P = 0.039), bacterial sepsis  or healthy controls . The spectrum of malarial retinopathy was similar to that previously described in African children, but no vessel discolouration was observed. The severity of retinal whitening correlated with admission venous plasma lactate (P = 0.046), suggesting that retinal ischaemia represents systemic ischaemia. In conclusion, retinal changes related to microvascular obstruction were common in adults with severe falciparum malaria and correlated with disease severity and coma, suggesting that a compromised microcirculation has important pathophysiological significance in severe and cerebral malaria. Portable retinal photography has potential as a valuable tool to study malarial retinopathy.A specific retinopathy has been described in African children with cerebral malaria, but in adults this has not been extensively studied. Since the structure and function of the retinal vasculature greatly resembles the cerebral vasculature, study of retinal changes can reveal insights into the pathophysiology of cerebral malaria. A detailed observational study of malarial retinopathy in Bangladeshi adults was performed using high-definition portable retinal photography. Retinopathy was present in 17/27 adults (63%) with severe malaria and 14/20 adults (70%) with cerebral malaria. Moderate or severe retinopathy was more frequent in cerebral malaria  than in uncomplicated malaria (3/15, 20%;  As images are recorded for later examination, each can be carefully scrutinised by multiple observers for much longer than would be possible with ophthalmoscopy; objectivity is therefore higher. The greater magnification and high resolution of modern fundus cameras and the possibility of adjusting colour and contrast with imaging software also allow subtle details to be detected more easily, which increases sensitivity. Currently, portable retinal cameras are available that can be used at the bedside, even in the sickest patients.An observational study was conducted using bedside portable high-resolution digital retinal photography in adults with severe and uncomplicated malaria compared with patients with sepsis and healthy volunteers in order to establish the full spectrum of malarial retinopathy.22.1This study was conducted at Chittagong Medical College Hospital, a large 1000-bed teaching hospital in Chittagong, Bangladesh, from June–August 2008. Malaria transmission is seasonal and of low intensity in this location.Consecutive adult patients (>16 years) with slide-confirmed severe or uncomplicated falciparum malaria, according to modified WHO criteria,2.2On admission, a full history and examination were carried out. Blood samples were obtained for haemoglobin, haematocrit, parasitaemia, platelet count, white cell count, plasma lactate levels, glucose levels and full biochemistry. Eye examination included pupillary reaction to light and accommodation and direct and indirect ophthalmoscopy. Ophthalmoscopy was done within 30 min of administration of two drops of 0.5% or 1% tropicamide. In addition, all patients had digital photographs taken of both retinas whenever possible. To obtain images including the peripheral retina, a minimum of nine overlapping photographs (corresponding to approximately 15 megapixels) were required from each retina. These photographs were then amalgamated and merged using Photoshop CS3 Extended software . Photographs were examined by a blinded investigator for evidence of malarial or other retinopathy, and changes were graded as mild, moderate or severe according to the classification of Beare et al.2.3Antimalarial drug treatment was with i.v. artesunate  at 2.4 mg/kg body weight on admission followed by 2.4 mg/kg at 12 h and 24 h and then every 24 h. When the patient was able to take food, treatment was switched to complete a standard six-dose course of oral artemether plus lumefantrine for a further 3 days. Supportive treatments were in accordance with the 2006 WHO guidelines2.4P for trend. The level of significance was P < 0.05.Analysis was performed using Excel 2007 , SPSS version 15.0  and Stata SE 10 . Correlations were assessed using Spearman's rank method for non-parametric data. Fisher's exact test was used to compare retinal severity scores between groups. The trend of increasing severity of retinopathy with increasing malaria severity was assessed by 3A total of 66 patients were enrolled. Of these, 42 patients had falciparum malaria, of whom 27 had severe malaria, including 20 with coma [Glasgow Coma Scale (GCS) score <11)], and 15 had uncomplicated malaria. In addition, 19 healthy volunteers and 5 patients with sepsis were recruited. One of the healthy volunteers appeared to have diabetes with associated retinopathy and was therefore excluded from further analysis. Nine patients died, all of whom had severe malaria. The mean ages of patients were similar in all groups: severe malaria 38.1 years (range 17–75 years); uncomplicated malaria 33.0 years (range 17–63 years); healthy controls 34.9 years (range 19–49 years); and sepsis controls 25.0 years (range 17–40 years). The number of males in each group was 21/27 (77.8%), 11/15 (73.3%), 14/18 (77.8%) and 1/5 (20.0%), respectively. The distribution of presenting severity symptoms in patients with severe malaria is shown in A mean of 11 (95% CI 9–14) usable photos were taken of each retina. For three patients with cerebral malaria and three patients with non-cerebral malaria, the photographs were judged to be insufficiently clear to exclude mild retinopathy, although their retinas appeared normal by indirect ophthalmoscopy. This loss of clarity was caused by rapid eye movements, which also made it difficult to perform adequate indirect ophthalmoscopy. In addition, two of the patients with severe malaria had severe corneal scarring in one eye so only the opposite retina could be examined.The technique of portable retinal photography was found to be highly practical in all groups of patients in this study. Clear images could also be obtained from patients with involuntary wandering eye movements often found in patients with cerebral malaria. Concordance of overall grading of retinopathy between the two blinded investigators was 100%.3.1P < 0.001). The severity of retinal findings correlated with severity of disease categorised from ‘healthy’ volunteers, uncomplicated, non-fatal non-cerebral severe, non-fatal cerebral to fatal malaria. This was true for the total retinopathy severity score (P for trend = 0.001), number of retinal haemorrhages (P for trend = 0.001) and severity of retinal whitening (P for trend = 0.001). The sensitivities and specificities of retinopathy for detecting malaria are shown in Features compatible with malarial retinopathy were present in 17/27 patients (63%) with severe malaria, 7/9 patients (78%) with a fatal course, 14/20 patients (70%) with cerebral malaria, 3/7 patients (43%) with non-cerebral severe malaria, 9/15 patients (60%) with uncomplicated malaria, 1/5 patients (20%) with sepsis and 0/18 (0%) of the ‘healthy’ volunteers . There wP = 0.039), bacterial sepsis (P = 0.038) and healthy controls (P < 0.001). There were no significant differences in the prevalence or severity grade of retinopathy between fatal, cerebral and non-cerebral severe malaria.Moderate or severe retinopathy was present in 12/27 patients (44%) with severe malaria, 5/9 patients (56%) with fatal malaria, 11/20 patients (55%) with cerebral malaria, 1/7 patients (14%) with non-cerebral severe malaria, 3/15 patients (20%) with uncomplicated malaria, 0/5 patients (0%) with sepsis and 0/18 healthy volunteers (0%) . Sensitir = 0.50; P = 0.009), and 16/21 patients (76%) with whitening also had haemorrhages. All patients with severe malaria who had whitening of the peripheral retina also had whitening of the macula. Admission venous plasma lactate levels in patients with severe malaria correlated significantly with the severity of retinal whitening  . Haematocrit on admission correlated inversely with the number of white-centred haemorrhages (P = 0.013). There were no correlations between admission GCS score, haematocrit, parasitaemia, venous lactate, venous bicarbonate or the number of presenting clinical features of severe malaria   and the  malaria  and the 4This study demonstrates that the high prevalence and spectrum of ocular fundus findings in Bangladeshi adults with severe malaria is similar to that found previously in African children. This result differs from previous studies on malarial retinopathy in adults describing lower prevalences.There is currently debate whether the pathophysiology of severe and cerebral malaria differs between adults and children.In the current study some differences were noticed between the findings in adults compared with previous studies of children in Africa. The first is that blood vessel discolouration, found previously in approximately one-third of children with cerebral malaria,Although this was a small study, a number of interesting correlations emerged. In accordance with previous studies, there was a significant trend of increasing severity of retinopathy with increasing severity of malaria, and moderate-to-severe retinopathy was more common in severe and cerebral malaria than in uncomplicated malaria and the control groups. A planned larger study will enable a more detailed comparison between subgroups of patients.Venous plasma lactate correlated with the severity of retinal whitening on admission. Lactate is a strong prognostic indicator in severe malaria and is caused by an increase in anaerobic glycolysis.The severity of retinal haemorrhages correlated with severity of malaria but also anaemia. This has been shown previously in adults with falciparum malaria.To our knowledge, this study is the first to examine malarial retinopathy systematically using high-resolution retinal photography. The methodology maximises objectivity and sensitivity in detecting retinal pathology. Owing to the underlying burden of corneal scarring and cataract disease, as well as involuntary eye movements typical of cerebral malaria, a small number of patients were difficult to examine and optimally clear retinal photographs were impossible to obtain. A limitation of this study was that many patients had limited tolerance to both retinal photography and indirect ophthalmoscopy when performed in quick succession, mainly due to the bright light involved in both techniques. For this reason, comprehensive indirect ophthalmoscopy was not performed in most non-comatose patients, excluding a comparative analysis between indirect ophthalmoscopy and retinal photography.Paediatric severe malaria patients have never been studied in detail in Asia and comparison with findings in adults is important in the assessment of possible differences in pathophysiology between these groups. Retinal changes associated with microcirculatory obstruction can also be compared with changes in microcirculatory blood flow elsewhere in the body using orthogonal polarising spectroscopy.In conclusion, this study describes a new technique to document objectively and to describe the retinal changes associated with severe falciparum malaria. Malaria retinopathy is very common in adults with severe malaria, particularly cerebral malaria. The absence of retinal vessel changes in adults was conspicuous and may indicate a difference in the calibre of vessels involved in sequestration between adults and children. The presence of retinal whitening and its association with coma suggest that a compromised microcirculation has important pathophysiological significance in severe and cerebral malaria.The Mahidol–Oxford Tropical Medicine Research Unit is funded by the Wellcome Trust of Great Britain.None declared.Ethical clearance for the study was obtained from the Oxford Tropical Research Ethics Committee (OXTREC) and the Bangladesh Medical Research Council.RJM, AMD, CCC and NAVB designed the study protocol; RJM, AAS, CCC and PC carried out the clinical assessments; MAF, AH, EBY, MGH and MUH helped with organisation and execution of the study; RJM carried out the analysis and interpretation of the data; RJM, AMD, NAVB, NJW, NPJD, AAS, MAF and CCC drafted the manuscript. All authors read and approved the final manuscript. AMD is guarantor of the paper.


ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients  compared to those with MSM  and HCs . ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold . ADMA was independently associated with decreased exhaled NO , but the causes of these processes are not fully understood. Asymmetrical dimethylarginine (ADMA), a competitive endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of mortality in other critical illnesses, and can impair vascular function in chronic disease. ADMA can be produced by both the host and malaria parasites. The major novel findings of this study in malaria are that ADMA is an independent predictor of death in falciparum malaria, and is associated with decreased availability of nitric oxide in at least two organ systems affected by malaria parasites, the lining of blood vessels and the lungs. This study contributes to knowledge of regulation and availability of pulmonary and endothelial NO in critical illness and identifies pathogenic processes which may contribute to death in severe malaria. Therapies which increase the availability of NO or which reduce ADMA levels may have potential for adjunctive therapy of severe malaria. Plasmodium falciparum causes ∼1 million deaths annually Severe malaria is associated with impaired nitric oxide (NO) bioavailability and blood mononuclear cell NO synthase (NOS) type 2 expression in both children Asymmetrical dimethylarginine (ADMA) is a non-specific endogenous NOS inhibitor which decreases vascular function in cardiovascular and renal disease Acute lung injury is a common but little-studied complication of severe falciparum malaria associated with high mortality In a prospective longitudinal study of Indonesian adults with malaria, we evaluated the hypotheses that concentrations of methylated arginines are independently associated with a) disease severity, b) reduced exhaled NO and vascular NO bioavailability and c) increased mortality.We measured asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA) and L-arginine in 49 patients with severe malaria, 78 with moderately severe malaria and 19 healthy controls. In the SM patients, 20 patients had only one criterion for severe disease  with the remainder having >1 criteria. In total, 34 of the patients with SM were treated with intravenous artesunate and the remaining 15 received intravenous quinine ADMA and SMDA concentrations were increased in SM patients  compared to those with MSM  and HC ; ANOVA p<0.001 for both ADMA and SDMA, s = −0.31, p = 0.003; s = −0.72, p = 0.01; s = −0.51, p<0.001) in malaria patients, but not in the SM group. FeNO remained inversely associated with ADMA, after adjusting for disease severity, creatinine and HRP2. FeNO was not associated with SDMA, the L-arginine/ADMA ratio, plasma hemoglobin or arginase.Exhaled nitric oxide concentration (FeNO) was significantly decreased in SM patients compared to MSM and HC; Table 2.s = −0.32; p<0.001; s = −0.35; p<0.001) concentrations. After adjusting for factors previously shown to be associated with RH-PAT index including plasma hemoglobin As reported previously, the RH-PAT index was significantly lower in SM patients compared to those with MSM and controls (p<0.001) s = 0.48 and 0.42 respectively; p<0.001; s = 0.54 and 0.52; p<0.001), and this was also apparent in the subgroup of SM patients; Patients with SM had significantly elevated plasma concentrations of Ang-2, ICAM-1 and E-selectin compared to those with MSM and HC; There was no significant correlation between ADMA or SDMA with E-selectin, and none between the L-arginine/ADMA ratio and Ang-2, ICAM-1 and E-selectin.P. falciparum histidine rich protein 2 (HRP2) and venous lactate were increased in SM compared to MSM , but not with ADMA (p = 0.06).The plasma creatinine, total bilirubin, d to MSM Table 2.In SM patients, ADMA and SDMA concentrations were significantly higher in the 8 patients who died  compared to the 41 survivors  and three-fold  increased risk of death, respectively. ADMA but not SDMA remained a significant risk factor for death after adjusting for other confounding factors, such as Ang-2, creatinine, parasite biomass, bilirubin, base deficit and lactate. A final model predicting a fatal outcome included ADMA, Ang-2, HRP2 and creatinine Table 4.The prognostic value of ADMA in predicting a fatal outcome was measured by the area under the receiver operating curve (ROC). ADMA ; approximately 20% is excreted unchanged in the urine The loss of DDAH-1 function in a murine model of sepsis increased ADMA, reduced NO signaling, and worsened vascular pathophysiology including endothelial function SDMA does not inhibit NOS, but competes with plasma L-arginine for intracellular uptake by the cationic amino acid transporters (CAT). Unlike ADMA, it is not metabolized by DDAH and is almost exclusively eliminated by the kidneys G-monomethyl-arginine (NMMA), another non-specific NOS inhibitor, in a phase 3 clinical trial of sepsis In critically ill adults with organ failure and severe sepsis, ADMA concentrations are associated with increased all-cause mortality and the severity of organ failure We have previously shown that hemolysis-related NO quenching by cell-free hemoglobin is associated with reduced vascular NO bioavailability in severe malaria in vitro, including inhibition of endothelial Weibel-Palade body (WPB) exocytosis and ICAM-1 expression NO has multiple regulatory functions that maintain endothelial quiescence Acute lung injury is a complication of severe malaria in adults associated with a high mortality rate in vitro studies with endothelial cells have shown that increasing extracellular ADMA results in five-fold increases in intracellular concentrations. This suggest that intracellular concentrations of ADMA in severe malaria may be higher, and may be adequate for meaningful inhibition of for all three NOS isoforms (IC50s ∼2-5µM) There are several limitations in our study. Measurement of exhaled NO was not possible in patients with coma and was possible in only half of severe malaria patients without coma. Our results may therefore not reflect the relationship between ADMA and exhaled NO in all syndromes of severe malaria. In patients who died, only 4 of 8 had at least one repeat blood sample, and the longitudinal data may not truly reflect the course of the methylated arginines in fatal cases. RH-PAT index is at least 50% dependent on endothelial NO release In summary, the endogenous non-selective NOS inhibitor ADMA is elevated in SM and is an independent risk factor for mortality. ADMA is also associated with decreased FeNO and vascular NO bioavailability, as well as increased endothelial activation and parasite biomass. Therapies which increase NO bioavailability or which diminish ADMA levels represent rational approaches for interventional trials of adjunctive therapy in severe malaria.The study was conducted at Mitra Masyarakat Hospital, Timika, Papua, Indonesia, a region with unstable transmission of multidrug resistant malaria Plasmodium falciparum malaria without P. vivax infection and with a hemoglobin level >60 g/L who had been prospectively enrolled in a study of endothelial dysfunction and exhaled NO P. falciparum parasitemia and ≥1 modified WHO criterion of severity (excluding severe anemia). MSM was defined as fever within the preceding 48 hours, >1,000 asexual P. falciparum parasites/µL, no WHO warning signs or severe malaria criteria and a requirement for inpatient parenteral therapy because of inability to tolerate oral treatment. Healthy controls (HC) were non-related hospital visitors with no history of fever in last 48 hours, intercurrent illness or smoking in last 12 hours, or evidence of parasitemia Patients were ≥18 years old with moderately-severe (MSM) or severe (SM) Solid phase extraction (SPE) of amino acids was followed by derivatisation with Accq-Fluor and separation on a Gemini-NX column at pH 9 Plasma concentrations of cell-free hemoglobin and the endothelial activation markers, ICAM-1, E-selectin and angiopoietin-2 were measured by ELISA as previously reported in this population a priori cutoff (1.67) for two consecutive days Endothelial function was measured non-invasively using peripheral arterial tonometry (EndoPAT) by the change in digital pulse wave amplitude in response to reactive hyperemia, giving a RH-PAT Index as reported previously Statistical analysis was performed with STATA 9.2 software. Intergroup differences were compared by ANOVA or Kruskal-Wallis test, where appropriate. Pearson's or Spearman's correlation coefficients were determined depending on normality of distributions. Multiple stepwise linear regression was used to adjust for confounding variables. Longitudinal associations were assessed by mixed effects modeling using generalized estimating equations. Logistic regression was used to determine the association between death and ADMA concentrations. Variables hypothesized, as well as those shown in previous publications


Plasmodium falciparum and P. vivax infections. The identification of sensitive and reliable markers of disease severity is fundamental to improving patient care. An intense pro-inflammatory response with oxidative stress and production of reactive oxygen species is present in malaria. Inflammatory cytokines such as tumor necrosis factor-alpha  and antioxidant agents such as superoxide dismutase-1 (SOD-1) are likely candidate biomarkers for disease severity. Here we tested whether plasma levels of SOD-1 could serve as a biomarker of severe vivax malaria.Severe outcomes have been described for both P. vivax transmission. Malaria diagnosis was made by both microscopy and nested PCR. A total of 219 individuals were enrolled: non-infected volunteers (n = 90) and individuals with vivax malaria: asymptomatic (n = 60), mild (n = 50) and severe infection (n = 19). SOD-1 was directly associated with parasitaemia, plasma creatinine and alanine amino-transaminase levels, while TNF-alpha correlated only with the later enzyme. The predictive power of SOD-1 and TNF-alpha levels was compared. SOD-1 protein levels were more effective at predicting vivax malaria severity than TNF-alpha. For discrimination of mild infection, elevated SOD-1 levels showed greater sensitivity than TNF-alpha , with higher specificity . In predicting severe vivax malaria, SOD-1 levels exhibited higher sensitivity than TNF-alpha . Neither SOD-1 nor TNF-alpha could discriminate P. vivax infections from those caused by P. falciparum.Plasma samples were obtained from residents of the Brazilian Amazon with a high risk for SOD-1 is a powerful predictor of disease severity in individuals with different clinical presentations of vivax malaria. Plasmodium vivax infection has been associated with fatal outcomes due to treatment failure or inadequate health care. The identification of sensitive and reliable markers of disease severity is important to improve the quality of patient care. Although not imperative, a good marker should have a close causative relationship with the disease pathogenesis. During acute malaria, an intense inflammatory response and a well-documented oxidative burst are noted. Among the free radicals released, superoxide anions account for the great majority. The present study aimed to evaluate the reliability of using an antioxidant enzyme, responsible for the clearance of superoxide anions, as a marker of vivax malaria severity. Thus, we investigated individuals from an Amazonian region highly endemic for vivax malaria with the goal of predicting infection severity by measuring superoxide dismutase-1 (SOD-1) plasma levels. In addition, we compared the predictive power SOD-1 to that of the tumor necrosis factor (TNF)-alpha. SOD-1 was a more powerful predictor of disease severity than TNF-alpha in individuals with different clinical presentations of vivax malaria. This finding opens up new approaches in the initial screening of severe vivax malaria cases.Despite being considered a relatively benign disease,  Plasmodium falciparum, but growing evidence also associates vivax malaria with severe complications Plasmodium vivax infection represents a global health threat. Therefore, identifying markers of disease severity is essential to improve clinical management. Plasma TNF-alpha levels have been described as a biomarker for the estimation of disease severity for P. falciparumP. vivaxSevere malaria presents a relevant public health problem worldwide, affecting the socio-economic development of many communities. The identification of predictors of disease severity is critical to improve patient care. Most of the actual knowledge regarding the immunopathological determinants of malaria severity refers to infection caused by 2O2), which can be removed by the catalase and glutathione systems P. vivax infection is highly endemic. The performance of SOD-1 as a predictor of disease severity even surpasses that of TNF-alpha.During malaria infection, reactive oxygen species (mainly superoxide anions) are produced at high levels, inducing parasite killing and tissue damage The objective of this study was to test whether the plasma level of SOD-1, an antioxidant enzyme, could predict vivax malaria severity with equivalent of better efficacy compared to the currently used marker TNF-alpha.P. malariae infection were identified and excluded from the study. In addition, 16 age-matched people infected with P. falciparum (uncomplicated forms) were invited to participate. In the last phase of the study, plasma samples from these individuals with P. falciparum malaria were used in order to assess if the markers compared were useful to discriminate P. vivax from P. falciparum infections.Plasma samples were obtained from individuals living in Buritis, a recently urbanized municipality in Rondônia, Brazilian Amazon, with a high risk for vivax malaria transmission P. falciparum were not included in the follow up. Infected individuals who remained without any presumptive malaria symptoms were considered asymptomatic; patients presenting clinical or laboratory signs of complicated malaria After obtaining the parasitological diagnosis, all vivax malaria positive cases were followed for 30 days. Individuals infected with Written informed consent was obtained from all participants, and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The project was approved by the institutional review board of the Faculdade de Medicina, Faculdade São Lucas, Rondônia, Brazil, where the study was performed.Plasmodium genus-specific primers named PLU5 and PLU6. Positive samples yielded a 1,200-bp fragment, which served as template for the nested reaction. The nested PCR amplification was performed with species-specific primers for 30 cycles at annealing temperatures of 58°C for P. falciparum , and 65°C for P. vivax (Viv1 and Viv2 primers) or P. malariae . The fragments obtained for P. vivax were of 120 bp, whereas for P. falciparum and P. malariae were 205 bp and 144 bp, respectively. The oligonucleotide sequences of each primer used are listed in The molecular diagnosis of malaria was performed using nested PCR, as described previously Plasma levels of TNF-alpha were measured using the Cytometric Bead Array - CBA®  according to the manufacturer's protocol, with all samples run in a single assay. The flow cytometric assay was performed and analyzed by a single operator, and standard curves were derived from cytokine standards. The minimum limit of detection was 3.7 pg/mL.Plasma measurements of creatinine, alanine amino-transaminase (ALT) and haemoglobin were made at the clinical laboratory of Faculdade São Lucas and at the Laboratório LPC .SOD-1 plasma concentrations were measured using the Cu/Zn Superoxide Dismutase ELISA Kit according to the manufacturer's protocol . Briefly, human serum was diluted 1∶200 in PBS and distributed in a sensitized 96-wells plate. The samples were incubated for one hour at room temperature with HRP-conjugated anti-Cu/Zn SOD antibody. A colorimetric substrate was added for ten minutes, being the system protected from intense light. The reaction was stop and the plate read at 450nm. The SOD activity assay was performed using the Superoxide Dismutase Colorimetric Assay Kit according to the manufacturer's protocol . Briefly, radical detector was added to a sensitized 96-wells plate. Pre-diluted (1∶50) samples were distributed in wells. The reaction was started using Xanthine Oxidase, and the plate was read after twenty minutes at 450nm. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.P. vivax and P. falciparum infections. Correlations between SOD-1 or TNF-alpha levels and severity factors were performed using the Spearman test. Receiver-operator characteristic (ROC) curves with C-statistics were used to establish the threshold value of SOD-1 and TNF-alpha able to discriminate between mild and severe infection. A p value<0.05 was considered statistically significant.The Kruskal-Wallis test with Dunn's multiple comparisons or linear trend analysis was used to compare SOD-1 and TNF-alpha levels according to different clinical presentations of vivax malaria infection. The Mann-Whitney test was used to verify differences between asymptomatic and symptomatic, between mild and severe vivax malaria or between P. falciparumP. vivaxP. vivax from P. falciparum malaria, we compared plasma samples from individuals presenting with mild symptomatic vivax malaria and age matched individuals with symptomatic P. falciparum infection. Neither SOD-1 nor TNF-alpha could differentiate between the infections  identification of patients with severe disease before the development of fatal outcomes and (ii) monitoring the success of therapy and clinical recovery. The viability of applying this methodology in the clinical practice will depend on its priority status in a diagnostic algorithm.This study is the first to examine the use of plasma SOD-1 levels as a surrogate marker of P. falciparum infections was not evaluated here, and should be tested in future investigations. However, in individuals presenting with mild disease, plasma SOD-1 levels could not differentiate between P. vivax and P. falciparum infections. This suggests that these two parasites may share more similar pathogenetic mechanisms than previously realized.Whether this anti-oxidant enzyme could be used as a marker of disease severity in P. falciparum-induced oxidative response and apoptosis in vitroP. bergheiP. falciparum or P. vivax malaria have less catalase activity then non-infected individuals but higher SOD activity 2O2, the release of hydroxyl radicals and increased tissue damage during severe malaria.SOD-1 represents an important defense against oxidative stress within a cell Although investigations analyzing more patients with broader clinical outcomes are necessary, SOD-1 plasma protein levels seems to represent a useful marker in predicting vivax malaria severity based on the oxidative response status.P. vivax malaria and highlights the likelihood of exploring the future use of the plasma SOD-1 levels as an effective marker of malaria severity. To validate our results, studies investigating samples from different endemic areas are crucial, as local health conditions such as co-infections may limit the effective use of a biomarker. A possible advantage of measuring SOD-1 levels as part of the clinical management in endemic areas cannot be assumed from our results. The use of the SOD-1 as a reliable marker also depends on future field interventions in which the pre-test prediction and cost-effectiveness should be considered. In addition, the specific role of SOD-1 in the immunopathogenesis of severe vivax malaria was not explored in this study and is still being addressed by our group.This study illustrates the possibility of using SOD-1 levels as a severity biomarker in human Checklist S1STARD checklist.(0.08 MB PDF)Click here for additional data file.Figure S1STARD flowchart.(0.02 MB PDF)Click here for additional data file.


PLoS Medicine that explores whether prenatal exposure to malaria is associated with increased susceptibility to malarial infection and anemia in infancy.Lars Hviid discusses a research article in  PLoS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1000116Malhotra I, Dent A, Mungai P, Wamachi A, Ouma JH, Narum DL, Muchiri E, Tisch DJ, King CL (2009) Can prenatal malaria exposure produce an immune tolerant phenotype?: A prospective birth cohort study in Kenya. PLoS Med 6(7): e1000116. In a prospective cohort study of newborns residing in a malaria holoendemic area of Kenya, Christopher King and colleagues find a subset of children born to malaria-infected women who acquire a tolerant phenotype, which persists into childhood and is associated with increased susceptibility to malarial infection and anemia.Plasmodium falciparum malaria every year, which cost 1–2 million lives. The majority of serious and fatal cases occur among children below the age of five years, as substantial protective immunity is gradually acquired following repeated infections. Adolescents and adults therefore rarely suffer clinical episodes of malaria.There are an estimated 250 million cases of P. falciparum parasites when they get pregnant, especially during a first pregnancy. This pregnancy-associated reappearance of susceptibility is related to the ability of the parasites to escape pre-existing clinical immunity by expressing particular pregnancy-specific antigens on the surface of infected erythrocytes Nevertheless, women in such areas again become highly vulnerable to infection by P. falciparum antigens in utero. Prenatal exposure can result in fetal acquisition of parasite-specific antibody and cytokine responses that can be measured in the infant's lymphocytes at delivery, but may also lead to tolerance and immunological anergy at subsequent re-exposure Placental parasitemia is associated with maternal anemia, prematurity, low birth weight, and excess perinatal morbidity and mortality P. falciparum infection at delivery than in offspring of mothers without. This discrepancy is often interpreted as the consequence of impaired acquisition of protective immunity in children exposed to antigen while in the womb. However, in all likelihood a multitude of factors contribute and interact in complex ways, which are rarely considered in much detail.The time to first parasitemia is generally shorter in offspring of mothers with placental PLoS Medicine, Christopher King and colleagues P. falciparum parasites in coastal Kenya. Venous and placental blood from the mothers as well as cord blood from the babies was examined by microscopy and PCR for presence of parasites to evaluate prenatal exposure to parasite antigens. The authors then tested the parasite antigen-specific immune reactivity in the offspring at delivery and every six months thereafter for the first three years of life. The antigens examined were parasite proteins involved in merozoite invasion of erythrocytes, targets of protective immunity, and vaccine candidates. Antigen-induced responses included lymphocyte proliferation and cytokine production. Furthermore, the authors measured plasma levels of IgG with specificity for some of the antigens used in the in vitro assays of cellular immunity.In this week's Once all data were collected, the authors compared the results in three sub-groups of the children. The first of these was composed of the 246 “sensitized” children, where cytokine responses (other than IL-10) could be detected in antigen-stimulated cord blood cultures. The second sub-group included the 120 “not sensitized” children, whose cord blood cells did not produce cytokine responses despite parasitological evidence of in utero exposure. Finally, the third sub-group consisted of the 220 “not exposed” children, where no antigen-induced cord lymphocyte cytokines were detected, but where parasitological evidence of prenatal exposure could not be obtained.P. falciparum antigens before birth were approximately 40% more likely to become infected during the follow-up period than either sensitized or unexposed children. In contrast to earlier studies The “not sensitized” children who appeared to have been tolerized to P. falciparum antigens before birth, and that this is of clinical importance because it increases their risk of parasitemia in the first few years of life. If this is indeed the case, it is of obvious importance to clinicians and health policy makers. However, there are issues that require further study and consideration by scientists in this field. First of all, it is intuitively surprising how such a disadvantage can persist despite the expected negative selection pressure against it. Secondly, it should be considered whether below average, Th1-type, “pro-inflammatory” cellular immune responses and low-level parasitemia are in fact adequate indicators of impaired clinical immunity, when several studies have shown that asymptomatic infection actually protects against clinical diseases episodes, and that eradication of such infections can be detrimental The authors conclude that their data show that a sizeable proportion of children in endemic areas are immunologically disadvantaged at birth as a consequence of being exposed to P. falciparum antigens. Hopefully, the study will inspire scientists in the field to study this complex and clinically significant issue further.In conclusion, the study by King et al. adds significantly to our understanding of prenatal exposure to


The efficacy of intravenous quinine, which is the mainstay for treating severe malaria in children, is decreasing in South East Asia and Africa. Artemisinin derivatives are a potential alternative to quinine. However, their efficacy compared to quinine in treating severe malaria in children is not clearly understood. The objective of this review was to assess the efficacy of parenteral artemisinin derivatives versus parenteral quinine in treating severe malaria in children.All randomized controlled studies comparing parenteral artemisinin derivatives with parenteral quinine in treating severe malaria in children were included in the review. Data bases searched were: The Cochrane Central Register of Controlled Trials , MEDLINE (1966 to February 2008), EMBASE (1980 to February 2008), and LILACS (1982 to February 2008). Dichotomous variables were compared using risk ratios (RR) and the continuous data using weighted mean difference (WMD).th day cure rate. One trial reported significantly more local reactions at the injection site with intramuscular quinine compared to artemether. None of the trials was adequately powered to demonstrate equivalence.Twelve trials were included . There was no difference in mortality between artemisinin derivatives and quinine . The artemisinin derivatives resolved coma faster than quinine , but when trials with adequate concealment only were considered this differences disappeared. There was no statistically significant difference between the two groups in parasite clearance time, fever clearance time, incidence of neurological sequelae and 28There was no evidence that treatment of children with severe malaria with parenteral artemisinin derivatives was associated with lower mortality or long-term morbidity compared to parenteral quinine. Future studies require adequately powered equivalence trial design to decide whether both drugs are equally effective. Malaria remains a major public health problem globally. It is estimated that each year there are about 350–500 million clinical cases of malaria, which result in more than 1 million deaths . TherefoPlasmodium falciparum resistance and which act faster than all known malaria drugs [P. falciparum malaria, particularly with metabolic acidosis, however, showed variable and unpredictable absorption of artemether after intramuscular injection a problem not encountered with intramuscular quinine [Over the years quinine has been the mainstay for treating severe malaria in children, but despite its high efficacy the case fatality rate for severe malaria in children can be as high as 40% . Additioia drugs . Pharmac quinine ,22.Because the findings from individual studies were inconclusive, two reviews have been conducted to clarify the efficacy of artemisinin derivatives versus existing drugs used for severe malaria ,24. The th day cure rate and incidence of adverse effects were the secondary outcomes.Studies were considered for inclusion in the review if they were randomized controlled trials comparing one parenteral artemisinin derivative  with parenteral quinine in children aged between 0 to 16 years or if trials stated that they included children only. The participants had to have severe malaria as defined by the World Health Organization (WHO) . Trials th annual scientific conference  of the Tanzania National Institute for Medical Research conducted between 4th to 7th February 1993, in Arusha, Tanzania. In addition, 26 malaria researchers across Africa, Asia, Europe and America were contacted to find out if they had any information on published, unpublished or ongoing trials for possible inclusion in the review. The reference lists of the retrieved articles were also searched for more articles.Electronic databases and other sources were used to search for studies to include in the review. The electronic databases searched were: The Cochrane Central Register of Controlled Trials , MEDLINE (1966 to February 2008), EMBASE (1980 to February 2008), and LILACS (1982 to February 2008). With the support of a Cochrane information specialist a comprehensive search strategy for each database was developed using both controlled vocabulary terms and free text words to ensure that all potential articles regardless of language or publication status were retrieved. The following key words were used: artemisinins, dihydroartemisinins, artemether, artesunate, β-arteether, artemotil, quinine, malaria, "cerebral, malaria", "severe malaria" complicated malaria, "malaria, falciparum", and randomized controlled trials. Other sources searched included: The proceedings of the 11After the literature search was completed the results were sorted to include abstracts, which had the potential of being included in the review. Complete articles of the potential abstracts were either retrieved or ordered. If a trial was published more than once, only one publication was presented for assessment and if an interim analysis of a particular major trial was published, only the final publication was presented for assessment. GP and AD assessed the articles for their suitability for inclusion in the review.Then all included studies were assessed by GP and AD for their quality in design and conduct. Generation of allocation sequence, allocation concealment, blinding and loss to follow up, were used to assess the quality of studies as recommended previously .GP extracted data using special forms, and then it was analysed using STATA version 8.2. Binary outcomes were compared by risk ratio (RR) and continuous outcomes by weighted mean difference (WMD). The 95% confidence interval (CI) was used and p value < 0.05 was assumed to be showing a statistically significant difference.th day cure rate; it was decided to explore the study setting (Asian versus non-Asian studies) as a possible source of heterogeneity. The rationale for choosing this characteristic was that in Asia, evidence suggested that the level of quinine resistance was higher than on other continents and therefore artemisinin derivatives were likely to be comparably more efficacious in Asia than on other continents; a situation that could have introduced heterogeneity in pooled data. Also sensitivity analysis based on adequacy of concealment was carried out for all outcomes. Data on adverse effects were summarized.To assess heterogeneity, forest plots were visually examined  and the chi-squared test for heterogeneity with p value < 0.05 was assumed to be showing significant heterogeneity among trials. The fixed effect model was employed in pooling data where there was no evidence of heterogeneity and where there was evidence of heterogeneity, the random effects model was used instead. Because there was significant heterogeneity in parasite clearance time, fever clearance time and 28Twelve randomized controlled trials involving 1524 children met the inclusion criteria and were included in the review -19. DetaMethods used in generation of allocation sequence were adequate in six trials ,12,14-16In three trials, there were no reports of loss to follow up or exclusion from analysis for the primary outcome ,17,19. INine trials ,14-16,19Six studies showed no statistically significant difference in mean parasite clearance times between artemisinin derivatives and quinine ,14-18, wOverall, pooled analysis showed that artemisinin derivatives did not clear parasites faster than quinine .  with quinine as assessed twice daily for seven days and then weekly for another three weeks. It showed that there was no difference in weakness, aphasia/speech disorder, deafness, fevers/rigors, anorexia, nausea, vomiting, diarrhoea, cough, pneumonia, conjunctivitis between groups .Another trial comparing artesunate with quinine found that patients on quinine suffered significantly more often from vomiting (30%), nausea (50%), headaches (40%) and tinnitus (20%) than patients on artesunate who had none of these adverse effects . One trith cure rates showed no difference among the two drug groups. Though data on adverse effects were inadequate, administration of intramuscular quinine was characterized by more local reactions at the injection site compared to artemether. In one study, it was found that compared to quinine, administration of intramuscular artesunate led to signs of transient bone marrow depression.This review suggests that artemisinin derivatives are not superior to quinine in preventing mortality from severe malaria in childhood. It also showed that though CRT was shorter with artemisinin derivatives, this difference was no longer significant when high quality studies were considered, indicating that the overall pooled results may have been biased by results from low quality trials. Other outcomes i.e. PCT, FCT, incidence of neurological sequelae and 28P. falciparum strains to quinine. Future reviews need to include subgroup analyses for Asian and African regions separately as long as quinine resistance is sporadic in Africa.Subgroup analysis of the individual artemisinins showed that none of the drug studied  in this review had lower mortality rate than quinine. It is important to note that despite this conclusion there were only two studies with a total of 194 children that compared β-arteether versus quinine, and only one study with 72 children that compared artesunate versus quinine, the number of subjects in these two drugs is too small to make a conclusion on their efficacy compared to quinine in terms of mortality. In contrast there were nine studies with 1258 children that compared artemether versus quinine and found it to be not different in effect on mortality. Previous reviews also came to a similar conclusion ,24. A moP. falciparum strains are known to be sensitive to quinine. However, since the efficacies between the two groups of drugs are comparable decision to replace quinine may be taken if other factors such as costs, user preference and logistics are taken into consideration.Based on efficacy; this review does not justify a replacement of intravenous quinine by artemisinin derivatives when treating children with severe malaria in Africa and Asian countries where Given the results of this review studies comparing artemisinin derivatives with quinine in treatment of severe malaria in children need to follow equivalence trial design. A sample size calculation for an equivalence trial taking mortality as a primary outcome, with a difference in mortality of five percent taken as range of equivalence and 85% survival as expected with standard quinine treatment of cerebral malaria, a sample size of at least 1071 in each treatment group would be required to demonstrate equivalence with a power of 80% with a two sided confidence interval of 95% for the difference in mortality . For comThere was no evidence that treatment of children with severe malaria with parenteral artemisinin derivatives was associated with lower mortality or long-term morbidity compared to parenteral quinine. Future studies require adequately powered equivalence trial design to decide whether both drugs are equally effective.The authors declare that they have no competing interests.GP, AD and ME contributed to the review design, GP extracted and analyzed the data. GP, AD and ME participated in interpretation of data and writing of the manuscript. All authors approved the final manuscript.


There is a paucity of information regarding glucose-6-phosphate dehydrogenase (G6PD) deficiency in endemic areas for malaria in Latin America.−. The deficiency was not associated with the ethnic origin (P = 0.571). In a multivariate logistic regression analysis, G6PD deficiency protected against three or more episodes of malaria (P = 0.049), independently of the age, and was associated with a history of jaundice (P = 0.020) and need of blood transfusion (P = 0.045) during previous treatment for malarial infection, independently of the age and the previous malarial exposure.This study determined the prevalence of the G6PD deficiency in 200 male non-consanguineous individuals residing in the Ismail Aziz Community, on the outskirts of Manaus (Brazilian Amazon). Six individuals (3%) were deficient using the qualitative Brewer's test. Gel electrophoresis showed that five of these patients were G6PD A− variant will help the clinical management of patients with drug-induced haemolysis. The history of jaundice and blood transfusion during previous malarial infection may trigger the screening of patients for G6PD deficiency. The apparent protection against multiple malarial infections in an area primarily endemic for Plasmodium vivax needs further investigation.The frequency of G6PD deficiency was similar to other studies performed in Brazil and the finding of a predominant G6PD A Plasmodium vivaxMalaria remains the most important tropical disease in tropical and sub-tropical areas of the world P. vivax infection may lead to severe clinical complications related to the side effects of the drugs, such as haemolysis induced by primaquine in individuals genetically deficient for G6PD. The worldwide distribution of this deficiency is a vital consideration in the development of new antimalarials that may also have the potential of causing haemolysis To obtain the radical cure of vivax malaria, primaquine is prescribed for all patients G6PD is an enzyme present in the cytoplasm of all cells, acting specifically in the maintenance of the integrity of the erythrocytes, preventing the oxidation of hemoglobin and other cellular proteins −), with new mutations already described only in non-endemic areas for malaria G6PD deficiency occurs most frequently in individuals of African descent, with a frequency ranging from 3.6 to 28.0% − standard on electrophoresis. One sample disclosed an indeterminate band in the gel, which did not permit a valid interpretation of the type of deficiency. All the patients were afebrile at the time of the thick blood smear collection, but three samples were positive for Plasmodium sp. , disclosing an infection prevalence of 1.5% (CI95% 0.49–2.51). In Of the 200 analyzed samples, six  were deficient for G6PD according to the first screening using the phenotypic Brewer's test. The deficiency was confirmed in all the samples by electrophoresis in agarose gel. Five samples with G6PD deficiency showed the type ASince G6PD deficiency is a recessive trait linked to the X chromosome, studying the population frequency of the disorder exclusively in men is an optimal approach The qualitative Brewer's test for the reduction of methemoglobin is a simple method that uses low-cost reagents. Although only yielding qualitative results, it is a good screening test and it has been widely utilized in various studies of selected populations − G6PD deficiency. To a certain extent this confirms findings in other Brazilian studies with different study designs − is usually a mild/moderate deficiency . In this case, G6PD activity is greatest in younger cells, so as the red cell population recovers, G6PD activity increases and hemolysis is controlled. This is what makes drug-induced G6PD deficiency-related hemolytic anemia in G6PD A− subjects a self-limiting problem in most cases The biochemical characterization of the isoenzymes demonstrated a high frequency of the phenotype A− variant, five were non-white (but not black) and one was white. This illustrates the impact of the racial mixing which occurred in the recent history of Brazil, especially in the Amazon region. A significant immigration from various parts of Brazil and abroad took place in the nineteenth century due to rubber extraction activities and during the twentieth century because of the creation of a tax-free zone and a growing industrial base in Manaus. The large number of immigrants from Portugal and Lebanon in Manaus probably explains this predominant variant found in the studied population, since G6PD A− is frequently seen in these groups Among the six individuals with the G6PD AP. falciparumP. vivax malaria, which is the major species in the area of the study. This potential protection conferred by G6PD deficiency needs further investigation.Epidemiologic studies have suggested that G6PD deficiency protects against malaria and severe malaria by P. vivax infection is reflected in their history of significantly more jaundice and more blood transfusions. This information could be useful as a first screening for G6PD deficient patients, to determine who should have a Brewer's test performed. This test is not routinely offered to the general population with a diagnosis of P. vivax malaria in Brazil even though they will receive a standard 7-day treatment regimen with primaquine (0.5 mg/kg/day) for radical cure of the parasite. G6PD deficiency therefore increases the morbidity of P. vivax infection itself and the side effects of primaquine. In both cases the costs of treatment and hospitalization are increased. These additional expenses threaten financial resources and difficulty achieving a radical cure complicates the control of P. vivax malaria.The impact of G6PD deficiency in this population affected mostly by P. vivax radical cure with new antimalarials having the potential of hemolysis in this population, such as tafenoquine Since only a few studies have been published estimating the prevalence of G6PD deficiency in malaria endemic areas of Latin America, this study adds information to the global mapping of the problem, despite a low external validity, as most of the literature on this issue. Understanding the burden of G6PD deficiency will guide local antimalarial treatment policies and enable the proper design of clinical trials for − was confirmed through electrophoretic isoenzyme detection in 5 of 6 patients after the deficiency was initially detected via screening with the qualitative Brewer's test. G6PD A− was not associated with a black ethnicity, probably because of the genetically mixed population of Manaus. The history of jaundice and blood transfusions associated with prior malaria infections was associated with G6PD deficiency and may be used as a simple clinical marker for this entity.Despite the small number of patients enrolled in this population-based survey, we detected a 3% prevalence of G6PD deficiency in an endemic area for malaria in Manaus. Accurate information about this deficiency and its relation to acute hemolysis with the use of primaquine in the Brazilian Amazon is scarce. G6PD AThe study was carried out in the municipality of Manaus (03°08′ S/60°00′ W), in the Ismail Aziz Community, with an estimated population of 1,500 inhabitants , including natives and immigrants . Census Ten milliliters of venous blood were collected from each person using EDTA tubes (BD Vacutainer®). A qualitative G6PD screening test by the methemoglobin reduction method was performed odds ratios (OR) with 95% CIs (CI95%) were estimated by univariate logistic regression analysis (2-test or the Fisher exact-test when indicated. Adjustment for multiple variables was performed by adding the covariates in a set of multiple logistic-regression models. Test for trend was conducted through the exact test for trend (modified Wilcoxon test for trend). A two-tailed value of P<0.05 was considered statistically significant.The statistical analyses were performed in Epi Info® 3.3 2004 (CDC/Atlanta). 95% CIs (CI95%) were estimated for all the observed frequencies of the study. To assess the extent to which ethnic origin, previous malaria exposure and previous clinical complications triggered by malarial infection were associated with G6PD deficiency, analysis , Table 2The study was approved by the Ethics Committee Board of the Tropical Medicine Foundation of Amazonas. Informed written consent was obtained from all participants.


The recent change of treatment policy for uncomplicated malaria from sulfadoxine-pyrime-thamine to artemether-lumefantrine (AL) in Kenya was accompanied by revised malaria diagnosis recommendations promoting presumptive antimalarial treatment in young children and parasitological diagnosis in patients 5 years and older. We evaluated the impact of these age-specific recommendations on routine malaria treatment practices 4–6 months after AL treatment was implemented.Cross-sectional, cluster sample survey using quality-of-care assessment methods in all government facilities in four Kenyan districts. Analysis was restricted to the 64 facilities with malaria diagnostics and AL available on the survey day. Main outcome measures were antimalarial treatment practices for febrile patients stratified by age, use of malaria diagnostic tests, and test result.Treatment practices for 706 febrile patients (401 young children and 305 patients ≥5 years) were evaluated. 43.0% of patients ≥5 years and 25.9% of children underwent parasitological malaria testing (87% by microscopy). AL was prescribed for 79.7% of patients ≥5 years with positive test results, for 9.7% with negative results and for 10.9% without a test. 84.6% of children with positive tests, 19.2% with negative tests, and 21.6% without tests were treated with AL. At least one antimalarial drug was prescribed for 75.0% of children and for 61.3% of patients ≥5 years with a negative test result.Despite different recommendations for patients below and above 5 years of age, malaria diagnosis and treatment practices were similar in the two age groups. Parasitological diagnosis was under-used in older children and adults, and young children were still tested. Use of AL was low overall and alternative antimalarials were commonly prescribed; but AL prescribing largely followed the results of malaria tests. Malaria diagnosis recommendations differing between age groups appear complex to implement; further strengthening of diagnosis and treatment practices under AL policy is required. Antimalarial treatment with highly efficacious but expensive artemisinin-based combination therapy (ACT) is a recent key strategy to reduce the public health impact of failing monotherapies in sub-Saharan Africa . In 2004To rationalize the use of expensive ACTs, the WHO promotes parasitological diagnosis, except for young children in high malaria risk areas where, pending further evidence, the clinical consequences associated with not treating potentially false negative test results may outweigh potential cost-saving benefits . To suppIn 2006 the new AL policy was implemented countrywide and evaluated approximately 4–6 months after AL was delivered to health facilities. Descriptions of the implementation process and overall paediatric malaria case-management results were published earlier . The detailed explanation of survey methods is presented elsewhere  had parasitological capacity for malaria diagnosis, more commonly providing malaria microscopy  than RDTs . Only four facilities had both diagnostic capacities. We analysed treatment practices for 706 febrile patients (401 < 5 years and 305 ≥ 5 years of age) presenting to the 64 facilities with diagnostic support where AL was in stock on the survey day. Of these patients, the majority in each respective age group was evaluated at facilities with microscopy (88.0% and 84.9% respectively). Among patients ≥5 years, only 43.0% (95% CI: 34.4–51.5) had a diagnostic test performed. At the same facilities, 25.9% of children <5 years of age (95% CI: 16.8–25.1) were tested. Positive malaria tests were routinely reported for 50.0% of children (95% CI: 39.3–60.7) and 52.7% of patients ≥5 years (95% CI: 42.8–62.5).The pattern of antimalarial treatments was similar among febrile patients below and ≥5 years . AL was Kenya's policy to promote presumptive malaria diagnosis in young children and parasitological testing in patients ≥5 years should have resulted in substantially different patterns of malaria diagnostic and treatment practices between these age groups. However, the only difference found was somewhat higher use of malaria testing in patients ≥5 years (43%) than in young children (27%). In both age groups, any antimalarial drug was prescribed to the majority of patients with a positive test (98–99%), negative test (61–75%) and those without test performed (50–64%). AL, however, was rarely prescribed for all patients’ categories (10–22%), except for those with a positive test result (80–85%).parasitological diagnosis is not prerequisite for the treatment’ in febrile children. Therefore, it was not surprising that we found similar age-specific practices characterized by suboptimal use of malaria diagnostics and prescription of AL predominantly for the small subset of test positive patients.The observed pattern is likely to reflect modalities of AL implementation process in Kenya. Prior to the introduction of ACTs, several studies in Kenya . SimultaAntimalarial treatment practices for test negative older children and adults deserve special attention. Although the use of malaria diagnostics was low, our previous concerns that health workers would massively overprescribe AL for these patients appear to be unfounded (The translation of revised diagnostic recommendations into effective malaria treatment practices under the AL policy in Kenya faces several challenges. Parasitological diagnosis is underused in older children and adults, young children are still tested and the overall use of AL is low in both age groups with marked tendency for alternative antimalarial prescriptions. Prescribing of AL, however, largely followed malaria test results. Different age-specific diagnosis recommendations appear complex to implement. The most suitable solution for policy implementers would be to have the same recommendations for all age groups. RDTs are a potential solution for young children, as suggested recently in urban setting in Uganda (


Plasmodium falciparum circumsporozoite protein (Pfcsp) are rather low and apparently stable in low endemic areas. This study sought to assess the pattern in a malaria endemic setting in Africa, using samples from Freetown, Sierra Leone.In the context of the development of a successful malaria vaccine, understanding the polymorphisms exhibited by malaria antigens in natural parasite populations is crucial for proper vaccine design. Recent observations have indicated that sequence polymorphisms in the C-terminal T-cell epitopes of the csp gene spanning the Th2R and Th3R epitopes was amplified and directly sequenced; sequences were analysed with subject parameters and polymorphism patterns in Freetown were compared to that in other malaria endemic areas.Filter-paper blood samples were collected from subjects at a teaching hospital in Freetown during September–October 2006 and in April–May 2007. The C-terminal portion of the PfP = 0.049). In contrast, the single-haplotype predominance (proportion > 50%) pattern previously identified in Asia was not detected in Freetown.Overall, the genetic diversity in Freetown was high. From a total of 99 sequences, 42 haplotypes were identified with at least three accounting for 44.4% (44/99): the 3D7-type (19.2%), a novel type, P-01 (17.2%), and E12 (8.1%). Interestingly, all were unique to the African sub-region and there appeared to be predilection for certain haplotypes to distribute in certain age-groups: the 3D7 type was detected mainly in hospitalized children under 15 years of age, while the P-01 type was common in adult antenatal females (Pearson Chi-square = 48.750, degrees of freedom = 34, csp in Freetown appeared to vary with age in the study population, and the polymorphism patterns were similar to that observed in neighbouring Gambia, but differed significantly at the sequence level from that observed in Asia. The findings further emphasize the role of local factors in generating polymorphisms in the T-cell epitopes of the P. falciparum circumsporozoite protein.Haplotype distribution of the T-cell epitopes of Pf Plasmodium falciparum circumsporozoite protein (Pf-CSP) is the most abundant protein on the sporozoite's surface and has a sequence comprising a central repeat that is flanked by polymorphic N-terminal and C-terminal non-repeat regions. The Pf-CSP antigen is the primary component of the RTS,S [Every year malaria kills more than a million people worldwide  and the he RTS,S , which ihe RTS,S  and chilhe RTS,S . Plans fhe RTS,S . Althougcsp gene, two distinct polymorphisms have been described: polymorphism in the number of NANP repeats and single nucleotide polymorphisms (SNPs) in non-repeat regions. Although polymorphisms in the number of NANP repeats can be extensive even in low endemic areas [csp gene in a typical African malaria-endemic setting in Sierra Leone. With no previous data on this important topic in the country, the main aim herein was to collect baseline sequence data and to characterize sequence polymorphisms of the T-cell epitopes of Pf-CSP among field isolates distributing in the capital, Freetown, with an ultimate aim of acquiring insight into host-parasite interaction(s). In addition, this study examined whether there was any significant difference in polymorphism patterns between African and Asian parasites. Results confirmed marked sequence diversity of African isolates as compared to Asia, with at least three major haplotypes in Freetown. Moreover, haplotype distribution in this endemic area appeared to vary with age, and presumably immunity status. Implications of findings are discussed with regards to malaria vaccine design and local malaria epidemiology.With regards to the Pfic areas , it has ic areas , or 10 yic areas . MoreoveAnopheles gambiae, Anopheles funestus, Anopheles melas, and Anopheles nili as efficient local malaria vectors, with P. falciparum being the dominant parasite species , as part of a study on G6PD deficiency polymorphisms, which has been described in . The PCMiewed in ). Previoiewed in  also ideiewed in .Hospital-based surveys were conducted in mid-September to mid-October 2006 and in mid-April to mid-May 2007. Participants in this study comprised two main groups of individuals residing in Freetown: (i) symptomatic children visiting and/or admitted at the PCMH, as well as (ii) antenatal out-patients visiting the PCMH microbiology laboratory for routine blood tests . Patients were introduced to the study and those consenting were screened for malaria via microscopy, and filter paper blood samples were collected for each subject. Filter paper blood spots were air-dried, sealed in tiny plastic bags and stored at room temperature (25–31°C), until later molecular analysis at Jichi Medical University, Tochigi, Japan. Prior to blood donation, subjects were interviewed and basic demographic information for each participant were recorded including age, sex, ethnicity, and whether they were admitted at the hospital (i.e. in-patients) or only visiting (out-patients). Falciparum parasitaemia (parasite density) was determined for each subject by local microscopists, but due to technical limitations encountered in the field parasite density data was not utilized further.P. falciparum csp was amplified by a semi-nested PCR using primers and cycle conditions described in [Genomic DNA was extracted from randomly selected filter paper blood samples using the GFX kit , regardless of microscopy results. The C-terminal portion spanning the T-cell epitope regions of the gene of ribed in . In briecsp sequence was treated as an independent entity, coded and statistical analysis was conducted using the SPSS 10.0 software for Windows. The 2-sided Pearson Chi-square test of independence for categorical variables was used to compare the respective frequencies of haplotypes with regards to subject parameters including age, ethnicity, sex, and "status" , as well as time of sample collection. This was done in an attempt to examine the hypothesis whether haplotype distribution was associated with age (and presumably immunity status) or any of the other parameters described above. Statistical significance was set at the 5% level (P = 0.05).Sequences were aligned using MEGA4 software  and manucsp sequences from Vietnam [n = 142), from Iran (n = 90) described in [n = 44) of field isolates from neighbouring Gambia [pi (π) [ST) [In an effort to examine geographical diversity patterns with other parasite populations, the C-terminal portion of recently published Pf Vietnam   , which i [pi (π) . In addi(π) [ST) , which m(π) [ST)  includincsp sequences obtained herein have been submitted to GenBank with accession numbers GQ119637–GQ119678.This study was approved by the Ethical Committees of the PCMH, University of Sierra Leone and Jichi Medical University, Tochigi, Japan, and the partial Pfcsp gene fragment of 321 base pairs (bp), and these were successfully sequenced. Sixty-six sequences were of clear electrophoregram peaks showing no or low (<1/3) background overlap of the dominant peak. These were referred to as "single dominant" sequences and coded as "single-clone" infections. Six samples revealed sequences with electrophoregram peaks overlapping at one known mutation site for which both possible amino acid sequences were deduced from the nucleotide sequence . Nine samples showed chromatogram peak overlap at two or more known mutation sites (termed "mixed-clone" infections), and were omitted in the analysis presented here.Table P < 0.0001) in the number of PCR-positive samples between isolates collected in September–October 2006 i.e. during the rainy season (81/102 or 79.4%) and in April–May 2007 i.e. the dry season (21/77 or 27.2%).Among blood samples collected during the April–May 2007 survey, an additional set collected from the Thirty Fourth Military Hospital (M-34) at Wilberforce  were also analysed. Of these 2007 samples, 12 of 47 (25.5%) from the PCMH and nine of 30 (30.0%) from M-34 amplified and were sequenced, and all revealed "single-dominant" sequences  from Freetown identified a total of 42 unique haplotypes for which the respective proportions are depicted in Figure Pooling the data and analysis of the whole set of 99 C-terminal P = 0.187), yet unexpected haplotype distribution pattern with respect to age. As shown in Figure P = 0.049), who were mainly pregnant women visiting for regular antenatal follow-up. However, repeating the analysis with one sequence per isolate (i.e. using a randomly selected sequence from the six double-clone infected samples) reduced the p-value slightly . The availability of fewer sequences in other sub-groups precluded further comprehensive analysis with respect to sampling time and place. Nonetheless, it appears that, in this African endemic setting, haplotype frequencies are likely to vary with time (e.g. season), as demonstrated between samples collected in 2006 (rainy season) and 2007 (dry season) in Figure Preliminary analysis for the whole set of 99 sequences revealed a non-significant , it is of interest to note that not finding a particular haplotype does not completely preclude its existence in the locality of interest but may simply suggest it might be of low frequency. Another striking feature was that all mutations were almost always restricted to the same positions in the T-cell epitope regions of the gene, regardless of parasite origin. What seemed important was the nature of the substituting amino acid, again suggesting strong functional constraints on the CSP molecule.Spatial polymorphism patterns were examined by comparing recently published sympatric  ountries ,15,16. Tountries ,15,16 unST, Dxy, Kxy and Da) indicate regional differences, with African parasites having similar indices than they are to Asian parasites. This data is probably reminiscent of differences in transmission intensities/dynamics between the respective geographic regions.As depicted in Table P. falciparum circumsporozoite protein among wild isolates circulating in the capital, Freetown. Contrary to previous data presented from moderate to low endemic settings of malaria [csp (Th2R/Th3R epitopes) in an African setting are markedly diverse, but again with some degree of restriction. In addition, the single-haplotype predominance of the "Asia type" sequence (proportion > 50%) previously documented in Asia . Since most of these antenatal females were of mild or asymptomatic infection, this finding inevitably warrants the speculation whether distribution of P. falciparum csp haplotypes could be associated with disease severity i.e. does naturally-acquired immunity select for certain CSP variants in malaria-endemic areas? While the data presented here may in part support this notion, it may also be argued that this study may have suffered due to limited sample size (thus underpowered), or the findings may have been simply due to chance . Whatever the case, this haplotype distribution pattern in an African endemic area is important in that it adds insight into a relevant issue in vaccine design and evaluation i.e. could there indeed be significant differences of vaccine effects in symptomatic versus asymptomatic malaria individuals, particularly in the context of an African endemic setting where transmission is usually intense? This might be a major issue for consideration in future large-scale longitudinal studies on malaria antigen polymorphisms of vaccine antigens in other endemic nations.Presented here is the first malaria molecular epidemiologic study in Sierra Leone on the genetic diversity of T-cell epitope regions in the C-terminal portion of the  malaria -8,15, recsp [Despite the documented polymorphisms in CSP, the mechanism(s) behind them are poorly understood. Proposed mechanisms include intragenic recombination and natural selection (human immune pressure- or mosquito-induced). In recent field trials of the most advanced CSP vaccine , it was demonstrated that vaccine-mediated immunity was not strain-specific , nor didcsp . Thus itcsp ,15. Howecsp , the rolcsp -26. TakeP. falciparum transmission intensity correlate with high parasite prevalence and high genetic diversity of the parasite, with resultant high proportion of mixed genotypes in a given infection. This would increase the probability that a feeding mosquito obtains genetically distinct haplotypes in its blood meal, which in turn increases the probability of out-crossing in the midgut. This is consistent with data presented here considering the numerous CSP haplotypes circulating in this African endemic setting, as well as the tendency for sympatric parasites appearing to be more closely related than their allopatric counterparts , transmission dynamics, strong functional constraints on the protein, as well as the reproductive nature of the parasite. Which factor(s) contribute a major role at any given time and place would depend largely on a "net effect" given the local epidemiologic situation. Generally, regions of high s Figure , Table 2s Figure  and, morcsp polymorphism, supplemented with well-coordinated entomologic and sero-immunological analyses, in other African endemic areas would shed more light on this issue, similar to what was recently documented for the P. falciparum merozoite surface protein-1 (MSP-1) in Mali [Hopefully future large-scale studies on Pf in Mali .This study highlights an apparent discordant haplotype distribution of the T-cell epitopes of Pf-CSP with respect to age. In addition, the observed sequence polymorphism patterns were similar to that in neighbouring Gambia, but differed significantly at the sequence level from that observed in Asia. Thus, the interpretation of genetic diversity data of malaria antigen genes should probably reflect not only the antigen but also the local epidemiologic situation. In the context of the RTS,S vaccine, its future deployment in Sierra Leone is envisioned to confer similar protection in children based on extrapolation of efficacy results from The Gambia and Mozambique, because the 3D7 haplotype appears to be prevalent in this endemic setting.The authors declare that they have no competing interests.AJ, MJ and HM participated in the field and molecular genetic studies, AJ performed the sequence alignment and drafted the manuscript. HM and AJ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.P. falciparum circumsporozoite protein of field isolates (N = 375) from Asia and AfricaSummary of C-terminal sequences of . The data presented represents a comparison of haplotype (H) proportions of sequences of P. falciparum csp of field isolates collected from different countries in Asia and Africa. Only the region of the gene spanning the Th2R and Th3R was used .Click here for file


TIRAP has recently been reported to be common in Europeans and Africans and to roughly half the risks of heterogeneous infectious diseases including malaria, tuberculosis, bacteremia, and invasive pneumococal disease in heterozygous mutation carriers.The Toll-like receptors (TLRs) mediate innate immunity to various pathogens. A mutation (S180L) in the TLR downstream signal transducer TIRAP S180L variant by melting curve and RFLP analysis in 1095 delivering women from malaria-endemic Ghana, as well as in a further 1114 individuals participating in case control studies on sepsis and leprosy in Germany, Turkey and Bangladesh.We assessed the TIRAP S180L polymorphism was virtually absent. In contrast, the mutation was observed among 26.6%, 32.9% and 12% of German, Bangladesh and Turkish controls, respectively. No significant association of the heterozygous genotype with sepsis or leprosy was observed. Remarkably, homozygous TIRAP 180L tend to increase the risk of sepsis in the German study (P = 0.04).In Ghana, the TIRAP S180L against infectious diseases per se is not discernible.A broad protective effect of  Almost sixty years ago, J. Haldane suggested that malaria had selected hemoglobinopathies to high gene frequencies in malaria-endemic areas due to the protection they confer against this life-threatening disease . This "mPlasmodium – can prevent or modulate disease, host proteins involved in pathogen recognition and defense may also be of pathophysiological importance in malaria [In vitro and in vivo data point to TLRs -2, -4 and -9 to be central for Plasmodium recognition and subsequent inflammatory host responses [TIRAP, also known as Mal) mediates downstream signaling of TLR-2 and TLR-4, eventually inducing pro-inflammatory responses [TIRAP has been reported to diminish TLR-2 signaling and to occur at a prevalence of some 30% among healthy subjects in the UK and in 2–6% among individuals from West and East Africa. Heterozygosity for this SNP has been claimed to confer protection against malaria and severe malaria to an extent comparable to HbAS, and in addition, to more than halve the risks of bacteremia and tuberculosis in African populations [TIRAP S180L in a Ghanaian population exposed to holoendemic malaria transmission, and examined the role of the SNP in Caucasian and Asian patients affected by sepsis or leprosy.While in case of malaria it is obvious that genetic variations of the erythrocyte – target of the malaria parasite  malaria  and othe malaria . Genetic malaria . In vitresponses -14. The esponses . Recentlulations . In an aTIRAP S180L (rs8177374) in four groups of individuals: i) 1095 delivering women were recruited at the Presbyterian Mission Hospital in hyper- to holoendemic Agogo, Ashanti Region, Ghana in 2000 and in 2006. Diagnosis of placental Plasmodium falciparum infection by PCR and malariological indices of the majority of women have been described elsewhere. Briefly, although roughly half of the woman were infected by Plasmodium falciparum as evidenced by specific PCR assays, only 4.5% were febrile. Still, anaemia (hemoglobin < 11 g/dL) and low birth weight (< 2500 g) occurred in 33% and 15%, respectively, of woman with live singleton delivery  [M. leprae was identified by the detection of acid-fast bacilli and specific histopathological changes. Differential diagnoses, including granulomatous disease, syphilis, tuberculosis, sarcoidosis, and deep fungal infection, were excluded by clinical and laboratory criteria. One hundred asymptomatic control subjects were recruited from the same centers and had neither a family history nor symptoms related to leprosy .We examined S180L rs87374 in fe 15–47) . 280 heaThe study protocols were reviewed and approved by the Committee on Human Research Publication and Ethics, School of Medical Sciences, University for Science and Technology, Kumasi, Ghana, and by the human subject review committees at Humboldt-University Berlin, Germany, and Akdeniz University, Antalya, Turkey, and the institutional guidelines were followed in conducting this study. For the Bangladesh study, written approval was granted by the Ethical Review Committee of Bangladesh Medical Research Council  using the following primers and probes: sense primer: GCCAGGCACTGAGCAGTAGT, antisense primer: GTGGGTAGGCAGC-TCTTCTG, anchor probe: Red640-GATGGTGCAGCCCTCGGCCCC, and sensor probe: AGGCCCAACAGCAGGG-FL. The melting peaks are at 53°C and 62°C for the wildtype and the mutated sequences, respectively. Due to secondary structures and allele biased amplification within the region of this SNP, analysis of heterozygous genotypes may sometimes result in false homozygous results. Therefore, all mutated samples were reanalysed by conventional RFLP method described by Khor et al. [DNA was extracted from blood by standard techniques. Genotyping for r et al. . Genomic2 or Fisher's exact test, as applicable.Comparison of proportions of heterozygous and wild type individuals was done by χTIRAP S180L among German controls of 26.6%  closely matched the reported figure from the United Kingdom [TIRAP S180L variant in only one (0.1%) out of 1095 individuals from malaria holoendemic Ghana. This asymptomatic woman had a placental P. falciparum infection with a mean of 28 parasites/microscopic high power field, and prematurely delivered a child of low birth weight. The mere absence of the SNP in more than 500 P. falciparum non-infected women rendered statistical analysis meaningless , also potentially indicating some degree of advantage of the heterozygous mutation for leprosy. The small study from Turkey showed controversial results. We observed heterozygous TIRAP S180L in 11% of control individuals, but more frequently, 22%, in leprosy patients (Table Both leprosy studies showed no significant association either of heterozygosity or homozygosity with leprosy Table . Again, TIRAP 180L in the African studies could suggest a state of balanced polymorphism due to a yet unknown disadvantage in homozygotes and acting against relevant allele frequencies. A homozygous disadvantage has been claimed for Caucasians with respect to a slightly increased risk for invasive pneumococcal disease and pneumococcal empyema [P. falciparum infection. Thus, potential protective features of the SNP could be missed in individuals with non-severe malaria. However, in The Gambia the risk reductions in terms of severe malaria and "general malaria" were very similar [TIRAP S180L in Ghana, where malaria endemicity is among the highest in the world argues against attempts to replicate its putatively protective effect: Given the observed prevalence of heterozygosity of 0.1%, a study able to confirm the reported risk reduction would need to comprise several tens of thousands of individuals to have sufficient statistical power. This, in turn, is even another argument against the presumed clinical relevance of this mutation in Africa.We here report the virtual absence of a putatively malaria-protective SNP in an area of holoendemic malaria transmission in West Africa. This not only contrasts the published prevalence among African populations of 2–6% , also, a empyema . One the similar . UltimatTIRAP S180L has been suggested to be protective not only against malaria but also against bacterial infections including invasive pneumococcal disease, pneumococcal empyema, overall and pneumococcal bacteremia, and tuberculosis [TIRAP S180L genotype seem to be influenced by co-existing diseases such as Diabetes and renal pathology, however, there is no significant difference (Table TIRAP S180L also failed to show any association of heterozygosity with tuberculosis in any population. Of note, this study also found a very low allele frequency for the TIRAP S180L of only 0.08% in Ghana [Heterozygosity for rculosis . In our ce Table . This isin Ghana .TIRAP in a highly endemic region arguing against a major role of this SNP in malaria. In our study populations with a relative high prevalence of the TIRAP S180L, we found a trend but no significant protection of heterozygosity in sepsis and leprosy. In contrast, in sepsis TIRAP 180L homozygosity appears to be a risk factor for disease. Taking together our data and those of Khor et al. [TIRAP S180L in several diseases. At the same time homozygous TIRAP 180L appears to be a risk factor for the same diseases. The specific role of TIRAP S180L needs further investigation.While the TLR system has been shown to be critically involved in susceptibility to and manifestation of malaria, we found a very low frequency of the S180L mutation in the TLR downstream mediator r et al. , we suggThe authors declare that they have no competing interests.Samples were collected by EA, OK, RPS, LO, GB-A, and FPM. Genotyping was performed by LH. Statistics were done by FPM and RPS. LH, UB, FPM, and RRS conceptualized and designed the study. LH, FPM, and RRS wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: