there are internal factors, within the community, which arise from the new goal of long-term malaria eradication. These bring additional challenges in drug discovery beyond those required by medicines that effect simple case control. First, the new medicines need to be able to reduce and, ideally, prevent transmission from one infected patient to the next , and registration in highly diverse countries means that the overall project timeline to launch in disease-endemic countries will probably remain at 12–15 years. With these long timelines it is essential to start out with a clear idea of what success will look like: what are the anticipated needs in the clinic at the time of launch, what is the ideal clinical candidate molecule, and how will these be combined, with existing therapies, into the ideal final product? This requires a clear hypothesis or hypotheses as to how the medical need will change over the next fifteen years. The description of the desired product is captured in what is known as the Target Product Profile (TPP). It is important also to underline that the target product profile can vary with different external factors. The clearest example here would be the spread of ‘artemisinin resistance’. Obviously, in countries or districts where no ACT is clinically effective, the TPP will be different from those where current therapy is still fully active. Since for malaria, the medicine, or target product will contain more than one active ingredient, the attributes of a target product profile can be divided into many different target candidate profiles (TCP), each of which is effectively the job description for a new molecule entering clinical development. Exactly how the overall list of attributes needed in the final product is divided between the different molecules it contains and indeed how many different components are needed will be a matter of much debate, and there are many different potential solutions. However, this debate will be anchored in reality when the activities of each candidate in patients is seen, a theme which is explored later in this paper. The other reality check in this process is provided by comparison to existing medications, which act as the gold standards for clinical trials in terms of safety, efficacy, potential for transmission-blocking and relapse prevention, cost of goods, and overall convenience. It is, therefore, important to ask continually which potential benefits a new molecule will bring compared with existing molecules , throughout the discovery and lead optimization process.Plasmodium in a patient, including the dormant liver stages or hypnozoites and asymptomatic sexual stages or gametocytes. This medicine should wherever possible be given as directly observed therapy (DOT), to ensure compliance, even in challenging field conditions; as such, the SERCaP represents the ideal treatment. It is important to underline that this may not be achievable, and so compromises will undoubtedly have to be made along the way. For this reason this paper provides two definitions, an ideal and a minimally acceptable product. A second class of medicine, a new generation of prophylactics was also suggested, which would be needed in countries which have eliminated malaria, but where a local resurgence of infection could occur.After some years of stagnation, the global pipeline of malaria medicines in development is now progressing as a result of significant investments over the past decades. This fresh acceleration raises an additional complication: that the minimum requirements for new medicines are not fixed, but are continually moving. As the number of molecules with the potential to meet a particular TCP increases, then the standards for new molecules will move higher. Conversely, if clinical candidates are lost from the portfolio due to safety considerations or the emergence of resistance, the bar may be lowered, though the shortcomings of the failed drug, whether safety or efficacy, will still need to be overcome. It is likely that compounds will be parked at various stages of the preclinical and clinical development processes such that they can then be reactivated in response to factors external to each individual project, such as spread of resistance to a mainstay therapy. This underlines the need for all drug discoverers to have access to a clear and accurate picture of all molecules in development. A detailed review of the global landscape was recently published . An onliHistorically, MMV played a role in coordinating proposals for TPPs describing both the ideal and minimally acceptable profile for new medicines. The last version concentrated more on the product profiles rather than the candidate profiles, and was produced with the External Scientific Advisory Committee of Medicines for Malaria Venture in 2010. In the present publication, definitions for the attributes of individual molecules (TCPs), and for the final combination product (TPPs) are laid out. The TPPs fall into two groups: medicines which can be used to cure patients, and medicines which can be used to protect populations from infections. The scientific justification and rationale behind these updated recommendations are presented, as well as some of the currently unanswered questions. Strategies for combining the individual candidate molecules to produce the products are discussed, and this is one area where there are potentially many solutions to the same problem. The malaria drug discovery portfolio certainly appears stronger than a decade ago. However, there are now enough data to characterize success rates. This enables a characterization of the unmet needs in terms of how many new molecules will be required in the future.Several characteristics of each candidate molecule are common across all of the different TCPs and can be discussed in general terms.in vitro parasite reduction rate), and the stages of the parasite lifecycle where the compound is active, in order to start to estimate how effective it will be in humans [Efficacy is initially established in a cell culture model of parasite activity, as close to the human infection as possible. As well as knowing the potency of a new molecule, it is important to also determine its speed of action  in artemisinin combination therapy cost between $100 and $1,000 /kg to produce , but some of these are used at total doses as high as 3 g in adults. If new medicines can be found with much lower human effective doses, for example around 30 mg, then the cost of the ingredient would be reduced by 100-fold . As a parenthesis, new generations of molecules with increased  per day . This isThe ease of synthesis and, ultimately, production is critical: a small number of synthetic steps, each with high yield, from low-cost available starting materials, will also play a role in reducing costs. Lowering the clinical dose generally also reduces variation in exposure and produces fewer side effects, especially the gastrointestinal irritancy caused by high-dose, poorly soluble medicines as discussed above. Packaging and manufacturing costs are often overlooked, and can represent a significant proportion of the overall costs. Compact, simple packaging helps reduce pricing. Figure P. vivax relapse, and can also, with a different dosing regimen, be used to prevent transmission of Plasmodium. Figure Plasmodium life cycle.The TCPs presented below summarize the four-five types of molecule that are sought to facilitate the elimination and eradication of malaria. Each profile describes a set of attributes for a single compound, for which there should be increasing confidence as a result of the regulatory preclinical studies, and which should be confirmed by the end of the human proof-of-concept  trial. Each TCP details a ‘Minimum Essential’ and an ‘Ideal’ profile. The ‘Ideal’ criterion builds on what is described in the ‘Minimum Essential’; therefore, criteria are not repeated unless there is a change. The ‘Ideal’ profile, as stated earlier, requires excellent safety as well as efficacy since administration to asymptomatics, under elimination tactics, is conceivable. It is possible that one molecule may fulfill all the requirements of two different TCPs. This is the case for primaquine which has good clinical activity against in vitro and in vivo studies can be used to predict how close any molecule in lead optimization is to the candidate definition, but these are only projections. The MMV experience has been that for success, compounds should typically have in vitro activities with an EC50 < 10 nM against laboratory-adapted strains and clinical isolates, and a single-digit mg/kg activity ED90 in the P. falciparum infected human erythrocyte-engrafted SCID mouse model [in vitro and in vivo can be used to predict this in humans, although these may be underestimated, since they do not allow for immunological or splenic clearance of damaged reticulocytes or erythrocytes [6-fold parasite reduction following a single oral encounter of one (or two) doses. For the minimum criteria, a medicine which produces the same effect over two to three days could still represent a clinically relevant alternative to current regimens though would need differentiating qualities to demonstrate advantage and justify investment. This reduction in parasite burden then needs to be confirmed clinically, measured by the proportion of patients who are cured as reflected by the ACPR at day 28. It is still not clear how large a response is required from a single agent to ensure an ACPR of >95% as part of a combination. Lessons from the artemisinins are instructive [in vitro against the blood stages of all five Plasmodium species which infect humans, although activity in P. falciparum and P. vivax are generally assumed to suffice, particularly since parasites and assays for the others are not readily available. TCP-1 can be summarized in the ideal case as a molecule that should be similar in activity to an artemisinin derivative but with pharmacokinetic properties such that it would allow for less frequent administration and even administration in a single sitting preferably with matched elimination PK and potency with a partner TCP-2 compound.The cornerstone of malaria treatment is the availability of at least one molecule capable of rapidly clearing the parasite load. In order to be effective, a compound addressing TCP-1  is impressive. Compounds such as mefloquine can maintain a blood concentration above the MPC for more than a month, which combined with a parasite reduction rate of 1.5 log units per day, gives an extremely impressive  maximum parasite reduction. The relationship between the duration that MPC is maintained and the post-treatment prophylaxis period is however not clear: mefloquine maintains these concentrations much longer than piperaquine, however piperaquine gives superior post-treatment prophylaxis [A candidate in this category is a long-acting compound, capable of killing the residual parasites not eliminated by the rapid-clearance TCP-1 medicine Table . Ideallyphylaxis . IdentifMost compounds used in screening come from diversity collections which have been specifically targeted against diseases in Western markets, for which the goal has often been once daily therapy. Furthermore, molecules with extremely long human half-lives  pose additional challenges in development, in terms of the design of toxicological and early clinical studies. A further problem is that the data on fast-killing molecules such as artemisinin, suggest that the logarithmic parasite reduction rates are not additive: a combination of two medicines does not necessarily increase the parasite clearance rate over the fastest compound alone. A simple way of viewing this is that the parasites can only be killed once. The second compound is needed to kill remaining parasites, and during the time that it is present as monotherapy there is a risk of resistance generation whilst the ‘resistance window’ is open. That is when the compound is still above its MPC for wild-type parasite, and hence providing a selective pressure, yet the concentration is below the MPC for any resistant parasite .P. vivax relapse due to its effects on hypnozoites, as well as its rapid gametocytocidal action [P. vivax hypnozoites for radical cure, and although it has been suggested this could be reduced to seven days by increasing the dose [As well as possessing erythrocytic-stage killing activity, an ideal combination would need to contain compounds which can prevent the relapse of dormant liver stages (hypnozoites) and the sexual stages of the parasite in the human host or in the mosquito vector. It is possible that a single molecule could be identified with all of these activities. The gold standard for this medicine is primaquine, which is the standard of care for preventing l action . Howeverthe dose , there athe dose ). There the dose ,42. Ideain vitro activity of EC50 <100 nM against hypnozoites in a validated assay would be desirable, although ultimately this number could be much higher if the compound has extremely high plasma exposure and is well tolerated in humans. Currently the only system available for testing new medicines uses Plasmodium cynomolgi infected primary rhesus hepatocytes, although assays for P. vivax infection of human cells are under development . The other challenge is the safety margin, in terms of plasma exposure at the EC90 and at the no-adverse effect level in pre-clinical species. Treatments should, therefore, be able to prevent relapses in a preclinical animal model or in man without toxicity. P. cynomolgi-infected rhesus monkeys have been the model of choice, with a gold standard 8-aminoquinoline as the reference drug [It is of course probable that no new single molecule will be found that can kill both the hypnozoites and prevent transmission, and that these two roles will have to be performed by different molecules in a combination medicine. The anti-hypnozoite attributes needed can be described by a subset of criteria, TCP-3a. Pragmatically, an lopment  allow fArtemisinin derivatives are capable of rendering stage V gametocytes inactive, but do not prevent malaria transmission clinically on their own due to their short half-lives, and so there is a need for a combination of anti-gametocyte activity combined with long-acting pharmacokinetics or dynamics. There is no generally agreed gold standard of activity required in an anti-gametocyte assay. The standard membrane feeding assay (SMFA) or mouse-mouse transmission models are currently the only intermediate assays that measure formal transmission to the mosquito vector and a mammalian host respectively ,50. The In Table Although in an ideal world, where the goal is a SERCaP, there may also be a place for a molecule with both transmission-blocking and anti-relapse activity as part of a three-day course of therapy, together with the current generation of artemisinin combination therapies. Hence for the minimal acceptable profile, a dose given over two or three days could have a role in malaria control and eradication.Ultimately, it would be better to prevent a population from becoming infected rather than treating the patients once they become symptomatic. In many disease areas, vaccines can provide such protection after a single injection providing protection for a large majority of subjects for as much as a decade. No such vaccine has ever been produced for a protozoan parasite, and the history of malaria control has relied successfully on chemoprotection from the earliest days of quinine therapy.There is a growing consensus on how malaria can be eliminated from affected countries. This strategy consists of active management of cases and their asymptomatic neighbours with first-line therapy in the early stages, followed by more intense programmes to break transmission. These would be followed by measures to contain reintroduction: either case detection by focal screening, or chemoprotection.As malaria incidence falls, the population in such countries could be expected to move from being semi-immune to being non-immune. Prior to such ‘end-game’ strategies, specific protection of sensitive populations such as pregnant women, infants, or children in zones with seasonal malaria has been shown to have significant impact ,16. The Chemoprotection can be achieved by: killing the sporozoite, killing the liver schizonts, or killing the parasites as soon as they emerge into the blood stream from the liver. Chemoprotection might be used to prevent an outbreak from spreading from an introduced index case to neighbouring households, or to protect sensitive populations. The current gold standards for chemoprotection are atovaquone/ proguanil and mefloquine, but both are far from ideal. The frequency with which an anti-malarial needs to be administered to achieve a high level of protection is key when the medicine is used for this purpose. A once per month dosing would provide a significant improvement over the current daily or weekly administrations. A medicine which only needs to be used once per outbreak would have a more significant advantage. Cost will be an important driver: atovaquone/proguanil is a combination daily prophylactic, with an adult cost of $5 per day, although these prices may fall now that the patent protection is expiring. Mefloquine, given one dose per week as mono-protection, is cheaper, costing around $1,000/kg to produce, and so the 500 mg adult weekly treatment costs around $0.50 in raw materials. Cheaper ways to make mefloquine have been developed , reducinAn alternative approach to the design of long-acting medicines is the production of a slow-release formulation. In the 1960s this was achieved with cycloguanil pamoate ,57, wherThe challenge of combining these candidates to design an ideal medicine against malaria Table  is formiSafety is clearly a paramount concern for any new medicine. The challenge with developing new medicines against malaria is that the current medicines are relatively safe, and serious adverse events rare . This means that any new medicine will be expected to measure up to such a standard, and that in turn requires extensive safety monitoring after launch of a new product. Confirmation that such safety has been achieved will only come with extensive pharmacovigilance, across a wide range of patient ethnicities. In countries planning malaria elimination there has been much discussion of strategies for mass drug administration, or mass screening and treatment . This is a combination of TCP-1, -2 and -3b. However, an ACT plus primaquine fails to meet the TPP because single dose primaquine does not prevent relapse. Other combinations are of course possible. The problem of leaving a partner to face the parasite alone is mitigated by having TCP1s with higher rates of parasite clearance than the artemisinins  or extended durations of exposure . The ultimate mitigation, however, is a strategy of matched pharmacokinetics and potency . Clinical data suggests that logarithmic additivity in parasite killing activity should not be assumed with anti-malarial combination treatments: for example, the parasite reduction over time with artesunate-mefloquine is no faster than artesunate [in vitro measurements of the parasite reduction rate with combinations . Thus, for compounds with matched pharmacokinetics where no logarithmic additivity is seen, both molecules are likely to need to achieve a PCR-corrected ACPR of greater than 95% as single agents. This additional stringency may make it difficult to identify suitable candidates. Should additivity be observed, as a result of complementary stage specific action then the individual ACPR will be less. In addition, a combination of two short-acting molecules will provide poor post-treatment prophylaxis and hence not deliver a formal SERCaP; operationally this could be a major disadvantage in high-transmission areas. Another interesting question is whether the gametocyte-killing activity needs to be in the TCP-3 molecule; a TCP-1 molecule with additional anti-gametocyte properties would allow a TCP-1/3b, TCP-2, TCP-3a combination, for example. Although several of the new fast-killing TCP-1 candidates have highly potent activity against stage V gametocytes, it is not clear yet whether this is sufficient to block transmission in a clinically meaningful way. Artemether is an excellent killer of gametocytes in culture, but artemether-lumefantrine does not successfully block transmission on its own, presumably due to the poor pharmacokinetics of the artemisinins [After considerations of safety and efficacy, the principal concern for a SERCaP will be to avoid the development of resistance. If the SERCaP is to help in driving malaria eradication it would be best if it did not have to be regularly cupgraded’, as happens with many vaccines against common bacterial or viral pathogens, and some drugs too. To avoid resistance, the key is to make sure that no one molecule is exposed to a large number of parasites on its own. The way this is achieved with an artemisinin combination therapy is that the artemisinin analogue reduces the parasite numbers by at least 4 log units over a three-day course, though this still leaves a maximum of 10tesunate . These omisinins ,59.In any disease eradication agenda, preventing the population from becoming infected is a key activity. In malaria this has been primarily achieved to date with bed nets. Vaccination is another strategy, but apicomplexan parasites have developed sophisticated immuno-evasive strategies. Chemoprotective medicines offer an additional approach to disease control Table . These mi.v. quinine will be reinstigated or the subset of TCP-1 molecules, already shown to be effective in uncomplicated malaria and for which an intravenous formulation is feasible, will be investigated. This is a special case: monotherapy would be considered adequate, since all patients would be treated afterwards with an oral combination therapy. Not all TCP-1 molecules will fall into this class, since some of them may not be sufficiently soluble for parenteral use. Third, while there is clearly a need for adjunct therapy to minimize the sequelae of severe malaria, these molecules will already have been shown to have clinical efficacy and high tolerability in studies before they are tested in children with severe malaria. The search for such medicines will largely come from investigators working in other therapeutic areas.The standard of care for severe falciparum malaria including cerebral malaria is now shifting from quinine to parenteral artesunate, based on recent clinical results obtained in South-East Asia and in sub-Saharan Africa ,61. The The increased investment in research and development of new medicines over the last decade has increased both the strength and the diversity of the global portfolio of anti-malarial medicines. The portfolio contains many new chemotypes currently being tested in regulatory non-clinical studies for the first time, all of which have been discovered in the last six years . In the n, or n = log(1 – P)/log(1 – s), as derived from the Negative Binomial Distribution. Using MMV’s empirical value for s  has remained mostly flat over the past 60 years, in spite of tremendous increases in expenditure on R&D . For negAnopheles and Plasmodium genomic information has not had an immediate impact; progress in biological understanding paradoxically has led to overshooting rational drug discovery efforts with excessive confidence in the power of reductionism. On the positive side, success rates for finding new chemical series have increased with improvements in high-throughput screening using live parasites and the use of large, wide-diversity compound collections, such that now there is an abundance of new chemical series to work on [The call for an Agenda for Malaria Eradication has set new challenges for all those engaged in the drug discovery process. New medicines are needed to back up the current gold standard ACT therapies, so as to provide immediate alternative control strategies should the reduced speed of action of artemisinin spread. In addition, new medicines are needed to prevent transmission and relapse, the causes of new disease episodes. Finally, all medicines have to be as safe and convenient and cost-effective as possible, which represents an additional challenge. These increased demands are set against a background of two additional difficulties. First, the overall resource of drug discovery in neglected diseases is still relatively small, and in any case the overall productivity of drug discovery  is decreasing. Second, the availability of human,  work on . These hThe availability of new chemical series to work on has underscored the need for clarity about the type of molecules which are needed to achieve the malaria eradication agenda. These goals are essential to guide the medicinal chemistry process needed to identify appropriate candidates. Building on existing therapies and future clinical needs, TCPs have been described for a rapid-onset molecule, a long-acting molecule, one that prevents relapse and stops transmission, and one that will act as a chemoprotectant. An analysis of the current success rates of drug development shows that for a 90% chance of registering a molecule as many as 20 different preclinical candidates may be required, of which some already exist. The challenge is that much of the existing portfolio is focused on the TCP-1, the rapid-clearance molecule. This has led to a relative dearth of new molecules with long half-lives, those which can kill the non-dividing forms of the parasite, and those active against sexual stages of the parasite. One of the priorities for anti-malarial drug discovery is to ensure that there is a standardized measurement of the activity of clinical candidates across the whole life cycle of the parasite, termed the Malaria Life cycle Fingerprint .The way in which candidate molecules are combined to fulfill the final target product profiles shows that there is a certain amount of flexibility depending on the actual attributes of the molecules themselves. Theoretically, several different ways of configuring a new combination medicine can be envisioned. These include the combination of the first three target candidate profiles , preferably with matching half-lives and even combinations which allow for one molecule having more than one attribute . However, in discussions of potential combinations the reality is often more simple than the theory. Discussions about potential partnering strategies for new molecules in phase IIa, such as the endoperoxide OZ439 ,7 or theHowever the process of reviewing potential partners underlines the need for other new classes of TCP-2 candidates for the future. These will not be easy to find, since the chemical diversity currently available for screening is focused more around medicines that can be given once per day. However, the availability of over 20,000 new hits which kill the parasite may allow the sub-selection or hit optimization of molecules with a long half-life; these types of prioritization will become increasingly important over the next few years. The other alternative is to combine two fast acting compounds, for example the two new agents OZ439 and NITD609. This has the advantage of using molecules which have never been exposed to malaria as single agents, which has a certain appeal. The plasma exposures of both molecules remain above the minimum parasiticidal concentration for around a week, and so it would not be expected that such a combination would provide the same post-treatment prophylaxis as the current ACT, although this is less of an issue in low-transmission settings since reinfection rates are lower.The choice of TCP-3 molecules (preventing relapse and transmission) is even more stark. Currently the only option is the 8-aminoquinoline primaquine, which requires 14 days of therapy. An analogue, tafenoquine, is in clinical trials to determine whether it can be efficacious and safer than primaquine as a single dose. New families of active molecules are starting to be prioritized, but so far none has reached clinical development. It is important to underline that even with substantial investment in this area there are unlikely to be new molecules with clinically proven activity within the next five years.Putting all these molecules together to achieve a single-exposure radical cure and prophylaxis is clearly the ideal situation, but may be difficult to attain. To ensure adequate coverage from a single exposure, the dose of each component will have to be high, and this inevitably reduces the safety margin of the product. It is important to underline that whereas an ambitious objective is laudable, then less dramatic improvements in the regimen  still represent a step in the right direction and should not be discarded. In the regions where ACT is failing to provide adequate treatment, then a three-day regimen would be clinically advantageous.The identification of TCP-4 as the cornerstone of the chemoprotection agenda is also a critical issue. Once again, there are few candidate molecules in the pipeline, and this is an issue that has to be redressed. However, there are also grounds for hope here. The fact that rapid onset of action and fast killing are not required means that there are already several scaffolds and target types which could be studied for their relevance to TCP-4, including previously discarded compounds with a delayed-death mode of action. New medicines for chemoprotection must be tested for their effects amongst people in malaria-endemic areas, and designed for use by people in those areas, rather than tourists or travellers.The Agenda for Malaria Eradication has set ambitious goals for the treatment and chemoprevention of malaria, which cannot be reached with the currently available medicines. The combination of the complexity of drug discovery and development, plus a timeline of over a decade from discovery to launch in the first country, means that clarity at the start of the process is critical for success. Acceptable and ideal TPP for a treatment and chemoprotection agent have been defined. These have then been broken down into constituent parts - defined by the respective TCPs. As with any retrosynthetic process, there are a number of different ways the product can be broken down, and the final one chosen will depend on the ease of identifying suitable molecules for each TCP. The definition of target candidate profiles has highlighted the extreme shortage of molecules for three of the four profiles. Whilst there is some ground for optimism that this gap will be closed over the next decade, it will require a focused effort by the whole malaria drug discovery community as well as a sustained source of funding. In addition, well-validated, robust, functional assays for hypnozoites and transmission blocking activity with proven clinical correlations are required. Keeping a continued focus of the community on such challenging end-goals, through the TPPs, helps to ensure that the final products are in line with the patient and public health needs of the future. With such a focus, the community should be able to partner to deliver new medicines with clinical improvements over the current gold standards, and lead the way in the eradication of malaria.ACPR: Adequate clinical and parasitological response; ACT: Artemisinin-combination therapy; API: Active Pharmaceutical Ingredient; ATQ: Atovaquone; CMR: Centre for Medicines Research; DHFR: Dihydrofolate reductase; DOT: Directly observed therapy; EC/D90: Concentration/dose that results in 90% suppression of parasitaemia; G6PD: Glucose-6-phosphate dehydrogenase; HIV: Human immune deficiency virus; ICH: International conference on harmonization; IPTc: Intermittent preventive treatment in children; IPTi: Intermittent preventive treatment in infants; IPTp: Intermittent preventive treatment in pregnancy; IRS: Indoor residual spraying; malERA: Malaria eradication agenda; MMV: Medicines for malaria venture; MPC: Minimum parasiticidal concentration; NOAEL: No adverse effect level; PRR: Parasite reduction ratio; RH: Relative humidity; SAEs: Severe adverse effects; SEC: Single exposure chemoprotection; SERCaP: Single exposure radical cure and prophylaxis; TB: Tuberculosis.The authors declare that they have no competing interests, beyond the fact that MMV is involved in supporting the development of some of these medicines.JNB, JJM and TNCW composed the TCP Tables - CO analyzed malaria drug discovery attrition, TNCW wrote the initial manuscript draft, and all authors contributed with further edits, comments and discussion. All authors read and approved the final manuscript.


Plasmodium falciparum infection in children living in an area of low endemicity. In three Senegalese villages, specific antibody (IgG) responses to 13 pre-erythrocytic P. falciparum peptides derived from Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, Csp and Pf11.1 proteins were simultaneously evaluated before (June), at the peak (September) and after (December) the period of malaria transmission, in children aged from 1 to 8 years.The evaluation of malaria transmission intensity is a crucial indicator for estimating the burden of malarial disease. In this respect, entomological and parasitological methods present limitations, especially in low transmission areas. The present study used a sensitive multiplex assay to assess the exposure to P. falciparum infected children compared to non-infected and this increase is significantly correlated with parasite density.Compared to other antigens, a high percentage of seropositivity and specific antibody levels were detected with Glurp, Salsa1, Lsa3NR2, and Lsa1J antigens. The seropositivity increased with age for all tested antigens. Specific IgG levels to Glurp, Salsa1, Lsa3NR2, and Lsa1J were significantly higher in The multiplex assay represents a useful technology for a serological assessment of rapid variations in malaria transmission intensity, especially in a context of low parasite rates. The use of such combined serological markers  could offer the opportunity to examine these variations over time, and to evaluate the efficacy of integrated malaria control strategies. Plasmodium falciparum malaria is a major cause of human morbidity and mortality in sub-Saharan Africa, and its transmission varies greatly in endemicity across the continent [Plasmodium parasitemia in human populations, present substantial limitations, e.g. reproducibility and can be time-consuming. In addition, both entomological and parasitological measures are affected by the seasonality and require a precise follow-up during longitudinal studies [Plasmodium infections, serological markers show greater sensitivity, as seroprevalence reflects cumulative exposure to infections and thus is less affected by the changes in parasite densities, which could be undetectable in the case of very low parasite density. Previous studies showed that serological measurements are robust to detect short term variations in transmission, and should be a pertinent tool for evaluating malaria exposure in the context of low transmission [ontinent . The expontinent ,3. Conse studies . For thi studies . In ordesmission .The Circumsporozoite protein (CSP: a protein expressed by sporozoites and early liver forms), has been frequently used for the serological estimation of MTI . ControvP. falciparum peptides [In the present study, we used multiplex fluorescent microsphere-based assays measuring simultaneously human Ab to thirteen peptides  to assespeptides ,14.The study was performed in the villages of Mboula , Gankette Balla  and Mbilor  in Northern Senegal, located along the Senegal Basin River [Plasmodium species and the number of malaria parasites was counted in 200 microscopic fields. Parasite density was defined as the number of P. falciparum parasites/μl of blood, corresponding to an average of 8000 leucocytes. In the same way, capillary blood was collected from each child at each passage for immunological assessments.Three cross-sectional surveys were performed in June, September and December 2004 in the three villages. For each passage, a sub-cohort of children aged from 1 to 8 years was randomly selected from an existing cohort of 450 children, as previously described . A totalThe present study followed ethical principles according to the Helsinki Declaration, and was approved by the Ethics Committee of the Ministry of Health of Senegal . Informed consent was obtained from the parents/tutors of the children.P. falciparum peptides , and the procedure for coupling Ags to beads, as previously described [P. falciparum peptides were synthesized with an added N-terminal cysteine residue and covalently coupled with bovine serum albumin by Genepep . These Ags were coupled to beads  and 50 μl/well was deposited at the final concentration of 80 beads/μl per peptide. Diluted individual sera (1/100) in equal volumes of PBS and MFIA (Multiplex Fluorescence ImmunoAssay) diluents  were added in duplicate using 50 μl/well. The Luminex system was set for reading a minimum of 100 beads per spectral address, and results were expressed as ΔMFI "median fluorescent intensity" value with ΔMFI = MFIAg - MFIBSA, where MFIAg represents the mean of individual MFI value for beads coupled with P. falciparum Ag, and MFIBSA the individual MFI value for each serum for beads without coupled Ag. For calculating the seropositivity threshold, the means and standard deviations (SD) of ΔMFI of individuals living permanently in a non-endemic area (i.e. negative control group - n = 19) were estimated for each Ag. The lower limit of positivity of Ab responses to one Ag was estimated as the mean of ΔMFI of the negative control group + 3.09 SD. Therefore, values higher than the cut-offs should be observed in less than 1% of sera of non-exposed individuals under the hypothesis of normally distributed of ΔMFI. The thresholds of positivity were 7466.6, 684.0, 725.0, 225.0, 344.2, 4593.1, 424.8, 439.7, 435.2, 933.6, 452.6, 380.2, and 302.5 respectively for Lsa1-41, Lsa1J, Lsa3NR2, Lsa3RE, Glurp, Glurp.P3, Salsa1, Salsa2, Trap1, Trap2, StarpR, Csp, and SR11.1. Individual time-period variations in Ab levels for each Ag were assessed by the ratio of ΔMFI values collected at different time intervals, i.e. between June - September; September - December or June - September. The significant increase in Ab level to each Ag was defined as a value superior to the mean + 3.09 SD of the ratio in ΔMFI in the negative control group collected two times at four months interval.The multiplex technique was performed using the same sequences of escribed . Briefly®  version 4. After checking for normal distribution, the proportions of Ab-positive individuals and Ab levels were analyzed using the chi-squared test, Mann-Whitney U-test, Kruskal-Wallis test, where appropriate. Spearman's correlation was used to check for correlations between continuous variables. Differences were considered significant at P < 0.05.All data were analyzed with Graph Pad PrismP. falciparum peptides are presented for all studied individuals , September  and December  and according to the village .The proportion of seropositive and the level of specific Ab (IgG) responses directed to different s Figure . A high s Figure  to theseIn the objective to explore the possibility that these potential serological markers could reflect exposure to malaria over a prolonged period, the proportion of seropositive to at least one peptide, and the number of peptides that showed seropositivity in children were analyzed according to age Figure . The serP. falciparum peptides is shown according to the 3 time-periods, i.e. between June - September , presented higher specific Ab response to Glurp, Salsa1, Lsa3NR2, and Lsa1J peptides  than negative children (n = 111). In P. falciparum infected children, 80.0%, 54.7%, 62.7%, and 50.7% were seropositive to Glurp, Lsa3NR2, Salsa1 and Lsa1J respectively. Moreover, 85.3% of P. falciparum infected children were seropositive for at least one of the candidate antigens . For other peptides, no significant differences of Ab response levels were observed between P. falciparum positive and negative children (data not shown). Moreover, the highest differences in specific IgG levels were respectively observed for Glurp, Salsa1, Lsa3NR2 and Lsa1J. The statistical correlation between specific IgG level and the P. falciparum parasite density indicated a positive and significant correlation for Glurp, Salsa1, Lsa3NR2, and Lsa1J . Altogether, these data suggested that Glurp, Salsa1, Lsa3NR2 and Lsa1J peptides seem to be closely associated with P. falciparum parasite presence and intensity, and could be therefore potential serological markers of malaria exposure. This positive association between P. falciparum infection and specific Ab response against plasmodial antigens is a consistent observation, and could result from a booster effect of sustained parasite-exposure [Specific Ab levels to n Figure . Positivexposure .P. falciparum isolates [P. falciparum prevalence. Nevertheless, further studies will be valuable for validating these potential serological markers. These studies could include future modelling exercises including covariates factors such as age, genetic background, village of residence, impact of vector/parasite control programmes and the period of transmission. This kind of tool could allow elaborating a precise picture of malaria epidemiology in large-scale areas, or be used as a complementary indicator for evaluating the efficacy of integrated malaria control strategies.Some of these antigens  appeared to satisfy several of the requirements expected for a relevant marker of MTI. These peptides are antigenic and provided a higher incidence rate of significant increases in IgG levels. Specific Ab levels to these Ags are closely associated with the presence and the intensity of malaria infection. Moreover, in children seropositive to at least one peptide, 86.3% (120 of 139) were seropositive to at least one out of these four peptides. Pre-erythrocytic Glurp, Lsa1, Lsa3 and Salsa Ags are mainly expressed in infected human hepatocytes, they present little polymorphism from geographically different isolates -26. Receisolates . Our resIn conclusion, the multiplex assay provides a useful tool to simultaneously measured Ab responses directed to several Ags used as potential markers of malaria transmission. In low transmission areas, serological measurements to various malaria antigens are needed for estimating short term and small scale variations in MTI. However, the present study suggests that the combined assessment of Ab levels to only Glurp, Lsa1, Lsa3, and Salsa Ags could be a pertinent serological marker for evaluating the MTI.The authors declare that they have no competing interests.All authors read and approved the final manuscript.JBS carried out the immunological assessments, statistical analysis, interpretation of data and drafted the manuscript. CS, SF, SG contributed to field activities, microscopic examinations, and initial statistical analysis. SC helped to draft the manuscript. EO-P, FR1, LK, TF and CR contributed to the design of the study, and choice of the peptides. GR, CR, FR2 provided the scientific supervision, contributed to the design of the methods, interpretation of the data, and revised the manuscript.


Malaria causes about 250 million cases of fever and 1 million deaths annually. It is one of the causes of poverty and a major hindrance to economic development. Malaria is caused by protozoa of the genus plasmodium. Early accurate diagnosis is essential to start treatment within 24 hours and to avoid empirical therapy with toxic drugs. This study is aimed at finding out the best diagnostic and screening method for malaria.Blood from 200 clinically suspected malaria patients were subjected to peripheral blood smear examination, quantitative buffy coat, immunochromatography and enzyme linked immunosorbent assay. The sensitivity and specificity were analyzed. The duration, equipment, expertise, electricity and economy needed were considered.Peripheral blood smear is highly reliable, species specific, requires expertise and is time consuming. Quantitative buffy coat is a reliable rapid test, but needs economy and experts. Immunochromatography is a rapid, economical, low sensitivity method. Enzyme linked immunosorbant assay is a economical, time consuming, low sensitivity method.Peripheral blood smear remains the gold standard technique while quantitative buffy coat is the most suitable rapid test in well equipped laboratories and blood banks with heavy work load. Immunochromatography and enzyme linked immunosorbent assay are suitable for population screening in endemic areas and to monitor anti-malarial treatment.


Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country.Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax.The prevalence of P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages.The P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.This is the first study that assessed the seroprevalence of  Plasmodium falciparum malaria cases is declining, even in Africa. Given this situation, in the late 1990s, WHO proposed a goal of controlling the disease and achieving elimination by 2015 in areas of low transmission. Policies, international and national initiatives have proliferated to help the neediest.According to the World Health Organization (WHO), approximately 3.3 billion people, nearly half of the world population, are at risk of malaria. Each year, approximately 250 million people contract the disease, and nearly one million people die. The inhabitants of the poorest countries are the most vulnerable. More than one in five infant deaths (20%) occurring in Africa are due to malaria. HoweverBased on the results of scientific research in all areas of malaria control and because of greater knowledge of the disease and its medical and social consequences, the proposed strategy is organized into two main phases: control and disposal. Among tAccording to the Roll Back Malaria project, malaria primarily concerns 109 countries, but 35 countries account for 98% of malaria deaths worldwide. Only five of these countries  represent 50% of deaths and 47% of malaria cases[Formerly known to be a malaria meso- to hypoendemic country with an unstable malaria transmission profile-8, this Djibouti has recently demonstrated its eligibility for the pre-elimination goal according to technical feasibility, i.e., the baseline domestic malaria transmission combined with the importation-related transmission and operational feasibility, which takes into consideration the country government status, health status and information on populations at risk. These oAccording to WHO, malaria control requires, at a national level, the expertise and development of databases containing information about the parasites found locally and information about changes in transmission levels and the status of resistance to anti-malarials. ControlPlasmodium falciparum and Plasmodium vivax in human populations and to assess epidemiological facts of the past and present[P. falciparum and P. vivax[Therefore, serological tools are widely used to assess the transmission level and thus the prevalence of  present,17. One  P. vivax-20.P. falciparum malaria, it was also necessary to study P. vivax malaria and mixed infections to gather enough information for elimination[Starting from this observation, it was necessary to perform a similar survey in the Republic of Djibouti. Because most of the previous studies have primarily concerned mination.P. falciparum, P. vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of hot spots of malaria infection and transmission within the country.The aim of the present study was to evaluate the prevalence rate of P. falciparum and P. vivax infections among adults aged 15–54 years living in the Republic of Djibouti was estimated using a anonymous non-correlated cluster sampling method between the 24th and the 31th of March 2002. In brief, 30 clusters were investigated in the city of Djibouti, and 25 clusters in the other districts of the country. The clusters were randomly selected proportionally to the population size according to the list of quarters used by the National Direction of Statistics in the city of Djibouti and the list of the towns used by the expanded programme of immunization in the other districts. In each selected site, a starting household was randomly selected, and the next nearest households were investigated until a total of 44 resident adults per cluster in the city of Djibouti or 35 resident adults per cluster in the other districts were obtained. A total of 1,910 blood samples were collected anonymously in accordance with the recommendations of the Djiboutian Ministry of Health, who gave the ethics clearance for the present study. Blood samples were stored at 4 °C until separation of plasma by centrifugation (less than 24 hours after collection) and freezing. Thirty sera samples from French adults who had never been to malaria-endemic countries were used as unexposed negative controls. For the seropositivity threshold, the means and standard deviations (SDs) of the antibody intensity of the negative control group for all antigens were estimated. The lower limit of positivity for each antibody was taken as the mean + 3 SD of the negative control group values and corresponded to a mean fluorescence intensity (MFI) of 1,000. For P. falciparum, a sample was considered to be positive if the reactivity to at least two different plasmodial antigens was > 1,000 MFI. For P. vivax, a sample was considered to be positive if the reactivity to PvMSP1-19 or to PvMSP1-42 was > 1,000 MFI.The prevalence rate of P. falciparum merozoite surface protein 1–19 (MSP119) and apical membrane antigen 1 (AMA1) were obtained using procedures previously described[P. vivax, MSP119 proteins were produced in a baculovirus[42 as described elsewhere[Eleven Peptides  and SR11.1) were synthesized with an added N-terminal cysteine residue and covalently coupled with BSA  by Genpep  and stored in aliquots at −20 °C. escribed. For P. ulovirus and MSP1et al.[Peptides and proteins were coupled to beads as described by Ambrosino et al., and an th and the 31th March 2002, self-administered questionnaires containing several items were filled out by Djiboutian inhabitants and validated by a member of the research team. Different types of independent variables were collected: the living area (the city of Djibouti or the rest of the country), the type of living area , having stayed in a malaria-endemic country for more than one year (yes or no), having had a fever during the last month before the study (yes or no), the utilization of bed nets , gender , schooling status (schooled or never schooled), educational level , wealth , and age.During the cross-sectional study between the 24A Geographic Information System was built in ArcGIS 9.2 . The layers were added as follows: i) map of inland water in Djibouti and the neighbouring countries  P. falciparum antigens, P. vivax antigens or both of them (mixed infection) were analysed as a dependant variable according to individual and cluster characteristics using a random effect mixed logistic regression model. The model was designed to take into account the intracluster correlations that could exist due to the sampling design (cluster effect as random effect). The logistic model was also adjusted using a generalized estimating equations (GEE) approach. Random effect and GEE regression models allow the estimation of cluster-specific and population-averaged effects, respectively[Data were recorded using Excel and were checked for consistency before statistical analysis using R software version 2.10.1) or STATA software (version 11). The seropositivity to ectively. First, .10.1 or ectively. A backwP. falciparum or P. vivax antigens could be quantified by MFI, another ordinal dependant variable in four classes was created: the level of immune response to P. falciparum antigens  and P. vivax antigens . The same type of analysis was applied to this dependent variable as described above for the seropositivity status, using the “svy” command of STATA  to take into account the cluster effect.As the immune response to several The results of the bivariate Bayesian ordinal multinomial regression analysis are presented in Additional filesP. falciparum peptides: Lsa1-41, Lsa1-J, Lsa3-NR2, Glurp, GlurpP3, Salsa1, Salsa2, Trap1, Starp-R, CS (NANP) and SR11.1. The proportion of sera that were positive for both and at least one of the two recombinant falciparum antigens, i.e., PfMSP1 and PfAMA1, were 13.24% and 29.98%, respectively. By taking into account the immune humeral response to at least two different peptides or recombinant proteins of the 13 P. falciparum antigens used in this study, the seropositivity rate to P. falciparum infection was 31.5% (602/1910 Djiboutian people), CI95% (29.4-33.7).The serological analysis showed that 25.90% of sera were positive for at least two of the following 11 P. falciparum malaria infection, according to the results of the multivariate logistic regression analysis .– The educational level .– Never having visited a malaria-endemic area for more than one year.– Not having had a fever during the last month before the study.P. falciparum was identified: older age (40 years to 55 years). There was a non-significant association of living near lakes and rivers (≤ 1.5 Km) with a higher risk of seropositivity.In contrast, only one risk factor for seropositivity for 42 or PvMSP119, the global seropositivity rate to P. vivax was 17.5% (334/1910 Djiboutian people), CI95% (15.8-19.3). Considering the potential determinants of the P. vivax malaria infection, according to the results of the multivariate logistic regression analysis .– Never having visited a malaria-endemic country for more than one year.P. vivax infection.Having no schooling was significantly and independently statistically associated with a higher risk of being seropositive for P. falciparum and P. vivax, showed that some variables were significantly and independently statistically associated with a lower risk of being seropositive for mixed infection:The global seropositivity rate of mixed infection was 10.2% (195/1910 Djiboutian people), CI95% (8.9-11.7).The results of the multivariate logistic regression analysis for predicting a mixed infection Table, i.e., s– The gender .– Never having visited a malaria-endemic country for more than one year.– Not having had a fever during the last month before the study.Some factors were significantly and independently statistically associated with a higher risk of being seropositive for mixed infection:– The type of living area (urban).– The educational level (never schooled).– The distance to rivers or lakes (≤ 1.5 Km).P. falciparum seropositives to the different peptides and recombinant proteins from the different L-groups  were illustrated in FigureP. falciparum antigens in the L1, L2 and L3 groups was presented in FigureThe proportions of P. falciparum antigens:According to the results of the Bayesian ordinal multinomial logistic regression analysis Table, some fa– The gender .– The educational level .– Never having visited a malaria-endemic country for more than one year.– Not having had a fever during the last month before the study.– Rarely to never used bed nets.P. falciparum antigens:In contrast, only one risk factor was significantly and independently associated statistically with a risk of having a high level of immune humeral response to – The distance to rivers (≤ 1.5 Km).P. vivax seropositives to the different recombinant proteins from the different L-groups  were illustrated in FigureP. vivax antigens in the L1, L2 and L3 groups was presented in FigureP. vivax antigens:The proportions of – The gender .– Never having visited a malaria-endemic country for more than one year.– Not having had a fever during the last month before the study.P. vivax antigens:Two risk factors were significantly and independently statistically associated with a risk of having a high level of immune humeral response to – The distance to rivers (≤ 1.5 Km).– The educational level (never schooled).P. falciparum and P. vivax seroprevalence were presented in FiguresP. falciparum, the city of Djibouti showed a clustering of low and medium prevalence areas on both sides of the Ambouli wadi. A hotspot was observed in Arhiba, in which more than half of the population (56.4%) , which globally showed a decreasing prevalence when the distance to Ambouli wadi increased. This tendency was also observed in the lower town, i.e., the other side of the Ambouli wadi quarter, with 4 hotspots  that had similar prevalence to Arhiba. The most prevalent cluster in the entire country was Balbala 4, in which almost two in three persons (67.5%) were seropositive for P. falciparum.The different clusters of P. falciparum. The situation in the South was more concerning, as 3 seropositivity hotspots were observed in Dagguirou (46.9%), Tammiro (64.7%) and As-Eyla (64.7%) in the Region of Dikhil, and 2 seropositivity hotspots were observed in Ali-Sabieh1 (52%) and Ali-Sabieh 3 (45.5%), the capital city of the Region of Ali-Sabieh.In the rest of the country  and the regional capital Tadjourah (Tadjourah Region). In southern regions, the seroprevalence rates were between 20% and 30%, such as in Dagguirou, Garabayis (or Gour’abouss) and As-Eyla for the Dikhil region and Doudoub Balaleh for the Ali-Sabieh region. Hotspots were observed in Tammiro, with a 32.4% seroprevalence rate (in the region of Dikhil), and in Ali-Sabieh 1, with a 40% seroprevalence rate .P. vivax seroprevalence rate in the Republic of Djibouti. Supplemental information on the P. falciparum situation in 2002 was also highlighted.The present study was the first to analyse the The use of different antigenic peptides was dictated by the fact that in countries where malaria transmission occurs, the results of serology may be ambiguous due to cross reactions with other parasitic infections, such as toxoplasmosis. Thus, bet al. observed a P. falciparum seroprevalence rate of 14.2% in adults above 50 years, 6.9% in children and an average of 9.9% when they tested the reactivity of 4769 sera to one or both serological markers PfMSP1 and PfAMA1 among Djiboutian population[P. falciparum malaria transmission have declined by at least a factor of three in the past seven years. These results were consistent with the needs and obligations that lead to a pre-elimination goal in which the reduction of transmission is the most important key to pre-elimination[In 2009, Noor mination.P. falciparum or P. vivax seropositivity status or the level of humeral immune response to P. falciparum or P. vivax antigens have shown that female gender, a high educational level and never having visited a malaria-endemic country more than one year were protective. Considering the mixed infection seropositivity status, the multivariate logistic regression model showed a protective effect of living at a distance > 1.5 Km from rivers and lakes, in a rural area and not having had fever during the last month before the cross-sectional study. Only the model that predicted the P. falciparum seropositivity status showed a protective effect of younger ages between 15 and 40 years. As serological tools reflect the cumulative exposition[P falciparum or P. vivax.All obtained models predicting the position, these oposition. Historiposition. Carteroposition,32. ThisIn the city of Djibouti, the seroprevalence rates for both parasite species revealed hotspots on both sides of the main wadi, i.e., Ambouli wadi and the quarters of Arhiba and Balbala 4. Arhiba and Balbala 4 are quarters with significant migrant populations who regularly travel to and/or from Ethiopia. There wP. falciparum seroprevalence rates were higher in the southern regions (Dikhil and Ali-Sabieh), and in particular, hotspots were found along the land routes to Ethiopia, i.e., Tammiro/As-Eyla and Ali-Sabieh. These roads are regularly used by professional truckers, private users and migrants because they are the only two terrestrial roads to Ethiopia[In the rest of the country, Ethiopia.Plasmodium vivax seropositivity status was more balanced throughout the Djiboutian territory, with hotspots in the same locations in the southern regions as for P. falciparum. Because of the possibility of liver persistence of hypnozoïtes, P. vivax can spread more widely across the entire country in the case of incomplete treatment. P. vivax can be found where P. falciparum is no longer detectable and can sometimes be more prevalent, as could be the case in the neighbouring countries[Anopheles vectors exist.ountries. This siPre-elimination is a combination of technical feasibility, i.e., the baseline domestic malaria transmission combined with the importation-related transmission, and operational feasibility, which takes into consideration the country’s government status, health status and information on populations at risk.P. falciparum, even though some hotspots were the same as those found in the Dikhil region. This work, once combined with recent information provided by Noor et al. has shown that the Republic of Djibouti is likely on the correct path to pre-elimination with benefits that are threatened by the persistence of hotspots such as those in the Dikhil Region. Finding the same hotspots seven years later constitutes a serious threat to the success of the announced goal. Taking into account technical and operational feasibility, pre-elimination is possible only insofar as efficient control methods are implemented at all administrative and executive levels of authority.Compared to one recent work, the preRecommendations emerge from the results of the statistical models. If the educational level of the population cannot be increased, pre-elimination will require increased awareness and health education for at-risk populations through methods that are appropriate to local realities. Although being female seems to be protective, health education should target mothers in particular to increase local knowledge, as has been previously done with HIV. RegionaP. falciparum seroprevalence rate was 25.90% in 2002, further studies with the same population would be required to assess if there was a real decrease in transmission of P. falciparum in the Republic of Djibouti since 2002.As seen in this study, the P. vivax in the Republic of Djibouti. It is necessary to improve our knowledge of this pathogen in order to create an effective elimination programme.This is the first study that assessed the prevalence of P. falciparum and P. vivax. For P. falciparum along, a protective effect was also observed for not having had a fever the last month, living > 1.5 km away from lakes and rivers and being younger in age.The protective effect of female gender, educational level and never having visited a malaria-endemic area for more than one year was observed for both P. falciparum and P. vivax transmission should be regularly monitored by serological methods or other tools and including children less than five years old.These findings should encourage authorities to improve efforts toward elimination and to begin the final assault against the few persistent hotspots. However, to assess the real pre-elimination status, the precise level of both The authors declare that they have no competing interests.BAK, SB, HB, CR and BP conceived and designed the experiments. BAK, LO, MAK, IHF, HMA, AAA, MKW, SNA, HYD and CR collected the data. HB, AP, BAK, SB, VM and CT contributed to reagents/materials/analysis tools. SB, BAK, HB, MAK, VM and CR analysed the data. BAK, SB, AP, VM, CR and BP wrote the paper. All authors read and approved the final manuscript.Blood samples were collected anonymously in accordance with the recommendations of the Djiboutian Ministry of Health, which also gave ethical approval for the study.Bivariate logistic regression analysis of P. falciparum infection’s seroprevalence for socio-economic variables.Click here for fileBivariate logistic regression analysis of P. falciparum infection’s seroprevalence for environmental, health and bed net use variables.Click here for fileBivariate logistic regression analysis of P. vivax infection’s seroprevalence for socio-economic variables.Click here for fileBivariate logistic regression analysis of P. vivax infection’s seroprevalence for environmental, health and bed net use variables.Click here for fileBivariate logistic regression analysis of P. falciparum and P. vivax mixed infection’s seroprevalence for socio-economic variables.Click here for fileBivariate logistic regression analysis of P. falciparum and P. vivax mixed infection’s seroprevalence for environmental, health and bed net use variables.Click here for fileBivariate ordinal logistic regression analysis of the serological response to P. falciparum antigens.Click here for fileBivariate ordinal logistic regression analysis of the serological response to P. vivax antigens.Click here for fileP. falciparum, P. vivax seroprevalences and geographical localization of the Djiboutian clusters.Click here for fileMap of clusters of P. falciparum and P. vivax seroprevalence in the capital of the Republic of Djibouti.Click here for fileMap of clusters of P. falciparum and P. vivax seroprevalence in the Republic of Djibouti.Click here for file


Rapid diagnostic tests (RDTs) are the current complement to microscopy for ensuring prompt malaria treatment. We determined the performance of three candidate RDTs  for rapid diagnosis of malaria in the Central African Republic.Blood samples from consecutive febrile patients who attended for laboratory analysis of malaria at the three main health centres of Bangui were screened by microscopy and the RDTs. Two reference standards were used to assess the performance of the RDTs: microscopy and, a combination of microscopy plus nested PCR for slides reported as negative, on the assumption that negative results by microscopy were due to sub-patent parasitaemia.We analysed 436 samples. Using the combined reference standard of microscopy + PCR, the sensitivity of Paracheck™-Pf was 85.7% , that of SD Bioline Ag-Pf was 85.4% , and that of SD Bioline Ag-Pf/pan was 88.2% . The tests performed less well in cases of low parasitaemia; however, the sensitivity was > 95% at > 500 parasites/μl.Overall, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan performed slightly better than Paracheck™-Pf. Use of RDTs with reinforced microscopy practice and laboratory quality assurance should improve malaria treatment in the Central African Republic. In countries in sub-Saharan Africa, malaria management is frequently based on clinical criteria, leading to overuse of antimalarial agents ,2. AfterPfHRP-2 gene [The World Health Organization (WHO) recommends parasitological confirmation for all patients suspected of having malaria before starting treatment. This recommendation includes high-quality microscopy or, when that is not available, use of easy-to-use rapid diagnostic tests (RDTs) . Panel sP-2 gene -21, the P-2 gene , cross-rP-2 gene ,24 and tP-2 gene . Local dP-2 gene ,26-28.PfHRP-2 and −3 found wide variation in PfHRP-2 sequences in samples from the Central African Republic. The authors speculated that this might reduce the sensitivity of RDTs for detecting malaria in people with very low parasite density [A recent multi-centre study assessing global sequence variation in  density . In this density . The Min density . The objPlasmodium falciparum, and malaria transmission occurs throughout the year, with peaks at the beginning and the end of the rainy season, although no data are available on the intensity of transmission. Malaria accounts for more than 40% of morbidity in the country .This cross-sectional study was conducted in August 2011 in Bangui, capital of the Central African Republic, in the Hospital de l’Amitié, the Complexe Pediatrique and the Institut Pasteur of Bangui. Bangui is located beside the Oubangui River, north of the Democratic Republic of the Congo . The climate is tropical, and rainfall peaks between April and November. The average temperature varies from 19°C to 32°C. The main malaria parasite is The Hospital de l’Amitié and the Complexe Pédiatrique are tertiary referral public health centres equipped with laboratories where thin and thick smears are analysed. The Institut Pasteur of Bangui is a private centre  for biomedical research, in which laboratory diagnosis for malaria is performed for patients referred by clinicians at health centres where this test is not available.P. falciparum-specific histidine-rich protein type 2 (PfHRP2), while Bioline Malaria Ag Pf/Pan contains antibodies targeting both PfHRP2 and lactate dehydrogenase specific to P. falciparum and other Plasmodium species . Blood samples from people attending each study centre for laboratory analysis were tested for the presence or absence of malaria parasites by microscopy and the RDTs. Samples from patients with negative microscopy (regardless of RDT result) were tested P. falciparum by nested PCR, on the assumption that false-positive results with RDTs are due to sub-patent parasitaemia [We analysed the performance of three RDTs: Paracheck™-Pf, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf and /pan . SD Bioline Malaria Ag Pf and Paracheck™-Pf contain antibodies against sitaemia .Consecutive patients of all ages presenting at each study site were approached by the study technician for recruitment. Each patient’s medical card was checked, and those with a request for smear analysis were considered eligible if they were febrile (axillary temperature ≥ 37.5°C) or had a history of fever in the previous 24 h. They were included in the study if they gave written consent; for patients aged ≤ 18 years, informed consent was provided by a parent or guardian.This project was reviewed and approved by the ethical committee of the University of Bangui. The Central African Republic Health Ministry also gave approval for this study.For an estimated annual malaria rate in Bangui of 40% , an expected sensitivity of Bioline RDTs of 95%  and a seIn each study centre, the laboratory team consisted of three technicians with university training, all of whom had been re-trained for diagnosing malaria in the laboratory by standard operational procedures . The tecP. falciparum. Parasite density was determined as the number of parasites per 200 leukocytes on the assumption of an average leukocyte count of 8000/μl of blood. A result was considered negative if no parasites were detected per 200 leukocytes. Each slide was read independently by two study technicians. In the case of a discrepant qualitative result (negative or positive), a third reading was done by the designated technician at the Institut Pasteur. All the laboratory technicians were blinded to the RDT results. The results of both microscopy and the RDTs were reported to the clinicians, who were advised to treat the patient for malaria if the results of these two analyses were discrepant.Finger-prick blood samples were obtained for slides and for testing with the RDTs. Blood smears were air-dried, stained with 4% Giemsa and analysed under a light microscope (× 100 oil immersion) to detect asexual forms of The RDTs were performed by the third technician at the study centre following the manufacturer’s instructions.For patients with negative microscopy, three drops of blood were collected on a piece of filter paper (Whatman®). The blood spots were air-dried and stored at 4°C in individual sterile plastic bags for PCR analysis.Plasmodium DNA [Parasite DNA extraction and PCR assays were performed at the Institut Pasteur. The blood-impregnated filter paper piece was washed with distilled water and placed directly in a PCR tube containing the PCR reaction components. Genomic DNA was determined in an assay based on nested PCR for dium DNA .Data were entered onto Excel spreadsheets and analysed with MedCalc®software .P) of 0.05.The first step of our analysis was to determine the performance of the RDTs according to the falciparum density in samples found positive by microscopy. Density was categorized as ≤ 100, 101–200, 201–500, 501–1000, 1001–5000, 5001–50 000 and > 50 000 parasites/μl. RDT results were compared (paired proportions) with the McNemar test, at a level of significance . According to the PCR results, the results of the RDTs were designated as TP2 if PCR was positive in FP1, FP2 if PCR was negative in FP1, FN2 if PCR was positive in TN1 and TN2 if PCR was negative in TN1. Combination of the results of the RDTs for samples positive by microscopy and of PCR for samples negative by microscopy gave the values TP = TP1 + TP2, TN = TN2, FP = FP2 and FN = FN1 + FN2, which were used to calculate the performance of the RDTs.The second step was to estimate the performance of the RDTs against the combined results of microscopy and nested PCR, expressed as true-positive (TP), true-negative (TN), false-positive (FP) or false negative (FN). The formulas used to calculate performance were TP/TP + FN for sensitivity, TN/TN + FP for specificity, TP/TP + FP for positive predictive value (PPV) and TN/TN + FN for negative predictive value (NPV). The results were interpreted with 95% confidence intervals (CIs). At baseline, the results of the comparison between the RDTs and microscopy were expressed as TPP. falciparum. In 234/235 (99.6%) cases of infection, P. falciparum was the only parasite species identified. One infected individual had a P. ovale mono-infection  and was hence excluded from the study, which was confined to detection of falciparum malaria.A total of 437 patients were recruited for this study between 8 and 22 August 2011. Microscopic analysis showed that 53.8% (235/437) of the blood slides were positive for The RDTs gave positive results in 52.3% (228/436) of cases with Paracheck™-Pf, 57.8% (252/436) with SD Bioline malaria Ag-Pf and 58.0% (253/436) with SD Bioline malaria Ag-Pf/pan.Comparison of the results of the RDTs with those of microscopy showed concordant positive results in 85.9% (201/234) of cases with Paracheck™-Pf, 92.3% (216/234) with SD Bioline malaria Ag-Pf and 92.3% (216/234) with SD Bioline malaria Ag-Pf/pan. Of the samples that were negative by microscopy, 13.4% (27/202) were found to be positive with Paracheck™-Pf, 17.8% (36/202) with SD Bioline malaria Ag-Pf and 18.8% (38/202) with SD Bioline malaria Ag-Pf/pan.p = 0.04)  by microscopy and 7 false-negative results (7/202 or 4.2%) by both microscopy and the RDTs. Overall, the combined results of microscopy and PCR showed a sensitivity of 85.7% for Paracheck™-Pf, 85.4% for SD Bioline malaria Ag-Pf and 88.2% for SD Bioline Ag-Pf/Pan. The specificity was 86.0% for Paracheck™-Pf, 86.3% for SD Bioline malaria Ag-Pf and 80.4% for SD Bioline malaria Ag-Pf/pan.The overall sensitivities, specificities, NPV and PPV for the three RDTs, evaluated against microscopy and microscopy and PCR are detailed in Table P. falciparum samples be at least 75% at 200 parasites/μl, although discrepancies between these scores and clinical sensitivity are limitations of these tests [The sensitivity and specificity of the tested RDTs found in this study appear to fall within the ranges quoted in other studies, which reported a sensitivity of Paracheck™-Pf of 82.9–100% and a specificity of 56.0–96.4% -42 and ase tests ,47.HRP2 during a malaria episode with low parasitaemia might not be detected by RDTs [In this study, some blood samples that were negative by microscopic examination gave negative results in all three RDTs, while some gave positive results in all RDTs. Factors that affect the sensitivity and specificity of RDTs, such as low parasitaemia, are particular challenges for malaria diagnosis with these new devices. We found that a parasitaemia between 40 and 400 parasites/μl in blood samples with positive results by microscopy gave negative results in all three RDTs. This finding corroborates those of other studies -52, whic by RDTs  because  by RDTs .HRP 2 gene may reduce the sensitivity of these RDTs [PfHRP2 sequences in samples from the Central African Republic might reduced the sensitivity of RDTs for detecting malaria at very low parasite density [HRP 2 in humans [Furthermore, deletions or mutations in the ese RDTs -21. It h density . Other f density  and the n humans , might ePfHRP2 from previous malaria infections [Cases incorrectly identified as positive by the RDTs might be due to the persistence of fections , cross-rfections ,24 or otfections .This study has some limitations. First, PCR was performed only on samples found negative by microscopy. It might be considered incorrect to use a different “gold standard” for slides found to be positive and those found to be negative by microscopy, by using PCR only for slides found negative by microscopy. However, the risk for false-positive microscopy results was considered low, as the slides were read independently by two experienced technicians. The fact that the microscopists counted only 200 negative leukocytes before declaring a slide as negative might have decreased the sensitivity of microscopic detection in this study. PCR is a useful gold standard because it is highly sensitive, can detect cases with low parasitaemia that are missed by other techniques and is easily reproducible . NeverthPlasmodium, even if they are less prevalent in the Central African Republic. Moreover, introduction of these tests will indisputably help to identify malaria cases in this area, where microscopy is still of poor quality. Microscopy should nevertheless complement RDTs for determining parasite density, and therefore reinforcement of microscopy skills by ongoing training of technicians and quality assurance of both RDTs and microscopy should be considered priorities in national malaria programmes. The results of this study also indicate that RDTs should be evaluated in each new setting before they are deployed, in view of possible variations in performance in different populations.This study showed that the performance of SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan was better than that of Paracheck™-Pf. SD Bioline malaria Ag-Pf/pan is the most useful test because it detects other species of The authors declare that they have no competing interests.DD and JMM conceived the study, with substantial contributions from AM and NM. DD, JI, JCG and GT participated in field data collection and laboratory analysis. AM, DD and SB interpreted the data and drafted the manuscript. All the authors have read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/109/prepub