Plasmodium falciparum merozoite antigens play a role in protection from malaria. The specific targets mediating immunity remain unclear. Evaluating antibody responses in infants naturally-exposed to malaria will allow to better understand the establishment of anti-malarial immunity and to contribute to a vaccine development by identifying the most appropriate merozoite candidate antigens.Substantial evidence indicates that cytophilic IgG responses to The study was based on parasitological and clinical active follow-up of infants from birth to 18 months of age conducted in the Tori Bossito area of southern Benin. For 399 infants, plasma levels of cytophilic IgG antibodies with specificity for five asexual stage malaria vaccine candidate antigens were determined by ELISA in infants’ peripheral blood at 6, 9, 12 and 15 months of age. Multivariate mixed logistic model was used to investigate the association between antibody levels and anti-malarial protection in the trimester following the IgG quantification. Moreover, the concentrations of merozoite antigen-specific IgG were compared between a group of infants apparently able to control asymptomatic malaria infection (CAIG) and a group of infants with no control of malaria infection (Control group (NCIG)). Protective effect of antibodies was also assessed after 15 months of malaria exposure with a Cox regression model adjusted on environmental risk.Cytophilic IgG responses to AMA1, MSP1, MSP2-3D7, MSP2-FC27, MSP3 and GLURP R2 were associated with increasing malarial infection risk in univariate analysis. The multivariate mixed model showed that IgG1 and IgG3 to AMA1 were associated with an increased risk of malarial infection. However infants from CAIG (n = 53) had significantly higher AMA1-, MSP2-FC27-, MSP3-specific IgG1 and AMA1-, MSP1-, MSP2-FC27-, MSP3 and GLURP-R2-specific IgG3 than those from NCIG (n = 183). The latter IgG responses were not associated with protection against clinical malaria in the whole cohort when protective effect is assessed after 15 months of malaria exposition.P. falciparum. This observation is encouraging in the context of the development of multitarget P. falciparum vaccines.In this cohort, merozoite antigen-specific cytophilic IgG levels represent a marker of malaria exposure in infants from 6 to 18 months of age. However, infants with resolution of asymptomatic infection (CAIG) seem to have acquired naturally immunity against  Plasmodium falciparum malaria remains a significant cause of infant mortality and morbidity in many parts of the world especially in sub-Saharan Africa. In 2017, there were an estimated 219 million cases of malaria and 435,000 deaths [Anopheles bites, infants develop immune responses that reduce clinical symptoms and life-threatening complications. Antibodies are known to be key components of naturally-acquired anti-malarial [malarial .®) is the most advanced malaria vaccine, targeting pre-erythrocytic stages, but it is suboptimal both in terms of efficacy and duration [An efficacious blood stage vaccine against malaria that remains potent in different transmission settings would greatly contribute to reducing the disease burden among endemic populations. RTS,S/AS01  1, 2, 3, apical membrane antigen 1 (AMA1) and glutamate rich protein (GLURP) [P. falciparum merozoite surface proteins have been associated with reduced risk of malaria and high parasitaemia in naturally-exposed children or adults [P. falciparum and they represent the main target in any malaria vaccination strategy. Previous work conducted in the Tori-Bossito cohort showed that factors such as age, past and current malaria infections, malaria antibody levels at birth, as well as exposure to Anopheles bites were significantly associated with the natural acquisition of anti-malarial IgG1 and IgG3 responses in 6- to 18-month old infants [P. falciparum candidate vaccine antigens in protection of infants against P. falciparum infection. IgG1 and IgG3 responses to five leading P. falciparum merozoite-stage vaccine candidate antigens  were quantified to investigate associations between these antibody responses and anti-malarial protection from 6 to 18 months of age.To date, a number of  (GLURP) . Numerour adults –19. Hower adults , 20–32.  infants . In the The data here were collected from a longitudinal malaria cohort study conducted in 9 villages of the district of Tori Bossito located in the Southern part of Benin described in detail elsewhere . BrieflyP. falciparum infection could be symptomatic or asymptomatic . All blood smears were stained with 10% Giemsa and were read by two independent technicians. A third reading was performed when discordant cases occurred.During systematic visits P. falciparum entomological inoculation rate was calculated in all villages, giving an average of 15.5 infected Anopheles bites/person/year [For each woman at delivery, a questionnaire collecting information on maternal age, parity, use of Intermittent Preventive Treatment during pregnancy (IPTp) and bed net possession was administered. In addition, thick and thin placental smears were made to detect placental malaria. An entomological, nutritional and biological follow-up was also performed as described in detail elsewhere . During son/year , with lason/year . Hence, son/year , 36. Alsson/year .Infant blood samples were collected in EDTA vials every 3 months during the follow-up and plasmas were conserved at − 80 °C for antibody quantification. In the present study, the main objective was to assess the protective role of infant antibody responses to malaria infection every 3 months following the blood draw. Antibody measurements at 0 and 3 months of age were excluded because of the content in those samples of antibodies from mixed maternal and infant origins. Therefore, only antibody measurements at 6, 9, 12 and 15 months were processed.Pichia pastoris and was produced by the Biomedical Primate Research Centre  [19  was expressed in a Baculovirus/insect cell system [Escherichia coli.The plasmodial antigens used in this study included a recombinant AMA1 , which was expressed in erlands) . MSP119 l system . It is cl system , 40. GLUThe standard operating procedures developed by the African Malaria Network Trust was used to assess cytophilic IgG1 and IgG3 concentrations by enzyme linked immunosorbent assay (ELISA) to a panel of recombinant proteins, as described previously . BrieflyR script based on ADAMSEL software   was usedp ≤ 0.10 in the univariate analysis.In the first approach, a logistic mixed model was performed to study the association between the antibody levels quantified at 6, 9, 12 and 15 month of age for 399 infants and the risk of malarial infection from 6 to 18 month of age. Each quarterly antibody measurement was associated with the presence or absence of at least one malaria infection during the following 3 months in the logistic mixed model. To focus specifically on antibody-mediated effects, the cytophilic IgG levels were first adjusted using a linear regression on the nuisance variables . The residuals of these adjustments were then used in the models. Moreover, all analyses were adjusted on maternal and newborn’s covariates known to potentially influence malarial infection: placental malaria, parity, prematurity, haemoglobin levels at delivery and birth, administration of IPTp, birth weight, ethnicity, age, environmental risk of exposure to malaria, gender, nutrition status, sickle cell trait and bed net use. Multivariate analysis was performed including the variables with P. falciparum without resolution of asymptomatic malaria infection. The control of asymptomatic infection was defined here as the disappearance of asymptomatic infections over time based on TBS examination. Infants from CAIG seem able to control not only the disease symptom (asymptomatic infection) but also the parasitaemia. NCIG was composed of infants at least exposed to two malaria infections during the follow-up. No difference in term of environmental risk of exposure to malaria (ERE variable) was observed between both groups. It is important to notice that the linear mixed model took into account IgG cytophilic levels at 6, 9, 12 and 15 months, respectively.In the second approach, a linear mixed model was used to assess the difference of the levels of cytophilic IgG between a group of infants apparently able to control asymptomatic malaria infections (CAIG (n = 53)) and a control group (NCIG (n = 183)) of infants infected by A linear mixed model was used to test the association between the antibody quantification at 15 months and the parasite density in the following quarter. The mean of the parasite density between 15 and 18 months  was calculated and antibody levels were divided into 4 categories according to the quartiles.Finally, a Cox proportional hazards regression model was used to test the protective effect of IgG responses previously associated with the phenotype of asymptomatic infection clearance (CAIG) at 15 months of age in the whole sample (CAIG + NCIG). Hazard ratios (HR) of clinical malaria were calculated on the 15–18 months period adjusted by ERE. To avoid a bias due to anti-malarial therapy, it has been considered a 14 days period after treatment where the infants were not considered at risk. Thus for infants who had a symptomatic malaria infection at blood sampling (n = 14) or within the 2 weeks before blood sampling, the starting point of survival analysis was shift to 14 days after treatment (n = 28). Those with an asymptomatic infection at sampling (n = 19) were excluded from analysis.p < 0.05.All statistical analyses except Cox model were performed using Stata, version 13.0 . Cox model were performed using the R package “survival” . StatistThe study protocol was approved by the Ethics Committee of the University of Abomey-Calavi (Faculté des Sciences de la Santé) in Benin and the Consultative Committee of Ethics of the Institut de Recherche pour le Développement. All women signed an informed consent before enrollment . All the methods were carried out in accordance with the approved guidelines.Newborns for whom plasma samples and clinical and parasitological data were available were included in the analyses (n = 399). Eighty-two per cent of the mothers declared having received IPTp at least once . Fourteen per cent of them were primigravidae and 10% had an infected placenta at delivery. Eleven per cent of the newborns had a low birth weight (< 2500 g) and 12% were born prematurely. The sex ratio was 0.98 and infants followed in the study belonged to different ethnic groups: Tori and Fon were the principal ethnicities represented with minorities of Aïzo and Yoruba. Sickle-cell trait (HbAS) carriers accounted for 22% of the study population. Details of the study participants are presented in Table A total of 834 uncomplicated malaria attacks (UMA) (from 1 to 11 UMA/infant) and of 168 asymptomatic infections (from 1 to 8 asymptomatic infections/infant) were recorded during the follow-up. Based on the definition cf. “”, 53 infp < 0.05) with the exception of antibody responses to GLURP R0 (Table p < 0.10). None of the other covariables showed an association with the risk of infection  in univariate analysis were included in the multivariate analysis   Table .Table 4Ap ≤ 0.05), whilst their levels of IgG3 with specificity for MSP2-3D7 displayed trends towards higher levels (p = 0.067)  the level of cytophilic antibodies directed to AMA1 were associated with an increasing risk of malarial infection during the complete cohort follow-up and (ii) a group of infants with resolution of P. falciparum asymptomatic infections (CAIG) had higher cytophilic IgG levels to AMA1, MSP1, MSP2-FC27, GLURP-R2 and MSP3.After birth, infants exposed to repeat malaria infections gradually acquire anti-malarial antibodies while their maternal antibodies wane in time. Numerous antigens on the merozoite surface have been identified as important targets of naturally-acquired immunity, and the need to develop an effective blood-stage vaccine against approach , its sam19 were significantly associated with an increasing malaria risk in two cohorts, each constituted by 40 children aged from 4 to 6 weeks in Burkina Faso and Senegal [First result suggests that IgG1 and IgG3 responses to AMA1—quantified every 3 months in all infants of the cohort—represent more a biomarker of exposure than a biomarker of protection from malaria. Most of the results observed in other infant cohorts have revealed similar associations between the antibody level and an increased risk of malaria. In a cohort study conducted by Kusi in Ghana on infants between 1 and 5 years old, authors found that anti-AMA1 antibodies were associated with a history of infection . Stanisi Senegal . Other s Senegal , 14. TheP. falciparum DNA could not be sufficient to prove the viability of the parasite as demonstrated by different groups [In order to better understand the acquisition of IgG to vaccine candidates leading to protection, a group of infants that experienced asymptomatic infections followed by a disappearance of these asymptomatic infections during the follow-up (CAIG) was defined. CAIG definition was based on microscopic examination of TBS although the sensitivity of this method is questionable. However, submicroscopic infections, even detectable by amplification of t groups . It coult groups . Anothert groups . AMA1 isPlasmodium strains circulating in this Beninese study area could lead to an underestimation of antibody responses directed to antigen of interest [P. falciparum could trigger blood monocytes to control parasite density known as Antibody-Dependent Cellular Inhibition (ADCI) [P. falciparum merozoite surface protein 1p19 could induce antibody-dependent respiratory burst (ADRB) in human neutrophils [Antigenic variations are often reported and often alter the vaccine efficacy. Therefore, potential differences between the strains used to produce the recombinant proteins and the interest . Howeverinterest , 14, 16.interest –51. The interest . The anainterest –55. Moren (ADCI) , 57 or atrophils –60.P. falciparum is highly dependent on parasite exposure and anti-malarial antibodies detected in plasma samples of the study population considered as a whole group represent rather a marker of exposure than a marker of protection. However the group of 53 asymptomatic infants who were able to control asymptomatic malaria infection presented higher antibody levels to AMA1, MSP1, MSP2-FC27, GLURP-R2 and MSP3 than infants from the symptomatic group, underlying the possibility of a very early establishment of naturally acquire immunity against clinical malaria. This last result is encouraging in the context of the development of multitarget P. falciparum vaccines.The findings presented here showed an association between high levels of cytophilic IgG responses to AMA1 and an increased risk of malaria infection from 6 to 18 months of life. Antibody acquisition to


Plasmodium falciparum malaria is associated with high mortality, yet the pathogenesis remains incompletely understood. The aim of this study was to determine the nature and source of metabolic acids contributing to acidosis in patients with severe falciparum malaria.Acidosis in severe P. falciparum malaria (n = 107) and healthy controls (n = 45) from Bangladesh using high-resolution liquid chromatography–mass spectrometry metabolomics. Additional in vitro P. falciparum culture studies were performed to determine if parasites release the acids detected in plasma from patients with severe malaria acidosis.A prospective observational study was conducted to characterize circulating acids in adults with P. falciparum parasites in vitro showed no evidence that these acids are released by the parasite during its life cycle. Instead, 10 of the plasma acids could be mapped to a gut microbial origin. Patients with malaria had low L-citrulline levels, a plasma marker indicating reduced gut barrier integrity. Longitudinal data showed the clearance of these newly identified acids was delayed in fatal cases.We identified previously unmeasured plasma acids strongly associated with acidosis in severe malaria. Metabolomic analysis of These data suggest that a compromise in intestinal barrier function may contribute significantly to the pathogenesis of life-threatening acidosis in severe falciparum malaria.NCT02451904. Acidosis in severe falciparum malaria is associated with an accumulation of unidentified acids in blood plasma. Clinical and in vitro metabolomic studies identify a potential role for translocation of bacterial acids from the gut into the bloodstream. Plasmodium falciparum parasite. Early diagnosis and treatment with parenteral artesunate reduces mortality, but case fatality rates still range between 10% and 30% depending on the extent of vital organ dysfunction  >1, Benjamini–Hochberg adjusted P < .01)   B. FurtheP < .01) C. The reA major difference was also observed in the levels of acetaminophen glucuronide in the plasma from patients who had malaria compared to healthy controls . Acetamiq values among patients with severe malaria. A significant association between SBDc and 36 organic acids was found . These organic acids are shown using their log2 normalized fold change stratified according to the degree of acidosis  data. We found an overrepresentation of organic acids from a microbial origin, and 10 of 36 (28%) metabolites were described as having a microbial source  . These pP. falciparum parasite–infected red blood cells release the acids identified in severe malaria acidosis, we performed an in vitro culture study using P. falciparum 3D7 strain parasites. A metabolomic analysis was conducted of temporal changes of metabolites in the spent culture media of Pf3D7 and nonparasitized red blood cell controls throughout the parasite life cycle. Culture media metabolites were extracted using the same methanol extraction techniques applied to patient plasma, and the samples were analyzed using the same UHPLC-MS platform (To determine if platform .Pf3D7 cultures. Except for lactate (parasites produce both D and L forms), none of these acids showed a cumulative increase during the parasite life cycle    36]. Ho. HoP. faWe analyzed the temporal profiles of plasma levels of gut microbial acids during the first 48 hours of hospitalization . All gutP. falciparum in vitro cultures at different stages of asexual development did not identify P. falciparum as a likely source for these newly identified acids. Patients with malaria had markers indicative of loss of gut barrier dysfunction. Clearance of microbial acids was delayed in fatal cases, supporting their prognostic significance in adult patients with severe malaria. Combined, these findings point toward a potential role for translocation of bacterial acids from the gut into the bloodstream, suggesting a novel pathogenesis for acidosis, contributing to disease severity and poor prognosis in patients with severe P. falciparum malaria.In this study, we characterized circulating acids in patients with severe falciparum malaria. LC-MS–based metabolomics revealed elevated levels of acids, which are known to be produced by gut bacteria. Assaying spent medium of PfHRP2 correlated positively with the newly identified organic acids, suggesting that an increased parasite biomass and subsequent sequestration was associated with translocation of microbial acids (Severe falciparum malaria predisposes to concomitant bacteremia , often wal acids . AlternaIn the presence of a compromised intestinal barrier, the composition of the gut microbiota may play a role in the pathogenesis of severe malaria acidosis. Recent mouse studies have found that the severity of malaria was affected by the composition of the gut microbiota . IncreasP. falciparum parasite, and the gut microbiota. We postulate that sequestration of parasitized red cells in the splanchnic microcirculation may cause intestinal barrier dysfunction, predisposing patients to translocation of gut microbial acids and enteric bacteria (Our findings suggest an interaction between the human host, the bacteria . If incrP. falciparum parasites [It should be noted that although L-citrulline is considered a marker of gut integrity, the observed reduction of L-citrulline in patients with malaria can be compounded by significant scavenging by arasites . Additioarasites . We do nPlasmodium falciparum clone 3D7 was selected for the in vitro studies because it is very well characterized, grows efficiently in vitro, and has been fully sequenced and well annotated, and the 3D7 intraerythrocytic developmental cycle transcriptome is available [vailable . While oIn conclusion, metabolomic profiling showed elevated plasma concentrations of gut microbial acids during severe malaria acidosis. Our data suggest a novel pathogenic pathway for acidosis in severe malaria, where loss of gut barrier function related to microcirculatory parasite sequestration may cause the translocation of microbial acids into the circulation.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary MaterialClick here for additional data file.


Plasmodium infections increase the likelihood of enteric bacteria causing systemic infections. Currently, it is not known whether Plasmodium infection impacts human gut microbiota as a prelude to bacteremia or whether antimalarials affect gut microbiota. Our goal was to determine to what degree Plasmodium infections and antimalarial treatment affect human gut microbiota.Gut microbiota were recently shown to impact malaria disease progression and outcome, and prior studies have shown that One hundred Kenyan infants underwent active surveillance for malaria from birth to 10 months of age. Each malaria episode was treated with artemether-lumefantrine (AL). Any other treatments, including antibiotics, were recorded. Stool samples were collected on an approximately biweekly basis. Ten children were selected on the basis of stool samples having been collected before (n = 27) or after (n = 17) a malaria episode and without antibiotics having been administered between collections. These samples were subjected to 16S ribosomal ribonucleic acid gene (V3–V4 region) sequencing.Bacterial community network analysis revealed no obvious differences in the before and after malaria/AL samples, which was consistent with no difference in alpha and beta diversity and taxonomic analysis at the family and genus level with one exception. At the sequence variant (SV) level, akin to bacterial species, only 1 of the top 100 SVs was significantly different. In addition, predicted metagenome analysis revealed no significant difference in metagenomic capacity between before and after malaria/AL samples. The number of malaria episodes, 1 versus 2, explained significant variation in gut microbiota composition of the infants.In-depth bioinformatics analysis of stool bacteria has revealed for the first time that human malaria episode/AL treatment have minimal effects on gut microbiota in Kenyan infants. Salmonella infections, this report demonstrates that Plasmodium infections and oral treatment with antimalarial drugs do not alter stool bacteria populations in Kenyan infants.Although malaria-induced dysbiosis of gut microbiota has been postulated to contribute towards increased susceptibility to Non-typhoid  Plasmodium ; P = .234, LMEM; 2 = 0.09, P = .044; 2 = 0.007, P = .6)  using Observed_OTUs (richness/number of species present) and Shannon index (richness and evenness), were analyzed between the before and after malaria/AL episodes. Neither Observed_OTUs ( P = .6) . Similar P = .6)  and D.P > .05, LMEM)  was calculated using (1) non-phylogenetic Bray-Curtis distance and (2) phylogenetic weighted and unweighted UniFrac distance for all samples  and paired samples  . The bac5, LMEM) . These aP < .05, Wilcoxon and Kruskal-Wallis test) . Consistently, analysis of the relative abundance of the top 12 bacteria family among the individual participants revealed no gross pattern between the before and after malaria/AL stool samples   . However samples . Similar samples . When ea t test) . Likewis t test)  and B of2 = 0.948, P < .0001)   . The LEf< .0001) . SV43, S< .0001) . However6, LMEM) . The topt test with the web portal, RNA-Seq 2G (http://rnaseq2g.awsomics.org). Nine and eight pathways were enriched in stools before and after malaria/AL using DeSeq2 and t test, respectively (P < .05)  (The metagenome in the stool samples was predicted using the online tool Piphillin. None of the top 20 most highly abundant KEGG pathways  were morP < .05) . Six patP < .05) . Only N- samples , but thi = .217) .t test using RNA-Seq 2G, resulting in 47 genes common to both tests (N-glycan biosynthesis (N-glycan biosynthesis pathway with Pathview (https://pathview.uncc.edu/), only 3 KO were inferred by Piphillin of 43 KO in the pathway  level was analyzed. The inferred metagenome at gene level were normalized with DeSeq2, and differentially abundant genes were calculated using DeSeq2 and th tests . All werth tests . Among tth tests . Inconsith tests , the patynthesis . Moreove pathway . However pathway  and S9. 2) explained by various covariates in this study was calculated using EnvFit implemented in vegan R package. Earlier, we showed no difference in alpha diversity among groups of various covariates     and C. H9, LMEM) .Plasmodium species (Plasmodium berghei ANKA and P yoelii nigeriensis) induced pathological changes in the intestine including infiltration of inflammatory macrophages, T cells, detachment of epithelia in the small intestine, increased expression of inflammatory cytokines, and intestinal permeability [P berghei ANKA-infected BALB/c mice [P yoelii nigeriensis infection in CBA mice induces anti-inflammatory interleukin (IL)-10 [Macaca mulatta) infected with Plasmodium fragile—the parasite suppressed gut inflammation via the induction of IL-10, which blunted the influx of neutrophils in the gut [Plasmodium species.This report provides the first longitudinal analysis of stool bacteria communities before and after a clinical malaria episode plus AL treatment in humans. The data identified minimal changes in gut microbiota of Kenyan infants due to malaria/AL. In contrast to these data in humans, 2 previous studies in rodent malaria models have reported larger shifts in the gut microbiota of mice before and after malaria infection associated with intestinal inflammation. Infection of mice C57BL/6) with rodent 7BL/6 wit (IL)-10 . Likewis the gut . Finally the gut , 18. TakP falciparum infection have been shown to have increased gastrointestinal permeability lasting for a couple of days depending on the severity of malaria and treatment, which reverts to normal during convalescence [Salmonella during malaria, including macrophage dysfunction due to ingestion of the malaria pigment hemozoin [Salmonella initially breaches the intestinal barrier to cause a systemic infection during malaria. It is well established that dysbiosis of the normal gut flora provides an opportunity for Salmonella to establish infection [P yoelii nigeriensis-induced dysbiosis resulted in increased susceptibility to Salmonella typhimurium enterica infection in C57BL/6 mice [P yoelii nigeriensis infections induce elevated production of intestinal IL-10, which facilitates increased translocation of S typhimurium enterica out of the intestinal tract [Plasmodium-induced changes in the anti-inflammatory status of the intestinal tract may be more likely to contribute to intestinal translocation of Salmonella rather than gut microbiota dysbiosis.Adults with lescence . In spitlescence . Varioushemozoin , impairehemozoin , dysfunchemozoin  among othemozoin . Howevernfection , and it al tract . Taken tP falciparum infection. Where slide is negative but RDT positive, suspicion may be lower. The RDT used detects the P falciparum-specific surface antigen histidine-rich protein 2 (HRP-2) in blood. The HRP2-based RDTs can remain positive for up to 42 days or beyond after the beginning of a treated clinical malaria episode [Of the 16 malaria episodes, all were RDT positive but only 11 were slide positive. Copresence of fever and slide positivity amongst infants in this moderate to high transmission intensity setting is strongly indicative of acute clinical malaria rather than incidental parasitaemia . Suspici episode . Partici episode . For parPrior studies have identified a positive correlation between microbial diversity, measured by alpha diversity, and age of infants . In contOnly the number of malaria episodes explained significant variation in the gut microbiome composition of Kenyan infants. It is important to note that this observation has been made in a small sample size, and that significance is lost when repetitive sampling is taken into consideration. Nevertheless, it raises the exciting possibility that differences in gut bacteria may contribute to differential outcomes of malaria in children. Clearly, this possibility will need to be examined in the context of a larger and more definitive study that determines the ability of gut microbiota to modulate the severity of malaria in African children.Plasmodium infections? Addressing these questions could make substantial improvements in developing novel approaches to prevent malaria-related fatalities.We demonstrate for the first time that human malaria episodes and AL treatment result in a minimal shift in the gut microbiota of Kenyan infants, which is in contrast to what was observed in some murine models of malaria. These results suggest that changes in the inflammatory nature of intestinal tissue, in contrast to gut microbiota dysbiosis, during malaria may contribute to translocation of enteric bacteria and progression to bacteremia. This study also leaves open exciting, yet unanswered, questions regarding interactions between human gut microbiota and malaria. For example, are there different gut microbiota between healthy individuals and patients with severe malaria? Given the increased appreciation of the gut-brain axis, is there an interplay between gut microbiota and cerebral malaria? Can machine learning algorithms predict the gut microbiota composition associated with children prone to The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiy740_suppl_Supplementary_Table_S1Click here for additional data file.jiy740_suppl_Supplementary_Table_S2Click here for additional data file.jiy740_suppl_Supplementary_Table_S3Click here for additional data file.jiy740_suppl_Supplementary_Table_S4Click here for additional data file.jiy740_suppl_Supplementary_Figure_S1Click here for additional data file.jiy740_suppl_Supplementary_Figure_S2Click here for additional data file.jiy740_suppl_Supplementary_Figure_S3Click here for additional data file.jiy740_suppl_Supplementary_Figure_S4Click here for additional data file.jiy740_suppl_Supplementary_Figure_S5Click here for additional data file.jiy740_suppl_Supplementary_Figure_S6Click here for additional data file.jiy740_suppl_Supplementary_Figure_S7Click here for additional data file.jiy740_suppl_Supplementary_Figure_S8Click here for additional data file.jiy740_suppl_Supplementary_Figure_S9Click here for additional data file.jiy740_suppl_Supplementary_MaterialClick here for additional data file.


Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India.Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of 19 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed.A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119.The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.These data suggest that natural acquired antibody response is higher for PvMSP-1 Plasmodium vivax is the most widespread human malaria parasite and a major contributor to the malaria burden outside Africa, accounting for approximately 100 million cases each year  higher than children (age < 15 years). Odds of being seropositive to PvAMA-1 for malaria diagnosed person using PCR was almost 13 [OR (95% CI) 13.02 (6.52–22.50)] times more than the person who do not have malaria  out of ten factors showed association with seropositive to PvMSP-119 in bi-variable analysis. In multivariable analysis four predictors  were identified significantly associated with seropositivity of PvMSP-119. Odds of being seropositive to PvMSP-119 for adult (≥ 15 years) was almost two fold [OR (95% CI) 2.70 (1.25–5.85)] higher than children (age < 15 years). Odds of being seropositive to PvMSP-119 for malaria diagnosed person using PCR was also almost two [OR (95% CI) 2.44 (1.29–4.63)] times more than the person who do not have malaria. Odds of being seropositive to PvMSP-119 of those who experienced fever in last 2 weeks five times [OR (95% CI) 5.70 (2.19–14.84)] more than those who were free from fever in last 2 weeks. Odds of being seropositive to PvMSP-119 of those who experienced malaria fever in last 12 months was three times [OR (95% CI) 3.65 (1.48–9.03)] higher than those who did not experience malaria in last 12 months , microscopy (38.0%) and PCR (48.7%) was observed in screening of P. vivax parasite prevalence. Lower parasite prevalence by RDT was observed at all three sites as compared to microscopy and PCR. Higher seroprevalence or antibody titre against these two antigens was observed at Chennai and Nadiad where P. vivax prevalence is high as compared to Rourkela . Heterogeneity in seropositivity against these two antigens was observed at all three study sites and the overall seroprevalence for PvMSP-119 was higher (62.4%) as compared to PvAMA-1 (40.6%). A total of 35.9% individuals were seropositive for both the antigens and seropositivity was higher in Chennai (45.4%) and Nadiad (41.3%) as compared to Rourkela (21%). Whereas, 32.9% individuals were not showing any antibody response against PvAMA-1 and PvMSP-119 antigens, might have slower immune response which stimulated antibodies but not up to the detectable level. This study observed PvMSP-119 to be highly immunogenic when compared to PvAMA-1 at all three study sites and similar observation was reported in previous immune-epidemiologic study conducted in north India [P. vivax and P. falciparum antigens conducted at these three field sites and in Goa (Southwest India) also reported merozoites surface proteins as the most immunogenic antigen in P. vivax and reported apical membrane antigen as most immunogenic in P. falciparum [19 could be its conserved genetic nature, which were reported in many studies [Pvama-1 gene found to be highly polymorphic at these three study sites (unpublished data) and also in various previous studies [19 on the surface of infected RBCs until the end of the intracellular cycle [P. vivax infected individuals against the PvAMA-1 antigen at all three study population. Interestingly, in Rourkela, seropositivity to PvMSP-119 antigen was slightly higher in P. vivax uninfected individuals  as compared to infected individuals , suggest antibody response against the PvMSP-119 antigen once acquired due to cumulative exposure of the parasite over time persist for so many years [P. vivax or P. falciparum malaria indicates earlier exposure of malaria parasite can enhance the immune response. Another possibility is mixed infections and cross-reactivity between antibodies formed towards P. falciparum MSP-119 [P. vivax MSP-1 were highly associated with being seropositive to P. falciparum MSP-1, described by a previous study [19 antigen in Rourkela where P. falciparum in dominant species. However, cross reactivity between antibodies formed against recombinant PvAMA-1 and PfAMA-1 antigen is very limited [P. vivax is dominant species reported strong IgG response towards P. falciparum antigens [P. falciparum and P. vivax co-exist. In Chennai and Nadiad population antibody response against the PvMSP-119 antigen was higher in P. vivax infected individuals. Prevalence of P. vivax relapse infections in an area may provide protective immunity [In India, insight to naturally acquired antibody response to leading th India  and otheth India , 37–39.  PvAMA-1 .6%. A tod Nadiad .3% as coRourkela %. Wherea studies –47 and t and PCR .7% was o studies . Anotherar cycle . Antibodny years . In Rour MSP-119 , 57. Chaus study  and this limited  and thatd Nadiad .3% as coimmunity , however19 antigen was significantly higher in adults as compared to children at each study site which clearly indicate age acquired immunity. The immune response increases with age due to repeated exposure of malaria parasite [19 and PvAMA-1 antigens in individuals having low parasitaemia was higher (insignificant) as compared to individuals having high parasitaemia in all three study sites. This may indicate that parasitaemia cannot be correlated with antibody response and antibodies against blood-stage antigens are developed in most individuals living in malaria endemic areas [Antibody response was compared in children and adults from two sites Nadiad and Rourkela as Chennai had an insufficient number of children enrolled. The antibody response against PvAMA-1 and PvMSP-1parasite , 61. In parasite , 63, whi19. Whereas, only two factors age and malaria positivity (by PCR) were significantly associated with seropositivity to PvAMA-1. Seropositivity to PvMSP-119 and PvAMA-1 was higher in individuals who were having fever within last 2 weeks in Chennai, Nadiad and Rourkela and were malaria positive (by PCR), which clearly indicates that individuals with malaria infection naturally produce antibodies against these two antigens at each site resulted in higher antibody response. Individuals with past history of malaria in last 12 months recalled by memory were significantly associated with seropositivity to PvMSP-119 as compared to individuals who were not infected by malaria in last 12 months at each site, indicates that frequent infection increase the antibody response and boosted with subsequent attack of malaria infection [19 antigens in logistic regression analysis but omitted in multivariable analysis and larger sample size may be required to observe the association. Other factors such as symptomatic and asymptomatic malaria cases, parasitaemia, fever suppressants, and anti-malarial drugs were not significantly associated with seropositivity for both the antigens at three sites. This study has few limitations; low number of study participants and the study has lower number of children at each study site as compared to adults.Total ten factors which could be associated with seropositivity namely sex, age, malaria positivity, symptomatic and asymptomatic malaria cases, parasitaemia, fever, fever suppressants, anti-malarial drugs, past malaria infection and heterogeneous malaria transmission sites were evaluated. Four factors age, malaria positivity (by PCR), fever (within last 2 week) and past malaria infection (in last 12 months) were significantly associated with seropositivity to PvMSP-1nfection . Fever wP. vivax endemicity [19 is highly immunogenic and recognized more strongly in different geographical population. The varied antibody response observed between two antigens in the present study could be human genetic background, antigenic polymorphism, structural differences in the antigens, differential responsiveness and differences in P. vivax transmission in the study sites, which needs further investigation.Limited studies were conducted in the Asia–Pacific region to identify immune response in a geographical diverse population with different demicity –68. BasiP. vivax MSP-119 is highly immunogenic during natural infection in individuals living in three geographically diverse malaria endemic regions of India. There is heterogeneity in antibody response to PvMSP-119 and PvAMA-1 antigen among three populations. Results obtained here have implications for understanding human immunity to malaria antigens in different populations and could be helpful in malaria elimination programme and vaccine development.In conclusion, it is evident that


Plasmodium falciparum antigens is associated with partial malaria protection in sickle hemoglobin (HbS) children. However, this response has been more studied in children with heterozygous sickle cell trait (HbAS) but little explored in those with homozygous sickle cell trait (HbSS). The current study was conducted to determine the IgG responses against specific Plasmodium falciparum antigens in children with homozygous sickle cell trait (HbSS) by comparing to those with normal hemoglobin (HbAA).High Immunoglobulin G (IgG) response to P value <0.05 was considered statistically significant.A cross sectional study was conducted between April and July 2018 in Dar es Salaam tertiary hospitals. Parents were consented for their child to give about 5 ml of venous blood. IgG concentration from the blood plasma of 220 children (110 HbAA vs. 110 HbSS) were determined using indirect Enzyme Linked Immunosorbent Assay (ELISA). Then IgG medians were compared between the groups with prism 5 software (GraphPad) using Mann Whitney U test. Where the differences in age, hemoglobin levels and body weight between the groups was analyzed using independent sample t test. Multiple linear regressions were used to control cofounding variables such as body weight, age and hemoglobin level using statistical package for social sciences software (SPSS version 23). p < 0.0001 for all IgG). In both groups; age, body weight and hemoglobin level had no impact on the levels of IgG responses to Plasmodium falciparum antigens except for HbAA group which showed a significant increase in IgG against Pfg27 by 0.004 ng/ml with 1 g/dl increase in Hb level (p = 0.028).The median IgG concentration to PfEBA-175, Pfg27, yPfs28C antigens were HbSS; 20.7 ng/ml  vs. HbAA; 2.3 ng/ml , HbSS; 2.76 ng/ml (IQR: 2.08–5.69) vs. HbAA; 1.36 ng/ml (IQR: 1.28–1.76), and HbSS; 26,592 ng/ml  vs. HbAA; 14,164 ng/ml  respectively (Plasmodium falciparum IgG responses in children with homozygous sickle cell trait than those with normal hemoglobin.This study found significant higher levels of specific  In 2016Plasmodium falciparum is partially inhibited in the sickle cell hemoglobin red cells, but still malaria is the commonest cause of hemolytic crises where less than 2% of the sickle cell disease children survive beyond 5 years of age with malaria morbidity ranging from 50 to 80% [While the development of 0 to 80% .Each year, 300,000 children are born with sickle cell disease worldwide, 70% of them in sub-Saharan Africa . Sickle Evidence from studies have reported the proportions of malaria in children with HbSS and HbAS are much lower when compared to children with normal hemoglobin (HbAA) , 7. InnaPlasmodium falciparum immunoglobulin G (IgG) responses have been shown to correlate with clinical protection in children with HbAS [Moreover, recent findings suggest that malaria resistance in sickle hemoglobin (HbS) may also involve humoral immune responses , 6–8. Hiith HbAS –12.However, the role of IgG in HbSS is less characterized. Therefore, this study aimed at shedding more lights by determining the magnitude of malaria specific IgG responses in children with HbSS by comparing with HbAA genotype.A cross-sectional study was conducted from April to July 2018 at Muhimbili National Hospital (MNH), Mwananyamala Referral Hospital, Temeke Referral Hospital and Mloganzila Academic Medical Center (MAMC) found in Dar es Salaam region. All these hospitals conduct a weekly sickle cell clinic in collaboration with Muhimbili University of Health and Allied Sciences (MUHAS) sickle cell programe.Children aged between 5 and 15 years tested malaria negative at the time of recruitment were enrolled to participate in this study. Consented parents/legal guardians of children with HbSS were requested to bring their siblings who are tested and known to have HbAA. In addition to that, children with hemoglobin levels below 6 g/dl, those treated for malaria infection a month prior to enrolment, and children participating in malaria vaccine trial were excluded from participating in this study.A total of 220 children were enrolled (110 HbSS and 110 HbAA) in this study. The multistage sampling was employed to first stratify hospital clinics  then participants were randomly selected from each clinic.Hemoglobin level was measured photometrically by using Hemocue Ltd., where children with hemoglobin concentration between 11.5 g/dl to 17.5 g/dl were designated as normal, 6 g/dl-11.4 g/dl were designated as mild anemic and below 6 g/dl were termed as severely anemic .Plasmodium falciparum indirect ELISA.Screening for malaria was done by using rapid diagnostic tests (mRDT) . Microscopy was performed for confirmation of malaria infection. For microscopy, thin and thick blood films were air-dried, Giemsa-stained, and examined by light microscopy  using 1000× oil- immersion lens for quantification and species identification. A participant who’s Hb counted above 6.0 g/dL and found malaria negative by microscopy was requested to give 5 ml of a venous blood for A total of 5mls of whole blood were collected in an Ethylene-diamine-tetra-acetic acid (EDTA) tube , followed by centrifugation for 10 min at 1000–2000x g using a centrifuge. The resulted supernatant (plasma) was transferred into a clean polypropylene tube using a Pasteur pipette. Then samples were maintained at 2–8 °C while handled. Where plasma was not analyzed immediately was stored at − 80 °C or lower.2SO4 . Absorbance was measured as optical density (OD) at 450 nm .Indirect ELISA was performed as explained in a study by Akpogheneta et al . BrieflyStandard curve was generated from a serial 5-fold dilution of purified human IgG starting at 1.0 ng/ml  and all samples were simultaneously assayed in duplicate.p value < 0.05 was considered statistically significant.Data from case report forms (CRFs) were recorded on Microsoft excel. In addition to that, data from the ELISA reader were exported directly into excel then to statistical package for social sciences version 23  and Prism 5 software  for coding and performing statistical analysis. IgG responses were compared between the different groups with Prism 5 software by using Mann Whitney U test. Where the differences in age, hemoglobin levels and body weight between the groups was analyzed using independent sample t test. Multiple linear regressions were used to control cofounding variables such as body weight, age and hemoglobin level using SPSS version 23. A two-tailed Plasmodium falciparum antigens. Additionally, children above 5 years were assented before enrolling them in this study. Children tested malaria positive and found anemic their results were immediately communicated to their attending physician for further management.Ethical approval was obtained from Muhimbili University of Health and Allied Sciences, Senate subcommittee for Research and Publications (MUHAS). A written informed consent was obtained from parents of all children for giving venous blood about 5 ml and test for IgG against specific p = 0.031. The mean body weight (±SD) of the participants was 24.9 ± 10.2 kg. Underweight was more prevalent in children with HbSS 22.1 ± 7.3 kg than in their counterparts 27.6 ± 11.8 kg, p < 0.0001. The mean hemoglobin level (±SD) of the participants was 10.1 ± 2.6 g/dl. Anemia was predominantly in children with HbSS 8.2 ± 1.5 g/dl than children with HbAA 12.0 ± 1.9 g/dl, p < 0.0001  of the participants was 9 ± 3.2 years. Children with HbSS were significantly younger 9. ± 3.1 years than HbAA whose mean age was 10 ± 3.2 years, P < 0.0001  compared to children with HbAA 2.3 ng/ml , p < 0.0001  compared to HbAA 1.36 ng/ml (IQR: 1.28–1.76) group, Higher levels of IgG responses were observed in HbSS than in HbAA, the median levels.of IgG responses to yPfs28C antigens were 26,592 ng/ml  compared.p < 0.0001  in HbAA, p = 0.028)  Table .Table 2TTo the best of our knowledge, this was the first study in our settings undertaken to determine the magnitude of IgG levels directed against PfEBA-175, Pfg27 and yPfs28C in children with homozygous sickle cell trait and those with normal hemoglobin.The magnitude of malaria remains high and affects up to about 40% of the world’s population with around 300–500 million people currently infected and mainly in the tropics –24, 28. This study has therefore shaded more light in understanding how the natural malaria protection (IgG levels) can be affected by differences in hemoglobin shape. Blood stage antigens PfEBA-175, Pfg27 and yPfs28C, which are also candidates for malaria vaccine, were employed to explore the differences –29.Plasmodium falciparum merozoites enhance the binding of parasites to the surface of a red blood cell during merozoites stage [PfEBA-175, a blood stage malaria vaccine candidate and a parasite ligand at the apical end of the es stage . Antibodes stage .The current study found the median IgG antibody responses to PfEBA-175 were lower 2.29 ng/ml (IQR: 1.21–3.04) in HbAA than 20.68 ng/ml (IQR: 18.1–25.64) in HbSS. These findings correlate from those reported by Howaida et al. which found an increased mean IgG level of IgG 13.3 pg/ml in children with sickle hemoglobin versus 4.5 pg/ml normal children . In addiP. falciparum and parasites lacking its gene fail to develop sexually [Pfg27, a gametocyte-specific protein is expressed early during sexual differentiation, this protein plays a crucial role in the sexual development of sexually . This stPfs28 a sexual malaria vaccine candidate which has shown to block malaria transmission in gut by preventing ookinete development during sexual stage . IgG agaThis study also found no association between age, body weight and hemoglobin and increase or decrease in IgG response against PfEBA-175, Pfg27 and yPfs28C in both HbSS and HBAA children except for HbAA children showed a significant increase in IgG against Pfg27 by 0.004 ng/ml with an increase in Hb levels. These findings differ from the study, which found a significant association between an increased IgG levels versus weight and hemoglobin level. IgG levels in this study increased with an increase in Hb levels and weight. These indicated that other factors such as weight, Hb levels apart from sickle cell type may also influence the increase in IgG, however the current study only found an association between Hb level and IgG against Pfg27 in HbAA where there was an increase of 0.004 ng/ml with an increase in Hb levels, the discrepancy may have been contributed by other factors such as use of different antigens, geographical area and study population –33.Furthermore, increased IgG levels in sickle cell individual have been associated with partial malaria protection; hence, treatment of individual with malaria, regardless of their sickle cell status, is recommended.This study was limited by unavailability of serum from a person who has never been exposed to malaria to define a cutoff for positive and negative response to each antigen. To mitigate this Ig concentration from both HbSS and HbAA were explored from the standard curve and compared.In addition to that, the conclusion of the findings from this study is limited to HbSS and HbAA children because HbAS were not included in this study.Plasmodium falciparum parasite antigens is more enhanced in children with HbSS than HbAA.In conclusion, this study found that humoral response (IgG) against specific Plasmodium falciparum and implications for vaccine development.This study recommends further study to characterize IgG subclasses against PfEBA-175, Pfg27 and yPfs28C. In addition to that, this finding suggests further study to be conducted to rule out the influence of hemoglobin polymorphism in natural immunity against


PfHRP2 based RDT, makes the management of febrile illnesses more challenging. This study aimed to assess the effect of the use of malaria RDT on antimicrobial prescription practices.Malaria rapid diagnostic tests (RDT) are widely used in endemic areas in order to comply with the recommendation that malaria treatment should only be given after the clinical diagnosis has been confirmed by RDT or microscopy. However, the overestimation of malaria infection with the use of A prospective study was conducted among febrile children under-5 years of age attending four health facilities and the referral hospital in the Nanoro Health District (Burkina Faso). To assess the effect of malaria RDT testing on the prescriptions of antimicrobials in febrile children, the initial diagnosis and antimicrobial prescriptions following a malaria RDT testing were recorded. The necessity of these prescriptions was subsequently checked by assessing the actual cause of fever by expert malaria microscopy and a microbiology analysis of blood, urine, stool and nasopharynx swabs that were collected from febrile cases to determine the actual cause of the fever episode.Malaria was diagnosed by nurses, who are the primary health care providers, with a malaria RDT in 72.7% (798/1098) of febrile children, but only 53.7% (589/1097) cases could be confirmed by expert microscopy. Health care workers were likely to prescribe antimalarials to malaria positive RDT compared to malaria negative RDT . Malaria negative RDT result had a significant influence on the antibiotic prescriptions . The risk of prescribing antimicrobials was higher in health facility level compared to referral hospital. By cross-checking of laboratory findings to antimicrobial prescriptions, an important part of children with positive bacterial infection have received antibiotic prescriptions although the majority without any infection have also received antibiotics.Despite the good attitude of health care workers to adhere to diagnostic test results, antimalarials and antibiotics remain inappropriate prescribed to febrile children. The low specificity of malaria RDT used could be an important cause of these practices. Acute febrile illnesses in children are globally one of the most common reasons to seek medical care and are associated with considerable morbidity and mortality , 2. HowePlasmodium falciparum histidine-rich protein-2 (PfHRP2), have a specificity problem and may result in a significant number of false positive test results when compared with the golden standard, expert microscopy  > Log10 or ratio > 2 in case of parasite density ≤ 400/µl and > 400/µl respectively), a decisive reading was performed by a third expert.Expert malaria microscopy on thin and thick blood smears was performed by two independent expert microscopists and in case of discordance , incubated in a BACTEC 9050 instrument (Becton–Dickinson) for a total of 5 days. In case of a positive growth signal a Gram straining was done and subsequent culture was performed at 35–37 °C for an additional 18–24 h.Salmonella and Shigella agar (SS agar). In addition, SD Bioline Rota/Adeno RDT  was used to detect Group A rotavirus or adenovirus serotype 40/41 in stool samples. Finally, fresh stool samples were used for parasitological examination for the presence of cysts, eggs, and vegetative forms of protozoa by microscopy. In case of suspicion of cysts, a second slide was prepared using lugol for confirmation.Stool cultures were performed on Eosin-Methylene Blue (EMB) agar (for children less than 2 years of age), Hektoen agar and inoculated in selenite of sodium broth. The culture media were incubated at 35–37 °C for 18–24 h, and the selenite broth at 35–37 °C for 4 h before putting in culture on Urine samples were tested with a dipstick  for the presence of leucocytes and nitrite. If positives for these parameters, a culture of the sample was done on Cystine Lactose Electrolyte Deficient (CLED) and EMB agar and incubated at 35–37 °C for 24 h.Streptococcus pneumoniae and Staphylococcus aureus species . Bacterial isolates from all cultures were identified by standard microbiological methods . The characteristics of study population are presented in Table A small percentage of the febrile children who received antimalarial prescription got first an injectable antimalarial (artesunate or artemether) followed by artemisinin-based combination therapy (artemether–lumefantrine or artesunate–amodiaquine) for subsequent home treatment 2.11% (17/805). The majority of malaria treatment 97.88% (788/805) was artemisinin-based combination therapy as previously mentioned. For the antibiotic prescriptions, 14.58% (125/857) and 1.17% (10/857) have received respectively 2 and 3 antibiotic prescriptions, and 84.24% (722/857) received a single prescription. All antiparasitics were single prescriptions . However, if malaria expert microscopy is considered as gold standard, the risk for febrile children tested with RDT-PfHRP2 to have their initial antimalarial prescription affected (modified) was statistically significant . Moreover, the likelihood of antibiotic prescription in case of a negative malaria RDTs was 3 times higher compared to positive malaria RDTs and statistically significant .Table It is apparent from Table By cross-checking the laboratory findings  with the antimicrobials prescribed by health care workers based on the routine practice , it is evident that a large part of the febrile children who received an antibiotic prescription did actually not need such a treatment. It was that at the rural health facilities all children with a positive bacterial bloodstream infection (bBSI) 25/25) or urinary tract infection (UTI) (8/8) and 80.39% (41/51) with bacterial gastro-intestinal infection (bGII) based to laboratory results did actually receive antibiotic prescriptions. But also 93.98% of the febrile children without any infection (confirmed by laboratory testing) in the present study too actually received antibiotic prescriptions. In contrast, at the referral hospital only 75% (30/40) of children with positive bBSI, 64.28% (9/14) with bGII and 100% (3/3) with UTI did actually receive antibiotic prescription. Moreover, 86.04% (74/86) of febrile children without (laboratory confirmed) infection did also got antibiotic prescriptions  in the current study area [A rapid diagnostic test based on the detection of udy area . This loudy area , 26–28. udy area . Due to udy area –16. Furtudy area , it is mThe inappropriate prescription of antibiotics and antimalarial treatments observed in the present study could be a consequence of the rightful fear of health care workers to miss a treatable bacterial infection. Indeed, a significant number of potentially treatable etiologies were missed in the present study and others , 30. DesAntiparasitics drugs were not much prescribed in this study. Based on the laboratory findings, there was a high under-prescription of antiparasitics as actually a significant number of febrile children who actually had gastro-intestinal parasites did not receive appropriate treatment. Most likely, the more obscure symptoms associated with a parasitic gastro-intestinal infection, which in most cases do not cause fever, are probably overlooked in favor of other febrile etiologies that are more readily diagnosed.Despite the recommendation of the WHO to treat all children with a positive malaria RDT or microscopy result, few cases (4.52%) of febrile children who had a malaria positive RDT did not receive antimalarials. The reason for this non adherence was not assessed in the present study. Possibly, complementary information on previous drug use provided by the parents/guardians to the attending nurses suggested that these children did not require antimalarial prescriptions and that the RDT were positive in these cases was interpreted as a consequence of persisting HRP2 antigen (up to 4 weeks or even more) after successful treatment for malaria –16. In cStaphylococcus aureus and Streptococcus pneumoniae, a relation of their presence with ongoing fever has not been established [A possible limitation of our study could be the lack of confirmation of pneumonia cases presumptively diagnosed by health care workers. Although the nasopharynx of some children was colonized by common bacterial pathogens, such as ablished . Therefoablished , 33, 34.ablished , 35, 36,ablished –39.Despite the correct attitude of health care workers to treat malaria according to a diagnostic test result, antimalarials and antibiotics are still inappropriately prescribed by nurses. The low specificity of the malaria RDT used and the absence of practical tools to diagnose bacterial infections could be important causes of this inadequate prescription practice. In addition, the fear to delay antibiotic treatment or to overlook a treatable bacterial infection, leads to the inappropriate prescription of antibiotics. The health system is likely to deal with the problem of inappropriate prescription if appropriate diagnostic tools are developed and implemented.


Reports on malaria and HIV coinfections in exposed infants from tropical countries arescarce.The case of a 2-month-old HIV-exposed Nigerian infant who presented with intermittentfever at a Nigerian tertiary hospital is reported. The rarity of the case and thechallenges associated with making the diagnosis informed our decision to report thecase.Diagnosing malaria in HIV-exposed infants in early infancy requires a high index ofsuspicion, good knowledge of the clinical presentation, and appropriate microbiologicalinvestigations for sepsis and malaria. Further studies need to be conducted on theassociation between malaria and HIV exposure. Malaria is rare in children aged below 3 months and considered an unlikely infection.Malaria should be considered as a possible cause of fever in HIV-exposed infants aged<3 months.Screening for malaria should be routine in HIV-exposed febrile children aged <3months.2 It is however rare in infants aged less than 6 months in malaria endemic settings. Werecorded this case of malaria in an HIV-exposed 2-month-oldinfant. The reasons adduced for the rarity of malaria in infants aged less than 3months include the protective placental barrier and passive transfer of antimalariaantibodies from the mother to fetal circulation across the placenta in semi-immune mothers.4 Another factor hindering the survival of malaria parasites in breastfed infantsinclude the deficiency of para-amino benzoic acid in breast milk, which is needed formalaria parasite nucleic acid metabolism. Fetal hemoglobin also inhibitsgrowth and survival of malaria parasites.3 The current Nigerian guidelines for management of HIV and previous research studieson this subject suggest that malaria is rare in HIV-infected or exposed infants aged below 6 months.47 The purpose of this report is to discuss the clinical presentation of malaria in anHIV-exposed infant and provide information that may aid diagnostic acumen and proper casemanagement. This report is also written with a view to canvass for better planned studies onthe association between malaria and HIV exposure or infection in infants.Malaria is a common cause of morbidity and mortality in Nigerian children.or history suggestive of dysuria in the patient.Neither was there a cough, rhinorrhea, or dyspnoea. There was no history suggestive ofprolonged rupture of membrane, chorioamnionitis, or maternal infections around the deliveryperiod. The infant had never been transfused with blood or blood products.The mother of the patient was a 38 -year-old nursing officer who wasdiagnosed to be HIV infected 16 years previously. Mum had adhered to her medications sincehighly active antiretroviral therapy (HAART) was initiated 7 years prior to the admission ofthe baby. The father, a 50-year-old accountant, was also HIV infected and had also initiatedHAART. The family setting is monogamous and the child is the second in a family of 3.A 2-month-old HIV-exposed male Nigerian infant presented at the pediatric antiretroviralclinic in the company of his mother for follow-up. His mother complained of fever of a weekduration occurring every 2 to 3 days, with the child appearing unwell when febrile and wellwhen afebrile. An episode of nonprojectile vomit containing recently ingested milk formulawas associated with this fever. There was no history of irritability, excessive crying,jaundice, reduced feeding, The index patient was delivered at term by elective cesarean section with a birth weight of2.8 kg and there was no history suggestive of birth asphyxia. The infant was initiallybreastfed for 2 weeks and thereafter fed with infant formula because the mother could notbear her neck pains due to cervical spondylosis. Nevirapine prophylaxis was administeredfrom birth for the first 6 weeks of life. Co-trimoxazole prophylaxis was then commenced atthe age of 6 weeks.3. Thedifferential lymphocyte and neutrophils counts were 84% and 16%, respectively. Blood filmfor malaria parasite screening was not done because malaria was not entertained. Bloodinvestigations for HIV by polymerase chain reaction were negative.General examination at presentation revealed an afebrile male infant with a temperature of37.5°C and a weight of 3.6 kg. He was pale but not jaundiced or cyanosed. He was wellhydrated and systemic examination revealed a heart rate of 144/minute with normal heartsounds. Both hepatic and splenic enlargement of 4 cm each were detected on abdominalexamination. No abnormalities were detected on examination of the respiratory or centralnervous systems. A diagnosis of sepsis was entertained and the patient was admitted andtreated with intravenous cefuroxime before complete blood count investigation. Feverresolved 2 days after commencing antibiotics and the patient was discharged home on oralcefuroxime. Complete blood count investigation conducted at the age of 2 months revealed apacked cell volume of 29% and total white cell count of 9800 mmPlasmodium falciparum in theperipheral blood film following microscopy. The infant was readmitted and treated with oralquinine. A remarkable improvement in his condition was thereafter noted with theintermittent fever resolving by crisis within 24 hours of administration of quinine. Thepacked cell volume of 26% at readmission increased to 32% on the fourth day of quininetherapy. The child remained well and was discharged home 2 days after.The mother re-presented a week later with complaints of fever recurring every 2 to 3 days.Examination findings were similar to those observed before discharge. The unresolved feverpattern was not reported on time by the mother because she felt that the fever would abatewith cefuroxime administration. A diagnosis of malaria was therefore entertained, which wasconfirmed after detection of trophozoites of P falciparuminfection and decreased maternal antimalaria specific antibodies.8 Early cessation of breast feeding and intake of infant formula at the age of 2 weekscould have further contributed to the predisposition of the index case to malaria. Breastmilk is deficient in para-amino benzoic acid which is needed to inhibit the thriving malaria parasites.3 The present case also shows that the antimalarial efficacy of cotrimoxazole is not 100%,10 due to the fact that tertian or quartan fever persisted from the sixth to the eighthweek of life of this infant despite adherence to the daily cotrimoxazole prophylaxis fromthe sixth week of life.The diagnosis of malaria in a 2-month-old infant of an HIV-infected mother resident in amalaria endemic setting is unusual. The HIV infection in the mothers could have probablypredisposed this infant to congenital malaria. Studies examining the association betweenmalaria and HIV exposure in children are scant, but a previous study has shown that maternalHIV infection was associated with increases in the cord blood P falciparum or malariae infection. Detection of Pfalciparum parasites in the peripheral blood film confirmed malaria infection inthe case reported. Clinical recovery to quinine monotherapy further supports the diagnosisof malaria. Sepsis is a common differential for febrile infants at the age of 2 months andit was considered as the most likely cause of fever in this infant, thus necessitatinginitial use of antibiotics only. Undue fixation of sepsis as the only differential of feverin infants may lead to a delay or missed diagnosis of malaria. This underscores theimportance of a good clinical acumen and high index of suspicion.The onset of tertian or quartan fever in a malaria endemic region is highly suggestive ofConfirming or ruling out sepsis can be a herculean task in resource restricted settingswhere diagnostics can be quite expensive and this may lead to being selective in carryingout tests indicated in screening for sepsis. Complete blood counts and blood film formalaria examination costs approximately US$5, while blood cultures costs as much as US$15 inour setting. Thus, selective and sequential running of tests is often the practice for thepurpose of cost-effectiveness, but the disadvantage lies in the consequences of delayed ormissed diagnosis of diseases. The lesson of this report is to extend the pragmatic practiceof examining blood films for malaria parasites in infants presenting with fever in malariaendemic communities to HIV-exposed newborns. Additional studies are required to explore theassociation between malaria and HIV in exposed infants.


Plasmodium falciparum isolates in uncomplicated and severe malaria infections may have different parasite genetic profiles. This study was conducted to assess differences in genetic diversity and allelic frequencies in P. falciparum isolates according to malaria severity and age of patients in the Gublack area, northwest Ethiopia.Malaria infection can present with a wide variety of symptoms, ranging from mild to severe. P. falciparum malaria patients with microscopically-confirmed infection were enrolled. Parasite DNA was extracted from filter paper blood spots and the polymorphic regions of the msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR with fragment analysis by gel electrophoresis.Cross-sectional health facility-based study conducted in Gublak, Ethiopia between July, 2017 and October, 2017. Symptomatic msp-1, the K1 allelic family was similarly prevalent in uncomplicated 42 (44.2%) and severe disease 12 (52.2%). In msp-2, FC27 was detected in 55 (57.9%) of uncomplicated infections and IC/3D7 in 14 (60.9%) of severe infections. 76 (64.4%) of the 118 isolates contained multiple genotypes; 56 (58.9%) in uncomplicated infections and 19 (82.6%) in severe infections. The overall of multiplicity of infection was 2.2 (95% CI 1.98–2.42) with 1.4 (95% CI 1.23–1.55) and 1.7 (95% CI 1.49–1.86) for msp-1 and msp-2, respectively. Multiplicity of infection was significantly higher in severe than uncomplicated infections (3.0 (95% CI 2.61–3.47) versus 2.0 (95% CI 1.83–2.23), respectively, p = 0.001). There was no difference in multiplicity of infection across age groups (p = 0.104).A total of 118 patients were enrolled including 95 (80.5%) with uncomplicated infection and 23 (19.5%) with severe disease. In P. falciparum genotypes and malaria severity in different malaria transmission areas.Patients with severe malaria were more likely to have multiclonal infections. Further studies are needed to describe the association between  Severe malaria is a major public health problem. Globally about 3.3 billion people live in malaria endemic areas, despite improvements in the implementation of effective control measures. Each year, there are an estimated 219 million clinical malaria cases and 435,000 deaths worldwide, the majority of which occur in sub-Saharan Africa [Plasmodium species that cause human malaria, Plasmodium falciparum is the main cause of severe malaria and malaria-associated deaths [P. falciparum than other malaria species and its propensity to sequester in capillaries leads to increased morbidity [Of the d deaths . Severe d deaths . The inforbidity , 7. HoweP. falciparum isolates using genetic polymorphisms as markers [msp) genes are the most commonly used markers of P. falciparum due to their high polymorphism. MSP-1 and MSP-2 are P. falciparum blood-stage malaria vaccine targets [P. falciparum parasite sub-populations. MSP-1 is major surface protein encoded by msp-1 on chromosome 9, which contains 17 blocks of sequences flanked by conserved regions. It plays a major role in erythrocyte invasion [msp-1, is grouped under three allelic families of K1, MAD20 and RO33 [msp-2 gene located on chromosome 2 and is composed of five blocks of which the central block is the most polymorphic. The msp-2 block 3 alleles are grouped into two allelic families, FC27 and IC3D7 [P. falciparum are important to determine diversity and multiplicity of P. falciparum infection [P. falciparum populations and MOI vary according to the intensity of transmission, outcome of infections and age in different geographical regions [Numerous studies have characterized strains of  markers –11. Mero targets  and are invasion  and is tinvasion . Block 2and RO33 . MSP-2 ind IC3D7 . Genotyp regions . In area regions , 18.P. falciparum genotypes have been associated with severe malaria in epidemiological studies [P. falciparum isolates from patients with severe malaria [P. falciparum in Ethiopia [msp-1 and msp-2 genes from isolates of patients with severe malaria in Ethiopia is important to obtain knowledge about parasite-factors associated with virulence in Ethiopia. This study aims to explore whether P. falciparum genetic diversity and multiclonality are associated with disease severity and age of patients in Gublack area, northwest Ethiopia.Some specific  studies . Severe  studies  and mult studies . In comp malaria , 21. OnlEthiopia –24. ThesP. falciparum is the predominant species and Anopheles arabiensis is the major vector. The Benishangul-Gumuz region had the highest risks of malaria compared to other regions, and also malaria burden is higher in-migrant labourers coming from nearby highland areas [A cross sectional health facility-based study was carried out in Gublak Health Center, Dangure district, Benishangul-Gumuz Regional State northwest Ethiopia Fig. . The stund areas , 26. Malnd areas . The aveP. falciparum infected blood samples were collected from children and adults presenting to the Gublak Health Center and enrolled into the study from July, 2017 to October, 2017. Patients were classified into uncomplicated and severe malaria according to World Health Organization (WHO) criteria [A total of 118 criteria . The inccriteria .®, Angelholm, Sweden). The blood was spotted onto Whatman 903® filter paper , air-dried and individually placed into a plastic bag with silica gel. These dried blood spots were transported and stored to the Malaria Research Laboratory  at Addis Ababa, Ethiopia.Capillary blood was obtained from a finger prick from each patient and used to prepare thick and thin blood smears. Thick and thin blood smears were air dried and stained with 10% fresh Giemsa for 15 min. All slides were examined by two independent laboratory technicians to determine malaria species and the parasite density. In case of discordance the slides were read by a third laboratory technician. The density of parasitaemia was expressed as the number of asexual parasites per microlitre of blood, assuming a leukocyte count of 8000/µL of blood . Blood s® (Bio-Rad Laboratories CA) method [P. falciparum merozoite surface protein 1 (msp1) (block2) and 2 (msp2) (block 3) was carried out as previously described [Parasite DNA was extracted from the blood spots collected on filter papers using the Chelex-100) method , with a escribed . Monoclomsp-1 and msp-2 allelic frequencies were calculated. The mean multiplicity of infection (MOI) was calculated for msp-1, msp-2 and overall. The mean MOI was compared between uncomplicated and severe malaria cases using the non-parametric Mann–Whitney U test. The proportion of alleles observed at each locus within each group were compared using Chi-square test statistic. Spearman’s rank correlation coefficient was used to assess the relationships between continuous variables. A p-value less than 0.05 was considered significant.Data were entered and analysed using SPSS version 20 . The The study received ethical approval from the Scientific and Ethical Review Office (SERO) of the Ethiopian Public Health Institute (EPHI). Written consent was obtained from the patients and guardians, and malaria positive individuals were treated in accordance with the national malaria guidelines .P. falciparum patients of whom 95 (80.5%) had uncomplicated malaria and 23 (19.5%) severe malaria. The major clinical manifestations of the patients are shown in Table The study population comprised 118 microscopically confirmed P. falciparum were genotyped using MSP1 (block 2) and MSP2 (block 3) by nested PCR and the magnitude of alleles were classified based on their fragments size  and IC/3D7 type was 49.2% (58/118).A total of 180 samples positive for P1 block  and MSP2msp-1 and msp-2 allelic families in uncomplicated and severe malaria patients is presented in Table msp-1 and msp-2, respectively. In severe malaria patients, a total of 11 different alleles were detected for msp-1 and 13 different alleles for msp-2. Of the 118 P. falciparum isolates, 76 (64.4%) harboured more than one parasite genotype . The mean MOI was highest  among the youngest age groups (< 5 years old) (Table p = 0.104). The youngest age group had the highest mean parasite density  compared to that of older age groups  (Table p = 0.09).The overall mean multiplicity of infection was 2.2 (95% CI 1.99–2.42). The mean MOIs for uncomplicated and severe malaria patients were 2.0 (95% CI 1.79–2.23) and 3.0 (95% CI 2.4–3.59), respectively, which was statistically significant  of patients with multiclonal infections, which is in line with the previous studies from Ethiopia  and SudaP. falciparum infection in individuals with severe malaria [The mean number of circulating genotypes in severe malaria patients was higher than from patients with uncomplicated malaria. Similar results have been observed among patients with severe malaria in Uganda . Studies malaria . These dAge is a key factor involved in the acquisition of immunity against falciparum malaria and has been found to influence MOI . In turnBed nets are an important tool for prevention of the malaria vector and are widely used in malaria endemic areas of the Ethiopia. Reduced MOI has observed to be associated with increased ITN use, which is consistent with being an indicator of transmission intensity . The preP. falciparum alleles and disease severity [Consistent with previous studies, the current study found no association between the frequency of specific severity , 35. Limseverity . In addiP. falciparum infections in severe malaria isolates in Gublack, North West Ethiopia. While the MOI was highest among young children, there was no significant difference between MOI amongst age groups. The high MOI and diversity of P. falciparum populations in this region could be related to the low utilization of the ITNs. These findings suggested that implementation of customized tools to prevent malaria transmission, by targeting migrant laborers, which substantially improves malaria control in the region. Further longitudinal studies on malaria severity with larger sample sizes in different malaria endemic areas of the country are needed to confirm whether severe infections are more commonly multiclonal across the country.The present study demonstrates a high multiplicity of Additional file 1: Figure S1a. Gel electrophoresis of K1 MSP-1 allelic families’ lane 1: 100 bp DNA ladder; lane 2: negative; lane 3: 130 bp; lane 4: 300 bp; lane 5, 6, 7: negative; lane 8 and 9: 280 bp; lane 10: negative control. Figure S1b. Gel electrophoresis of MAD20 MSP-1 allelic families’ lane 1: 100 bp DNA ladder; lane 2: 150 bp; lane 3: 130 bp; lane 4: negative; lane 5: 180 bp; lane 6: 130 bp; lane 7 and 8: negative; lane 9: 180 bp; lane 10: 150  bp. Figure S1c. Gel electrophoresis of RO33 MSP-1 allelic families’ lane 1: 100 bp DNA ladder; lane 2 and 3: 150 bp; lane 4: 180 bp; lane 5: negative control; lane 6 and 7: 150 bp.Additional file 2: Figure S2a. Gel electrophoresis of FC27 of MSP-2 allelic families, lane 1: 100 bp DNA ladder; lane 2: 400 bp; lane 3: 350 bp; lane 4: 350 bp; lane 5: 380 bp; lane 6: 380 bp; lane 7: 320 bp; lane 8: negative; lane 9: 300  bp; lane 10: negative; lane 11: negative control. Figure S2b. Gel electrophoresis of IC/3D7 MSP-2 allelic families’ lane 1: 100 bp DNA ladder; lane 2: 400 bp; lane 3: 380–500 bp; lane 4: negative; lane 5: 380–520 bp; lane 6: 380 bp; lane 7: 300 bp; lane 8: 300 bp; lane 9: negative control.


Across millennia of coevolution, malaria parasites have mediated their human hosts’ patterns of settlement, authored their historical milestones, and shaped their genome. The human genome harbors archived responses to the pressure of severe, life-threatening malaria, and chief among these are innate variants of erythrocytes, which serve as principal cellular hosts for the parasite. Since JBS Haldane first speculated that red blood cell (RBC) variants may confer some resistance to malaria, numerous studies using diverse approaches have identified an array of innate variants, which can be grouped as disorders of the erythrocyte cytoskeleton , variation in erythrocyte surface antigens , enzymatic aberrations , and mutants of α- or β-globin proteins, either as deletions  or point mutations . To varying degrees, each of these variants of erythrocytes have been selected over generations owing to enhanced fitness when human populations are exposed to intense malaria transmission.These variants can provide striking protection: as one example, in African children, heterozygosity for sickle Hb reduces the risk of severe, life-threatening malaria by over 90% . This deAnopheles spp. mosquitos and is caused by five species of Plasmodium spp. parasites—Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum and P. vivax are distributed most widely throughout the tropics and cause most human disease. P. vivax is very rare in most of Africa because most Africans lack expression on their erythrocytes of the Duffy antigen receptor for chemokines (DARC), which enables the efficient invasion of red cells by P. vivax. Globally, P. falciparum is the most common and the deadliest species, and it kills over 400,000 people annually, mostly children in sub-Saharan Africa.Malaria is transmitted between humans by Table 1). Hb is formed by a tetramer of two α- and two β-globin proteins; mutations of Hb result from either deletion of globin gene copies  or in coding point mutations in β-globin. A wide variety of point mutations in β-globin have been described, and most important for malaria protection are the substitutions of glutamate at position 6 for either valine (to produce HbS) or lysine (HbC). The inheritance of two mutated β-globins (as either HbSS or HbSC) produces sickle cell anemia and significant multisystem morbidity; in contrast, the inheritance of either sickle-cell trait (HbAS) or homozygous HbC (HbCC) produces little morbidity but substantial protection.After a short stage in hepatocytes, the principal target in humans is the erythrocyte, which serves as the primary platform for parasite propagation. Consequently, variants of erythrocytes are the principal known means by which humans have developed protection, most notably through mutations in Hb , uncomplicated malaria (31%), and asymptomatic parasitization (none) [Interestingly, both HbAS and HbCC specifically protect children from severe disease, suggesting that they attenuate the specific pathogenic mechanisms of either the parasite or host while allowing children to be infected with malaria 9%, uncomp malaria 1%, and aNumerous in vitro studies have reported the effects of Hb variants on parasite cellular phenotypes. Generally, in vitro studies have rejected the hypothesis that parasites are unable to invade hemoglobinopathic RBCs: invasion has been reported as normal for HbAS , HbCC 55, HbAC , as wellP. falciparum erythrocyte membrane protein 1 (PfEMP1), repetitive interspersed families of polypeptides (RIFIN), and subtelomeric variable open reading frame (STEVOR). This cytoadherence allows iRBCs to sequester in the microvasculature and avoid splenic clearance, attenuate immune activation [P. falciparum’s ability to interact with extracellular ligands contributes significantly to their protection from severe malaria.Cellular mechanisms of pathogenesis are principally governed by interactions between infected RBCs (iRBCs) and extracellular ligands on host cells, including endothelium, leukocytes, and uninfected RBCs; these interactions are enabled by the expression of parasite-derived proteins on the RBC surface, including the hypervariable protein families tivation , and acttivation . In vitrtivation , 17. Thetivation . Intrigutivation , mirroritivation . These ativation . Taken tP. falciparum exports a large number of proteins to the iRBC surface, and a principal component of the export machinery is Maurer’s clefts, the parasite-derived Golgi-like membranous structures that serve as transport hubs for PfEMP1 and other exported proteins. As the parasite matures in the RBC, these Maurer’s clefts are conveyed along a scaffold of host-cell actin that the parasite has repurposed to assemble a transport network; in HbCC and HbSC RBCs, Maurer’s clefts are dysmorphic, the actin assemblage is disorganized [Box 1).As noted above, rganized , Maurer’rganized , and prorganized . Some ofrganized . In a serganized , suggestHow does reduced PfEMP1 expression and cytoadherence modify the effects of hypoxia-induced arrest of maturation in HbAS RBCs?Do host miRNAs influence the quality or quantity of surface antigen expression or cytoadherence in HbAS RBCs?How do β-globin variants disrupt the assembly of the actin cytoskeleton and Maurer’s clefts during ring-stage development?Do β-globin variants attenuate the interaction of infected RBCs with host immune cells?P. vivax malaria. As noted above, many Africans are protected from P. vivax because the absence of DARC on the erythrocyte surface prevents RBC invasion by P. vivax. This model of protection enabled the identification of the parasite-derived binding partner for DARC: P. vivax Duffy binding protein (PvDBP). In populations exposed to P. vivax, naturally occurring antibodies to PvDBP are associated with protection from vivax malaria (in DARC-positive people), and this apparent functional protection has prioritized PvDBP as an advanced candidate antigen for vivax-specific vaccination strategies [P. falciparum malaria. Epidemiologically, pregnant women are at high risk in endemic areas for falciparum malaria but develop over successive pregnancies acquired immunity to its consequences, including placental malaria. Placental malaria results from interactions between chondroitin sulfate A in the syncytiotrophoblast and a parasite-derived surface protein VAR2CSA; this essential interaction, and the model of acquired immunity to it, enabled the identification of functional antibody responses that attenuate placental malaria. Currently, two early-phase vaccines are being developed to generate responses directed against VAR2CSA and protect women and their offspring from placental malaria.Just as pathogen drug-resistance phenotypes furnish a model by which to better understand drug mechanisms of action, human disease resistance enables us to more efficiently explore pathogen mechanisms of disease and thereby identify targets for intervention. There are two models for this approach. The first model relates to rategies . The secAdaptive evolution in human populations has furnished to us natural strategies by which to neutralize malaria parasites in the form of erythrocyte variants. By using the protective phenotypes to infer essential mechanisms of disease, we can rationally develop new approaches to preventing and treating this ancient and immiserating disease.


Plasmodium species. This study sought to determine the distribution of non-falciparum malaria and assess the performance of diagnostic tests for Plasmodium falciparum in Western and Southern Provinces of Zambia, two provinces planned for early malaria elimination.Zambia continues to make strides in reducing malaria burden through the use of proven malaria interventions and has recently pledged to eliminate malaria by 2021. Case management services have been scaled up at community level with rapid diagnostic tests (RDTs) providing antigen-based detection of falciparum malaria only. Key to national malaria elimination goals is the ability to identify, treat and eliminate all A sub-set of individuals’ data and samples from a cross-sectional household survey, conducted during peak malaria transmission season in April and May 2017, was used. The survey collected socio-demographic information on household members and coverage of malaria interventions. Malaria testing was done on respondents of all ages using blood smears and RDTs while dried blood spots were collected on filter papers for analysis using photo-induced electron transfer polymerase chain reaction (PET-PCR). Slides were stained using Giemsa stain and examined by microscopy for malaria parasites.P. falciparum infections were co-infections with Plasmodium ovale or Plasmodium malariae.From the 1567 individuals included, the overall prevalence of malaria was 19.4% (CI 17.5–21.4) by PCR, 19.3% (CI 17.4–21.4) by RDT and 12.9% (CI 11.3–14.7) by microscopy. Using PET-PCR as the gold standard, RDTs showed a sensitivity of 75.7% (CI 70.4–80.4) and specificity of 94.2% (CI 92.8–95.4). The positive predictive value (PPV) was 75.9% (CI 70.7–80.6) and negative predictive value (NPV) was 94.1% (CI 92.1–95.4). In contrast, microscopy for sensitivity, specificity, PPV, and NPV values were 56.9% (CI 51.1–62.5), 97.7% (CI 96.7–98.5), 85.6% (CI 80.0–90.2), 90.4% (CI 88.7–91.9), respectively. Non-falciparum infections were found only in Western Province, where 11.6% of From the sub-set of survey data analysed, non-falciparum species are present and occurred as mixed infections. As expected, PET-PCR was slightly more sensitive than both malaria RDTs and microscopy to detecting malaria infections. Plasmodium falciparum is the major cause of malaria in Africa, while P. vivax is the most widely distributed species outside Africa , specificity [(TN)/)], positive predictive value (PPV) [TP/(TP + FP)]and negative predictive value (NPV) [TN/(TN + FN)]. The results were interpreted with 95% confidence intervals (CIs) GraphPad prism  was used to calculate Cohen’s Kappa agreement coefficient.Table P. falciparum mono-infections with 85.7 and 97.8% in Western and Southern, respectively. No mixed/co-infections were found in Southern Province, but 11.6% of all positives in Western had more than one species present  and 2 P. malariae infections, one in each Province ] and a specificity of 94.2% [1190/1263 (95% CI 92.8–95.4%)], with a PPV of 75.9% [230/303 (95% CI 70.7–80.6%)] and NPV of 1190/1264 [94.1% (95% CI 92.1–95.4%)]. The observed agreement percentage was 90.97%, and Cohen’s Kappa was 0.71 (CI 0.66–0.75). Microscopy was observed to have a sensitivity of 56.9% [173/304 (95% CI 51.1–62.5)], specificity of 97.7% [1234/1264 (95% CI 96.7–98.5)], PPV of 85.6% [173/202 (95% CI 80.0–90.2)] and an NPV of 90.4% [1234/1365 (95% CI 88.7–91.9)]. The observed agreement percentage was 89.79% and a Cohen’s Kappa of 0.63 (CI 0.57–0.68)  in Western Province and two  in Southern Province. Interestingly, the diversity of parasite species found in a province broadly correlated to the malaria prevalence, i.e., the higher the prevalence the greater the number of species found. From these data alone, it seems clear that non-falciparum infections are under-reported due to the use of P. falciparum-specific RDTs and challenges in achieving high quality microscopy [This study identified the presence of four croscopy , 32.P. falciparum, of which 89% were mono-infections and 11.6% co-infections and very few mono non-falciparum infections were identified . These findings are in agreement with previous studies which reported 10.6% mixed infections and 88% P. falciparum [P. falciparum reported by Wolfe [Anopheles gambiae is able to transmit all Plasmodium species [P. falciparum transmission is local and how much is imported through travel [P. malariae in Southern Province is maintained through importation, while there is more robust local non-P. falciparum transmission in Western Province. No association was found between travel history and presence of non-falciparum infections however, only travel in the last month was recorded. Considering non-falciparum malaria infections may be chronic or dormant for long periods of time, it is not possible to determine the source of infections in this study. Genotyping of the parasite population may help dissect this relationship by defining the parasite population diversity and relatedness of different infections.Overall, 97% of all malaria infections contained lciparum , and areby Wolfe . The fin species  and is fh travel . ConsideP. falciparum-specific RDTs as observed in approximately 63% of health facilities in Zambia [P. falciparum [Often the non-falciparum species are under-reported or not identified due to a number of factors: the use of n Zambia  and micrlciparum , 32. Othlciparum .P. vivax transmission exists [P. vivax infections due to carrying the Duffy FyFy genotype [P. vivax, albeit in only one person. There is evidence of low-level P. vivax endemicity in sub-Saharan Africa [P. vivax [While evidence for n exists , the majgenotype . It was,n Africa , 40. EviP. vivax .The sensitivity value was below the 95% threshold for both RDTs and microscopy when compared to PCR, while specificity was above the recommended 90%. The findings of RDT sensitivity of 75.5% using PCR as gold standard is consistent with other publications on RDT sensitivity e.g., 88.6% in mainland Tanzania , 76.5% iIn this study RDTs and microscopy had a relatively low PPV (< 90%), meaning that many positives were not infected, which in turn affects malaria morbidity, prevalence and incidence estimates . FurtherResults of this study should be interpreted keeping some limitations in mind. Firstly, there was no similar pre-2017 analysis of species in Southern Province to indicate whether there was previously a higher level of mixed and non-falciparum species present. This makes it difficult to be certain if the elimination activities in the province are responsible for the low positivity rate. Secondly, these data cannot be generalized to the whole country, as this analysis is for two provinces, despite representing lower and higher transmission zones (low meaning an area with parasitaemia less than 5% and high meaning an area with parasitaemia above 15%), and the moderate transmission zone is not represented. Thirdly, an element of selection bias cannot be excluded as clusters with zero RDT prevalence were excluded. It is possible that the clusters may have had non-falciparum species leading to underestimation of the species. Finally, the study used secondary data and the authors have little or no control of RDT results, which may have affected the sensitivity and specificity results. Four samples from participants that were RDT-positive and PCR-negative, who were given treatment within 14 days, were included in the analysis. It is possible that the positive RDTs could have been due to lagging antigenaemia. Although on recalculation of sensitivity and specificity, new figures were within the same range as those previously calculated.P. falciparum. While this study confirms that P. falciparum dominates, a not-insignificant 9.9% of these infections were mixed with another species. Encouragingly, where malaria is closest to elimination , non-falciparum infections identified in this study were minimal (1 case). This may suggest that interventions that reduce P. falciparum transmission also impact non-falciparum species. It is important to expand surveillance activities to monitor non-falciparum infections and ensure this remains the case.There is a concern that other species could continue to drive transmission but remain undetected, as the RDTs currently used in Zambia detect only P. falciparum and non-falciparum species).From the finding, recommendation made include: the national malaria programme consider increasing capacity to diagnose and detect non-falciparum species. This can be done through strengthening diagnosis quality assurance, increase access to functional microscopy, and provide refresher training in malaria microscopy for all laboratory staff, and in addition, introduce Pan RDTs (RDTs able to diagnose