Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here.The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing  P. falciparum and dormant stages of Plasmodium vivax (hypnozoites). Despite being an intrinsic part of most national and international malaria treatment guidelines, the use of primaquine in the treatment of P. vivax as well as P. falciparum is often deferred by attending physicians and healthcare providers. Lingering uncertainty regarding the efficacy and tolerability of primaquine is the most likely reason for this under-utilization. Yet the unique features of primaquine could be critical to tackle three of the most important challenges to malaria therapeutics: 1) The interruption of malaria transmission: despite massive gains made in reducing the burden of malaria in Asia as well as Africa, interruption of the residual transmission has been challenging. Expanding the use of primaquine has been proposed to reduce the transmission potential in both symptomatic patients and asymptomatic parasitaemia. A World Health Organization (WHO) expert committee came to the conclusion that primaquine can be safely used as a single dose (0.25 mg base/kg) even in individuals with G6PD deficiency [P. falciparum strains: one of the greatest threats to current treatment strategies is the emergence and spread of P. falciparum strains resistant to artemisinin derivatives [P. vivax: although current malaria control efforts have been successful in reducing the transmission of falciparum malaria they have had a more limited effect on the transmission of vivax malaria, resulting in a relative increase of the proportion of malaria cases due to P. vivax in areas where effective malaria control programmes have been implemented [P. vivax infections and the difficulties in eliminating the dormant liver stages. To address this problem malaria control programmes need to be able to deploy effective radical cure of P. vivax, to kill both erythrocytic and exo-erythrocytic stages of the parasite. The only drugs currently available with activity against all stages of P. falciparum gametocytes as well as P. vivax hypnozoites are the 8-aminoquinoline drugs, of which only primaquine and the prodrug bulaquine are currently licensed. Bulaquine is produced by Central Drug Research Institute (CDRI), Lucknow and licensed in India only [The 8-aminoquinolines, a class of drugs discovered almost 80 years ago, remains unique among anti-malarial drugs in having activity against the sexual stages (gametocytes) of ficiency . 2) Reduivatives . Artemisivatives -6. Severlemented . This redia only . A long-Despite the therapeutic advantages of the 8-aminoquinolines, the wider use of drugs such as primaquine is restricted by their toxicity profile. The most important adverse effect of 8-aminoquinolines is dose-related haemolysis. In people with an inherited deficiency of glucose-6-phosphate dehydrogenase (G6PD) a single dose of primaquine can trigger a severe haemolysis reaction. The first report of 8-aminoquinolines-related haemolysis was recorded in 1926 following the administration of pamaquine, the earliest available 8-aminoquinoline, also known as plasmoquine or plasmochin: “On the fourth day of treatment, after the fever had disappeared the patient developed profound anaemia, leucocytosis, jaundice, nausea, vomiting, and somnolence. He died within 48 hours of the onset of this sudden attack. The toxic influence of plasmochin compound was suspected to have played an important role in the cause of death” . It tookHaemolysis in individuals with G6PD deficiency has been reported to follow therapy with a range of anti-malarial drugs , sulphones (dapsone), sulphonamides , analgesics , non-sulpha antibiotics , methylene blue and naphthalene . In addiFollowing administration of a standard 14-day course of primaquine (0.25 mg/kg body weight per day) the majority of treated people experience some degree of subclinical haemolysis regardless of G6PD status . The cliThere is little if any information on the safety of primaquine use during pregnancy. Since the unborn child is not accessible to testing there is a risk of exposing a G6PD deficient child to primaquine which at least in theory could result in hydrops fetalis. Women who perceive themselves at risk of being pregnant should not be treated with primaquine. There is a need for pharmacovigilance when primaquine is used in populations which could include pregnant women .The incidence of severe adverse reactions secondary to primaquine administration is difficult to determine since most reports do not provide certainty regarding the numerators or the denominators involved. A major review of the safety data related to primaquine and its predecessors plasmoquine or plasmochin archived by the WHO and the League of Nations detected 13 deaths associated the administration of primaquine. Acknowledging the methodological limitations in estimating the denominator the investigators calculated “the risk of death associated with primaquine ingestion at approximately 1 in 692,307” . The risUncertain about the potential risks of primaquine administration, policymakers and practitioners are often reluctant to put patients at risk of the potential consequences of primaquine toxicity. Few patients will blame their healthcare provider for a malaria relapse months after treatment in contrast to an episode of severe haemolysis immediately following the start of treatment. From the practitioner’s perspective it may appear advantageous to withhold primaquine therapy, particularly if patients are not requesting it.Primaquine toxicity in patients with G6PD deficiency is a significant barrier to the effective delivery of therapies essential to combating the endemic malarias. A more satisfactory approach to the detection and management of malaria patients with G6PD deficiency would unlock a much broader anti-malarial armamentarium. This review discusses the key gaps in understanding the G6PD deficiency problem and provides strategies for its detection and management.The gene encoding the G6PD enzyme is located at the q28 locus on the X-chromosome. As a result, the phenotype due to genetic mutations is more likely to be manifest in males. Heterozygous females display varying levels of enzyme deficiency due to the partial inactivation of one female X chromosome a phenomenon first described by Mary Lyon in 1961 and hence known as lyonisation . HomozygI. Severe deficiency (<10% activity) with chronic (non-spherocytic) haemolytic anaemia;II. Severe deficiency (<10% activity), with intermittent haemolysis;III. Mild deficiency (10-60% activity), haemolysis with stressors only;IV. Non-deficient variant (60 - 100% activity), no clinical sequelae;V. Increased enzyme activity (>100% activity), no clinical sequelae.With the exception of rare sporadic WHO class I individuals, most G6PD-deficient individuals are asymptomatic. One hundred and eighty-six genetic determinants of G6PD deficiency have been described, including point mutations, deletions, and insertions ,23. DiffIt has been estimated that more than 400 million people worldwide are affected by G6PD deficiency . IndividHowes and co-workers have identified 1,734 community G6PD surveys globally of which 1,289 (74%) were conducted in malaria-endemic countries and used this evidence-base to model a continuous prevalence map of the deficiency . The preThe available tests to assess an individual’s G6PD activity status can be broadly divided into genetic and phenotypic/biochemical approaches  and complexity of the G6PD gene makes comprehensive re-sequencing challenging. However, new high-throughput genotyping platforms enable rapid and cost-effective screening of predefined variants in hundreds of individuals . Genotyp® and CareStart®. Phenotypic tests can be divided into four broad categories which are summarised in Table Phenotypic testing of the enzymatic activity of G6PD on freshly collected venous blood remains the most widely used diagnostic method and the most informative indicator of the level of enzyme function . There aDirect tests measure the enzymatic activity of G6PD. The standard measurement of such enzymatic activity remains spectrophotometry [a) otometry . The basotometry . Commercotometry , but theotometry . The preotometry . BesidesIndirect phenotypic tests include the methaemoglobin reduction test (MRT) [b) st (MRT) . After tCytochemical assays assess the G6PD status of individual erythrocytes and hence can be used to detect hemizygous-deficient males, homozygous-deficient females, and heterozygous-deficient females [Cytofluorometric assays can provide valuable data based on individual cells which neither genotyping nor biochemical assays can provide. In practice such methods are limited by the need for extensive laboratory capacity including expensive equipment and skilled staff. These tests are useful in well-equipped laboratories, but are of limited use in field settings.c)  females . Cytoche females ,44. CytoRapid, point-of-care tests allow healthcare providers to diagnose G6PD deficiency in the clinical setting and could influence the case management of vivax malaria. Several tests have been developed; the more recent products approach the ease of the chromatographic malaria tests, which have revolutionized the diagnosis of malaria.d) ®, Japan) is an improvement on Hirono’s method. This is an enzymatic method which utilizes a tetrazolium salt WST8 and a PMS hydrogen carrier, 1-methoxy PMS [The Hirono–1-methoxy PMS Sephadex method requires a substrate mixture  and a mixture of the tetrazolium salt MTT with the phenazine methosulfate (PMS) which are dissolved in water and mixed in a Sephadex gel . While thoxy PMS . Specifi® G6PD test and CareStart® G6PD deficiency screening test, the latter introduced more recently. The BinaxNow® G6PD deficiency screening test is a qualitative enzyme chromatographic test which in a laboratory setting distinguished accurately between samples with G6PD activity less than 4.0 U/g of haemoglobin and those with greater enzyme activity [® G6PD deficiency screening test is a qualitative enzyme chromatographic test based on the reduction of light yellowish tetrazolium dye to purple-coloured formazan. The version of the test used in published studies aims to distinguish samples with G6PD activity less than 2.7 U/g of haemoglobin from those with greater enzyme activity [® G6PD deficiency screening test is considered promising but, like the BinaxNow product, the currently commercially released version of the test needs to be optimized and validated for use in field settings. Upgrades of the CareStart® G6PD deficiency screening test are in development including a version which allows quantitative estimate of G6PD activity using a reader.Currently two chromatographic test are licensed and commercially available: BinaxNowactivity . The tesactivity . The CarThese biochemical approaches offer a functional analysis of G6PD deficiency, but are often difficult to interpret, especially for mosaic female heterozygotes, who may also haemolyse following treatment with 8-aminoquinolines. The interpretation of results of these tests should further take into account the duration of malaria infection, anaemia of the patient and other haematologic parameters. It is possible that an acute febrile episode drives haemolysis. Measured G6PD levels may be reduced during acute malaria episodes. Serial evaluations of G6PD activity during the acute malaria episode and following recovery are on-going in Thailand (see below) and will help define the dynamics of this process. A major challenge for these tests is to ensure their sensitivity in detecting vulnerable populations, choosing an appropriate cut-off value for deficiency and ensuring affordable scale-up in field settings. Neither genetic nor gross biochemical approaches capture the full picture of G6PD activity and hence a combination of assays may be needed for a more complete understanding of G6PD deficiency. Treatment decisions in contrast may be based on rapid diagnostic tests, which are currently in development and will be of critical importance for the safe deployment of primaquine and other 8-aminoquinoline anti-malarials.Testing along the Thai-Burmese border around Mae Sot showed a prevalence of G6PD phenotypic deficiency between 9% and 18% among healthy men of Karen and Burmese ethnicity, using the fluorescent spot test . ApproxiP. falciparum and as such a crucial location for deploying primaquine safely as part of strategies aimed at containing resistant parasites [® G6PD test in this region, suggests a role for this test in the region [Pailin, Cambodia is considered an epicentre of the emergence of multidrug resistant arasites ,53,54. Parasites . Mass trarasites . Althougarasites -59. The arasites . Accordie region .In the Philippines routine screening of newborns for G6PD deficiency using direct tests is part of the national newborn screening programme. Espino and co-workers recently evaluated two assays in Puerto Princesa City, Palawan. The direct fluorescent spot test was compared to the rapid colorimetric test WST-8 in their ability to detect G6PD deficiency in 537 high school students. The investigators found that both assays were difficult to implement in field conditions and were unsuitable for screening in the Philippines. In addition the investigators found discrepancies between test results based on venous compared to capillary blood. The discrepancies may be due to higher haemoglobin levels in capillary blood.® G6PD deficiency screening test in the field setting in Sumba. Possible explanations include the high humidity, high temperature, and an unstable supply of electricity, all of which rendered the quantitative G6PD gold standard subject to likely error.A range of surveys in the Indonesia suggests a prevalence of genotypic G6PD deficiency between 0.6% and 14% ,45,60-70Relatively little is known regarding G6PD deficiency in the Solomon Islands . The WSTThere is a growing awareness that robust and reliable G6PD tests that can be used in remote, malaria-endemic regions are needed. The primary demand for G6PD tests comes from malaria control programmes in vivax-endemic countries, where prolonged and higher doses of primaquine are required for radical cure. A single low-dose primaquine (0.25 mg base/kg) can be safely administered even in individuals with G6PD deficiency and is sufficient to kill gametocytes as mentioned earlier .Besides the malaria control programmes, the pharmaceutical industry is interested in reliable, affordable point-of-care tests for G6PDd to license and safely roll-out new 8-aminoquinolines. Tafenoquine, the putative successor of primaquine, has been in development for over three decades . Being aEqually important for the development and regulatory approval of rapid diagnostic tests to prevent haemolysis may well be the unresolved question of which level of enzyme activity carries an unacceptable risk for severe haemolysis. The absence of a consensus over what represents an unacceptable level of haemolysis is not only the major challenge for the development of tests to diagnose G6PD deficiency but also a critical barrier to the regulatory approval of candidate 8-aminoquinolines.Several institutions and companies are currently involved in developing new generation G6PD testing kits. The Program for Appropriate Technology in Health (PATH), an international, non-profit global health organization, has identified the development of diagnostic tests to detect G6PD deficiency as an urgent priority. Other institutions include the Foundation for Innovative Diagnostics (FIND) as well as the UK Department for International Development (DFID) and the Bill and Melinda Gates Foundation (BMGF) have indicated interest in supporting the development of diagnostics to detect G6PD deficiency.A first step toward the development of a robust clinical test is the description of a target product profile (TPP) of the end product Table . Major cThe broader benefits of a reliable point-of-care test are likely to be considerable, but benefits to individuals and to society need to be weighed against the potential costs and risks of the overall objective. Such an analysis should consider the alternatives to the introduction of testing, including the status quo (in most cases no primaquine and no testing), as well as the introduction of different primaquine treatment regimens without prior G6PD testing. For the latter, the potential benefits of testing in terms of decreased risk of primaquine-induced haemolysis needs to be balanced against the cost of diagnosis and the potential negative consequences of introducing the tests, including those due to poor test accuracy. The potential impact of rolling out primaquine on endemic parasite populations has not been measured, but may be rationally presumed to be very significant given its broad therapeutic range. Reliable, accurate, affordable and easy to use G6PD testing may dramatically improve primaquine therapy by permitting relatively high dose and short duration regimens rather than the current standard regimen of 14 days’ duration. Ultimately, at the societal level the accumulated costs and consequences of continued malaria transmission, morbidity and mortality need be considered in a global economic analysis.The exploration of safe but efficacious doses of 8-aminoquinolines for the radical cure of vivax malaria and the reduction of falciparum transmission has a high priority in global malaria research (see also Table The most relevant phenotype regarding potential primaquine sensitivity is residual G6PD enzyme activity but the evidence that informs any correlation between G6PD and primaquine sensitivity phenotypes remains crude. The first step towards a better understanding is the collection of data on the reduction in enzyme activity that results in a significant risk of haemolysis following administration of a standard regimen of an 8-aminoquinoline. A major challenge for G6PD testing is heterozygous females. Lyonization of the trait renders expression of the mutant gene anywhere between 0% and 100% and is a random event. This can result in unrecognized heterozygous females being at risk for haemolysis. This problem requires careful study, applying cytochemical characterization of the erythrocyte populations of girls and women. There is no consensus on what level of enzymatic activity is required to completely exclude severe haemolysis. The current WHO classification suggests a rather arbitrary cut-off of less than 10% of enzyme activity is indicative of a significant risk of severe haemolysis. Neither is there agreement on what constitutes a clinically significant fall in a patient’s haemoglobin concentration. To understand the fraction of the drop in haemoglobin attributable to 8-aminoquinoline therapy it would be critical to understand the level of haemolysis attributable to uncomplicated malaria without 8-aminoquinoline treatment. For a broader understanding between the genotype, the phenotype, the threshold for haemolysis and what represents an unacceptable risk, clinical observations need to be combined with sequence-based analysis of the G6PD gene, cytofluometric assay and candidate diagnostic tests.The uncertainties regarding the regional distribution of G6PD genotypes suggest a need for phenotypic and genotypic surveys in areas with sparse or outdated data as well as correlates with haemolysis phenotypes. It is not sufficient to conduct such surveillance in selected, established study centres. Specifically, understudied malaria-endemic regions require collection of high-quality data on G6PD deficiency. Current plans to conduct multicentre studies of 8-aminiquinolines could serve as a platform for the in-depth evaluation of G6PD diagnostic kits.P. falciparum the focus of G6PD tests will be the detection of individuals at risk for haemolysis following 8-aminoquinoline therapy targeting hypnozoites.The 8-aminoquinoline doses required to kill the metabolically inactive hypnozoites for the radical cure of vivax malaria are multi-fold higher than doses required to kill gametocytes for the interruption of falciparum malaria transmission . A serieP. vivax burden now look to the scientific community to finally solve the 60-year-old primaquine toxicity problem and provide a more effective chemotherapeutic management of the many species, stages, and clinical manifestations known collectively as the endemic malarias [These strategies target a critically important global health problem: the inability to safely treat a potentially life-threatening infection. The consequences of morbidity and opportunities for the transmission caused by the relapses of vivax malaria have been operationally and scientifically neglected. A better understanding of the risk benefit ratio of deploying or withholding primaquine regimens is crucial for the wider use of 8-aminoquinolines. The Asian nations carrying the bulk of the global malarias ,74.The authors declare that they have no competing interests.LvS and RP organised the workshop. LvS wrote the first draft of the manuscript. All authors contributed to the content, read and approved the final manuscript.


Congenital malaria has been considered a rare event; however, recent reports have shown frequencies ranging from 3% to 54.2% among newborns of mothers who had suffered malaria during pregnancy. There are only a few references concerning the epidemiological impact of this entity in Latin-America and Colombia.The aim of the study was to measure the prevalence of congenital malaria in an endemic Colombian region and to determine some of its characteristics.Plasmodium spp. infection by thick smear from the umbilical cord and peripheral blood, and followed-up weekly during the first 21 days of postnatal life through clinical examinations and thick smears.A prospective, descriptive study was carried out in the mothers who suffered malaria during pregnancy and their newborns. Neonates were clinically evaluated at birth and screened for P. vivax and one by P. falciparum), two in umbilical cord blood and three in newborn peripheral blood. One case was diagnosed at birth and the others during follow-up. Prevalence of congenital infection was 4.3%. One of the infected newborns was severely ill, while the others were asymptomatic and apparently healthy. The mothers of the newborns with congenital malaria had been diagnosed with malaria in the last trimester of pregnancy or during delivery, and also presented placental infection.116 newborns were included in the study and 80 umbilical cord samples were obtained. Five cases of congenital infection were identified (four caused by Congenital malaria may be a frequent event in newborns of mothers who have suffered malaria during pregnancy in Colombia. An association was found between congenital malaria and the diagnosis of malaria in the mother during the last trimester of pregnancy or during delivery, and the presence of placental infection. Plasmodium spp. is usually transmitted by Anopheles spp. mosquitoes, but can also be transmitted from mother to child causing congenital malaria (CM) [Plasmodium spp. are frequent entities in malaria endemic regions and have been extensively studied in Africa, Asia and Oceania; in contrast, there is little information regarding CM. The first studies by Covell and Bruce-Chwatt in the midst of the 20th century reported an incidence of CM between 0.18% and 0.3% in newborns from mothers suffering from malaria during pregnancy in hyper-endemic regions [Plasmodium vivax, one of them with congenital infection based on epidemiological criteria [Most neonatal and perinatal deaths are preventable. According to The State of the World's Children report, published in 2009, 3.7 million children die annually during the first 28 days of postnatal life, 75% during the first week and 36% due to severe infection . Malaria regions , and onl regions . The few regions -15, and  regions . In Colo regions  and therP. vivax [This investigation was carried out in the Caribbean region of Urabá . This region is close to the Panama border, with tropical rainforest ecosystems and a population of 509,409 people, most of them African descendants (51.5%) and under 25 years of age (59.0%). Banana growing and cattle farming are the main economic activities in this region . The stuP. vivax . An inciP. vivax .Plasmodium spp. infection by thick smear at each prenatal visit and during delivery, and/or when they consulted due to a febrile syndrome. At birth, newborns from mothers who had a diagnosis of malaria in pregnancy (MIP) were clinically assessed and thick smears from peripheral blood and umbilical cord were analysed for the presence of the parasite. Weekly clinical and parasitological follow-up was carried out for 21 days in newborns, according to the criteria proposed by Henrys for CM in unstable transmission areas [This was a prospective, descriptive study that was part of a larger epidemiological study on gestational malaria that included 2,117 pregnant women and was carried out from 2005-2007 by the Malaria Group of the Universidad de Antioquia in Colombia. Pregnant women were screened for on areas , such asThe appropriate sample size was calculated  based onCongenital malaria: 1) newborn of a MIP mother with an infection caused by the same Plasmodium species at birth or during the 21-days follow-up period or 2) Plasmodium spp. infection of the umbilical cord [cal cord .Adolescent: under 20 years of age [s of age .Low birth weight (LBW): less than 2,500 g [ 2,500 g .Preterm birth (PB): birth before 37 weeks of gestation [estation .Small for gestational age: below the 10th percentile of weight for gestational age, according to Lubchenco curves [o curves .Gestational age: determined by ultrasound or the first day of the mother's last menstrual period.P. falciparum parasitaemia was calculated counting ring forms, while P. vivax parasitaemia was calculated counting all asexual forms. A sample was considered negative if after the examination of 200 microscopic fields, no parasites were observed or when only P. falciparum gametocytes were observed [capillary puncture was performed and the first drop of blood was discarded; the following drops were used for diagnosis . Thick sobserved .1 mL of blood was extracted from the umbilical vein during delivery . Two thi3 incision was made in a cotyledon close to the umbilical cord, from where placental blood samples were collected [placentas were cleaned with 50 mL of 0.9% saline solution. A 1-cmollected .Newborn data from birth, physical exam and anthropometric measurements were registered in a standardized form (Latin-American Centre for Perinatology) . RegisteStatistical analyses were made with EpiInfo software, version 6.04 . A descriptive analysis was made to summarise and present the information. Median and interquartile range (IQR) or mean and confidence interval were calculated for continuous variables , and absolute frequency and percentages were used for categorical variables. Mann Whitney's test and chi-squared test were used to compare maternal and neonatal characteristics between the newborn groups with and without CM. A significance level of 5% was chosen.The project was approved by the Ethics Committee of the Faculty of Medicine, Universidad de Antioquia. The Colombian Ministry of Health research standards (resolution 008430/1993), the WHO ethical standards for research on human beings and the 2002 Helsinki declaration were taken into account. Informed consent was obtained from all the mothers of the newborns or from their legal guardians.From a total of 220 pregnant women who were diagnosed with MIP during prenatal control, only 157 gave birth in a health facility and were accessible; 116 of them were selected to be included in the study after fulfilling the criteria. The general characteristics of the mothers and newborns included (n = 116) and not included (n = 41) in the study are shown in Table 37.9% of the mothers included in the study were adolescents and 6.4% were over 35 years of age; 21.8% were primigravida, 19.5% reported 3 or more previous pregnancies, and 69.8% had been living in malaria risk areas for 10 years or longer. 91.4% of the pregnant women were attending a prenatal care program with a median of 4 visits, 34.5% reported having at least one episode of malaria in the previous year, and 12.7% had PB; 9.1% of the newborns had LBW and 18.2% were small for gestational age.P. vivax and 30 by P. falciparum) were diagnosed among the 116 mothers . The median for the gestational age at the time of diagnosis of the first malaria episode was 30.1 weeks (IQR: 22.2-35.8). Twenty-seven episodes of malaria were diagnosed in the mothers during delivery, 21 caused by P. vivax and six by P. falciparum; the median parasitaemia was 2,400 parasites/μL .One hundred and sixty-eight episodes of malaria  with a median parasitaemia of 200 parasites/μL (IQR: 80-920). Two mothers who were reported as negative for Plasmodium infection in peripheral blood samples during the prenatal control and delivery, had parasites in the placentas .Ninety-four placental blood samples were obtained from the 116 mothers; 17 (18%) had P. vivax. The 116 newborns were followed-up for 20.5 ± 1.1 days with an average of 2.8 ± 1 postnatal controls per neonate. During follow-up, three (2.6%) neonates were diagnosed with Plasmodium spp. infection; two by P. vivax (one at birth and one on day 14), and one by P. falciparum (on day 8). CM was diagnosed in five cases, for a prevalence of 4.3%, three diagnosed in the 116 newborns and two in the 80 umbilical cords. None of these results were positive in both units of analysis simultaneously.Of the 80 blood samples collected from the umbilical cords at birth, two (2.5%) were positive for There were no significant differences in the characteristics of mothers of newborns with and without CM (p > 0.05) Table . The medth percentile according to the Lubchenco curves.There were no significant differences in the anthropometric characteristics and neonatal adaptability among neonates with and without CM Table . MediansP. falciparum presented clinical and laboratory signs compatible with severe malaria , and required hospitalization in a high complexity health institution in the immediate postpartum period. The mothers of the other CM cases, caused by P. vivax, were classified as having acute non-complicated malaria; three were diagnosed at the time of delivery and had concomitant placental infection, and one was diagnosed in the third trimester of pregnancy during the prenatal control .Newborns with se Table , case 5.P. vivax malaria is the most frequent specie. This study found a prevalence of CM of 4.3% among newborns from mothers with MIP in the Urabá region. Such frequency was similar to that found in Nigeria by Falade et al, who identified a 5.1% parasitaemia in samples from newborns whose mothers had suffered from MIP [et al in Honduras in 2001 [et al in Brazil, who found just one case of CM in 2,781 cases of malaria between 1980 and 1994 in the Campinas region [P. vivax, the Plasmodium responsible for most of the malaria cases in the Americas [CM is one of the least known and studied adverse events of MIP . The prefrom MIP , and to  in 2001 , but gres region . 80%  and of 2.6% (3/116) would have been obtained for malaria positive samples from the umbilical cord and neonatal peripheral blood, respectively.et al and, recently, Mwangoka et al have found in P. falciparum endemic regions that when using thick smears to diagnose malaria from umbilical cord samples, the frequencies range from 0.4% to 6.0%, and when PCR is used the frequencies increase to 20% to 61% for samples taken from the same site [The prevalence of CM reported here was obtained using thick smear as the diagnostic test; however, a greater prevalence might have been found if more sensitive techniques, such as PCR, had been used. Kamwendo ame site ,34.Plasmodium spp. and low socioeconomic level have all been associated with greater CM frequency [Plasmodium trans-placental transmission risk, with a higher infection frequency and placental damage [et al in 2002, highlighted the strong relationship between maternal parasitaemia at birth and positive placental samples with congenital infection [Low parity, fever during the last trimester of gestation, maternal malaria at birth, placental infection by requency ,35,36. Al damage -40. Falanfection .P. vivax and one by P. falciparum, where the mothers had malaria during delivery; placental infection was also found in the three CM cases by P. vivax. The other mother had P. vivax malaria 10 weeks before giving birth. A previous report by our group on neonatal malaria , showed that two of the four positive mothers were in their first or second gestation [The study identified four cases of CM, three by estation ; the preestation ,41, the P. falciparum during pregnancy and at birth, which is explained by a combination of intrauterine growth restriction and PB [P. falciparum weighed less than 2,500 grams and was born at week 30 of gestation. Such PB could have been due to the mother presenting P. falciparum infection at birth. Infection by this species has been associated with a greater risk of PB due to the magnitude of the induced inflammatory response. Many reports have associated LBW with MIP caused by P. falciparum [P. vivax on birth weight. Nosten et al reported an increased risk of LBW associated with P. vivax infection [LBW has been associated with infection by n and PB  in both n and PB . In thislciparum , but onlnfection . The absPlasmodium spp. is difficult and incomplete, so there is little passage of antibodies through the placenta [In low endemic and unstable transmission areas the acquisition of immunity against placenta . In thisplacenta ,18,44. Iplacenta ,46.P. vivax in the same region, in which the newborns presented severe disease [This study location had unstable transmission of malaria, which has thus hampered the induction of effective immunity against the parasite. In contrast to a previous report on cases of neonatal malaria by  disease , in the P. falciparum presented a very serious disease requiring treatment in a neonatal intensive care unit outside the endemic zone. Other conditions including sepsis by Escherichia coli, hyaline membrane disease and PB converged in this patient and can explain the extreme severity of the clinical picture. Research in severe malaria has shown that some cases of extreme clinical severity in both children and adults can be explained by co-infection with bacterial agents [The newborn with CM by l agents  which, el agents ,49.1) CM is a frequent event in newborns of mothers who have had malaria during pregnancy in a coastal region of Colombia;2) The main maternal characteristics in these cases of CM were having malaria during the last trimester or during delivery, and placental infection.The authors declare that they have no competing interests.JGPJ: conceiving and designing the research proposal, analysing the data and writing the manuscript; GA: analysing the data and writing the manuscript; AT: designing the investigation, coordinating the field research and critically reviewing the manuscript; MA: designing the research, acquiring the data and critically reading the manuscript; SC: acquiring the data and critically reading the manuscript; SB: conceiving and designing the research proposal, interpreting the data and writing the manuscript.All authors read and approved the final manuscript.


Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) and to determine the multiplicity of infection in clinical isolates collected from children living in the Southern district of Brazzaville in the Republic of Congo.The characterization of malaria parasite populations circulating in an area is part of site characterization, as a basis for evaluating the impact of malaria interventions on genetic diversity, parasite species, and multiplicity of infection. The present study was aimed at analysing genetic diversity of msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR.A total of 125 isolates from patients with uncomplicated malaria attending Terinkyo and Madibou health centres were collected between January and June 2005 while evaluating the therapeutic efficacy of amodiaquine-artesunate combination. DNA was extracted and msp-1 and 20 msp-2 genotypes were identified. For the msp-1 gene, K1 family was the predominant allelic type carried alone or in association with RO33 and Mad20 types, whereas the 3D7 family was the most prevalent in the msp-2 gene. Overall, the mean multiplicity of infection was 2.2. Out of 125 samples, 104 (83%) harboured more than one parasite genotype. There was no statistical significant difference in the multiplicity of infection by either sex or age of patients. However, a statistically significant correlation was found between parasite densities and the number of genotypes.Out of 468 distinct fragments detected, 15 P. falciparum clinical isolates from Brazzaville was high and mainly of multiple clones. The basis for the positive association between parasite densities and multiplicity of infection is discussed.Polymorphism in  Plasmodium falciparum, is a major public health problem. The global burden is estimated at 225 million malaria cases every year resulting into 781,000 deaths [Malaria, a disease mostly caused by Data collected from 2000 to 2003 by the University Teaching Hospital of Brazzaville during studies on chloroquine resistance showed that 22% of children deaths at pediatric health facilities were due to malaria -6. ConseP. falciparum is the extensive genetic diversity in parasite populations limiting the efficacy of acquired protective immunity to malaria [P. falciparum genotypes per infected individual.Despite policy change, there is still need to collect more data on the epidemiology of malaria in the country to facilitate the development and implementation of other malaria control interventions such as the search for alternative drugs, vaccine trials and indoor residual spraying. In this regards, characterization of malaria parasite populations circulating in an area is part of site characterization before any intervention, as a basis for evaluating the impact of the intervention on genetic diversity, parasite species, and multiplicity of infection (MOI) in the human or vector host. One of the limitations to the development of an effective malaria vaccine against  malaria . MOI and malaria -11. In tP. falciparum parasite sub-populations [msp-1 and msp-2 has been reported in several parts of world including in Central Africa [P. falciparum populations from the Republic of Congo.Asexual blood stages antigens, such as merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) are considered prime candidates for the development of malaria vaccine and are also suitable markers for the identification of genetically distinct ulations . Polymorl Africa -15. HoweP. falciparum genes mutations associated with anti-malarial drugs resistance, such as those found in P. falciparum chloroquine resistance transporter, dihydrofolatereductase, dihydropteroate synthase have been conducted in the Republic of Congo since 2003, before introducing artemisinin-based combination treatment (ACT) in 2006 [P. falciparum infections and MOI in a group of children with uncomplicated malaria in Brazzaville [Molecular studies to identify  in 2006 ,16,17. Bzzaville . Followimsp-1 and msp-2 and determined the multiplicity of infection in clinical P. falciparum isolates collected from children living in the southern district of Brazzaville, the capital of the Republic of Congo. The Ministry of Public Health of the Republic of Congo approved the study ethically and scientifically.The present characterized allelic polymorphism of P. falciparum.The study materials were collected from patients attending Centre de Santé Intégré (CSI) in Terinkyo and Madibou hospitals, located in the southern part of Brazzaville. This urban area has stable and highly endemic malaria with perennial transmission  with an P. falciparum infected blood used in this study were collected from children and adults presenting to the two health centres and enrolled into the study for evaluating the therapeutic efficacy of artesunate-amodiaquine combination  from February to June 2005. The inclusion criteria of participants were: auxiliary temperature ≥ 37.5°C measured with an electronic thermometer, malaria parasite  in the thick blood smears (≥ 800 parasites/μL), no symptoms of severe malaria or febrile conditions caused by other diseases. Informed consent was obtained from adult patients and from parents/guardians for the children. Additionally, at inclusion into the study and before treatment, three drops of blood from each patient were blotted on to 3 MM Whatman filter paper, dried and stored in individual sealed envelopes until used for DNA extraction.A total of 125 Genomic DNA was extracted from blood samples collected on filter paper using QiaAmp DNA mini kit  according to the manufacturer's instruction. Then, the DNA was recovered in 100 μl of elution buffer from the kit. All parasites DNA extracted were stored at - 20°C until use.P. falciparum using the nested polymerase chain reactions (PCRs) technique. The highly polymorphic loci, msp-1 block 2 and msp-2 central region were used as markers for this genotyping as described previously [msp-1, and for FC27 and 3D7 allelic families of msp-2. Allelic specific positive controls and DNA free negative controls were included in each set of reaction. Five microliters of each of the PCR products were loaded on 2% agarose gel, stained with the Syber Green, separated by electrophoresis and visualized under UV trans-illumination. Individual alleles were identified by fragment length and by the corresponding allele-specific primers used and the size of the PCR products were estimated using a 100 bp DNA ladder marker . The size polymorphism in each allelic family was analysed; assuming that one band represented one amplified PCR fragment derived from a single copy of P. falciparum msp-1 or msp-2 genes. Alleles in each family were considered the same if fragment size were within 20 bp interval. The minimum number of genotypes per isolate was estimated to be the highest number of fragments identified for either msp-1 or msp-2.All samples were genotyped for eviously -13. The P. falciparum, all samples were subjected to Plasmodium species genotyping as described elsewhere [Plasmodium malariae and Plasmodium ovale species were investigated; P. falciparum being already done by msp-1 and msp-2 genotyping. The PCR products were loaded on 2% agarose gels and visualized under UV trans-illumination. The DNA positive controls for P. malariae and P. ovale as well as DNA free negative controls were included in each set of reaction.To confirm that all study participants were infected with only lsewhere . At the msp-1 and msp-2 allele frequency was calculated as the proportion of allele found for the allelic family out of the alleles detected in isolates. The multiplicity of infection was defined as the minimum number of P. falciparum genotypes per infected subject and estimated by dividing the number of amplified PCR fragments reflecting parasite genotypes by the number of positive samples. The chi-square test was used to compare proportions. Spearman's rank correlation coefficients were calculated to assess association between MOI and geometric mean parasite densities and age. Statistical significance was defined as P values < 0.05.The P. falciparum infection were enrolled in the study. Sixty-seven and 48 were children aged between seven months to five years of age, and between six to 15 years of age, respectively; ten patients were more than 16 years old. Sixty-eight patients were males and 57 were females.A total of 125 patients with clinical symptoms of malaria and microscopically confirmed The geometric mean parasite density was 62,170 parasites/μl of blood with the range of 830-914,000 parasites/μl  and Mad20 (29%) families, respectively. The RO33 (23%) family did not show any polymorphism, with only one variant (180 bp) detected. The K1 family was the predominant allelic type carried alone (23.4%) or in mixed infection with RO33 (17%) and Mad20 (20%) types as well as when the tree types are observed in the same isolates  of the isolates carried both msp-2 allelic types. Ten genotypes (5%) could not be assigned to any family.The msp-1 and msp-2 genes separately, the overall multiplicity of infection was 2 and 1.7 respectively, while eighty-nine (72%) and 67 (54%) of isolates carried more than one parasite genotype respectively. As presented on the Tables msp-1 or msp-2 alleles did not show any impact on the parasite density. Moreover, no statistical significant difference was observed in the multiplicity of infection and parasite density according to the age . The correlation was higher when msp-1 and msp-2 alleles are taken together , since the number of genotypes per isolate was calculated as the highest number of fragments for either msp-1 or msp-2  harboured more than one parasite genotype. When considering % harbourP. falciparum populations in an area is likely to affect acquisition of immunity to malaria [The level of antigenic diversity of  malaria . TherefoP. falciparum msp-1 and msp-2 genotyping has shown that malaria parasite population in Brazzaville is highly diverse. However, the number of alleles may have been underestimated due to the limit of the technique. Indeed, fragments within the length interval of less than 20 bp could not clearly be distinguished. Thus, a minimum of fifteen alleles of msp-1 were observed, in which, K1 allelic family was predominant, consistent with the previous study in the same locality and others studies in the central Africa sub-region [msp-2 locus, alleles belonging to 3D7 family were mostly detected, both in mono-infection and mixed infection with FC27 alleles. This finding differs from those of the previous study [P. falciparum malaria in Gabon [Allelic specific b-region ,13,22,23us study , where Rus study , Brazil us study  and in Ius study . Three aP. falciparum infections were detected in 83% of Congolese isolates with an overall mean multiplicity of infection of 2.2 (with 2 and 1.7 in msp-1 and msp-2 respectively). This corroborates reports from other areas of Central Africa with intense malaria transmission [Multiple genotypes of smission ,28. In tsmission . IrrespeP. falciparum infection in this symptomatic population shows a significant correlation between parasite density and the number of parasite genotype. This suggests that symptomatic P. falciparum infection in this population is associated with the presence and growth of recently inoculated parasite strain(s) to which individuals have either not previously been exposed to or have not fully developed immunity [msp-1 and msp-2 genes appeared polymorphic, and both taken together, the relation between parasite density and multiplicity of infection become more pronounced. This finding supposed that in area with high transmission where individuals are exposed to several different clones, disease-inducing strains may have similar growing capacity leading to the increase of their parasite densities. Consequently, with the technique used, the high density may increase the probability to distinguish more clones in the complex infections.However, in contrast with the first report in Brazzaville , P. falcimmunity ,30. Thisimmunity  and in Mimmunity . The mspDespite the lack of recent entomological data from Brazzaville, the latest estimation being in 1985 , the genP. falciparum + P. malariae and P. falciparum + P. ovale, confirming high sensitivity of molecular tools in detecting sub-microscopic infection.With regard to malaria speciation, two isolates had mixed infections of P. falciparum genes associated with anti-malarial drug resistance would be useful developments.The description of malaria parasite population remains a priority malaria research area in the Republic of Congo for a better understanding and evaluation of on-going malaria control interventions. This study was conducted before the implementation of the new policy for the treatment of uncomplicated malaria using ACT. Assessment of the impact of ACT on malaria parasite population and characterization of mutations in The authors declare that they have no competing interests.PIM and FVM conducted molecular genetic analysis. The four authors participated in the design of the study, analysis and interpretation of the results, as well as in preparing the manuscript. All authors read and approved the final manuscript.


Malaria in the western Kenya highlands is characterized by unstable and high transmission variability which results in epidemics during periods of suitable climatic conditions. The sensitivity of a site to malaria epidemics depends on the level of immunity of the human population. This study examined how terrain in the highlands affects exposure and sensitivity of a site to malaria.The study was conducted in five sites in the western Kenya highlands, two U-shaped valleys , two V-shaped valleys  and one plateau (Shikondi) for 16 months among 6-15 years old children. Exposure to malaria was tested using circum-sporozoite protein (CSP) and merozoite surface protein (MSP) immunochromatographic antibody tests; malaria infections were tested by microscopic examination of thick and thin smears, the children's homes were georeferenced using a global positioning system. Paired t-test was used to compare the mean prevalence rates of the sites, K-function was use to determine if the clustering of malaria infections was significant.The mean antibody prevalence was 22.6% in Iguhu, 24% in Emutete, 11.5% in Shikondi, 8.3% in Fort-Ternan and 9.3% in Marani. The mean malaria infection prevalence was 23.3% in Iguhu, 21.9% in Emutete, 4.7% in Shikondi, 2.9% in Fort-Ternan and 2.4% in Marani. There was a significant difference in the antibodies and malaria infection prevalence between the two valley systems, and between the two valley systems and the plateau (P < 0.05). There was no significant difference in the antibodies and malaria infection prevalence in the two U-shaped valleys (Iguhu and Emutete) and in the V-shaped valleys (Marani and Fort Ternan) (P > 0.05). There was 8.5- fold and a 2-fold greater parasite and antibody prevalence respectively, in the U-shaped compared to the V-shaped valleys. The plateau antibody and parasite prevalence was similar to that of the V-shaped valleys. There was clustering of malaria antibodies and infections around flat areas in the U-shaped valleys, the infections were randomly distributed in the V-shaped valleys and less clustered at the plateau.This study showed that the V-shaped ecosystems have very low malaria prevalence and few individuals with an immune response to two major malaria antigens and they can be considered as epidemic hotspots. These populations are at higher risk of severe forms of malaria during hyper-transmission seasons. The plateau ecosystem has a similar infection and immune response to the V-shaped ecosystems. The U-shaped ecosystems are transmission hotspots. Malaria epidemics have occurred in the highlands of western Kenya since the late 1980s, often resulting in high morbidity and mortality . Apart fThis study was conducted in five sites, two U-shaped valleys (Iguhu and Emutete), two V-shaped valleys (Marani and Fort Ternan) and one plateau (Shikondi). Iguhu  is located in Kakamega district, western Kenya. The slow flowing river Yala runs through the flood prone flat valley. Emutete village  is located in Emuhaya district in Western Kenya. The area is characterized by large flat bottomed valley with slow running streams. Marani  is located on the highland plateau adjacent to the Lake Victoria Basin and 17 km north of Kisii town. The fast flowing Marani river runs along the shallow gorge. Fort Ternan  is located in Kericho District. River Kipchorian bisects the hills and runs a long a deep gorge. Shikondi  is a plateau located 4 Km from Iguhu in Kakamega district, western Kenya  who had given consent to participate in the study. The homes of the 600 children were geo-referenced, mapped, and tested for clustering. The human population density in all the study sites is very similar and so equal numbers of children were allocated to each site. Of the final sample size of 425 children, each site was allocated 85 children.Plasmodium falciparum surveys. The cohorts were followed monthly for 16 months (from May 2009- August 2010). Before the children were allowed to participate in the study, consent was obtained from their parents or guardians. Children aged between 6-15 years residing in the study sites and with no reported chronic illness, except malaria, were allowed to participate in the study. Children who were found having fever at the time of sampling were taken to the nearest government clinic for treatment.Cohorts of 170 children in the two U-shaped valleys, 170 children in the two V-shaped valleys and 85 children at the plateau aged 6-15 years were recruited for monthly P. falciparum capture antigens  on the test band P. falciparum region and with recombinant malaria P.vivax capture antigens  on the test band P.vivax region. The recombinant malaria Plasmodium vivax (P.v)./Plasmodium falciparum (p.f). antigen  -colloidal gold conjugate and serum sample moves along the membrane chromatographically to the test region  and forms a visible line as antibody-antigen gold particle complex forms with 91.3% sensitivity and 98.5% specificity. The control line is used for procedural control. Blood samples were collected by a standard finger prick and drawn into capillary tubes, then centrifuged at 1,800 revolutions per minutes for 5 minutes to obtain the serum. After the test kit had been brought to room temperature, a drop of serum was added in to the sample window and allowed to soak in, and then two drops of the diluent were added into the sample window. The positive results were read after 10 minutes and the negative results confirmed after 20 minutes as indicated by the kit manufacturer.The SCIMEDX  rapid diagnostic antibody test kit contains a membrane strip, which is pre-coated with recombinant malaria Blood samples were collected by the standard finger-prick method and thick and thin smears prepared on labeled slides. The smears were allowed to air dry then fixed in methanol and stained in 4% Giemsa for 30 min. The stained smears were then examined using the magnification of × 1,000 (oil immersion) to identify and count the parasite species. Random checks were carried out on the slide counts by independent microscopists to ensure quality control. Parasite density was scored against 300 leukocytes in positive slides. Parasite densities were converted to number of parasites per microliter of blood, assuming a leukocyte count of 8,000 cells/μl [The homes of the participants were geo-referenced using a global positioning system (GPS) unit. A database was created with participants' names, ages, GPS location, and malaria parasite infection results. Participants' names were coded for confidentiality. ArcView 3.3  was used to create spatial-distribution maps of infected and uninfected participants.Mean monthly rainfall, maximum and minimum temperature for the study sites was obtained from the Kenya Department of Meteorology.Plasmodium falciparum infections were tested for clustering by household with the global spatial statistic, the K function, weighted by parasite density [L(d), examines the spatial dependence of malaria infection by household over a wide range of scales of pattern [L(d) function values were tested against the null hypothesis that the spatial distribution of all infected residents in the study area was random, by using 1,000 Monte Carlo iterations. The global spatial cluster analysis was conducted separately for the five sites.Site specific prevalence rates were determined by expressing positive blood smears as a percentage of the total blood smears examined. Only positive slides were considered when the geometric mean parasite density was calculated for each site. The paired t-test was used to determine differences of the prevalence of antibody and malaria parasite infections among the sites. To determine spatial distribution patterns of malaria infections in the study area, ArcView 3.3  was used to create Spatial-distribution maps of infected and uninfected participants for the 5 sites.  density -11 by us pattern . The obsScientific and ethical clearance was given by Kenya Medical Research Institute. Inclusion criteria were provision of informed consent, age >5 years at recruitment, and no reported chronic or acute illness, except malaria.Rainfall among the three districts, Kakamega, Kisii and Kericho were not statistically different. However Kisii received 435 mm more rainfall than Kakamega Table . The meaThus rainfall and mean annual temperatures were not significantly different between Kakamega and Kisii districts.P. falciparum prevalences during the 16 months study period were Iguhu, 23.3%, Emutete 21.9%, Marani 2.4% Fort Ternan 2.9% and Shikondi 4.7%  and so was the difference between the U-shaped valley populations and Shikondi the plateau (p << 0.05 t = 10.98). Seasonal variation in parasite prevalence was observed in populations living in the U- shaped valleys and was responsive to rainfall. Above normal El Niño rains were experienced in December 2009 which is normally a dry period ; p > 0.5 t = -1.678  was not significantly different (p > 0.05 t = -0.765), but was significantly different between the U-shaped valleys and V-shaped valley (Fort Ternan) (p < 0.05 t = 4.064) and between the U-shaped valleys and the plateau, Shikondi (p < 0.05 t = 5.058). However the prevalence between the V-shaped (Fort Ternan) and the plateau, Shikondi was not significantly different  Table .2 = 0.994; p = 0.00191) , and between the plateau and U-shaped valleys . There was no significant difference in antibody prevalence in the two U-shaped valleys (Iguhu and Emutete) (p > 0.05 t = -0.346), in the two V-shaped valleys (Fort Ternan and Marani) (p > 0.05 t = 0.352) and between the plateau (Shikondi) and the V-shaped valleys . An increase in antibody prevalence levels was observed after the rains in September 2009 and these declined rapidly in March 2010  Figure  at the p) Figure .L (d), was used to examine the spatial distribution of malaria infections by household over an interpoint distance of 100-1,400 m for all the sites. Figure L (d) and the 95% CI plotted for various values of interpoint distance for the surveys in Iguhu and Emutete respectively. The spatial distribution of infections was considered evenly dispersed if the observed K function values were below the lower limit of the 95% CI, clustered if above the upper limit, or random if within the 95% CI. The weighted K function indicated that the malaria infection distribution pattern was significantly different than expected under complete spatial randomness in the U-shaped valley but was random in the V-shaped valleys and the plateau  while the spatial variation is a function of the terrain characteristics. Furthermore these two drivers of malaria transmission have a direct impact on the development of an immune response to malaria infections. In this study we examine how the terrain characteristics are associated with prevalence levels of asexual and sexual stages of P. falciparum malaria spatial distribution of infections and the immune response. Knowledge gained has contributed towards differentiation of malaria epidemic and transmission hotspots which will further lead to a better understanding of the evolution of malaria epidemics.Temporal variation of P. falciparum malaria parasite prevalences, immune response and spatial distribution of malaria infections. The U-shaped valleys system had 8.5-fold more malaria infections and 2.6-fold greater prevalence of antibodies compared to the V- shaped valleys systems. This observation has implications in terms of predicting the outcome of weather-driven malaria epidemics and their prevention and control. In the highlands, most severe malaria cases during an epidemic come from human populations that have not been regularly exposed to malaria infections [Rainfall was similar among all the sites and the mean annual temperatures were not significantly different between Kakamega (Iguhu) and Kisii (Marani) which have U and V-shaped valleys respectively. Despite having similar weather, the two sites had very different fections . Physicafections . Thus whThe early malaria epidemic prediction model developed by Githeko and Ndegwa  detects During late 2009, El Niño type of rains started in September and continued to December. An increase in the proportion of children with antibodies increased by 24.2% in the U-shaped valleys system and this was associated with a 13.4% decrease in malaria prevalence in December. In the V-shaped valleys system an increase of 20.8% in the prevalence of antibodies was associated with only 1.7% decrease in malaria prevalence in November. These observations suggest that the immune response in the U-shaped valley systems may be more robust than that of the V-shaped valley systems. Besides a higher prevalence in malaria infections in the U-shaped valleys system, children were infected 3-fold more frequently than in the V-shaped valley system which may contribute to a better development of an immune response to malaria. Our results also showed a high positive correlation between site-specific mean antibodies and site specific mean parasite prevalence rates; this indicates that the site-specific malaria infection prevalence is predictive of the site-specific prevalence of CSP and MSP antibodies. While this relationship was strong at the population level, the relationship at the individual level was much less obvious and could have been affected by the sensitivity of kit, antigenic polymorphism and delayed immune response. Other studies have shown a log-linear relationship between exposure and antibodies prevalence predicted by mathematical models assuming exposure-dependent immunity . The logThe high gametocytes prevalence rates in the U-shaped valleys compared with the V- shaped valleys indicate that the reservoir of malaria infections in the U-shaped valleys is higher than in the V-shaped valleys and the plateau. While the population living in the U-shaped valley maintains a large reservoir of infectious gametocytes, the people living in the V-shaped valley comprise a high proportion of susceptible individuals. Under permissive climatic conditions the infectious vector population could increase, leading to higher rates of malaria prevalence. Earlier studies indicated that the mean gametocyte prevalence in Kericho district was 1.8% , 2.8% inSpatial analysis of the malaria antibodies and infections indicated that there was a significant positive clustering of malaria infections in the flat bottomed U-shaped valleys resulting from the high availability and stability of vector breeding habitats. In contrast malaria infections were randomly distributed in the V-shaped valleys, and this can be explained by the fact that these valley systems are characterized by fast running rivers at the valley bottoms, and steep slopes that provide good drainage in the area and so there are few vector breeding habitats in these ecosystems. We also observed less clustering in the plateau though it was not statistically significant. Earlier studies in the same area indicated that malaria was mesoendemic at the plateau at 26.7% prevalence . Our resOngoing studies in the same areas indicate that the V-shaped valley ecosystems require anomalously high temperatures and rainfall over an extended period for epidemics to occur. These epidemics are defined by the numbers of people infected, severity of the disease and mortality. In Kericho district Western Kenya out of 254 malaria deaths 31% were due to cerebral malaria, 37% severe anemia and 32% due to malaria with gastroenteritis or pneumonia . It is iAs a strategy for epidemic prevention special attention should be focused on the V-shaped valley and plateau ecosystems as they constitute epidemic hotspots. It is likely that malaria could be eliminated in the current epidemic hotspots however the current transmission hotspots may convert to epidemic hotspots as the current malaria control efforts reduce transmission.This study has provided evidence that the V-shaped ecosystems have very low malaria prevalence and few individuals with an immune response to two major malaria antigens. This ecosystem can be regarded as an epidemic hotspot. These populations are a higher risk of severe forms of malaria during periods that support hyper-transmission of the parasites. When predicting malaria epidemics this factor should be taken into consideration. Plateaus with water bodies may also behave like the V-shaped valleys. It is critical that ecosystems sensitivities are taken into account in predicting and controlling malaria.The authors declare that they have no competing interests.CLW participated in the study design, supervision of the field work, data analysis and drafted the manuscript. AKG designed the study, extracted the rainfall and temperature data from the metrological stations contributed to writing the manuscript, GZ did the mapping of the study sites and spatial analysis, JW participated in the designing of the study and contributed to writing of the manuscript. All authors have read and approved the final manuscript.


Plasmodium vivax mono-infection, which was confirmed by molecular methods. A serological panel with the most prevalent infectious agents known to cause myocarditis was performed, and specific anti-cytomegalovirus (CMV) IgM and elevated levels of anti-CMV IgG were also detected in the serum. After treatment for malaria, there was improvement of respiratory distress, although cardiac function did not recover. She was discharged home with drugs for cardiac insufficiency and is currently under follow-up with a paediatric cardiologist as an outpatient. This report presents a young child with several episodes of vivax malaria who suffers from cardiac insufficiency, probably related to CMV-induced myocarditis.A child living in the Brazilian Amazon region who had had vivax malaria at the age of 11 months was admitted three months later with a history of progressive dyspnoea and fever, which culminated in respiratory distress and severe dilated cardiomyopathy at hospital admission in a malaria-free area. She received treatment for cardiac insufficiency and was tested for malaria with two thick blood smears, which were negative. There was general improvement of cardiorespiratory function in the next two weeks, but in the third week of hospital admission, there was re-appearance of fever, severe anaemia, severe plaquetopaenia, and respiratory distress. A third thick blood smear was positive for  Vivax malaria is an important disease responsible for up to 400 million infections in endemic areas of Asia, Latin America, and some parts of Africa. In recent years, it has been shown that this so-called benign malaria can result in severe presentation with multiple organ dysfunction and life-threatening disease. The majority of severe vivax malaria cases have been reported in India -4, but mVivax malaria is an important morbidity in children living in countries with endemic transmission. At the same time, children are also susceptible to other infections, such as viral diseases, because they still lack specific immunity to most of the pathogens in their first years of life, and co-morbidities can occur, especially in underdeveloped and developing regions, such as the Amazon.This is the report of a case of acute myocarditis and dilated cardiomyopathy associated with cytomegalovirus (CMV) infection and vivax malaria in an infant residing in the Brazilian Amazon region.th, 2013 with a complaint of shortness of breath for approximately 30 days prior to the admission. Her parents referred to productive cough and fever in the preceding days. Her medical history (referred by parents) included a three-day hospital admission at the age of 11 months (June 2013) for respiratory symptoms, which were considered to be ‘pneumonia', according to parents report. Since she had fever and lived in an area of malaria transmission, she was tested for malaria. Malaria records showed that she was diagnosed with vivax malaria, confirmed by microscopy. Malaria treatment was initiated with oral chloroquine (25 mg/kg over three days) and primaquine (0.5 mg/kg/day over seven days) while she was in the hospital, and primaquine treatment was completed later at home, according to parents’ report. She had been born full-term, 3,695 kg in weight and 50 cm in length, from caesarean surgery. Her mother denied attending prenatal care during pregnancy.A 13-month old girl living in the rural area of Cruzeiro do Sul, in Acre state  was admitted to a local hospital facility on September 9th. According to the protocol stipulated by the Ministry of Health, 100 high power fields were examined before a negative diagnosis was issued. Unfortunately, rapid diagnostic tests for malaria were not available at the hospital at this time.At hospital admission, she had signs of respiratory distress on physical examination, with wheezing, crackles, and nasal flare. Heart rate was 129 bpm, respiratory rate was 26 ipm. She measured 72 cm in length and her weight was 9 kg. There was mild hepatomegaly. During the next three days, her heart rate escalated to 162 bpm, respiratory frequency varied between 26 and 50 bpm, and axillary temperature ranged from 37 to 37.8°C. Based on signs of respiratory distress and productive cough, there was an initial diagnosis of pneumonia and she was started on ceftriaxone on the day of admission. The electrocardiogram showed sinus rhythm, left ventricular overload and left atrial overload  and a regular tachycardic heartbeat (140 bpm), with hypophonetic heart sounds. The first echocardiogram, performed on September 14th, showed dilated cardiomyopathy with severe systolic dysfunction: mildly dilated left atrium (27.2 mm), severely dilated left ventricle (60 × 50 mm), ejection fraction of 35%, severe mitral insufficiency, mild aortic valve insufficiency, and no signs of cardiac malformation. Laboratory examinations showed normal erythrocyte sedimentation rate, negative C-reactive protein test, normal CPK , increased value of CK-mB , AST 42 U/l, ALT 21 U/l. A haemogram revealed anaemia (haemoglobin 9.69 g/dL) and mild leukocytosis , with 57% of granulocytes and 46% of lymphocytes, with normal platelet count . Another blood thick smear for malaria was performed on September 13rd, using the same criteria described before, which was also considered to be negative. She continued to receive ceftriaxone, and she was started on carvedilol, digoxin, furosemide, captopril, spironolactone, and oxacilin.Because of her critical conditions, she was referred to a larger hospital in another city (Rio Branco) on September 13rd and 17th she remained in stable condition with gradual decrease of heart rate (from 130 to 100 bpm) and respiratory rate (from 50 to 28 ipm). There was also disappearance of other clinical signs of respiratory distress.Between September 13th, she had diarrhoea with liquid stools which lasted nine days, with up to seven evacuations per day, accompanied by low-grade fever (37.2-37.6°C). On 19 September, the CKmB levels returned to normal (19.20 U/l). Ceftriaxone was terminated on September 20th but the patient continued to present low-grade fever in the next days.On September 17rd, because fever did not cease and clinical conditions were getting worse day by day even after a full course of Ceftriaxone, making the clinicians very worrisome about her clinical status Diarrhoea ceased after three days of vancomycin, but axillary temperature continued to raise and reached 38.5°C despite antibiotic administration. On September 29th, she was started on dipirone every six hours; haemoglobin values were 8.3 g/dL. OnOctober 3rd haemoglobin values had decreased to 6.6 g/dL, when she received a blood transfusion because of the acute drop in haemoglobin levels. There was worsening of dyspnoea (from 30 to 42 ipm) and tachycardia (from 110 to 124 bpm), and on October 4th, another thick blood smear for malaria was performed, which was positive for P. vivax, with approximately 10,000 parasites/mm3. Molecular diagnosis of Plasmodium species was performed using nested polymerase chain reaction [P. vivax , and six days later, IgM titres increased to 9.34 (cut-off > = 1.0). Laboratory investigation for other infectious agents  were negative on two separate samples (October 4th and October 10th). It was decided not to use gancyclovir because there was no evidence of possible clinical benefit at this point and since this drug can cause bone marrow toxicity, it may have exacerbated the anaemia and thrombocytopaenia if it had been used [A panel of serologic tests for the most common infectious agents known to cause myocarditis in children was performed. Anti-CMV antibodies were detected on October 4een used .th) and dyspnoea improved from 7th October onwards. There was a progressive significant clinical improvement with disappearance of respiratory distress between October 7th and 14th. Despite this clinical improvement, cardiac echocardiogram remained mostly unchanged. A second echocardiogram performed on October 1st showed dilated cardiomyopathy with severe systolic dysfunction, increased dilation of left atrium (31.9 cm), severe distension of left ventricle (48 cm × 39.5 cm), ejection fraction of 36% and severe mitral valve insufficiency. CMV IgM was 1.44 on October 25th. The third echocardiogram performed on November 1st showed severe dilation of left atrium , severe dilation of left ventricle , moderate mitral valve insufficiency secondary to ventricle dilation, severe systolic dysfunction with an ejection fraction of 41% and myocardial performance index of 1.03 (defined as the sum of isovolumetric contraction and relaxation time divided by the ejection time) [This one-year old child had a vivax malaria episode in June and another episode in October. Since she had a prolonged admission stay (23 days) with two negative thick blood smears and there is no malaria transmission in Rio Branco , it is highly possible that the patent parasitaemia detecting on 4 October, and the preceding seven days of fever, were the result of hypnozoite reactivation. Vivax malaria treatment was administered orally and it is possible that treatment with primaquine at home was not adequate in June, which would favour early relapse when facing a systemic disease . It is let al. estimatet women) .th, on the other hand, is directly related to the severity of vivax malaria [rd, one day before she had a positive thick smear form malaria. Although it is not a correlate of severe disease, lymphopaenia is frequent in malaria patients [It is described that repeated episodes of malaria can cause the removal of both infected and uninfected red blood cells, which could explain the anaemia observed at the second hospital admission, together with some degree of iron deficiency caused by low intake. The substantial drop of haemoglobin levels around October 4 malaria , and canpatients .Clostridium difficile-associated diarrhoea. Clostridium difficile diarrhoea is the most common cause of diarrhoea associated with antibiotic usage, and the most common symptoms in children are liquid stools that resolve in 30 days, while adults tend to have pseudomembranous colitis much more frequently [rd showed increased leucocyte count with increased segmented neutrophils, supporting the hypothesis of an antibiotic-associated C. difficile diarrhoea [The aetiology of diarrhoea was not formally investigated, but it is possible that she had an antibiotics-associated colitis, or even equently . This chiarrhoea .Technical limitations that are common in the Amazonian health care system prevented a more detailed investigation of the case. Malaria smears should have been performed earlier and more often during the course of admission in this case, and maybe this would have prevented the acute drops in haemoglobin levels and platelet numbers, and the worsening of cardiorespiratory function. The aetiology of diarrhoea was not properly investigated either. Social conditions will make the follow-up of this child with cardiac insufficiency more difficult, since at her home area there is no cardiologic health care available. It is worth pointing out that in malaria-endemic settings, socio-economic inequalities can facilitate the occurrence of co-morbidities and at the same time may hinder proper health care.This case portrays the concomitance of two important infectious diseases in a young child. While CMV infection is a cosmopolitan infectious agent, vivax malaria is a disease associated with social inequities, and should be investigated in all patients with similar settings. Health care provided to this patient at the beginning of clinical course was limited because of inherent health care access in the Amazon, but despite that, medical efforts were able to keep her health condition stable.Written informed consent was obtained from the parents of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare they have no competing interests.Plasmodium infection. MCVR and RBR performed the cardiological evaluation and diagnosis and provided final health care to the patient. MdaSN participated as an infectious disease consultant expert. ACM and MdaSN conceived the case report, participated in its design and coordination and wrote the manuscript. MCVR and RBR helped to draft the manuscript. JBL, LMNS, TCM, LNF, and RSM revised the manuscript critically for important intellectual and clinical content. All authors approved the final manuscript.ACM, JBL, LMNS, and TCM provided clinical care to the patient and pursued the initial clinical investigation, interpretation of exams and malaria diagnosis. LNF and RSM carried out the molecular diagnosis of


GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative are working in partnership to develop a malaria vaccine to protect infants and children living in malaria endemic regions of sub-Saharan Africa, which can be delivered through the Expanded Programme on Immunization. The RTS,S/AS candidate vaccine has been evaluated in multiple phase I/II studies and shown to have a favourable safety profile and to be well-tolerated in both adults and children. This paper details the design of the phase III multicentre efficacy trial of the RTS,S/AS01 malaria vaccine candidate, which is pivotal for licensure and policy decision-making.The phase III trial is a randomized, controlled, multicentre, participant- and observer-blind study on-going in 11 centres associated with different malaria transmission settings in seven countries in sub-Saharan Africa. A minimum of 6,000 children in each of two age categories  have been enrolled. Children were randomized 1:1:1 to one of three study groups: (1) primary vaccination with RTS,S/AS01 and booster dose of RTS,S/AS01; (2) primary vaccination with RTS,S/AS01 and a control vaccine at time of booster; (3) primary vaccination with control vaccine and a control vaccine at time of booster. Primary vaccination comprises three doses at monthly intervals; the booster dose is administered at 18 months post-primary course. Subjects will be followed to study month 32. The co-primary objectives are the evaluation of efficacy over one year post-dose 3 against clinical malaria when primary immunization is delivered at: (1) 6-12 weeks of age, with co-administration of DTPwHepB/Hib antigens and OPV; (2) 5-17 months of age. Secondary objectives include evaluation of vaccine efficacy against severe malaria, anaemia, malaria hospitalization, fatal malaria, all-cause mortality and other serious illnesses including sepsis and pneumonia. Efficacy of the vaccine against clinical malaria under different transmission settings, the evolution of efficacy over time and the potential benefit of a booster will be evaluated. In addition, the effect of RTS,S/AS01 vaccination on growth, and the safety and immunogenicity in HIV-infected and malnourished children will be assessed. Safety of the primary course of immunization and the booster dose will be documented in both age categories.This pivotal phase III study of the RTS,S/AS01 candidate malaria vaccine in African children was designed and implemented by the Clinical Trials Partnership Committee. The study will provide efficacy and safety data to fulfil regulatory requirements, together with data on a broad range of endpoints that will facilitate the evaluation of the public health impact of the vaccine and will aid policy and implementation decisions.NCT00866619Clinicaltrials.gov  The past decade has seen unparalleled advances in the fight against malaria, and numerous public and private organizations are contributing hundreds of millions of dollars to malaria infection and disease research ,2. MalarPlasmodium falciparum malaria vaccine, RTS,S/AS01, which is currently in phase III clinical trials in infants and children living in malaria-endemic regions of sub-Saharan Africa [A safe and affordable vaccine would be a valuable addition to existing control measures. GlaxoSmithKline (GSK) Biologicals has been working towards the development of a safe and effective malaria vaccine for more than 20 years and has developed a candidate n Africa . It is iP. falciparum parasite. It contains the RTS,S antigen and is formulated with a novel proprietary Adjuvant System (AS). Clinical trials of the vaccine formulated with closely related Adjuvant Systems - AS01 or AS02 - have been conducted, and the RTS,S/AS01 formulation has been selected for phase III development based on comparative clinical studies [O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21 [The candidate malaria vaccine targets the pre-erythrocytic stage of the  studies ,8. AS01 and QS21 .A series of phase II clinical trials have been conducted to determine the safety, immunogenicity and efficacy of the RTS,S/AS vaccine in the target population of children at high risk of the disease. A proof-of-concept study in children aged 1-4 years in Mozambique showed that the RTS,S/AS02 vaccine was well-tolerated, with a vaccine efficacy of 35% against clinical malaria and 49% against severe malaria over 18 months ,11. SubsThe licensure claim of vaccine efficacy will be based principally on a large phase III clinical trial. This paper describes the overall design of the phase III multicentre efficacy study of the candidate RTS,S/AS01 malaria vaccine. The aims of this study are two-fold. Firstly, it will provide pivotal efficacy and safety data to support regulatory approval of the vaccine by the European Medicines Agency (EMA) and African national regulatory authorities and to facilitate pre-qualification by the World Health Organization (WHO). Secondly, it includes a broad range of endpoints that will allow assessment of the full public health impact of the vaccine. This information will be required to support a recommendation by the WHO and implementation decisions by local policy makers, and is key to ensure uptake of the vaccine following licensure . MeasureStreptococcus pneumoniae vaccines, which are expected to become part of the EPI program in the near future, and the second with three lots of RTS,S/AS01 vaccine in order to demonstrate lot-to-lot consistency.The population of the phase III trial mirrors as closely as possible the population of children who usually attend EPI visits. Low-birth-weight infants, malnourished children, and HIV-infected children were eligible. For safety reasons, those that were critically sick were excluded: in particular any child who required hospital admission or had an advanced stage of HIV disease (WHO classification grade 3 or 4). A dedicated phase III study will assess safety and immunogenicity in children exposed to HIV (NCT01148459). Two further studies will evaluate the safety and immunogenicity of RTS,S/AS01: the first in co-administration with rotavirus and Clinical development of the RTS,S/AS01 vaccine is undertaken in a public private partnership between GlaxoSmithKline and the PATH Malaria Vaccine Initiative (MVI), which receives funding from the Bill and Melinda Gates Foundation. The trial is also supported by the Malaria Clinical Trials Alliance (MCTA), an African-led organization that aims to build capacity and share best practice for the conduct of clinical trials. This multi-centre efficacy trial was designed by the Clinical Trials Partnership Committee (CTPC), which has membership representing each of the academic institutions participating in trial conduct, GSK Biologicals and MVI.This is a phase III, randomized, controlled, multicentre, participant- and observer-blind study. Enrolment occurred between May 2009 and February 2011. Follow up is currently on-going at 11 centres covering a wide range of transmission settings in seven countries in sub-Saharan Africa  or the left deltoid (children aged 5-17 months); all children receive the booster injection in the left deltoid. Neither the study subjects and their parents/guardians nor the study personnel involved in evaluation of the study endpoints are aware of the group allocation of the subjects. Because the study vaccines differ in appearance, the study staff responsible for their preparation and administration is aware of treatment allocation and therefore perform no other role in the trial.There is no routine testing for HIV infection in this study, HIV tests are performed only if clinically indicated. Voluntary counselling and testing, Highly Active Antiretroviral Therapy (HAART) and Prevention of Mother to Child Transmission (PMCT) are available at all study centres according to national policies.In accordance with national policies, all centres use artemisinin-based combination therapy (ACT) as first line treatment for malaria cases. The use of insecticide-treated bed nets is optimized at all centres by the close collaboration between research staff and malaria control program managers or through distribution at screening. Other control interventions such as intermittent preventive treatment in infants (IPTi) and indoor residual spraying (IRS) are not currently part of policy in the study areas, but if this changes during the trial, their use will be recorded.The overall sample size has taken into account the recent description of falling rates of malaria disease in several parts of Africa. To be assured of meeting the primary endpoints, conservative rates of disease were applied in the calculation of sample size and a case driven approach selected . To contInclusion and exclusion criteria are shown in Table P. falciparum circumsporozoite (CS) protein central tandem repeat and carboxy-terminal regions fused to the amino-terminus of the S antigen of hepatitis B virus (HBsAg). The vaccine is formulated with the AS01 Adjuvant System.The candidate malaria vaccine is RTS,S/AS01 . The RTS,S antigen is a hybrid recombinant protein consisting of the S. pneumoniae, which is a common cause of pneumonia in children in Africa. The reasons for not selecting pneumococcal vaccines were that they were expected to be implemented as policy in some countries prior to the enrolment and so would not provide additional benefit. In addition, there is a poorly understood interaction between malaria and pneumococcal infections. In paediatric hospital admissions, pneumonia and malaria co-occur more often than expected by chance [The choice of comparator vaccines was guided by the need to offer potential benefit to the control group without compromising the evaluation of study endpoints. The pros and cons of a number of options were debated and consensus reached by the CTPC after taking into account regional epidemiology and EPI programs. Rabies vaccine was chosen as the control for the 5-17 month age category because of the high burden of rabies across all of sub-Saharan Africa, its high fatality rate and the particular risk to children . Rabies y chance . This may chance . The cary chance . Meningoy chance -25 and, Clinical malaria cases are detected through passive surveillance at local health facilities. A blood sample for evaluation of malaria parasites is taken from all children with axillary temperature of ≥37.5°C or those reported to have had a fever within 24 hours of presentation. All subjects attending hospital emergency departments in the study areas are evaluated as potential cases of severe malaria following an algorithm, and case assessment is standardized across centers . The algTwo cross-sectional surveys will be conducted at study months 20 and 32 to assess vaccine efficacy against prevalent parasitaemia and anaemia. Data will be collected on potential covariates which may be included in the analysis of efficacy. These are bed net usage by direct observation, application of IRS, administered doses of IPTi, distance from nearest inpatient health facility, distance from nearest outpatient health facility, pneumococcal/Hib vaccination status, ethnicity, anthropometric measurements and feeding history.Full quality systems are in place for all laboratory tests in the trial and these are described in a companion paper . Anti-CSSerious adverse events (SAE) are collected for all subjects for the entire study period. Completeness of SAE reporting is strengthened by monthly visits of field workers to the children's homes. SAEs are defined as AEs resulting in death, which are life-threatening or require hospitalization or prolongation of existing hospitalization or those that result in disability or incapacity. Unsolicited AEs occurring during the 30 days after each vaccine dose and solicited AEs occurring during the seven days after each vaccine dose are collected for the first 200 children enrolled in each age category at each centre. In the remainder of children, only AEs that are considered to be related to vaccination or those resulting in study withdrawal are recorded. Investigators will grade all AEs and SAEs as mild, moderate or severe based on a scale of interference with normal daily activities, and assess the relationship to vaccination.Seizures occurring within 30 days of vaccination are also required to be reported as SAEs. For seizures occurring within seven days of vaccination, an analysis will be performed based on the Brighton Collaborations guidelines, which captures the features of the seizure and classifies the level of diagnostic certainty . In the Solicited local (injection site) AEs recorded are pain, swelling and redness; grading of symptoms is on a scale of 0-3. Solicited general AEs recorded are drowsiness, fever, irritability/fussiness and loss of appetite; intensity of symptoms (except fever) are graded on a scale of 0-3 based on interference with normal daily activities; fever is defined as axillary temperature ≥37.5°C. Methods have been fully described previously .Verbal autopsies are carried out for all children who die outside a health facility to ascribe the cause of death. The questionnaire used is based on the INDEPTH standard and adapted to be locally appropriate . At studSafety and immunogenicity will be described in the special sub-populations of malnourished and HIV-infected children. Weight at enrolment will be used to determine a subset of children who are low weight for age (weight for age z-score ≤-2) and very low weight for age (weight for age z-score ≤-3). HIV infections known at enrolment or diagnosed during the trial are recorded.The co-primary objectives of the study are efficacy over 1 year post-dose 3 against clinical malaria when primary immunization starts at: (1) 6-12 weeks of age, with co-administration of DTPwHepB/Hib and OPV antigens; (2) 5-17 months of age  map of malaria risk in Africa in Figure This pivotal phase III efficacy study of the RTS,S/AS01 candidate malaria vaccine in African children was designed and implemented by the Clinical Trials Partnership Committee. The study design is in full accord with the conclusions of the WHO expert panel on the measurement of malaria vaccine efficacy in phase III clinical trials . The desAL, JV, MLi, OOA, CC, WRB, LV are employees of GlaxoSmithKline Biologicals. AL, JV, and WRB hold shares in GSK, WRB holds several patents, including one for RTS,S/AS.GSK and all participating institutions declare to have received a grant and travel funds from PATH-MVI for/relating to the clinical trial described in this manuscript. Employees of PATH-MVI  report their institute received a grant by the Bill and Melinda Gates foundation for this trial. All GSK employees, SA, PB, and PN declare their institution has received grants or has grants pending from PATH-MVI for previous clinical trials. CSS, JFT declare no potential conflicts of interest.All authors and members of the Clinical Trials Partnership Committee contributed to the design of the phase III trial through discussions and document review. AL led the writing of the manuscript coordinating the incorporation of all reviewer comments. SOA, SA, EM, and PN lead the review process by chairing the CTPC and leading the scientific debate and review process around trial design. AL, JV, OOA, CC, ML, and WR wrote the study protocol from which the tables and graphics are drawn. BS and CL coordinated the input of the MVI team. All authors read and approved the final manuscript.