Despite the protection provided by several factors, including maternal antibodies, the burden of malaria in young infants may be higher than previously thought. Infants with congenital or neonatal malaria may have a different clinical presentation than older children, and diagnosis may be confused with other neonatal diseases due to an overlap of clinical manifestations. In addition, there is little information on the use of artemisinin-based combination therapy in young infants. There is the need for a more accurate estimate of the parasite prevalence and the incidence of clinical malaria in infants under 6 months old, as well as a better characterization of risk factors, pharmacokinetic profiles, safety and efficacy of currently available anti-malarial treatments, in order to develop evidence-based treatment guidelines for this population. Plasmodium falciparum malaria in children aged under five years is well documented [Although the burden of cumented , there icumented -4, despicumented . Young icumented ,7 and locumented  than oldcumented , and maycumented .in utero[P. falciparum erythrocyte membrane protein-1 (PfEMP-1) mediates cytoadherence of infected red blood cells to the endothelial cells lining the blood vessels. HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized erythrocytes in the first few months of life by altering PfEMP-1 display on HbF RBCs, and by binding PfEMP-1 and preventing sequestration of parasitized RBCs, respectively [P. falciparum malaria increases [Neonatal malaria is considered a rare occurrence due to the protective effect of maternal immunity after birth. Maternal immunoglobulin G (IgG) antibodies are acquired by the fetus via the placenta in utero, althougin utero. IgG levin utero. Neonatein utero. Parasitin utero. Haemoglin utero, can inhin utero and can ectively . HbF levectively , and as ncreases .Iron-deficiency anaemia is common in sub-Saharan Africa and can cause impaired cognitive and motor development, growth impairment and anorexia. Consequently, in many areas, iron supplementation is recommended in children below two years of age. However, iron status has been shown to have an effect on the risk of malaria in young infants, with iron deficiency potentially causing a decrease in malaria morbidity and mortality .In Mozambique and Benin, placental malaria has been associated with shorter time to first episode of infant malaria ,18. The Malaria in infants is classified according to the time of infection. Congenital malaria, defined as asexual parasites detected in the cord blood or in the peripheral blood during the first week of life , is due in utero is also reflected by the prevalence of splenomegaly at one month of age, which can be as high as 80%, indicating an early development of a splenic response to the infection [Recent reports suggest that malaria in infants under six months of age may not be uncommon, although data on prevalence and outcome are still contradictory . Prevalenfection ,35.In Mozambique, clinical malaria incidence in infants aged 1– < 6 months was substantial  . ReportsMalaria infections  were common in Ghanaian infants less than 6 months of age , although clinical malaria symptoms were rare or uncommon  . In MozaA systematic review of age-patterns of malaria revealed that as transmission increased, there was a shift of clinical malaria towards younger age groups, regardless of seasonality . As tranSigns and symptoms of congenital malaria include fever, anaemia, splenomegaly, hepatomegaly, jaundice, vomiting, diarrhoea, poor feeding, restlessness, drowsiness, pallor, respiratory distress, cyanosis, and possibly convulsions ,56.Malaria in young infants may be asymptomatic ,7, or itIn areas with limited resources, the capacity to diagnose malaria in young infants may be limited ,58, and Due to the dynamic developmental changes experienced by infants aged under 6 months, the pharmacokinetic profiles of anti-malarials may be different than in older children, and age-dependent dose adjustments may be necessary . For exaP. falciparum malaria. Thus, oral anti-malarials recommended as first- and second-line therapies are frequently used off-label, based on the recommended mg/kg dosing schedule for older children [As the burden of disease in infants under 6 months of age is not well defined, this age group has been excluded from previous clinical trials and national treatment guidelines on uncomplicated children .There is little information on the use of artemisinin-based combination therapy (ACT) in young infants, to the extent that many of them carry label restrictions for this age group . AccuratWith these issues in mind, it must be considered how ACT could be used to treat malaria in infants aged under six months. There are no official data on how to use ACT in this age group, despite the fact that malaria can occur at a very young age and that ACT offers greater efficacy and tolerability compared with quinine, which is often used in infants with clinical malaria. Furthermore, the use of a unified first-line therapy could offer benefits for healthcare providers with similar anti-malarial use for the whole population, as well as logistical benefits in terms of procurement and distribution. The safety profile of these combination therapies in young infants should be established.Previous studies have shown that chemoprophylaxis in infants has the potential to reduce malaria-related morbidity and mortality . In The Some studies of intermittent preventive treatment (IPT) in infants (IPTi) have timed the doses of anti-malarial to coincide with routine vaccinations delivered by WHO’s Expanded Programme on Immunization (EPI) at 2, 3 and 9 months. In a pooled analysis of six randomized, placebo-controlled trials of IPTi using sulphadoxine-pyrimethamine delivered at the same time as EPI immunizations, a protective efficacy against clinical malaria of 30.3% was reported .Seasonal malaria chemoprevention (SMC), previously referred to as IPT in children (IPTc), involves the administration of full anti-malarial treatment courses during the malaria season to children aged 3–59 months . The WHOThe candidate malaria vaccine RTS,S/AS01 administered to infants aged 6–12 weeks at the time of the first vaccination provided modest protection against both uncomplicated and severe malaria , castingThere are several ongoing or planned clinical studies aiming to obtain more accurate estimates of the malaria burden and parasite prevalence in infants aged less than 6 months living under different endemic conditions, as well as the safety and efficacy of currently available ACT in this population. Future longitudinal studies should also characterize infant iron status in relation to subsequent malaria risk, and relate findings to the occurrence of placental malaria.A multicentre descriptive study is underway, aiming to assess malaria morbidity  and corresponding risk factors in young infants, compared with their siblings, in areas of seasonal low (The Gambia) and high (Guinea) malaria transmission, and in an area with intense, perennial transmission (Benin).P. falciparum malaria. This trial, sponsored by Novartis and in association with MMV, commenced in 2012 and involves sites in Benin, Burkina Faso, the Democratic Republic of the Congo, Nigeria and Togo.A multicentre trial will evaluate the safety, pharmacokinetics and efficacy of dispersible artemether-lumefantrine tablets in infants weighing under 5 kg with uncomplicated Another study planned at the College of Medicine at the University of Malawi will assess the population pharmacokinetics and the safety and efficacy of ACT in infants weighing under 5 kg or aged less than 6 months.Knowledge gained from such studies will assist in guiding the development of evidence-based treatment guidelines for this population.The perception that malaria is uncommon in young infants has resulted in the paucity of information currently available and the lack of evidence-based treatment guidelines in this population. Many children are dying before malaria is diagnosed. In resource-constrained settings, diagnostic techniques may not be available, or malaria may not be recognized due to overlapping symptoms with other neonatal illnesses such as sepsis . MalariaACT is being used off-label in this vulnerable patient group, with physicians using a pragmatic approach to guide dosing. At this time, a clear recommendation on the use of ACT could be difficult, due to a lack of data in infants with a body weight of less than 5 kg (less than 4.5 kg for artesunate plus amodiaquine). A more accurate estimate of the disease burden and parasite prevalence, risk factors for infection including iron status, as well as the pharmacokinetic profiles, safety and efficacy of currently available ACT in early infancy from data provided by clinical trials that are planned or underway will contribute to the development of evidence-based treatment guidelines for this population, as well as aid the research and development of new drugs. It is definitely worth providing malaria control strategies that are aimed directly at this vulnerable age group, in a bid to reduce infant mortality.ACT: Artemisinin-based combination therapy; EPI: Expanded Programme on Immunization; HbF: Haemoglobin F; HIV: Human Immunodeficiency Virus; IgA: Immunoglobulin A; IgG: Immunoglobulin G; IPT: Intermittent preventive treatment; IPTc: Intermittent preventive treatment in children; IPTi: Intermittent preventive treatment in infants; MMV: Medicines for Malaria Venture; pABA: Para-aminobenzoic acid; PCR: Polymerase Chain Reaction; PfEMP-1: Plasmodium falciparum erythrocyte membrane protein-1; SMC: Seasonal malaria chemoprevention; WHO: World Health Organization.UD has been invited as speaker at symposia organized by Sigma Tau and Novartis Pharma, and has received a research grant from Sigma Tau. KH is an employee of Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA. This declaration is made in the interest of full disclosure and not because the author considers this to be a competing interest. The other authors have no competing interest to declare.All authors met International Committee of Medical Journal Editors criteria for authorship. All authors contributed to the development of the outline, revised the manuscript critically, and read and approved the final manuscript.


Plasmodium falciparum and Plasmodium vivax malaria among adults in Africa.There have been few published reports on severe P. falciparum and P. vivax  were described in adult patients admitted to Kassala Hospital, eastern Sudan.Clinical pattern/manifestations of severe P. falciparum  or P. vivax  malaria. Manifestations among the 139 patients included hypotension , cerebral malaria , repeated convulsions , hypoglycaemia , hyperparasitaemia , jaundice , severe anaemia , bleeding , renal impairment  and more than one criteria . While the geometric mean of the parasite count was significantly higher in patients with severe P. vivax than with severe P. falciparum malaria , the different disease manifestations were not significantly different between patients with P. falciparum or P. vivax malaria. Three patients (2.2%) died due to severe P. falciparum malaria. One had cerebral malaria, the second had renal impairment, jaundice and hypoglycaemia, and the third had repeated convulsions and hypotension.A total of 139 adult patients  with a mean (SD) age of 37.2 (1.5) years presented with severe P. falciparum and P. vivax malaria is an existing entity among adults in eastern Sudan. Patients with severe P. falciparum and P. vivax develop similar disease manifestations.Severe malaria due to  Plasmodium falciparum causes approximately 600,000 deaths each year and the vast majority of the burden of malaria mortality is in sub-Saharan African countries [P. falciparum is due to its ability to induce severe malaria, and in some cases, multiple organ dysfunction [P. falciparum malaria and there have been no published data on severe Plasmodium vivax infection among African adults. Until recently, P. vivax was considered a benign parasite compared with P. falciparum. The current study was conducted at Kassala Hospital in eastern Sudan to investigate the manifestations and epidemiology of severe P. falciparum and P. vivax malaria among adult patients, so as to add to the previous studies on severe malaria in Sudan [P. falciparum (95%) was the main species in the area. In contrast, P. vivax was considered rare and constitutes only 3% of the Plasmodium species in the area [P. vivax malaria is increasing in this setting [Malaria is a big health problem and is endemic in an area where around two billion people live. ountries . The higfunction . The prefunction . In areafunction . Yet thefunction -5. Furthin Sudan . Such dalciparum 5% was th setting .P. falciparum or P. vivax  to investigate the presentation/manifestation of severe malaria caused by P. falciparum or P. vivax malaria according to World Health Organization (WHO) criteria were enrolled [All patients, or their relatives in the case of comatose patients, provided informed consent, and their socio-demographic and clinical data  were collected using standard questionnaires. Patients with one or more manifestations of severe enrolled . Pregnanenrolled .Thick and thin blood films were prepared from capillary blood, stained with Giemsa, and 100 oil immersion fields were examined. The parasite density was evaluated by counting the number of asexual parasites for every 200 leukocytes, assuming a leukocyte count of 8,000 leukocytes/μl. All slides were double checked in a blinded manner and only considered negative if no parasites were detected in 100 oil immersion fields. If gametocytes were seen, then the count was extended to 500 oil immersion fields. Haemoglobin concentrations were estimated using a HemoCue haemoglobinometer . Blood glucose was measured at baseline before quinine infusion and two hours after quinine infusion if there was clinical suspicion of hypoglycaemia using the bedside device Accu-Chek™ Multiclix . The Accu-Chek™ machine was calibrated weekly and a new box of test strips was opened each time.Resuscitation and supportive management were given according to WHO guidelines  includinTreatment during the first malaria season of 2011 consisted of quinine . During the second season, intravenous artesunate was used with quinine in an open clinical trial. Artesunate was given as 2.4 mg/kg body weight at 0, 12, and 24 h, and then daily. Each 60mg vial of artesunic acid  was dissolved in 1mL of 5% sodium bicarbonate to form sodium artesunate and then mixed with 5mL of 5% dextrose. Details of the response to treatment will be described in a future report.t-test and analysis of variance were used for normally distributed data to compare between two or more than two groups, respectively. Proportions were compared by χ2-test. P-values <0.05 were considered significant.Statistical analysis was performed using SPSS software  version 16.0) and double checked before analysis. Data were checked for normality, and the Student The study received ethical approval from the Research Board at the Ministry of Health Kassala, Sudan.P. falciparum  or P. vivax  malaria  and those in the 2012 season .Of the total 9,559 adult patients presented to the hospital during the study period, 2,124 (22.0%) patients had malaria. Among 2,124 patients with malaria, 1,985 patients had uncomplicated malaria, 139 patients  with a mean (SD) age of 37.2 (1.5) years presented with severe P. falciparum or P. vivax infection, the frequency of symptoms were as follows: hypotension , cerebral malaria , repeated convulsions , hypoglycaemia , jaundice , hyperparasitaemia , severe anaemia , bleeding , renal impairment  and more than one criteria   and hypoglycaemia; patient 2: cerebral malaria with repeated convulsions and hypotension; and, patient 3: hypotension, severe anaemia and jaundice. Five patients developed severe anaemia and jaundice. There may be an overlap between the three main manifestations  of severe malaria. While there was no significant difference in levels of blood glucose, white blood cells and parasite counts between the patients presenting with hypotension, convulsion and cerebral malaria, their age  and haemoglobin were significantly higher in patients with cerebral malaria  died due to severe P. falciparum to P. vivax malaria (4.3 : 1.0) in patients from eastern Sudan. In addition, the predominant manifestations of severe malaria were hypotension (27.3%), cerebral malaria (16.5%), and convulsions (13.0%), and their frequency was not significantly different between patients with P. falciparum or P. vivax malaria. It has been demonstrated that severe malarial anaemia was the most common complication (45.4%) of P. falciparum malarial infection among patients in a hospital (Gedarif) in eastern Sudan [The current study demonstrated a high ratio of rn Sudan . The difrn Sudan . Anaemiamalaria 1.5%, and Interestingly, it has been observed that 36.2% of adult patients in the same study area had anaemia regardless of their age, sex, education level or infection with malaria . In TanzP. falciparum and P. vivax malaria in this report. Previously, P. vivax was considered the cause of tertian benign malaria that rarely led to a severe form of the disease. However, increasing evidence has shown an increased risk of mortality and morbidity owing to P. vivax malaria [P. vivax malaria are from Southeast Asia and India, and there have been few published data on severe P. vivax from Africa. It has been documented different manifestations of severe P. vivax malaria among children in a nearby hospital  [P. vivax in India presented with cerebral malaria, severe anaemia, renal failure, hypoglycaemia, jaundice, acute respiratory distress syndrome, shock, and death [P. vivax malaria, but according to the WHO guidelines, it should be considered as severe as P. falciparum malaria [The manifestations of severe malaria were not significantly different between  malaria ,19-22. Gw Halfa) . The clind death . However malaria .P. falciparum malaria. In Tanzania, the fatality rate of severe malaria was 3.2% and the majority of deaths occurred in children younger than five years [In the current study, three patients (2.0%) died owing to severe ve years . High move years . Likewisve years . Low deave years . It is aThere are some limitations of this study; manifestations of the disease itself were overlaps and might not be strictly followed e.g. hypotension is only severe disease if not responsive to fluids, bleeding by itself is not severity as well, unless it is followed by circulatory collapse and hyperbilirubinaemia is considered a severity criterion if its followed by other organ dysfunctions. Furthermore PCR was not performed in any sample of the patients to confirm the parasite species as has been done in some other setting .P. falciparum or P. vivax malaria is an entity that exists among adults in eastern Sudan, with similar manifestations between severe P. falciparum and P. vivax.Severe malaria due to The authors declare that they have no competing interests.TMA and IA designed the study and shared in the statistical analyses. MTA and ISA conducted the clinical work. HZH and MMM conducted the biochemical work. All the authors shared in the draft and approved the manuscript.


Intermittent preventive treatment of malaria in children less than five years of age (IPTc) has been investigated as a measure to control the burden of malaria in the Sahel and sub-Sahelian areas of Africa where malaria transmission is markedly seasonal.IPTc studies were identified using a systematic literature search. Meta-analysis was used to assess the protective efficacy of IPTc against clinical episodes of falciparum malaria. The impact of IPTc on all-cause mortality, hospital admissions, severe malaria and the prevalence of parasitaemia and anaemia was investigated. Three aspects of safety were also assessed: adverse reactions to study drugs, development of drug resistance and loss of immunity to malaria. Twelve IPTc studies were identified: seven controlled and five non-controlled trials. Controlled studies demonstrated protective efficacies against clinical malaria of between 31% and 93% and meta-analysis gave an overall protective efficacy of monthly administered IPTc of 82% (95%CI 75%–87%) during the malaria transmission season. Pooling results from twelve studies demonstrated a protective effect of IPTc against all-cause mortality of 57% (95%CI 24%–76%) during the malaria transmission season. No serious adverse events attributable to the drugs used for IPTc were observed in any of the studies. Data from three studies that followed children during the malaria transmission season in the year following IPTc administration showed evidence of a slight increase in the incidence of clinical malaria compared to children who had not received IPTc.IPTc is a safe method of malaria control that has the potential to avert a significant proportion of clinical malaria episodes in areas with markedly seasonal malaria transmission and also appears to have a substantial protective effect against all-cause mortality. These findings indicate that IPTc is a potentially valuable tool that can contribute to the control of malaria in areas with markedly seasonal transmission. Intermittent preventive treatment of malaria (IPT) refers to the administration of a full therapeutic course of an anti-malarial drug to the whole of a population at risk, whether or not they are known to be infected, at specific times, with the aim of preventing mortality or morbidity from malaria Plasmodium falciparum parasitaemia and anaemia when administered to children under five years of age compared to placebo or no intervention. The same studies were also used to assess the effect of IPTc on the prevalence of markers of resistance to SP, as well as any rebound effect in clinical malaria or anaemia in the year following IPTc administration. Non-controlled studies were used additionally to assess the efficacy of IPTc against all-cause mortality and to assess the toxicity of the drugs used.A Cochrane review published in 2008 reviewed the efficacy and safety of chemoprophylaxis and IPT in children A systematic literature search for IPTc studies  was carried out on 25th March 2009 and updated on 19th August 2010. A single investigator (ALW) developed and conducted the search. Studies were identified using database searches, citation searches of selected articles and contact with investigators. The electronic databases searched were: Pubmed (1965-present), Web of Science (1970-present), and Global Health (1910-present). Articles in English, German or French were selected for review and no date restrictions were applied to the search. The search was conducted using free-text terms and standardised subject terms appropriate to the specific database. Combinations of the following search terms were used; intermittent, prevention, presumptive, season, therapy/treatment, malaria and mass drug administration . OngoingStudy eligibility was assessed by a single investigator (ALW) in an un-blinded manner. Studies were retained if they met the following criteria; i) seasonal administration of more than one therapeutic course of anti-malarial drugs, whether or not subjects were known to be infected, ii) study subjects were children, aged less than 5 years, resident in a malaria endemic area, and, iii) the objective of the study was evaluation of the effect of drug administration on clinical malaria . Studies which enrolled children older than 5 years were included if data on children under 5 years were available for analysis. All study designs were accepted, although only controlled studies were used for analysis of primary efficacy outcomes. Studies were excluded if their aim was to evaluate; i) the effect of a sustained protective drug concentration against infection (chemoprophylaxis), or, ii) the effect of drug administration on interruption of transmission (mass drug administration). Studies in population subgroups such as anaemic children were also excluded.Data from eligible studies was extracted based on the intention-to-treat principle into a purpose built database by a single investigator (ALW) . We did not have access to data from individual children and, instead, combined results taken from published papers were used. Investigators were contacted directly if any information was unclear or not specified in the published articles.P. falciparum parasitaemia at any density. An alternative definition of clinical malaria using a locally defined threshold concentration of P. falciparum parasitaemia was also used for comparison. Severe malaria was defined as per the WHO definition P. falciparum at any density. Molecular markers of SP resistance assessed were the dhfr triple  mutations and the dhps 437 mutation, associated with resistance to pyrimethamine and sulphones respectively. Adverse events (AE) and Serious Adverse Events (SAE) were defined as per standard ICH definitions Clinical malaria was defined as an illness accompanied by an axillary temperature of greater than or equal to 37.5°C or a history of fever within the previous 24 or 48 hours and the presence of asexual forms of All trials used in the meta-analysis, whether published or unpublished, had been approved by an ethics committee. The risk of bias in the studies was assessed by a single investigator (ALW) in an un-blinded manner using a tool developed by the Cochrane Collaboration  2 statistic The effect of IPTc on malaria incidence  was expressed as a rate ratio. Person time at risk was expressed in years  and was defined as date of exit from the study minus date of enrolment into the study. Date of exit for children who died was the date of death and was defined as the date of the last contact with active or passive malaria surveillance systems if a child was lost to follow up. Person time at risk was reduced by 28 days  was tailored to the duration of the malaria transmission season. In all studies, except for one (Tivaouane), the intervention was implemented for only one transmission season. A variety of anti-malarial drugs were used, either alone or in combination. Standard adult tablets were used throughout. All studies reported that study drugs had greater than 90% efficacy when used for treatment of patients (children or adults) with uncomplicated but symptomatic malaria in the study area. Two efficacy studies Hohoe  utilisedIn the majority of the studies, two cross-sectional surveys were carried out during the intervention year; one at baseline and another at the end of the intervention period. Either passive Controlled studies were generally of high quality . Four stThe PE of IPTc with SP monotherapy administered every 2 months against uncomplicated clinical malaria (any parasite density) was 35% (95%CI: 17–48%) in Ghana and 69% (95%CI: 54–80%) in Mali . ArtesunRandom effect meta-analysis of all studies gave a summary rate ratio of 0.25 (95%CI: 0.17–0.36)  and restData on severe malaria were available only for two trials . However, as the prevalence of parasitaemia in the children who received IPTc was overall much lower than that in children in the placebo group, the estimated prevalence of drug resistant parasitaemia among study children was lower in the SP+AS arm than in the placebo arm . Data on the prevalence of drug resistance markers in the second year of follow-up was only available for the study conducted in Niakhar. In this study, the difference in the prevalence of markers of resistance to SP was lost at the end of the second year of follow-up . In the Kati Region, the prevalence of the dhfr triple and dhps mutations was higher in the IPTc arm compared to the placebo arm at the post intervention survey dhfr triple mutation SP+AQ 5%, placebo 6% and dhps 437 mutation SP+AS 6%, placebo 8%). In a further two studies (Boussé and Hohoe), the proportion of parasites carrying SP resistance markers was similar in IPTc and placebo arms at the post-intervention survey A concern of any form of community wide drug administration is that it will encourage the emergence of drug resistant parasites. In the study conducted in Niakhar, the prevalence of the n period  [20]. AtA further safety concern for IPTc is that it will impair the development of natural immunity making children more susceptible to malaria after treatment is stopped. At the time of this review, data on clinical outcomes in the year after administration of IPTc were available for only three studies As with any retrospective study of this kind, this review has a number of limitations. It is possible that the literature search may have missed some studies. However, we consider that this is unlikely as the group of scientists involved in the study of IPTc is small and personal enquiries from those known to be active in the field, as well as formal literature searches, were undertaken. Heterogeneity in study design and findings makes some comparisons, for example those on the occurrence of AEs, difficult. Since the reviewers did not have access to individual patient data, the effect of study/patient characteristics, such as baseline parasitaemia, age or ITN usage, on clinical outcomes could not be assessed. Study quality was variable but the small number of IPTc studies identified limited the extent to which sensitivity analysis could be conducted. However, an attempt was made to increase the robustness of the meta-analysis by assessing the effect on PE of using different malaria definitions and using both fixed and random effect meta-analysis. The meta-analysis focused on total malaria episodes, the important public health outcome, rather than first episodes so it may have overestimated PE for this endpoint. Despite these limitations we believe that this review provides a valid overview of current knowledge on the potential of IPTc as a malaria control tool.All of the controlled studies identified demonstrated a protective effect of IPTc against clinical episodes of malaria during the malaria transmission season ranging from 31% to 93%; the overall PE of IPTc administered monthly was 82%. Analysis of the efficacy trials allows some conclusions to be made about the efficacy of individual drugs and dosing regimens in preventing episodes of clinical malaria. The highest PE was observed using two drugs with a long half-life in combination, SP+AQ or SP+PQ. Long acting drugs used alone or in combination with short acting drugs, such as AS or DHA showed lower PEs. No advantage was seen from the use of artemisinin combination therapies, which should probably be reserved for treatment of clinical malaria when the rapid action of artemisinins is of particular benefit. As would be expected, bimonthly administration of SP or AS+AQ demonstrated a lower PE than monthly IPTc administration. The results add weight to the growing body of evidence which suggests that IPT works largely by providing a period of post-treatment prophylaxis and that the length of this period of protection is determined by the pharmacodynamics of the drugs used None of the trials was individually powered to demonstrate an effect on severe malaria or all-cause mortality. However, pooled data from Kati Region and Boussé showed a PE of 82% against severe malaria and data from twelve studies suggests that administration of IPTc reduced overall mortality in children aged 3–59 months by more than 50% during the intervention period. These results are supported by a study in The Gambia, which deployed fortnightly chemoprophylaxis with pyrimethamine/dapsone during the malaria transmission season, and demonstrated an approximately 40% reduction in overall mortality in children in the same age range Two of the five studies which assessed the effect of IPTc on the prevalence of anaemia and mean Hb levels demonstrated a beneficial effect. The lack of effect in other studies may have been due in part to the fact that children were kept under close observation, so episodes of malaria or anaemia were detected and treated promptly during the course of the intervention period, and partly due to the varying importance of malaria as a contributory factor to the pathogenesis of anaemia in different epidemiological settings Three aspects of the safety of IPTc have been considered in this review – immediate toxicity, facilitation of the spread of drug resistance and impairment of naturally acquired immunity.No SAEs which could be attributed to administration of drugs for IPTc were recorded. In particular, no cases of Stevens Johnson syndrome were observed in the 32,757 children who received SP or SP-containing regimens. These observations confirm reports of the safety of SP when used for IPT in infants The most commonly recorded AEs were fever and vomiting, which were reported most often following administration of SP+PQ, DHA+PQ and SP+AQ. Vomiting may have been misreported when children spat out AQ or PQ tablets in reaction to their bitter taste. In the study conducted in Jasikan, Ghana Extensive use of anti-malarials for prevention adds to drug pressure and carries some risk of facilitating the emergence and spread of drug resistant parasites. Drug pressure will be higher when a drug is used for IPTc, covering up to 20% of the population, than when used for IPT in infants or pregnant women (2–5% of the population). Some evidence to support the view that IPTc with SP-containing regimens will select resistant parasites was obtained from the study in Niakhar, Senegal where the proportion of parasites carrying markers of resistance to SP was higher at the end of the malaria transmission season in children who had received IPTc than in children who had received placebo. However, although the proportion was higher, the prevalence of resistant parasitaemia in children who had received IPTc was lower than that in control children because of the effect of IPTc in reducing the overall prevalence of parasitaemia in these children. Which of these variables is most relevant to the spread of drug resistance is uncertain. It was reassuring that by the following year the difference between groups had been lost, suggesting that the parasites carrying resistance markers were at a biological disadvantage in the absence of drug pressure. Nevertheless, if IPTc is sustained for a period of many years it will exert significant drug pressure on the parasite population; use of a combination of drugs rather than a monotherapy should help to mitigate this risk and use of the same drugs for both first-line treatment and IPTc should be avoided whenever possible.P. falciparum per µL was reduced from 82% during the high transmission season to 63% during the whole 12 month period in Mali and similarly from 70% to 49% during the whole 12 month period in Burkina Faso.As for any other successful malaria control measure, an effective IPTc regimen will reduce exposure to malaria parasites and thus has the potential to impair the development of naturally acquired immunity. Meta-analysis indicated a small increase in the incidence of clinical malaria episodes during the malaria transmission season in the year following IPTc administration among children who received IPTc compared to control children. In Niakhar, a small increase in the prevalence of anaemia was observed in children who had received IPTc in the previous year but no significant effect on mean Hb concentration was observed so this may have been a chance finding. Unpublished results from Kati Region, Mali and Boussé, Burkina Faso also indicate a slight rebound effect on clinical malaria among children who received IPTc . The 12 month incidence of clinical malaria in the placebo arms of the studies conducted in Kati Region and Boussé indicates that approximately 10% of episodes occur outside the high transmission season. Taking these two factors into account, the PE of IPTc against clinical malaria defined using a parasite threshold of 5000 asexual forms of A limitation of the efficacy studies is that IPTc was only administered for only one transmission season. It is likely that if children are given IPTc each year for their first five years of life this will have a greater impact on the development of naturally acquired immunity to malaria. This was observed in The Gambia when fortnightly chemoprophylaxis during the rainy season was given each year for many years Based on the data reviewed, IPTc offers young children a high level of protection against clinical malaria and appears to substantially reduce mortality during the malaria transmission season. Widespread deployment of IPTc will inevitably cause some drug related side effects, may enhance drug resistance and, like all effective malaria control measures, may lead to some loss of naturally acquired immunity. Balancing these benefits and risks will require careful assessment of the burden of malaria in a particular community, its seasonality and the sensitivity of the prevalent parasites to the drugs currently available for use in IPTc. Implementation of IPTc should now be considered as part of an integrated malaria control strategy in areas of seasonal malaria transmission. In considering introduction of IPTc as a malaria control policy it will also be important to assess the acceptability, feasibility, sustainability, cost and cost effectiveness of the intervention. Research that investigates some of these aspects of IPTc is underway Table S1Search Strategy for Pubmed and Web of Science Databases(DOC)Click here for additional data file.Table S2PRISMA Checklist(DOC)Click here for additional data file.Table S3IPT Studies Excluded From Review(DOC)Click here for additional data file.Table S4Incidence of Most Common Adverse Events During the Intervention Period(DOC)Click here for additional data file.


The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria™ RDT (AccessBio®) which is currently used in Cambodia, and real-time PCR (as “gold standard”).Plasmodium falciparum and non-P. falciparum were with 20–30 min reading times  and were similar to those for the CareStart Malaria™ test.The best performances of the VIKIA Malaria Ag Pf/Pan™ test for detection of both This new RDT performs similarly well as other commercially available tests , and conforms to the World Health Organization’s recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas. Plasmodium falciparum parasites[Now that artemisinin derivatives in combination with partner drugs (artemisinin combination therapy (ACT)) are being used, parasitological confirmation has become essential before treatment in routine malaria case management, in most countries endemic for malaria,2. This arasites, particuarasites-7.Microscopic examination of blood films still remains the gold standard for malaria diagnosis despite the requirement for high-quality microscopes and equipment, and for qualified personnel. HoweverP. falciparum and detect the histidine-rich-2 protein (HRP-2). Others combine HRP-2 detection with the detection of antigens common to all species, such as lactate dehydrogenase or aldolase (combo RDTs). Combo RDTs can diagnose infections by P. falciparum or by non-P. falciparum malaria parasites [The malaria RDTs available are all based on the same principle and like other lateral flow immune-chromatographic tests, there are various formats . They contain antibodies conjugated to colloidal gold particles, which bind specifically with parasite antigens. Some of them are specific for alariae).© is a company which aims to address public health requirements by developing reliable diagnostic tools responding to the constraints associated with use in the field for populations in developing countries at affordable prices[IMACCESSe prices. The comIn this context, the objective of the study presented here was to assess the performances of this new immunochromatographic test by following a conventional field evaluation design including microscopic examination (thick and thin blood film) and molecular detection  as gold standard methods for malaria diagnosis of febrile, malaria-suspected patients seen at health centre level. In addition, the VIKIA Malaria Ag Pf/Pan™ test was also compared to another RDT widely used in Cambodia and elsewhere .©, Ref. 41 2499, Lot Number: V110727601, Lyon, France), containing one package insert, 25 test cassettes in individual sealed pouches with a disposable specimen pipette and a dessicant and one vial of running lysis buffer, was used according to the manufacturer’s instructions. Briefly, a 5 μL aliquot of blood drawn from patients was added to the sample well. Five drops of lysis buffer were then dispensed into the buffer well  was also performed according to the manufacturer’s instructions and used to diagnose patients as malaria infected.The VIKIA Malaria Ag Pf/Pan™ test kit  were selected Figure: Veal VePlasmodium species all co-exist in the Cambodia and P. falciparum is the most frequent cause of malaria infections . The distributions of Plasmodium species have been changing over recent years, with a particularly significant increase in the incidence of P. vivax malaria cases (from 8% in 2000 to 23% in 2007). In low transmission areas, a proportion of infections that are P. vivax is commonly up to 50%[Malaria remains a public health problem at these sites, and is mainly linked to the forests. Forest villagers in the eastern and northern provinces are at high risk of malaria, whereas elsewhere in the country malaria is an occupational disease with particular groups being at high risk, including forestry workers, new settlers and mobile/migrant populations moving into forested areas, and soldiers and their families serving in the forests. The prevalence is between 15% and 40% in villages near or in forested areas, and between 0% and 3% in the plains and rice field areas. The five human p to 50%.P. falciparum malaria according to the definition of the World Health Organization (2001) were excluded[In the four sites, patients with clinical symptoms of malaria (fever ≥37.5°C) and with a prescription for malaria diagnosis from a health care worker, agreeing to be enrolled in the study (by signing the consent form), were included. Pregnant women and patients with signs of severe and complicated nization 01 were eBlood samples were collected by finger-prick and used for malaria testing . In addition, two drops of blood were spotted onto Whatman 3 M filter paper™ and individually stored in an envelope and a plastic bag. Slides for thick and thin microscopic examination were also prepared, and stored in appropriate boxes. Clinical and biological information were recorded on an anonymous form to ensure patient confidentiality. Filter papers and slide boxes were sent to the Institut Pasteur du Cambodge (IPC) in Phnom Penh every week.At IPC, thick and thin blood smears were stained with 3% Giemsa for 30 min and analysed by light microscopy by two experienced technicians without reference to RDT results. A minimum of 200 consecutive fields was checked in the thick blood film before classifying a slide as negative. Parasites in thick blood films were counted against 200 white blood cells. The parasite density was estimated assuming 8,000 white blood cells/μL of blood. One in 10 slides selected at random, and all slides corresponding to discordance between the first two readings, were read a third time by a third expert microscopist.©, Marnes la Coquette, France), according to the manufacturer’s instructions. Molecular detection and identification of Plasmodium parasites involved two steps: Plasmodium was detected by a “screening real-time PCR” with primers targeting the Plasmodium cytochrome b gene. Secondly, DNA samples identified as positive for Plasmodium were analysed for malaria species by PCR with several pairs of specific primers  targeting the same gene . From August to December 2011, 1,000 patients  with an age range of one to 70 years  were recruited. The male/female ratio was 2.1/1. The mean ± SD axillary temperature was 38.1 ± 0.9°C (range = 37.5–41.5°C) and the mean ± SD parasitaemia density was 13,455 ± 52,694 parasites/μL .Plasmodium falciparum was present in 39.5%, P. vivax in 58.8% and both P. falciparum and P. vivax (mixed infections) in 1.5% of the positive specimens. A total of 92 discordant results (9.2%) were found between microscopy and real-time PCR . In addition, 59 samples were misclassified by microscopy: 54 were classified as single infections by microscopy  but found to be mixed infections by real-time PCR ; three cases classified as mixed P. falciparum/P. vivax infections were identified as single P. falciparum infections by real-time PCR; two cases classified as single P. vivax infections were found to be single P. falciparum infections by real-time PCR.Microscopy results showed that 389 (38.9%) of the 1,000 patients had malaria. Plasmodium spp. by real-time PCR, microscopy, CareStart Malaria™ and VIKIA Malaria Ag Pf/Pan™ tests at several reading times  are shown in TablePlasmodium spp. parasitaemia are summarized in TableP. falciparum infections and FigureP. falciparum infections.Details concerning patients positive for vs 42.2%), CareStart Malaria™ test  or VIKIA Malaria Ag Pf/Pan™ test . A similar trend was also found for a greater sensitivity of microscopy by expert microscopists than malaria RDTs: 2.0% more positive samples by microscopy than CareStart Malaria™ test (38.8% vs 38.1%) and 2.8% (60 min reading time) to 20.8% (10 min reading time) more than with the VIKIA Malaria Ag Pf/Pan™ test .This study reports the first evaluation of the performance of the VIKIA Malaria Ag Pf/Pan™, a new immunochromatographic test developed by IMACCESS®, using a combination of microscopy and real-time PCR as reference methods for classification of samples. This approach had the advantage of combining the high sensitivity  and specificity (to correctly identify the parasite species) of real-time PCR, with the possibility of estimating parasite density by microscopy. As expected, more positive samples were found using real-time PCR than microscopy  to 94.2% for P. falciparum detection and 82.7% for non-P. falciparum detection (60 min). The specificity  decreased with reading time from 99.3% for P. falciparum detection and 99.5% for non-P. falciparum detection (10 min) to 98.1% for P. falciparum detection and 98.9% for non-P. falciparum detection (60 min).The sensitivity  of the VIKIA Malaria Ag Pf/Pan™ test increased with reading time, from 89.7% for P. falciparum and non-P. falciparum detection were observed at 20–30 min . Using these reading times, the performance of the VIKIA Malaria Ag Pf/Pan™ test was similar to those of the CareStart Malaria™ test or those found in previous studies[The best performances of the VIKIA Malaria Ag Pf/Pan™ test for both  studies-26.P. falciparum, the sensitivity of the both tests started to decrease at levels of parasitaemia < 500 parasites/μL (100% above 500 parasites/μL) and for non-P. falciparum, the sensitivity of the CareStart Malaria™ test and the VIKIA Malaria Ag Pf/Pan™ test started to decrease below 1,000 parasites/μL or below 10,000 parasites/μL, respectively. False negative results for both malaria RDT were only found in P. falciparum samples containing <250 parasites/μL and in non-P. falciparum samples with <1,000 parasites/μL.Moreover, as has been previously reported, the sensitivities of both malaria RDT decreased with the level of the parasitaemia,26-30: fAccording to WHO recommendations for RDT performances, sensitiIn conclusion, this new RDT performs similarly well as other commercially available tests , and conforms to WHO recommendations for RDT performance. It appears to be a satisfactory alternative tool for the diagnosis of malaria in endemic areas.The authors declare that they have no competing interests.MC, SK and DM contributed to the design and coordination of the study, assisted with data entry and interpretation and prepared the manuscript. SK supervised the field study. NK, SC, SS, DD and LC were involved in laboratory work. MC, SK, CN and DM helped to write the manuscript and gave constructive advice. All authors read and approved the final manuscript.


Plasmodium falciparum infection on the rate of acquisition of immunity to malaria and on the immune correlates of protection has proven difficult to elucidate. A randomized, double-blind, placebo-controlled trial using monthly chemoprophylaxis with sulphadoxine-pyrimethamine plus artesunate was conducted to modify the age of first P. falciparum erythrocytic exposure in infancy and assess antibodies and malaria risk over two years.The impact of the age of first 19, AMA-1, and EBA-175 were measured by ELISA, and IgG against variant antigens on the surface of infected erythrocytes by flow cytometry. Factors affecting antibody responses in relation to chemoprophylaxis and malaria incidence were evaluated.Participants (n = 349) were enrolled at birth to one of three groups: late exposure, early exposure and control group, and were followed up for malaria morbidity and immunological analyses at birth, 2.5, 5.5, 10.5, 15 and 24 months of age. Total IgG, IgG subclasses and IgM responses to MSP-119. Previous and current malaria infections were strongly associated with increased IgG against MSP-119, EBA-175 and AMA-1 (p < 0.0001). After adjusting for exposure, only higher levels of anti-EBA-175 IgG were significantly associated with reduced clinical malaria incidence .Generally, antibody responses did not vary significantly between exposure groups except for levels of IgM to EBA-175, and seropositivity of IgG1 and IgG3 to MSP-1P. falciparum infection did not influence the magnitude and breadth of IgG responses, but previous exposure was critical for antibody acquisition. IgG responses to EBA-175 were the strongest correlate of protection against clinical malaria.Overall, the age of first NCT00231452.ClinicalTrials.gov:  Plasmodium falciparum different individuals experience different outcomes; while some may die of severe malaria others may experience mild disease to asymptomatic infection [Malaria is among the leading causes of morbidity and death in children, despite being a preventable disease. The World Health Organization estimates that 219 million cases and 660,000 deaths occurred in 2010, with approximately 81 and 91%, respectively, being in sub-Saharan Africa [nfection . Howevernfection . In endenfection ,6. Humannfection ,8.in vitro by promoting phagocytosis of P. falciparum-infected erythrocytes [in vitro for some blood stage antigens that are candidates for malaria vaccines. First, the recombinant 62 kDa apical membrane antigen-1 (AMA-1), involved in the re-orientation of merozoites prior to invasion of the erythrocyte [19), the most abundant merozoite surface protein, thought to be involved in the initial attachment of the merozoite to the erythrocyte surface [In people living in endemic areas, malaria infection induces strong humoral immune responses, involving production of predominantly immunoglobulin (Ig) M and IgG ,10. Cytohrocytes ,9,11,12.hrocytes . Parasitthrocyte ; second,throcyte ,15; and, surface . Sero-ep surface .P. falciparum as well [P. falciparum erythrocyte membrane protein 1 (PfEMP-1), is highly variant and equipped with several binding sites mediating adhesion of infected erythrocytes to vascular endothelium of capillaries and post-capillary venules thus avoiding destruction in the spleen [Variant surface antigens (VSA) expressed on the parasitized erythrocyte membrane are thought to induce protective responses to  as well . The pre as well . The gene spleen -21. Folle spleen ,6. Anti-e spleen .P. falciparum during the first six months of life was not relevant for acquisition of immunity, whereas exposure in the second six months could be critical for acquisition of immunity, and conducted a clinical trial to test this. The age of first exposure to P. falciparum blood stage infection was controlled by chemoprophylaxis with sulphadoxine-pyrimethamine (SP) plus artesunate (AS) during different periods of the first year of life in Mozambican children who were then followed up to 24 months for clinical malaria [Longitudinal studies in Indonesia  and Tanz malaria . The con19, AMA-1, EBA-175 and VSA antigens was conducted in the children participating in the clinical trial during their first two years of life. Risk factors affecting antibody levels were examined at five cross sectional visits and upon P. falciparum infection and convalescence, and the association between the magnitude and breadth of antibody response and subsequent incidence of malaria.A detailed assessment of antibody responses to MSP-1P. falciparum malaria is perennial with two well-demarked seasons, a dry season (May-October) and a rainy season (November-April). The entomological inoculation rate estimated in 2002 was of 38 infective bites/person/year [The study was conducted in the District of Manhiça (Maputo Province) in Southern Mozambique, in the neighbourhoods of Maragra and Maciana in the town of Manhiça. Characteristics of the area have been described elsewhere . Brieflyson/year . A demogson/year .The study was a double-blind, randomized, placebo-controlled trial, described in detail elsewhere . BrieflyApproval for the study was obtained from the National Mozambican Ethics Review Committee (Ref 05/CNBS/05) and the Hospital Clínic of Barcelona Ethics Review Committee. Children were enrolled in the study after their mothers gave a written informed consent.P. falciparum parasite in vitro cultures. Parasites were cryopreserved by adding 1.7 volumes of glycerolyte dropwise and at a continuous agitation, and then kept at -80°C. For recovery, parasite aliquots were thawed using two sodium chloride thawing solutions  following the protocol described elsewhere [In previous work by the same group, a panel of parasite isolates  was tested for immunogenicity. The laboratory line (R29) was found to be the most immunogenic in the study area . Stocks lsewhere .P. falciparum VSA has been described elsewhere [P. falciparum) and one dilution (1/20) of five negative control plasmas  were included in each plate. Samples were then acquired in a four-colour FACSCalibur  using CellQuest 3.3 software. Up to 1,000 T-iE (or ethidium bromide positive events) were counted per sample, and the geometric means (GM) of the fluorescence intensities were calculated.The protocol for quantifying IgG against lsewhere . Briefly19 , AMA-1 , and EBA-175  from the ICGEB  [2HPO4 0.512 M + 1 mg/ml of o-phenylenediamine chromogen  with 0.012% H2O2 substrate were added per well for 5 min, and the colorimetric reaction was stopped with 25 μL of 3 M H2SO4. The specific reactivities of plasma samples were obtained as optical density (OD) values . In addition, antibody levels were categorized using a cut-off OD value (the arithmetic mean of negative controls plus three standard deviations) for statistical analyses.All plasma samples were assayed for total IgG, isotypes  and IgM against the recombinant proteins MSP-1, India) . Ninety-Plasmodium falciparum infections were diagnosed and quantified by microscopy at 2.5 and 24 months of age, and in non-programmed visits whenever a child attended the hospital with fever or history of fever in the preceding 24 hours. Thick and thin blood films were Giemsa-stained and read as previously described [P. falciparum. The probe used was labelled with 6-carboxy-flourescein. A standard curve was produced using known concentrations of 3D7 strain P. falciparum parasites in parasite culture. Importing this curve into subsequent qPCR runs achieved quantification of parasitaemia with an ABI Prism 7500 apparatus (Applied Biosystems).escribed . Cases wescribed . BrieflyClinical malaria was defined as axillary temperature ≥37.5°C or reported fever in the preceding 24 hours, plus positive parasitaemia by microscopy. For anti-VSAs antibodies the sample fluorescence intensity was defined as the difference between the GM of the fluorescence emitted by the T-iE and the GM of the fluorescence emitted by the non-infected erythrocytes. Antibody values for ELISA  were logarithmically transformed and averaged within the groups and presented as GM plus 95% confidence intervals (CI). Distribution of antibody responses to each antigen at each cross-sectional visit, acute malaria and convalescence, are presented as weighted scatter plots. Differences in antibody prevalence (percentage of positive responders) between treatment groups at different time points were estimated by Chi-square and Fisher’s exact test, as appropriate, and differences in continuous antibody values among treatment groups were evaluated by ANOVA, evaluated using a likelihood ratio test and a global p-value was used for significance. Breadth of antibodies at the cross-sectional visits was defined as the number of antigens with positive IgG or IgM antibodies responses as defined by the categories above. Mixed-effects linear regression models with a random-intercept at the subject level were used to analyse factors independently associated with antibody levels at the cross-sectional visits, and the variables used to estimate models were: intervention group, age, season (dry/rainy), neighbourhood , gender, previous infection, current infection, low birth weight, weight for age score, maternal peripheral infection, placental infection and inflammation, parity, maternal age, maternal anaemia, congenital infection (parasites in cord blood by qPCR), use of insecticide-treated nets (ITN) and documented indoor residual spraying (IRS) in the household. Selection of the variables for the multivariable model was done through a forward-stepwise procedure, where the criterion for including a variable to the model was p-value ≤0.05 from the likelihood ratio test. In addition, mixed-effects linear regression models were used to analyse the effect on antibody responses of the following variables selected for the highest biological plausibility to influence Ig levels: intervention group, age, season, neighbourhood, previous infection, current infection, placental infection and inflammation, congenital infection and low birth weight. Antibody levels and breadth of response at the cross-sectional visits in relation to incidence of malaria were assessed by negative binomial regression models. First, the control group (no treatment) was evaluated in two risk intervals from 2.5 to 12 months and from 2.5 to 24 months. Second, all treatment groups were assessed in three risk intervals: from 5.5 to 24 months, from 10.5 to 24 months and from 15 to 24 months. The strength of the association between antibody levels or breadth and malaria risk was assessed first unadjusted and second after adjusting by treatment, age, season, neighbourhood, current infection, previous infection, maternal infection, congenital infection, placental inflammation, ITN use and IRS. Statistical significance was defined at P < 0.05. Crude p values reported in this exploratory study were not adjusted for multiple comparisons and were interpreted for internal coherence, consistency of results and biological plausibility. Data analysis was performed using STATA 11 .P. falciparum did not differ significantly between intervention arms at most study visits  for the majority of serological markers evaluated  and after the intervention period (second year). Antibody responses to  Figures  and 2. Ovs 0.42, 0.38-0.46, respectively, p = 0.0469; AMA-1: 0.40, 0.37-0.43 and 0.40, 0.37-0.42 vs 0.35, 0.32-0.39, p = 0.0543; MSP-119: 0.43, 0.39-0.47 and 0.46, 0.41-0.52 vs 0.40, 0.36-0.43, p = 0.0885) , and similarly for levels of IgG3 . Non-cytophilic antibodies (IgG2 and IgG4) yielded consistently low level responses and are not reported here.In contrast, IgG levels did not significantly vary after chemoprophylaxis and thus there was no evidence for supporting differences in IgG levels between the three exposure groups at the cross-sectional visits Figures  and 3. T19, EBA-175 and AMA-1 at 15 and 24 months of age than Control children who had been under continuous exposure to P. falciparum .In the second year, no significant differences in antibody levels were detected overall according to intervention group. However, children with prior malaria exposure  who had received SP + AS chemoprophylaxis any time during year 1 (early exposure and late exposure groups) had higher IgG to MSP-1 Figures , 4 and 5There were no statistically significant differences in IgG responses to VSA among the three groups at any of the sampling visits , heterogeneity of exposure, proximity to swamps, and higher previous exposure are significant predictors of higher antibody responses ,32,35. IIn these very young children, anti-VSA IgG responses were not prominent. Levels were very low and dependent on neighbourhood and age, with an overall pattern showing higher maternally derived IgG waning over time. This was in contrast to studies in a different endemic area of Tanzania where the level of antibodies increased with age from five to 24 months . The resBreadth of antibody response has been suggested to be an important predictor of protection from clinical malaria in Kenyan children , with anP. falciparum exposure seemed not to be critical for acquisition of protective immunity as evaluated by acquisition of antibody responses to blood stage antigens that are candidate targets for malaria vaccine development. Overall, the breadth and magnitude of antibody responses were strongly influenced by previous exposure and age. Only levels of IgG to EBA-175 were associated with reduced incidence of malaria in the second year of life. It remains to be assessed whether the intervention might have an impact on the quality of antibody responses such as affinity/avidity, fine specificity or functionality, and on cellular immune responses also thought to contribute to naturally acquired immunity.The age of first The authors declare that they have no competing interests.PA, JJA and CD conceived and designed the field trial: CG, QB, AN, MNM, AB, and MHR conducted the field trial and follow-up of participants; CD and AM conceived and designed the experiments; CEC produced recombinant proteins; AN, AJ, MNM, RA, MHR, AB, and PC processed samples and performed laboratory analysis; LQ and JJA analysed the data; AN and CD wrote the paper. All authors contributed to the interpretation of the results and writing of the manuscript. All authors read and approved the final manuscript.Comparison of the anti-variant surface antigens (VSA) Immunoglobulin (Ig) G antibody responses. Differences in anti-VSA IgG antibodies levels among chemoprophylaxis groups, at the five sampling study visits; assessed by ANOVA.Click here for fileVariables associated to antibody levels assessed by multilevel regression model. Analyse of variables associated with antibody levels assessed by mixed-effects linear regression models including the following variables: intervention group, age, season, neighbourhood, previous infection, current infection, low birth weight, placental infection, placental inflammation and congenital infection.Click here for fileAssociation between different neighborhoods and antibody levels. Proportional differences and P-values of the association between neighbourhood and the magnitude of antibody response by linear regression analysis.Click here for fileDistrict map of the Manhiça study area. Group of study regions and distribution of study participants per neighbourhood (bairros).Click here for fileMagnitude of antibody response related to incidence of clinical malaria only in control group. Association between levels of antibodies (2-fold increment) at 2.5 months of age and the incidence of malaria up to 12 and 24 months of age, only for the placebo control group. Analysis done by negative binomial regression models adjusted by season, neighborhood, current infection, previous infection, maternal infection, congenital infection, placental inflammation, insecticide treated bednet use and indoor residual spraying.Click here for fileBreadth of antibody response related to incidence of clinical malaria only in control group. Analysis done by negative binomial regression. Participants of control group were assessed in five risk intervals: from 2.5 months to 12 months, from 2.5 months to 24 months, from 5.5 months to 24 months, from 10.5 to 24 months and from 15.5 to 24 months. The strength of the association between breadth of response and malaria risk was assessed first unadjusted and after adjusting by treatment, age, season, neighbourhood, current infection, previous infection, maternal infection, congenital infection, placental inflammation, insecticide treated bednet use and indoor residual spraying.Click here for file


P. falciparum infection, and is often the primary endpoint in clinical trials and epidemiological studies. Subjective and objective fevers are both used to define the endpoint, but have not been carefully compared, and the relative incidence of clinical malaria by active and passive case detection is unknown.Febrile malaria is the most common clinical manifestation of We analyzed data from cohorts under active and passive surveillance, including 19,462 presentations with fever and 5,551 blood tests for asymptomatic parasitaemia. A logistic regression model was used to calculate Malaria Attributable Fractions (MAFs) for various case definitions. Incidences of febrile malaria by active and passive surveillance were compared in a subset of children matched for age and location.Active surveillance identified three times the incidence of clinical malaria as passive surveillance in a subset of children matched for age and location. Objective fever (temperature≥37.5°C) gave consistently higher MAFs than case definitions based on subjective fever.The endpoints from active and passive surveillance have high specificity, but the incidence of endpoints is lower on passive surveillance. Subjective fever had low specificity and should not be used in primary endpoint. Passive surveillance will reduce the power of clinical trials but may cost-effectively deliver acceptable sensitivity in studies of large populations. P. falciparum infection and is the endpoint most commonly used to measure the public health burden of the disease, and to assess the efficacy and effectiveness of preventative interventions such as bed nets Childhood febrile disease is the most common clinical manifestation of Defining clinical malaria in malaria endemic countries is difficult because individuals may carry parasites without symptoms, and coincidental febrile episodes may have etiologies other than malaria. The malaria-attributable fraction method uses population data to estimate the frequency of true febrile malaria among all febrile cases by fitting the risk of fever as a function of parasite density using a logistic regression modelIn active surveillance, individuals are visited regularly  and assessed for malaria infection or disease status. Provision is usually made to identify febrile episodes between visits. This additional provision is often referred to as “passive surveillance”, and hence the combination may be called “active and passive surveillance”. However, the likelihood of presentation between visits often rises as a result of the frequent contact It has been argued that active surveillance is more likely than passive surveillance to misdiagnose asymptomatic parasitaemia with a coincident, non-malaria cause of fever as a true malaria case, leading to over-estimation of burden of malaria Comparison between surveillance methods can be confounded by the location, malaria endemicity, age of individual being followed and period of follow up. Therefore a formal comparison of the relative incidence of endpoints detected by passive and active surveillance requires matching by these variables, and has not previously been undertaken.Regardless of the surveillance method used, cases are often defined by malaria parasitaemia in association with either an objective fever (i.e. temperature>37.5) or a subjective fever. Use of subjective fever in the case definition may lack specificity, and has been avoided in some studiesWe therefore analyzed data across four different cohorts in Kilifi District, Kenya, in order to describe the sensitivity and specificity of different case definitions from active and passive surveillance. We also performed a formal comparison of specificity and incidence of mild malaria endpoints from active and passive surveillance in a sub-group of children who were matched by age, period of follow up and location .Previous studies have suggested that a threshold of 2,500 parasites per µl adds specificity to the case definition P. falciparum malaria, in order to describe the sensitivity and specificity of case definitions within cohorts and compare the specificity and incidences of the endpoints from active and passive surveillance. The cohorts were located in Chonyi, Ngerenya, Junju and Pingilikani sub-locations of Kilifi District, on the coast of Kenya between January 1998 and June 2009 . Only children aged 0–5 years old were included in the analysis of general MAF. Age specific MAF were estimated to investigate variation of MAF with age for each cohort. Children who had objective fever and also were reported to have subjective fever were included in the sub-analysis of “objective fever” but not “subjective fever”.Active surveillance in the Junju cohort located in Pingilikani sub-location overlapped with passive surveillance at Pingilikani dispensary. This overlap applied to the 105 children aged 5–17 months who were under active surveillance between May 2007 and April 2008. In order to define a comparable cohort under passive surveillance at Pingilikani dispensary, we used the demographic surveillance system to identify age- and location-matched children during the same period. However, we did not include children sharing a homestead with a child under active surveillance in this matched cohort, to avoid a possible contaminating effect of the homestead being visited. Incidence rates of malaria clinical episodes were calculated by counting the number of clinical episodes of malaria divided by the total time at risk expressed as total person years at risk.The detailed characteristics of each cohort are shown in Using a subjective history of fever in the case definition was associated with a consistently lower MAF than objectively elevated temperature (temp≥37.5°C) independent of age. This difference was more marked in the higher transmission cohorts (Chonyi and Pingilikani) In the Junju cohort, where blood smears were only made on objectively febrile children, on only 8 out of 532 occasions (0.02%) were children with a history of fever but no elevated temperature found to have febrile malaria on a return visit conducted 6–12 hours later. The children who had no objective fever at 6–12 hours follow-up had a similar risk of a subsequent malaria episode as the children (matched by season and age group) who had no history of fever .Older children (2.5–5 years) had lower MAF than younger children (0–2.5 years) which increased after using a parasite threshold of >2500/µL (data not shown). However this difference was less marked in a low transmission cohort (Ngerenya).Children under passive surveillance in Pingilikani had a significantly higher geometric mean parasite density than children under active surveillance in Junju . Furthermore, there was no variation in geometric mean parasite density with distance from dispensary for the Pingilikani dispensary cohort within 10 kilometers of the dispensary suggesting that distance to healthcare was not a factor that determined the likelihood of presentation .Part of the Junju active cohort and the Pingilikani dispensary-based cohort overlapped geographically . Among aClinical malaria is the endpoint most commonly used in the field to measure the efficacy of interventions to prevent malaria, and is often used to assess the public health burden. In a subset of concurrent location- and age-matched cohorts, we found that purely passive surveillance without specific prompts for visits detected about one third of the episodes that would have been identified by active surveillance yet MAFs were similar. Furthermore, cases with subjective fever but no objective evidence (temperature <37.5°C) had persistently lower Malaria Attributable Fractions, even when a parasite density threshold was used.It is surprising that the difference between rates for active and passive case detection in matched subset of children was not larger. Pingilikani dispensary serves a population of 4000 children under 5, and there had been no contact at the homestead or village level to encourage attendance. In contrast, active surveillance required one local fieldworker per 30–40 children monitored with frequent contact at the homestead level . The large numbers of children using Pingilikani dispensary could be attributed to the availability of adequate staff seven days a week, good malaria diagnostic facilities and a constant antimalarial drug supply in contrast to frequent supply problems reported elsewhere in Kenya Nevertheless, fewer cases of malaria were identified at the dispensary than would have been seen on active surveillance, and in comparison children brought to the dispensary had significantly higher mean temperatures when they were febrile but similar symptomatic blood stage parasitaemia. This suggests that parents were inclined to postpone presentation at the dispensary until more acute overt illness. This was not simply the parents' inability to identify illness, since when field workers were made highly accessible in the subset of Junju cohort children located in Pingilikani sub-location, the majority of malaria episodes were identified by assessments initiated by the mothers between regular weekly visits. There was no gradient of increasing parasite density with distance from the dispensary, suggesting that the distance parents had to travel with their children did not delay their treatment seeking. We conclude that, in our setting, there is a barrier to approaching medical staff in a health facility that does not exist with more familiar, local field workers. Cases of febrile malaria not presenting to the local dispensary may resolve without treatment or be treated by anti-malarials bought by the parents from shopsPassive surveillance in health care facilities has been reported to be insensitive outside Africa The proportion of fevers attributable to malaria was persistently lower in children with subjective fever than in children with objective fever. Fever diagnosis by parents is unreliable Other factors may be relevant in defining the endpoint of a study. Although the main outcome for most Phase IIb vaccine trials in children would be clinical malaria, cluster randomized trials of transmission blocking vaccines might appropriately include all parasitaemic cases identified regardless of the definition of clinical malaria.However, approximately half of all cases presenting had only subjective fever, and so the loss in power of the study must be balanced against the need to include endpoints of high specificity. As an illustration, consider a hypothetical example where the incidences of subjective and objective febrile malaria were both 10 per 100 children, and the respective MAFs were 54% and 85% . A study would have 90% power to detect 50% vaccine efficacy on including 1800 children using objective fever alone as the endpoint, falling to 1300 children using objective plus subjective fever. However, the estimate of efficacy would be 43% using objective fever, but 35% using objective fever plus subjective fever.When we followed-up 532 children with subjective fever in Junju cohort 6–12 hours later, we found only 4% (19/532) developed an objective fever despite not receiving treatment and only 8 (0.02%) had parasitaemia. Furthermore, children who remained afebrile at 6–12 hours of follow-up had a similar risk of subsequent malaria episodes and other adverse events as other children matched by age and time of follow up. This was done in a clinical trial where we carefully monitored children and ensured 24 hour access to health care, and would not be practical for routine healthcare or larger studies, but demonstrates that subjective fever alone does not identify children at a high risk of serious disease.When estimating MAFs in the Pingilikani cohort, cross-sectional surveys from neighboring Junju were used. Since malaria transmission can vary significantly within short distances Our results of the comparative analysis between passive surveillance and active surveillance apply in the context of our transmission patterns  and age group compared, and extrapolation to other transmission patterns and age groups must be done with caution. Ngerenya and Chonyi cohorts operated identical surveillance methods, but the difference between objective fever and subjective fever was greater in Chonyi (at moderate transmission intensity) than in Ngerenya (at low transmission intensity). We have avoided comparisons between surveillance in Junju and Ngerenya/Chonyi because both the surveillance methodology and transmission intensity are different. Another limitation of our study is its retrospective nature which is prone to unmeasured confounding and bias.In conclusion, the Malaria Attributable Fractions are similar between active and passive surveillance, and passive surveillance at the dispensary underestimates malaria occurrence substantially. Cases of malaria identified on the basis of reported fever alone should not be included in the primary endpoint of a clinical trial since the specificity of the endpoint is low. Phase III studies intended to lead to marketing licenses should test vaccines in the standard setting of dispensary surveillance, but active surveillance should be preferred in Phase IIb studies. Further studies are needed to examine the potential factors inhibiting attendance at health facilities. Improved malaria surveillance at local levels is particularly important for supporting current malaria control efforts and eventually, its elimination.


Plasmodium knowlesi in Thailand. Diagnosis was confirmed by the small subunit ribosomal RNA and the mitochondrial cytochrome b sequences. The occurrence of simian malaria in human has signified the roles of wild primate populations in disease transmission in some malaria-endemic areas.We describe a case of naturally acquired infection with  Plasmodium circulate among primate populations . The parasite structure was compatible with that of P. malariae. The parasite density inferred from the number of malarial parasites per 500 leukocytes in thick blood smear yielded 1,155/μL or equivalent to parasitemia 0.03%. The patient was treated with 10 mg/kg of oral chloroquine initially, followed by 5 mg/kg, 6 hours later on the day 1, and 5 mg/kg/day for the next 2 days. On day 2, with a temperature of 37.5°C, he came to the hospital. Parasitemia decreased to 137/μL. Complete defervescence was observed on day 3, and parasitemia could not be detected. Two weeks and 2 months later, his blood smears were negative for malaria. Fever did not recur.Upon examination, his temperature was 38.5°C, and pulse rate was 90 beats per minute. His hemoglobin was 14.0 g/dL, hematocrit was 0.4, and erythrocyte count was 4.2 x 10P. falciparum, P. vivax, and P. malariae and four isolates of P. ovale were used as positive controls. Results showed that all isolates gave concordant positive PCR products with those diagnosed by microscopy except an isolate from this patient (data not shown). Retrospective examination of blood smears has shown several developmental stages of malaria parasites similar to those typically seen in P. malariae. However, some erythrocytes that harbored mature asexual parasites possessed fimbriated margins. The cytoplasm of some young trophozoites appeared spread out into the network of irregular pseudopodia, and the chromatin was distributed into fragments, conforming to the tenue forms. Pinkish dots varying from fine to large irregular masses called Sinton and Mulligan's stippling developed intracorpuscularly with the maturation of some parasites  targeting the small subunit ribosomal RNA (SSU rRNA) genes of all four species of human malaria as reported   (Phylogenetic tree showed that analysis . ConsistP. knowlesi is prevalent among crab-eating macaques, Macaca fascicularis, in the Malaysian peninsula and the Philippines (,M. nemestrina, and leaf monkeys, Presbytis melalophos (,P. knowlesi isolated from monkey could be infectious to humans, the first naturally acquired human infection with P. knowlesi was not reported until 1965 (P. knowlesi has been identified from Malaysian Borneo (P. knowlesi to a forest in Thailand near southern Myanmar border, where wild populations of crab-eating macaques, despite being considered endangered, are still substantial.P. knowlesi found in the first naturally acquired human case led to the initial diagnosis of P. falciparum, while the mature parasites could masquerade as those of P. malariae, as we encountered in this patient (P. knowlesi have been delineated, we were unable to find the ring form with double chromatin dots (P. malariae variant found in Myanmar (P. knowlesi with one or more of the four human malaria species was not supported by our PCR detection. The structure of P. knowlesi is highly dependent on the host erythrocytes, i.e., resembling P. vivax in M. fascicularis, P. falciparum in rhesus monkeys, and P. malariae in humans (,,,P. knowlesi–infected blood smears of Sarawak's patients, the presence of Sinton-Mulligan stippling in infected erythrocytes in this study is in accord with the report by Fong et al., in which erythrocytic stippling served as one of the diagnostic feature (,The prevalence of naturally acquired primate malaria in humans can be underestimated from examination of blood films. The reported abundance of ring stages of P. knowlesi in human and its natural macaque host requires ≈24 hours, coinciding with a quotidian fever pattern. However, fever pattern per se may not be a precise indicator for differentiating malaria caused by P. knowlesi and P. malariae. Although the merogony cycle of P. malariae has been generally known to be 72 hours, fever patterns might not be strictly quartan (P. vivax has reportedly conferred partial resistance to induced infection during malariotherapy (P. vivax can reduce symptoms in P. knowlesi infection requires further investigation.The complete asexual erythrocytic cycle of P. knowlesi population. Analysis of the SSU rRNA gene from the isolate in this study has shown minor difference from those of P. knowlesi from monkeys and patients in Malaysian Borneo (,P. knowlesi could lose or increase its virulence on blood passage in humans, which suggests that strain difference could occur in wild populations and might effect humans differently (P. knowlesi could contribute to the reemergence of simian malaria in Thailand and southeast Asia, where its vectors, Anopheles leucosphyrus group, are abundant (To date, little is known about the extent of variation in the