Non-malaria febrile illnesses such as bacterial bloodstream infections (BSI) are a leading cause of disease and mortality in the tropics. However, there are no reliable, simple diagnostic tests for identifying BSI or other severe non-malaria febrile illnesses. We hypothesized that different infectious agents responsible for severe febrile illness would impact on the host metabololome in different ways, and investigated the potential of plasma metabolites for diagnosis of non-malaria febrile illness.We conducted a comprehensive mass-spectrometry based metabolomics analysis of the plasma of 61 children with severe febrile illness from a malaria-endemic rural African setting. Metabolite features characteristic for non-malaria febrile illness, BSI, severe anemia and poor clinical outcome were identified by receiver operating curve analysis.The plasma metabolome profile of malaria and non-malaria patients revealed fundamental differences in host response, including a differential activation of the hypothalamic-pituitary-adrenal axis. A simple corticosteroid signature was a good classifier of severe malaria and non-malaria febrile patients . Patients with BSI were characterized by upregulated plasma bile metabolites; a signature of two bile metabolites was estimated to have a sensitivity of 98.1% (95% CI: 80.2–100) and a specificity of 82.9% (95% CI: 54.7–99.9) to detect BSI in children younger than 5 years. This BSI signature demonstrates that host metabolites can have a superior diagnostic sensitivity compared to pathogen-detecting tests to identify infections characterized by low pathogen load such as BSI.This study demonstrates the potential use of plasma metabolites to identify causality in children with severe febrile illness in malaria-endemic settings. In the tropics, malaria is commonly attributed to be the cause of most childhood fevers, while in fact this condition is more commonly caused by other pathogens that are clinically indistinguishable from malaria. These so-called non-malaria febrile illnesses include bacterial bloodstream infections, which are associated with a higher mortality than malaria. Most health care facilities in the tropics have malaria diagnostic tests available, but tests for non-malarial febrile illnesses are extremely limited. There is the critical need for new tests that can address the question ‘if a febrile patient is not suffering from malaria, then what is it and what treatment will be effective?’ Using metabolomics, we have comprehensively screened the biochemical profile of patients with severe febrile illness for biological markers of non-malaria febrile illness. The results show that severe malaria and non-malaria febrile illness trigger a distinct metabolic response in the host. We demonstrate that this pathophysiological difference can be exploited for differential diagnosis of severe febrile illness and identification of patients with bacterial bloodstream infections. The introduction of malaria rapid diagnostic tests (RDT) has revealed that febrile illnesses in the tropics and subtropics are more commonly caused by non-malaria pathogens than by malaria –5. BacteHowever, in most malaria-endemic regions, children with non-malaria severe febrile illness are not easily diagnosed, which reflects a number of deficiencies in current WHO guidelines that still rely largely on malaria diagnostic tests. First of all, health care workers do not have access to tests to diagnose non-malaria febrile illness, which would inform the most effective treatment. For instance, BSI diagnosis still relies on traditional microbiology, which requires laboratory infrastructure and highly trained staff. Even when available, it takes two to three days for a result, which is too slow to timely inform case management. This can lead to ineffective first-line treatment choices that result in poorer survival outcomes , or alteAn ideal test to assess severe febrile illness in malaria-endemic settings should detect more than a single pathogen. In particular, it should provide information on the type of infecting pathogen  in order to immediately inform on the best treatment and referral options. Such information is unlikely to be captured by a single biological measurement or molecule (biomarker), but is expected to require a combination of molecules (signature). With the development of ‘omics’ profiling approaches it is now possible to measure hundreds of biological analytes simultaneously and thus enable assessment of the diagnostic performance of a molecular signature. This study utilized metabolomics, which in contrast to studies of DNA, RNA and proteins, enables characterization of the metabolome that is the final product of all cell regulatory processes. Hence, a metabolome profile provides a read-out of the (patho)physiological status of a patient at the time of sampling that cannot be obtained directly from the genome, transcriptome, or proteome. In addition, metabolomics analyses only require small sample volumes (20–50 μL) to determine a comprehensive metabolome profile, which is a particular advantage when studying pediatric populations. We hypothesized that the pathophysiological processes triggered by malaria and non-malaria febrile illness induce distinctive changes in the > 4,000 blood metabolites and explored for the first time whether such characteristic metabolites can be used for differential diagnosis of severe febrile illness.The study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the national ethics committee of Burkina Faso, the institutional review board of the Institute of Tropical Medicine Antwerp, the ethics committee of the University Hospital of Antwerp and the human research ethics office of the University of Western Australia. Written informed consent was given by all parents or guardians of enrolled children.Patients were recruited at the district hospital Centre Médical avec Antenne Chirurgicale Saint Camille in malaria-endemic Nanoro, Burkina Faso. Admitted children < 15 years of age presenting with axillary temperature ≥ 38°C, or clinical signs of severe illness were enrolled. Medical history, physical examination and outcome of febrile episode were registered on a standardized form. At time of hospital admission, venous whole blood for blood culture, malaria diagnosis, full blood count, glucose measurements, plasma metabolome analysis, and 16S rRNA deep sequencing were collected from all participants by trained study nurses. Details of sample processing and diagnostic procedures are provided in Plasmodium falciparum parasites in blood smear confirmed by microscopy. All patients with a negative blood smear were classified as non-malaria. Recent malaria was defined as positive malaria HRP-2 RDT (which can remain positive up to 6 weeks after successful treatment), but a negative blood smear [Malaria was defined as the presence of asexual od smear ,14. Conf8 column liquid chromatography mass spectrometry (C8-LC-MS) in positive ionization mode, and C18 column ultra-high pressure liquid chromatography mass spectrometry (C18-UHPLC-MS) in positive and negative ionization mode. These three analytical techniques each capture a different part of the plasma metabolome due to the different chromatography column chemistries used, and thus provide a very broad coverage of the metabolome. Detailed procedures for sample preparation and data acquisition are provided in Mass spectrometry-based metabolomics data were collected for all patients with 3 complementary analytical platforms: gas chromatography mass spectrometry (GC-MS), CAll data processing and statistical analyses were performed using the R software environment (version 3.1). This language comprises a selection of packages suitable for the implemented statistical methods: multivariate analysis  regression), receiver-operating curve (ROC) analysis, correlation analysis, hierarchical cluster analysis, biomarker validation and Bayesian latent class modeling. The specific statistical methods and R packages used are explained in We studied the plasma metabolome profile in 61 children with severe febrile illness. All were tested for BSI and malaria. The final diagnosis and characteristics of the patients are shown in metabolite features showed a minimal degree of variation (relative standard deviation < 15%) in the study sample  patients with non-malaria febrile illness, (ii) malaria patients, (iii) BSI patients, (iv) non-survival patients and (v) severe anemia patients . The detPlasmodium parasite density (Pearson’s correlation: 0.38–0.45). The heatmap of the 26 metabolite features characteristic for non-malaria/malaria further clarifies the distinct metabolic character of the two patient groups  and a disruption of fatty acid ß-oxidation (e.g. lower concentration of plasma acylcarnitines).The metabolome features that had lower concentrations in BSI patients compared to non-BSI patients were consistent with a lower rate of lipolysis  which had a very good AUC of 0.86, and a cut-off value that was indeed almost two-fold higher than the cut-off value to predict non-malaria febrile illness . A test 27H44O5 C27 bile acid, ID_6663_ON in S1 data) and bile alcohol  for the BSI signature. The individual diagnostic performance of the bile alcohol was AUC = 0.82 (95% CI = 0.69–0.93), adjusted p-value = 0.07, fold change = 2.54; and for the bile acid AUC = 0.79 (95% CI = 0.63–0.92), adjusted p-value = 0.1, fold change = 2.8. The ROC-curve analysis for the 2 bile metabolites combined including 51 patients  had a good AUC of 0.85 . If our findings can be validated in large-scale studies, then such a simple metabolite signature could be the basis of a new rapid diagnostic test that could potentially reduce BSI mortality by facilitating early diagnosis and timely hospital referral of BSI patients, and reduce empirical antibiotic usage.Severe malaria was shown to affect the plasma lipid profile, with triglycerides and phospholipids being most significantly changed . HypertrP. falciparum parasites. Further research is needed to understand this phenomenon, but our results already suggest that whatever the cause of the HPA dysfunction in malaria, it may be overcome by concomitant infections. Indeed, patients with the presumptive co-infections in our study were characterized by both the typical malaria lipids and the immunoregulatory metabolites found in non-malaria illness  axis by the immune system when sensing challenges like infectious agents . During  illness .The immunoregulatory metabolites also appeared to be predictive of poor clinical outcome  when present in high concentrations . There ie.g. dipstick tests to detect bacterial toxins, pesticides, veterinary drugs) whereby metabolite-binding antibodies are designed with computer-assisted molecular modelling [BSI patients were marked by increased plasma levels of bile acids and alcohols, which appeared to be independent of the infecting bacterial species. Elevated plasma levels of bile acids points to endotoxemia-related cholestasis in the liver ,28, and odelling .The design of this study has several limitations. We acknowledge that the presented results are hypothesis-generating and large-scale validation studies are essential to validate the performance of the candidate diagnostic signatures for BSI and non-malaria illness. In this discovery study, we did not include a diagnostic test panel for viral and non-malaria parasitic infections, which could have helped to better characterize the non-malaria patient group. We did not check the identified candidate diagnostic signatures in an asymptomatic control population and in patients with non-severe febrile illness. These 2 groups should be included in future validation studies. However a recent study conducted in Tanzania reported that over 70% of pediatric febrile patients without severe clinical signs had a viral disease , thus miIn conclusion, this study demonstrates that malaria and non-malaria patients with severe febrile illness have some fundamental differences in host response that could be exploited for differential diagnosis of severe febrile illness. In combination with the current malaria RDT, a rapid test assessing the plasma levels of 3–4 metabolites could inform on the likelihood of malaria, non-malaria illness, BSI, and survival and could thus empower primary health care centers in making informed treatment and referral decisions.S1 DataThe excel sheet comprises the following information in the columns for each metabolite feature: (1) patient group (characterized by metabolite feature), (2) Feature ID (study ID of metabolite feature), (3) area under ROC curve (AUC), (4) sensitivity, (5) specificity, (6) sensitivity + specificity, (7) p-value Mann-Whitney test comparing cases and controls adjusted for multiple comparisons, (8) fold change , (9) median signal intensity cases, (10) median signal intensity controls, (11) mass (determined by mass-spectrometry), (12) RT (chromatographic retention time in sec), (13) CoDa-DW , (14) rsd clin. samp. , (15) rsd pbqc samples , (16) formula , (17) putative ID 1 (putative identification 1), (18) KEGG 1 (KEGG entry of putative identification 1), (19) HMDB 1 (HMDB entry of putative identification 1), (20) putative ID 2 (putative identification 2), (21) KEGG 2 (KEGG entry of putative identification 2), (22) HMDB 2 (HMDB entry of putative identification 2), (23) putative ID 3 (putative identification 3), (24) KEGG 3 (KEGG entry of putative identification 3), (25) HMDB 3 (HMDB entry of putative identification 3), (26) class , (27) remarks on ID , (28) remarks on signal , (29) MSI scoring , (30) RT min (chromatographic retention time in min), (31) platform (platform with which the metabolite feature was detected). Metabolite features for which no plausible identification could be found are shaded in grey. Metabolites with a red Feature ID were upregulated in the considered patient group, metabolite features with a blue Feature ID were downregulated in the considered patient group. The putative identifications for the lipids were annotated with the abbreviations PC, PE, SL, MG, DG or TG for phosphatidylcholines, phosphatidylethanolamines, sphingolipids, monoacylglycerides, diacylglycerides and triacylglycerides respectively, and the total number of acyl side-chain carbons and the double bonds in the side-chains.(XLS)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 Results(DOCX)Click here for additional data file.


Plasmodium falciparum and Plasmodium vivax. Second, we describe and compare different parasite genotyping methods commonly used in malaria epidemiology and population genetics. Finally, we discuss potential applications of molecular epidemiological tools and methods toward malaria control and elimination efforts.Molecular epidemiology leverages genetic information to study the risk factors that affect the frequency and distribution of malaria cases. This article describes molecular epidemiologic investigations currently being carried out by the International Centers of Excellence for Malaria Research (ICEMR) network in a variety of malaria-endemic settings. First, we discuss various novel approaches to understand malaria incidence and gametocytemia, focusing on  Malaria-endemic areas have traditionally been classified in terms of transmission intensity, from hypo- to holoendemic. However, malaria epidemiology cannot be characterized solely on a one-dimensional scale. Prevalence of specific clinical manifestations of disease or parasite species, the onset of natural immunity, the spread of antimalarial drug resistance, and vectors involved in transmission, among others, vary across endemic areas.The ICEMR provide an opportunity to follow endemic areas over time and space, either longitudinally or by successive cross-sectional sampling at different spatial scales. These site-based research projects generate information and resources at regional scales, including epidemiologically contextualized molecular data and specimens, with the expectation that globally generalizable knowledge will emerge and guide evidence-based malaria elimination programs.Molecular methods have been used in malaria epidemiology for almost two decades.From an operational point of view, molecular tools allow 1) more sensitive estimations of prevalence and incidence that include subclinical cases of parasitemia; 2) assessment of the effectiveness of intervention strategies on the occurrence, complexity, and duration of infections; 3) differentiation between recrudescent, relapsing, and new infections; 4) estimation of the effect of interventions on the allele frequency of the targeted gene ; 5) estimation of the differential contribution of individual hosts to transmission by targeting gametocyte-specific genes; and 6) assessment of demographic patterns within parasite populations (gene flow–migration–colonization of new areas and population expansions), especially when transmission is driven by specific groups of particularly mobile subclinically infected individuals or migration across borders.A variety of coordinated efforts have focused on understanding geographic patterns of malaria transmission.Diagnostics remain fundamental to molecular epidemiology. Although many specific aspects of malaria diagnostics are discussed separately in this supplement,Plasmodium vivax infections were missed by microscopy, as compared with molecular detection, in hypoendemic areas of Brazil as the incidence of clinical cases declined.pfhrp2 and/or pfhrp3 genes (the antigens targeted by the RDTs) in the Americas,P. vivax.Light microscopy based on Giemsa-stained thick blood smears has limited the sensitivity for detecting low-density infections, hampering its use for measuring actual transmission.Plasmodium falciparum that target repetitive genomic sequences, substantially increasing diagnostic sensitivity without requiring laborious sampling of large blood volumes or elaborate sample processing.In addition, the ICEMR is developing new approaches to detecting low-level malaria parasite infections. At the southwest Pacific ICEMR, two new quantitative polymerase chain reaction (qPCR) assays have been developed for Determining the different contributions of groups of patients  to malaria transmission requires information about gametocytemia. Asymptomatic infections, for example, represent a “hidden” parasite reservoir that can sustain transmission.P. falciparum infections and parasite infectivity to mosquitoes has been investigated in Africa,P. vivax or low endemic regions outside Africa, where other vector species are involved in malaria transmission. The two Latin American ICEMR sites have characterized P. vivax transmission dynamics in symptomatic and asymptomatic volunteers. Molecular assays  found comparable expression levels of Pvs25 in symptomatic and asymptomatic volunteers.Anopheles mosquitoes  without statistically significant differences between direct feeding and membrane-feeding assays  indicating that asymptomatic carriers can infect mosquitoes in this setting. Studies to quantify the transmissibility of subpatent and asymptomatic parasitemia to Anopheles darlingi in Peru are ongoing .The relationship between asymptomatic Plasmodium species by qPCR and qRT-PCR.P. falciparum gametocytes, including new and existing markers .P. vivax have not yet been identified.A major challenge to understanding parasite population biology and epidemiology is the paucity of data characterizing the different elements of intra-host dynamics.Understanding the genetic diversity and structure of malaria parasite populations is the key for predicting the emergence and spread of phenotypes of interest, such as new antigenic or drug resistance variants. Whereas population genomics is an area of investigation reaching maturity in malaria,msp2 in P. falciparum, msp3α in P. vivax, and msp1 and csp in both species.31Traditional genotyping methods for malarial parasites rely on the size polymorphism of genes encoding surface antigens with variable number of tandem repeats, such as A second approach involves using partial or complete gene sequences from nuclear or organellar genomes. Such analyses have usually aimed to better understand the diversity of a targeted gene under consideration as a vaccine candidate, or because it harbors mutations linked to drug resistance.Finally, a major trend across ICEMR is the use of multi-locus genotyping that targets non-antigenic loci . This ap43P. falciparum genome; an average of one microsatellite locus is found per 2–3 kb of sequence. Microsatellite genotyping has revealed a wide range of population structures in P. falciparum isolates from four continents. Diversity and recombination rates are highest in holoendemic Africa,P. vivax,P. vivax was found to vary worldwide, with highest levels in south and southeast Asia57Microsatellites are highly abundant in the P. vivax microsatellite loci for use across sites. ICEMR India and the non-Amazonia Latin America ICEMR have developed their own sets of microsatellite loci that complement those identified by others.ICEMR has been working on standardizing different sets of microsatellite loci to mitigate problems derived from these complex patterns of evolution. Particularly important are the efforts by Amazonia and southwest Pacific ICEMR sites to standardize The high reproducibility of SNPs allows global comparisons and exploration of patterns over long time scales.P. falciparum and P. vivax to identify SNPs that can distinguish between parasites from different geographic areas. These “region-specific SNPs” will provide a means to predict the origins of outbreaks and to estimate the contribution of imported infections to overall transmission in areas where transmission has decreased to very low levels. The southwest Pacific ICEMR is developing algorithms to identify the most informative markers so that minimal numbers of markers can be developed as a parasite “barcoding tool”; other ICEMR sites are expected to follow suit. Regional molecular barcodes also include elements of the global barcodeAs with microsatellites, multiple ICEMR sites  are developing SNP typing protocols for P. falciparum have been selected from different chromosomes so that demographic processes including origin of an infection can be studied.59The previously established term “molecular barcode” refers to a small standardized set of SNPs used for genotyping.P. vivax) or recrudescence of a previously undetected minority clone may lead to a false conclusion.In clinical trials of antimalarial drugs, genotyping can help to distinguish between parasite recrudescence  and new infections . A problem reported by ICEMR in hypoendemic areas was that the circulating parasites may be closely related, making it difficult to separate recrudescences from new infections.P. falciparum or P. vivax genotypes over time to determine the number of new infections on a background of preexisting parasite clones. This molecular measure of the force of infection (defined as the number of distinct parasite clones acquired over time) provides a marker for individual exposure and transmission and thus is suitable for measuring outcomes of interventions.28A novel application developed by the southwest Pacific ICEMR follows individual Determining whether all infections had a single origin (outbreak) or multiple origins is crucial for designing proper containment strategies, and DNA analysis can help in this task.46P. vivax microsatellites carried out by the southwest Pacific and Amazonia ICEMR sites included 841 isolates from four continents collected in 1999–2008, which were genotyped with 11 microsatellite markers.P. falciparum and P. vivax can be maintained over time, requiring a higher number of hypervariable microsatellites to achieve the same discrimination.60A common theme across the ICEMR settings is that the number and type of loci needed for genotyping should be tailored to the objective of the epidemiologic investigation in the setting where such studies will be carried out. For example, a global study of P. vivax and P. falciparum in the same endemic settings. Whereas in some areas the two parasites show similar patterns,54Population structure is the result of common processes in nature, including inbreeding and geographic isolation.P. vivax performed by the southwest Pacific and Amazonia ICEMR sites showed that parasite populations from southeast Asia, where transmission was intermediate but the migration of infected hosts was high, were more diverse than populations sampled from South America.P. vivax in the Americas, as a region, may be comparable to that in Asia and Oceania.35A collaborative study of the geographic population structure of P. vivax malaria in the Americas have resulted in patterns of low local but high regional genetic diversity where several populations are isolated from each other. Thus, if only a handful of populations were sampled in the Americas, one could observe low regional genetic diversity. However, when aggregated, P. vivax in South America is the result of a complex demographic history with limited gene flow within and among some regions.21Unlike Asia where human migration increases local genetic diversity, the combined effects of the geographic structure and the low incidence of When interventions lead to reduction in transmission, it is expected that parasite population diversity, overall, will be reduced. This process will yield signatures that can be captured with molecular data . There aMOI is the result of two ecologically distinct processes that are hard to differentiate by genotyping only: coinfections (two or more genotypes being transmitted simultaneously by a mosquito) and superinfections (a patient acquiring multiple but independent infections). Both of these processes relate to transmission intensity.62P. falciparum indicate that the prevalence of multiclonal infections diminishes with a reduction in malaria transmission.P. falciparum, but an analysis across sites is still pending. Despite this clear trend, the relationship between MOI and transmission is not linear,P. vivax, where multi-clone infections remain common even in low-transmission areas  is expected to rise when transmission decreases, so inbreeding is likely to increase.62P. falciparum and P. vivax observed throughout the ICEMR sites and elsewhere.Estimates of genetic diversity may show even more complex patterns since they depend on the effective population size (see below in this section) and the mutation rate of the loci under study.Ne.Ne is not equivalent to heterozygosity but rather predicts loss in heterozygosity. It relates to the uneven reproductive success of parasite lineages.Ne has the properties of the harmonic mean, so its value is affected by the smallest population size.Ne concept has important implications in malaria epidemiology if we consider that changes in genetic diversity in a set of loci are observable at a time scale that is relative to their mutation rates. For example, after a reduction in Ne that might occur after a sustained intervention, heterozygosity would be expected to recover faster at microsatellite loci than SNPs simply because the former have a higher mutation rate. Such a difference can be informative in terms of evaluating the long-term effect of interventions.Finally, some studies have explored estimating changes in the parasite effective population size, Ne, each one measuring different aspects .Ne and malaria incidence is likely not linear as has been shown in other pathogens,Ne. Third, Ne estimates might be inflated by migration or population substructure.Ne has the property of the harmonic mean,Ne than the total number of cases in a given year or the number of cases during the high-transmission season.Ne.Although a reduction in the effective population size is expected if transmission is reduced, many factors could make such an outcome difficult to observe. First, there are different ways to measure The use of technologies that capture genetic information from the parasite, vector, and patients permits characterization of malaria transmission in ways that were unthinkable 10 years ago. That intra-host dynamics characterized by a combination of RNA- and DNA-based measurements illustrates major advances in malaria epidemiology from incorporating molecular tools. The ICEMR sites are currently taking advantage of such methods to better characterize malaria transmission across study sites with divergent epidemiologic characteristics. How to translate complex population genetic data into epidemiologically relevant information on malaria transmission is an ongoing discussion in the ICEMR network. Molecular methods provide information on malaria prevalence/incidence that considers subclinical/asymptomatic infections and better characterizes gametocytemia. Population genetic parameters will likely provide useful information if they are interpreted properly; nevertheless, population genetic approaches require further validation by evidence gathered by all ICEMR sites and comparisons worldwide.


In vitro studies have documented an inhibitory effect of angiotensin II on Plasmodium growth, while studies using chemical inhibitors of angiotensin II in mice showed protection against experimental cerebral malaria but not major effects on parasite growth. To determine whether the level of angiotensin II affects Plasmodium growth and/or disease outcome in malaria, elevated levels of angiotensin II were induced in mice by intradermal implantation of osmotic mini-pumps providing constant release of this hormone. Mice were then infected with P. berghei and monitored for parasitemia and incidence of cerebral malaria. Mice infused with angiotensin II showed decreased parasitemia seven days after infection. The development of experimental cerebral malaria was delayed and a moderate increase in survival was observed in mice with elevated angiotensin II, as confirmed by decreased number of cerebral hemorrhages compared to controls. The results presented here show for the first time the effect of elevated levels of angiotensin II in an in vivo model of malaria. The decreased pathogenesis observed in mice complements a previous human genetic study, reinforcing the hypothesis of a beneficial effect of angiotensin II in malaria.Angiotensin II, a peptide hormone that regulates blood pressure, has been proposed as a protective factor against cerebral malaria based on a genetic analysis.  Plasmodium falciparum infected red blood cells and host endothelial cells plays a key role in cerebral malaria. Mature stage parasites express ligands (PfEMP1) on the surface of infected erythrocytes that interact with host endothelial cell receptors  lead, ICAM-1 .Angiotensin II (Ang II) is a peptide hormone with well-characterized effects on circulatory homeostasis, where it induces vasoconstriction that results in increased high blood pressure. Ang II is derived from angiotensinogen through sequential enzymatic cleavages: first renin cleaves angiotensinogen, forming Ang I that is then converted to Ang II by angiotensin converting enzyme (ACE). Circulating Ang II not only contributes to increase blood pressure, but it is also involved in key inflammatory events, including the activation of endothelial cells to express higher levels of leukocyte adhesive molecules and the increase in vascular permeability .P. falciparum in vitro [A protective role for Ang II against cerebral malaria was proposed based on a gene polymorphism analysis of angiotensin-related enzymes in patients with severe or mild malaria, suggesting that elevated levels of Ang II would reduce the incidence of severe disease . Additioin vitro .in vivo, an experimental model in mice exposed to increased levels of circulating Ang II was developed. Since Ang II has a rapid turnover [To study the effect of Ang II in malaria turnover , continuThis study 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 (IACUC#110910) was approved by the Institutional Animal Care and Use Committee of New York University School of Medicine, which is fully accredited by the Association For Assessment and Accreditation Of Laboratory Animal Care International (AAALAC).P. falciparum 3D7 erythrocytic asexual cultures were maintained at 5% hematocrit in complete media , supplemented with 25 mM sodium bicarbonate and 0.5% Albumax II at 5% oxygen, 5% carbon dioxide and 90% nitrogen. Parasite cultures were synchronized using magnetic separation of schizont stages with MACS cell separation column (Miltenyi Biotec). P. falciparum infected erythrocytes in late stages were added to 96-well plates at 1.9% parasitemia 5% hematocrite and incubated for 24 h in the presence of different concentrations of Ang II . 10 μl from each well were smeared on glass slides and stained with Giemsa before blind microscopic quantification of parasites. To asses whether Ang II interferes in the process of erythrocyte rupture after the completion of the infection cycle, 5x105 schizont stages per well (96% purity) were added to 96-well plates in the presence of different concentrations of Ang II (n = 8/dose) and incubated for 16 h and therefore analyzed independently to quantify the remaining non-ruptured erythrocytes in a standard hemocytometer.Plasmodium berghei-ANKA 106 infected erythrocytes in 200 μl of sterile PBS i.p. Determination and severity of experimental cerebral malaria was scored based on appearance  and behavior . This scoring system is a modification of a method described before [Mice 5–6 weeks old C57BL/6 were purchased from Taconic and infected with Mice were systemically infused with saline buffer (0.25 μl/h) or Ang II (100 or 500 ng/kg/min at 0.25 μl/h)  using osmotic mini-pumps that provide sustained 14-day delivery . Mini-pumps were implanted subcutaneously on the animal back right before infection.2 of brain histological sections per mouse using the Slidepath Digital Image Hub (DIH) software.Paraffin-embedded brain sections were stained with H&E and scanned on the Leica Biosystems SCN400 whole slide scanner. Experimental cerebral malaria induced histopathological alterations in the brain were assessed by counting and measuring the number and maximal diameter of petechiae and hemorrhages in an area of 41.6 ± 4.9 mmData on every experiment was analyzed to determine if it follows a normal distribution and if variance between groups is not different using Shapiro-Wilk and Levene’s tests for normality and homoscedasticity, respectively. Parasitemia levels in P. falciparum cultures in vitro. A dose-dependent inhibition of growth was observed, confirming previous reports in P. gallinaceum [P. falciparum [To first characterize the anti-Plasmodium effect of Ang II, the activity of Ang II was tested against linaceum  and P. flciparum . It was in vivo, the P. berghei-ANKA infection of C57BL/6 mice model of malaria was used. This combination of mouse strain and parasite species induces experimental cerebral malaria, a syndrome that mimics the pathogenesis of human cerebral malaria, despite differences in the initial mechanism of erythrocyte sequestration in the brain [To determine whether Ang II affects Plasmodium growth and disease outcome he brain .P. berghei in mice  previously shown to have no detectable effects in mouse blood pressure or other apparent pathology , 15. WheAng II moderately reduced the development of experimental cerebral malaria and increased survival. There was also a delay in the onset of symptoms of 1 to 2 days and a corresponding increase of survival time of these mice .Hemorrhages in the brain of each mouse were quantified in histological sections . Mice wiP. berghei infection, other authors observed that a treatment that inhibits Ang II generation  or one that inhibits one of Ang II receptors  resulted in decreased T cell responses and partial protection from cerebral malaria [P. berghei infection, on one side inhibiting parasite growth as observed in vitro in this study and others [P. falciparum growth in vitro, while the effect in mice parasitemia is only observed after day 5 of infection may indicate differences in the Ang II effect on the two Plasmodium species and/or possibly that the growth-inhibitory effect of Ang II is not relevant in vivo and the late decrease of P. berghei parasitemia is due to indirect effects of Ang II on the immune response rather than direct killing of the parasite.It is possible that the Ang II-induced decrease in parasitemia contributed to the delay on the onset of experimental cerebral malaria, since the development of this syndrome is dependent on the levels of parasitemia . Using t malaria . Howeverd others , 11, on d others , or evend others  and humad others –21. The A protective role for Ang II against human cerebral malaria has been proposed based on a gene polymorphism analysis of angiotensin-related enzymes in patients with severe or mild malaria. It was observed that an insertion/deletion polymorphism of ACE, that is associated with increased levels of Ang II, was also strongly associated with mild malaria. Similarly, a mutation on angiotensin converting enzyme 2, that catalizes the conversion of Ang I or Ang II into Ang 1–7 and may result in increased Ang II in women, was also associated with mild malaria [It was observed that elevated levels of Ang II have a beneficial effect against malaria-induced pathology in mouse models. Elevated Ang II moderately decreased the levels of parasite and the pathogenesis of the disease, resulting in lower number of hemorrhages and increased survival rates. These results are complementary to the previous observations in the human genetic study , suggestS1 Table(XLSX)Click here for additional data file.


Plasmodium falciparum carriers are only partially understood. Among them, the role plays by the self-reactive antibodies has not been clarified yet. In this study, the relationship between repertoires of circulating self-reactive and parasite-specific immunoglobulin G (IgG), their correlation with cytokine levels, and their association with protection against malaria was investigated in asymptomatic Plasmodium falciparum-infected Gabonese children.Mechanisms of acquired protection to malaria in asymptomatic P. falciparum-specific antibody repertoire was analysed using a protein micro-array immunoassay, the total auto-antibody repertoire by quantitative immunoblotting and circulating cytokine levels were measured by ELISA in endemic controls (EC) and P. falciparum-infected children from Gabon with asymptomatic (AM) or mild malaria (MM). The association of self- and parasite-specific antibody repertoires with circulating cytokines was evaluated using single linkage hierarchical clustering, Kruskal – Wallis tests and Spearman’s rank correlation.The diversity of P. falciparum-specific IgG were similar to those in the MM group. Moreover, the asymptomatic children had increased levels of autoantibodies recognising brain antigens. In addition, a correlation between IL-10 levels and parasite load was found in AM and MM children. These two groups also exhibited significant correlations between plasma levels of IL-10 and IFN-γ with age and with total plasma IgG levels. IL-10 and IFN-γ levels were also associated with auto-antibody responses in AM.Children with AM exhibited an IgG response to merozoite surface protein 3 (MSP3) but not to MSP1-19, although their levels of total P. falciparum infection in Gabonese children.Altogether, these results indicate that a self-reactive polyclonal response associated with increased IgG to MSP3 and high plasma levels of IL-10 and IFN-γ may contribute to protective immune mechanisms triggered in asymptomatic The online version of this article (doi:10.1186/s12936-015-0658-7) contains supplementary material, which is available to authorized users. Plasmodium falciparum infection can lead to asymptomatic malaria (AM), mild malaria (MM) or severe malaria (SM) [ria (SM) . Despiteria (SM) , and sucria (SM) -5. Howevria (SM) . There iP. falciparum.Malaria is associated with hypergammaglobulinemia and the production of self-reactive antibodies that recognize self-antigens, such as phospholipid, cardiolipin, ssDNA, dsDNA, and rheumatoid factor -9, althoP. falciparum-infected children with either AM or MM and in healthy children (endemic control (EC)). The global analysis of antibody repertoires was done using a microarray immunoassay [Several mechanisms may contribute to auto-antibody production in malaria. These include polyclonal B cell activation by parasitic mitogens , stimulaunoassay  and quanunoassay -18 couplResults clearly show that IgG autoreactivity was greater in AM than in MM children. In addition, the auto-antibody response was correlated significantly with the presence of anti-merozoite surface protein antigen 3 (MSP3) IgG and high levels of IL-10 in the AM group. By contrast, anti-MSP1 IgG was predominant in MM patients. Interestingly, IL-10 levels were also associated with parasite load in AM and MM groups.P. falciparum carriers.Altogether, these results strongly support a protective role for auto-antibodies triggered by the parasite which correlate with plasma IL-10 levels and with anti-MSP2 and anti-MSP3 IgG levels in clinical immunity observed in asymptomatic P. falciparum thin blood smears and no clinical symptoms were included in the asymptomatic malaria (AM) group and were defined as AM carriers and children with no clinical symptoms and a negative thin blood smears were enrolled in the endemic control (EC) group. All AM and EC children were from the same area of Libreville city. MM were recruited at Owendo Pediatric Hospital (OPH) and Libreville Hospital Centre. AM and EC children were examined daily for clinical symptoms. Parasitaemia were determined on days 0 , 7 and 30. Oral amodiaquine (25 mg/kg) was administered for three days from day-0 to MM patients and at day 30 to AM carriers. No patient death occurred during the recruitment period.The ethics committee of the Gabon Health Office approved this study. Between 1996 and 2000, 97 children (age range two months to six years) living in Libreville Hospital Centre (Gabon) were included in the study after obtaining parental informed consent. All patients presenting diseases other than malaria were excluded from the study. Malaria patients were categorized on the basis of World Health Organization (WHO) guidelines for the definition of uncomplicated malaria. Mild malaria (MM) presented fever with positive thin blood smears. Individuals with positive Venous blood was collected in EDTA on days 0 (before treatment), 7 and 30. Plasma was separated and stored at −80°C until use. Parasitaemia (expressed as the percentage of infected erythrocytes) was determined by microscopic examination of Giemsa-stained thin blood smears.Plasmatic levels of IFN-γ, TNF and IL-10 were measured in EC, AM and MM at day 0, 7 and 30 using a sandwich-type ELISA  according to the manufacturer’s recommendations.2O2 in 0.05 M phosphate-citrate buffer, pH5 and the product was quantified from the optical density (OD) at 450 nm. The mean of OD value was fitted into the sigmoidal standard curve using a specific ELISA programme running in Igor version 3.16 .The quantification of total IgG in children plasma from all groups was done by ELISA. At day 0, 7 and 30 after their inclusion in the study. Briefly, 96 microwell plates  were coated with 5 μg/ml of purified sheep polyclonal anti-human IgG  followed by overnight incubation at 4°C. After blockage with 1% gelatine and washing with 1% gelatin-PBS, serial dilutions of plasma samples were incubated overnight at 4°C. Bound IgG was detected using a peroxidase-conjugated polyclonal anti-human-IgG . Binding was revealed using 0.5 mg/ml O-phenylenediamine (OPD) substrate  and 0.03% HP. falciparum specific plasmatic IgGs were quantified using ELISA. Briefly, ninety-six-well plates  were coated overnight at 4°C with 5 μg/ml of P. falciparum 3D7 schizont-stage protein extract. Plates were blocked with 1% gelatine. One-hundred ml of 1:100 dilution of patient plasma was added to each well in duplicate, incubated for one hour at 37°C, and developed using anti-human IgG-HRP and OPD . Ten per cent sodium dodecyl sulphate was added after 30 min and the OD measured at 450 nm. Concentration of P. falciparum-specific IgG in the samples was calculated from a standard curve using 0.001 − 2 mg/ml of purified human IgG. For every sample tested a cut-off value was estimated using a pool of plasma from children living in Europe who had never been exposed to P. falciparum infection.Total P. falciparum erythrocyte membrane protein 1 (PfEMP1), and circumsporozoite protein epitope NANP-4. Arrays were incubated with 100 μl of 1:100 dilution of plasma for 15 min at 37°C and washed for 5 min in PBS-Tween. Arrays were then incubated with Alexa Fluor 546-labelled anti-human IgG monoclonal antibody  for 20 min. Alexa 555 diluted 1:1 in PBS was used as signal control. Ten mg/ml bovine serum albumin was used as a negative control. Anti-human IgG  was used as carry-over control. Arrays were washed twice in PBS-Tween, dried under nitrogen, and scanned with a GMS 428 scanner . Acquired images were analysed with the GenePix image analysis software . The mean intensity of duplicate spots was analysed using the linear range of the individual slide standard micro-array [The plasma parasite-specific antibodies were profiled using a protein micro-array immuno-assay . Each arro-array .The PANAMA-Blot method was used to determine the antibody repertoire to brain antigens as previously described ,16. PANA2 test. The association between continuous quantitative parameters was assessed by Spearman’s rank correlation. The trend over time was tested by the paired t-test; p-values <0.05 were considered significant. Linear discriminant analysis was performed on log-transformed data, and samples with incomplete information were rejected. The significance of contributing parameters was calculated by backward elimination, using partial F tests on variances explained by models with or without the respective parameters. The significance criterion was a Bonferroni-corrected p-value <0.05. Single linkage hierarchical clustering of the antibody response against the 20 P. falciparum antigens tested in the different groups was performed using the hclust function in R software. For the heatmaps, fold change of the antibody response and was calculated using the formula Fc = μ1/μ2 for μ1 > μ2 and Fc = −μ2/μ1 for μ1 < μ2, where μ1 is the mean of the first group and μ2 the mean of the second group compared. The same methods were used for brain self-reactive IgG responses clustering and heatmaps.Immunoblot data were analysed by multivariate statistical methods, using IGOR software . After baseline subtraction, the standard migration scale was divided into sections around individual peaks of immunoreactivity. Section-wise absorbance values were subjected to principal component analysis (PCA). The first component (PCA-1) was the one-dimensional fitting vector that accounted for the largest proportion of variance and factor 2 (PCA-2) was the second-best orthogonal one-dimensional vector fitting the data. Quantitative comparisons between groups were done either with the Mann–Whitney (between two groups) or Kruskal-Wallis tests (>two groups). Qualitative association was tested by Pearson’s cversus 4.5% (range 0.6 − 48.6); p <0.001), , compared to 17% in AM and 11% in EC ,  compared to the EC group, whereas IgG to MSP1-B2 PAR distinguished MM from AM , whereas MSP3 and MSP2 antigens are predominantly recognized by IgG of AM carriers.The differences in fold change of s Figure F. These P. falciparum infection was investigated by measuring auto-antibodies against against brain protein extract using a quantitative immunoblots. Differential repertoire analysis was done on all groups using Principal Component Analysis (PCA). PCA-1 represents a score of total anti-brain activity. Both in non-adjusted and adjusted assays, PCA-1 was higher in the AM group than in MM and EC groups, particularly in patients older than 18 months in whom the presence of maternal IgG could be excluded  IgG reactivity. With the exception of two, all study participants fell into three sub-groups: α) high self-reactive IgG and low P. falciparum-specific IgG; β) low self-reactive IgG and high P. falciparum-specific IgG; and, δ) low self-reactive IgG and low P. falciparum-specific IgG    and anti-G Figure A. Partict-test), when no parasites were detectable in peripheral blood    Figure A. The sa) Figure B. In add) Figure C.Figure P. falciparum infection recognize parasite antigens; the rest are against unrelated or host antigens [An infinitesimally small fraction of antibodies produced during antigens ,19. To gantigens ,17.P. falciparum and Plasmodium vivax patients [P. falciparum-specific antibodies were lower in AM than in MM. Antibody responses to MSP1-19 and MSP1-B2 PAR antigens were predominant in MM, whereas AM was associated with low levels of antibody to MSP1-19 but higher levels of antibodies to MSP3 and MSP2. These observations suggest that parasite control does not depend upon anti-P. falciparum antibody levels but is linked to the nature and effector functions of those antibodies – such as cytophilic antibodies – that contribute to parasite elimination [P. falciparum transmission are known to develop a strong antibody response to PfEMP1 [Surprisingly, the asymptomatic carriers displayed greater self-reactive IgG response than MM children. The auto-antibody response was correlated with age and total IgG levels, suggesting a link with premunition ,11,12. Tpatients . Howevermination -22. Antimination ,24. Chilo PfEMP1 ,26. HoweP. falciparum-specific IgG, high plasma levels of IFN-γ and IL-10, while MM was associated with high levels of IL-10 and specific anti-falciparum antibodies but low self-reactive response with low TNF secretion.A correlation between elevated self-reactivity to brain antigens and plasma levels of IFN-γ and IL-10 was observed in the AM group. IL-10 is an anti-inflammatory cytokine with a controversial role in malaria; high concentrations of IL-10 are associated with both severe disease and protection ,27. In aP. falciparum infection may not be exclusively attributable to cross-reactivity between parasite and host antigens or to polyclonal B cell activation. The origin of the self-reactive antibodies induced during malaria remains unclear. Auto-antibodies associated with AM might be derived from CD27+ marginal zone B cells that are expanded in some auto-immune diseases. Alternatively, some of these auto-antibodies could be ‘natural antibodies’ that are produced in the absence of exogenous antigenic stimulation and that are characterized by low affinity for the antigen and polyreactivity [There was no correlation between parasite-specific and self-reactive antibody responses in the infected groups. However, a self-antigen candidate was detected in the AM group. This suggests that the self-reactive response during activity ,10,23. Aactivity -24. Indeactivity -36.P. falciparum-specific IgG levels and low parasitaemia in AM children provide new clues into the mechanisms underlying AM. They also strongly suggest a role for the auto-antibody response in protective mechanisms against clinical disease in P. falciparum-infected individuals living in endemic areas.The findings of a strong correlation between autoreactivity and plasma cytokine profile, low


Malaria endemicity in the archipelago of Indonesia varies substantially across regions. Following the government’s plan for a malaria elimination programme in Indonesia, baseline malaria surveys were conducted in Mamuju District, West Sulawesi Province, Indonesia to re-assess the malaria situation prior to the establishment of an evidence-based malaria elimination programme in the area. The present study aims to determine the antibody response to three merozoite antigens among the inhabitants of the district.Antibodies were measured following elution from filter-paper blood spots collected during cross-sectional surveys in the dry and wet season in 2010. Enzyme-linked immunosorbent assays using three merozoite antigens, MSP2, EBA175 and PfRh2a were conducted. A positivity threshold was determined by samples from unexposed individuals and the difference in antibody level against each antigen and correlation of antibody level in different age groups and seasons were statistically analysed.Plasmodium falciparum infection status of the subjects.A total of 497 subjects, 248 in dry and 249 in wet season, aged between 0.6 and 78 years were included. The prevalence of positive antibody responses to MSP2, EBA175 and PfRh2a antigens in dry season were 27.82, 27.42 and 25.81%, respectively. In wet season, the antibody prevalences were 64.26, 64.66 and 61.45%. The antibody levels to the three antigens were all higher in older age groups and also significantly higher in the wet season. The antibody levels also correlated positively with the P. falciparum infection and as markers for malaria exposure.MSP2, EBA175 and PfRh2a induce antibody responses among the subjects in Mamuju District, and the prevalence is significantly higher in wet season. The level of antibody also correlates significantly with age and malaria positivity. The overall results indicate the antigens might be used as a target for vaccines against  Plasmodium falciparum and Plasmodium vivax are the major causative species of malaria in Indonesia. Plasmodium malariae and Plasmodium ovale occurred infrequently, mainly in eastern Indonesia.Malaria remains one of the major public health problems in many tropical countries around the world, including Indonesia. In 2013, of the 97 countries with ongoing malaria transmission, 11 were in pre-elimination phase and seven in elimination phase [in vivo by inhibiting merozoite invasion of erythrocytes, opsonizing merozoites for phagocytosis, and inducing antibody-dependent cellular inhibition.Malaria symptoms in humans are exclusively caused by the erythrocytic stage of the parasite and therefore vaccine development is mainly focused to prevent the invasion and development of the parasite in this stage. Children and pregnant women are the most vulnerable groups in areas of stable malaria, and after repeated exposures individuals develop immunity to the blood stage parasite, thereby reducing the risk of clinical symptoms and life-threatening complications. To date, various merozoite proteins have been implicated in the erythrocyte invasion, and some of these have been identified as targets for vaccine development and markers for epidemiologic studies . AntibodP. falciparum parasites that mediates erythrocyte invasion. EBA-175 is found in the micronemes of merozoites, which secrete EBA-175 to bind erythrocytes that are ready to be invaded [Invasion of the erythrocytes by the malarial parasite involves several families of merozoite proteins during the initial step of attachment, reorientation, penetration, and formation of the parasitophorous vacuole. This includes merozoite surface protein family (MSP), erythrocyte binding-like protein (EBP) and the reticulocyte binding-like or reticulocyte homologue proteins (RBL or PfRh). Many of the proteins are being evaluated as targets for anti-malarial vaccine candidates , 5. Mero invaded , 7. Anal invaded .Plasmodium is one of the classes of type 1 transmembrane ligand parasites localized in the rhoptry. A recent study indicated that antibodies that target the binding domain of PfRh2a inhibited the invasion of the erythrocyte by the merozoites [Reticulocyte binding-like protein (protein RBL) of rozoites .The present study aims to measure antibody responses against merozoite proteins MSP2, EBA-175 and PfRh2a in Mamuju District, West Sulawesi, a malaria-endemic region in eastern Indonesia and to evaluate the association between antibody levels, malaria infection, age, and season.The study was a part of research activities to establish an evidence-based malaria control programme in the Mamuju District, West Sulawesi Province, Indonesia through assessment of the baseline malaria prevalence in the area. Mamuju District is located within 2°40’- 3°39’ south latitude and 118°46’- 119°25’ east longitude and divided into 15 subdistricts. The District occupies an area of 8,014 sq km along the midwest coastal region of the island of Sulawesi with a total population of 336,973  and rainy season (December), respectively, in 2010. The samples were further selected for immunological assays using all confirmed falciparum malaria cases by microscopy and polymerase chain reaction amplification (PCR). In addition, 5% of the total negative samples, selected randomly and representing proportionally all the villages’ population, were also included.The parasite DNA was extracted from the filter paper using Chelex-100 ion exchanger method as described previously . The DNAElution of the antibodies from the filter paper was essentially done as that described previously with slight modification , 14. PlaRecombinant MSP2, EBA175 and Rh2A9 proteins were all gifts from Prof J Beeson of the Burnet Institute, Australia. Total immunoglobulin (IgG) responses to MSP2 , EBA175 and Rh2a were measured using enzyme-linked immunosorbent assay (ELISA) as described previously . BrieflyStatistical analysis was performed using software of IBM SPSS Statistics 22. To test for differences in antibody levels for the three antibodies, MSP2, EBA175, and PfRh2a, based on sampling time, malaria positivity and age, Mann–Whitney test was used. The median differences of the three antibodies based on age group were determined using Kruskal Wallis test. Spearman correlation test was used to determine the association between the level of MSP2, EBA175 and PfRh2a antibodies with age.P. falciparum based on the results of PCR  and 64.26% , respectively (Table Antibody response to EBA-175 antigen is shown in Table Antibody response to PfRh2a antigen in the two collected seasons is shown in Table The prevalence of antibody response to MSP2, EBA-175 and PfRh2a in different age groups is shown in Table versus those <12 years old (Table Based on the age group, the median levels of IgG response to MSP2 and EBA-175 were slightly higher in subjects ≥12 years old ld Table . The difversus uninfected subjects. The difference in antibody level to MSP2, EBA-175 and PfRh2a is not statistically significant.Of the 497 subjects included in this study, 59 were confirmed to be falciparum infected, based on the PCR detection method (Table P. falciparum merozoite proteins MSP2, EBA-175 and PfRh2a were detected in subjects living in the malaria-endemic areas of Mamuju District, West Sulawesi Province, Indonesia and the responses are significantly higher in the wet season. The findings indicate that the three antigens might potentially be used as markers for exposure to malaria. Antibody responses to various merozoite proteins have been well documented in different population studies, but only few of the antigens was found to confer protection against clinical malaria [P. falciparum merozoite antigens were observed in many children during the rainy season. The levels of antibody to AMA-1, EBA-175, MSP119 and MSP2 increased at the end of the rainy season in children who had experienced clinical symptoms of malaria [The antibody responses to the  malaria . In Indo malaria . A previ malaria , 20. In  malaria .P. falciparum-positive subjects although this difference was not statistically significant. Further study involving larger sample size and specific IgG subclasses may provide a more conclusive result in this regard.Antibody responses to MSP2 and EBA-175 have also been associated with protection from clinical malaria in adult subjects . In thisAntibody response to the PfRh2a antigen was also observed, and the levels tend to constant with age. PfRh2a and PfRh2b proteins are identical for 2700 N-terminal amino acids and differed only in a C-terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain and cytoplasmic domain . It is tA study involving 206 children in Papua New Guinea demonstrated that the IgG1 and IgG3 levels were predominantly higher in children who had parasitaemia detected by PCR than those who did not have malaria. Interestingly, they found that antibody responses to PfRh2a were associated with age and active infection. High levels of IgG to PfRh2 were strongly associated with protection from malaria symptoms and parasitaemia .Antibody responses against three merozoites proteins were observed in the population of Mamuju District, West Sulawesi Province, Indonesia. The increase of the antibody response during the wet season indicates the high malaria transmission during the period. Therefore, MSP2, EBA-175 and PfRh2a are potentially useful as markers for malaria exposure. Among the three merozoite proteins examined, only the MSP2 showed protective efficacy as indicated in the higher level in the older age group and malaria-positive subjects. This study has a limitation as the assays were performed using archived filter-paper blood spots collected during the baseline malaria survey. Analysis of the total samples collected during the survey, although it would be laborious and require more logistics, may provide more conclusive results.


Glucose-6-phosphate dehydrogenase (G6PD) is a metabolic enzyme involved in the pentose phosphate pathway, its especially important in red blood cell metabolism. Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive hereditary disease characterised by abnormally low levels of G6PD. About 400 million people worldwide have a deficiency of this enzyme. The remarkable geographic correlation of G6PD deficiency distribution with historical endemicity patterns of malaria has led to suggestions that the two could be linked. Some studies have concluded that G6PD deficiency confers resistance to malaria.P. falciparum infection among children in Uganda.To determine the prevalence of G6PD deficiency, and determine its relationship with prevalence and incidence of This was longitudinal study involving 245 children, 135 were actively followed up for 12 months. G6PD status was assessed for using PCR-RFLP method. A thick smear was done to determine presence of plasmodium trophozoites and parasite densities.P falciparum infection. There was no statistically significant difference in prevalence and incidence rates of malaria infection among the different G6PD genotypes with prevalence among heterozygous, homozygous, and wild type being 29%, 42.6% and 43% respectively (p = 0.11) and incidence among heterozygous and wild type being 0.56 and 0.52 episodes/year (p = 0.5). The heterozygous G6PD A- females had a lower parasite density compared to the wild type .A total of 245 children between 6 months and 9 years were recruited. Of these 46.5% were males. Overall prevalence for the X-linked G6PD A- mutation was; 79.59% wild type, 12.65% heterozygous and 7.76% homozygous or hemizygous. Among the males 14% were hemizygous. At baseline, 40.8% had asymptomatic P. falciparum infection incidence and prevalence rates are similar among the G6PD genotypes though, once infected, P. falciparum parasite densities are lowest among G6PD A- heterozygous females. This suggests differences in P. falciparum infection rates and severity of disease could be mediated by differences in parasite densities among the different G6PD genotypes.This study showed that 20.41% of the population in this part of Uganda carry the G6PD A-mutation, within the range of 15-32% seen in other parts of Africa.  Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that is present in the cytoplasm of all cells of the body. It plays a pivotal role in the generation of nicotinamide adenine di-phosphosphate-H (NADPH) in cells. G6PD is involved in the first step of the Pentose Phosphate Shunt, where it catalyzes the oxidation of Glucose-6-Phosphate to 6-Phosphogluconolactone. This is the first step in a process that results in formation of NADPH and eventually reduced glutathione (GSH).G6PD deficiency is the most frequent inherited human enzyme defect in red blood cells. G6PD is an X-linked enzyme. Mutations in the G6PD gene result in different levels of enzyme activity, consequently causing a wide range of biochemical and clinical phenotypes ,2. Up toIt is estimated that up to 400 million people worldwide have this deficiency. The high incidence of G6PD deficiency in Africa is likely due to malaria. The striking geographic correlation of G6PD deficiency distribution with the historical endemicity patterns of malaria has led to suggestions that two are linked. Studies to confirm this link have been equivocal ; this miP. falciparum infection prevalence (asymptomatic infection), incidence and parasite density.This was a sub-study of an ongoing cohort study of children for potential malaria vaccine trials. The objective of this sub-study was to determine the prevalence of G6PD deficiency and determine its relationship with This study was a longitudinal study conducted starting October 1st 2008 to 31st December 2009. Field methods:A total of 245 children between 6 months and 9 years where recruited by stratified random sampling from Iganga district in eastern Uganda. Majority of the inhabitants are from the ethnic Basoga tribe. Only one child per household was recruited to minimize clustering and if there was more than one potential subject in the household then a ballot was used. After informed consent, blood samples were collected. The children were followed up for 1 year with 114 and 135 in active and passive arms respectively. Allocation to each arm was done randomly. Those in the active arm were visited at home fortnightly and had a blood smear and body temperature taken on each visit. They were also asked to report to the study clinic if any illness occurred. Those in passive arm were asked to report to the study clinic in case of any illness. All study participants who developed malaria were treated with Artemether/lumefantrine in standard doses as per Uganda treatment guidelines.DNA was analyzed for the presence for the presence of one of the common G6PD mutations G → A at nt 202 using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. DNA was extracted from a prepared buffy coat using E.Z.N.A blood DNA kits (Omega Bio-Tek Inc. Doraville. GA 30362. USA). PCR amplification was done using primers F – 5’-CCA CCA CTG CCC CTG TGA CCT-3’ and R- 5’-GGC CCT GAC ACCACC CAC CTT-3’. Details of the PCR-RFLP process are described elsewhere .Thick blood smears were done to assess for malaria parasites using equal volumes of venous blood and stained with 2% Giemsa. All the blood smears were then double read independently by trained laboratory technicians. Where results between the two expert microscopists were discordant, a third microscopist re-read the blood slide and the majority decision was accepted as the final result. Parasite densities were determined from thick blood smears by examining the smears under × 100 objective. The average number of parasites/oil immersion field was then determined and the parasite density in number/μl was calculated by multiplying the average number of parasites per field by a microscope factor of 1/0.002. Samples collected at baseline were used to determine prevalence and G6PD status of the participants.STATA statistical software version 10.0  was used for analysis. Descriptive statistics were expressed as proportions and means ± SD (Range). For the relationship between G6PD status and P. falciparum infection, two by two tables were used to calculate Chi squared tests and odds ratio with 95% confidence intervals. The t test for unequal variances was used for comparison between malaria parasite density and different genotypes. Spearman’s correlation coefficient was used to test the relationship between parasite density and age. Incidence rates and rate ratios were calculated from the follow up data. A p-value < 0.05 was taken as statistically significant.Study was approved by the School of Medicine, Research and Ethics Committee of Makerere College of Health Sciences and Uganda National Council for Science.A total of two hundred forty five (245) children were recruited into the study. Of the children approached, 97.5% of those eligible were enrolled. One hundred and fourteen subjects (46.5%) were male. The age ranged from six (6) months to nine (9) years with a mean age of 4.4 (±2.3) years. The majority 232 (95%) of the study population were of the Basoga ethnic group which is the predominant tribe in Iganga. Ninety two percent (225/245) of the study participants reported to have used an Insecticide Treated Net in the night preceding the interview. The average household size was five inhabitants. Of the children recruited into the study the median family position was third. Fifteen percent 20/134) did not complete the anticipated follow up period of one year due to migration out of the study site. Overall twenty four percent (50/245) of the study participants had a G6PD A-mutation. The distribution of the prevalence of G6PD A genotypes among the study participants are shown in Table 34 did noThis prevalence of asymptomatic P falciparum infection was 40.8% 100 of 245) among the children recruited at the baseline. The P. falciparum gametocyte carriage rate was 22% (55/245) among the study participants at baseline Table 45 among During the one year follow up, a total of 64 cumulative malaria episodes of symptomatic malaria were recorded among the 245 study participants. 55.4% (31/56) had only one malaria episode, 28.5% (16/56) had two, 12.5% (7/56) suffered three episodes while 3.6% (2/56) had four malaria episodes during the one year follow up period.P. falciparum infection among the G6PD A- heterozygous females compared to the wild type and homozygous but this was not statistically significant; 29.03%, 42.6%, and 43% respectively OR 0.55, . The percentage of G6PD A hemizygous males harboring malaria parasites was not statistically different from those with the wild type G6PD , p = 0.9.The relationship between G6PA mutations and presence of malaria parasites was assessed for by comparing proportions using chi square test. There was a lower prevalence of P. falciparum infection, the geometric mean parasite density was 2285 (range 250 – 41150) parasites/μl. There was a statistically significant difference in mean parasite densities between the wild types and the heterozygote’s, with lower parasite densities among the female G6PD A- heterozygote’s compared to wild type with; 941 parasites/μl versus 2505 parasites/μl respectively with P = 0.024, using a t test with unequal variances). There was a weak negative correlation between malaria parasite density and age, with r = - 0.33 (p = 0.0008).Malaria incidence rates were 0.56 episodes/year for G6PD wild-type, and 0.52 episodes/year for G6PD A - female heterozygote’s (p = 0.5) and there was only 1 homozygous male followed up actively. Of the children who had It is now apparent that genetic influence of malaria susceptibility in humans is very important. Understanding these interactions may influence the different interventions especially with drug therapy for malaria. This is important in clinical trials especially vaccine trials as this would impact on the outcome endpoints. The prevalence of G6PD was not known in our study site.This study reveals that the prevalence of the G6PD A- genotype is high in this part of Uganda at 20.4%. This rate is within the range of 15-30% that has been found elsewhere in sub-Saharan Africa -14 and cP falciparum infection. This rate however should be interpreted in context of the period of recruitment that coincided with a period of prolonged dry weather as this would result in lower infection rates. The rate of asymptomatic P. falciparum infections suggests that this is an area of moderate malaria transmission. Higher asymptomatic P. falciparum infection rates of greater than 70% are seen in areas of high transmission and rates of lower than 10% are seen in areas of low transmission [P falciparum infection probably plays an important role in maintaining malaria endemicity by providing a continuous source of parasites that helps perpetuate malaria transmission [P falciparum infection might play an important role in the development of malaria specific immunity [Of the 245 children studied, 40.82% were found to have asymptomatic smission ,15-17. Asmission ,17. In asmission ,19. The immunity .The lack of a significant difference in the both prevalence and incidence rates of infection among the different G6PD genotypes suggests this mutation might not influence susceptibility to malaria. On the other hand, the significant difference in parasite densities among the heterozygous females and the wild types points towards the fact that this mutation might influence disease severity as parasite density is a known factor in disease severity. This is in keeping with longitudinal studies on the relationship between malaria and G6PD polymorphisms that have shown that G6PD A- individuals have lower incidence of severe malaria ,9,14. ThP falciparum asymptomatic infection could have resulted in a lower prevalence as a PCR assay would have been more accurate for this and only one host factor that influences susceptibility to malaria i.e. G6PD status was studied hence no multivariate analysis was done.Limitations of this study include; Use of microscopy to determine P. falciparum infection incidence and prevalence rates are similar among the G6PD genotypes though, once infected, P. falciparum parasite densities are lowest among G6PD A- heterozygous females. This suggests differences in P. falciparum infection rates and severity of disease could be mediated by differences in parasite densities among the different G6PD genotypes. Given this high prevalence of the G6PD A- mutation, screening for this mutation is warranted in the work up of patients with acute hemolytic anemia in this population. It has also shown that up to 40% of otherwise healthy children harbor malaria parasites. It is likely that the interaction between malaria and these polymorphisms is complex thus a clear impact for one of them might not be easily discernible from a single cross sectional study. A large longitudinal study to assess the role and interaction of each of these is necessary.This study showed that 20.41% of the population in this part of Uganda carry the G6PD A-mutation, within the range of 15-32% seen in other parts of Africa. DNA: Deoxyribonucleic acid; G202A: Mutation in the G6PD gene with exchange of guanine for adenine at position 202; GSH: Reduced glutathione; G6PD: Glucose-6-phosphate dehydrogenate; NADPH: Reduced nicotinamide adenine dinucleotide phosphate; PCR: Polymerase chain reaction; RFLP: Restriction fragmentation length polymorphism; TNF: Tumor Necrosis factor.We declare that we have no competing interests.BD: Participated in participant recruitment and conducted the G6PD assays. HD and KM participated in the study conception and supervision of the study. FK Conceived the study supervised the data collection and ensured quality of the laboratory results. All authors participated in the writing and review of the manuscript. All authors read and approved the final manuscript.


Haemolytic conditions may contribute to disease pathogenesis and severe clinical manifestations through the liberation of free haemoglobin (Hb) and production of toxic free haem. Thus, free Hb and haem should be associated with altered MetHb and COHb levels in malaria as in other conditions.This study comprises data collected at three different sites: (i) a retrospective analysis of the first arterial blood gas result (ABGS) of any patient during 2010 at the University Hospital in Lisbon; (ii) a retrospective analysis of ABGS from patients with severe malaria admitted to the intensive care unit in Berlin, Germany; and (iii) a prospective study of non-invasive MetHb measurements in children with and without malaria in Lambaréné, Gabon.In Lisbon, the mean MetHb level was 1.4% (SD: 0.5) in a total of 17,834 ABGS. Only 11 of 98 samples with a MetHb level of >3.0 referred to infections. COHb levels showed no particular association with clinical conditions, including sepsis. In 13 patients with severe malaria in Berlin, the mean MetHb levels on admission was 1.29%, with 1.36% for cerebral malaria and 1.14% for non-cerebral malaria (P > 0.05). All COHb measurements were below 2.3%. In Lambaréné, Gabon, 132 healthy children had a mean MetHb level of 1.57%, as compared to 150 children with malaria, with a value of 1.77% and 2.05% in uncomplicated and complicated cases, respectively (P < 0.01).The data appears consistent with the methaemoglobin/haem hypothesis in malaria and sepsis pathogenesis. However, although MetHb was significantly different between healthy controls and children with malaria in Africa, the difference was rather small, also when compared to previous studies. Still, non-invasive bedside MetHb testing may warrant further evaluation as it could be a simple adjuvant tool for prognosis in resource poor settings. Conditions which cause haemolysis and lead to free haemoglobin (Hb) appear to contribute to disease pathogenesis and often severe clinical manifestations, such as renal impairment, vascular disease or inflammation . One proConsequently, one would expect that the presence of free Hb and haem, as well as the increase of HO-1 should be associated with altered MetHb and COHb levels in malaria and possibly other severe infections. Indeed, in an experimental cerebral malaria model in rodents, MetHb levels were significantly raised as compared to non-infected controls . AlthougPatients with overt haemolysis as in sickle cell crisis often have noticeably increased MetHb and COHb levels ,13. ThreMetHb and COHb are usually invasive tests, which require a blood sample. In affluent countries they are determined as part of the arterial blood gas analysis (ABGS), using sophisticated instruments, often not available in malaria endemic areas. A new type of pulse oximeter, the RAD-57™  allows to measure MetHb non-invasively Figure  if approThe objective of the study was to establish values for MetHb and COHb, measured invasively, in (i) a general patient population of a European tertiary care hospital (Lisbon) with or without evidence of infection and/or sepsis and (ii) in a retrospective cohort of patients with severe malaria in Europe (Berlin). The Lisbon data served to establish baseline values for MetHb and COHb, while the Berlin study served to determine which would be best parameters to be measured in Gabon, based on the observed alterations in severe malaria. Based on these results, (iii) the study prospectively investigated the usefulness of non-invasive measurement of MetHb in Gabonese children with malaria.This study comprises data collected during three studies at three different sites: (i) a retrospective analysis of all arterial blood gas results performed during 2010 at the University Hospital in Lisbon and for which MetHb and COHb was also measured, (ii) a retrospective analysis of ABGS from patients admitted to the intensive care unit at Charité University Medical Center in Berlin, Germany, with severe malaria in the years from 2003 to 2007, and (iii) a prospective study of MetHb measurements in children with and without malaria at CERMEL/HAS, Lambaréné, Gabon during the year 2012. Each part of the study was approved by the ethics committee at the respective study site in Lisbon, Berlin and Lambaréné. In Lambaréné informed consent was obtained from parents/guardians prior to inclusion.In Lisbon, data from all those arterial blood gas analyses for which MetHb and COHb results were also available were obtained from the Laboratory Information System (LIS) for a one-year period. Only the first result in a series of results of the same episode was used, thought to represent the result on presentation. Information on the location/department of request and clinical information which accompanied the request were recorded. Arterial blood gases were analysed on a ABL 800 FLEX  as described by the manufacturer. All results were analysed to establish associations between MetHb, COHb and clinical information.In Berlin, results were retrieved retrospectively for a cohort of patients with severe malaria who had been cared for on the Intensive Care Unit (ICU). MetHb and COHb mean values for the first day on ICU and in the clinical course were analysed for differences between groups. Arterial blood gases were determined on a ABL 800 Flex instrument .At the Hospital Albert Schweitzer in Lambaréné (HAS), children suffering either from non-complicated malaria or severe malaria were prospectively included in the study. Control subjects from the local kindergarden of the HAS and a primary school in town were invited to participate. Children with sickle cell disease were excluded. Sickle cell status was determined by electrophoresis. MetHb levels were measured non-invasively using a RAD-57™, pulse oximeter with a pediatric MetHb rainbow® finger probe . The measurement involves placing the finger probe on the third finger and immobilizing the hand during the measurement for about 45–60 seconds Figure . Each meData was analysed in SPSS version 15.0 . Results were analysed using the student t-test for unpaired data, the Chi-Square test or Fisher’s Exact test and differences of p < 0.05 were considered to be statistically significant.In Lisbon, 17,834 arterial blood gas results from patients  with a mean age of 61.5 years  were retrieved. Overall, the mean MetHb level was 1.4%  and the distribution for infectious diseases, including sepsis are shown in Figure In Berlin, ABGS from 13 patients who fulfilled the WHO criteria for severe malaria admitted to the intensive care unit (ICU) were retrospectively retrieved. The patient’s main characteristics are shown in Table In Lambaréné, Gabon, a total of 282 children were included, 132 controls and 150 with malaria. The characteristics of the study population and the mean values for MetHb are shown in Table The central chain of events in the haemolysis/MetHb/haem hypothesis ,7,8 is bIt should be noted that reference values can vary a lot, especially for COHb, from 1.5%-3% in non-smokers and up to 10% in smokers, highlighting the impact that environmental factors can have on these values -22. RefeThe results obtained in Lisbon show that the distribution of infectious diseases, including sepsis, is similar to that seen in all other conditions, both for MetHb as well as COHb Figures . This coFurthermore, the recent study in 117 Indonesian adults also showed significantly raised COHb levels in moderately and severe malaria, with mean values of 7% and 10%, respectively, as compared to the 3.6% in healthy controls (HC). Interestingly, this contrasts with a much larger study in 1,520 Kenyan children , making As a baseline, in Lisbon MetHb levels were very similar between any infection and other clinical conditions, while only sickle cell disease showed a clear increase Figure . AlthougSeveral studies have investigated MetHb levels in malaria, yet the results are often discrepant with regards to the absolute values as well as the magnitude of difference between the studied disease categories and HC. One study in 132 Tanzanian children reported significantly increased MetHb levels in uncomplicated and complicated malaria, with mean values ranging from 4.1%-5.8% as compared to HC with a mean MetHb level of 2%. However, no significant difference between the various forms of malaria were found . In anotversus other manifestations of severity, although the small sample size is clearly a limitation.In this present study, MetHb differed significantly between children with malaria (complicated or uncomplicated) as compared to HC. However the magnitude of this difference was modest, with some overlap of values in both categories Figure . The corPlasmodium falciparum positive individuals with a mean of 2.7% as compared to negative controls, with a mean of 2% [Thus, the results presented here appear to be more in line with two studies in Nigeria which reported more modest increases in MetHb levels in malaria: one study in 228 blood donors found a significantly higher MetHb level in an of 2%  while anan of 2% .The presented results still appear consistent with the haemolysis/methaemoglobin/haem hypothesis, and do not provide data to falsify this hypothesis . Yet, thHowever, the non-invasive measurement was rapid and easy to perform and studies have confirmed that the method appears to be reliable as a screening tool for measuring MetHb and COHb ,30,34. TData from this study still appear to be consistent with the central chain of events in the haemolysis/MetHb/haem hypothesis, and they do not provide evidence to refute this hypothesis. However, the results show that MetHb and CO-Hb levels in infectious diseases, including sepsis, are similar to those seen in a broad range of other medical conditions, both for MetHb as well as COHb. With regard to malaria, although MetHb is significantly different between healthy control and uncomplicated or complicated cases in African children, the magnitude of the difference is very discrete. Nevertheless, the simplicity of the non-invasive measurement makes bedside MetHb testing a simple option. The current study was not designed to address if this measurement might be valuable adjuvant tool for prognosis determination in resource poor settings, an issue to be addressed in future studies.ABGS: Arterial blood gas analysis; SD: Standard deviation; FBC: Full blood count; Hb: Haemoglobin; MetHb: Methaemoglobin; COHb: Carboxyhaemoglobin; CO: Carbon monoxide; NS: Statistically not significant (p > 0.05); HC: Healthy controls; LIS: Laboratory information system.The authors declare that they have no competing interests.TH and MPG designed the study, analysed data and prepared the manuscript. TH collected the data in Lisbon. TZ collected the data in Berlin. TG, SL and AF collected the data in Lambaréné. JMC assisted in designing the study. All authors have contributed to and approved of the manuscript’s final version. All authors read and approved the final manuscript.COHb values in an unselected hospital population in Lisbon. Distribution of Carboxyhaemoglobin values retrieved retrospectively from the laboratory information system for a one year period from an unselected population at the University Hospital, Lisbon . Stratified by clinical information accompanying the request into major groups.Click here for fileCOHb in patients with complicated malaria admitted to an Intensive Care Unit in Berlin. Carboxyhaemoglobin values retrieved retrospectively from patients admitted with malaria to the Intensive Care Unit at the Charité University Hospital in Berlin (n = 13). All patients had criteria for complicated malaria as defined by the WHO, with eight patients having cerebral malaria (lines) and five patients having other criteria, as shown in Table rlin n = 3. All paClick here for fileCorrelation of MetHb and parasitaemia levels in children with malaria in Lambaréné, Gabon. Dot plot of MetHb (Y-axis) versus parasitaemia (parasites/μl) (x-axis).Click here for fileCorrelation of MetHb and Hb levels in children with malaria in Lambaréné, Gabon. Dot plot of MetHb (Y-axis) versus Hb (g/dL) (x-axis).Click here for file