Plasmodium falciparum malaria in part by enhancing naturally-acquired immunity to this disease. To investigate this hypothesis, we compared antibody levels to four merozoite antigens from the P. falciparum 3D7 clone  in a cohort of 103 HbAA, 73 HbAS and 30 HbAC children aged 3 to 11 years in a malaria-endemic area of Mali. In the 2009 transmission season we found that HbAS, but not HbAC, significantly reduced the risk of malaria compared to HbAA. IgG levels to MSP1 and MSP2 at the start of this transmission season inversely correlated with malaria incidence after adjusting for age and Hb type. However, HbAS children had significantly lower IgG levels to EBA175 and MSP2 compared to HbAA children. On the other hand, HbAC children had similar IgG levels to all four antigens. The parasite growth-inhibitory activity of purified IgG samples did not differ significantly by Hb type. Changes in antigen-specific IgG levels during the 2009 transmission and 2010 dry seasons also did not differ by Hb type, and none of these IgG levels dropped significantly during the dry season. These data suggest that sickle-cell trait does not reduce the risk of malaria by enhancing the acquisition of IgG responses to merozoite antigens.Heterozygous hemoglobin (Hb) AS (sickle-cell trait) and HbAC are hypothesized to protect against  Plasmodium falciparum malaria remains one of the greatest global health problems 6 Glu to Val) in Africa. This hypothesis is supported by epidemiological studies showing that heterozygosity for ‘adult’ HbA and ‘sickle’ HbS  protects children from developing severe, life-threatening malaria syndromes P. falciparum malaria, this has not been a consistent finding P. falciparum malaria have verified the importance of immune IgG in the clearance of blood-stage parasitemias 6 Glu to Lys). The effects of HbAC on the incidence of uncomplicated and severe P. falciparum malaria are less well established than those of HbAS The HbC variant is also produced by a single amino acid substitution  deficiency, ABO/Rh blood group antigens). Plasma samples were collected at the start of the transmission season (May 2009), at the end of the transmission season (December 2009), and at the end of the subsequent dry season (May 2010). We measured antigen-specific IgG levels to four merozoite antigens  and analyzed their relationship to malaria incidence in the 2009 transmission season.To investigate whether IgG responses to In May 2008, we initiated a 4-year longitudinal cohort study of Malian children (aged 0.5 to 17 years) in three villages  where malaria transmission is intense and seasonal (June to December/January). The details of the study site and cohort have been described elsewhere P. falciparum parasites on microscopic examination of a thick blood film, and no other etiologies of febrile illness discernible on clinical examination. Severe malaria was defined by WHO criteria At the start of the sub-cohort study (May 2009), we obtained plasma samples and stored them at −80°C until use. The parents or guardians of all children enrolled in the cohort study were activity encouraged to bring their child to our study clinic if they developed symptoms of malaria. Uncomplicated malaria was defined as axillary temperature >37.5°C (or history of fever in the past 48 hours) with or without other symptoms of malaria , any density of asexual The cohort and sub-cohort studies were approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odontostomatology, University of Bamako, and the Institutional Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID). Adult parents or guardians of children gave written informed consent if they could read French. If they could not read French, oral consent was obtained by the study physician and a finger print obtained from the consenting adult. A literate third-party individual witnessed both informed consent processes and signed the informed consent document. The Ethics Committee at University of Bamako and the IRB of NIAID approved the informed consent procedure. The study is registered with Clinicaltrials.gov, number NCT00669084.405 = 1 in a standardized assay. Apical membrane antigen 1 (AMA1)-3D7 The standardized method for performing the ELISA was described previously To measure anti-tetanus toxoid IgG titers and determine IgG subclass, we randomly selected plasma samples from eight pairs of matched HbAA and HbAS children in each age group  and tested them (n = 48) at a 1∶500 dilution. Tetanus toxoid was purchased from List Biological Laboratories, Inc. .P. falciparum parasites (3D7 clone) and culture medium were added to 96-well tissue culture plates in triplicate and cultured for ∼40 h. Relative parasitemias were quantitated by biochemical determination of parasite lactate dehydrogenase. Percent inhibition of test IgG was calculated as 100 - [/ ×100].The growth inhibition assay (GIA) was conducted with protein G-purified total IgGs and the standardized method was described previously 10-transformed ELISA units were used as variables. To determine whether there was a significant interaction between age and Hb type on the time to first malaria episode, a Cox regression analysis was performed with age, Hb type and the cross-product of these two variables. Data were analyzed using Prism 5  or JMP8 . p-values <0.05 were considered significant.Categorical variables were compared using a chi-squared test. Times to first malaria episode were compared by a log-rank test. Continuous variables between two groups were compared by a Mann-Whitney test, and those between three groups were compared by a Kruskal-Wallis test . For paired comparisons, a Wilcoxon signed-rank test was used. Spearman's correlation test was used to assess correlation between two data sets. We used a nominal logistic fit of data for malaria experience , a Cox proportional hazards regression for time to first malaria episode and a longitudinal analysis for number of malaria episodes. For these multivariate regression analyses, age (as a continuous variable), Hb type (HbAS or non-HbAS) and logG6PD*A- and α-globin−3.7kb genotypes) was matched as closely as possible  at enrollment. A total of 206 children were followed passively for malaria episodes throughout the 2009 transmission season . Increasing age and HbAS were significantly associated with reduced malaria risk, as measured by: (i) whether or not malaria was experienced  and HbAC (n = 30) children in a cohort of Malian children from two villages. For each HbAS or HbAC child, we enrolled a HbAA child who was completely matched for age. The erythrocyte polymorphism profile  between all combinations of antigen-specific IgG levels (G6PD*A- and α−3.7kb genotypes) were not associated with antigen-specific IgG levels (data not shown).To explore whether enhanced humoral immunity contributed to the malaria-protective effects of sickle-cell trait, we quantified plasma IgG levels to four merozoite antigens in HbAA, HbAS and HbAC children just prior to the transmission season (May 2009). In the entire sub-cohort of children, we found strong correlations . We found no differences in these IgG levels between HbAA and HbAS children (data not shown). In these paired HbAA and HbAS plasma samples, we also found no differences in IgG subclass distributions. Regardless of Hb type, IgGs = 0.27–0.49; p<0.0001 for AMA1, EBA175 and MSP2; p = 0.0002 for MSP1). The median growth-inhibitory activity of purified IgGs was 23.0%  in HbAS children, 23.0%  in HbAC, and 36.0%  in HbAA. The difference among the three Hb types did not reach significance .Antibody functionality was evaluated by a growth inhibition assay (GIA) using protein G-purified IgGs. We found significant correlations between antigen-specific IgG levels and growth-inhibitory activities  since malaria risk did not differ between HbAC and HbAA children . After aP. falciparum parasitemia (25–1875/ µl whole blood). The IgG levels to all four merozoite antigens were higher in December 2009 than in May 2009 . The increases in these IgG levels did not differ significantly by Hb type , HbAS  and HbAC  children in December 2009. At this time, 18 HbAA (18.6%), 12 HbAS (17.6%) and 5 HbAC (19.2%) children had asymptomatic  Hb type  and did P. falciparum parasitemia. The IgG levels to all four merozoite antigens were not significantly different between December 2009 and May 2010 (p>0.05). These changes in antigen-specific IgG levels did not differ significantly by Hb type , HbAS  and HbAC  children in May 2010. At this time, none of these children had microscopically detectable  Hb type . Taken ts = 0.59–0.89, p<0.0001) between these IgG levels in May and December 2009 , environmental factors  and/or antigens used for assay. Using highly standardized ELISA and GIA assays, we have showed that HbAS children had lower IgG levels to EBA175 and MSP2, and a reduced risk of P. falciparum malaria, compared to HbAA children. HbAS children also had lower IgG levels to AMA1 and MSP1, but these did not differ significantly from those in HbAA children. These data suggest that HbAS does not confer protection against malaria in our study population by enhancing IgG responses to merozoite antigens.Previous studies investigating the impact of HbAS and HbAC on the development of P. falciparum antigens generally. Cholera et al. reported that levels of P. falciparum erythrocyte membrane protein 1 (PfEMP1) are lower on the surface of HbAS compared to HbAA erythrocytes et al., submitted). If HbAS causes quantitative or qualitative changes in merozoite antigens exposed to the immune system during erythrocyte invasion, these changes may explain the lower IgG levels to merozoite antigens in HbAS children. However, to our knowledge, studies have not yet compared antigen levels on the surface of merozoites released from parasitized HbAS, HbAC and HbAA erythrocytes. Alternatively, some studies have reported that the parasite density in HbAS children was lower than that in HbAA children While we measured IgG levels to only four merozoite antigens, the strong correlations between these IgG levels within individuals suggest that HbAA and HbAS children differ in their exposure to Observations that IgG titers to a single antigen can correlate significantly with clinical protection in one study, but not in another Previous studies have suggested a contribution of acquired immunity to protection in HbAS children, evidenced by the observation that the level of protection against malaria increases with age to a greater extent in HbAS than in HbAA children In this study, HbAC and HbAA children did not differ in malaria risk or IgG levels to four merozoite antigens. There were relatively fewer HbAC children (n = 30) than HbAS children (n = 73) in this study site, which might reduce the power to detect the differences between HbAC and HbAA children. However, we did not see any trend  in HbAC children. In addition, since the effects of HbAC on malaria risk P. falciparum-specific IgG responses.We found no evidence that antigen-specific IgG levels are lower in HbAS children because they increase less during the transmission season or decay more rapidly in the dry season than in HbAA and HbAC children. These IgG levels increased in all children during the 2009 transmission season, but the fold-increase in IgG levels did not correlate with the number of malaria episodes the children experienced. This finding suggests that increases in antigen-specific IgG levels during a transmission season do not necessarily result from the development of a malaria episode. Instead, asymptomatic infections may play an important role in developing P. falciparum-specific antibodies are short-lived  P. falciparum-specific antibodies to be relatively longer, especially in adults Once they increased, antigen-specific IgG levels did not decay over the 5-month dry season. This finding is surprising since others have reported that ex vivo, do not always correlate with the level (or presence) of antigen-specific antibodies P. falciparum infection was very uncommon in our study population during the dry season. However, the prevalence of sub-patent parasitemia could have been substantially higher if we used a more sensitive method of detection, such as polymerase chain reaction. Further study is required to uncover the mechanism of sustained antigen-specific IgG levels during the dry season.The mechanisms of long-term antibody maintenance by memory B cells and long-lived plasma cells are poorly understood, especially in humans. In part, this is because the predominant site of antibody production is the bone marrow where up to 90% of long-lived plasma cells reside In summary, HbAS and HbAC differentially associated with malaria risk and merozoite antigen-specific IgG levels in Malian children. HbAS children had lower IgG levels to four merozoite antigens, suggesting that these IgG responses – two of which correlated with reduced malaria incidence – do not mediate the malaria-protective effects of HbAS in 3- to 11-year-old children. The development and decay of IgG levels through the transmission and dry seasons did not differ between HbAS and HbAA children, suggesting that the differences in their IgG levels may have been established before 3 years of age.Figure S1Significant correlations between merozoite antigen-specific IgG levels. IgG levels in individual plasma samples against two different merozoite antigens are compared in each panel. All responses below the limit of detection (44 ELISA units) were assigned a value of 22 ELISA units. All correlations shown are significant .(EPS)Click here for additional data file.Table S1Detail of Malian children pairs.(XLSX)Click here for additional data file.Table S2Demographic characteristics and erythrocyte polymorphisms of Malian children, stratified by hemoglobin type.a p-values were calculated using Chi-square tests. b α-thalassemia genotypes for two HbAS children were not determined.(DOCX)Click here for additional data file.


P.falciparum resistance gene markers have been investigated during the malaria transmission October 2011 to February 2012. More than 1500 children and 700 pregnant women were recruited. The prevalence of malaria was comprised between 9% and 30% depending of the localization of the health center. It was found that acceptability of technicians to use rapid diagnostic tests was low and microscopy is still considered as the reference. A comparison between health centers and reference lab technicians showed similar level of in malaria diagnosis performance. The P.falciparum genetic diversity  and multiplicity of infection (1.7) do not show any reduction but parasite densities were lower than reported in studies conducted before the introduction of ACTs. As a conclusion: in order to improve quality of care and the acceptability of RDT, there is a need to provide a targeted training to heath workers.Since the introduction of artemisin-based combination therapies in the Republic of Congo, limited number of investigations have been conducted to evaluate the burden of the disease at the district level. The main goal of this study was to document laboratory-confirmed cases using rapid diagnostic tests of malaria in children and pregnant women attending health facilities located in Northern districts of Brazzaville and Pointe Noire which is the second main city of the Republic of Congo. As objective2, the malaria diagnostic performance of each health facility has been assessed and as objective3, genetic diversity, multiplicity of infection and the prevalence of


Plasmodium falciparum malaria are at increased the risk of invasive bacterial disease, largely enteric gram-negative organisms (ENGO), which is associated with increased mortality and recurrent morbidity. Although incompletely understood, the most likely source of EGNO is the bowel. We hypothesised that as a result of impaired gut-barrier function endotoxin (lipopolysaccharide), present in the cell-wall of EGNO and in substantial quantities in the gut, is translocated into the bloodstream, and contributes to the pathophysiology of children with severe malaria.Children presenting to hospital with recent or current We conducted a prospective study in 257 children presenting with malaria to two hospitals in Kenya and Uganda. We analysed the clinical presentation, endotoxin and cytokine concentration.Endotoxaemia (endotoxin activity ≥0.4 EAA Units) was observed in 71 (27.6%) children but its presence was independent of both disease severity and outcome. Endotoxaemia was more frequent in children with severe anaemia but not specifically associated with other complications of malaria. Endotoxaemia was associated with a depressed inflammatory and anti-inflammatory cytokine response. Plasma endotoxin levels in severe malaria negatively correlated with IL6, IL10 and TGFβ .P. falciparum-infected erythrocytes within the endothelial bed of the gut has been observed in pathological studies and may lead to gut-barrier dysfuction. The association of endotoxaemia with the anaemia phenotype implies that it may contribute to the dyserythropoesis accompanying malaria through inflammation. Both of these factors feasibly underpin the susceptibility to EGNO co-infection. Further research is required to investigate this initial finding, with a view to future treatment trials targeting mechanism and appropriate antimicrobial treatment.Endotoxaemia is common in malaria and results in temporary immune paralysis, similar to that observed in patients with sepsis and experimentally-induced endotoxaemia. Intense sequestration of  Endotoxin is a complex lipopolysaccharide (LPS) present in the cell walls of gram-negative bacteria and in substantial quantities in the bowel, and a potent trigger of innate immunity due to activation of Toll Like Receptor (TLR) signalling . The effPlasmodium falciparum malaria are characterised be excessive inflammation, organ damage and metabolic dysregulation, a picture similar to that observed in patients suffering from severe bacterial sepsis [P. falciparum infection are non-typhoidal Salmonellae (NTS), E. coli and other enteric organisms with gram-negative organisms (EGNO) becoming increasingly more predominant across the severity spectrum: from those with evidence of recent malaria infection , intercurrent malaria and greatest in those with severe malaria and hyperparasitaemia [. Although the mechanisms underlying the increased susceptibility are uncertain, they may include disordered gut barrier function secondary to intense sequestration of parasitized red cells in the intestine [Salmonella typhimurium bacteraemia [Severe forms of l sepsis . Malariasitaemia -12. Althntestine -15 and/oteraemia . These oteraemia .We hypothesised that either translocation of endotoxin alone or translocation of gram-negative bacteria and subsequent release of endotoxin into the blood stream may play a role in pathogenesis of severe malaria and its complications such as shock and organ dysfunction. We prospectively examined endotoxin levels and its relationship to disease severity and cytokine activation in children with malaria at two hospitals in East Africa.P. falciparum malaria and the acute paediatric ward at the Mbale Regional Referral Hospital (MRRH), Eastern Uganda were recruited. In Kilifi District malaria transmission is now mesoendemic due to a steady decline in transmission over the last 10 years [Between August and December 2009 paediatric admissions to Kilifi District Hospital (KDH), Kenya with 10 years . In Mbal10 years .P. falciparum malaria with signs of severe illness but without life-threatening clinical syndromes (n=51). Second, children with severe, life-threatening malaria (n=206) included children with respiratory distress (deep breathing or indrawing), impaired consciousness (prostration or coma) or severe anaemia  [Three groups were considered based on the severity of clinical symptoms: First, children hospitalised with alaria n=6 include ≤5g/dl) . A propo ≤5g/dl) . The thiMalaria was diagnosed by a rapid malaria test (DiaMed OptiMAL®) and a blood film. In Mbale, parasite blood films were screened for the presence of parasites but parasite densities not reported whereas parasite densities were reported in Kilifi. Clinical history, presentation and laboratory values where recorded for all children using the FEAST Case Report Form. Blood cultures were done on all study participants in KDH but not in MRRH. At KDH, only two children were blood culture positive whereas in all other children no growth was reported. Children presenting with severe anaemia (haemoglobin ≤5g/dl) were transfused 20mls/kg whole blood over 3 h. Moderate anaemia was defined as an Hb>5 and ≤7g/dl. All children received parenteral antimalarials and antibiotics.The study was approved by the Ugandan Ethical Review Committee (REIRC 002/2009) and the National Ethical Review Committee in Kenya (SSC nos. 1135 and 1247). Parents or guardians of all children recruited to take part in the study provided written informed consent.Escherichia coli 055:B5 LPS) with high endotoxin levels  related to prognosis [We used an Endotoxin Activity Assay  to detect the presence of endotoxin in whole blood within 3 h of venepuncture according to manufacturers instructions. This is a novel chemiluminescent assay that relies on the detection of Lipid A component of lipopolysaccharide (LPS), which is not susceptible to microbial contamination. It measures endotoxin activity in whole blood by the priming of host neutrophil respiratory burst activity via complement opsonised LPS-IgM immune complexes and has shown good correlation with Gram-negative infection and with clinical sepsis, which was not apparent using limulus amoebocyte lysate (LAL) assay . The cutrognosis .Plasma was separated and stored in aliquots at -80C. The cytokine concentration of TNFα, IL6, IL10, and TGFβ was determined using Quantikine ELISA Kits (R&D) according to the manufacturer’s instructions. The plasma concentration of the intestinal fatty acid binding protein (I-FABP)  was dete2. Continuous values such as age and cytokine concentrations were compared between groups using the Mann Whitney U test or Kruskal Wallis H test. Spearman’s rho correlation coefficient was used to determine a relationship between continuous variables. Statistical analyses were performed using SPSS 18 (IBM SPSS Statistics) and Stata version 11 (StataCorp LP).The presence or absence of clinical parameters in the different study groups were determined using Pearson χWe recruited 257 children, 205 at MRRH and 52 at KDH, the basic clinical presentation of all children is described in Table Endotoxaemia (EAA≥0.4) was detected in 16 (31.4%) children with non-severe malaria, 15 (28.3%) children with severe malaria but not detected in any of the 25 healthy children recruited as a control group. The prevalence of endotoxaemia, in the subgroup of children with severe malaria and shock was 40/154 (26%), with 3/10 (30%) in fatal cases and 37/144 (25.6%) in non-fatal cases suggesting that the presence of endotoxaemia was neither associated with disease severity nor outcome.The proportion of children with severe malaria complicated by endotoxemia was lower in the cohort recruited at KDH than in the cohort recruited at MRRH, as was the number with high endotoxin levels Table . This co2 = 1.17, p=0.299). We found no association between endotoxin with other clinical subgroups or with outcome.Endotoxaemia (EAA≥0.4) was more likely to be present in children with moderate anaemia (Hb>5 and ≤7g/dl) or severe anaemia (Hb≤5g/dl) compared to children without anaemia (Hb>7g/dl) Table . By contU test, p<0.016). Fatal cases had significantly higher plasma concentrations of IL6 than those who survived (Mann Whitney U test p=0.022).In critically ill patients, endotoxin acts as a potent stimulus for the release of pro-inflammatory cytokines followed by a phase of immune paralysis characterised by control of inflammation. We speculated that children with malaria and endotoxaemia could show either elevated or depressed levels of pro-inflammatory cytokines. Plasma concentrations of TNFα, IL6, IL10 and TGFβ are presented in Table th and 75th percentile) with endotoxaemia: 0 pg/ml (0–54.3) without endotoxaemia: 26.7 pg/ml (0–73.9) Mann Whitney U test, p=0.2).Of note, in all subgroups of children with severe malaria plasma concentrations of IL6, IL10 and TGFβ were reduced in the presence of endotoxaemia compared to those without endotoxaemia , a marker for acute enterocyte death and putative intestinal permeability, in 198 samples Table . NeitherTo our knowledge this is the first study to document the prevalence of endotoxaemia in children suffering from malaria. Approximately 30% of children with malaria had detectable levels of endotoxin (LPS) in their blood. Surprisingly, and contrary to findings in other critical illnesses but not all paediatric studies , we founIn our study, the most notable clinical parameter associated with endotoxaemia was anaemia. The origins of malaria anaemia are multiple. Next to acute vascular haemolysis of infected erythrocytes during acute malaria, both acute and chronic infection cause inflammation resulting in dyserythropoiesis due to changes in iron storage metabolism and cytokine-mediated suppression of the generation of progenitor cells -26. It sP. falciparum-infected erythrocytes within the endothelial bed of the gut, particularly at the tip of intestinal villi [We hypothesised that the most likely source of endotoxaemia is the gastrointestinal tract. Gastro-intestinal symptoms are common in malaria yet there has been little recent interest in gut barrier dysfunction despite evidence provided over 2–3 decades ago demonstrating histopathological changes and impaired absorption ,14,31,32al villi ,14 and tal villi  has beenal villi  and inteal villi . SequestSequestration is mediated by parasite molecules expressed on the surface of infected erythrocytes that mediate their adhesion to receptor expressed on endothelial cells . InteresTogether these data suggest that parasite-mediated sequestration and inflammation contribute to both anaemia and changes in gut permeability. However, particularly TLR-mediated induction of inflammation leads to altered responsiveness of leukocytes to additional signals such as endotoxin-tolerance. In our study, endotoxaemia was associated with decreased plasma concentrations of TNFα, IL6 and IL10 compared to those without endotoxaemia . In sepsThere is strong evidence that recent or current infection with malaria increases the risk of systemic bacterial infection with high associated mortality rates in several sub-Saharan countries ,11,47,48Our study is limited by the absence of blood culture data and parasite density data, which were available only for a very small number of children. All but one child with blood culture data were negative for gram-negative organisms suggesting that endotoxaemia resulted from endotoxin translocation due to gut barrier dysfunction. Despite the small number of children with blood culture data, this appears plausible since the prevalence of endotoxin observed in this study was much higher than previous reports of bacterial co-infection, which are in the order of 5-12% -12; but We suggest the following hypothesis: A proportion of children suffering from malaria experience loss of the barrier function of the gut due to parasite sequestration (and reduced microcirculatory flow), inflammation or both. This could be augmented by other pathophysiological processes, specific to falciparum malaria including severe anaemia and those leading to microvascular obstruction. In some children, loss of gut barrier function might be sufficient to allow translocation of endotoxin and translocation of gram-negative bacteria with subsequent release of endotoxin into the bloodstream. Endotoxin and other TLR–ligands expressed by gram-negative bacteria could further increase malaria-induced inflammation followed by regulatory immune responses that result in temporary immune paralysis as has been described for sepsis. Then children, having entered a phase of immune paralysis, might not be able to mount an adequate immune response controlling bacteraemia. This hypothesis is testable through investigation of gut-barrier function in children suffering from malaria, characterisation of the cytoadhesion profile of clinical parasite isolates from children with malaria and endotoxaemia and by analysing differences in the innate and adaptive immune response pathways in children with severe malaria in the presence or absence of endotoxaemia.EGNO: Enteric gram negative organisms; NTS: Non-typhoidal Salmonellae; I-FABP: Intestinal fatty acid binding protein; KDH: Kilifi district hospital; MRRH: Mbale regional referral hospital.None of the authors report a conflict of interests.Design of the study: POO, KM, BCU. Patient recruitment: JK, JN, PO, SA, AT. Laboratory analysis: HMF, HK, EG, PB. Statistical analysis: JJ, BCU. Manuscript preparation: POO, KM, BCU. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/117/prepub


During natural infection NK cells exhibit a cytolytic effect and regulate dendritic cells, macrophages, neutrophils as well as affect antigen specific T and B cell responses. To characterize the NK cells in P. falciparum infected patients of a highly endemic region of Colombia, the degree of NK proliferation and production of IFN gamma and TNF production in these cells were explored.In ColombiaP. falciparum malaria patients from the Northwest region of the country were recruited in the study. In addition, 20 healthy controls were included: 10 from Medellin (no-transmission area) and 10 from the Uraba region . Immunophenotypic analysis of peripheral mononuclear cells was performed by FACS to detect total number of NK cells, subtypes and intracellular IFNγ and TNF production by NK cells in the different patient groups.Seventeen patients with acute and three with severe +/CD3- NK cell proportions in acute and severe malaria subjects were 9.14%  and 19.62% , respectively, in contrast to healthy controls from endemic  and non-endemic area . Analysis of basal IFNγ and TNF levels confirmed the CD56bright NK population as the main cytokine producer (p < 0.0001) in the groups affected with malaria, with the CD56dim NK cell exhibiting the highest potential of TNF production after stimulus in the acute malaria group.The total mean CD56bright but also of CD56dim NK cell populations as producers of the two cytokines in malaria patients in Colombia.The results confirm the important role of not only CD56 The clinical presentation of malaria depends on the confluence of diverse factors, including the degree of natural and acquired specific immunity, host's genetic composition, age, occupation and social and economic factors of the population .Malaria in Colombia is highly endemic in the north-west, Pacific Coast and Amazon regions, all among the most deprived of the country due to social-political conflicts resulting in migrations and poverty. Previous studies in the northwest of Colombia, confirmed that children below 12 years of age are highly susceptible to malaria with a mean seven years of age for clinical presentation with malaria .Plasmodium falciparum infection, severe or fatal malaria cases are rare in the country. Out of the 79,909 malaria cases  reported in 2009, 307 were severe  and the fatality rate was 0.04%[Despite the high frequency of was 0.04%. In Colowas 0.04%. This isP. falciparum infection has been known. In this sense, both innate and adaptative immune responses, constitute a key component in subsequent Plasmodium challenges by reducing parasitaemia during the acute phase of the disease [For many years, the importance of effective acquired immune response to protect against severe  disease . After i disease .dim representing the vast majority of human NK cells and a small distinct population of CD56bright NK cells [bright NK cells fail to express CD16 and exhibit a weak cytotoxic activity. In healthy humans, CD56bright cells comprise up to 10% of all NK cells. Granules of CD56dim cells are richer in perforin and granzyme A, which results in a more effective cytolysis compared to CD56bright NK cells. However, CD56bright NK cells are more efficient in producing the pro-inflammatory cytokines IFNγ and TNF [The evidence gathered so far confirms that NK cells can positively ,10 or neNK cells ,15. AlmoThe presence of severe malaria has been strongly attributed to an exaggerated immune response of the host towards parasite antigens and several authors reported on the positive correlation between high TNF production and cerebral malaria in humans as well as in animal models . ParasitP. falciparum-infected patients of a highly endemic region of Colombia are described. In addition, the degree of NK proliferation and production of IFNγ and TNF production by these cells was explored, with the aim of providing a first glimpse into their profile in naturally-infected populations on this continent.Based on the limited and controversial knowledge on the function of NK cells during the inflammatory process leading to severe malaria and the important role of TNF and IFNγ in the pathogenesis of the complicated disease, the study proposed to correlate the production of pro-inflammatory cytokines by these cells and their subsets, and the clinical outcome in malaria patients in Colombia. The characteristics of NK cells in P. falciparum malaria patients were recruited. Individuals were included immediately after diagnosis of P. falciparum infection was performed by microscopy. Inclusion criteria for this group were: permanent residency in the endemic area, presence of symptomatic malaria and absence of clinical viral infections within 2 months of attendance to the malaria clinic. The degree of clinical impairment was assessed by an experienced medical officer. Patients were classified as having acute malaria or severe malaria according to the WHO criteria[The study took place in Medellin-Colombia, where patients from the northwest region of the country attended the malaria clinic of Grupo Salud y Comunidad, Universidad de Antioquia. A total of 23 acute (non-severe) and seven severe  criteria. ConcomiVolunteers were notified of the risks and their rights and enrolled after providing informed consent. Ethical clearance was granted by the Ethics Committee of the Centro de Investigaciones Medicas, Faculty of Medicine, Universidad de Antioquia (Medellin-Colombia).Plasmodium species, parasite density, and schizontaemia. Parasite density was measured by counting the number of asexual parasites per 200 leukocytes, based on a mean count of 8,000 leukocytes per microlitre of blood . A slide was considered negative after examination of at least 300 microscopic fields [Giemsa-Field stained thick/thin blood films were examined with a 100× objective to identify presence of parasites, c fields .Blood samples from volunteers were obtained from peripheral vein and, in the case of malaria subjects, before malaria treatment was administered. Samples of 16 mL were collected into EDTA vacutainer vials from patients and controls and peripheral blood mononuclear cells were separated by density centrifugation (Ficoll-Hypaque 1077). Mononuclear cells were collected from the interface, washed twice by centrifugation with EDTA-PBS at 640 g and 160 g, respectively, for 10 min at 4°C. Cells were then suspended in complete RPMI 1640 medium buffered with 25 mM Hepes and supplemented with 12 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 5% foetal bovine serum. Cell viability was assessed with 0.1% trypan blue in PBS pH 7.3.Immunophenotypic analysis of cells was performed using four-colour analysis on a FACSCalibur  with CELLQuest Pro software (Becton Dickinson). Cells were stained with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD56 (SIGMA) and Cychrome (Cy)-anti-CD3 (SIGMA) to determine the percentage of NK cells within the sample. Gating was performed according to size and granulation using forward-scatter versus side-scatter (FSC/SSC) dot plots.6 freshly isolated mononuclear cells per well were incubated with phorbol myristate acetate (PMA) 10 mg/ml plus calcium ionophore 250 ng and brefeldin A 0.4 mg/mL for 6 h at 37°C in 5% CO2. Analysis included a description of the total NK cell and subtypes populations according to IFNγ and TNF production in the different groups of study: acute malaria, severe malaria, and healthy volunteers from endemic and non-endemic area. Results were processed using Prisma®, and mean values and standard deviations were compared using t-student.Production of INFγ and TNF by NK cells was determined by flow cytometry. For this, 10Natural killer population analysis in the malaria affected group was successfully performed in 17 acute malaria and three severe malaria volunteers. Samples from 10 subjects from Medellin (non-endemic) and 10 from Uraba (endemic region) were processed as controls.3 . All patients with severe malaria reported fever, chills, sweating, general malaise, headache, vomiting, two reported dizziness and one had abdominal pain. Parasitaemia in each of these patients was 154,000, 171,000 and 195,000 rings/mm3 . The diagnosis of severe malaria was based on the high number of vomiting episodes per day and hyperparasitaemia.The male/female ratio in the experimental group was 16/4 and the age ranged from 18 to 59 years old (mean 34 ± 14). The male/female ratio in the controls was 13/7 and the age ranged from 18 to 49 years old (mean 33.3 ± 9.9). Among the patients with acute malaria, the most common symptoms were fever (100%), chills (88%) and sweating (47%), while other complaints such as diarrhoea and vomiting were rare. Parasitaemia in these patients ranged between 80 and 9,440 rings/mm+/CD3- NK cell population was 9.1 ± 0.3% of which 7.1 ± 0.4% were CD56dim and 2.0 ± 0.2% were CD56bright. Meanwhile, in patients with severe malaria, the total population of CD56+/CD3- was 19.6 ± 0.6% and the mean CD56dim and CD56bright populations were 16.0 ± 0.3% and 3.6 ± 0.9%, respectively. These proportions of NK cells in acute malaria-infected and severe patients were statistically similar. After PMA and calcium ionophore addition, cells from complicated malaria patients showed higher levels of both CD56dim and CD56bright populations when compared to acute malaria patients  than the CD56dim population  than the CD56dim population  can produce IFN-γ. In malaria, the main cellular sources of the cytokine are T cells, γδ T cells, and NK cells. However, in some subjects, there is little or no evidence of an NK cell response after exposure to ng cells ,38. Contcytokine . Such prulations ,38 with ulations , among oulations ,41. In aulations ,42. Howebright cells exhibiting the highest production of the cytokine in both infected groups.TNF is involved in the immune response to malaria as well as in the pathogenesis of severe disease,44. Dysrbright but also of CD56dim NK cell populations as producers of the two cytokines in naturally infected malaria patients in Colombia. Future studies should include exploration of the status of activation of other lymphocyte populations and their relationship with NK cells in infected patients with malaria by P. vivax and P. falciparum exhibiting different clinical presentations.The results confirm the important role of not only CD56The authors declare that they have no competing interests.OA and JB designed and performed the NK analysis in blood samples. AV conceived the project and designed the experiments. AM designed the experiments, supervised overall design and development and wrote the manuscript. All authors read and approved the final manuscript.


Dear Sir:We read with great interest the study on the abdominal computed tomography (CT) scan during acute vivax malaria in South Korea by Kim and others.1Plasmodium vivax. We therefore agree with Kim and others that infection with P. vivax—less prevalent worldwide than Plasmodium falciparum—likely carry a higher risk of splenic complications than infection with P. falciparum. Acute splenic enlargement, which is as frequent in P. vivax as in P. falciparum infection despite lower mean parasitemia in the former,7In our recent analysis of 55 cases of pathological rupture on the spleen in malaria,The mechanistic connections between malaria, splenic infarction, and pathological splenic rupture are not clearly established. Although splenic infarction leads to splenic rupture in a small number of patients, 53 of the 55 malaria patients with pathological splenic rupture had no obvious signs of pre-existing infarction.8Not least, pathological rupture of the spleen and other splenic complications may occur well beyond the acute phase of the malaria attack .


Plasmodium falciparum malaria. Thin and thick smears are routinely used for this purpose.Counts of malaria parasites in peripheral blood are important to assess severity of ® Malaria Test, an easy-to-perform rapid diagnostic test, with Histidine Rich Protein-2 (HRP-2) and aldolase as diagnostic markers, was used for semi-quantitative assessment of parasitaemia of P. faciparum.In this study the Binax NOWP. falciparum malaria, reactivity of aldolase increased with higher parasitaemia. In all patients with a parasitaemia above 50,000 asexual parasites/μl (> 1%) co-reactivity of HRP-2 and aldolase was observed. Absence of aldolase reactivity in the presence of HRP-2 was a reliable predictive marker to exclude high (> 1%) parasitaemia in P. falciparum malaria.In 257 patients with imported Assessment of HRP-2 and aldolase co-reactivity can be of help in clinical decision making in the acute care setting of returning travellers suspected of having malaria. The global burden of malaria is largely carried by the world's malaria-endemic regions with as many as 225 million cases and a death toll of more than 750,000 individuals in 2009 . In striPlasmodium falciparum malaria, RDTs are not thought to provide sufficient information about parasitaemia, one of the major determinants of disease severity [® Malaria Test allows a semi-quantitative assessment of parasitaemia and rapid exclusion of high P. falciparum parasitaemia, which may facilitate clinical decision making in the acute care setting.Recent studies in non-endemic industrialized countries showed that rapid diagnostic tests (RDTs) for malaria provide an excellent tool for diagnosis of malaria as compared to peripheral blood smears . Althougseverity . In the P. falciparum load in routine clinical practice, an operational laboratory study was conducted at two hospital-based laboratories with expertise in malaria diagnosis in The Netherlands . Of all patients detailed demographic, clinical and laboratory data were available, as well as the outcome measures severe malaria and death. Severe malaria was diagnosed according to predefined WHO criteria in travellers [In order to assess the utility of this RDT as a semi-quantitative measure of avellers . In both® Malaria Test was used as RDT and performed according to the manufacturer's instructions. The Binax NOW® Malaria Test uses monoclonal antibodies that target the histidine-rich protein 2 (HRP-2) antigen specific to P. falciparum (the 'T1' line) and the pan-malarial antigen aldolase (the 'T2' line), common to all five Plasmodium species that can be detected in humans  [The RDTs were performed on fresh blood samples, simultaneously with microscopy of the blood slides. Every RDT and blood slide was read by two independent, experienced laboratory technicians. The Binax NOWnowlesi) . Aldolasum the 'T' line anFor external validation of the reproducibility of the RDT findings in the Dutch centers, a reference center for malaria diagnosis participated , Antwerp, Belgium). In this setting in thick and thin blood films, stained with Giemsa , parasite densities were determined by counting asexual parasites against 200 white blood cells (WBC) in thick blood films and converting this number to parasites/μl using the actual white blood cell count [P. falciparum malaria were included. All patients had HRP-2 reactivity in the Binax NOW® Malaria Test. The general characteristics and laboratory findings of 184 evaluable patients from Amsterdam  and Rotterdam  are provided in Table A total of 257 patients with Co-reactivity of HRP-2 and aldolase was observed in blood specimens with both low and high parasitaemia, ranging from 56 to 558,000 (Amsterdam), 23 to 1,380,600 (Rotterdam) and 26 to 400,000 asexual parasites/μl (Antwerp), respectively. The proportion of aldolase co-reactivity substantially increased with increasing parasitaemia  in absence of aldolase reactivity, proved a reliable predictive marker for a low (< 1%) ® in 80% of patients with P. falciparum parasitaemia > 40,000 parasites/μl [P. falciparum parasitaemia [Co-reactivity of aldolase and HRP-2 was present in all patients with a parasitaemia above 50,000 asexual parasites/μl (corresponding to approximately 1% infected red blood cells). Others reported co-reactivity with Binax NOWsites/μl . The autsitaemia . In the The data of the present study suggest that aldolase and HRP-2 co-reactivity is present in all patients with severe malaria. Apparently HRP-2 and aldolase reactivity, which depends on the load of these antigens in the blood specimen, is preserved because these antigens are derived not only from circulating viable and non-viable malaria parasites, but also from sequestered parasites that are abundantly present in severe malaria. As a consequence of this sequestration, microscopic determination of peripheral blood smears might underestimate the total parasitaemia.P. falciparum parasitaemia, as observed with Binax NOW® in this study, could be dependent of the process of manufacturing. Other RDTs with a similar three-band configuration, therefore, should be studied in detail, to assess about the precise relationship between P. falciparum parasitaemia and aldolase reactivity.The relation between aldolase reactivity and Plasmodium infection. In a recent study of 2,847 cases of imported malaria in the Netherlands [P. falciparum whereas the remainder was caused by P. vivax (15%), P. ovale (7%), and P. malariae (3%), respectively. Only 0,7% of all infections was attributable to mixtures of species, mostly involving P. falciparum. Thus, in the Dutch setting, HRP-2 and aldolase co-reactivity is far more likely to reflect a mono-parasitic P. falciparum infection rather than a mixed infection.HRP-2 and aldolase co-reactivity may also be a feature of a mixed herlands , 75% of P. falciparum malaria [P. falciparum infection in Africa; other malaria-endemic continents like South-East Asia and South-America were underrepresented. Caution is warranted with extrapolating the applicability of the current findings to imported malaria acquired outside Africa. False negative results have been suggested for certain genetic polymorphisms of HRP-2 geographically confined to the Asia-Pacific region [P. falciparum isolates from South America lacking HRP-2 [® Malaria Test), resulting in a false low HRP-2 signal, whereas aldolase reactivity was not affected [P. knowlesi infection, which may give rise to severe disease and fatal complications as well [P. knowlesi infections, later studies demonstrated that P. knowlesi was reactive with the aldolase band in the Binax NOW® Malaria Test, but not with HRP-2 and that aldolase reactivity depended on the P. knowlesi parasitaemia [Results of RDT's may facilitate clinical decision making in patients suspected of having malaria. There are however also some drawbacks to consider. First, these tests cannot replace clinical assessment of the ill-returning patient and results of RDT tests should always be confirmed by thin or thick blood smears, including parasite counts in case of  malaria ,12. Secoc region  and for ng HRP-2 . In addiaffected . Finally as well . Even thsitaemia .® Malaria Test allows a rapid semi-quantitative assessment of P. falciparum load in travellers with malaria returning from the tropics, especially for exclusion of high (>1%) parasitaemia in the acute care setting. This may facilitate clinical decision making for subsequent oral anti-malarial treatment or timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment.In conclusion, the RDT Binax NOWThe authors declare that they have no competing interests.TvG and PvG designed the study, acquired, analysed and interpreted data, drafted and revised the manuscript, MvW acquired and analysed data, and revised the manuscript, RK, PvT, JJ and JvH acquired, analysed and interpreted data, and revised the manuscript. All authors read and approved the final manuscript.


Malaria is the commonest cause of childhood morbidity in Western Kenya with varied heamatological consequences. The t study sought to elucidate the haemotological changes in children infected with malaria and their impact on improved diagnosis and therapy of childhood malaria.Haematological parameters in 961 children, including 523 malaria-infected and 438 non-malaria infected, living in Kisumu West District, an area of malaria holoendemic transmission in Western Kenya were evaluated.The following parameters were significantly lower in malaria-infected children; platelets, lymphocytes, eosinophils, red blood cell count and haemoglobin (Hb), while absolute monocyte and neutrophil counts, and mean platelet volume (MPV) were higher in comparison to non-malaria infected children. Children with platelet counts of <150,000/uL were 13.8 times (odds ratio) more likely to have malaria. Thrombocytopaenia was present in 49% of malaria-infected children and was associated with high parasitaemia levels, lower age, low Hb levels, increased MPV and platelet aggregate flag. Platelet aggregates were more frequent in malaria-infected children  and associated with thrombocytopaenia rather than malaria status.Plasmodium falciparum malaria exhibited important changes in some haematological parameters with low platelet count and haemoglobin concentration being the two most important predictors of malaria infection in children in our study area. When used in combination with other clinical and microscopy, these parameters could improve malaria diagnosis in sub-patent cases.Children infected with  Plasmodium falciparum[P. falciparum malaria in Kisumu, western Kenya. Haematological parameters  of children less than 5 years infected with P. falciparum are compared with uninfected children from the same community. In this study, haematological patterns and their possible predictive values of malaria infection are identified.Blood is the most easily accessible diagnostic tissue. Changes in haematological parameters are likely to be influenced by any disease condition which affects the haemopoetic physiology at any level. This is likely to happen with an endemic disease such as malaria that affects the host homeostasis at various fronts resulting in a myriad of clinical presentation. Malaria is a major cause of morbidity in the tropics. Two hundred and forty seven million cases were reported worldwide in 2006 . Haematoalciparum-6. This alciparum. Presumpalciparum,8. Microalciparum-9. Haemaalciparum,10,11. FThis study was approved by Kenya Medical Research Institute (KEMRI) Scientific Steering Committee and Ethical Review Committee, Walter Reed Army Institute of Research (WRAIR) Scientific Review Committee, US Army Medical Research and Materiel Command (USAMRMC), Human Subjects Research Review Board (HSRRB), and the PATH Human Subject Protection Committee (HSPC). The parents/guardians of the study participants gave a written informed consent before being enrolled into the study. The consent included long-term secure storage of collected samples and information generated during the study and their use for research. The study was conducted at the Kombewa, Kenya Medical Research Institute – US Army Medical research Unit-Kenya (KEMRI/USAMRU-K) Clinical Trails Unit in Kisumu West District of western Kenya an area of intense malaria transmission. The data used in this study were collected during the screening and follow-up phases of a malaria vaccine trial in March – December 2005 . The stuP. falciparum malaria and 438 non-malaria infected children from the same community.Laboratory records of children enrolled in the vaccine trial were reviewed. Nine hundred and sixty one children (age <5yrs) were included in this analysis. This included 523 children diagnosed with The investigations were performed on venous blood sample drawn into EDTA tubes for preparation of the thick and thin smears for malaria parasites and automated determination of Complete Blood Counts (CBCs). Blood counts were performed using ACT5 Diff Haematology Analyzer  as per local SOPs within 1 hour.Daily Quality Assurance checks were performed and recorded; commercial controls were used in accordance with manufacturer’s recommendations. The Analyzer provided data on WBCs, RBCs, haemoglobin level, platelet counts, Mean platelet volume, red cell distribution width (RDW) and five part differentials. The Analyzer also detected and flagged as platelet aggregation and cold agglutinins based on the particle size using a 256-channel pulse–height analyzer of platelet histogram region.Two blood slides were prepared and stained with Giemsa. One slide from each study participant was examined independently by two experienced microscopists. They determined presence or absence of malaria parasites, the species and the number of asexual parasites. Parasite enumeration was performed on thick film/200 WBCs or thin film/2,000 RBCs, depending on the parasite density. Results of the two primary readers were averaged if concordant and used for the calculation of parasite density. Non-concordance in species or parasite counts between the primary readers was referred to a third microscopist (tie breaker) whose determination of species and parasite count was considered final. Parasite densities were calculated as parasite/µL of blood . Percent parasitaemia was calculated as follows; parasite count per µL of blood/RBCs per µL x 100. These blood samples were also used for determining the presence of haemoglobinopathies .P-values, whereas data not conforming to normal distribution were compared by Mann-Whitney U test. Categorical variables were compared using Fisher’s exact test. Association between two continuous variables was assessed by Spearman’s rank correlation. Diagnostic accuracy of haematological parameters was measured by computing sensitivity, specificity, predictive values and odds ratios. Precision of these parameters was evaluated using 95% confidence intervals.Data analysis was performed using GraphPad prism software . Normally distributed continuous data were compared using student’s t-test with 2 tailed P. falciparum malaria confirmed by microscopy while the remaining were negative and were used as controls.Nine hundred and sixty one children were included in this study. The mean age was 32 months (5-48) and 56% were males. Fifty four percent (n=523) of the subjects had The mean values of selected haematological parameters for the non-malaria infected group (controls) were determined for this population stratified by sex. T-test showed no significant difference in Hb, platelets, total WBCs, RDW, and RBC counts between males and females. As a result, subsequent analysis of continuous variables was not stratified by sex.Haematological parameters of the malaria parasitemic group were compared with those of the controls using t-test (Mann-Whitney U test). Median values for Haemoglobin, Platelet count, RBC count lymphocyte and eosinophils counts were significantly lower for the parasitemic group compared with the controls. Conversely, the Mean platelet volume (MPV), Monocyte and neutrophil counts were significantly higher in the parasitemic group. There was no significant difference in total WBC and RDW between the parasitemic and the non-parasitemic groups Table .<5g/dl. Severe malaria anaemia was consequently defined as Hb<5g/dl in the presence of hyperparasitaemia  [P<0.0001). Three hundred and twelve (60%) of the malaria-infected children had anaemia with 18(3%) being severe compared to 118 (27%) in the non-malaria infected group all being mild anaemia. There was poor correlation between parasitaemia and haemoglobin .Anaemia was defined as haemoglobin level <10g/dl for both males and females and further classified as severe if Hb ites/μL) . The med-=4% and α*=16%) of non anaemic children  compared to 25% . Thrombocytopaenia was reported in 255 (49%) of the malaria-infected children with 25 (5%) being severe as compared to 16 (4%) in the non-malaria infected group which had no cases of severe thrombocytopaenia (Table Thrombocytopaenia was defined as platelet count <150 x 10P=0.0003) and age . There was a strong inverse correlation between platelet counts and mean platelet volume  and Non-infected  group. There was an inverse relationship between parasitaemia and platelet counts   ranged from 0.0004 to 29 with a mean of 1.9 . Most children (60%) had parasitaemia ≤1%, 157 (30%) had values between 1 - 5%, 37 (7%) had values between 5.1-10%, and 17 (3%) had a parasitaemia >10% . Thirteen percent of children with normal platelet counts had platelet aggregation.Platelet aggregation refers to the clumping together of platelets in the blood and comprises homogenous clusters of 3-12 individual platelets. When present in the blood sample they are detected by the Analyzer on the basis of their size using a 256-channel pulse–height analyzer. Platelet aggregate flag is generated by the Analyzer when platelet aggregation is detected. Significantly more children in the malaria-infected group had platelet aggregation . Stratifying the data by different platelet count revealed an inverse relationship between platelet aggregation and platelet count irrespective of malaria infection status  was comparable with that of children with normal platelet counts 36%). The presence of cold agglutinins was correlated with malaria infection as 64% of the children with cold agglutinins also had malaria (Table %. The prp=0.0028), there was no significant difference in total WBC between the two groups . However leukocyte components were significantly affected. Neutrophil count , monocyte count  were all significantly elevated in the malaria-infected group as compared to the non-malaria infected group. Eosinophil count  and lymphocyte count  were significantly reduced in the malaria-infected group as compared to the non-infected group (Table 3/uL)  compared to those not infected. Malaria infected children were also more likely to have lymphocytopenia defined as count <2.0 x 103/uL compared to the non-infected ones . Monocyte count was positively correlated with parasite density  and negatively with age . There was no demonstrable relationship between total WBC, neutrophil or lymphocyte counts with parasite densities or age.Leucocytosis was defined as total white cell count>17,000/μL. Although leucocytosis was frequently seen in the malaria-infected group than the non-infected group  times more likely to have malaria than children with normal platelet Table .A combination of thrombocytopaenia and anaemia with high sensitivity 80%), specificity (84%) and odds ratio (22) makes it the best predictor of malaria  [This study confirms that haematological abnormalities considered hallmark of malaria infection are common and more pronounced in on (DIC) ,11,14.P. falciparum as compared to those not infected (Table - α*) could be the most important in the current study singly or in combination as shown in . Additionally monocyte count was positively associated with parasitaemia and negatively with age. The high monocyte count had been reported in patients with uncomplicated malaria [In this study, although leucocytosis was frequently seen in the malaria-infected children, no significant difference in WBC was found between the two groups. In contrast, other studies have demonstrated leucopaenia ,7,19 or  malaria  and subj malaria . None of malaria ,21,23 ch malaria ,14 the d malaria . Lymphoc malaria . This ma malaria .Plasmodium during malarial attack, produce inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 and interleukin-6 . These cytokines stimulate the hepatic synthesis of acute phase inflammatory proteins, including CRP, which increase in malaria.Mononuclear cells, which are activated by Phagocytosis of malaria pigments, infected and uninfected RBCs by monocytes, and neutrophils, has been observed in blood films of patients with malaria and is associated with disease severity ,11,21,23p=0.0003) as previously reported from a study in Nigeria [Plasmodium vivax parasitaemia and increased cytokine production [Platelet abnormalities in malaria are both qualitative and quantitative. In this study, platelet counts were significantly reduced in malaria-infected children. Thrombocytopaenia occurred in 49% of malaria cases and was inversely related to parasite density Figure . Platele Nigeria , which soduction . In the P. falciparum infected erythrocytes [Thrombocytopaenia seems to occur through peripheral destruction , excessihrocytes . This cohrocytes  possiblyPlatelet clumping was the most important platelet functional abnormality observed in this study. A large number of small platelets are seen mixed or clamped with a few giant platelets possibly due to the cytokine interference of megakaryopoiesis . The finp=0.6) Table et al[Significantly more samples in the parasitaemic group had platelet aggregation suggesting that the aggregation was associated with malaria. However, when the data was stratified according to different platelet count ranges, a different pattern emerged suggesting that the platelet aggregation frequency was mainly related to platelet count rather than malaria infection status given that the number of thrombocytopaenic samples with the platelet aggregation in the malaria infected group increased in parallel with that of thrombocytopaenic samples in the non-malaria infected group  (Table 4 % (TablThe present study demonstrates that low haemoglobin levels (OR=5) and low platelet counts OR=13.8) are the two most reliable haematological parameters in predicting falciparum malaria in children from endemic areas. Children with thrombocytopaenia were 13.8 times more likely to have malaria than those with normal platelet count while anaemic children were 5 times more likely to have this outcome than non-anemic children. Thrombocytopaenia and anaemia with sensitivity of 90% and 74%, specificity 61% and 65% respectively, would result in use of anti-malarial drugs in 35%-39% of malaria negative cases, but would only result in 10-26% of true malaria cases not being treated (Table .8 are thWhen haematological parameters were evaluated in combination, anaemia and thrombocytopaenia still had the greatest power in predicting malaria infection Table . This stP falciparum produces significant changes of haematological parameters in children living in malaria endemic regions. The most commonly affected parameters are platelets, haemoglobin, absolute monocyte counts and MPV. Thrombocytopaenia and monocytosis are associated with parasite density and, therefore, may be a marker of disease severity. The reduced platelet counts observed in some children may be related in part to pseudo-thrombocytopaenia but immune mediated thrombocytopaenia may not be ruled out. Although there was a trend towards platelet aggregation being associated with thrombocytopaenia but the frequency may still be primarily associated with malaria parasitaemia.In conclusion, infection with Presence of thrombocytopaenia in combination with anaemia and monocytosis in children from endemic areas may be useful as supportive diagnostic criteria for malaria in circumstances where definitive microscopic or RDT may be sub-optimal, as may be the case with low parasite density. Therefore, when used in addition to clinical and microscopy parameters, it can significantly improve malaria diagnosis and ideally prompt timely initiation of anti-malarial therapy. Further studies are required to characterize the cold agglutinins, platelet aggregates and explain their association with malaria.The large sample size used in this study gives this analysis power to detect differences between malaria infected and non-malaria infected cases. Limitations include lack of previous medical history including anti-malarial treatment for the non-infected cases, which could potentially affect the interpretation of the results. Additionally no further investigations were done to rule out other infections such as bacterial and viral that could produce such haematological alterations since diseases such as HIV are associated with thrombocytopenia . HoweverNone of the authors had conflict of interests in the results of the study.Study was funded by the PATH-Malaria Vaccine Initiative, the Malaria Vaccine Development Program of the U.S. Agency for International Development, and the US Army Medical Research and Materiel Command.The views of the authors do not necessarily reflect the position of the Kenya Medical Research Institute or the Department of the Army or the Department of Defense of USA.RNM – coordinated specimen collection and analysis of clinical specimens, statistical analysis, manuscript writing and participated in study design. GH – participated in specimen collection and oversight for malaria microscopy. JW- coordinated Hb typing. DW, CG, LO and DJ participated in data analysis and manuscript writing. BRO Conceived the study, participated in study design, data analysis, manuscript writing and was the lead study investigator. All authors read and approved the final manuscript.


Plasmodium falciparum and Plasmodium vivax co-exist and malaria rapid diagnostic test (RDTs) is vital in rendering parasite-confirmed treatment especially in areas where microscopy from 2008 to 2010 is not available. CareStartTM Malaria Pf/Pv combo test was evaluated compared to microscopy in Butajira area, south-central Ethiopia. This RDT detects histidine-rich protein-2 (HRP2) found in P. falciparum, and Plasmodium enzyme lactate dehydrogenase (pLDH) for diagnosis of P. vivax. The standard for the reporting of diagnostic accuracy studies was complied. Among 2,394 participants enrolled, 10.9% (n=87) were Plasmodium infected (household survey) and 24.5% (n=392) health facility-based using microscopy. In the household surveys, the highest positivity was caused by P. vivax , P. falciparum , and the rest due to mixed infections of both . In health facility, P. vivax caused 78.6% (n=308), P. falciparum caused 20.4% (n=80), and the rest caused by mixed infections 1.0% (n=4). RDT missed 9.1% (n=8) in household and 4.3% (n=17) in health facility-based surveys among Plasmodium positive confirmed by microscopy while 3.3% (n=24) in household and 17.2% (n=208) in health facility-based surveys were detected false positive. RDT showed agreement with microscopy in detecting 79 positives in household surveys (n=796) and 375 positives in health centre survey .Malaria is a major public health problem in Ethiopia. Plasmodium and P. falciparum (77.2%) in health centres; and Plasmodium (76.7%) and P. falciparum (87.5%) in household surveys. NPV was low in P. vivax in health centres (77.2%) and household (87.5%) surveys. Seasonally varying RDT precision of as low as 14.3% PPV (Dec. 2009), and 38.5% NPV (Nov. 2008) in health centre surveys; and 40-63.6% PPV was observed in household surveys. But the influence of age and parasite density on RDT performance was not ascertained. Establishing quality control of malaria RDT in the health system in areas with low endemic and where P. falciparum and P. vivax co-exist is recommendable. CareStartTM RDT might be employed for epidemiological studies that require interpreting the results cautiously. Future RDT field evaluation against microscopy should be PCR corrected.RDT performance varied in both survey settings, lowest PPV (64.3%) for  Plasmodium falciparum and Plasmodium vivax are epidemiologically the most important malaria parasites in the country [Plasmodium falciparum has been the major causes of high case fatality. Since 2005, the National Malaria Control Programme intensified the deployment of key malaria interventions including artemisinin-combination therapy (ACT), malaria rapid diagnostic tests (RDTs), and vector control measures [In Ethiopia, approximately 68% of the recently estimated 92 million total population, about 63 million people, is at risk of malaria infection ,2. Typic country . Plasmodmeasures ,8, and lP. falciparum, those which target the parasite enzyme lactate dehydrogenase (LDH) and aldolase that can detect non- falciparum from mixed infection. LDH test uses either monoclonal antibodies which react with LDH of all species including P. falciparum (or known as Pan-LDH), or antibodies specific for P. falciparum LDH [P.vivax-specific LDH.Malaria RDTs were introduced as one of the diagnostic methods following the revision of malaria diagnosis and treatment guideline in 2004 . The priarum LDH -13. RDTsP. falciparum were recommended [P. vivax was documented in some highland areas [P. falciparum, and consequently P. vivax may be misdiagnosed as malaria negative and treated based on clinical grounds. Moreover, World Health Organization (WHO) recommends that malaria case management be based on parasite-based diagnosis [P. falciparum and P. vivax is helpful in improving the early diagnosis and treatment of malaria at peripheral areas. CareStart™ Malaria Pf/Pv combo test was among multi-species detecting RDT according to WHO product testing [RDTs for detection of only ommended , and havommended ,15. Howeommended ,16. Follnd areas ,18. At ciagnosis . Thus, t testing . Various testing -25. Howe testing -28.There is paucity of information regarding the precision of malaria RDTs in the low-endemic highland zones of Ethiopia. Therefore, the present study was aimed at evaluating the performance of CareStart™ at health centres and concurrent surveys in highland areas of south-central Ethiopia.kebeles  part of the Demographic Surveillance site (DSS) of Butajira Rural Health Programme (BRHP) [This study was conducted in Butajira area, situated 130 km south of Addis Ababa, in six of ten e (BRHP) , and adme (BRHP) .kebele has a health post. Drug venders are also available in Butajira Town.There were 58,335 people living in the BRHP-DSS in 2008 and half  of the population were female. Of those 26,834 (46%) people lived in the study area. Malaria is endemic in the area with frequent outbreak that caused significant cases and deaths as documented previously . The locP. falciparum. Thus, CareStartTM Malaria Pf/Pv combo test was among the options for the field evaluation before introducing to malaria control program of the country.This study was conducted while the National Malaria Control Program of Ethiopia has initiated various local academic and research institutions for field evaluation of RDTs that could detect multi-species and replace RDTs that only detect A prospective household and health facility-based surveys were conducted to assess the performance of CareStart™ Malaria Pf/Pv combo test in reportedly febrile cases between 2008 and 2010. Six rural kebeles such as Hobe, Bati Lejano, Dirama, Shershera Bido, Yeteker and Wurib were randomly selected. A total of 750 households were selected randomly from those kebeles, using probability proportion to size (PPS) sampling. Individuals with the history of fever in the previous 48 hours of the survey period were included in both household and health facility-based surveys. Febrile cases visiting outpatient department (OPD) suspected of malaria cases at Butajira and Enseno health centres were recruited. In general, this study complied with Standards for Reporting of Diagnostic Accuracy guidelines (STARD) to improve the quality of reporting .Six cross-sectional surveys were performed on the same households for two consecutive years. The surveys were conducted in October-November 2008 (a month after the main rainy season), January-February 2009 (dry season), June-July 2009 (main rainy season), October-November 2009, January-February 2010, and June 2010 in the household surveys. The health facility-based survey was done during October 2008, November 2008, August 2009, September 2009, October 2009, November 2009, and December 2009 .Data collection was done using pre-tested structured questionnaire and format prepared for this purpose. Pre-testing of the data collection tool was performed in an adjacent The health facility-based survey was organized as a research team of four health personnel assigned from the same health centre. Overall, the study was supervised by the principal investigator (AW), a laboratory technician, and two OPD staffs were the member of the team. A health centre head was responsible for coordination of the survey. Those OPD staffs each assigned to screen malaria suspected febrile cases at pediatric and adult OPD separately. During the data collection, participants were requested for blood specimen to be screened for malaria parasite using CareStart™ Malaria Pf/Pv combo test . In addition, thin and thick film was prepared immediately for microscopic examination. CareStart™ RDTs are individually packaged with alcohol swab, lancet, capillary tube, buffer, and test device.Both blood slides and the RDT devices were given identical code for later comparison of results. Specimen collection and reporting result for the malaria RDT was performed using the recommended procedures by the company . First, Plasmodium positive, asexual parasite count was performed using standard procedures of light microscopy as recommended by World Health Organization [Then, thin and thick films were prepared and Giemsa-stained for comparison using light microscopy. Blood specimen processing, examination, and reporting of results were performed using standard guidelines . In brienization . The numnization .P. vivax. Other lines (test line 1) is printed with a monoclonal antibody specific to HRP2 of P. falciparum, and the control area (line 3). So, the CareStart™ Malaria Pf/Pv combo test is designed for the differential diagnosis between P. vivax and P. falciparum[P. falciparum; ii) two colour bands (one band in the control area and another band in the “2” area) indicates a positive result for P. vivax; iii) three colour bands  indicates a positive result for mixed infection for P. falciparum and P. vivax; iv) only one band in the control area within the result window indicates a negative result; and v) the test is invalid if the line in the control area does not appear were used in the interpretation of RDT results [CareStart™ Malaria Pf/Pv combo test contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line 2) is specific to pLDH of alciparum. The fol results . In addiA malaria microscopist temporarily stationed at BRHP-DSS , did theIn order to assure quality of the microscopic examinations, in both surveys, the entire positive and 10% of the negative slides were sent to senior malaria microscopist and re-examined at Adama Malaria Control Laboratory, Adama Town, Ethiopia. In the household surveys, seven of 2,094 slides (0.3%) showed discordant results. A third reader, blinded to the previous results, re-examined the seven discrepant slides . Similar procedure was followed for the survey held at health centres. Each reader was blinded to the RDT results and others’ slide reading results. To ensure maximum participation, households with absentees were re-visited once.This study obtained ethical approval from Institutional Review Board of the College of Health Sciences of the Addis Ababa University, and from the Ministry of Science and Technology of Ethiopia. Local health authorities at all levels also gave permission to conduct the study. Individual informed consent was obtained from adults, and from the parents or guardians of children aged less than 18 years. In addition, minors gave verbal assent. All study participants including minors had the right to refuse. Blood specimens were collected aseptically using an alcohol swab and disposable blood lancets by trained staff. All people found to be malaria positive during the survey were treated directly according to the national guideline .Plasmodium vivax positives were treated with chloroquine, 25 mg/kg for three days . Artemether-lumefantrine (20 mg artemether plus 120 mg lumefantrine in a fixed dose combination) was administered, based on body weight, two times a day for three days to Plasmodium falciparum-positive patients [Febrile patients were screened for malaria parasites on the spot using the RDT, and blood slides were also collected for the survey to investigate the nature of the malaria parasite using light microscopy. Malaria positive cases detected using malaria RDTs were treated immediately. Moreover, those found to be malaria positive on microscopy were also treated. Data entry and cleaning was done using Epi Info version 6  and exported to SPSS. Data analysis was performed using IBM SPSS Statistics 20. Sensitivity, specificity, positive and negative predictive values of the test was computed with 95% confidence interval (CI) by taking malaria microscopy as reference.The kappa statistic or agreement between the test and microscopy was calculated using data observed and probability of expected as recommended . A kappaTM performance was based on self-reported 2,394 febrile cases from public health facilities  and household surveys . The participants’ mean (±SD) age was 13.9 ± 13.0 (from one month to 70 years), and median 10 years in health centre surveys, and 19.7 ± 18.2 (from one month to 99 years) with median 13 years in household surveys. More participants were recruited from Enseno Health Centre  than from Butajira Health Centre  in health facility-based surveys. A total of 31 infants  were suspected for malaria screened (Data not shown).The evaluation of CareStartSeasonal variation of malaria suspected cases observed in both survey settings, which was higher during October-November 2008 in household surveys, and October 2009 and November 2009 at health centres Figure .Plasmodium positive in the household surveys. Of those, 83.9% (n = 73) were caused by P. vivax, 15.0% (n = 13) were due to P. falciparum, and the rest 1.1% (n = 1) were mixed infections due to both vivax and falciparum malaria. Similarly, 24.5% (n = 392) of people were malaria infected among febrile cases visited health centres. Among them, 78.6% (n = 308) were P. vivax, 20.4% (n = 80) were P. falciparum, and the rest 1.0% (n = 4) were mixed infections due to both vivax and falciparum malaria  of people examined were Plasmodium positive confirmed by microscopy. Similarly, 3.3% (n = 24) in household and 17.2% (n = 208) in health facility-based surveys were detected false positive. No invalid test results were observed  in household and 4.3% (n = 17) in health facility-based surveys among 3.3% n = 4 in housPlasmodium infection varied among different age groups, and males were more infected than females in both survey settings. Higher proportion of the malaria infection occurred during peak malaria transmission in both survey settings. About 67% of the total positives cases were screened during October-November 2009 and about 20% during October-November 2008 in household surveys. Moreover, above half of the positives during October 2009 and one-fifths during August and November 2009 each, in the health facility-based surveys  and P. falciparum (87.5%) in household survey; and in P. vivax (92.8%) in health facility surveys. Similarly, low specificity observed in overall Plasmodium (82.7%), and P. falciparum (92.8%) in health facility surveys, and in P. vivax (87.5%) in household surveys.Table Plasmodium (64.3%) and P. falciparum (77.2%) in health facility; and overall Plasmodium (76.7%) and P. falciparum (87.5%) in household surveys. Negative predictive value of the test was good in both overall Plasmodium and P. falciparum. However, lowest NPV was found in P. vivax in both health facility (77.2%) and household (87.5%) surveys. The test and microscopy showed almost perfect agreement for household and health centres surveys  of P. falciparum with asexual parasite count of ≥ 500 p/μl were not detected by CareStart™ RDT. Moreover, the RDT failed detecting 10.3% (n = 7) of vivax malaria, five of those with ≥ 500 p/μl and the rest two with <200 p/μl in household survey. Only a single (1 of 13) falciparum case was missed by RDT in household survey (Table P. vivax (n = 15) (data not shown).In health facilities, 4.5% (n = 10) of microscopy confirmed P. falciparum and P. vivax in “active” and clinical settings of highland-fringe areas of south-central Ethiopia. A greater proportion of RDTs than slides gave positive result for Plasmodium. In the survey, the test showed low sensitivity and high specificity for P. falciparum, but vice versa for P. vivax. The test revealed lower PPV for P. falciparum and lower NPV for P. vivax. Overall, season substantially influences RDT precision with good sensitivity at health facilities compared to household survey. However, specificity was found to be low in both settings with comparable results. Interestingly, better NPV obtained unlike lowest PPV in both survey settings. RDT showed low sensitivity in children aged below 10 years. No strong evidence for contribution of low parasite density for missed Plasmodium detection by the test product.This study found varying precision of CareStart™ RDT to detect This study has got some limitations. This study used light microscopy for comparison of performance of RDT, in spite of its shortcomings related to variable techniques in specimen preparation and slide reading, and mainly the level of expertise of the examining microscopist . Thus, PP. falciparum (87.5%) and P. vivax (92.8%) using CareStart™ RDT is comparable with a study conducted in south-west  and North-West  Ethiopian highland areas [P. falciparum and P. vivax); North-East ; and South-West  Ethiopia [The present finding of low sensitivity for nd areas ,24. But Ethiopia ,23,25. TEthiopia , and in Ethiopia .The varying RDT performance in different seasons in the present household survey is generally consistent with another study in hypoendemic highland zones in Kenya and Uganda . MoreoveVarious factors such as clinical and epidemiologic characteristics of the study populations, reference standards and products of different lots influence the results of RDT-based diagnosis ,13,16. TP. vivax  and P. falciparum (survey) disagrees with other studies that consistently found multi-species RDTs to better detect P. falciparum infection than non-falciparum infection, most probably P. vivax[P. vivax may be interpreted as due to low performance of multi-species RDTs for this species. The low sensitivity obtained in this study may be explained by several factors pertinent to the manufacturing process and environmental conditions [The present finding of low sensitivity of  P. vivax,42. The nditions ,16,20. TIn addition, low-prevalence, or hypoendemic, malaria poses particular diagnostic challenges since low population prevalence reduces the PPV of tests . MoreoveP. falciparum and P. vivax for survey and health facility might be interpreted as the difference in clinical condition of patients. Study participants visiting health facilities might be clinically more specific than otherwise. Review evidences showed that higher RDT sensitivity in studies involving patients seeking relief from moderate to severe disease in clinical settings while low RDT sensitivity in studies that enrolled patients by means of “active” case finding [The varying performance of sensitivity for  finding ,41.P. vivax at both survey and health centres. A past study showed that less sensitive for non-P. falciparum than for P. falciparum[. There is rich evidence that extremely low sensitivity for both HRP-2 and p-LDH tests and batch specific problems were suspected [P. falciparum is likely due to sequestration [The RDT had low NPV for alciparum. There iuspected . This imuspected . The prestration . False pstration ,20. Crosstration .P. falciparum and P. vivax co-exist. CareStart™ RDT could be used for epidemiological studies and results interpreted cautiously by considering its seasonal variation. Future research targeted to RDT evaluation should consider the use of a more sensitive reference standard such as PCR.In conclusion, it appears plausible to put malaria RDT quality control system in place for health systems operating in highlands with low endemicity and seasonally varying transmission where The author(s) declare that they have no competing interests.AW contributed to conception and design, data acquisition, analysis and interpretation, and drafting the manuscript. WD substantially contributed to conception and design of the study and reviewing the manuscript, revisiting it critically for important intellectual content. AA substantially contributed to conception and design of the study, reviewing the manuscript, and revisiting it critically for important intellectual content. BL substantially contributed to conception and design, analysis and interpretation of data, reviewing the manuscript, revisiting it critically for important intellectual content. BL, AA, WD and AW reviewed the paper and all authors approved the final version.


Insecticide-treated bed nets (ITN) reduce malaria morbidity and mortality consistently in Africa, but their benefits have been less consistent in Asia. This study’s objective was to evaluate the malaria protective efficacy of village-wide usage of ITN in Western Myanmar and estimate the cost-effectiveness of ITN compared with extending early diagnosis and treatment services.Plasmodium falciparum and Plasmodium vivax infections, and the biting behaviour of Anopheles mosquitoes in the area were studied concurrently. The trial data along with costs for current recommended treatment practices were modelled to estimate the cost-effectiveness of ITNs compared with, or in addition to extending the coverage of early diagnosis and treatment services.A cluster-randomized controlled trial was conducted in Rakhine State to assess the efficacy of ITNs in preventing malaria and anaemia in children and their secondary effects on nutrition and development. The data were aggregated for each village to obtain cluster-level infection rates. In total 8,175 children under 10 years of age were followed up for 10 months, which included the main malaria transmission period. The incidence and prevalence of , malaria infections, spleen rates, haemoglobin concentrations, and weight for height, did not differ significantly during the study period between villages with and without ITNs, with a weighted mean difference of −2.6 P. falciparum episodes per 1,000 weeks at risk . In areas with a higher incidence of malaria there was some evidence ITN protective efficacy. The economic analysis indicated that, despite the uncertainty and variability in their protective efficacy in the different study sites, ITN could still be cost-effective, but not if they displaced funding for early diagnosis and effective treatment which is substantially more cost-effective.In aggregateIn Western Myanmar deployment of ITNs did not provide consistent protection against malaria in children living in malaria endemic villages. Early diagnosis and effective treatment is a more cost effective malaria control strategy than deployment of ITNs in this area where the main vector bites early in the evening, often before people are protected by an ITN. Plasmodium falciparum have been high in this region for decades , 2,408 children visited the clinics with complaints of fever and were confirmed by microscopy to have malaria; 1,102 Twelve children  developed severe falciparum malaria; in Dabhine, six severe patients were reported from the NN-villages and two from the ITN-villages. In Myothugyi, one severe patient was reported from the NN-villages and three from the ITN-villages. Thus, overall seven children developed severe malaria in NN villages and five in ITN villages.P. falciparum this was 8.4% (341/4066) in ITN villages and 15.2% (618 /4109) in NN control villages respectively, a weighted difference of −6.9% , p = 0.23, and for P. vivax, 9.9% (401/4066) in ITN villages versus 12.3% (505/4109) in NN villages, a weighted difference of −2.6% , p = 0.25 , p = 0.35  had at least one vivax infection during the 10 month study period (13.7% in ITN villages and 21.0% in NN villages) and children in their second year of age had 185 (19.4%) infections (18.1% in ITN villages and 20.7% in NN villages). After the second year the proportion of children with an episode of vivax malaria decreased steadily by age and only 3.7% of 9-year-old children (3.8% in ITN villages and 3.7% in NN villages) had an episode of vivax malaria in the same period.Of the 8175 children registered, 7764 (95%) were present at all three ‘prevalence’ surveys. Of the other 411 children, 46 children reportedly died during the study-period ,  and 365 had moved out of the area or were absent during one or two of the follow-up surveys .P. falciparum and P. vivax, and also in the splenomegaly prevalences among the villages  in NN villages and 4.2% (161) in ITN villages. Some protective efficacy from impregnated bed nets was seen in six village pairs, while four village pairs showed the opposite effect; overall difference - 1.9% , p = 0.30  in NN and 11.6% (446) in ITN villages. Four village pairs suggested protection by ITN while five did not, and in one village pair, both clusters, had no vivax malaria at all; overall difference = −2.8% ; p = 0.30. For splenomegaly prevalence, six village pairs suggested protection by ITN, while four did not; overall difference = − 7.3% , p = 0.23. The overall splenomegaly prevalence was 14% in bed net villages and 21% in non-bed net villages.The prevalences of children with malaria and splenomegaly were measured at the end of the study (January – early February 1999), which coincided with the end of the peak transmission season. A total of 7,828 children were present at this survey. There was considerable variation in malaria prevalence, both for es Table  and 4. Tnd 4.2% 11 in ITN The prevalences of anaemia (Hb <10 g/dL) and malnutrition  at baseline in the ITN and NN villages were similar. The weighted mean difference in the prevalence of anaemia was −5.2%  and the weighted mean difference in the prevalence of malnutrition was 0% . The mean haemoglobin level of children during the first cross-sectional survey was lower in Myothugyi  than in Dabhine . The proportion of anaemic children (Hb < 10 g/dL) decreased during the study period in all but two clusters (1 NN and 1 ITN village). At the final cross-sectional survey, the decrease in the proportion of children with anaemia in bed net villages was more pronounced than in control villages in 7 cluster pairs while 3 pairs showed the opposite trend  decreased over the study period in 14 of the 20 villages; 8 ITN villages and 6 NN villages Table . At the . The model output suggests that in the absence of ITN or EDAET, 1.4 DALYs are accumulated per 1,000 person-weeks at no cost to the provider. The use of ITN would add a cost of $35 and reduce the number of DALYs to 0.69, or an incremental cost-effectiveness ratio (ICER) of $51/DALY averted. The use of EDAET alone would cost $23 per 1000 person-weeks and reduce DALYs to 0.19, or an ICER of $19 per DALY averted (i.e. nearly three times less). While both options are considered cost-effective, the implementation of ITN instead of EDAET would incur higher costs and avert less than a third of the number of DALYs. Where EDAET is already in place the introduction of ITN would avert an additional 0.2 DALYs at a cost of $148 per DALY averted. The cost-effectiveness acceptability curves  services are less costly and substantially more effective than ITN, when comparing each of these to a baseline of no intervention, which is still the reality in some areas in Myanmar. Once EDAET is implemented effectively, ITNs can still provide additional benefit.During the extended interval since this study was carried out there has been a consistent and substantial decline in malaria incidence across the region, including many areas in Myanmar even when allowing for improved reporting rates . While tITNs are very popular with international donor agencies. Millions of dollars are spent on ITN programmes in this populous malaria endemic region. ITNs are both provided free by donor organizations or promoted through social marketing, encouraging the population to purchase their own ITN. In this malaria endemic region of Myanmar, the economic modelling based on the study data and contemporary prices suggests that early-diagnosis and effective treatment is substantially more cost-effective than deployment of LLINs as a malaria control measure. This argues for careful evaluation in each region before large-scale deployment. The findings here provide some evidence of a protective efficacy of ITNs in areas of higher endemicity within this region, suggesting that they can be a cost-effective intervention in the context of improving child health. If, however, donor funding is targeting specifically the control of malaria and ultimately its elimination from the region it seems likely that improving preventive, diagnostic and curative interventions for all ages and particularly in adult, often migrant males, could offer better returns on investment.Anopheles mosquito vectors [Malaria infections, palpable splenomegaly, haemoglobin concentrations, and weight for height, did not differ significantly during the study period between villages with and without ITNs. The limited efficacy of ITNs may be explained by the biting behaviour  of the most common  vectors . Given tThe authors have no conflicts of interest concerning the work reported in this paper.FS, JAS and NJW conceived the idea for the paper. JAS, KS, MKK and FS conducted the analysis. YL conducted the economic evaluation. FS and NJW wrote the paper with input from all other authors. All authors have approved the final version of this manuscript.Change in prevalence of falciparum malaria, vivax malaria, and spleen enlargement from before to after the intervention. Table showing change in prevalence of falciparum malaria, vivax malaria, and spleen enlargement comparing the prevalence before (December 1997) and after (January 1999) the intervention and the relative change between ITN and NN villagesClick here for filePrevalence of falciparum malaria before the study. Description Prevalence of falciparum malaria before the start of the study comparing ITN and NN villages.Click here for filePrevalence of vivax malaria before the study. Description Prevalence of vivax malaria before the start of the study comparing ITN and NN villages.Click here for filePrevalence of splenomegaly before the study. Description Prevalence of palpable spleens before the start of the study comparing ITN and NN villages.Click here for filePrevalence of falciparum malaria after the study. Description Prevalence of falciparum malaria after the study comparing ITN and NN villages.Click here for filePrevalence of vivax malaria after the study. Description Prevalence of vivax malaria after the study comparing ITN and NN villages.Click here for filePrevalence of splenomegaly after the study. Description Prevalence of palpable spleens after the study comparing ITN and NN villages.Click here for fileEffects on anaemia. Description Change of proportions of children with anaemia (Hb < 10 g/dL) from the 1st to the 3rd cross-sectional survey in ITN and NN villages.Click here for fileEffects on malnutrition. Description Change of proportions of children with malnutrition (Wt/Ht < −2Z) from the 1st to the 3rd cross-sectional survey in ITN and NN villages.Click here for file