Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.FoxP3+ regulatory CD4 T cells . Prior human studies have shown that Tregs are induced by malaria parasites both in vivo and in vitro, but the role of these cells in immunity in children who are chronically exposed to malaria remains unclear. In this study, we assessed the frequency and features of Tregs among children from areas of high malaria transmission in Uganda. We found that this regulatory T cell population declined markedly with increasing malaria episodes. This loss was associated with decreased expression of TNFR2, which is a protein implicated in stability of Tregs. Additionally, T cells from highly malaria exposed children demonstrated a reduced propensity to differentiate into Tregs following parasite stimulation. Together our data suggest that repeated episodes of malaria alter Treg homeostasis, which may influence the development of immunity to malaria in children.In malaria endemic regions, immunity is slow to develop and does not provide substantial protection against reinfection. Rather, following repeated exposure, older children and adults eventually develop protection from most symptomatic manifestations of the infection. This may be due in part to the induction of immunoregulatory mechanisms by the  In the setting of infection, Tregs help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Preserving this equilibrium requires a complicated balance between regulatory and effector T cell activity. For instance, in the murine leishmania model, Treg-mediated suppression of effector immune responses interferes with complete parasite clearance—but paradoxically, the resulting pathogen persistence fosters the long-term maintenance of effector immune responses that are required for protection from reinfection [reg activity must be fine-tuned for promote resolution of the effector immune response only after control of the pathogen has been achieved. Malaria, like many other parasite infections, has been reported to induce an expansion of the Treg population [reg homeostasis in the setting of P. falciparum infection, which in high transmission regions is characterized by both recurrent symptomatic episodes in young children and persistent asymptomatic infection in older individuals, remain unclear, as does the role of Tregs in the immunopathogenesis of malaria.FoxP3+ regulatory CD4 T cells  were associated with increased risk of malaria in one prospective cohort study [reg induction was associated with increased parasite replication rates [regs in individuals who had asymptomatic infection compared to symptomatic malaria or healthy controls, suggesting dampened activation of Tregs may be associated with decreased risk of disease [regs may serve a protective role in preventing immunopathology during infection [regs [regs, different depletion regimens, or different parasite strains have failed to demonstrate a consistent host-protective role  310 bites ppy [reg frequencies were measured as the percentage of CD4 T cells that were FoxP3+CD25+  did not differ from that of uninfected children (Wilcoxon ranksum p = 0.951). Furthermore, there was no relationship between the frequency of Tregs and the duration of time since the last malaria episode, which might be expected if Tregs transiently increase in response to acute malaria , similar to what has been shown in malaria-naïve adults [P. falciparum-infected red blood cells from blood samples obtained concurrently , but we observed no statistical relationship between the frequency of Tregs and other effector or regulatory T cell populations, including cells producing IFNγ (p = 0.65), TNFα (p = 0.17), or the recently described IL10-producing “self-regulatory” CD4 T cells (p = 0.99) [To investigate the relationship between Tites ppy ). In botreg frequencies or an increased susceptibility to symptomatic malaria among children whose Treg frequencies are inherently low. To distinguish between these possibilities, we measured Treg frequencies in a third cohort of children residing in the same high transmission Nagongera, Tororo District , likely because older children do not develop symptomatic clinical malaria with each P. falciparum infection, and thus malaria incidence is not a good measure of total P. falciparum exposure beyond early childhood. There was, however, a strong inverse relationship between Tregs and age  were calculated by normalization to absolute CD4 cell counts in a subset of children , but at the low transmission Jinja District , in who, n = 91 . In thisites ppy ). Among reg frequencies within individual subjects over time correlates with their malaria incidence, we measured Treg frequencies longitudinally in 41 subjects at 16, 24 and 36 months of age . Subjects were stratified into tertiles based on their number of malaria infections between 16 and 36 months. Among children in the highest tertile of malaria incidence (n = 14), there was a consistent decline in Treg frequencies between 16 and 36 months of age . Repeated-measures analysis using generalized estimating equations confirmed that changes in Treg frequencies over time differed between the exposure groups . Children in the high transmission Tororo District experienced a much higher malaria incidence (median 3.6 vs. 0 ppy) and a much shorter duration since last infection (median 62 vs. 230 days) than children in the low transmission Jinja District (full details in reg frequencies (FoxP3+CD25+CD127dim) were higher in children from the high transmission district compared to the lower transmission district across all age groups . These data suggest that TNFR2 expression is transiently up-regulated during parasitemia but declines over time following repeated malaria episodes. This decrease in TNFR2 expression could contribute to the loss of FoxP3+ Tregs from peripheral blood by decreasing the stability of FOXP3 expression [We next assessed expression of TNFR2 on Tronments ,32. Furtcapacity ,33,34, acapacity . We founpression ,32.regs in children heavily exposed to malaria could be explained by changes in Treg homeostasis such as decreased induction, increased loss (due to apoptosis or downregulation of FOXP3), or both. Therefore, we next examined whether heavy prior malaria infection altered the propensity for Treg induction or apoptosis. It has previously been shown that in vitro stimulation of adult PBMCs with P. falciparum antigen induces regulatory T cells [regs in response to parasite antigen is influenced by age and/or prior malaria exposure, we measured induction of Tregs following in vitro stimulation with P. falciparum schizont extracts (PfSE) in malaria-naïve adults, malaria-exposed children (28 months of age), and malaria-exposed adults from the high incidence district of Tororo (gating strategy and ex vivo Treg frequencies shown in regs (regs was reduced compared to naïve adults (reg induction (fold change >1), only 55% of children with high prior malaria exposure (>6 episodes ppy) induced Tregs following PfSE stimulation , as did the level of CD95 expression  (regs declined with age (Rho = -0.266 p = 0.035)  were measured on Tregs both ex vivo and following stimulation with camptothecin (an activator of apoptosis) or PfSE in 28-month infants with low or high prior malaria incidence (PROMOTE no-chemoprevention control arm). There was no difference in sensitivity to apoptosis as measured by any of the markers either ex vivo or following stimulation with camptothecin or parasite antigen; the frequencies of positively stained cells ex vivo, and the fold change of apoptosis staining, were the same regardless of prior malaria exposure  . Convers= 0.035) . Howeverexposure . Togetheregs influences susceptibility to malaria. We assessed the influence of Treg frequencies on protection from malaria in both the 2-year-old PROMOTE no chemoprevention control arm and 4-year-old TCC cohorts using two methods; a time-to-event analysis , and negative binomial regression of the relationship of Treg frequencies to malaria incidence in the year following assessment. Among 2-year-olds, we found that higher Treg frequencies were associated with an increased time to next malaria episode and a lower future malaria incidence in univariate analysis , suggesting that the decline in circulating Tregs may be associated with the acquisition of clinical immunity.Given their immunoregulatory role, is also possible that Tablished ,2,15,19.measures ,39. Compregs in peripheral blood are influenced by repeated exposure to malaria. While children in settings of intense exposure have higher Treg frequencies during early childhood, frequencies decline throughout childhood in settings of high (but not low) exposure, and the extent of Treg loss correlates with the intensity of P. falciparum exposure. We provide both in vivo and in vitro evidence that among children in high exposure settings, there is a reduction of parasite induced Treg expansion during infection. Further, we show a down-regulation of TNFR2, which is required for stabilization of the FoxP3+ regulatory phenotype in inflammatory environments [reg homeostasis in vivo, which may have a downstream impact on the acquisition of immunity and control of infection.Here, we have shown through both cross-sectional and longitudinal studies that the percentage and absolute number of FoxP3+ Tronments . These dreg induction and homeostasis differ markedly between high and low malaria transmission settings. Although children residing in high transmission areas had higher Treg frequencies overall, perhaps in response to a parasite-driven Treg expansion in early childhood or in utero [decline in Treg frequencies beginning at 1–2 years of age, which appeared to be driven by persistent parasite exposure. Further, among highly exposed children, we saw no association between Treg frequencies and current or recent infection, suggesting that Tregs have reduced in vivo induction during infection of these chronically exposed children. Consistent with this, we demonstrated that induction of Tregs following parasite stimulation of PBMC was diminished in heavily exposed adults and children, providing in vitro evidence that chronic antigen exposure may blunt the proliferative expansion of Tregs in response to malaria. This is in contrast to published studies suggesting that Tregs expand in response to malaria in vivo and in vitro [regs may be induced in initial encounters with parasites, induction capacity is diminished after repeated parasite exposure and instead Tregs undergo a steady decline in the periphery. The induction of Tregs by Plasmodium is believe to occur through activation of latent membrane-bound TGFβ [P. falciparum thrombospondin-related adhesive protein (PfTRAP) [reg induction mechanism is shared by related protozoal pathogens Toxoplasma and Leishmania [regs in heavily malaria exposed children suggests that the host may be able to circumvent parasite induction of Tregs, potentially enabling enhanced control of infection.Our data indicate that the dynamics of Tin utero –30, we oin vitro –13,18,19und TGFβ ,40, whic(PfTRAP) . This Trishmania  and may regs among children chronically exposed to malaria is via loss of FOXP3 expression by “unstable” Tregs, which has been reported to occur in highly inflammatory immune environments [FOXP3 by Tregs is critical for maintenance of regulatory function [regs can become “unstable” and lose FOXP3 expression in response to cues in the microenvironment, although the significance, extent, and triggers of this phenomenon remain subject to considerable debate [hi Tregs to lose both FOXP3 expression and suppressor function [FOXP3 expression and the conversion to pro-inflammatory cytokine producing cells in natural Tregsin vitro [reg destabilization in malaria infection, as recurrent exposure resulted in down-regulated Treg expression of TNFR2, which has been shown to be critical for both the proliferative expansion of Tregs and stabilization of their FOXP3 expression in inflammatory environments [regs have previously been shown to have higher FOXP3 expression and enhanced suppressive function [regs have significant plasticity in response to inflammatory environments such as that observed in malaria infection, which may culminate in loss of FOXP3 expression and suppressive function.Another potential mechanism for the observed decline in Tronments –45. Sustfunction . Severale debate ,45,47–52function , and evefunction . Furtherin vitro . Our datronments ,32,55. Ifunction . Thus ouregs in peripheral blood. Because Tregs track to sites of inflammation, it is possible that Tregs induced by P. falciparum traffic to the liver, spleen, or secondary lymphoid organs during infection. Invasive sampling was not possible in our study cohorts; therefore we were unable to exclude a preferential sequestration of Tregs in tissues or lymphoid organs. However, we did not observe any statistical relationship of Treg frequencies with the presence of parasitemia, nor with the amount of time elapsed since the last P. falciparum infection, as might be expected if Tregs migrate to sites of local inflammation during active infection. Alternatively, loss of Tregs through apoptosis might contribute to their decline following repeated malaria infections. However, we observed no relationship between prior malaria incidence and the expression of the pro-apoptotic molecule CD95 or the anti-apoptotic marker Bcl2 on Tregs (after controlling for age), nor did we observe a differential susceptibility towards apoptosis ex vivo, or following in vitro re-stimulation with parasite antigens.Several additional processes might contribute to the observed loss of Treg frequencies with increasing prior malaria incidence contrasts with that of another regulatory T lymphocyte population, IL10-producing Th1 cells, which we have recently shown to dominate the P. falciparum-specific CD4 T cell response among heavily malaria-exposed children, including among children from both the TCC and the PRISM Nagongera, Tororo cohorts tested here [regs, in contrast to an earlier small cohort study that reported a positive correlation between these two regulatory cell populations [regs is not directly compensated by increased frequencies of IL10 producing CD4 responses. In addition, we did not observe any statistical relationships between Treg frequencies and P. falciparum-specific CD4 effector responses. In prior studies, FOXP3 mRNA levels measured during acute malaria were shown to correlate inversely with the magnitude of the subsequent Th1 memory response to P. falciparum measured 28 days after infection [FOXP3 expression among malaria-naive adults following experimental sporozoite vaccination was shown to correlate inversely with the Th1 memory response measured >100 days later [regs are likely to influence the development of parasite-specific T cell memory responses, no relationship between these populations could be demonstrated through our concurrent measurements in peripheral blood, which maybe due in part to the chronicity of malaria exposure in these children and/or the substantial heterogeneity within the cohort with regard to time elapsed since the last infection. Furthermore, additional parameters of Treg function that cannot readily be measured in peripheral blood in large cohorts, such as suppression of T cell proliferation and modulation of APC function, are likely to influence the cellular immune response to malaria, but could not be assessed in the present study.The observed decline in Tted here ,56. Thisted here . We obseulations . This suys later . Thus, wregs in children, the role of these cells in protection from malaria and the development of immunity remains unclear. We observed no association between Treg frequencies and future malaria incidence or time to next malaria episodes in any of our cohorts. However, our data suggest that although children with the lowest Treg frequencies had a higher monthly probability of infection, they were less likely to become symptomatic once infected compared to children with the highest Treg frequencies over the entire study period. While these data suggest that clinical immunity is acquired as Tregs decline, the role of Tregs in mediating clinical immunity remains unclear, and may not be causal—rather, declining Treg frequencies may coincide with other immune changes that mediate protection. Because all children in our study cohorts have easy access to dedicated study clinics and prompt antimalarial drug treatment, the incidence of severe malaria was extremely low, preventing assessment of the potential role of Tregs in protection from severe disease. We were similarly unable to assess the impact of Treg activity on pathogen persistence following infection, because all cases of symptomatic malaria were promptly treated with potent artemisinin-based drugs, thus altering the natural course of infection. In other protozoal infections, such as leishmaniasis and toxoplasmosis, pathogen-induced Tregs have been reported to curb the inflammatory response, allowing long-term pathogen persistence [reg-mediated immune suppression has been shown to be a requirement for immunity to re-infection [P. falciparum infection that is commonly observed among adults in high-transmission areas [regs in mediating this process is not known. Although we did not observe higher frequencies of peripheral blood Tregs among children with asymptomatic P. falciparum infection, which is not routinely treated in Uganda, this does not exclude a role for Tregs in maintaining this state of host-parasite equilibrium.While our results clearly suggest that repeated malaria impacts peripherally circulating Tsistence ,2,15. Inon areas  may reprregs from the peripheral blood of children following chronic repeated malaria infections, accompanied by downregulation of TNFR2 and diminished in vitro induction of Tregs by parasite antigen. Together these data demonstrate that the impact of chronic malaria antigen exposure on the FoxP3+ regulatory T cell population is quite different from that of acute infection of malaria-naïve individuals. Our findings also add to mounting data suggesting that the stability and homeostasis of FoxP3+ Tregs are perturbed under highly inflammatory conditions. The implications of this pathogen-driven Treg loss for pathogen clearance, host-parasite equilibrium, and the development of clinical immunity in regions of intense malaria transmission require further investigation.In conclusion, we observed a progressive loss of TWritten informed consent was obtained from the adult individual or parent/guardian of all study participants. Study protocols were approved by the Uganda National Council of Science and Technology and the institutional review boards of the University of California, San Francisco, Makerere University and the Centers of Disease Control and Prevention.Samples for this study were obtained from children enrolled in 3 longitudinal childhood malaria cohort studies conducted in Tororo District and Jinja District of eastern Uganda. Cohort characteristics are described in The PROMOTE-Chemoprevention Study was conducted from 2010–2013 and enrolled 400 children who were randomized to receive chemoprevention with monthly sulfadoxine-pyrimethamine (SP), daily trimethoprim-sulfamethoxazole (TS), monthly dihydroartemisinin-piperaquine (DP) or no chemoprevention (control arm) from 6–24 months of age, then followed for an additional year after the intervention ended. Results of this trial have been published . In thisThe Tororo Child Cohort Study (TCC) was conducted from 2007–2012 in Tororo district, and enrolled children at approximately 6 months of age and followed until age 5. Results of this study have previously been published . SamplesThe PRISM cohort was initiated in 2011 and is ongoing. This longitudinal observational cohort consists of 200 households across two study sites, the Nagongera sub-county in Tororo district and the Walukuba sub-county in Jinja district. Description of the study and results have been published in  In all hFor the PROMOTE-Chemoprevention, TCC and PRISM Nagongera high transmission area, the estimated entomological inoculation rate (aEIR) is approximately 310 bites ppy. In contrast, at the PRISM Walukuba low transmissions site the aEIR is estimated at 2.8 .regs parasite infection was assessed via PCR from dried blood spots as previously described [On enrollment all study participants were given an insecticide treated bed net and followed for all medical care at dedicated study clinics. Children who presented with a fever (tympanic temperature ≥38.0°C) or history of fever in the previous 24 hours had blood obtained by finger prick for a thick smear. If the thick smear was positive for malaria parasites, the patient was diagnosed with malaria regardless of parasite density and treatment with artemether-lumefantrine or dihydroartemisinin-piperaquine for all episodes of malaria. Incident episodes of malaria were defined as all febrile episodes accompanied by any parasitemia requiring treatment, but not preceded by another treatment in the prior 14 days. The incidence of malaria was calculated as the number of episodes per person years (ppy) from the time of enrolment into the cohort. In a subset of PRISM cohort children used to assess TNFR2 expression on Tescribed .reg frequencies were enumerated from whole blood and fresh and cryopreserved PBMCs as indicated below. For enumeration of Tregs from whole blood , 100 μl of fresh whole blood was stained with BD Pharmingen anti-CD3-FITC (UCHT1), anti-CD4-PE-CY7 (SK3), and CD25-APC (M-A251) for 20 minutes and then lysed and permeabilized with eBioscience RBC lysis buffer. Cells were washed and then incubated with eBioscience anti-FoxP3-PE (PCH101). Samples were acquired on Accuri C6 Cytometer.Tregs from fresh PBMCs (PRISM Nagongera cohort), PBMCs were isolated by Ficoll density gradient centrifugation and rested over night in 10% fetal bovine serum. PBMCs were stained with BD Pharmingen anti-CD3 PerCP (SK7), anti-TNFR2-Alexa646 (hTNFR-M1), anti-CD95-PECy7 (DX2) and Biolegend anti-CD4-APC-Cy7 (OKT4), anti-CD25-BrillantViolet510 (M-A251), anti-CD127-PacificBlue (A019D5). Following surface staining, cells were fixed and permeabilized with eBioscience FoxP3 staining set and intracellular stained with FoxP3-PE (PCH101) and BD Pharmingen anti-Bcl2-FITC (Bcl-2/100) as per manufacturers protocol. Samples were acquired on three laser BD FACsCantoII with FACSDiva software.For analysis of Tregs from frozen PBMCs , cryopreserved PBMCs were thawed using standard methods, and immediately stained with the following panels of antibodies; BD Pharmingen anti-CD3-FITC (UCHT1), anti-CD4-PE-CY7 (SK3), CD25-APC (M-A251) and Biolegend anti-CD127 Pacific Blue (A019D5); or Biolegend anti-CD3-BrilliantViolet650 (OKT3), anti-CD4-PerCP (OKT4), anti-CD127-FITC (A019D5); or Biolegend anti-CD3-PerCP (OKT3), anti-CD4-APC-Cy7 (OKT4), anti-CD25-BrillantViolet510 (M-A251), anti-CD127-PacificBlue (A019D5), anti-TNFR2-APC (3G7A02). Live/dead aqua amine (Invitrogen) was included in all panels. Following surface staining, cells were fixed and permeabilised with eBioscience FoxP3 staining set and intracellular stained with FoxP3-PE (PCH101) as per manufacturers protocol. Samples were acquired on LSR2 three laser flow cytometer (Becton Dickinson) with FACSDiva software.For analysis of Tregs counts , peripheral blood CD4 T cell concentrations were measured from whole blood using counting beads, and Treg frequencies were calculated by normalization to total CD4 T cell numbers.For calculation of absolute TP. falciparum infected RBCs via intracellular cytokine staining was performed as previously described [P. falciparum infected RBCs or uninfected RBCs and CD4 T cell production of IFNγ, IL10, and TNFα were measured via intracellular staining.Analysis of CD4+ T cell responses to escribed ,56. PBMC6 PBMC were labeled with 1 ml of 1.25 mM 5,6-carboxyfluorescein diacetate succinimidyl ester  for seven minutes. CFSE-labeled PBMC were incubated in 96-well, deep-well culture plates  at 106 PBMC/ well in 1 ml for 7 days with P. falciparum schizont extract (PfSE) (W2 strain) or protein extract from uninfected RBCs (uE) at a effector to target ratio equivalent to 1:1 PBMC:infected RBC. PHA (1μg/ml) was used as a positive control. PfSE extracts were made from the W2 stain grown in standard culture conditions and confirmed to be free of mycoplasma contamination using MycoAlert (Lonza). Mature stage parasites were magnet purified from culture using MACs purification columns. Purified parasites or uninfected RBCs were freeze thawed 3+ times (via snap freezing on liquid nitrogen and then transfer to 37°C water bath) to produce PfSE and uE and stored at -20°C. Following culture of PBMCs with protein extracts, cells were treated with 100 units of DNase I (Invitrogen) in culture media for 5 minutes and then surface stained with Biolegend anti-CD3-BrilliantViolet650 (OKT3), anti-CD4-PerCP (OKT4), anti-CD25-PE-Cy7 (BC96), anti-CD127 Pacific Blue (A019D5), BD Pharmingen anti-CD8-ABC-H7 (SK1) and Live/dead aqua amine (Invitrogen cells). Following surface staining, cells were fixed and permeabilized with eBioscience FoxP3 staining set and stained with FoxP3-PE (PCH101). Proliferation with PHA was used to ensure cell viability, and cells incubated with uE were used as a background control. Of the infants tested, the prior median malaria incidence was 1.2 episodes ppy in the low exposed group and 8.5 episodes ppy in the high exposed group.PBMCs from PROMOTE subjects  and adults from the high malaria transmission region of Tororo were thawed and washed in 10% Human serum (AB) media (Gemini), and 3–6X10P. falciparum schizont extract (PfSE) or protein extract from uninfected RBCs (uE) at an effector:target ratio of 3:1. To test for induction of apoptosis, stains for AnnexinV (Biolegend) or YoPro (Invitrogen), or activated Caspase 3 FITC (BD) were used according to the manufacturer’s instructions in combination with the following antibodies: AnnexinV and YoPro staining—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) APC-Cy7, CD127 (A019D5) APC, CD25 (BC96) PE-Cy7 from Biolegend; for Caspase3—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) PerCP, CD127 (A019D5) Pacific Blue, CD25 (BC96) PE-Cy7. Tregs were gated as CD3+CD4+CD25+CD127dim. Sensitivity to apoptosis was measured ex vivo (untreated control), after induction with camptothecin (fold change compared to untreated), and after stimulation with P. falciparum schizont extract (fold change comparing PfSE to uE).PBMCs from PROMOTE subjects  were thawed and rested overnight either in standard media (untreated), 5uM camptothecin (Sigma) or Unless otherwise indicated, samples were acquired on an LSR2 flow cytometer (Becton Dickinson) with FACSDiva software. Flow cytometry data were analysed using FlowJo software . Color compensation was performed using single color cell controls or beads stained for each fluorochrome. Gating strategies are outlined in Supplementary Figures. Fluorescence minus one controls were used for gating of CD25, CD95, HLA-DR and Bcl2. For FoxP3 staining, an anti-Rabbit-Isotype control was used.reg frequencies and other continuous variables  were assessed using Spearman’s correlation. Changes in Treg frequencies within an individual over time were assessed using the Wilcoxon signed rank test. All other two-group comparisons of continuous variables were performed using the Wilcoxon rank sum test. Repeated measures analysis of longitudinal changes in Treg frequencies was performed using generalized estimating equations, with adjustment for concurrent parasitemia, age and duration since last malaria episode. Categorical variables were compared using Chi sq test. Associations between Treg frequencies and time to next malaria episode were evaluated using the Kaplan-Meier product limit formula, and a multivariate cox proportional hazards model was used to adjust for surrogates of malaria exposure . Negative binomial regression was used to estimate associations between Treg frequencies and the prospective incidence of malaria in the following year  and prevalence of asymptomatic parasitemia in the following year , adjusting for malaria exposure as above. Two-sided p-values were calculated for all test statistics and p<0.05 was considered significant. In the PRMOTE and TCC cohorts, associations between the highest and lowest tertiles of Treg frequencies and the monthly risk of parasitemia, probability of symptoms if parasitemic, and incidence of malaria, stratified by year of age, were evaluated using generalized estimating equations with robust SEs accounting for repeated measures in the same patient, for the period of the inter study (6 months to 3 or 5 years of age) [Data analysis was performed using Stata version 12  and PRISM version 6 (Graph Pad). Associations between T of age) .S1 Table(PDF)Click here for additional data file.S1 FigA. For 2yo, whole blood was stained and cells analyzed on four-color Accuri flow-cytometer. B. For 4yo samples, PBMCs were thawed and stained and analyzed on a LSRII. See also Frequencies of regulatory T cells were enumerated by staining whole blood samples (2yo cohort) or frozen PBMC samples (4yo cohort). (PDF)Click here for additional data file.S2 Figdim expressing CD4+ T cells, normalized to CD4+ T cell absolute counts, from 1 to 11 year old children , declined with increasing age.Absolute count of regulatory T cells, analyzed as the percent of FoxP3+CD25+CD127(PDF)Click here for additional data file.S3 FigFrequencies of regulatory T cells expression TNFR2 were enumerated by staining PBMCs. Gating for FoxP3+CD25+CD127dim regulatory T cells was as for (PDF)Click here for additional data file.S4 FigA. PBMCs were incubated with P. falciparum schizont extract (PfSE), or uninfected RBC extract (uE) for 7 days, then the frequency of FoxP3+CD25+CD127low cells among CD4+ T cells was measured. B. Ex vivo frequencies of FoxP3+CD25+CD127low regulatory T cells in samples used for stimulation assays. C. In PBMCs from malaria-naïve adults, incubation with PfSE resulted in increased frequencies of Tregs compared to PBMCs cultured with uE. See also (PDF)Click here for additional data file.S5 Fig(A) and AnnexinV (B) staining of Tregs from 28 month old children  and high (>6 episodes ppy) prior malaria incidence was measured ex vivo and following stimulation with camptothecin (an activator of apoptosis) or P. falciparum antigen.Activated Caspase 3 (PDF)Click here for additional data file.


Children with severe malaria are at increased risk of invasive bacterial disease particularly infection with enteric gram-negative organisms. These organisms are likely to originate from the gut, however, how and why they breach the intestinal interface in the context of malaria infection remains unclear. One explanation is that accumulation of infected red blood cells (iRBCs) in the intestinal microvasculature contributes to tissue damage and subsequent microbial translocation which can be addressed through investigation of the impact of cytoadhesion in patients with malaria and intestinal damage.Using a static adhesion assay, cytoadhesion of iRBCs was quantified in 48 children with malaria to recombinant proteins constitutively expressed on endothelial cell surfaces. Cytoadhesive phenotypes between children with and without biochemical evidence of intestinal damage [defined as endotoxemia or elevated plasma intestinal fatty acid binding protein (I-FABP)] was compared.The majority of parasites demonstrated binding to the endothelial receptors CD36 and to a lesser extent to ICAM-1. Reduced adhesion to CD36 but not adhesion to ICAM-1 or rosetting was associated with malarial anaemia (p = 0.004). Increased adhesion of iRBCs to ICAM-1 in children who had evidence of elevated I-FABP (p = 0.022), a marker of intestinal ischaemia was observed. There was no correlation between the presence of endotoxemia and increased adhesion to any of the recombinant proteins.Increased parasite adhesion to ICAM-1 in children with evidence of intestinal ischaemia lends further evidence to a link between the cytoadherence of iRBCs in gut microvasculature and intestinal damage. Plasmodium falciparum malaria are at increased risk of concomitant invasive bacterial infection (IBI) particularly with enteric gram-negative organisms (EGNOs) [P. falciparum malaria (describing IBI) across sub-Saharan Africa, the mean prevalence of IBI co-infection was 6.4 % (95 % CI 5.81 −6.98 %). Bacterial co-infection results in higher case fatalities compared to children with severe malaria alone (24.1 versus 10.2 %). An estimated one-third of all severe malaria deaths in African children are attributable to bacterial co-infection [Children with severe  (EGNOs) . In a syEscherichia coli, in children with malaria is highly suggestive of an intestinal process involving translocation of intraluminal microbes across a compromised gut barrier. There is patchy evidence, which attests to gut barrier damage in malaria disease. Adults with acute malaria have reduced absorptive capacity as well as increased intestinal permeability measured using a differential sugar absorption test, the highest ratios being noted in severe disease [The preponderance of gram-negative bacteraemias, including non-typhoidal salmonellae and  disease . Up to 5 disease . In addi disease , 7. In 5P. falciparum malaria, can result in increased intestinal permeability even in the absence of enterocyte damage [l-arginine deficiency, a hallmark of malaria parasite infection, which has been shown to potentiate intestinal inflammation and permeability in mice [P. falciparum malaria is thought to be the adherence of infected red blood cells (iRBCs) to receptors on small vessel endothelial cells. The resultant sequestration of iRBCs in specific organs is most notable in the case of cerebral malaria where iRBCs accumulate in brain microvasculature. Moreover, findings from histopathological sections taken at autopsy from children who died of severe malaria suggest that intense sequestration is also manifest in the gut [There are several mechanisms, not necessarily mutually exclusive, that might impair gut barrier integrity and function in children with malaria. Firstly, hypoperfusion may occur in the context of severe malarial anaemia and shock; the resulting hypoxic damage to the intestinal epithelium can lead to increased permeability with subsequent translocation of intestinal contents . Secondle damage . This ma in mice . Thirdly the gut , 12. TheTo further explore the contribution of parasite sequestration to microbial translocation in the gut, parasite adhesion in children with both malaria and endotoxemia or acute gut injury was assessed. Adhesion was quantified in vitro by measuring rosetting of iRBCs with uninfected erythrocytes as well as their binding to receptors constitutively expressed on endothelial cell surfaces.The study population has been described in detail elsewhere . In brieThe study was approved by the Ugandan Ethical Review Committee (REIRC 002/2009). Written informed consent was provided by all parents or guardians of children recruited into the study.The plasma concentrations of TNF  and I-FABP (Hyglos GmbH) were determined using ELISA according to the manufacturer’s recommendations.An endotoxin activity assay, EAA™  was used to quantify endotoxin levels in EDTA blood, carried out within 3 h of venepuncture according to the manufacturer’s recommendation. A cut off value of 0.4 EAA units for clinically relevant endotoxemia was used, as evaluated in the MEDIC trial .P. falciparum iRBCs cultured to maturity according to standard procedures [Samples were blinded and remained so until all the data had been collected and analysed. RBCs were thawed and ocedures . Mature Image-Pro Analyser and adhering parasites counted using QCapture Pro (QImaging) software. Results are presented as the mean number of parasites/mm2 protein adjusted to 3 % parasitaemia.Recombinant ICAM-1-Fc (donated by Alister Craig’s laboratory) and CD36-Fc (R&D Systems Inc.) have been used before in static adhesion assays. Two less recognized proteins, fractalkine and MadCAM  were included, both of which are implicated in intestinal pathology. Fractalkine expression is enhanced in inflammatory Crohn’s disease lesions  and MadCi, Japan) [For the rosetting assay, 20 μl of parasite suspension in binding medium containing 5 μg/ml acridine orange (Sigma–Aldrich) and 20 % AB serum was rotated for 30 min at room temperature. The rosetting frequency of each isolate was scored by determining the percentage of 200 trophozoite-infected erythrocytes bound to two or more uninfected erythrocytes using a fluorescence microscope  .Continuous variables were compared between groups using the Mann–Whitney U test or Kruskal–Wallis H test. Spearman’s rho correlation coefficient was used to determine a relationship between continuous variables. Statistical analyses were performed using Stata version 12 (StataCorp LP).P. falciparum malaria were cultured to allow iRBCs to grow to maturity. Of the 109 isolates, iRBCs of 48 (44 %) samples grew to maturity. The high failure rate may be explained by the use of anti-malarials prior to hospital admission, which is commonplace in this region of Uganda. There was no significant difference with respect to haematological and clinical data between the two groups and, therefore, the samples evaluated for adhesion can be considered representative of the overall group of children  and a marker of acute enterocyte damage (I-FABP ≥183 pg/ml plasma). Adhesion of iRBCs to CD36 and ICAM-1 as well as rosetting was similar in children with or without endotoxemia Fig. . By contIn children with malaria and acute gut injury, adhesion of mature iRBCs to ICAM-1 under static conditions was significantly increased. These data suggest that acute enterocyte damage is associated with the binding of sequestered iRBCs to ICAM-1 expressed on the surface of gut endothelial cells. Without a healthy enterocyte barrier, it follows that intestinal pathogens or their products such as endotoxin may pass unimpeded into the systemic circulation further complicating malaria infection.There is a well-established link between adhesion of iRBCs to ICAM-1 and cerebral malaria. Histological examination of brains of patients who died of cerebral malaria have shown that iRBCs co-localized with ICAM-1  and bindOverall, the data presented here correspond with other published studies on the binding of parasitized RBCs under static conditions ; the majThis study is the first to examine in vitro the role of parasite adhesion in malaria subjects with evidence of intestinal compromise. I-FABP, a small water-soluble protein present in mature enterocytes, is abundantly expressed on villous tips in the small intestine and is therefore a useful marker of acute enterocyte damage. It has been used to investigate intestinal damage in other cohorts including children undergoing surgery for congenital heart disease and neonates with suspected necrotising enterocolitis and meningococcal septicaemia where it can delineate between early sepsis and specific gut damage –27. In tThere were several limitations to this study. First, the high fall out rate when thawing and culturing iRBCs, comparable with other adhesion studies , halved These findings lend further evidence to a link between cytoadhesion and intestinal damage mediated by the sequestration of iRBCs in gut microvasculature. However there are other pathways that may also contribute to gastrointestinal damage in the context of malaria. Systemic inflammation, which is associated with severe disease, but also a hallmark of the sepsis syndrome can lead to gut epithelial cell damage . The corIn conclusion, the increased parasite adhesion to ICAM-1 in children with evidence of intestinal damage is an important finding, which highlights the complex aetiology behind increased susceptibility to gram-negative bacteraemia in children with malaria. Further research is required to unpick the relative contribution of iRBC sequestration and ICAM-1 binding towards gut barrier compromise. Only then the precise sequence of events in the pathogenesis of malaria-IBI co-infection and progress towards adjunctive therapies including antibiotics can be understood.


Microscopy is the gold standard for diagnosis of malaria, however, manual evaluation of blood films is highly dependent on skilled personnel in a time-consuming, error-prone and repetitive process. In this study we propose a method using computer vision detection and visualization of only the diagnostically most relevant sample regions in digitized blood smears.P. falciparum ring-stage trophozoites (n = 27) and uninfected controls (n = 20) were digitally scanned with an oil immersion objective (0.1 µm/pixel) to capture approximately 50,000 erythrocytes per sample. Parasite candidate regions were identified based on color and object size, followed by extraction of image features  used as input to a support vector machine classifier. The classifier was trained on digital slides from ten patients and validated on six samples.Giemsa-stained thin blood films with P. falciparum. The method achieved a diagnostic sensitivity and specificity of 95% and 100% as well as 90% and 100% for the two readers respectively using the diagnostic tool. Parasitemia was separately calculated by the automated system and the correlation coefficient between manual and automated parasitemia counts was 0.97.The diagnostic accuracy was tested on 31 samples (19 infected and 12 controls). From each digitized area of a blood smear, a panel with the 128 most probable parasite candidate regions was generated. Two expert microscopists were asked to visually inspect the panel on a tablet computer and to judge whether the patient was infected with We developed a decision support system for detecting malaria parasites using a computer vision algorithm combined with visualization of sample areas with the highest probability of malaria infection. The system provides a novel method for blood smear screening with a significantly reduced need for visual examination and has a potential to increase the throughput in malaria diagnostics. P. falciparumAccurate malaria diagnosis is a key to successful management of the disease; yet in the African region, in 2012, up to 40% of suspected malaria cases attending public health facilities did not receive a diagnostic test The two diagnostic methods for routine detection of malaria parasites are microscopy and rapid diagnostic tests (RDTs). The choice between using microscopy or RDTs is dependent on several factors including local malaria epidemiology, skills of interpreters, caseload, and availability of microscopy for differential diagnostics. High-quality microscopy still remains the gold standard providing parasite quantification, allowing species and stage differentiation, enabling monitoring of antimalarial drug response and identification of other disease causing agents. Furthermore, in areas where the caseload of febrile patients is high, microscopy is a more cost-effective diagnostic option compared to RDTs According to recommendations, at the community level or outside formal health care services where high quality microscopy generally is not available, RDTs may be feasible for diagnosing malaria Microscopy can be cost-effective and multipurpose. Although quality assurance programs have now been established; the quality of microscopy-based diagnosis is frequently inadequate Recent development in imaging techniques enables digitization of large parts of a microscope sample or even an entire blood film at a high optical resolution, e.g. 20×, 40×, or even >63× with oil immersion. The obtained digital sample can then be viewed on a computer screen in a manner similar to conventional microscopy, i.e. panned and zoomed to different magnification Most of the published computer vision algorithms for identification of malaria parasites have been developed and tested using a relatively small number of patients and, due to different study end-points, comparisons between the performances of the algorithms are difficult to perform P. falciparum ring-stage trophozoites and thus assist in the diagnostic process. Moreover, the method can also quantify the level of parasitemia and thus potentially provide a diagnostic system for antimalarial drug screening programs.We here propose and evaluate a novel diagnostic aid based on a computer vision algorithm that analyzes an average of more than 50,000 erythrocytes in a thin blood film, ranks sample areas according to probability of infection and presents a small subset (approx. 100) of the highest scoring areas as a single panel to the user. This means that the most suspicious regions from a sample area corresponding to 500–600 high-power fields-of-view in a microscope are collected into a panel corresponding to a single field-of-view. The tool can aid the human evaluator in the identification of Routinely collected, anonymized thin blood films from 47 patients (27 cases and 20 controls) were obtained from the Helsinki University Central Hospital Laboratory . Written informed consent was not required according to the Ministry of Social Affairs and Health, Finland's Act On the Medical Use of Human Organs, Tissues and Cells (Amendments up to 277/2013 included) because the samples collected contained no personal identity codes.P. falciparum, as assessed with a conventional microscope by an expert microscopist according to WHO guidelines Thin blood films were prepared from EDTA-anticoagulated blood and Giemsa stained according to standard procedures 2. For the current study, selected rectangular areas with an average size of 6 mm2 (range 5.28–7.40) from monolayer regions of Giemsa-stained thin blood films were digitized with a whole-slide scanning system that consists of a microscope  equipped with a motorized stage , a 63× oil immersion objective , an 1.0 camera adapter, an RGB LED illuminator , a microscope camera  and software for whole slide scanning . The scanner captured 475–667 (average 549) fields-of-view  per sample and these were stored as uncompressed R, G and B channel TIFF (Tagged Image File Format) image files and then stitched and compressed into an ECW  file format and uploaded to a whole-slide image management platform  running image server software . Finally the images were annotated and for the analysis downloaded as 1500×1500 pixel tiles from the server in a lossless Portable Network Graphic (PNG) format  fields-of-view per thin blood smear sample should be screened to exclude a malaria infection ) format .The digitized blood films (n = 47) were divided into three separate sets; a training set (n = 10), validation set (n = 6) and test set (n = 31). The training set consisted of five thin blood films with a malaria infection and five uninfected control samples. The validation set consisted of three infected and three uninfected blood films and was used for optimization of the algorithm. Finally, the test set consisted of blood films from 19 patients with a malaria infection and 12 uninfected controls and used for assessment of accuracy.http://fimm.webmicroscope.net/research/momic/mamicErythrocytes suspected to be infected were manually labeled by one of the researchers (N.L.) into two classes: certain parasites (label 1) or uncertain parasites (label 2) using a previously described annotation tool P. falciparum detection. The rationale of the selected two-step approach is to first identify potential parasites using simple morphological parameters such as color, shape and size. Further detailed inspection of the remaining candidate regions is then performed with computationally more expensive algorithms aiming to detect the actual malaria parasites among the candidates (The parasite detection method consists of two stages: 1) a candidate region segmentation and 2) ndidates .h of has a bimodal distribution where the first mode m1 represents the background with maximum value P, while the second mode m2 represents the presence of red blood cells with maximum value Q . The histograms  value Q . Two thrBT separates an image into background (≥TB) and foreground (<TB) regions. While ST defines strongly stained regions (<TS) in the foreground.B), all objects with an average area less than 10 µm2 e.g. platelets and debris were removed. Further, from the strongly stained regions of the foreground (<TS), objects larger than (40 µm2) were filtered out, e.g. leukocytes, aggregated platelets. The remaining strongly stained regions in the foreground defined the parasite candidate regions, which typically represent only a small fraction of the whole 1500×1500 pixel tile (for example 0.64% of the tile in From the foreground (<TEach candidate region was divided into partly overlapping image windows of 64×64 pixels from which the below described features were extracted and used as input to the support vector machine (SVM) classifier. The windows were defined by their center points, which were sampled from the candidate regions using a sliding window-sampling step of eight pixels . The sizThe characteristics of the windows were mathematically modeled using a joint distribution of local binary pattern and rotation invariant local contrast features (LBP/VAR) and scale invariant feature transform (SIFT) features. Both LBP/VAR and SIFT features are widely used for object and face recognition in state-of-the-art computer vision applications P pixels in a neighborhood with a radius R. The intensity value of the central pixel is used to threshold the neighborhood pixels, thus forming a binary code. The LBP operator detects variations in the structure of the texture pattern, e.g. line ends, dots, borders, and curves. LBP is combined with a rotation invariant local contrast (VAR). The VAR represents the variance of the gray values of the neighborhood pixels. The LBP codes and the quantized VAR values were combined to an LBP/VAR feature vector. In the current study, the LBP and VAR features were computed from the gray scale images using parameter values P = 16 and R = 2 and with mapping to uniform and rotation invariant LBP patterns and VAR values quantized to four levels The LBP operator compares each image pixel to The SIFT descriptor is an image region descriptor capturing the local image gradients. The descriptor is a three-dimensional histogram composed of gradients in quantized orientation. For the purpose of the current study, he SIFT descriptors were computed using the settings originally proposed 2 kernel was applied to the features. Before the feature mapping the final feature vectors had 456 bins (72+128+128+128), and 3,192 bins after the mapping.A linear support vector machine (SVM) classifier The training set comprised of five patients with different parasitemia levels (7–16%) and five uninfected cases. To ensure a sufficient number of parasite examples, cases with high parasitemia counts were selected for training. The training samples correspond to windows (64×64 pixels) representing parasites or non-parasite objects  present in the candidate region.The positive training samples, i.e. windows containing a parasite, were extracted from the malaria-infected cases using an annotation mask . The annotation mask was defined by expanding the annotation points that were assigned by the human observer approximately at the center of the trophozoite to circles of 16 pixels in diameter. In addition, the positive samples were extracted in four different orientations: . Only annotations labeled as a certain parasite (label 1) were used for training and annotations labeled as an uncertain parasite (label 2) were excluded from the training set. Twenty thousand malaria positive training windows were chosen randomly within each of the positive training samples (n = 5) and, resulting in a total of 100,000 windows containing a parasite.Ten thousand malaria negative training windows were extracted from the candidate regions of all ten training samples (randomly from both the infected and uninfected patient samples), giving a total of 100,000 malaria negative windows. Areas near an annotation labeled as a parasite were excluded when extracting negative training samples, so that positive and negative training windows could maximally have an overlap of 5%.The total number of training windows from which the LBP/VAR and SIFT features were extracted was therefore 200,000, with 50% positive and 50% negative windows.−10, 2−8, …,28,210). The parameter was evaluated based on areas under the receiver operating characteristics (ROC) and precision recall (PR) curves for classification on a window level. The best performance on the validation set was achieved with a C value of 24.The cost parameter C of the linear SVM classifier was optimized on windows extracted from the three infected and three negative controls in the validation set with exponentially growing parameter values: C = was applied to the classification maps. Thus, the highest classification scores were identified and scores closer than 4 µm (40 pixels) to the local maximums were suppressed. The remaining windows with associated classifications scores were entitled detections. The detections were ranked from the highest to the lowest according to the classification score and thereby the probability of containing a parasite.http://demo.webmicroscope.net/montagetest.aspx) and presented to the expert in a random order blinded from the ground truth. No time limit for evaluation was set but the experts received a few of minutes of introduction to the decision support system before starting the interpretation. The introduction to the expert microscopist included guidance regarding how to swipe from one case to the next on the tablet computer and advice for viewing the detection areas and corresponding heat maps.To interpret the decision support tool on a patient level, two expert malaria microscopists evaluated the 128 highest-ranking detections in the test set samples  on a tablet computer  . The detS), large objects (larger than 40 µm2) were removed. Second, objects with a roundness, B) foreground were removed from the mask, after which only the objects with a defined roundness and size remained. This was done to ensure that the detected cells represent only individual erythrocytes. Roundness P): σ (0.06 micrometer) of the average erythrocyte size (7.52 micrometer) in the training set were removed.Accurate segmentation of erythrocytes is necessary to assess parasitemia i.e. the ratio of infected erythrocytes. Automated erythrocyte segmentation was applied to the malaria-infected samples to define a subset of well-defined erythrocytes. We decided to segment only well-defined erythrocytes since segmentation of clustered cells requires computationally expensive algorithms and approximations that introduce uncertainty to the infection status of individual cells. The erythrocytes were segmented from the earlier defined foreground mask  as previAfter the segmentation, the erythrocytes were scored based on the classification scores that were previously calculated for the detection of parasites . The claROCAUC) and area under PR curve (RRAUC) respectively. The ROC curve is defined with false positive rate  on the x-axis and true positive rate  on the y-axis, and the PR curve is defined as a curve with TPR on the x-axis and positive predictive value (PPV or precision) on the y-axis. The F-measure is defined as a harmonic mean of precision and recall: 2*/ and used in selection of the threshold for erythrocyte classification. The above-mentioned metrics are based on the outcomes of a binary test (true positive (TP), false positive (FN), true negative (TN) and false negative (FN)): FPR = FP/(FP+TN), TPR = TP/(TP+FN) and PPV = TP/(TP+FP). Respectively, the accuracy is calculated as a ratio: (TP+TN)/(TP+FP+TN+FN) and negative predictive value (NPV) as a ratio: TN/(TN+FN). The agreement between the diagnostic tool and automated methods in the assessment of erythrocyte and patient-level infection status was estimated by percent-agreement and kappa-statistics. Kappa values were categorized as suggested previously in the literature: <0 as disagreement, 0–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1 as almost perfect agreement The detection accuracy of the classifier was evaluated with the area under the ROC curve . The Central Laboratory for the Hospital District of Helsinki and Uusimaa, HUSLAB, Helsinki, Finland approved the study protocol (VLE82M0005). According to the Ministry of Social Affairs and Health, Finland Act On the Medical Use of Human Organs, Tissues and Cells (Amendments up to 277/2013 included), written informed consent was not required because no clinical records were retrieved and the study contained no personal identifiers.The proposed method is evaluated from two perspectives: 1) as a decision support tool that detects parasite-like objects in a digitized thin blood film and presents the most suspicious sample regions to a human observer and 2) as an automated parasitemia estimation tool by classifying segmented erythrocytes as infected or un-infected.κ = 0.94). The ten strongest detections in each of the samples are shown in http://demo.webmicroscope.net/montagetest.aspx.To evaluate the malaria algorithm on a patient level the panels with the 128 highest detections out of an average of 5,782 detections  in the 31 samples  were shown separately to two skilled malaria microscopists on a tablet computer . The micFor parasitemia estimation, automated erythrocyte segmentation was applied to the malaria-infected samples and the segmented cell was classified into infected and not infected. On average 29,066  erythrocytes per sample were segmented, representing approximately 56% of all erythrocytes in the samples.The sensitivity of the erythrocyte level classification was 84.9% (95% CI 78.5–91.3), the specificity 99.9% (95% CI 99.8–100.0) the negative predictive value (NPV) 99.9% (95% CI 99.8–100.0) and the positive predictive value (PPV) 74.2% (95% CI 61.5–86.9) respectively.ROC and AUCPR of the classification was 0.997 and 0.901 respectively. Based on the classification results, parasitemia was calculated for all test samples as a ratio of infected erythrocytes to all segmented erythrocytes and the correlation coefficient between the automated and human observer-based parasitemia counts was 0.97 .Two clear outliers are seen in the parasitemia calculation  and 7. TP. falciparum). A decision support system, which aids the microscopist in malaria diagnostics, is proposed. We show that image analysis combined with visualization of selected sample areas and a human expert decision on diagnosis is feasible and in the current patient series achieves an accuracy comparable to the malaria diagnostic quality criteria defined for a qualified microscopist The present study describes a computer vision assisted method for screening of digitized thin blood films to detect ring-stage malaria parasites (early stage trophozoites of 2) as compared to the total digitized area of a sample that contained in average 549 fields-of-view (6 mm2). The diagnostic tool achieved a sensitivity of 95% and 90% for each of the two microscopists, with a specificity of 100% on a patient level in the series of 19 malaria-infected samples and 12 uninfected controls.In our study, each of the two experienced microscopists evaluated a panel of the 128 top ranking detections out of an average of approximately 6.000 detections identified by the algorithm in each thin blood film sample in the test set. The sample area presented in the panel corresponds approximately to one high power (100×) field-of-view in a conventional microscope (112×107 µm = 0.12 mmhttp://demo.webmicroscope.net/montagetest.aspx). The prior sample also contained debris and the latter a very high platelet count. One of the microscopists was doubtful regarding the diagnosis of this particular case and thus, in a true clinical setting confirmation would typically involve a tie-breaking smear read from the most senior microscopist, if the original two readers cannot agree on the sample positivity, species speciation, and parasitemia.Of the two microscopists who assigned the diagnosis using the decision support system, one made a single and the other two false negative decisions, i.e. they incorrectly interpreted positive malaria samples as uninfected. Both microscopists missed a malaria diagnosis in the same malaria positive case. The two misclassified samples are case numbers 57516 and 57551 and both had a very low number of annotated parasites, representing the second and third lowest levels of parasitemia in the test series (0.025–0.127%).  or erythrocyte level and only a few on a patient level. We were not able to find previous studies that would have used a semi-automated approach similar to ours, where a human observer makes the diagnostic decision based on screening with image analysis. In one study on a fully automated approach showing patient level results, thin and thick blood films from 174 patients were analyzed with a computer algorithm based on pattern, color and shape features, and a sensitivity of 92% and specificity of 90% was reported Another study on two sets of 20 and 41 thin smears from patients with early stage ring-form parasites reported a sensitivity of 100% in both series and specificities of 50% and 88%, respectively P. falciparum and P. vivax and achieved a sensitivity of 99.0% and a specificity of 99.8%. Our somewhat lower sensitivity as compared to other similar studies could partly be explained by the large number of erythrocytes analyzed - in average more than 50,000 erythrocytes per sample and an area that corresponds to 500–600 high-power (100×) fields of view in a conventional microscope, and a total of more than 900,000 cells.In contrast to the limited number of publications regarding the detection of malaria parasites on a patient level, there are a number of machine vision applications for detecting parasites on an erythrocyte level. Previous reports have included results on analysis of 2,000–30,000 erythrocytes for identification of malaria parasites In addition to aiding the malaria diagnosis on a patient level, the method described here allows automated calculation of parasitemia. Parasitemia was only calculated for cases that were considered positive based on visual inspection of the panel of sample areas with the highest probability of malaria infection. Our approach shows a high level of agreement (correlation coefficient 0.97) between the parasitemia as counted by a human observer and the level estimated by the computer vision method. Parasitemia was calculated on erythrocytes that were easily segmented, i.e. approximately 56% of erythrocytes. A potential drawback of our parasitemia estimation method is that if the segmented erythrocytes differ in their parasite count compared to the unsegmented erythrocytes, it might give rise to a biased parasite count. This is possible if parasite-containing erythrocytes are morphologically different compared to uninfected red blood cells. Other possible sources of bias in the parasitemia calculation include debris on top of the erythrocytes and throphozoite rings with aberrant morphology. It can be challenging for the algorithm to account for these artifacts and the problem might be best tackled by a general sample quality check, before running the parasitemia calculation.Previous studies regarding automated calculation of malaria parasitemia reported correlation coefficients ranging from 0.97 to 0.99 Parasite clearance rates are essential for measuring treatment outcome of antimalarial drug efficacy particularly in clinical trials assessing artemisinin resistance. A robust and accurate automated method for determination of parasite clearance profiles could significantly reduce the workload also in this context, since readouts are usually performed by manual microscopy and requires at least four data points P. falciparum stage in a sample of peripheral blood from an individual with a malaria infection. The trophozoite ring-stage is the most common P. falciparum stage in peripheral blood samples from malaria infected individuals but late-stage tropohozoites, schizonts and gametocytes are occasionally seen. Thus, blood films with falciparum other than early ring stages as well a vast number of staining and sample qualities need to be trialed. Also, in future proof-of-concept studies, the time breakdown for capturing and processing the required number of images per slide, and how the method compares to routine slide examination should be addressed. Due to the visual similarity of the trophozoite in different malaria species, the method presented here most probably has the capability to also detect rings of vivax, ovale and malariae, but use of the method for speciation remains to be explored in further research.The trophozoite ring-stage is the most common Diagnostic tests for the detection of malaria should be rapid and have a high degree of accuracy. Furthermore, tests must be of low-cost, easy to use and easily available We used a high-resolution scanning microscope with a 63× oil immersion objective to acquire the images. The digitization hardware is a challenge when implementing automated vision methods in low resource settings or in small and medium sized diagnostic centers in developed countries. The costs of high throughput scanners for sample digitization have so far prevented transfer of the new technology to low-resource settings. However, the system described here will be further assessed also in combination with less expensive digitization systems. Motorized scanning systems have been described that can be integrated with a conventional light microscope An interesting alternative to our approach is a large-scale public experiment where images from malaria suspected blood films where classified by 1000 untrained, crowd sourced interpreters worldwide and combined into a diagnostic decision, resulting in an accuracy comparable to those of expert microscopists Positive effects of increased diagnostic throughput are improved case management, a more rational anti-malarial drug use, including increased access to treatments since a larger proportion of patients will be verified, and reductions in the development of resistance to antimalarial combination therapies. The massive investments in antimalarial drug development P. falciparum microscopy using an automated machine vision method combined with a panel display showing regions of interest from a virtual thin blood smear of patients with suspected malaria infection. From each blood smear, an area equivalent to roughly 500 microscope high-power fields-of-view, covering an area with approximately 50.000 red blood cells, is compressed into a single easy-to-read panel showing malaria suspected areas of interest to aid the microscopist in assessing the malaria diagnosis.We present a novel system for decision support in


Plasmodium infections tend to remain asymptomatic yet causing significant problems like maternal anemia, low birth weight, premature births, and still birth. This study was conducted to determine the prevalence and predictors of asymptomatic Plasmodium infection among pregnant women in the rural surroundings of Arba Minch Town, Southern Ethiopia.In Sub-Saharan African countries, including Ethiopia, malaria in pregnancy is a major public health threat which results in significant morbidities and mortalities among pregnant women and their fetuses. In malaria endemic areas, Plasmodium infection was diagnosed by using Giemsa-stained blood smear microscopy and a rapid diagnostic test .A community based cross-sectional study comprising multistage sampling was conducted between April and June, 2013. Socio-demographic data were collected by using a semi-structured questionnaire. Plasmodium species by microscopy and rapid diagnostic tests (RDTs), respectively. The geometric mean of parasite density was 2392 parasites per microliter (μl); 2275/ μl for P. falciparum and 2032/ μl for P. vivax. Parasitemia was more likely to occur in primigravidae (Adjusted odds ratio (AOR): 9.4, 95% confidence interval (CI): 4.3–60.5), secundigravidae , using insecticide treated bed net (ITN) sometimes , not using ITN at all  compared to multigravidae and using ITN always, respectively.Of the total 341 pregnant women participated in this study, 9.1% (31/341) and 9.7% (33/341) were confirmed to be infected with Plasmodium infection and be treated promptly via the antenatal care (ANC) services.Asymptomatic malaria in this study is low compared to other studies’ findings. Nevertheless, given the high risk of malaria during pregnancy, pregnant women essentially be screened for asymptomatic  Malaria is the most deadly tropical infectious disease disproportionately affecting the poor, children under the age of 5 years, and pregnant women. There were an estimated 216 million episodes of malaria worldwide in 2010, of which approximately 81% or 174 million cases were in the African region [Plasmodium species [Plasmodium species in placenta is believed to be associated with low birth weight, preterm delivery, miscarriage, and still birth [In malaria endemic areas, a significant proportion of individuals have asymptomatic infection with  species , 3 among species . The seqll birth , 6. Besill birth , 8.Malaria during pregnancy remains a major public health threat in sub-Saharan Africa where about 125 million pregnancies are at risk of malaria each year, and up to 200,000 babies die as a result .Use of long-lasting insecticidal nets (LLINs), the administration of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP), and appropriate case management through rapid and effective therapy of malaria in pregnant women are the current strategies recommended by The World Health Organization (WHO) . CurrentPlasmodium falciparum and Plasmodium vivax are the main species accounting for roughly 60 and 40% of malaria cases, respectively [falciparum to vivax in highland areas [In Ethiopia, an estimated 55.7 million people (68% of the population) are at risk of malaria, and three fourth of the land mass is considered malarious . The Fedectively  though rnd areas , 16.In Ethiopia, early diagnosis and prompt treatment is one of the key strategies in controlling malaria. Blood smear microscopy and malaria rapid diagnostic tests (RDT) represent the two diagnostics most widely used. RDT is used at health post level in rural areas where microscopy cannot be used. The mass distribution of insecticide-treated bed nets (ITNs), together with increased utilization of long-lasting ITNs (LLINs), indoor residual spraying (IRS) and adoption of Artemisinin-based combination therapy (ACT) resulted in substantial declines in malaria-related deaths in Ethiopia .To the best of our knowledge there has been no published study to attest the epidemiological data regarding asymptomatic malaria parasitemia among pregnant women in the study area. Thus, the present study determined the prevalence of asymptomatic malaria parasitemia and its predictors among pregnant women in the rural District surrounding Arba Minch Town, Southern Ethiopia.This study was conducted at the rural district surrounding Arbaminch Town, which is one of the Districts in the Southern Nations, Nationalities, and Peoples' Region of Ethiopia. The Arba Minch town lies in tropical climatic zone with an altitude of 1,200–1,300 meter ASL, an average annual temperature of 29.7°C and rainfall of 700 mm. The District consists of 30 villages, of which 11 have been known malarious villages with intense transmission pattern .A community based cross-sectional study was conducted among apparently healthy pregnant women. Pregnant women with absence of disease symptom/sign within the past 48 hours, axillary temperature ≤ 37.5°c, permanent residents in the study area, and those willing to participate in the study and signed the informed consent were included. Individuals having taken anti-malarial drugs in the past six weeks prior to data collection, those undergoing any kind of long term medical treatments, and unwilling individuals were excluded from the study.The required sample size for this study was calculated using a formula for a single population proportion. Taking expected malaria prevalence of 11.59% , 95% conMultistage sampling of the study subjects in eight Study villages, from the known 11 malarious villages in the District, were selected by simple random sampling using the lottery method. The sample size was then distributed proportionally to the villages based on the size of their pregnant women population . The selAt the village level, the households were selected by simple random sampling by using the sampling frame which was prepared after having identified the pregnant women in the households by the preliminary assessment through active house-to-house visits. Computer generated random numbers were used for the random selection of the study households or study participants. In the cases where more than one eligible woman was encountered in a single household, a lottery method was used to select a woman to be recruited to the study. The health extension workers guided us in the preliminary assessment by indicating the houses where pregnant women were living.A pre-tested semi-structured questionnaire was administered by trained interviewer to obtain data on socio demographic characteristics of the pregnant women. Capillary blood samples were collected by a finger pricking using disposable lancet.μl, 1000–9999/μl, and >10,000/μl, respectively, following the method described elsewhere [Giemsa stained blood smear microscopy and RDTs were employed for the diagnosis of asymptomatic malaria parasitemia. Parasite density per microliter (μl) was determined by counting the number of parasites per 200 white blood cells on a thick blood film assuming a total standard WBC count of 8000/μl. The degree of parasite density was graded as mild, moderate, and severe when the counts were between 1–999 parasites/P. falciparum, and pLDH (Plasmodium lactate dehydrogenase) specific to P. vivax in a human blood sample.The rapid diagnostic test used in this study was SD BIOLINE Malaria Ag P.f/P.v POCT standard diagnostics, Inc., Korea. This test is one step, rapid, qualitative, and differential test for the detection of HRP-II (Histidine-rich protein II) specific to Plasmodium parasite were referred for treatment and medical consultation in the ANCs of nearby health facilities and followed up to ensure appropriate treatment.The study protocol was approved by the ethical review board of Jimma University, Ethiopia. Community agreement and local oral consent was obtained from community leaders. Written informed consent was obtained from all of the study participants. For individuals under the age of 18, written informed consent was obtained from their husbands. Data collected from each study participant and results of laboratory tests were kept confidential and used only for the research purpose. Result of participants with parasitic infection was addressed to the study participants. The pregnant women who were found to be infected with Plasmodium infection in pregnant women. Binary logistic regression was employed to assess the predictors of asymptomatic Plasmodium infection. Variables significant at P-value of 0.25 in the univariate logistic regression were selected for Multivariate logistic regression analysis model. Odds ratios with 95% confidence intervals were calculated and P- value < 0.05 was considered to be statistically significant. Cohen’s kappa coefficient (k) was used to determine the degree of agreement between the diagnostic methods. Shapiro-Wilk test of normality was applied for continuous variables.Data were coded, entered into, cleaned and analyzed using SPSS for windows version 16.0. Both descriptive and inferential statistics were employed for the analysis of data. Frequencies were used to determine the prevalence of asymptomatic Two experienced laboratory technologists individually examined the microscopic slides. Hundred microscopic fields of the thick smear were examined before concluding as negative. Discrepancy between the first and second readings was settled by a third senior microscopist, whose readings were considered final. The manufacturer’s instruction was strictly followed for the RDTs. Blood smear microscopy readers were blinded to the result of RDTs.A total of 341 pregnant women were involved in this study giving a response rate of 98.6%. The age of the participants ranged from 17 to 40 years with a median age of 25 (Inter-quartile range: 23–29), majority (41.1%) of them between 21–25 years. Majorities (95%) of the women were married and about 70% of the women completed their primary and secondary school education. The mean family size in this study was 4.5 (SD ±1.9). Twenty six percent of the subjects were primigravidae, 34.6% of the pregnant women were in the third trimester and more than 80% of the subjects attended ANC .P. falciparum, 15(48.38%) P.vivax, and 4(12.9%) mixed infections. Mixed infection was observed among primigravidae, secundigravidae, and women in their 2nd trimester (μl (95% CI: 1790–3165); 2275/ μl for P. falciparum, and 2032/ μl for P. vivax. Of the 31 diagnosed of asymptomatic Plasmodium infection; 5(16.1%), 23(74.2%), and 3(9.7%) had mild, moderate, and severe parasitemia, respectively. Severe parasitemia occurred among primigravidae and individuals in their 1st and 2nd trimester  P. vivax, and 4(12.1%) mixed infections.The prevalence of asymptomatic plasmodium infection was determined as 9.1% (31/ 341) and 9.7% (33/341) by using microscopy and RDTs, respectively. The species identified from thin blood smear include: 12(38.71%) rimester . The denrimester . The speP.f/P.v POCT was assessed by taking blood smear microscopy as a reference method. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of the RDTs used in the present study were 100%, 99.3%, 93.9%, 100%, and 99.4%, respectively. A good measure of agreement (k = 0.96) was observed between the SD BIOLINE Malaria Ag P.f/P.v POCT and light microscopy.The diagnostic performance of SD BIOLINE Malaria Ag Plasmodium infection at P-value of < 0.05.Socio-demographic characteristics and other health related factors were analyzed using binary logistic regression for possible association and presented in Plasmodium was 9.4 times (95%CI: 4.3–60.5) higher among primigravidae, 6.3 times (95% CI: 2.9–27.3) higher among secundigravidae as compared to multigravidae  higher among women who were using ITN sometimes and 4.6 times (95% CI: 1.4–14.4) higher among those who did not use ITN at all as compared to those who were using ITN always . Yet it was unable to independently predict the risk of asymptomatic Plasmodium infection after adjusting for others confounders   .nd trimester where 10.7% (15/140) were positive, followed by those in 3rd trimester with 8.5% (10/118) positive, and the lowest rate was in the 1st trimester with 7.2% (6/83) were found to be positive. However, these differences were not statistically significant .Parasitemia was more common in the 2Plasmodium infection among pregnant women in the rural District surrounding Arbaminch town, Southern Ethiopia. The study revealed prevalence of asymptomatic infection with P. falciparum or P. vivax among the women as 9.1% and 9.7% by using Giemsa stained blood smear microscopy and the SD BIOLINE Malaria Ag P.f/P.v POCT test, respectively. This prevalence is in agreement with the report of malaria prevalence among pregnant women ranged from 10% to 65% in malaria endemic areas [The present study aimed at assessing asymptomatic ic areas .The finding of the present study corroborates the findings of similar studies reported from other parts of Ethiopia , 23. HowIn the present study, a good measure of agreement between microscopy and RDTs was obtained. The SD BIOLINE Malaria Ag P.f/P.v POCT test had a sensitivity of 100% and a specificity of 99.35% for the detection of asymptomatic malaria compared to microscopy. This study results agree with findings from similar studies conducted elsewhere , 27.The ownership of ITN in the present study is 60.4%, though usage pattern differs among the study subjects. This is lower than the report of 89.6% by Getachew and his colleagues .Plasmodium infection was significantly higher in primigravidae than the multigravidae [Plasmodium infected erythrocytes sequester in the maternal placenta by producing surface antigens, mainly variant surface antigen, that adhere to chondroitin sulphate-A (CSA) receptors expressed by syncytiotrophoblasts in the placenta. Primigravidae and secundigravidae are more susceptible to infection, as they lack these anti-adhesion antibodies against CSA-binding parasites, which develop only after successive pregnancies [There was a strong association between increasing gravidity and decreasing rates of parasitemia. This agrees with findings of similar studies from sub-Saharan African countries where the prevalence of asymptomatic gravidae , 29. It gnancies .In the present study there was no significant difference in the rate of asymptomatic malaria parasitemia with respect to age of the pregnant women. However, the findings of some similar studies , 31 suggPlasmodium infection [The fact that this study is community based and built-in multistage probability sampling technique increases the chance of generalization to the whole pregnant population in the district. Besides, incorporation of the two diagnostic methods in detection of asymptomatic malaria among the pregnant women is also one of the strong side of this study. However, the use of more sensitive and specific technique such as nested PCR is recommended for detection and identification of asymptomatic nfection .Plasmodium infection should be the way forward to fight against malaria in pregnancy and its consequences on fetus.Malaria has a low to moderate transmission pattern in Ethiopia. In countries where malaria has moderate to high transmission, WHO recommends intermittent preventive treatment with sulfadoxine pyrimethamine (IPTp-SP) for pregnant women as part of the ANC services, which does not apply for Ethiopia. Hence, Plasmodium infection among the pregnant women. Hence a longitudinal study covering both the major and minor malaria transmission season and with an objective of assessing the outcome of asymptomatic Plasmodium infection is needed.The present study has its own limitations; the study period was during the minor malaria transmission season. The wider confidence interval in the calculation of odds ratio might indicate the inadequacy of the sample size. Besides, due to the study design  it could not underscore the health impact of asymptomatic Plasmodium infection followed by prompt malaria treatment in the ANC service package. Such initiatives not only safeguard the pregnant women from malaria associated morbidities and mortalities but also the fetus and the newborns. As part of ANC service package, educating on the appropriate usage and benefits of the bed nets, and encouraging early ANC attendance among pregnant women could enhance benefits for the women’s health. RDTs can be used sufficiently for diagnosis of the asymptomatic malaria in areas where there is no access to light microscopy.The findings of the present study suggests the need of further malaria control strategies to screen the pregnant women for asymptomatic


Plasmodium falciparum and Plasmodium vivax during an elimination programme (2012) in Chabahar District, Sistan and Baluchistan Province, south-eastern Iran.Iran has achieved a substantial decline in malaria incidence over the past decades. A common feature of malaria-endemic settings is the requirement for more sensitive techniques to describe levels of low transmission. In this study, serological and parasitological methods were used to measure transmission levels of P. falciparum and P. vivax blood-stage antigens were assessed by ELISA, while microscopy and molecular testing were used to determine parasite carriage by species. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCR).Participants were randomly selected from 64 different geographical clusters in Chabahar city and surrounding villages. Antibody responses to P. falciparum seroconversion was one with a change in exposure 21 years before sampling was done in Chabahar city (P = 0.018) and 4 years in the villages (P = 0.039). There was a higher level of recent P. vivax transmission compared to P. falciparum, based on the SCRs, in both the city and village settings.There was no evidence of recent transmission in the study areas, indicated by an absence of parasite infections in all ages and low or absent serological responses to either species in young children. The best model for age P. falciparum transmission in the urban areas of Chabahar, consistent with a previously described decrease in malaria in the early 1990s, demonstrating the utility of this approach to reconstruct exposure history. At present, it remains unclear whether the P. vivax antibody responses reflect active transmission due to new infections or relapse infections. The absence of parasitological and serological evidence of recent malaria transmission in Chabahar District is viable evidence for certification of elimination.Serological analysis identified a decline in The online version of this article (doi:10.1186/s12936-016-1432-1) contains supplementary material, which is available to authorized users. Following global decline in malaria over past decades , the impIn Iran, a national strategic plan for the elimination of malaria was approved by High Council for Health and Food Security in 2010 to stop local transmission by 2025. In this regard, Iran achieved a substantial decline in malaria incidence according to WHO Malaria Reports . Areas oPlasmodium vivax and Plasmodium falciparum to be 1 % or less , indoor residual spraying, the distribution of LLINs, larviciding, and improved diagnostic capacities in health facilities (microscopy and rapid diagnostic testing) [This might be explained by the up-scaling of different interventions during elimination strategies , such as active case detection, case management, early diagnosis, prompt and effective treatment [artemisinin combination therapy (ACT) plus single dose of primaquine on day 3 as the first-line recommended therapy for testing) . In additesting) .P. vivax, with values similar in both rural and urban settings. This may be due to ongoing exposure to infection with this parasite or boosting of antibody levels by hypnozoite-derived infections. However, it may also be due to small sample size in addition to low seroprevalence [Moreover, no change in SCR was seen for evalence . This reP. falciparum and P. vivax seroprevalence presented here  was lower than previously described levels in other countries in the Eastern Mediterranean region, such as Somalia (18 and 19 %) [The overall nd 19 %) , Djiboutnd 19 %) , and Yemnd 19 %) . These sP. vivax remained twice as high for villagers in comparison with city dwellers in the adjusted model. It seems that ‘previous’ P. vivax transmission was higher in the villages surrounding Chabahar city than in the city itself, indicating higher potential receptivity for the re-introduction of malaria in the villages in this district. More geospatially explicit work would allow specific targeting of these areas to make their use more cost-efficient [P. falciparum uncomplicated malaria, and improved diagnostic capacities. These could help maintain the absence or prevent the re-introduction of malaria transmission in the study areas with very active human population movements between Iran, Afghanistan and Pakistan, common parasite-vector fauna, as well as similar economic-cultural inhabitants, which provides an environment that is in favour of parasite transmission. Further monitoring of seroprevalence in residents of the study areas is considered highly useful given the low levels of transmission and population movement between malaria-endemic areas of Pakistan and Afghanistan that might affect transmission in these settings.As no parasite infections were detected with either microscopic or nPCR methods, risk factors to describe exposure to malaria could only be examined using serological outcomes. The increased likelihood of seropositivity to fficient . Iran hafficient , vector P. falciparum, but not P. vivax. This observation might be due to P. vivax antibody levels being predominantly associated with relapses rather than new infections, as recently described in Papua New Guinea [P. vivax  seroconversion curves between city and village settings as well as the fact that there was weak to absent parasitological or serological evidence of recent P. vivax transmission in children, support this hypothesis. However, the latter may also be due to a higher risk of P. vivax in older individuals (e.g. due to work). It would be useful to confirm these findings in other P. vivax endemic, but eliminating, areas. These assumptions would be strengthened by inclusion of a broader range of antigens, ideally including those related with hypnozoite carriage (as yet unidentified).Interestingly, SES influenced the trend for seropositivity to w Guinea , and thuP. falciparum or P. vivax. Secondly, a change point in P. falciparum transmission in Chabahar city was detected that overlaps with a previously described dramatic drop in cases since the early 1990s [P. vivax transmission compared to P. falciparum transmission based on the SCRs in both the city and village settings. Whether these antibody responses reflect active transmission due to new infections or responses to relapse infections is unclear. The weak to absent evidence of recent malaria transmission in Chabahar District, even though this study targeted malaria prone areas, is promising in the context of elimination.This study aimed to determine malaria transmission patterns in Chabahar District, southeastern Iran. As entomological endpoints are challenging to define correctly at very low transmission levels , 33, thely 1990s , 25. Las


Plasmodium falciparum infection in a 17-year-old school girl with altered sensorium, seizures and phobic spasms.Cerebral malaria is arguably one of the most common non-traumatic encephalopathies in the developing world. Unless the diagnosis of cerebral malaria is made promptly, the consequence could be disastrous. Even though the diagnosis of cerebral malaria can be made relatively easily in majority of cases atypical presentation can often lead to misdiagnosis or delayed diagnosis. We report a case of an uncommon presentation of P. falciparum and peripheral blood smear) were positive for P. falciparum. She was treated as for cerebral malaria with intravenous quinine and discharge from hospital with no residual neurological deficit.A previously healthy 17-year-old school girl was admitted to our hospital with acute condition characterised by comatose state, recurrent seizures and phobic spasms. She initially presented to a local hospital with agitation and over talkativeness and was diagnosed as having an acute psychotic state. Few days later she became drowsy and developed recurrent seizures and marked phobic spasms which prompted the treating physician to diagnose human rabies. However, further investigations carried out in our unit (including rapid antigenic test for Atypical presentation of cerebral malaria can often lead to misdiagnosis. This patient presented with encephalopathic illness with phobic spasms was initially misdiagnosed as human rabies. Therefore, the physicians in malarial endemic areas should be vigilant of similar presentations and should consider cerebral malaria as a possibility. Cerebral malaria is a life threatening complication of falciparum malaria especially when managed inappropriately. It is characterised by rapid onset of unarousable coma associated with or without seizures . Few decPlasmodium falciparum infection in a 17-year-old Sri Lankan school girl, who initially presented with acute psychotic state followed by altered sensorium and phobic spasms (hydrophobia and aerophobia) typically observed in human rabies [We report a case of an uncommon presentation of n rabies .A previously healthy 17-year-old school girl was admitted to our hospital with acute condition characterised by comatose state and seizures. The family reported that she fainted while in the school 2 weeks earlier and was referred to the General Hospital of Hambanthota, in Southern Sri Lanka. Following unremarkable examination and baseline investigations, the patient was discharged. However, following day morning her family noticed some unusual behaviour of the patient including agitation and over talkativeness. As her agitation worsened over next few days, she underwent psychiatric consultation, in which an acute psychotic state had been diagnosed and antipsychotic was prescribed. She did not have symptoms such as fever, headache, chills or rigors at this stage of the illness. Over next 2 days she gradually became drowsy and developed several episodes of seizure. With these new symptoms she was readmitted to the same regional hospital. On admission she was reported to be drowsy with Glasgow Coma Scale (GCS) of 8/15, and had hypertonia with opisthotonus posture. She had exhibited phobic spasms including hydrophobia and aerophobia (positive fan test). Furious rabies was suspected as the most likely diagnosis on the basis of her acute encephalopathic state, phobic spasms and an unconfirmed dog bite history around a month ago. As there was no definitive treatment she was managed conservatively and the family was informed the poor prognosis. Devastated by this acute illness which has no cure; her family sought second opinion from our hospital.On admission to our unit, she was febrile with a temperature of 39.6 °C, heart rate of 120/min, respiration of 38/min, and blood pressure of 134/82 mmHg. She was drowsy with a fluctuating level of consciousness, incoherent, and not cooperative with the examination. Pupils were equal (3 mm) and reactive to light. Fundoscopic examination revealed no abnormalities. No obvious jaundice, enlarged lymph nodes, spleen, or cranial nerve palsies, were observed. She had opisthotonus posture with hypertonia and exaggerated reflexes. She also had obvious phobic spasms including phobic spasm with air (positive fan test). Involuntary jerky movements suggestive of myoclonic jerks were observed at frequent intervals throughout the hospital stay.−1, creatinine 38 μmol/l and erythrocyte sedimentation rate 32 mm/h. Urgent non contrast computed tomography showed no significant abnormality and cerebral spinal fluid examination was unremarkable with protein of 43.4 g/l and 2–4 lymphocytes/mm3. The electroencephalogram showed frequent generalized 4- to 6-Hz spikes in the back ground of slow-wave discharges. A rapid antigenic test for P. falciparum and peripheral blood smear for malarial parasite were performed as the patient was from geographical area traditionally considered as malarial endemic area. Antigen test was positive and the peripheral blood smear revealed the presence of P. falciparum ring forms (Fig. Investigations showed white blood cell count of 7300 cells/μl with 74.3 % neutrophils and 18.3 % lymphocytes, haemoglobin concentration 13.2 g/dl, platelet count 156,000 μlSoon after the diagnosis was made, the patient was started on intravenous quinine with intravenous dextrose. She also was treated with sodium valproate to control the seizure activities. She started to show signs of improvement on the following day and her confused mental state gradually improved over 3 days. On Day 07, she was discharged with a good mental status. The patient was reviewed monthly for 3 months and during the follow up visits she did not have any residual neurological deficit or cognitive impairment. Sodium valproate was stopped 3 months later as she did not have seizures anymore.P falciparum [P. vivax infection [Malaria is one of the most important parasitic diseases of humans, and its main neurological complication, cerebral malaria is arguably one of the most common non-traumatic encephalopathies in the world , 6. Cerelciparum . Howevernfection . The mecnfection , 6, 8.Even though malaria is common in most of the tropical countries affecting 234 million people with 2.5 million deaths per year, there has been a dramatic reduction of case load in Sri Lanka during last decade . Number This case illustrates the difficulty in diagnosing atypical presentation of condition which is getting rarer and rarer. Absence of any obvious pointers favouring the diagnosis of cerebral malaria is a reason for difficulty in diagnosis. Our patient had many atypical features. First she had an acute psychotic state without any prodromal symptoms such as fever, body ache or headache. Fever is a cardinal symptom of malaria and most patients with malaria experienced fever before the onset of other symptoms related to cerebral dysfunction . HoweverThe most unusual manifestation observed in our patient is phobic spasm which is typically described in patients with human rabies , 14, 15.This case illustrates difficulty in diagnosing atypical presentation of cerebral malaria presenting as an encephalopathic illness with phobic spasms. Phobic spasms which are typically seen in human rabies were observed in our patient with cerebral malaria. This is the first time that presence of phobic spasm as a significant manifestation was described in a patient with cerebral malaria. Physicians in malarial endemic areas as well as rabies endemic areas should vigilant of patients presenting with encephalopathy and should consider cerebral malaria as a possible diagnosis in patients presenting with phobic spasms and encephalopathy.