P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1.Antibodies to P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies.We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1. Plasmodium falciparum malaria eventually develops after repeated exposure to infection, and is characterised by control of blood-stage parasitemia and prevention of clinical illness and severe complications P. falciparum merozoites invade erythrocytes during blood stage replication, and antibodies that inhibit invasion by targeting merozoite antigens are believed to be important for acquired immunity Immunity to A containing recombinant AMA1 of the 3D7 strain, there was no significant protection against clinical malaria overall, but there was a significant reduction in risk of clinical malaria caused by parasites expressing vaccine-like AMA1 alleles, suggesting strain-specific protective efficacy The merozoite antigen apical membrane antigen 1 (AMA1) is a leading vaccine candidate and appears to be an important target of acquired immunity. It plays a key role in erythrocyte invasion The crystal structure of AMA1 reveals a long hydrophobic trough in domain I that appears to be a binding site for proteins forming an erythrocyte invasion complex comprised of AMA1 and RON proteins P. falciparum. However, to date, it has not been possible to directly measure AMA1-specifc inhibitory antibodies among individuals in relation to protection from clinical malaria, and better understand the acquisition of inhibitory antibodies. Furthermore, knowledge on the acquisition of antibodies to polymorphic and conserved epitopes in relation to immunity is limited P. falciparum AMA1 by standard methods in vitro growth of 3D7 and D10 parasite strains, but not HB3 or W2mef Antibodies to AMA1 are thought to contribute to protective immunity by inhibiting erythrocyte invasion and blood-stage replication of Using the 1F9 and 2C5 mAb in competition ELISAs we were able to indirectly measure naturally acquired antibodies to two polymorphic epitopes of AMA1, one of which is recognised as a functional epitope and a potentially important immune target. Our aims were to define the acquisition of antibodies to these epitopes in relation to age, parasitemia, and markers of exposure to malaria, examine the association between antibodies and risk of malaria in a longitudinal cohort of children, and assess the relationship between antibodies to the inhibitory 1F9 epitope and growth inhibitory antibodies.Ethical clearance was provided by the Ethics Committee of the Kenya Medical Research Institute, the Human Research Ethics Committee of the Walter and Eliza Hall Institute and the Alfred Hospital Human Research and Ethics Committee (for the Burnet Institute). Written informed consent was provided by all subjects or their guardians.Samples were obtained from two villages, Chonyi and Ngerenya, located in Kilifi district, Kenya In the Chonyi cohort, participants were enrolled and finger-prick blood was collected in October 2000 for baseline antibody measurements and blood smears for microscopy. Asymptomatic parasitemia was not treated. Participants were followed by active and passive case detection for symptomatic illness for 210 days. Blood-smears for malaria diagnosis were performed on any symptomatic individuals. Malaria was defined as a temperature greater than 37.5°C with a parasitemia above 2500 parasites/µl. The cohort has been described in detail elsewhere Samples were collected from subjects in the Ngerenya study site in September 1998, including 150 children and adults  Monoclonal antibodies (mAb) to the AMA1 epitopes 1F9 and 2C5 were used Escherichia coli using the full ectodomain of 3D7 (21). Schizont protein extract was prepared from P. falciparum (A4) by sonicating highly synchronous parasites on ice Measurement of serum antibodies to the 3D7 allele of AMA1 and schizont extract was carried out using standard enzyme linked immunosorbent assay (ELISA) Epitope-specific antibodies to AMA1  were measured indirectly using competition ELISAs, which measured the ability of serum antibodies to compete with binding of epitope-specific monoclonal antibodies, 1F9 and 2C5. ELISA plates were coated with 0.5 µg/ml of AMA1 overnight at 4°C. Wells were washed three times and incubated with blocking buffer at 37°C for two hours. The optimal concentrations of mAb 1F9 and 2C5 were established in preliminary experiments by performing serial dilutions of each mAb. Standard curves were plotted and the optimum amount of each mAb was selected as 0.2 µg/ml. This value was close to saturation but still on the linear part of the curve such that there was enough mAb to occupy most specific sites in the absence of competing antibody. Sera were diluted in sample buffer together with the appropriate mAb . The serum/mAb mix was added to duplicate wells and incubated at 37°C for one hour. The plates were washed three times and incubated with secondary antibody (HRP conjugated rabbit mouse IgG diluted at 1∶2500 in sample buffer) for one hour at room temperature. After three washes, plates were incubated in the dark with ABTS substrate system  for 15 minutes. The development reaction was stopped using 1% SDS. Optical density was measured at 405 nm. Results were expressed as the percentage inhibition relative to binding of the monoclonal antibody in the absence of serum ((OD no serum – OD serum)/OD no serum x100), where high % inhibition values indicated high levels of epitope-specific antibodies in the serum sample. The cut-off used to define inhibitory samples was the mean +2 standard deviations of negative control sera tested in parallel.In vitro inhibition of P. falciparum growth by human serum was tested by growth inhibition assay (GIA) with the growth of parasites expressed as a percentage of the maximal growth achieved in control wells. Assays were performed using established methods P. falciparum 3D7 parasites. The cultures were diluted to a starting parasitemia of 0.4–0.8% and 1% hematocrit at a volume of 25 µl per well with 2.5 µl of sample sera added into duplicate wells. Positive controls of pharmaceutical grade heparin (100 µg/ml) All analyses were performed using STATA version 9.2 . Median antibody levels were compared using the Mann Whitney or Kruskal Wallis test. Chi square tests were used to assess associations between antibody prevalence and age or exposure. Correlations between antibody levels and parasite growth inhibition were determined using Spearman’s rank correlation.P. falciparum infection status at enrolment had a significant influence on the association between antibodies and risk of malaria Associations between antibodies and risk of symptomatic malaria (with parasitemia >2500/µl) were examined in children aged 1–10 years from the Chonyi cohort. Analysis was performed until day 210 of follow-up, with the first 14 days excluded to reduce the effect of baseline parasitemia accounting for malaria episodes in the follow-up period. Although some children had multiple episodes of malaria, only the first episode was included in the analysis. Survival analysis was performed among children from 1 year to 10 years of age (n = 228), because prior studies have shown that associations between antimalarial antibodies and protective immunity in this population are restricted to this age range, and adults were excluded to reduce the potential confounding effect of age and because few adults experienced malaria during follow-up  in Chonyi and 27 older children and adults for comparison. The prevalence of AMA1 IgG was high at 82.3% (95% CI 77.6–87.1%). Antibodies to specific epitopes were less common, with 20.7% (95% CI 15.9–25.5) of children showing inhibition of 1F9 binding and 52.0% (95% CI 45.8–58.3%) inhibiting 2C5 binding by competition ELISA. Median anti-1F9 activity  was lower than anti-2C5 activity . Some samples showed negative values of inhibition, which suggests enhanced binding of the monoclonal antibody to its epitope in the presence of serum (discussed further below). Inhibition of mAb binding to the 1F9 and 2C5 epitopes correlated with levels of antibodies to the AMA1 ectodomain . There was also a positive correlation between anti-1F9 and anti-2C5 activity .P. falciparumThe prevalence of antibodies to the AMA1 ectodomain increased with increasing age  and 96% individuals over 10 years were classified as positive for AMA1 IgG . AntibodAdditional studies on antibodies to the AMA1 ectodomain and the 1F9 epitope were performed among 100 children (aged 2–14 years) and 50 adults of the Ngerenya cohort to allow comparison between antibodies to AMA1 and growth inhibitory antibodies (there was insufficient sample volume available to measure growth inhibitory antibodies in the Chonyi cohort) and to further evaluate antibody acquisition among children versus adults. The prevalence of IgG to the AMA1 ectodomain was lower than for Chonyi at 73.3% (95% CI 65.7–79.8%), despite inclusion of adults in this sample set, reflecting lower transmission intensity in this population. When the population was divided into three age groups , levels of AMA1 IgG were significantly different between groups, with lower levels amongst 2–5 year olds . At the time of sampling, 39.3% of the population were parasitemic, and there was a significant difference in the level of IgG to AMA1 between aparasitemic and parasitemic individuals .The level of inhibition of 1F9 binding (%) shown by most samples was low , with negative values suggesting enhanced binding of 1F9 to its epitope. Thirty one (23.9%) individuals were defined as 1F9 inhibitors (inhibition of 1F9 binding>(mean +2 standard deviations of negative controls = 6.19%)). There was a moderate correlation between anti-1F9 activity and IgG to the AMA1 ectodomain . When including all Ngerenya samples, neither levels of anti-1F9 activity or prevalence of 1F9 inhibitors was significantly different between the three age groups. However, after stratifying by parasitemic status, levels of anti-1F9 activity were significantly different between age groups amongst aparasitemic individuals , but not amongst parasitemic individuals . The medThe longitudinal follow-up of children in the Chonyi cohort allowed us to prospectively examine the association between antibodies at baseline and risk of clinical malaria during 6 months active follow-up. Two hundred and twenty eight children between the ages of 1 and 10 years were included in this analysis. Only children 1–10 years of age were included, since this is the group acquiring immunity in this community  = 0.02), the 1F9 epitope , or the 2C5 epitope  . The Cox proportional hazards model was used to calculate risk estimates and adjust for potential confounding. In the univariate analysis, younger age (P<0.001) and concurrent parasitemia at sampling (P = 0.04) were associated with significantly higher risks of clinical malaria. Associations between antibody levels and protection from malaria were assessed, with the dataset stratified by parasitemic status and the multivariate analysis adjusted for age. When antibody responses were analysed without taking into account parasitemic status, there was significantly less risk of a clinical malaria episode at any point during the 6 months after sampling in those with a high antibody response to AMA1  . However = 0.06) . Among t = 0.06) , althougTo further understand the relevance of the different antibody responses in immunity, we examined associations with protection for combinations of responses. The risk of experiencing a clinical episode at any time within 6 months of sampling was investigated in Chonyi children with high responses to combinations of AMA1 responses compared to their low responder counterparts using Cox proportional hazards; analysis compared children who were high responders to a combination of two responses  versus those who were low responders to the same two responses. There was a greatly reduced risk of a clinical malaria episode during follow-up amongst children with high responses to AMA1 and 1F9  or high responses to AMA1 and 2C5  ; the hazP. falciparum , we then restricted our analysis to individuals with higher reactivity to schizont protein extract ; we reasoned that those with low levels of antibodies to AMA1 would have little growth inhibitory activity. Amongst individuals with high schizont reactivity, there was a significant correlation, with decreasing parasite growth as AMA1 IgG levels increased   . High AMf growth . When thA negative correlation between parasite growth and serum antibody reactivity to the 1F9 epitope was expected, as the 1F9 monoclonal antibody inhibits parasite invasion. Although parasite growth in the presence of serum ranged from 10% –65% (relative to control), most individuals had low levels of antibodies to1F9 such that there was no significant correlation between anti-1F9 activity and parasite growth when including all samples  . HoweverP. falciparum and antibodies to full-length AMA1, and were generally higher in individuals with active parasitemia, although the pattern and strength of associations differed for the two epitopes. The demonstration that acquired human antibodies target the 1F9 epitope is particularly interesting, because 1F9 MAb blocks AMA1 function and inhibits invasion. Our findings suggest that individuals acquire antibodies that have a similar function to that of 1F9 mAb and target similar epitopes to inhibit invasion. Consistent with this, antibodies to the 1F9-epitope correlated with antibody growth-inhibitory activity, suggesting that people do acquire AMA1-specific growth-inhibitory antibodies through natural exposure. Presently it is not possible to quantify AMA1-specific inhibitory antibodies among individuals and examine the prevalence and pattern of acquisition of these antibodies. Therefore, these findings provide important insights into understanding the functional activity of antibodies to AMA1, which are believed to play a role in protective immunity In this study, we have shown that individuals living in malaria-endemic settings acquire antibodies to polymorphic epitopes of AMA1, the inhibitory 1F9 epitope and the 2C5 epitope. Antibodies to both epitopes were acquired in association with increasing age and correlated with a marker of exposure to blood-stage High-level antibodies to AMA1, the 1F9 epitope and the 2C5 epitope were associated with significantly reduced risk of clinical malaria during follow-up in Chonyi children, and the association was strongest for antibodies to the inhibitory 1F9 epitope. However, antibodies were significantly and positively associated with increasing age, and associations with protection were no longer statistically significant after adjusting for age. Therefore, data on the associations between antibodies and protection should be interpreted with caution, and further studies are needed to investigate this association in other populations. These findings are broadly consistent with published studies on the same cohort, which showed antibody levels to the full length ectodomain of AMA1  were associated with a reduced risk of malaria Interactions between antibodies of differential specificities  may be synergistic, antagonistic or neutral in their influence on disease outcome, and analysis of combinations of antibodies may reveal associations with protection that are not identified when responses to individual antigens are assessed Age is often considered as a potential confounding factor in studies assessing the association between immune responses and protection from malaria. In our study, we limited our survival analysis to children aged 1 to 10 years of age because prior studies have shown that this was the age range during which malaria immunity was acquired in this population The prevalence of 1F9 antibodies was low in both populations (less than 25%) despite the high prevalence of antibodies to the AMA1 ectodomain (above 75%), and was lower than prevalence of antibodies to the 2C5 epitope. The affinity of mAb 1F9 appears to be high (about one hundred times that of 2C5) The lower prevalence of antibodies to 1F9 and 2C5 epitopes compared to the AMA1 ectodomain could reflect their polymorphic nature and the incidence of exposure due to 3D7-like alleles of AMA1. Although we do not have specific data on the prevalence of the 3D7 AMA1 allele at the study sites, 3D7 appears to be one of the more common AMA1 variants found in Africa  P. falciparum and that naturally acquired human antibodies could compete with 1F9 mAb binding in competition ELISAs  The ability of serum to inhibit 1F9 mAb binding correlated with growth inhibitory activity, although the association was only evident in sera with higher overall levels of antibodies to AMA1. This is the first study to demonstrate a link between naturally acquired human antibodies to the 1F9 epitope of AMA1, or any functional epitopes of AMA1, and growth inhibitory activity in a large set of human sera. Our findings support earlier studies which showed that the 1F9 mAb inhibits in vitro growth of A AMA1 vaccine Further studies to confirm that antibodies to the 1F9 epitope are protective are needed and will have important implications for development of an AMA1 vaccine, since growth inhibitory antibodies targeting the 1F9 epitope are likely to be predominantly strain specific. The strain specific nature of vaccine-induced protective antibodies targeted to the region encompassing the 1F9 epitope was suggested by the recent clinical trial of the FMP2.1/AS02In conclusion, our studies suggest that naturally-acquired antibodies target the invasion inhibitory 1F9 epitope and non-inhibitory 2C5 epitope of AMA1, are acquired with increasing exposure to malaria, and show some association with a reduced risk of clinical malaria. The ability of acquired antibodies to inhibit the binding of mAb 1F9 and the correlation between these antibodies and growth inhibitory activity suggest that AMA1 is a target of naturally acquired growth-inhibitory antibodies in humans. Together, these findings provide further evidence supporting AMA1 as an important target of protective immunity. Competition ELISAs provide a useful tool for measuring the fine specificity of human malaria antibodies, and could be used in future studies of acquired immunity and AMA1 vaccine trials to assess invasion inhibitory antibodies targeting the 1F9 epitope. Additionally, these approaches may be valuable as markers of immunity for monitoring and surveillance of malaria in populations. Futures studies to further investigate these epitopes as targets of acquired and vaccine-induced immunity, particularly the inhibitory 1F9 epitope, define other functional and important epitopes, and better understand antigenic diversity of AMA1 will be valuable to further quantify the importance of AMA1 as an immune target and its priority as a vaccine candidate, and facilitate the development and testing of AMA1 vaccines.


Plasmodium falciparum histidine-rich protein 2 can be used to distinguish between severe malaria and other severe febrile illness in African children with malaria.Arjen Dondorp and colleagues investigate whether the plasma level of  Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria.In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental PfHRP2 was measured prospectively in African children  aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine . In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611  ng/mL in fatal cases (n = 381) versus 1,046  ng/mL in survivors , without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log10 plasma PfHRP2 and risk of death. Mortality increased 20% per log10 increase in PfHRP2 above 174 ng/mL . A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61  in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile . A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings.Admission plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of “true” severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children.Plasma Please see later in the article for the Editors' Summary. Plasmodium parasite species that cause malaria in humans, with one species, Plasmodium falciparum, causing the most severe disease. However, diagnosing severe falciparum malaria in children living in endemic areas is problematic, as many semi-immune children may have the malaria parasites in their blood (described as being parasitaemic) but do not have clinical disease. Therefore, a positive malaria blood smear may be coincidental and not be diagnostic of severe malaria, and unfortunately, neither are the clinical symptoms of severe malaria, such as shock, acidosis, or coma, which can also be caused by other childhood infections. For these reasons, the misdiagnosis of falciparum malaria in severely ill children is an important problem in sub-Saharan Africa, and may result in unnecessary child deaths.Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected mosquitoes. In 2010, malaria caused an estimated 655,000 deaths worldwide, mostly in Africa, where according to the World Health Organization, one African child dies every minute from the disease. There are four P. falciparum histidine-rich protein-2 (PfHRP2)—is a measure of the total number of parasites in the patient. Unlike the circulating parasites detected on a blood film, which do not represent the parasites that get stuck in vital organs, PfHRP2 is distributed equally through the total blood plasma volume, and so can be considered a measure of the total parasite burden in the previous 48 hours. So in this study, the researchers assessed the prognostic value of plasma PfHRP2 in African children with severe malaria and whether this protein could distinguish children who really do have severe malaria from those who have severe febrile illness but coincidental parasitaemia, who may have another infection.Previous studies have suggested that a parasite protein—PfHRP2 in 3,826 out of a possible 5,425 African children who participated in a large multinational trial  that compared the anti-malarial drugs quinine and artesunate for the treatment of severe malaria. All children had a clinical diagnosis of severe malaria confirmed by a rapid diagnostic test, and the researchers used clinical signs to define the severity of malaria. The researchers assessed the relationship between plasma PfHRP2 concentrations and risk of death taking other well established predictors of death, such as coma, convulsions, hypoglycaemia, respiratory distress, and shock, into account.The researchers assessed levels of plasma PfHRP2 was detectable in 3,800/3,826 (99%) children with severe malaria and that the average plasma PfHRP2 levels was significantly higher in the 381 children who died from malaria than in children who survived . Plasma PfHRP2 was also significantly higher in children with severe malaria signs and symptoms such as coma, acidosis, and severe anaemia. Importantly, the researchers found that high death rates were associated with either very low or very high values of plasma PfHRP2: the odds (chance) of death were 20% higher per unit increase in PfHRP2 above a specific threshold (174 ng/ml), but below this concentration, the risk of death increased with decreasing levels, probably because at lower levels disease was caused by a severe febrile disease other than malaria, like septicemia. Finally, the researchers found that in children within the highest PfHRP2 tertile, the chance of death when treated with the antimalarial drug artesunate versus quinine was 0.61 but that there was no difference in death rates in the lowest tertile, which supports that patients with very low plasma PfHRP2 have a different severe febrile illness than malaria. The researchers use mathematical modeling to provide cut-off values for plasma PfHRP2 denoting the proportion of patients with a diagnosis other than severe malaria.The researchers found that PfHRP2 levels taken on admission to hospital can differentiate children at highest risk of death from severe falciparum malaria from those likely to have alternative causes of severe febrile illness. Therefore, plasma PfHRP2 could be considered a valuable additional diagnostic tool and prognostic indicator in African children with severe falciparum malaria. This finding is important for clinicians treating children with severe febrile illnesses in malaria-endemic countries: while high levels of plasma PfHRP2 is indicative of severe malaria which needs urgent antimalarial treatment, low levels suggest that another severe infective disease should be considered, warranting additional investigations and urgent treatment with antibiotics.These findings suggest that in areas of moderate or high malaria transmission where a high proportion of children are parasitaemic, plasma http://dx.doi.org/10.1371/journal.pmed.1001297.Please access these Web sites via the online version of this summary at small study in PLOS ONE explores the relationship between plasma PfHRP2 and severe malaria in Tanzanian childrenA previous WHO website and the website of Malaria No More have comprehensive information about malariaThe  Severe falciparum malaria in children presents a major diagnostic challenge in malaria-endemic countries where a high proportion of children is parasitaemic at any time. A positive malaria blood smear is therefore not specific for severe malaria, and neither are clinical signs, which are similar to those of other severe childhood infections Plasmodium falciparum histidine-rich protein 2 (PfHRP2) is a water-soluble protein found inside the malaria parasite and host erythrocyte, and that circulates free or bound to proteins or antibodies in the plasma compartment PfHRP2 production peaks during the trophozoite stage, and approximately 90% is released during schizont rupture PfHRP2 is distributed through the total plasma volume, plasma PfHRP2 can be considered a measure of total parasite burden of the preceding 48-hour asexual parasite life cycle PfHRP2, disease severity, and outcome PfHRP2 in African children with severe malaria and tested the hypothesis that its assessment could distinguish children with “true” severe malaria, in need of urgent antimalarial treatment, from those with non-malarial severe febrile illness and coincidental peripheral blood parasitaemia, in whom alternative diagnoses and additional treatment need to be considered.In the current study we assessed the prognostic significance of plasma ISRCTN 50258054), undertaken between October 2005 and July 2010 P. falciparum lactate dehydrogenase (pLDH)-based rapid diagnostic test were included, provided their parents or caregivers gave full written informed consent. Severity was defined by clinical criteria . Slide reading was performed by expert microscopists at the Mahidol-Oxford Tropical Medicine Research Unit, and parasites/µl was calculated from thin film  or thick film (count/200 WBC×40) The study was part of a large multinational trial comparing quinine and artesunate for the treatment of severe malaria in African children , according to the manufacturer's instructions with minor modifications P. falciparum parasitaemia >200,000/µl was calibrated with recombinant PfHRP2 standard  and used to construct standard curves. Concentrations in duplicate plasma dilutions  were determined according to the linear segment of the standard curve, with re-assay in cases where duplicates differed by more than 50%. Plasma samples for PfHRP2 were received from nine of the 11 “AQUAMAT” research sites in seven countries . The study site in Ghana did not collect samples, and the samples from Nigeria defrosted during transportation.Plasma PfHRP2 has been described in detail in Asian adults with severe malaria and requires incorporation of an elimination half-life estimate PfHRP2), which has a higher level in high transmission settings, and PfHRP2 production is parasite strain dependent PfHRP2 half-life was assessed in 30 patients from Tanzania from samples taken on admission and after 3 and 7 days following treatment. Separate ethical approval for this sub-study was obtained from the Ethics Committee of the National Institute for Medical Research, Tanzania. These data were analysed using WinNonlin statistical package . Individual PfHRP2 concentration-time curves were fitted according to a first-order elimination model. From this, a mean (95%CI) plasma elimination half-life (t½) was estimated as 1.10 (0.91 to 1.29) days, or 0.55 erythrocytic cycles. Half-life was not significantly different between treatment arms, and was not correlated with renal function (estimated by blood urea nitrogen [BUN]). A parasite multiplication factor of 3 immediately before peak parasitaemia was assumed, based on in-vitro and Saimiri monkey studies of African parasite strains causing severe malaria tot = 7.3×PfHRP2×(1−Hct)×body weight [kg] ×1013, with PfHRP2 in g/L PfHRP2 half-life and parasite multiplication rates. The circulating parasite burden was calculated from the peripheral blood: parasites/µl×106×blood volume ( = 0.08×weight [kg]) Estimation of the total body parasite burden from plasma 10-normalized variables were compared using a Student's t-test, the remainder by Wilcoxon rank-sum test. For lowest, middle, and highest tertiles of plasma PfHRP2, comparisons were made between peripheral blood parasitaemia, sequestration index, and treatment effect  following artesunate versus quinine treatment.Data were analysed with STATA, version 10 . Categorical variables were compared between survivors and fatal cases with Chi-squared or Fisher's exact test. Normally distributed or logPfHRP2, a logistic regression model was constructed with in-hospital death as the dependent variable and PfHRP2 as the independent variable. Since the risk of death showed a non-linear association with log10PfHRP2 ; (2) a probability of severe febrile illness due to non-malaria, which decreased exponentially with increasing logPfHRP2: Prnon-malaria = exp(−k4logPfHRP2); (3) a risk of death in patients with non-malaria infection equal to 0.3, independent of plasma PfHRP2: Prdeath|non-malaria = 0.3 non-malaria/Deathtotal = 0.2 PfHRP2 stratum is then given by:A mechanistic model was constructed to describe the observed U-shaped relationship between al death , top, manon-malaria = Prdeath|non-malaria×Prnon-malaria×Casestotal and the number of deaths due to malaria by Deathmalaria = Prdeath|malaria××Casestotal. For more details, see DeathPfHRP2 was measured in 3,826 patients. PfHRP2 could not be measured in 1,600 (29%) patients because the sample was either not collected or not received in optimal condition. Patients without PfHRP2 data did not differ from the remainder regarding malaria slide positivity rate, geometric mean parasitaemia, or case fatality rate. Baseline clinical and laboratory characteristics according to outcome are summarized in Of the 5,425 children with pLDH-based rapid diagnostic test RDT confirmed falciparum malaria included in the “AQUAMAT” trial, plasma PfHRP2 was detectable in 3,800/3,826 (99%) patients with severe malaria. A detectable plasma PfHRP2  with a negative blood slide result  was found in 36 (0.9%) children. Geometric mean plasma PfHRP2 (95%CI) in survivors was 1,046 ng/mL  versus 1,611 ng/mL  in fatal cases .PfHRP2 concentrations in relation to established features of severe falciparum malaria are summarized in PfHRP2 was significantly higher in patients with coma, acidosis, and severe anaemia but not in those with shock.Plasma PfHRP2-derived total parasite burden was 7.5×1011 (7.2×1011 to 7.9×1011) parasites/body ; this was greater in fatal cases  than in survivors  . A sensitivity analysis varying the multiplication factor and PfHRP2 plasma half-life is shown in Geometric mean (95% CI) <0.0001) . In contPfHRP2 and risk of death with a nadir in case fatality rate at a logPfHRP2 of 2.24  above a threshold logPfHRP2 value of 2.24 ( = 174 ng/mL). Below this concentration, risk of death increased with decreasing plasma logPfHRP2 . The final model was adjusted for plasma BE, BUN, coma, convulsions, hypoglycaemia, peripheral blood parasitaemia, and antimalarial treatment .There was a U-shaped association between plasma PfHRP2  , the probability that death resulted from malaria fell below 50%, corresponding to overall proportions of malaria-attributable severe disease <90%  and above, the absolute risk of death due to malaria exceeded 8% with a probability of “true” severe malaria >95% and a probability that a death was caused by severe malaria >85% , the odds ratio (OR) for death in patients treated with artesunate versus quinine was 0.61 . In patients in the lowest PfHRP2 tertile, there was no difference in mortalities with an OR for death of 1.05  are biologically plausible, and were significantly higher in fatal cases. In contrast, less pathogenic circulating peripheral blood parasite numbers were not correlated with a fatal outcome. The calculated sequestration index was 17 in surviving patients and 30 in non-survivors, which is similar to the median (IQR) sequestration index of 40 (9.9–273.8) calculated directly from post-mortem blood vessel counts in 50 Thai and Vietnamese adults who died from cerebral malaria Firstly, plasma PfHRP2 and risk of death fits with the assumption that with low PfHRP2, death is caused by non-malarial febrile illnesses (including sepsis) which are independent of the low parasite burden, whereas in patients with plasma PfHRP2 above this nadir the probability of death increases with PfHRP2, representing “true” severe malaria with increasing sequestered parasite burdens. The mechanistic model based on these assumptions had a close fit with the observed data. An alternative explanation could be the presence of highly virulent parasite strains causing severe disease independent of a high total parasite burden. However, this would result in a PfHRP2-independent mortality at the left side of the curve and cannot explain the U shape that was actually observed. Assumptions in constructing the mechanistic model included an alternative cause of death in 20% of patients and a risk of death in non-malaria disease of 30%, based on published autopsy and clinical microbiology data Second, the U-shaped curve with a nadir at 174 ng/mL describing the relationship between PfHRP2 tertile, and strongest in the highest tertile . Since injectable artesunate can benefit only patients with “true” severe malaria, this provides strong supportive evidence that patients with high PfHRP2 do represent this group, and patients with low PfHRP2 do not. The OR of 0.61 in the highest PfHRP2 tertile is remarkably close to the OR of 0.60 (95%CI 0.45 to 0.79) reported in the large SEAQUAMAT trial comparing artesunate with quinine in the treatment of severe falciparum malaria in 1,461 patients in low-transmission settings in Asia Third, the treatment benefit of artesunate over quinine was absent in patients in the lowest PfHRP2 should prompt investigation of alternative diagnoses including septicaemia, early administration of parenteral broad spectrum antibiotics , and intensive monitoring. Often antibiotics are given only after a disappointing clinical response to antimalarials, which may be too late. High plasma PfHRP2 concentrations should not discourage antibiotic treatment combined with antimalarial treatment, because of the high proportion of concomitant invasive bacterial disease PfHRP2, which indicates “true” severe malaria with a poor prognosis, should be monitored closely, preferentially in a high-dependency or intensive care unit. As a tool in the design of clinical trials, plasma PfHRP2 is substantially better than peripheral blood parasitaemia in assessing the malaria-attributable fractions and defining the group of patients with “true” severe malaria and a high risk of death  denotes a probability >95% of “true” severe malaria with an overall case fatality rate of 11.6% (95%CI 10.3 to 12.9). Defining populations with “true” severe malaria and high mortality is thus critical information for clinicians as well as researchers. In contrast, a plasma PfHRP2 concentration <100 ng/mL (8.1% of cases in our cohort) denotes a probability >15% that severe non-malarial illness is the cause of illness, warranting additional investigations.Identification of children with slide-positive severe febrile illness but who do not have severe malaria is important for patient management, since overdiagnosis of severe malaria is associated with increased mortality PfHRP2 is sensitive to the assumed parasite multiplication factor. In the current study the multiplication rate was assumed to be 3, based on in vitro data comparing multiplication rates and multiplication potency of parasites obtained from African children compared to Asian adults. The multiplication rate of 8 used in the original model in Asian adults was based on non-immune adult patient data from the era of malaria therapy of neurosyphilis, and comparable information is obviously not available for our patient group. Applying this higher multiplication rate in this study results in an implausibly high estimated total parasite burden. In addition to differences in parasite multiplication rates, the calculated total parasite burden is dependent on the assumed half-life of plasma PfHRP2, which can vary between patients, and on the amount of PfHRP2 released per parasite per cycle, which can vary between strains PfHRP2 in the current study was shorter than observed in adult patients in Southeast Asia (mean 1.1 versus 3.7 days) PfHRP2 antibody titres could increase plasma clearance of PfHRP2 PfHRP2 and disease severity PfHRP2 values (median 456 ng/mL), whereas patients in that study considered to have uncomplicated malaria had lower plasma bicarbonate concentrations as a measure of acidosis than those with severe malaria. In the present study, <1% cases had undetectable plasma PfHRP2 concentrations, despite presence of P. falciparum on the blood slide. This could have been caused by genetic variation in PfHRP2 PfHRP2 gene have been reported in field isolates from the Amazon region and in a single report from sub-Saharan Africa PfHRP2 gene in parasites from all patients in the current study who had low plasma PfHRP2 concentrations is underway.Limitations of this study include the inherent dependency of the models on certain assumptions. Estimating the total parasite burden from PfHRP2 provides a tool in areas of moderate and high malaria transmission to distinguish “true” severe falciparum malaria from severe febrile illness with incidental malaria parasitaemia. Plasma PfHRP2 concentrations are a valuable prognosticator in African children with severe falciparum malaria.In conclusion, admission plasma Text S1Description of enrolment criteria for severe falciparum malaria.(DOC)Click here for additional data file.Text S2The mechanistic model and sensitivity analysis .(DOC)Click here for additional data file.Text S3PfHRP2 half-life .Sensitivity analysis of the estimated total parasite burden as a function of parasite multiplication factor and (DOC)Click here for additional data file.Text S4PfHRP2 and parasitaemia according to outcome .Plasma (DOC)Click here for additional data file.


Plasmodium falciparum is the species responsible for the most severe form of malaria leading to different complications. Beyond the development of cerebral malaria, impairment of renal function is a mortality indicator in infected patients. Treatment with antimalarial drugs can increase survival, however the long-term effects of malaria on renal disease, even after treatment with antimalarials, are unknown. The aim of this study was to evaluate the effect of antimalarial drug treatment on renal function in a murine model of severe malaria and then evaluate kidney susceptibility to a second renal insult. Initially, mice infected with Plasmodium berghei ANKA achieved 20% parasitemia on day 5 post infection, which was completely abolished after treatment with 25 mg/kg artesunate and 40 mg/kg mefloquine. The treatment also decreased plasma creatinine levels by 43% and partially reversed the reduction in the glomerular filtration rate induced by infection. The urinary protein/creatinine ratio, collagen deposition, and size of the interstitial space decreased by 75%, 40%, and 20%, respectively, with drugs compared with untreated infected animals. In infected-treated mice that underwent a second renal insult, the plasma creatinine level decreased by 60% and the glomerular filtration rate increased compared with infected animals treated only with antimalarials. The number of glomerular cells, collagen deposition and the size of the interstitial space decreased by 20%, 39.4%, and 41.3%, respectively, in the infected group that underwent a second renal insult compared with the infected-treated groups. These functional and structural data show that renal injury observed in a murine model of severe malaria is partially reversed after antimalarial drug treatment, making the kidney less susceptible to a second renal insult.Malaria is a worldwide disease that leads to 1 million deaths per year.  Plasmodium falciparum is the most severe form leading to the development of cerebral damage , respiratory distress, and kidney injury Malaria is a global disease that affects about 500 million people every year; about 1.5 million people die from malaria every year Plasmodium falciparum, indicating that impairment of renal function is a great risk factor for these patients infected with malaria The development of experimental cerebral malaria (ECM) coincides with the accumulation of the parasite in different organs such as the heart, lungs, spleen, liver, gastrointestinal tract, and the kidneys Plasmodium berghei ANKA (PbA), a murine model of severe malaria, is associated with a decrease in cerebral edema after treatment with antimalarial drugs Usually, survival of mice infected with Another important factor is the tubule interstitial injury, which is a crucial step in the progression of renal disease. In this context, tubule interstitial lesions are observed in acute kidney injury induced by malaria infection Two main questions are addressed: (1) Does the treatment of PbA-infected mice with antimalarial drugs lead to complete recovery from AKI even after treatment has been stopped? (2) Are PbA-infected mice treated with antimalarial drugs more susceptible to a second kidney insult? The BSA challenge tubule interstitial injury animal model is used as a second kidney insult. In this animal model, the glomerular flow rate (GFR) is not changed.This work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Ethics Committee of Federal University of Rio de Janeiro (permit number CEUA-CCS-098) and by The Committee on Ethical Use of Laboratory Animals of Fundação Oswaldo Cruz (permit number L004/08).6 erythrocytes infected with PbA in a final volume of 200 µl, as a model of severe malaria; and/or (b) animals were subjected to intraperitoneal injections of saline or 10 g/kg/day bovine serum albumin (BSA) for 7 days, a BSA challenge tubule interstitial injury animal model C57BL/6 male mice (6–8 weeks old) were used. When indicated, the animals were (a) infected or not by intraperitoneal injection of 105d); (2) infected and untreated mice (infected5d); (3) noninfected mice that were treated with antimalarial drugs following the protocol described by Clemmer et al 21d); (4) infected and treated mice (infected+treated21d). Untreated mice and treated mice were euthanized at day 5  and 21, respectively.Two experimental protocols were developed: (1) to determine renal function in PbA-infected mice treated or not with antimalarial drugs ; (2) to 21d); (2) noninfected, untreated, and BSA i.p. injection (control+BSA21d); (3) noninfected, treated, and BSA i.p. injection (control+treated+BSA21d); (4) infected, treated, and no BSA i.p. injection mice (infected+treated21d); (5) infected, treated, and BSA i.p., injection (infected+treated+BSA21d). All animals were euthanized on day 21 p.i.In the second experimental protocol, the animals were randomly divided into five groups: (1) noninfected, untreated, and no BSA i.p. injection  and xylazine (5 mg/kg body weight) before blood collection via cardiac puncture.Parasitemia was determined daily in all infected groups throughout the period in the metabolic cage . For thig for 5 min, and the supernatant was separated and stored at −20°C until use. The levels of protein, urea, and creatinine were determined colorimetrically in urine samples using specific Gold Analisa kits . Blood samples were collected via cardiac puncture in heparinized tubes and centrifuged at 600×g for 5 min to separate plasma. In these samples, the level of plasma creatinine and blood urea nitrogen (BUN) were determined. The glomerular filtration rate (GFR) was determined by creatinine clearance (CCr).After an adaptation period of 24 h, urine volume was determined and collected for the last 24 h before euthanasia on days 5 and 21. Urine samples were clarified at 600×On the days of euthanasia, the kidneys were analyzed histologically as described by Landgraf et al The results are expressed as means ± standard error. Differences between the control and experimental groups were analyzed by two-way analysis of variance followed by the Newman-Keuls test for multiple comparisons.5d group. Urine creatinine was unchanged in all groups tested (data not shown). These effects are correlated to the decrease in CCr, a marker of the GFR (21d group) reversed all these effects. However, the antimalarial treatment did not reverse the effect of infection on the BUN level. In addition, antimalarial treatment (control+treated21d) per se increased the urinary flow, BUN, and CCr, but the serum creatinine level was not changed. The ratio between BUN and serum creatinine was increased in both the control+treated21d and infected+treated21d groups  did not change the UPC ratio, indicating that antimalarial treatment does not promote kidney injury. These results were obtained by analyzing the profile of renal protein excretion using sodium dodecyl sulfate polyacrylamide gel electrophoresis (The urinary protein/creatinine (UPC) ratio is a well-established marker of renal injury 5d group . This inphoresis .5d group) showed a higher number of glomerular cells (21d). Antimalarial treatment (treated21d group) did not change the renal histomorphological parameters measured , the serum creatinine and BUN levels returned to the levels observed in mice subjected only to kidney insult (control+BSA21d and control+treated+BSA21d groups). The serum creatinine level was not changed but BUN was increased in the control+BSA21d group. Moreover, the CCr value was not changed when the animals were subjected to kidney injury (control+BSA21d group) but it was increased when the mice were treated with antimalarial drugs , renal injury was attenuated but still observed. The reason for this relies on exacerbated production of proinflammatory cytokines such as interferon-γ, tumor necrosis factor α, and interleukin-6, -1, and -8 produced during malaria infection. The same cytokines are involved in the pathogenesis of malaria-induced AKI and are correlated with the progression of proteinuria It is well established that malaria-induced renal injury is a consequence of parasite adhesion to renal endothelial cells as well as activation of the host immune response In patients or an animal model with malaria, combined treatment with artesunate and mef loquine is sufficient to abolish parasitemia and attenuate cerebral malaria, contributing to improvement in survival One important question in relation to the treatment is the side effects of antimalarial drugs. Campos et al 21d) and kidney insult (control+BSA21d) groups.An interesting result obtained in the present work is the observation that infected mice treated with antimalarial drugs are less susceptible to a second kidney insult. Antimalarial drugs have been used in the treatment of other pathologies besides malaria. The administration of artemisinin protects against sepsis in mice challenged with lipopolysaccharide by decreasing the secretion of proinflammatory cytokines Malaria and septic shock have important similarities in relation to induction of AKI Plasmodium falciparum remains high, despite the advances in therapy and prevention of disease P. falciparum infection or the combination of different pathologies in endemic areas.The pathology of severe malaria has been extensively explored, however mortality induced by Figure S1Effect of antimalarial treatment on urinary protein. Animals were euthanized on day 5 or day 21 post infection and urine samples were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein analysis was based on the intensity of Coomassie blue staining. Urinary samples were collected from the different experimental groups as depicted in the figure.(TIF)Click here for additional data file.


We identified SNP allele frequency differences between the 2 ethnic groups in CD36, IL4, RTN3 and ADCY9. Moreover, polymorphisms in FCER1A, RAD50, TNF, SLC22A4, and IL13 genes were correlated with antibody production . Further work is required to understand the mechanisms underpinning these genetic factors.Malaria still remains a major public health problem in Mali, although disease susceptibility varies between ethnic groups, particularly between the Fulani and Dogon. These two sympatric groups share similar socio-cultural factors and malaria transmission rates, but Fulani individuals tend to show significantly higher spleen enlargement scores, lower parasite prevalence, and seem less affected by the disease than their Dogon neighbours. We have used genetic polymorphisms from malaria-associated genes to investigate associations with various malaria metrics between the Fulanai and Dogon groups. Two cross sectional surveys  were performed. Healthy volunteers from the both ethnic groups (n=939) were recruited in a rural setting. In each survey, clinical  and parasitological data  were collected, as well as blood samples. One hundred and sixty six SNPs were genotyped and 5 immunoassays  were performed on the DNA and serum samples respectively. The data confirm the reduced malaria susceptibility in the Fulani, with a higher level of the protective O-blood group, and increased circulating antibody levels to several malaria antigens (p<10 Malaria remains the major public health problem in more than 90 countries inhabited by more than 40% of the world’s population, with at least one million deaths every year. More than 90% of all malaria cases occur in sub-Sahara Africa . In MaliMalaria is a complex disease with many genetic and environmental determinants influencing the natural variation in response to infection, progression and severity. Several factors are important for the different phenotypes observed, such as parasite genetic make-up, and host age, state of immunity and host genetic background . There aPlasmodium falciparum malaria antigens  in a context of similar malaria exposure  and NANP [NANP4]) were coated on ELISA plates (Immulon4 ELISA plates/Dynatech), and incubated at 4°C overnight. Plates were washed 3 times with PBS-0.05% Tween 20 (PBS/T) before adding 200 µL of blocking solution (2% skimmed milk in PBS/T) and then incubated for 3 hours at ambient temperature before washing 3 times with PBS/T. Serum samples were added in duplicate and incubated overnight at 4°C. Following washing 6 times with PBS/T, 50 µL of horseradish peroxidase-conjugated rabbit anti-human IgG (DAKO) diluted 1/5,000 in PBS/T was added and incubated for three hours at room temperature. The plates were again washed 6 times with PBS/T and OPD substrate solution (100 μL/well) was added and left at room temperature for 10 to 15 minutes for the assay to develop. Twenty-five microlitres of H2SO4 (2M) were added to stop the reaction and the plates were read at 492 nm in an ELISA reader. The cut-off value of the assay was determined by calculating the arithmetic mean of the absorbance of negative control samples obtained from European individuals who had never been exposed to malaria and adding three standard deviations to that value (mean OD+3SD). Using standard positive  and analysed by ELISA for antibodies against four malarial antigens . Genotypic deviations from Hardy-Weinberg equilibrium (HWE) were assessed using a χ2 statistical test. SNPs were excluded from analysis if they had at least 10% of genotype calls missing or there was significant deviation from HWE (p<0.0001). The SNP association analysis for the (binary) malaria and clinical phenotypes used a logistic regression model including age group and season as covariates. The AMA1, MSP1, MSP2, CSP assay results were logarithmically (base 10) transformed in order to obtain Gaussian distributions approximately (see 10 transformed) immunological titre phenotypes used linear regression models, which included age group and season as covariates. In all regression models, SNP data were included by fitting a series of genetic models , and the minimum p-value reported. Performing multiple statistical tests leads to inflation in the occurrence of false positives. In our setting a Bonferroni correction would be too conservative because many of the SNPs are from a chromosome 5 region and the same genes. We applied a permutation approach that accounted for the correlation between tests, and estimated that a p-value cut-off of 0.003 to ensure a global significance level of 5%. All association analyses were performed on each ethnic group separately. The Fst metric [http://www.r-project.org).All clinical and meta- data were double-entered from a case-report form, and underwent range checks, where any outlier was checked with written records and site staff. Mann-Whitney-Wilcoxon sum-rank tests were used to assess overall ethnic differences with continuous background (e.g. age in months) and phenotypic  variables. Similarly, Pearson’s χtely see S1. The t metric  was usedThe study enrolled 939 participants of which 53.8% were Dogon and 46.2% are Fulani 1. Data -5, adjusting for ethnicity and age). CSP, AMA1 and IgE titres show no differences between seasons, but MSP1 and MSP were significantly lower during the dry season . There were modest (Spearman) correlations between the AMA1, CSP, MSP1, and MSP2 immunoassay measurements , and less between these assays and total IgE  . The rs3211938 polymorphism , with four others greater than 0.1  (see We compared the study characteristics between rainy and dry seasons S3. As e. 0.190) S4. Ther25Y/stop ) had the-5), but not with spleen enlargement (P=0.133). We then considered the role of the polymorphisms on malaria outcomes, spleen enlargement and parasite positivity in each ethnic group (adjusted for age and season) . The rs3091336 (IL3) polymorphism was associated with asymptomatic and any malaria in the Dogon , whilst the rs2304081 (SLC22A4) SNP was associated with these phenotypes in the Fulani ethnic group . For clinical malaria, only significant associations were found in the Fulani (rs17047661 (CR1), OR 5.327, 95% CI 1.733-16.376, P=0.0008; rs2075820 (NOD1), OR 0.364, 95% CI 0.188-0.704, P=0.001). The rs1128127 polymorphism (DERL3 gene) in the Dogon group was the only SNP associated with spleen enlargement . Whilst there were five polymorphisms associated with parasite positivity: (i) rs17047661 ; (ii) rs2304081 ; (iii) rs3212227 ; (iv) rs569108 ; and (v) rs10775349 . A combined analysis (adjusting for ethnicity) identified no polymorphisms associated with spleen enlargement, but one with parasite positivity .There were no strong effects of immunological variables on clinical outcomes (P>0.03) . The associations detected (P<0.003) include: (i) CSP , Dogon; rs3093662, TNF, Dogon), (ii) AMA1 , (iii) MSP1 , (iv) MSP2 , and (v) total IgE . A combined analysis  revealed the following polymorphisms: (i) CSP , (ii) MSP1 , and (iii) total IgE . There were no putative associations for AMA1 and MSP2 in this combined analysis.The effect of the candidate genetic polymorphisms on the immunological assays was considered  living in close proximity in Mali. Using standardized and robust genetic and immunological assays, we found that the Fulani ethnic group had significantly higher levels of total IgE against crude malaria antigens relative to the Dogon ethnic group. This confirms results from other studies in Burkina Faso comparing the Fulani and Mossi ethnic groups , in keeping with those reported in Burkina Faso . We also found strong allele frequency differences between ethnic groups in the rs3211938 , rs2243in Ghana ,; observin Ghana ), rs5429in Ghana ). CD36 . LTA and TNF have long attracted attention as candidate genes for susceptibility traits for malaria, and several of their polymorphisms have been found to be associated with severe malaria phenotypes [We identified genetic loci associated with CSP , AMA1 (IL5), MSP1 , MSP2 , and total IgE .  malaria -36. IL3  malaria ,38. The  malaria . Many im malaria ,41. Furt malaria , and wit in Mali . Worm inenotypes . It has enotypes .It is important to highlight some study limitations. First, it was not possible to assess ethnic differences in seroprevalence from the antibody titre data, as there was not a control population for normalization . Second,These potential limitations withstanding, our study confirms and provides additional insights into the reduced malaria susceptibility in the Fulani ethnic group. An unresolved issue is whether pregnant Fulani women compared to Dogon are more resistant to malaria. In general, further understanding of the mechanisms underlying the reduced susceptibility of the Fulani to malaria could provide essential information for the rational design of malaria vaccines, monitoring of their trial, or implementation of therapeutic strategies to improve public health.P. falciparum malaria infection not only in parasite positivity but also in parasite density than the Dogon. The results of our study suggest that immunogenetic factors may be responsible for the observed differences in malaria–related phenotypes between the Fulani and Dogon ethnic groups. These factors may be helpful in understanding the precise mechanisms of resistance in the nomadic Fulani population. This study was performed in a rural setting and identifies some human genetic factors that are involved in malaria susceptibility and resistance, and thus strengthens previous observations.Our study showed that during the high and low transmission seasons the Fulani tend to be less susceptible to Table S1List of 166 Single Nucleotide Polymorphisms (SNPs).(DOCX)Click here for additional data file.Table S2Squenom Multiplexes.(XLSX)Click here for additional data file.Table S3Study characteristics by season.(DOCX)Click here for additional data file.Table S4Spearman correlations between antibody titres.(DOCX)Click here for additional data file.Table S5Fst).Population differentiation ((DOCX)Click here for additional data file.Table S6Effects of immunology on malaria and clinical outcomes  *.(DOCX)Click here for additional data file.Table S7Genetic association tests for malaria and clinical phenotypes by ethnic group (All Results).(TXT)Click here for additional data file.Table S8Genetic association tests for malaria and Immuno-Assay by ethnic group (All Results).(TXT)Click here for additional data file.Figure S1Transformed titre values and correlations.(DOCX)Click here for additional data file.Figure S2Transformed antibody levels by age group* and ethnicity.(DOCX)Click here for additional data file.


Pulse wave velocity (PWV) is an indirect measure of vascular stiffness. Higher PWV is a recognised cardiovascular risk marker. Magnetic resonance imaging (MRI) is a non-invasive method of assessing PWV. Assessing maternal influences on offspring PWV is important as reduced fetal nutrient supply and impaired early development are linked with an increased risk of cardiovascular disease in adulthood. In multiparous women, changes in the uterine spiral arteries arising during previous pregnancies result in increased fetal nutrient supply. Rat studies have shown that changes in maternal fatty acid intake in pregnancy are associated with increased offspring arterial stiffness. Some studies of human adults suggest omega-3 fish oils reduce arterial stiffness. The objective of the study was to measure vascular stiffness using MRI, and examine maternal influences on vascular structure in children aged 9 years.Aortic PWV was assessed in 125 children aged 9 years  using velocity-encoded MRI as part of a study of developmental influences on cardiovascular structure and function. Maternal diet had been assessed in early and late pregnancy, and the child's diet at age 9 years, using administered food frequency questionnaires.PWV values fell within previously reported childhood ranges. Childhood aortic PWV was lower in offspring of multiparous mothers . Higher maternal oily fish intake in pregnancy was associated with lower childhood aortic PWV , but there was no association with the child’s current fish intake.MRI measurements of childhood aortic PWV indicate that the mother’s parity, and normal variations in maternal oily fish intake in pregnancy, may alter vascular development in utero - changing arterial structure and function with long-term consequences for cardiovascular risk in later life.This work was supported by funding from the British Heart Foundation and the National Institute for Health Research .


Hypertension is an increasing health issue in sub-Saharan Africa where malaria remains common in pregnancy. We established a birth cohort in Nigeria to evaluate the early impact of maternal malaria on newborn blood pressure (BP).timing of malaria parasitaemia as ‘No Malaria’, ‘Malaria during pregnancy only’ or ‘Malaria at delivery’, and parasite density as low (<1000 parasites/µl of blood) and high (≥1000/µl).Anthropometric measurements, BP, blood films for malaria parasites and haematocrit were obtained in 436 mother-baby pairs. Women were grouped to distinguish between the ≤0.005). In babies of mothers who had ‘malaria at delivery’, their SBPs adjusted for other confounders were lower respectively by 4.3 and 5.7 mmHg/kg compared with ‘malaria during pregnancy only’ or ‘none’. In contrast the mean newborn systolic (SBP) and diastolic BPs (DBP) adjusted for birth weight were higher by 1.7 and 1.4 mmHg/kg respectively in babies whose mothers had high compared with low parasitaemia.Prevalence of maternal malaria parasitaemia was 48%, associated with younger maternal age (p<0.001), being primigravid (p = 0.022), lower haematocrit (p = 0.028). High parasite density through pregnancy had the largest effect on mean birth indices so that weight, length, head and mid-upper arm circumferences were smaller by 300 g, 1.1 cm, 0.7 cm and 0.4 cm respectively compared with ‘No malaria’ (all pAs expected, prenatal malarial exposure had a significant impact on fetal growth rates. Malaria at delivery was associated with the lowest newborn BPs while malaria through pregnancy, which may attenuate growth of the vascular network, generated higher newborn BPs adjusted for size. These neonatal findings have potential implications for cardiovascular health in sub-Saharan Africa. Hypertension is now a public health and economic problem in Sub-Saharan Africa with a prevalence up to 33% in urban areas in Nigeria In numerous global studies, adult risk of hypertension and other chronic disease is associated with LBW In newborns, their blood pressure (BP) correlates with birth weight Therefore, we tested the hypothesis that BP at birth would be higher and birth size would be smaller in babies whose mothers had malaria in pregnancy, defined by its timing during pregnancy and/or at delivery and the magnitude of parasite density. This in turn may affect the rate of BP rise through childhood and set the scene for hypertension in later life.Ethical approval was obtained from the joint University of Ibadan / University College Hospital Ethics committee and the University of Manchester Ethics committee. The study protocol and the rationale for the study were explained carefully in appropriate language, most commonly Yoruba or English, with questions answered as needed and written informed consent was obtained from all participants. After the delivery of their babies, another written informed consent was also obtained for the participation of their babies in the study.A semi-urban community, Yemetu-Adeoyo, Ibadan in Southwest Nigeria, where malaria transmission is perennial, was the site for the study. Families come from a range of socioeconomic backgrounds. The local community hospital, Adeoyo Maternity Hospital (AMH), the oldest maternity hospital in Nigeria dating from 1927, provides primary and secondary medical care with over 4000 deliveries each year.Healthy women aged 18–45 years presenting at AMH before 36 weeks gestation and residing within the catchment area of the study centre for at least 2 years were recruited. Singleton babies born at ≥37 weeks gestation were included.At booking, women were tested for sexually transmitted infections and HIV and those positive were excluded and those with chronic diseases such as hypertension and diabetes. Preterm deliveries, babies with known syndromes, metabolic defects, congenital abnormalities or severe birth trauma were excluded.Women were enrolled over one year to cover both wet and dry seasons.In the year, 3496 women booked for ante-natal care, but many were not planning their delivery at AMH so 659 women were eligible, of which 624 were recruited but 161 still did not deliver at AMH; thus the final cohort included 463 mother-baby pairs. Of these, 27 were excluded due to 4 (0.9%) maternal deaths, 11 (2.4%) still births, 5 (1.1%) miscarriages and 7 (1.5%) neonatal deaths, leaving 436 pairs. There were no significant differences in the socio-demographic and clinical data of excluded women.Standard operating procedures (SOPs) were developed. Informed consent, using forms translated into Yoruba, was taken at booking, then demographic, obstetric, family and health details, malarial history and use of antimalarial drugs was recorded. All women were issued with prescriptions for sulphadoxine-pyrimethamine (SP) for Intermittent Preventive Therapy (IPT) for malaria according to standard hospital practice.Standardized measures of anthropometry and BP were taken at every antenatal visit until delivery, weight to the nearest 0.1 kg , height on a stadiometer to the nearest 0.1 cm, both without shoes, according to the SOP and training video.2 areas. Total leucocyte number was calculated as 50× the number of cells counted.At each visit throughout pregnancy, 2 ml of blood was obtained in EDTA tubes for haematocrit, leukocyte count and blood film for MP. For haematocrit, a capillary tube was filled to about 80% with blood, sealed with a flame and centrifuged for 5 minutes at 10,000 revolutions per minute. The haematocrit value was read with a heamatocrit reader. For the leucocyte count, anticoagulated blood at a one in 20 dilution in turk's solution was mixed for about 1 minute and placed in the counting chamber. The sample was left to settle, and the white cells were counted using a microscope (10× objective lens) in the four outer 1 mmBlood films were prepared, stained with 3% Giemsa at pH 7.2 and examined for malaria parasites (MP) under light microscopy. Thick smears were recorded as negative only after 200 high-power microscope fields had been scanned. In those with malaria, absolute parasite counts were determined as previously reported For quality control, 30% of negative samples and 40% of positive samples were re-examined by two different trained microscopists.Women were followed up until delivery based on routine ante-natal practice, determined by gestational age. Repeat MP blood films were obtained every visit and at delivery when a film was also prepared from cord blood. The placenta was weighed, turned to the maternal surface, cotyledons exposed and 1 ml of blood obtained from the intervillous space for a placental malarial blood smear. Detailed delivery information was recorded.Babies were weighed naked to the nearest 0.1 kg, length was measured from crown to heel on an infant stadiometer to the nearest 0.1 cm and occipito-frontal circumference (OFC) around the widest circumference of the head using a non-stretchable tape to the nearest 0.1 cm. Skinfold thicknesses  were measured using Holtain calipers on the left side to the nearest 0.1 mm. Measurements were obtained in duplicate or triplicate if disagreeing by >15%. All babies were examined within 72 hours of birth.Maternal and newborn BPs were taken according to a standard protocol and SOP. Before the BP reading, the woman was comfortably seated and relaxed with the back and arm supported, the legs uncrossed, for at least 5 minutes and not moving or speaking. Her upper arm was supported at the level of the heart with no tight clothing constricting the arm. The measurement was carried out on the left arm with validated Datascope BP monitor using appropriate-sized cuffs .Before performing the BP reading, the baby was comfortably lying on the mother's lap for at least 5 minutes, and many times they were asleep. The measurement was again done with the Datascope BP monitor, specifically validated for infants, using appropriate newborn cuffs on the left arm. Babies were measured within 72 hours of life. In both mother and child, measurements were repeated three times at least one minute apart and the mean of the last two readings used for analysis.Three nurses trained in anthropometry and BP methods, based on the WHO manual (1995) and SOPs, carried out all measurements throughout the study on the same equipment. Inter-observer and within-observer error were minimized through 3-monthly refresher training sessions.Anaemia was defined as packed cell volume (PCV)<30%.Malaria was defined as asexual blood stages of Plasmodium falciparum in peripheral blood or placenta of the pregnant women or cord blood at delivery. All visits during pregnancy and at delivery were taken into account.For this study, women were first grouped into 2 categories:‘No Malaria’ - no parasites detected throughout pregnancy or at delivery (n = 225).‘Malaria present’ - parasites present at least once during pregnancy and/or at delivery (n = 211).timing of malaria through all visits in pregnancy and at delivery:Women with malaria were then stratified to distinguish between the ‘Malaria during pregnancy only’ - presence of malaria parasites at least once during pregnancy but not at delivery (n = 138).‘Malaria at delivery’ - mothers with parasites present in the placenta and/or their peripheral blood sample at delivery and/or in the cord blood (n = 73).To examine effects of parasite load during pregnancy and at delivery, parasite density was classified into low (<1000 parasites/µl), or high (≥1000/µl) based on the highest density recorded at any time point'.Data were analysed using SPSS version 14 . Socioeconomic index scores were based on occupations and educational attainment of both parents on scales I to V, as previously All women recruited had at least 2 antenatal clinic attendances, 94% attended 3 times, 80% four times and 63% five times before delivery. The median (range) durations of the pregnancy at booking and at delivery were 28 (12–36) and 39 (37–42) weeks respectively with 28% of women being primigravida.timing, 138 (31%) had malaria parasitaemia at some time during pregnancy only and 73 (17%) at delivery.Parasitaemia was present at least once in pregnancy and/or delivery in 211 of the 436 recruited mothers . Classified by 2 = 5.276, p = 0.022) and associated with nearly a 3-fold increase in risk of having malaria .56% of those with parasitaemia were primigravid, so malaria and first pregnancy were significantly associated .About half of the women reported the use of preventive measure such as chemoprophylaxis / insecticide spray or coil / bed nets / netted windows but these were not associated with protection from malaria (p>0.05). Social class and maternal malaria parasitaemia were also not associated , they were younger  and more likely to be anaemic than those without parasitaemia Table 1.There were no differences in other clinical characteristics such as pregnancy duration at booking, weight, height, body temperature, SBP and DBP in women with and those without malaria Table 1. and Anthropometric variables and skinfolds of infants born to women with malaria parasitaemia were globally lighter than those of women without  and 3a.parasite density, birth weight, length, OFC, and MUAC of newborns born to women with high parasitaemia were smaller by 300 (95% CI 100–400)gm, 1.1 (0.5–1.6)cm, 0.7 (0.3–1)cm and 0.4 (0.2–0.6)cm respectively compared with those without parasitaemia  and DBP (p = 0.049) than those with parasitaemia Table 2.In contrast when evaluating the effect of parasite density through pregnancy , mean SBIn regression analyses, newborn SBP was independently associated with birth weight and gestational age and inversely associated with birth length and maternal height. Malaria parasitaemia timing, specifically at delivery was also a determinant of infant SBP Table 7.This study illustrates the continuing impact of malaria in otherwise healthy pregnant Nigerian women, almost half of whom (48%) were affected. The rates confirm recent reports in pregnant Nigerian women For those women reporting the use of preventive measures against malaria, including the 53% who said they used chemoprophylactic drugs, there was no difference in malaria parasitaemia frequency. Previous findings have shown that netted windows, insecticide sprays, mosquito repellent and insecticide-treated nets were effective in protecting against malaria In the suburban women in this study, malaria parasitaemia was not associated with social class, similar to a Kenyan report In this study, diagnosis of malaria was based on microscopy of peripheral, placental and cord blood samples. Malaria in pregnancy still presents diagnostic challenges and has relied mainly on microscopy in studies in sub-Saharan Africa. Microscopy, though valuable, requires well-trained and skilled staff Babies of mothers with parasitaemia were globally smaller, most marked among babies of mothers with high parasite density during pregnancy. Similarly, mean birth weights in Burkina Faso, Tanzania, Mali and Pakistan for babies born to mothers with malaria in pregnancy were lower by 105 gm Birth weight is the single most important determinant of neonatal and infant survival and health, and malaria reduced all growth parameters, Some of the mechanisms linking small birth size and adult hypertension are poor maternal nutrition and maternal iron deficiency anaemia, which reduces vascular elasticity In this cohort we found that babies whose mothers had malaria parasitaemia at delivery had lower SBP and DBP . This obOverall, malarial load through pregnancy had the greatest impact on birth size . Thus thduring pregnancy leads to general growth restriction and smaller birth size and relatively higher BP for size. The more limited vascular tree of lighter infants may not be able to meet end-organ oxygen and nutritional demand without reactive peripheral vasoconstriction and higher BPs over time. Marginally but progressive higher BPs in infancy and early childhood may result, leading to a higher risk of hypertension in later life. Recurrent post-natal malaria will intermittently alter peripheral blood flow, but again at the expense of optimal supply to particular organs, hence restricting growth. The balance between the overall size of the fetus' vascular tree, how well particular organs grow during pregnancy, notably the renal glomeruli with their afferent and efferent arterioles, and continuing environmental hazards  or opportunities  will determine vascular performance, now measurable by pulse wave velocity This leads to the question of how these two contrasting observations on BP related to timing and parasite density are mediated. Our hypothesis is that in these LBW babies, placental parasitaemia is associated with significant inflammation in the placental and infant arterial tree leading to more acute placental changes and significant infant vascular dilatation as an initial protective mechanism and hence lower BP at birth in those with parasitaemia at delivery. In contrast, parasitaemia There is a high incidence of malaria and anaemia in this apparently healthy cohort of pregnant women, particularly the younger mothers and primigravids. Malaria in pregnancy adversely affects birth size, with high parasite density during pregnancy having the greatest impact on all growth parameters and being associated with higher BP corrected for weight. Follow-up studies to extend these observations into early childhood and to provide a better understanding of the influence of maternal malaria on BP in this cohort are underway.


Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels  and weakly with parasitemia . In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability, and this effect correlated strongly with plasma L-arginine levels . Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P.falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature.The mechanisms underlying reduced red blood cell (RBC) deformability during  Plasmodiumfalciparum malaria34278P. falciparum (PfRBC)7891078The reduced deformability of the red blood cell (RBC) contributes to the pathogenesis of several hematological disorders1P. falciparum malaria compared to healthy subjects33PfRBCs and, quite importantly, of uRBCs as well3Ecktacytometry measurements showed that whole peripheral RBCs were less deformable during acute Several factors may drive the reduced deformability of uRBCs during malaria, including rigidifying substances released upon schizont rupture, uptake by the membranes of uRBCs131617192122NO is produced by nitric oxide synthase (NOS) which catalyzes the conversion of L-arginine to L-citrulline. Human RBCs possess an active endothelial NOS2122242526P. falciparum infection has been intensively investigated16282826P. falciparum cultures18The role of NO during P.falciparum malaria. We analyzed the relationship between L-arginine and nitrite levels, parasitemia and RBC deformability in patients with acute uncomplicated P. falciparum malaria, and explored the effect of patient plasma on the deformability of P. falciparum ring-stage cultures in vitro.The work reported here aims at clarifying the role of L-arginine and NO pathway on the deformability of RBCs during P. falciparum uncomplicated malaria at admission (day0). The clinical and biological characteristics of the malaria patients are summarized in We evaluated the deformability of whole blood from 30 individuals with PfRBCs) RBCs fractions, with a contribution to the overall values proportional to their respective sizePfRBCs exclusively contribute to the reduced deformability of whole RBCs) displayed the lowest EI max values. (per se (the fraction of PfRBCs in the sample) is not the sole explanatory variable for the reduced EI values of the whole RBC population of patients during malaria episodes and that uRBCs may also be involved, confirming conclusions of studies with patients living in endemic regions3RBC deformability of patients estimated by the Ecktacytometer at day0 is a summation of the cellular deformability of both uninfected (uRBCs) and infected  compared to day28  or healthy controls  . The plaP. falciparum malaria, and measured their EI by Ektacytometry  for 2 hours at 37 or 41°C with the cryopreserved plasma samples collected at day0 from patients with uncomplicated ytometry . Eight pytometry . 9 of 12ytometry . The medytometry . This su/or 41°C  that can/or 41°C .There was no correlation between the plasma concentrations of L-arginine or nitrite and EI max values of cultured rings incubated at 37°C with patient plasma . HoweverPfRBCs. First, we studied whether labeling with the fluorescent indicator 4,5-diaminofluorescein diacetate (DAR-4M AM)PfRBCs incubated with L-arginine showed intense fluorescence within the parasite (red color), but not in the host RBC cytoplasm or cytoplasmic membrane (PfRBCs did not fluoresce in the absence of DAR-4M AM (not shown). Preincubation of PfRBCs with 3 mM L-NAME, a competitive inhibitor of NOS, caused a significant reduction of the intra-parasite fluorescence . We found intense intra-parasite fluorescence at different parasite developmental stages when this assay was conducted on P. falciparum samples freshly isolated from malaria patients and incubated in vitro during 48 hours  staining to localize NOS in fixed cells34We next investigated whether NOS could be detected in uRBCs and PfRBCs, a polyclonal antibody raised to a region conserved in the murine NOS isoforms was used for immunofluorescence assay , deformability of the peripheral RBCs was reduced and plasma levels of L-arginine were low during the acute attack. L-arginine plasma concentrations had returned to basal levels four weeks later, as did RBC deformability. A similar reduction of cellular deformability could be induced upon incubation of P. falciparum ring-stage cultures with patients' plasma collected during the clinical attack. This effect was particularly marked when incubation was carried out at 41°C, i.e., a body temperature frequently observed in febrile malaria patients. Plasma-induced alterations of cellular deformability of P. falciparum cultures correlated with L-arginine concentrations. Taken together, these results suggest that the reduced deformability of RBCs during malaria attacks may be due to the lower plasma concentration of L-arginine. This hypothesis is substantiated by the enhanced deformability of healthy RBCs or cultured trophozoites upon incubation in vitro with L-arginine. This effect of L-arginine on RBC deformability was at least in part NOS-dependent, as L-NAME, a specific inhibitor of mammalian NOS consistently reduced the phenomenon.The present study aimed to evaluate the role of L-arginine-dependent production of NO on the RBC deformability during P. falciparum cultures may stem from several factors. In vitro, this could result from the high parasite arginase activity26P. falciparum culturesin vitro depended upon the presence of parasites in the culture, since the deformability of mock-cultured RBCs incubated with the malaria plasma samples was not affected  measures a minority of endogenous NO, as most nitrite is oxidized in vivo to nitrate. Nitrite may not be a good surrogate marker of NO, because the plasma nitrite levels are known to be influenced by a variety of NOS-independent factors131415PfRBCs and uRBCs in malaria patients.We found a weak correlation between plasma levels of nitrite and deformability of whole RBCs during malaria attacks or of in vitro experiments is supraphysiological . Therefore, the results are of uncertain physiological significance. Thirdly, the fact that the centrifugation and freezing of plasma occurred within 2 hours, may result in underestimating the plasma concentrations of L-arginine since about 10% of L-arginine might be loosed within half an hour after blood collection due to artefactual ex vivo metabolismP. falciparm malaria.Some methodological limitations must be taken into account in this study. Firstly, the fact that the contribution of RBC diseases known to affect RBC deformability, such as α,β- thalassemia5353P.falciparum malaria probably limits NO production and contributes to reduced deformability of RBCs. Strategies to increase L-arginine and/or NO production could improve outcome by restoring RBC deformability thereby facilitating their circulation through systemic capillaries and splenic slits. Drugs aimed at improving RBC deformability in P.falciparum malaria are a promising area for future research.In conclusion, the results presented here suggest that hypoargininemia during P. falciparum monoinfection, parasitemia > 0.1%, no signs or symptoms of severe malaria, and providing written informed consent were included in this observational study approved by the Necker Hospital Investigational Review Board . Parasitemia was determined on Giemsa-stained smears as per routine procedure. Except for the collection of 25 ml of venous blood at days 0 and 28, the capture of clinical data, current laboratory parameters and RBC deformability measurements, the study did not induce any modification of medical care. Antimalarial therapy was administered immediately after blood sampling as per decision of the attending physician. Blood samples were centrifuged within less than 2 hours after collection, and plasma samples were aliquoted and stored at −80°C.Adult patients (age range: 25 to 61 y) with a RBC deformability was measured by ektacytometry using a laser-assisted optical rotational cell analyzer  as previously described7Plasma L-arginine concentration was measured by high performance liquid chromatography at the CERBA Laboratory  on a thawed plasma sample. Nitrite concentration was measured using the Griess reaction as describedP. falciparum FUP-CB - alias FCR3-NADPH-diaphorase (NADPHd) activity was assayed as describedThe following reagents were used to detect the human RBC NOS enzyme and/or the intraparasitic NOS: a mouse monoclonal antibody against human eNOS , a rabbit polyclonal antibody raised to a region conserved in the murine NOS isoforms , designated universal NOS (uNOS)P. falciparum cultures at different stage of development  were incubated with PBS-albumax supplemented with L-arginine (3 mM) and 1 μM DAR-4M AM  for 30 min at 37°C and then washed three times with PBS before microscopic observation.This was performed as describedThis assay was performed as describedThe Wilcoxon signed-rank test for matched pairs was used to compare continuous outcomes at different time points. The Mann Whitney U test or OneWay ANOVA with a Tukey posthoc analysis was used to compare continuous outcomes of different groups at the same time point. The correlation between different continuous measures was determined using the Spearman correlation coefficient. All p-values were two-sided, and p-values of less than 0.05 were considered statistically significant. All statistical analyses were performed with SPSS statistical software (PASW statistic version 18).J.R. performed research and analyzed data; P.A.B. designed research, performed research, analyzed data and wrote the paper; L.C. analyzed data, contributed vital analytical tools; O.M.P. and G.M. designed research, analyzed data and wrote the paper; I.S. designed research, performed research, analyzed data and wrote the paper. All authors reviewed the manuscript.NEW SI FILE