Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk  and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions.Populations exposed to  Plasmodium falciparum infections in children in sub-Saharan Africa  and DDC [3 association hits]), we assessed the level of LD within and flanking the gene to inform a haplotype-based analysis. For the chromosome 4 region containing FREM3 and USP38 , there was high LD within the genes  and some limited evidence that this linkage extended to SNPs within different genes  . Similarly, for the genomic region on chromosome 7 containing DDC and IZKF1 , there were several high-LD blocks , including 2 covering the association hits  .For the 2 loci with multiple association hits  revealed a 55% reduced risk of severe malaria between the 2 major haplotypes  (Table P = .0032) (Table P = .012). These results are consistent with the heterozygous advantage effects from the single SNP analyses.A haplotype analysis using the DDC block 1, which includes the rs880028 polymorphism, revealed no significant associations (P > .091) (Table P = .0088) (Table P = .841). The analysis of severe malaria and DDC block 2 also captured the heterozygous advantage effects of the rs10249420 and rs7803788 polymorphisms when haplotype combinations were considered  (Table P = .0185).Severe malaria haplotype analysis of 1) Table  although8) Table . HoweverD metric was used to determine whether the heterozygous association effects in the FREM3 and DDC loci were due to balancing selection. This seems to be the case for DDC  but not for FREM3 (−0.965 vs −1.245), for which increasing positive values are indicative of balancing selection. The magnitude of the DDC effect is at the extreme positive tail of an observed negatively centered Tajima's D distribution for African populations [USP38, DDC, FREM3, SDC1, and LOC727982) for severe malaria risk (P > .129).The Tajima's ulations , where pulations . Such baulations . There wUSP38 (8.1%), DDC (3.8%), FREM3 (3.0%), SDC1 (2.6%), and LOC727982 (1.7%) all accounting for more variation than ABO blood group and α-thalassemia but in aggregate less than the sickle HbAS polymorphism. Overall, the newly identified polymorphisms explain an additional nominal 4.3% of the variation in risk of severe malaria over and above the combined 7.4% for HbAS (6.3%), ABO blood group (0.2%), α-thalassemia (0.3%), and G6PD (0.5%).Finally, we assessed the proportion of variation in the severe malaria phenotype that is explained by the new candidate loci Figure . Of the Plasmodium parasite and Anopheles mosquito factors, both genetic and environmental, and their interactions with the human host. Application of genomic technologies in large, well-designed studies with detailed characterization of samples and metadata are required to disentangle these confounding effects. This Tanzanian case-control study, with complete metadata on a large number of participants, standardized phenotypic definitions and high-quality genotyping data, provides a solid platform for genomic discovery.As noted for historical candidate genes studies, GWAS findings are not always replicated across studies or populations, leading to reporting of false-positive associations and suspicion of the validity of novel associations . In studUSP38, FREM3, SDC1, DDC, and LOC727982). We could not confirm that polymorphisms in the ATP2B4 locus . It is possible that the ATP2B4 effect established in a West African population [We sought to test, in a setting of high malaria transmission, the evidence for 40 relatively new loci (114 SNPs) that have been associated with severe malaria risk in 4 recent GWASs –6. We coDDC locus was associated with severe malaria through a mechanism underlying the pathogenesis of cerebral malaria and, although the number of cases was low (n = 99) compared with other phenotypes, it demonstrates potentially strong genetic effects mediated through serotonin- and catecholamine-related pathways. The DDC SNP data, including rs10249420 with its heterozygous advantage effect, are consistent with those from analysis of cerebral malaria in a Gambian study [DDC locus [P = .00007), and we observe some evidence of association in our Tanzanian data .The an study . Other sDC locus . In the DDC and its surrounding regions followed by association testing is required. Our data suggest that polymorphisms in IZKF1 (60 kb from DDC) are unlikely to explain the associations with severe malaria, thereby reducing the amount of sequencing or genotyping required. DDC was found to be under balancing selection, a potential mechanism of maintaining protection from severe malaria in a population, while retaining minimal putative deleterious effects for the host. Balancing selection has also been observed at the HBB [G6PD loci [A full genetic survey of  the HBB  and G6PD6PD loci .USP38, chromosome 4) was associated with pleiotropic protection from acidosis, severe malarial anemia, and severe malaria per se. SNPs in USP38 have been associated with susceptibility to asthma [GYP-E/A/B gene cluster as well as the SMARCA5 [FREM3 loci. Several SNPs in FREM3 were associated with acidosis and severe malaria in our study. A study comparing human and chimpanzee sequences identified FREM3 as being under ancient balancing selection [SDC1) and rs10188961 (LOC727982) polymorphisms were associated with respiratory distress and acidosis, respectively, as well as with severe malaria per se. Both loci are located in regions with sparse reporting of any GWAS signals. Further characterization of the genetic variation in these regions in Tanzanian individuals through sequencing could help impute other genotypes for more detailed SNP association analysis.The rs4266246 polymorphism , FREM3 (0.7%), SDC1 (0.6%), DDC (0.8), and LOC727982 (0.4%)—explain another further 4.3% of the variation. The contributions of the new loci tend to be greater than the established effects of ABO blood group (0.2%), α-thalassemia (0.3%), and G6PD (0.5%), making them enticing targets for further investigation, including functional studies.Our final statistical model included factors with direct or indirect genetic underpinning to assess the overall heritability of susceptibility to severe malaria and apportion this between the various protective loci. Our estimate that genetic differences accounted for approximately 22% of the variation in the severe malaria phenotype in the Tanzanian population is very is similar to an estimate from a more robust family-based study . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


Mechanisms and outcomes of host-parasite interactions during malaria co-infections with gastrointestinal helminths are reasonably understood. In contrast, very little is known about such mechanisms in cases of malaria co-infections with tissue-dwelling parasites. This is lack of knowledge is exacerbated by misdiagnosis, lack of pathognomonic clinical signs and the chronic nature of tissue-dwelling helminthic infections. A good understanding of the implications of tissue-dwelling parasitic co-infections with malaria will contribute towards the improvement of the control and management of such co-infections in endemic areas. This review summarises and discusses current information available and gaps in research on malaria co-infection with gastro-intestinal helminths and tissue-dwelling parasites with emphasis on helminthic infections, in terms of the effects of migrating larval stages and intra and extracellular localisations of protozoan parasites and helminths in organs, tissues, and vascular and lymphatic circulations.The online version of this article (doi:10.1186/s40249-015-0070-0) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi parasites infect humans under normal conditions [P. falciparum and P. vivax being the major species that cause morbidity and mortality in children under five years of age, pregnant women and travellers from non-malarious areas [Malaria is a deadly infectious disease and one of the main health problems facing developing countries in Sub-Saharan Africa (SSA) and Asia. Globally, 3.4 billion people are at risk of new malaria infections, and there are around one million deaths annually –3. Plasmus areas , 6.P. knowlesi malaria infections [In SSA, morbidity and mortality due to malaria is decreasing despite a lack of a malaria vaccine, emergence of parasite resistance to available anti-malarial drugs, the anopheline mosquito being resistant to insecticide residual spraying and a poor socio-economic situation that hinders malaria control and management –9. Efforfections –14 as wefections , 16. Trifections –23. The fections . Parasitfections –36.Epidemiological studies have shown that the largest burden of malaria infections is felt by communities living in poor regions of developing countries –39. In tIn the majority of co-infection studies, tissue-dwelling parasites, prevalent in SSA, have not been adequately considered. The hypothetical arguments presented are sketchy, making it difficult to clearly predict disease outcomes during malaria interaction with tissue-dwelling parasites. In this review, we discuss and summarise the available information and research gaps in studies undertaken on the interactions between malaria and tissue-dwelling parasites.The online bibliographic databases, MEDLINE/PubMed, EMBASE, Web of Science, Cochrane Library and Google Scholar were searched for studies on host-parasite interactions of malaria co-infection with tissue-dwelling helminths (up to May 2015). Bibliographic lists and references of the selected papers and previous reviews were used as leads for identification of additional studies.The search was conducted using predefined medical subject heading (MeSH) terms, Boolean operators  and truncation symbols used in combinations of direct key words: malaria, protozoa, co-infection, nematodes, tissue-dwelling parasites, cestodes, trematodes, intracellular parasites, helminths AND all permutations of MeSH terms in all fields.Studies were included in the review if they explicitly reported on immune responses and disease outcomes during malaria co-infection with: (i) tissue and organ-dwelling parasitic protozoa; (ii) migrating parasitic helminths and protozoa; and (iii) vascular and lymphatic circulation dwelling parasites. One hundred and sixty-eight (168) studies were retrieved from the search of published work, of which 13 were excluded because of duplication and 123 were irrelevant because they were dealing with malaria co-infections and soil-transmitted parasites. Therefore, 32 studies including abstracts, reviews and reports on malaria co-infection with tissue-dwelling parasites were selected and reviewed. No grey literature was included. All articles were managed using Mendeley Desktop reference manager version 1.13.3 . The results of the analysis of the full papers read are described below.Trichinella larvae [Poor hygiene and sanitary conditions, and dysfunctional health delivery systems in developing countries predispose people living in these areas to STH infections , 54. Fora larvae , or gasta larvae , 56, 57.a larvae –61. The a larvae  through a larvae . The Th2a larvae , 64. ButP. chabaudi malaria parasites are capable of altering Th2 immune responses and initiation of pulmonary tissue repair in BALB/c mice co-infected with Nippostrongylus brasiliensis. This indicates that the presumption that helminths always alter malaria severity and immunity is not always correct. Several laboratory experiments have also shown that malaria parasites are capable of suppressing helminth-associated immunological activation thereby exacerbating pathological outcomes caused by the ML [Trichinella sp. as the tissue-dwelling helminth is proposed because of its adaptability to laboratory animals as well as its ease of maintenance. For example, studies could be done on the migratory pathways taken by Trichinella sp. and how the Trichinella sp. may ameliorate allergic and autoimmune diseases in mono- and co-infections with malaria [Trichinella sp. with tropical infectious diseases. In SSA, although very few human cases have been reported, trichinellosis is considered as an emerging/re-emerging zoonotic disease that has been reported to infect a variety of mammals [T. zimbabwensis ameliorate and supress P. berghei infection. This is attributable to the comparable levels of interferon gamma (IFN-γ) secreted during co-infection and correlated with protective immunity [Several co-infection studies have been designed and conducted on the assumption that chronic helminths may alter malaria severity and immunity either through Th2/T regulatory lymphocyte immunomodulation, altered antibody dependent cellular inhibition, immunosuppression of pro-inflammatory activity or presence of cross reactive antibodies , 65. On y the ML . Therefo malaria , 68, 69. mammals , 20, 70. mammals  establisimmunity . HoweverGiardia lamblia and Entamoeba histolytica have been reported to be major causes of severe intestinal disorder mostly in children, and HIV/AIDS and immunocompromised patients [Intestinal protozoans, patients –75. The patients . The undpatients .Isospora belli, Cryptosporidium sp. and Cyclospora sp. also cause severe diarrhoea, morbidity and mortality [Cryptosporidium parvum infections are increasing due to environmental contaminations by pets, poultry, domestic animals and infected humans [Coccidian parasites, ortality . Despiteortality . In deved humans –81. In ld humans –85. Theid humans , 87, beid humans . Entericd humans .Transmissions of vector-borne parasitic infections are on the rise due to changes in climate and global trends, human behaviour, vector behaviour and prey/host switching , 89. TheP. yoelii or Trypanosoma brucei have been shown to block resistance to Echinostoma revolutum parasite infection. [a) Trypanosomes and malaria: In SSA, the tsetse fly transmits extracellular protozoan parasites cause debilitating human African trypanosomiasis (HAT) and nagana in livestock . The disfection. . This sufection. , 98, 99.Babesia sp. and malaria: Due to increased human-wildlife and livestock-wildlife interactions, a severe recrudescence of malaria-like babesiosis in humans and livestock has been reported [Babesia sp. infections might be misdiagnosed as Plasmodium sp. because of their overlapping similarities in symptoms [Babesia and Plasmodium parasites confer cross-protection to mice during co-infection. A human case report from Korea showed that Babesia parasites prolonged severity of malaria-induced haemolytic anaemia during co-infection [Plasmodium sp. and Babesia sp. parasites exhibited markedly enhanced malaria severity [B. microti infection showed that B. microti parasites were able to suppress P. cynomolgi infection [B. microti parasites either provoke induction of immune responses that either ameliorates or exacerbates malaria infection. However, these few available studies have not provided enough insight into immunology and cellular mechanisms that are involved during mono- and co-infection.b) reported . In SSA,reported –104. In symptoms –109. Thisymptoms , 110. Dusymptoms . Clark asymptoms  establisnfection . A childseverity . On the nfection . TherefoLeishmania sp. and malaria: In Sudan and Uganda, Leishmania donovani complex parasites and malaria have been reported to co-infect humans. The co-infections showed a synergistic immunological interaction characterized by enhanced Th1 immune responses [L. donovani complex parasites naturally colonise macrophages to initiate counter regulation of host immune responses resulting in a release of anergic/dysfunctional T-cells and blocking of intracellular cytokine signalling in macrophages and dendritic cells [Leishmania parasites. This shows that during co-infection the control of malaria will be impaired in that the effector cells are used by the Leishmania parasites for immunoregulation. This will result in the exacerbation of malaria disease and suppression of Leishmania parasites or, conversely, the parasites will impact the host immunity and influence infection and pathophysiological responses of both parasites. The role of Leishmania parasite mediators and vector saliva components in mediating immunosuppression of host regulatory immune responses are still unknown [c) esponses , 117. Thic cells . Current unknown , 119. ThToxoplasma gondii and malaria: Toxoplasma gondii is a cosmopolitan intracellular apicomplexan parasite that causes ocular, congenital, neurological and systemic infections in approximately one third of the world’s population [T. gondii infections [T. gondii and Plasmodium parasites utilise similar cellular mechanisms and biochemical pathways for their nutrition, metabolism, pathology and immunomodulation [Toxoplasma parasites sequester in the placenta resulting in placental disc plate damage thus influencing foetal and pregnancy outcomes [T. gondii.d) pulation –122. Humpulation . Stray dfections , 125. Thfections . A questfections . Howeverfections , 128–130dulation , 132. Tdulation , severidulation , 134–136dulation , 138, 98outcomes . DespiteWuchereria bancrofti, Brugia malayi, Onchocerca volvulus and Loa loa are endemic in SSA [Plasmodium parasites are transmitted by the same anopheline mosquito vector making co-endemicity a common phenomenon [Plasmodium parasite infectivity within the mosquito vector. However, these studies have not determined the immunological changes that occur during deworming and its benefits towards integrated control strategies in malaria-endemic areas.Lymphatic filariasis (LF) caused by c in SSA , 141. Thenomenon , 143 Thienomenon  and haveenomenon –146. Theenomenon , 148. Onenomenon . In epidenomenon , 150. Hoenomenon , thus neenomenon  establisLarval stages of some tapeworms cause fatal liver, brain and lung metastasis in humans and livestock , 154. InFasciola hepatica and F. gigantica in humans are emerging infections and occur in malaria endemic areas despite the parasites not being considered to have relevance in malaria co-infections [Schistosoma haematobium and S. mansoni, and these are becoming emerging or re-emerging infections in developing countries of SSA due to climate changes that are influencing spatial distribution of fresh water snails [fections –169. Oner snails , 171. Thr snails . Thus, rSeveral laboratory experiments and epidemiological studies have been conducted and meta-analysis reviews have been done to determine the host-parasite interactions of malaria and schistosomes during co-infection . The stuThere are few studies that are directed towards elucidating the host-parasite interactions and disease outcomes that are elicited by tissue-dwelling parasites during co-infection with malaria. This has created a glaring paucity of data on understanding the mechanisms and outcomes of tissue-dwelling parasites and mono- and co-infection with malaria. This has also hampered diagnosis, vaccine development, drug discovery, and management and control of these emerging and re-emerging parasites. Therefore, further studies are imperative to address this lack of data and the heterogeneity of results reported during STHs, schistosomes and filarial worm co-infection with malaria. These future studies should be designed and controlled towards elucidating cellular and molecular pathways as well as migratory pathways that are utilised by migrating tissue-dwelling helminths and protozoa. The utilisation of different study designs and approaches, as well as different tissue-dwelling helminths and protozoa will provide vital information that can be extrapolated to humans. These studies and experiments will also provide information on non-immunological aspects, timing and order of parasite infections. The disease outcomes across broad range of hosts and parasites will show evidence of parasite-host-parasite interactions at the phenotypic level. This data will be useful in explaining the actual cellular and molecular mechanisms and signalling pathways that influence conferring of protective immunity, exacerbation and/or amelioration of disease outcomes that have been observed in concomitant and concurrent infections. In the long term, the studies will provide the thrust for deworming, surveillance, diagnosis, vaccination campaigns and vaccine trials in areas of SSA where tissue-dwelling parasites are co-endemic with malaria.


There are growing data supporting the differences in susceptibility to malaria described between sympatric populations with different lifestyles. Evidence has also been growing for some time that nutritional status and the host’s metabolism are part of the complex mechanisms underlying these differences. The role of dietary advanced glycation end-products (AGEs) in the modulation of immune responses (innate and adaptive responses) and chronic oxidative stress has been established. But less is known about AGE implication in naturally acquired immunity and susceptibility to malaria. Since inflammatory immune responses and oxidative events have been demonstrated as the hallmark of malaria infection, it seems crucial to investigate the role of AGE in susceptibility or resistance to malaria. This review provides new insight into the relationship between nutrition, metabolic disorders, and infections, and how this may influence the mechanisms of susceptibility or resistance to malaria in endemic areas. The outcome of this primary encounter is driven by many related factors. Virulence of the microbe, infectious burden, route of inoculation, the host’s immunity, susceptibility to infection, and previous drugs used, are among these well-documented factors. Compelling evidence has been growing for some time that the nutritional status of the host is part of this complex relationship. Parasitism has some specificity in terms of the host-pathogen relationship that confers selectivity for the host to the parasite (adaptation).The nature, magnitude, and effectiveness of the immune response induced by the malaria parasite determines the clinical outcome of the current malaria episode, as well as the susceptibility to subsequent episodes , 41. TheStreptococcus infection, peritonitis, and sepsis , 60D-gluIn vivo, AGEs act as damage-associated molecular patterns (DAMPs) with their specific receptors known as receptors for advanced glycation end-products (RAGE).Advanced glycation end-products are generated endogenously when the blood sugar level is chronically high (during diabetic) or oxidative stress, as well as exogenously during the cooking process of meals , 54, 61.The receptor for advanced glycation end-products (RAGE) is a transmembrane protein on the cellular surface that recognizes tridimensional molecules, instead of amino-acid sequences. RAGE contains two constant (C-type) and a variable (V-type) immunoglobulin-like domains. Different variants of RAGE exist, including the soluble one (sRAGE). sRAGE only has the extracellular domain of the receptor. sRAGE acts as a decoy receptor for AGEs and may prevent inflammation –80. OtheNF-kappa B, leading to the production of pro-inflammatory cytokines, chemokines, adhesion molecules, and oxidative stress. The recruitment of RAGE by AGE also leads to endothelial cell activation [The activation of RAGE triggers a series of cellular signaling events, including the activation and translocation into the nucleus of transcription factor tivation . Upon bitivation . The exptivation , 78–80.3.Homo erectus domesticated fire and started cooking meat about 400,000 years ago [Homo sapiens could have co-evolved with AGEs by developing genetic polymorphisms on the RAGE gene. The gene encoding RAGE is located on the major histocompatibility complex region that comprises the HLA gene loci [RAGE gene polymorphism and susceptibility to malaria in endemic areas.ears ago . During ene loci . Since tene loci , it seemp < 0.001); and significantly lower serum soluble RAGE (sRAGE) levels. In contrast, 82GS polymorphism is associated with higher serum sRAGE levels [The involvement of some genetic polymorphisms (single nucleotide polymorphism: SNP) on RAGE promoters in the susceptibility or resistance to some metabolic infectious diseases suggests that RAGE is sensitive to natural selective pressure. SNPs 374T/A and 429T/C in the RAGE promoter region are involved in the regulation of RAGE expression by acting on the nuclear protein-binding site . This poE levels . Higher E levels , but sevE levels , 38.P. vivax, ovale, and malariae) are more adapted to certain human subpopulations in endemic areas by developing host immune tolerance [Homo sapiens infection with Plasmodium malariae can stay lifelong in the host.The malaria parasite has co-evolved with human beings for about 100,000 years and the host has developed many protective strategies  , 41. Whiolerance , 87, sugDifferences in susceptibility to malaria are well established in sympatric populations with different lifestyles in sub-Saharan Africa , 13, 23.4.There is evidence that the interplay between some nutrient-derived metabolites  and their nuclear receptors plays a key role in maturation of the lymphoid organs and modulation of the immune system , 72. TheStreptococcus pneumoniae infections by impaired CD4+ and Th17 cell memory responses [influenza A virus pneumonia [RAGE is a cellular receptor that interacts highly with nutrient-derived metabolites. Growing evidence supports the relevance of the RAGE signaling pathway in the pathogenesis of many diseases like diabetes and its associated complications, but also in certain clinical settings. RAGE, as a pattern-recognition receptor, seems to have a crucial role in early host defense against invading pathogens by activating innate immune response, which primes acquired immunity, particularly by driving induction of a Th1-type response . Many stesponses . Experimesponses . Inhibitesponses . Blockadesponses  and inflneumonia . Howeverneumonia . The facneumonia . The appT helper 2 (Th2) cytokine production, which is frequently downregulated in T cells expressing RAGE, is critical for host defense against blood stage malaria [The innate immune response induced by RAGE when activated by AGE is a sterile immune reaction. The sterile inflammatory immune response is similar to infection immune response, including the recruitment of neutrophils and macrophages, the production of inflammatory cytokines and chemokines, and the induction of T cell-mediated immune responses . The mag malaria .The role of RAGE-AGE in the susceptibility or resistance to malaria remains unknown and needs to be investigated. Increased susceptibility to clinical malaria has already been described in 2-year-old children with higher plasma levels of advanced oxidative protein products (AOPP), compared to those with lower plasma AOPP .falciparum malaria and was suspected of being an amplification signal in disease pathogenesis, and thus illness [P. falciparum (Pf-HMGB1/2), which has 98% similarity with human HMGB1, has been found to be able to activate RAGE and induce secretion of pro-inflammatory cytokines by human immune cells [But no study has directly investigated the relationship between AGE-RAGE signaling and malaria susceptibility. During sepsis, high-mobility group box-1 (HMGB1), a DNA- and heparin-binding protein with pro-inflammatory activity, enhances and prolongs inflammatory processes. HMGB1 is one of the ligands of RAGE and shares part of the RAGE-AGE pathway. More than a decade ago, HMGB1 was shown to be increased in  illness . Further illness , 30. A Dne cells .Chronic inflammatory immune response that results in immune tolerance (by upregulation of negative feedback control), cell dysfunction, tissue destruction, and exhaustive immune cells.Depletion of Th2 immune response and impairment of the Th1/Th2 immune response balance, with deleterious effects on the host-pathogen interaction.Chronic production of elevated HMGB1, which could recruit new RAGE and maintain a vicious circle of “RAGE overexpression → AGE/HMGB1 production → RAGE activation and overexpression”.Chronic oxidative stress with generation of reactive oxygen species (ROS), oxidation and glycation of proteins, nucleic acids, and lipids; with auto-amplification of AGE and AOPP generation.Selection of SNPs on the gene coding for RAGE with protective effects against malaria.Malaria is endemic in some countries and sometimes leads to genetic diversity (blood group polymorphism) in endemic areas by positive selection. We hypothesize that in malaria endemic areas where populations are exposed to high levels of AGE in food, the chronic activation of RAGE by dietary AGE could lead to :Chronic 5.Plasmodium falciparum infection [Natural resistance to malaria has been described among pastoral populations (Fulani) compared to their neighboring farmers (Dogon) in Mali, or Mossi in Burkina Faso , 64. FulThese populations living in malaria endemic areas are exposed to the same risk of malaria infection and disease. The main difference between the two population groups is their dietary habits. While Fulani have milk and couscous as their main food, Dogon have at least three well-cooked meals a day. Cooked foods contain more AGEs than milk and couscous. Even though the protective role of diet against malaria in Fulani has not yet been thoroughly investigated , these oIt is well documented in the literature that reduction of dietary AGE consumption protects against loss of innate defense . There iMore studies comparing the serum levels of AGE, sRAGE, RAGE expression, and RAGE polymorphism in these populations could provide new insight into the mechanism of malaria susceptibility or resistance.6.In malaria endemic areas, people are exposed to multiple malaria infections. This exposure leads to the development of innate and adaptive immune responses against malaria, and genetic polymorphism in the long term.In populations with AGE-rich cooked foods as their main food source, chronic activation of RAGE by AGE may induce and maintain pro-inflammatory cytokine production, oxidative stress, reactive oxygen species (ROS), and AOPP production, and result in innate immune tolerance, inadequate adaptive immune response, cellular dysfunction, and depletion of anti-oxidative mechanisms. The disturbance of host defense systems may increase susceptibility to malaria in these populations.Targeting the dietary AGE-RAGE signaling pathway as a new research perspective in populations with different lifestyles could contribute to understanding the mechanism of the differences of susceptibility to malaria observed in some African populations. Such new strategies could contribute to reducing morbidity and mortality related to malaria in endemic countries by acting on lifestyles of populations, as already implemented in the control of some chronic metabolic diseases like diabetes and hypertension. The determination of blood levels of AGE, sRAGE, and cell expression of RAGE and AGER1, as well as polymorphism of genes encoding for these receptors, will provide relevant information on malaria susceptibility.The authors declare no competing interest.


Plasmodium falciparum and Plasmodium vivax protein microarrays to identify a panel of the most informative antibody responses in diverse malaria-endemic settings representing an unparalleled spectrum of malaria transmission and malaria species mixes before and after interventions to reduce malaria transmission.A blood test that captures cumulative exposure over time and assesses levels of naturally acquired immunity (NAI) would provide a critical tool to monitor the impact of interventions to reduce malaria transmission and broaden our understanding of how NAI develops around the world as a function of age and exposure. This article describes a collaborative effort in multiple International Centers of Excellence in Malaria Research (ICEMRs) to develop such tests using malaria-specific antibody responses as biosignatures of transmission and immunity. The focus is on the use of  The ability to accurately and rapidly monitor the effectiveness of malaria interventions to reduce transmission and the burden of disease is essential for an effective control program. Current methods usually measure the prevalence of parasitemia in children, and either passive or active case detection of symptomatic malaria. Although these methods are useful, they can be resource intensive and imprecise, especially in areas with low or changing transmission. A blood test that captures cumulative exposure, recent exposure, and assesses levels of naturally acquired immunity (NAI) against malaria would be extremely valuable. This article describes a collaborative effort in multiple International Centers of Excellence in Malaria Research (ICEMRs) to develop such a test using malaria-specific antibody responses as biosignatures of transmission and immunity. The rationale of using antibodies comes from the observation that antibodies against specific parasite antigens persist in time and at reasonably stable concentrations, even when transmission is seasonal. The antibodies in sera are relatively easy to measure through experiments using basic techniques such as enzyme-linked immunosorbent assay or more advanced approaches such as bead arrays or protein microarrays on a genomic scale.Plasmodium spp. induces potent antibody responses that increase with subsequent exposure and include three basic characteristics: 1) circulating immunoglobulin, especially immunoglobulin G (IgG); 2) plasma cells, predominantly located in the bone marrow, that produce these antibodies; and 3) memory B cells that can rapidly expand to generate additional antibody-producing plasma cells, more memory B cells, and potentially immunoregulatory B cells. These three components are often correlated, but sometimes may not be.Human infection with Immunological memory to malaria as measured by the presence of serum antibodies to plasmodia blood-stage antigens can persist for years as can the presence of circulating memory B cells,Plasmodium falciparum (Pf)/Plasmodium vivax (Pv) protein microarray designed to measure biosignatures of transmission and acquired immunity across a large number of malaria-endemic sites. Protein microarrays of the format used in the cross-ICEMR survey have been previously used to measure antibody responses to malaria antigens in the context of exposure and acquired immunity.Pf, exclusively Pv and locations where both parasites are transmitted.The strength of cross-ICEMR studies is the ability to use standardized assays to measure antibody responses in a broad variety of epidemiological settings ranging from areas with high transmission to those with low or unstable transmission. Moreover, many of the ICEMR sites are undergoing malaria control measures such that the effects of the reduction in malaria transmission on infection and disease can be evaluated. This study describes the development of a A detailed discussion of the ICEMR study sites and epidemiology related to these sites are indicated in other articles in this volume. An overview of the studies and related epidemiology and the number of individuals whose sera was used to probe the arrays is provided in Ethical approval was obtained from all ICEMR centers in the United States and the FWA-accredited ethical review boards in the respective malaria endemic areas.Pf clone library. Each protein contains N-terminal poly-His and C-terminal hemagglutinin tags for quality control. Serum samples were diluted 1:200 in Protein Array Blocking buffer  supplemented with 10% Escherichia coli lysate , and incubated overnight on arrays at 4°C. After incubation with secondary antibody and wash steps, slides were scanned on a GenePix 4200AL  and spot intensities quantified using ScanArray Express software .All sequences for this particular array were derived from a single reference genome (3D7). This approach allowed the evaluation of a greater breadth of antigens, but limited evaluation of the effect of antigenic variation on the antibody response. Separate studies outside the ICEMR program to evaluate the latter are ongoing. Microarrays  were printed and probed following previously published methods.www.r-project.org) to calculate two-sided t test and P values adjusted for false discovery. The fold-over control (FOC) normalization reduces variation in signals that could potentially arise between probing operations performed at different times. FOC normalization provides a relative measure of the specific antibody binding to the nonspecific antibody binding to the IVTT controls. Thus, with the normalized, log2-transformed data, a value of 0.0 means the intensity is not different from background and a value of 1.0 indicates a doubling with respect to background. However in some analyses, straight mean fluorescence indices (MFI) minus IVTT background were used.The mean pixel intensity for the spot was subtracted by the local background around the spot and a floor of 100 was set for the data. The IVTT protein spot intensities were normalized either by dividing by or subtracting the multiple IVTT control spots (IVTT reactions lacking plasmid template) that are printed throughout the chip. Statistical analyses were performed on the base-2 logarithm of the normalized data using the base stats package in R (www.tm4.org). In this analysis, only positive serological responses that were significantly different from the U.S. controls were counted as seropositive.The number of seropositive antigens in different locations was determined relative to North American controls using significance analysis of microarray implemented in the MultiExperiment Viewer microarray software suite from TM4 (http://plasmodb.org/plasmo/) that provides free access to information on Plasmodium genomes and individual genes identified for the ICEMR array.All data were uploaded on PlasmoDB (Pf and Pv (Pf500/Pv500) for the trans-ICEMR serological survey. Antigens to be included on this array were down-selected from larger arrays probed with highly reactive sera representative of different populations around the globe. Thus for the Pf features, an array of well-known Pf genes with 4,528 features (Pf4528) was probed with adult samples from Papua New Guinea (PNG), Kenya, Mali (N = 20 each), and the United States as a negative control (N = 10). Kenya and Mali are high-transmission areas with ostensibly only Pf transmission, while PNG has high transmission of both Pf and Pv and reacts very strongly with antigens from both species on the protein arrays. For Pv, Pv4441N = 15 each), Peru (N = 22), Thailand (N = 10), and the United States (N = 10). Seroreactive antigens in each country were defined as those antigens with an average signal that was above a cutoff defined as the average plus two standard deviations of the U.S. control population. From the compiled list of seroreactive antigens, the top 500 Pf and 500 Pv antigens were selected using hierarchical filtering. Highest priority was given to antigens seroreactive in all countries tested, with the remaining positions allocated by descending order of reactivity across the countries (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL18316). On the basis of the information from plasmodb.org, the distribution of Pf life stage of maximum expression represented on the array were as follows: merozoite 24%, early ring 22%, late schizogony 14%, early trophozoite 14%, early schizogony 11%, late trophozoite 8%, late ring 5%, and null 2%, representing almost exclusively blood-stage antigens.P. vivax proteins.To develop a protein array that was most informative about exposure and immunity as well as cost effective, we designed an array containing approximately 500 antigens from both Mali N =  each, anPf features on the ICEMR array, > 300 were reactive with sera from PNG (Pacific southwest ICEMR). Adults from Uganda , Thailand (Tak Province), and Santa Emila in Peru detected similar numbers of reactive antigens to that observed in PNG. Overall, sera from sites with higher transmission recognized more antigens in the array compared with areas with lower transmission.Pv transmission , sera recognized many Pv features suggesting cross-reaction between antigens (see “Species specificity of assays” below). Similarly in areas with predominantly or only Pv transmission , sera also recognized Pf antigens. Overall sera recognized fewer Pv than Pf features. This may represent higher parasitemia levels associated with Pf infections and stronger boosting of antibody responses to blood-stage antigens.In areas with little or no Plasmodium species-specific genes may give rise to species-specific antigens. However, the majority of genes in Pf and Pv are orthologs of each other, and therefore, it is likely they share epitopes for antibody recognition. This portends antigenic cross-reactivity that will complicate the interpretation of antibody recognition of malaria proteins, especially in areas of the world where there is coincident exposure to both Pf and Pv. Through the ICEMR program, it has been possible to assess serology in essentially mono-exposed populations. Thus, the antibody profiles in samples from sub-Saharan Uganda, Senegal, Malawi, and Zambia primarily represent Pf mono-infections, although a low level of Pv exposure might occur in some of these populations.Pf antigens, there was also strong reactivity to a subset of Pv antigens, which we attribute to antigenic cross-reactivity . Here too there was cross-reactivity to a subset of Pf antigens. As might be predicted, many orthologous genes encode cross-reactive antigens and reciprocally non-orthologous antigens are more abundant among those that are species specific. However, there were exceptions as shown in Pf that have shared orthologs in Pv, but which were recognized only in Pf infections  and/or Plasmodium ovale (Po) are also co-endemic in some ICEMR study sitesPf and/or Pv.Intuitively, vity see . Reciprofections . Similarspecific . Presuma species  and C. STo determine which antibody responses are likely to be most informative in different epidemiologic settings, it is critical to understand the natural variation in responses across these settings. Shown in y axis value for each antigen represents the mean reactivity to that antigen in the particular population and the x axis the average reactivity for all sites. Note that the United States has the lowest slope and provides a useful baseline.P. falciparum antigens are represented, whereas for Colombia and Amazonia only P. vivax antigens are represented. The slopes generated by such analyses for all ICEMR sites tested to date are provided in It should be emphasized that an analysis of this kind is highly dependent on the sampled populations, and it is possible to find a range of seroreactivity within the same country, particularly in low-transmission areas, which may be the reason why the slopes of the lines are low  for Senegal and Zambia. For example, Pf antigens for sites that had predominantly falciparum malaria  with the aim that most or all malaria infections and illnesses are captured. Biosignatures of protection are measured in serum or plasma samples and are related to risk of clinical malaria, accounting for varied exposure, over the course of follow-up. Such studies are labor intensive and require populations with sufficiently high transmission levels such that enough clinical events are captured to have sufficient statistical power. An example of this approach using the microarray and correlates of immunity (non-ICEMR) was recently published.Pf antigens on the microarray were significantly less likely to acquire symptomatic malaria compared with those with lower overall serological responses during the transmission season.Pv in Remansinho, Brazil ICEMR.N = 24) by passive case detection and asymptomatic (N = 14) P. falciparum malaria infections  in carefully age-matched (nested) cohorts were probed on the Pf824 microarray to identify differentially recognized antigens that may be associated with protection. The number of prior malaria episodes was similar between the two groups as well as the median age . Median parasite densities, however, were more than log-fold higher in symptomatic individuals compared with asymptomatic individuals, which may have contributed to consumption of antibodies. On the basis of mean MFI, 52 antigens were found to be differently reactive between samples from these two different patient groups , an established vaccine candidate, showed the highest reactivity among the differentially reactive antigens. Other vaccine candidates found were EBA175 (MAL7P1.176) and liver-stage antigen 3 . Ring erythrocyte surface antigen (PFA0110) was also differentially reactive; this protein has been reported to be involved in modifying the biophysical properties of infected erythrocytes and the effect seems to be enhanced by increasing temperature to fever levels. Two heat shock proteins, HSP40 (PFE0055ce4) and HSP70 (MAL7P1.228), were differentially recognized. Several other differentially recognized proteins are involved in interactions of infected red blood cells (RBCs) with uninfected RBCs and endothelial cells: ring-exported protein 1 (ID: PFI1735c), early transcribed membrane proteins (E-TRAMPs) 4,5,10.1, and 14.1 .The second are serial cross-sectional studies in the same population. Often the same individuals are observed repeatedly, but surveys are less frequent than cohort studies and missing data on individuals is much more common than in the cohort studies . With good passive surveillance, most symptomatic malaria cases are captured. The dynamics of asymptomatic infections are missed however. An example of such a study  showed that children with higher overall serological reactivity to It should be emphasized that the majority of antigens on the array did not show any difference between the two groups and some of these had the highest overall seroreactivity that could be potential biosignatures of exposure. Examples of highly seroreactive antigens in both groups are PfMSP10, PfRh2, MSP1, and LSA3.Much of the data presented in this article provide the basis for more detailed studies to identify optimal antigens for biosignatures of transmission and NAI. In the following discussion, we describe these preliminary findings in the broader context of the present understanding and approaches to identify biosignatures of transmission and acquired immunity.Plasmodium have been used for decades to assess exposure to infecting parasites and thus characterize transmission. To date, the most standardized and widely validated method for evaluating exposure via serology is the seroconversion rate (SCR), which appears to be informative across a broad range of transmission settings. Typically SCR is calculated from age-specific seroprevalence, with seropositive individuals representing both presently infected and previously exposed individuals depending on the kinetics of antibody responses to the particular antigen.Antibody responses to Calculation of an SCR extends seroprevalence analysis by calculating the rate at which individuals in a population become seropositive. SCR is analogous to the force-of-infection and is derived from the analysis of seroprevalence as function of age.31Pv circumsporozoite protein accurately stratified risk between the three sites; in Uganda, estimates of SCR using responses to Pf apical membrane antigen 1 also accurately stratified risk between the three sites, in contrast with parasite prevalence.34Several studies have demonstrated the utility of SCR as a malaria epidemiological measure, which can be collected from a variety of survey types  and that shows good agreement with classic measures of malaria transmission intensity such as the entomological inoculation rate.One important question being addressed in the ICEMR program is defining the optimal antigenic targets for evaluating exposure, including how these may vary in different epidemiologic settings, and when evaluated in different age groups. This is important as the dynamics of antibody acquisition and maintenance vary based on exposure intensity, thus the degree to which some serologic markers are informative of exposure will likely vary in these contexts. Moreover, the different kinetic properties of antigens should allow a more refined characterization of the time elapsed since exposure occurred, for example, using antigens with short half-lives to reflect more recent exposure.7Pv and Pf antigens with ultimate aim to identify a set of novel serological assays as biosignatures of transmission and NAI using antibodies targeting a suite of particular proteins. The serological assays that identify transmission versus those for acquired immunity may differ, although there may be some overlap.In this study, we developed a targeted protein microarray to examine antibody response to both Overall, the array identified antigens that were generally more reactive in subjects residing in high- versus low-transmission conditions, however this relationship was imprecise and some individuals living in high-transmission areas had lower overall responses compared with those living in lower transmission areas. Since malaria transmission can be highly heterogenous and vary greatly with age, more precise analyses are currently underway to better control for these variables and to better select the more informative antigens.Pf and Pv are endemic is important. This may not be of great significance where one species is much more prevalent compared with the others, for example, Pf in sub-Saharan Africa and Pv in many areas of Latin America; however, there are many areas where both are co-endemic. We show some initial approaches to identification non-cross-reactive antigens using serum samples from areas that are only Pf or Pv endemic from careful studies in the ICEMR. As might be expected, non-orthologous gene products are most likely to be non-cross-reactive between Pf and Pv, although some orthologous genes are cross-reactive. Studies are currently underway to identify the most informative species-specific antigens.The issue of cross-reactivity between antigens especially in areas where both Another important question being addressed is evaluating whether alternative methods for estimating transmission from serology can provide more information from fewer subjects, and/or information not easily obtainable from current methods, in particular, quantifying recent changes in exposure or defining heterogeneity in exposure at a more granular level. Whether the same antigens are associated with clinical immunity as determined by a similar study design or cohort studies in other malaria-endemic settings are a goal of ongoing ICEMR studies.Pf in Zambia and Zimbabwe (South Africa ICEMR), biosignatures of Pv in Peru, Brazil (Amazonia ICEMR), in PNG and Solomon Islands (south west Pacific ICEMR), and in Thailand and Columbia where transmission levels are very low.A separate protein microarray study, targeted specifically at identifying serologic biomarkers of exposure, is testing the hypothesis that individual-level estimates of exposure can be obtained from a limited number of properly chosen antibody responses. This study is leveraging detailed cohort studies from the east African ICEMR to obtain individual-level exposure data, and using flexible, data adaptive models to identify the most informative biosignatures. Data from these ongoing studies suggest that antibody responses to three properly chosen antigens can provide detailed information regarding recent exposure in individuals and can be aggregated to provide accurate estimates of population-level exposure within and between communities. Protein production of these promising antigens is underway, with plans to evaluate the kinetics of these responses and their ability to inform exposure across multiple epidemiologic settings, taking advantage of the rich epidemiologic data and sample bank being collected as part of the ICEMR program. In addition, separate studies to perform novel candidate discovery across individuals from distinct groups of age and exposure settings are planned. Other studies are currently underway to examine biosignatures of exposure to Pf and Pv infection, but also the pathological processes that underlie the clinical manifestations of infection. Improved understanding of the development and maintenance of NAI and the ability to cope with the severe manifestations of malaria are now particularly important, since effective public health interventions that reduce transmission are being deployed. Knowledge of the underlying mechanisms of NAI will also be invaluable in the design and evaluation of additional tools that prevent and treat malaria-related illness, most especially in constructing vaccines that ultimately eliminate the disease. Moreover, the identification of a biosignature of NAI can help determine whether populations are at a high risk of disease with malaria infection and whether this risk changes as interventions successfully reduce transmission.NAI is now recognized as a major determinant of the incidence and prevalence of malaria infection and disease in endemic areas.Anti-parasite and anti-disease immunity are often correlated, but develop at different rates and likely differ at least in part in their underlying mechanisms of action. Indeed, an ongoing analysis of cohort data from the Ugandan and southwest Pacific ICEMRs aims to quantify the relationships between each of these types of immunity with age and exposure and their relationship to each other. In general, low parasitemia levels in an immunologically naive individual will cause disease so the presence of malaria infection in otherwise clinically asymptomatic individuals reflects immunity to disease, suggesting malaria-exposed individuals develop a type of “tolerance” to malaria infection. What comprises this tolerance remains poorly understood, but likely comprises several components. First is acquisition of immunity to components of the malaria parasites that trigger innate inflammatory responses, for example, antibodies that may interfere with pro-inflammatory glycoprotein to Toll-like receptors.The mechanisms that govern NAI remain elusive, although our knowledge has expanded greatly over the past several decades. Studies performed over 50 years ago showed that transfer of IgG from clinically immune adults to children symptomatic with malaria markedly attenuated their infection and disease, demonstrating the critical role of antibodies in blood-stage immunity.The overall goal of the trans-ICEMR studies is to identify a constellation of malaria antigen-specific antibodies whose levels are consistently associated with NAI using different epidemiological approaches. However, it may be that the correlates of exposure vary in different epidemiologic settings or age groups. Importantly, as the frequency of clinical malaria diminishes resulting from interventions that reduce malaria transmission, the development of NAI will also diminish or shift to older individuals. As a consequence ideal biosignatures may also change in accordance with epidemiological studies monitoring changes in the incidence and/or prevalence of clinical malaria. The aim is to identify serological tests that can identify individuals lacking adequate NAI resulting in an increased risk of clinical malaria. Such monitoring can alert the public health system when NAI in the population declines below a critical level, leaving the population prone to epidemics with potentially high morbidity. Initial results from the southwest Pacific ICEMR suggest a critical threshold can be defined based on seroreactivity to relatively small group of antigens.49Because of its relative simplicity, the primary focus of ICEMR efforts has been on serological biosignatures of transmission and immunity; however, effector mechanisms of immunity are complex. For example, the ability of antibodies to opsonize malaria-infected erythrocytes or merozoites might better correlate with clinical immunity.Pf and Pv to inform malaria vaccine development and identify potential new biosignatures associated with exposure and protection worldwide. These data will improve and broaden our understanding of how NAI develops around the world as a function of age and exposure and will be useful to monitor eradication and control measures underway in the different ICEMRs. The project may also contribute to the discovery of vaccine antigen candidates to boost NAI to asexual and sexual stage parasites.The knowledge obtained from ICEMR-related studies will begin to address critical gaps in our understanding of naturally acquired humoral immune response against


P. vivax malaria.Angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are biomarkers produced during activation and dysfunction of the vascular endothelium in several infectious diseases. The aim of this study was to determine the serum levels of Ang-1 and Ang-2 and to establish their relationship with the main indicators of worst-case prognosis in patients with This is a retrospective case-control study nested within a cohort of symptomatic malaria patients. A potentially severe case was defined as a patient that presented at least one of the main indicators of the worst-case prognosis for falciparum malaria, as established by the World Health Organization. Ang-2 and Ang-1 and the Ang-2/Ang-1 ratio were used to analyze the role of angiopoietins as biomarkers in signaling potentially severe vivax malaria. ROC curves were generated to identify a cut-off point discriminating between the angiopoietin concentrations that were most strongly associated with potential infection severity.P. vivax malarial infection, was larger in the subgroup of patients with platelet counts <75,000/µL.The serum levels of Ang-2 and the Ang-2/Ang-1 ratio were higher in the case group. In contrast, the serum levels of Ang-1 were lower in the cases than in the control patients. The blood count for platelets showed a positive correlation with Ang-1 and a negative correlation with Ang-2 and with the Ang-2/Ang-1 ratio. The area under the ROC curve (AUC) for serum angiopoietins, as an indicator of worst-case prognosis in a potentially severe P. vivax malaria have lower Ang-1 and higher Ang-2 serum levels  than controls. Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.This study showed that patients with predictors of worst-case prognoses for  Plasmodium vivax malaria has increased, moving away from the conventional idea that this infection is benign P. vivax malaria, multiple syndromes have been described, similar to those caused by P. falciparumP. vivax as those proposed by the World Health Organization P. falciparumOver the past few years, the number of publications on severe P. falciparum, P. vivax has a greater capacity to induce an inflammatory response, which results in a lower pyrogenic threshold P. falciparum, are less frequent in severe vivax malaria P. falciparum and severe infections caused by P. vivax, of which severe anemia Compared to P. vivax have been described, including those for endothelial lesions, hemolysis, and oxidative stress. Numerous substances have been evaluated as biomarkers for the severity of malarial infections such as proteins associated with iron metabolism and anti-oxidant enzymes, as well as various cytokines and chemokines P. vivax infections, and its association with severe cases has been established A number of laboratory markers have been used to assess inflammatory patterns in response to malaria and their relation to the severity of the infection An understanding of the relationship between the presentation of the disease and the production of a biomarker may help in clinical decision-making and could minimize the risk of disease complications and consequently, the suffering of patients P. falciparum infections, as well as that between the levels of these proteins and the occurrence of cerebral malaria, placental malaria, retinopathy, and death P. vivax infections P. vivax malaria.Recent studies have elucidated the relationship between disease severity and plasma levels of angiopoietins in This retrospective case-control study was performed within a cohort of malaria patients treated at the Hospital Julio Müller, in the city of Cuiabá, state of Mato Grosso, Brazil, between December 2011 and November 2013. The hospital is a reference center for the diagnosis and treatment of malaria in people living and traveling in the south and southeast of the Brazilian Amazon. Participation in the study was voluntary, with a free and informed consent form signed by the patient, or in the case of children, by the parents. This study was approved by the Ethics Committee of the Julio Müller School Hospital (Document 130/CEP/HUJM/2011).P. vivax, as confirmed by microscopy of a thin blood smear and polymerase chain reaction (PCR) analysis. Following the confirmation of the diagnosis, the patients underwent clinical examination, as well as hematology and blood biochemistry tests, including those for serum levels of Ang-1 and Ang-2. To analyze the role of angiopoietin as a biomarker for potentially severe vivax malarial infections, a subgroup of patients were classified as potentially severe cases, if they presented at least one of the main indicators (for children and adults) of the worst-case prognosis for falciparum malaria, as established by the World Health Organization Patients with symptomatic malaria were eligible to participate in the study if they had a confirmed monoinfection with g. The serum was removed and aliquots were stored at −80°C. The serum was defrosted only once prior to the assay for angiopoietin.Ang-1 is present in platelet granules and is released upon platelet activation The hematological evaluation of the patients was performed using a Cell Dyn Ruby multi-parameter automated hematology analyzer . Blood biochemistry was analyzed by photometry using a BT-3000 Plus automated chemistry analyzer .Angiopoietin levels in the serum samples were assessed by enzyme-linked immunosorbent assay (ELISA) using standard commercial kits  for Ang-1, the Quantikine Angiopoietin-1 kit (Lot 306702), and the Quantikine ELISA Human Angiopoietin-2 kit (Lot 308323) for Ang-2, according to the manufacturer’s instructions.U test was used to compare the distribution of the classic indicators for potentially severe malarial infection between the cases and the control patients. Spearman’s correlation test was used to analyze the association between angiopoietins and platelet counts. Because of heteroscedasticity, we used the non-parametric Kruskal–Wallis test to compare the values of the levels of angiopoietin and classified the patients into three groups according to their platelet counts: severe thrombocytopenia , moderate thrombocytopenia , and no thrombocytopenia . Cuzick’s test for trend was used to analyze the tendency of quantitative variables between platelet counts of the different groups analyzed. We generated receiver operating characteristic (ROC) curves and calculated the area under the ROC curve (AUC) to evaluate the diagnostic accuracy of angiopoietins for discriminating between patients with and without the indicators of the worst-case prognosis for potentially severe vivax malaria. The Youden ‘J’ index was used to define the cut-off points corresponding to the sensitivity and specificity values with the lowest probability of having occurred randomly Because of the non-normality of the data, non-parametric statistics were used for the analysis. The Mann–Whitney P. vivax monoinfection were included in the study. Of the 80 patients, 58 (72.5%) were men and 22 (27.5%) were women, with ages ranging from 5 to 78 years, with a median (1st–3rd quartile) of 39 (26–49) years. All had presented with fever within the 24 h prior to entering the study. Altered hematologic and biochemical parameters were infrequent except for platelet counts, for which the median (1st–3rd quartile) was 98,000  platelets/µL for all patients. Severe and moderate thrombocytopenia were observed in 10 (12.5%) and 45 (56.2%) patients, respectively. Only 25 (31.3%) of the patients showed normal platelet counts. The clinical and laboratory characteristics of patients according to platelet count are shown in Eighty patients with symptomatic malaria caused by PCR-confirmed Eighteen patients were classified as potentially severe cases in this study, showing one or more of the main indicators of the worst-case prognosis for malaria: severe anemia (n = 1), elevated blood urea nitrogen (BUN) (n = 3), hyperbilirubinemia (n = 10), and elevated serum transaminases (n = 4). The ages of the potentially severe cases did not differ from those of the controls (P = 0.870). As expected, however, the parameters signaling severe malarial infection were altered in the potentially severe cases, with significant differences in serum bilirubin levels (P = 0.008), aspartate aminotransferase (AST) (P<0.001), alanine aminotransferase (ALT) (P = 0.002), and parasitemia (0.010). No significant differences in hemoglobin (P = 0.760), hematocrit (0.471), serum urea (0.179), serum creatinine (P = 0.061), platelet count (P = 0.097), or axillary temperature (P = 0.126) were observed between cases and controls .Angiopoietin levels were not associated with other factors known to be related to the severity of malaria, such as age, level of parasitemia, or axillary temperature. When the analysis of the serum levels of angiopoietin was stratified according to the patients’ platelet counts, we found that the median value of Ang-1 was lower (P = 0.0001) in the patients with thrombocytopenia than in those with normal platelet counts. The median values for Ang-2 and the Ang-2/Ang-1 ratio, on the other hand, were higher  in the patients with thrombocytopenia. As the platelet count increased, so did the levels of Ang-1 (P<0.001) but the levels of Ang-2 decreased (P = 0.003) as did the Ang-2/Ang-1 ratio (P<0.001). In summary, regardless of whether or not the patient met the worst-case prognosis criteria, the blood count for platelets showed a positive correlation with Ang-1  and a negative correlation with Ang-2  and with the Ang-2/Ang-1 ratio  .ROC curves were generated to identify a cut-off point to discriminate between angiopoietin concentrations that were most strongly associated with the potential severity of the case, including the subgroups of patients stratified according to the platelet blood count. From the ROC curve, a highly accurate cut-off level for serum angiopoietins was defined as an indicator for a worst-case prognosis of a potentially severe malarial infection. In the analysis of all patients, the AUC was 0.667 and the cut-off point for Ang-2 was 5.9 ng/mL (77.8% sensitivity and 50.0% specificity), whereas for the Ang-2/Ang-1 ratio, the AUC was 0.737 and the cut-off point was 0.75 (61.1% sensitivity and 83.9% specificity). Considering only the subgroup of patients with platelet counts <75,000/µL, the ROC curve showed an AUC of 0.833 and a cut-off point of 8.4 ng/mL for Ang-2 (87.5% sensitivity and 66.7% specificity), whereas for Ang-2/Ang-1, the AUC was 0.881, with a cut-off point of 1.2 (75.0% sensitivity and 90.5% specificity). In patients with platelet counts >75.000/µL, the AUC was 0.527 and the cut-off point was 3.85 ng/mL for Ang-2 (80.0% sensitivity and 29.3% specificity), whereas for Ang-2/Ang-1, the AUC was 0.645 and the cut-off point was 0.30 (60.0% sensitivity and 68.3% specificity) .P. vivax infections. In P. falciparum malaria, the production of angiogenic factors is principally associated with an increase in the cytoadherence of infected erythrocytes to the vascular endothelium P. vivax promotes cytoadhesion P. vivax infections remain unclear. Only two studies have described the relationship between angiopoietins and malaria caused by P. vivax, and these presented conflicting results. One report showed lower levels of Ang-2 in patients infected by P. vivax than in patients infected by P. falciparumP. vivaxIn this study, low levels of Ang-1 and high values of Ang-2 and the Ang-2/Ang-1 ratio were associated with criteria that indicate the worst-case prognosis in patients with symptomatic In vitro and in vivo studies have demonstrated that higher concentrations of Ang-2 are associated with elevated production of TNF-α, nitric oxide, vascular endothelial growth factor (VEGF), and hypoxia Angiopoietins are proteins that are intimately involved in the stability of the endothelium P. vivax malaria patients, whereas a negative correlation was detected between platelet counts and Ang-2 and Ang-2/Ang-1 levels. Although not a defining feature of severe malaria, vivax-associated thrombocytopenia occurs in more than 24% of patients with vivax malaria In this study, a positive correlation was observed between platelet counts and Ang-1 levels in P. vivax and the elevation of Ang-2 levels.The association between Ang-1, Ang-2, and the Ang-2/Ang-1 ratio was stronger in patients with thrombocytopenia. Some possible explanations for this association are given below. Firstly, Ang-1 is secreted by platelet granules, and thus its concentration in the plasma is dependent on the number of circulating platelets P. vivax malaria patients have not been identified to date. Disequilibrium in the expression and production of angiogenic factors is associated with the severity of malaria caused by P. falciparumP. falciparum malaria than lactate Although useful for clinical decision-making, biomarkers that predict the appearance of criteria indicating the worst-case prognosis for P. vivax malaria, especially in patients with low platelet counts. Once incorporated into routine hospital laboratory investigation, the systematic assay of angiopoietins could help isolate patients with the potential for developing severe malaria during clinical management of vivax malaria.To the best of our knowledge, this study provides the first evidence of the relationship between changes in angiopoietin levels and criteria indicating the worst-case prognosis for The main limitation of this study is the small number of patients examined, thus restricting the possibilities for extrapolating the results to other populations. It is also important to point out that the comorbidities of the patients studied may explain the findings suggesting a worst-case prognosis, as already shown in the literature P. vivax malarial infections have lower serum Ang-1 and higher serum Ang-2 levels  than controls. Furthermore, a positive correlation was observed between platelet count and Ang-1, and a negative correlation was observed between platelet count and Ang-2 (and the Ang-2/Ang-1 ratio). Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.This study showed that patients with worst-case prognoses for


In children, the prevalence rates of anemia , malaria , and acute and chronic inflammation  were high. However, the prevalence of vitamin A deficiency  was moderate, and the prevalence of iron deficiency  and iron-deficiency anemia  were low. Malaria and inflammation were associated with anemia, yet they explained only 25% of the population-attributable risk. In women, 44.8% , 35.1% , and 23.6%  were affected by anemia, malaria, or inflammation, respectively. The prevalence rates of iron deficiency , iron-deficiency anemia , vitamin A deficiency  and vitamin B12 deficiency  were low, while folate deficiency was high . Iron deficiency, malaria, and inflammation were significantly associated with anemia, but explained only 25% of cases of anemia. Anemia in children and women is a severe public health problem in Sierra Leone. Since malaria and inflammation only contributed to 25% of anemia, other causes of anemia, such as hemoglobinopathies, should also be explored.To identify the factors associated with anemia and to document the severity of micronutrient deficiencies, malaria and inflammation, a nationally representative cross-sectional survey was conducted. A three-stage sampling procedure was used to randomly select children <5 years of age and adult women from households in two strata . Household and individual data were collected, and blood samples from children and women were used to measure the prevalence of malaria, inflammation, and deficiencies of iron, vitamin A, folate, and vitamin B Anemia, defined as low hemoglobin concentration, is estimated to globally affect 43% of children under five years of age, 29% of non-pregnant women aged 15–49 years), and 38% of pregnant women. These estimates are considerably higher for Central and West Africa  [–49 yearsSierra Leone is one of the poorest countries in the world with slow economic growth despite increasing political stability . Sierra The survey protocol, questionnaires , and conData collection for the Sierra Leone Micronutrient Survey (SLMS) was conducted in November and December 2013. The SLMS was a cross-sectional national survey designed to produce estimates of micronutrient deficiencies in children aged 6–59 months and women of reproductive age for two strata—urban and rural. Two- or three-stage sampling was carried out, depending on the target group. Using a random starting point, 30 enumeration areas (EA) were selected probability proportional to population size within each stratum . Twenty-four households were then randomly selected in each EA from a list of all households in an EA compiled just prior to the field work. Within a household, all pregnant women were enrolled, followed by an additional stage of sampling to select one child <5 years old and one non-pregnant women using a Kish table [Prior to data collection, all field workers  participated in a one-week training on proper data and specimen collection procedures. The training consisted of two days of theoretical training and one day of role play to familiarize field workers with the survey procedures, instruments, and equipment. As part of the role play, phlebotomists drew blood specimens from field workers, and laboratory technicians practiced processing and labeling samples. At the end of the training, a written test was administered to team members. As more trainees were recruited than were needed, only the best performing individuals were selected for actual data collection, and the best performing and most experienced interviewers were hired as team leaders. Following the classroom training, two-days of field testing in two EAs not included in the SLMS  was conducted to familiarize teams with logistical procedures, team dynamics, and real-world scenarios.As part of the SLMS data collection, a household questionnaire was administered first, followed by the child and women questionnaires. If present, the head of the household responded to the household questionnaire, and if not present, another adult in the household responded. The selected woman responded to the women questionnaire, and the caretaker of the selected child provided responses to the child questionnaire. Questionnaires were administered in either Krio, Themne, Mende, or English, depending on the mother tongue of the respondent. If the required individuals were not present at the household during the first visit, at least two follow up visits were made to interview the selected household or individual.™ analyzer  [The household questionnaire contained modules related to household demographics, socio-demographic variables, water, sanitation and hygiene, and household consumption of wheat flour and vegetable oil. In addition, each household was requested to provide a salt sample for the quantitative testing of iodine concentrations using colorimetric method on the iCheck IodineGermany) . In addiGermany) . The salGermany) .™, Orchid Biomedical systems, Goa, India). The malaria test assess if the blood contains histidine-rich protein-2, a protein found in the blood stream during a current malaria episode and remains in the blood stream a up to one month after infection has passed [Children and women were invited for blood sampling in a nearby phlebotomy site established for the survey. Non-fasting blood was drawn in an EDTA-coated tube from children and non-pregnant women; for pregnant women, only a few drops of blood were collected to measure hemoglobin concentration and current and/or recent malaria status. Hemoglobin concentration was measured using a portable hemoglobinometer  and malaria status was checked using a dipstick antigen rapid diagnostic test  and protected from light. Later in the day, these samples were centrifuged and the plasma was separated and frozen at -20°C. This temperature was maintained during domestic transport and sample sorting. Samples were packed on dry ice (-70°C) during shipment to international laboratories where they were analyzed for iron, vitamin A, folate, vitamin B12 concentrations using the Cobas e411 analyzer (Roche Diagnostics USA). Because of concerns that Ebola virus may be present in some samples, samples analyzed for plasma folate and vitamin B12 were heated to 60°C for 60 minutes. This same heating procedure was conducted on 32 samples with known values and only a negligible deterioration in folate or vitamin B12 was observed. The coefficient of variation between non-heated and heated folate and vitamin B12 samples was 5.5% and 3.2%, respectively.Specifically, plasma was analyzed in one run for retinol-binding protein (RBP), ferritin, C-reactive protein (CRP) and a1-acid glycoprotein (AGP) at the VitMin-Lab  using the sandwich ELISA method of Erhardt et al . In addi12 deficiency was defined as a plasma concentration <150pmol/L [any inflammation.In children and pregnant women, hemoglobin concentrations <70 g/L defined severe anemia, 70–99 g/L defined moderate anemia, and 100–109 g/L defined mild anemia. In non-pregnant women, anemia was defined as hemoglobin<120 g/L; hemoglobin <80 g/L, 80–109 g/L, 110–119 g/L denoted severe, moderate, and mild anemia, respectively . To clas2 test was applied.All field data were doubly entered using a pre-programmed data-entry screen (CSPro v. 5.0) and then merged and cross-checked. Laboratory data were either auto-generated or doubly entered . Data analysis was done using SPSS version 22 with the complex survey module. Statistical weighting accounted for the unequal selection probability in the two strata. Standardized sampling weights were calculated for each stratum by dividing the overall sampling fraction for the entire survey sample by the sampling fraction within that stratum. Histograms and the Kolmogorov-Smirnov test were used to check for normality of data distribution. The statistical precision of all prevalence and mean estimates were was assessed using 95% confidence limits, which were calculated accounting for the complex sampling. The independent samples median and Kruskal-Wallis tests were used for to compare of groups of non-normal data. For binomial data, the chiChild age was initially calculated in days by subtracting the child's date of birth from the date of interview. Subsequently the child's age in months was calculated using an average number of 30.4375 days per month. Pregnant women were identified during the household interview, and pregnancy status was confirmed by self report during the interview with each selected woman.Using data on household characteristics and assets, principal component analysis was used to calculate an index of household wealth; households were subsequently classified into wealth quintiles base on approaches employed by UNICEF MICS, the World Bank, and the World Food Programme ,22. WHO The household response rate was 97%, leading to completed household interviews of 1,354 households. Children <5 years old had an individual response rate of 87%, and 52.2% were female. Complete questionnaire and biomarker data were available for 654 children 6–59 months of age, and slightly higher sample sizes were obtained for hemoglobin (n = 710) and malaria (n = 723) status. As shown in any inflammation accounted for 12.7% anemia. Nearly 75% of the anemia in children was not explained by the factors explored in our analysis.When those factors showing statistically significant associations in bivariate analysis see  were inc12 deficiency (0.5%). Inflammation and current or recent malaria were found in approximately one-quarter and one-third of non-pregnant women, respectively.Eighty-two percent of the randomly-selected non-pregnant women participated in the SLMS and had complete questionnaire and biomarker data. About one-half were anemic . Severe,Among pregnant women, the prevalence of anemia was 70.0% ; 2.3%  had severe anemia, 39.6%  had moderate anemia, and 28.2%  had mild anemia. There was no statistically significant difference in the prevalence of anemia by age, residence, region, education, or household wealth. Malaria testing was positive in 28.6%  of pregnant women, and the prevalence of malaria was statistically significantly higher in rural areas, 34.3%, compared to 20.6% in urban areas (p <0.05). Age, educational level, and household wealth were not statistically significantly associated with malaria.In bivariate analyses, no statistically significant associations were found between demographic factors and anemia in non-pregnant women . On the No statistically significant associations were found between iron deficiency and the available demographic, nutritional, and health factors, most likely because the number of women with iron deficiency was relatively low. For folate deficiency, only consumption of multi-vitamin tablets or syrup in the past six months was significantly associated, with women consuming supplements displaying slightly less folate deficiency than those who did not take supplements.Due to the small number of factors associated with iron and folate deficiencies and the very low prevalence of vitamin A deficiency, we only conducted multivariate analysis for anemia. Iron deficiency, malaria status, and inflammation were all highly significantly associated with anemia in the multivariate model . As in tThe population attributable risk estimates of each risk factor's contribution to anemia demonstrate that iron deficiency causes 11.2% of anemia while current or recent malaria causes 13.8% of anemia. The status “any inflammation” accounted for no cases of anemia. As with children, the variables included in this analysis failed to explain about 75% of the anemia in non-pregnant women.Among children, malaria and inflammation were the only factors consistently associated with anemia. Nearly 40% of children had concurrent malaria and inflammation, and this co-morbidity was found in half of all anemic children . Anemia Concurrent malaria and inflammation was found in only 5.0% of all women, and present in 11.1% of anemic women. Malaria (without inflammation) affected similar proportions of anemic (14.6%) or non-anemic (11.9%) women, and inflammation  affected a greater proportion of non-anemic (9.1%) than anemic (5.2%) women. Most notably, 20.1% of anemic women tested negative for inflammation and had a negative malaria rapid test.The prevalence of anemia in Sierra Leonean children and women exceeds 40%, denoting a severe public health problem according to WHO classifications . The aneThe high overlap of anemia and malaria in Sierra Leonean children has been previously observed elsewhere in Sub-Saharan Africa. A systematic review of observational studies from Sub-Saharan Africa found that malaria was more highly associated with anemia than measures of under-nutrition (i.e. stunting and wasting) . As malaAmong the statistically significant risk factors for anemia in children, malaria and inflammation can be modified in order to prevent anemia. Although the risk factor age is non-modifiable, it does help target interventions to the age groups in greatest need. The peak of anemia prevalence among children 6–11 months of age, the typical period when complementary foods are introduced, may result from insufficient nutrient quality and/or quantity of complementary foods, increased exposure to infectious diseases, and/or reduced nutrient absorption. Because the standard infant and young child feeding indicators are calculated for relatively narrow age groups , they were not examined in this analysis. The role of infant and young child feeding in micronutrient malnutrition should be investigated in greater detail.Contrary to children, only a small proportion of women (5%) exhibited concurrent anemia, malaria, and inflammation. This suggests that malaria and inflammation are independent predictors of anemia in women. As 20.1% of anemic women did not display malaria or inflammation, there is likely other factors causing the anemia observed. Concurrent and independent malaria and inflammation was also found in non-anemic women, indicating that malaria and inflammation do not necessary result in anemia.Iron deficiency in Sierra Leone was relatively uncommon among children and women, in contrast to the general belief that iron deficiency accounts for approximately half of anemia in most populations . Iron-deSerum ferritin was used as the indicator of iron deficiency. However, ferritin acts as an acute-phase reactant which increases in concentration during inflammation. The adjustment for inflammation that was applied prior to data interpretation may not have corrected for the entire effect , thus ouThe prevalence of vitamin A deficiency is considered a public health problem  in childIn addition to biannual supplementation of vitamin A, increasing the regular dietary vitamin A intake in children is also warranted; promoting vitamin A-rich foods through food diversification and expanding the fortification of commercially-produced vegetable oil with vitamin A are options. However, as average daily consumption of vegetable oil was only about 4–5 grams in children and disproportionately reaches the less poor and urban households, adequately fortified vegetable oil may fail to reach a high proportion of children at greatest risk . Biofort12 are quite different. These deficiencies were not, however, associated with low consumption of folate- and vitamin B12-rich foods. The SLMS found that in the 24-hours prior to being interviewed, 83.3% of women had consumed foods rich in folate  and 71.5% of women had consumed foods rich in vitamin B12 . Howevenot a major contributor of anemia in developing countries [Despite the high prevalence of folate deficiency in Sierra Leone, there was no association between folate deficiency and anemia. This finding is consistent with other, albeit sparse, evidence that suggest folate is ountries . Althougountries . Plasma ountries .12 deficiency reported (0.5%) was based on the WHO-endorsed deficiency cutoff of <150 pmol/L. Herbert [12 depletion, or marginal vitamin B12 status. Using this alternative cutoff, 3.8%  of women would be considered as having marginal and deficient vitamin B12 status. This finding illustrates that for the vast majority of women, vitamin B12 concentrations are at normal or higher levels. Vitamin B12 deficiency in Sierra Leone is considerably lower than that found in nationally-representative surveys in Côte d’Ivoire [The prevalence of B Herbert  suggested’Ivoire  and Camed’Ivoire , which fSierra Leone's 2013 Malaria Indicator Survey (MIS), which was conducted approximately eight months prior to the SLMS, determined that 46.2% of children 6–59 months had a positive rapid diagnostic test for malaria . The MISThe government of Sierra Leone has identified malaria is one of the main public health problems affecting the country, and malaria-related illnesses account for 38% of all mortality in children < 5 years of age . The govBecause our results indicate malaria is a key contributor to anemia, malaria prevention may contribute to anemia prevention. As of 2015, the Ministry of Health and Sanitation established an anemia working group to take a multi-sectoral approach to addressing anemia. The efforts of this working group will compliment the efforts of government and its stakeholders to treat and prevent malaria.Given the high prevalence of anemia and the relatively minor contribution of iron and vitamin A deficiencies, inflammation, and malaria, a thorough investigation of the etiology of anemia in Sierra Leone is needed before a comprehensive prevention and control strategy can be formulated. It is recommended that those potential contributing factors not measured by the SLMS be investigated, including hemoglobinopathies, such as sickle cell and thalassemia traits. Previous studies in Sierra Leone found that the prevalence of the sickle cell trait ranged from 22%  to 29%  and variFollowing a thorough investigation of the etiology of anemia, large-scale programs to address the factors associated with anemia should be developed. Due the varying severity of anemia by residence, region, age, education, and wealth quintile, programs should be tailored and targeted at specific population groups.The prevalence of anemia is a severe public health problem for children and women in Sierra Leone, and based on our analysis, acute and chronic inflammation and malaria are the most consistent risk factors of anemia in both groups. In addition, vitamin A deficiency poses is a moderately severe public health problem in children, and high levels of folate deficiency were observed in women. Continued programmatic efforts should address the risk factors of anemia, reduce micronutrient deficiencies, and conduct further research to further describe the determinants of anemia.S1 Appendix(PDF)Click here for additional data file.