Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC’s are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis. Plasmodium falciparum infections are responsible for about 283 million malaria cases and 584,000 deaths annually, primarily in Sub Saharan Africa .–18.+ celpression –21. In 2pression , 23. By-pression , 23, 24.Recent reports have associated decreased circulating EPC with poor prognosis of severe malaria . Underst-CD34+VEGFR2+ cells in vivo, and the EPC precursor population, CD34+ hematopoietic stem and progenitor cells, characterized as CD34+-HSPC in vitro. CXCL10 and TLR4 expression in these cells were assessed after exposure to heme at different, physiologically relevant concentrations (10–60 μM). We hypothesized that CD34+-HSPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by induction of CXCL10 and TLR activation. Here, we report that free heme activates TLR4 expression and induces over production of CXCL10 resulting in apoptosis and decreased bioavailability of EPC and HSPC precursors.The objective of this study was to determine the effects of free heme on EPC, characterized as CD45Heme was purchased from Frontier Scientific . Camptothecin (CPT) and Lipopolysaccharide (LPS) were purchased from Sigma-Aldrich , TLR4 inhibitor/antagonists monoclonal anti-CD14 antibody and Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1 -carboxylate,  were purchased from InvivoGen , 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Life Technologies .+ hematopoietic stem and progenitor cells (CD34+-HSPC) isolated from human bone marrow obtained from StemCell Technologies, Vancouver, Canada.Human brain microvascular endothelial cell line  was graciously provided by Dr. Bond’s laboratory at Morehouse School of Medicine. Their characteristics include expression of endothelial lineage markers VEGFR2, von Willebrand factor, CXCL10 and corresponding receptor CXCR3. Primary human CD34All study subjects were enrolled after written informed consent was obtained from them or their guardians. Informed consent and human subject research guidelines of the National Institutes of Health (NIH), and the Centers for Disease Control and Prevention (CDC) in the United States were followed. The IRB committees at Morehouse School of Medicine (USA) and the University of Ghana approved this study.P. falciparum is the most prevalent in the country with occasional mixed infection with P. malariae [The study participants were recruited from the Greater Accra region which accounts for 4% of all malaria cases among children under 5 years and 27% of all outpatient department (OPD) malaria cases in Ghana . The stumalariae .Plasmodium Lactate Dehydrogenase/Histidine Rich Protein-2 (pLDH/HRP-2) Antigen Combo Card rapid diagnostic test  were recruited into the study after informed consent. Parasitemia was evaluated microscopically on the number of parasites per field  and at least 100 fields/slide were examined to rule out any negative thick film slide. Enrollees in this group had no evidence of impaired consciousness, seizures, past history of mental illness, meningitis or head injury.Malaria patients with both confirmed thick film slides and P. falciparum parasitemia were recruited and classified as non-malaria subjects.Individuals with negative pLDH/HRP-2 RDT and no Relevant data relating to age, sex, complete blood counts and available medical history were obtained from medical records as well as a survey administered in native language of the subjects . Venous th subject. Selection of EPC was based on dual positive CD34+CD309+ events [-CD34+CD309+ or CD45-CD34+CD133+, to account for immature EPC [- events from leukocyte Forward/Side Scatter dot plot, to exclude lymphoid cells, followed by gating for CD34+CD309+ or CD34+CD133+ double positive events for EPC quantification or CD34+CD284+ double positive events for EPC expression of TLR4 .in vitro, we examined plasma in patients and supernatants from HBVEC and CD34+-HSPC cell culture using commercially available Human CXCL10/IP-10 Quantikine ELISA kit  and Human HO-1 Enzyme Immunoassay kit . CXCL10 and HO-1 levels were measured using optimal concentrations of standards and antibodies according to the manufacturer's instructions. The data was analyzed at 450 nm wavelength using a Spectra Max 190 fluorescence micro plate reader .To determine the relationship between malaria infection, plasma CXCL10 and HO-1 expression levels in malaria negative versus positive subjects and between free heme and CXCL10 +-HSPC were isolated from human bone marrow mononuclear cells and include both hematopoietic stem and progenitor cells . CD34+-HSPC’s were isolated using immunomagnetic positive selection from human adult bone marrow. The cells were cultured in StemSpan SFEM II basal media supplemented with StemSpan 100 expansion cocktail, both from StemCell Technologies. Both HBVEC and CD34+-HSPC were passaged at 70–90% confluence, plated at a density of 2×105 cells/mL and incubated at 37°C in 5% CO2 until ready for treatment.Human brain microvascular endothelial cells (HBVEC) were cultured in endothelial basal media supplemented with 2% Fetal Bovine Serum and growth factors to obtain endothelial growth media . Human primary CD34+-HSPC were seeded at a density of 1×105 cells/mL in 96-well plates. Fresh heme was prepared in 0.02 M NaOH. Cells were serum-starved for 4 hr, followed by exposure to 60 μM heme, vehicle (0.02 M NaOH) or positive control agent, 57 μM camptothecin, (CPT) for 18 hr. The Guava easyCyte flow cytometry system was used to quantify apoptosis with the TUNEL Kit for Flow Cytometry . Cells were fixed and permeabilized using Guava TUNEL solution and apoptotic events were counted if they emitted a nucleated cell fluorescent signal, and exhibited the forward light scatter (FSC) intensity appropriate for a particle the size of a cell. Debris events with low FSC signal were not counted. All population events were analyzed using CytoSoft version 2.0 software. Data corresponds to three experiments run in parallel.HBVEC and CD34+-HSPC using Qiagen RNeasy kit  and quantified using the Nanodrop N-1000 by Agilent Biosystems . The Qiagen QuantiTech Reverse Transcription kit was used to synthesize cDNA according to manufacturer instructions . The reverse transcription reactions were carried out in 20 μL volumes at 42°C for 15 min followed by 95°C for 3 min. TLR4 and CXCL10 expression were analyzed by quantitative RT-PCR and was performed using iQ SYBER Green Supermix  in 25 μL reaction volumes with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control. The CFX96 Real-Time PCR System  was used to perform qRT-PCR and analyzed the cycle threshold data obtained using thermocycling conditions: 95°C for 15 min, 95°C for 15 sec, 55°C for 30 sec, and 72°C for 30 sec for 40 cycles. Primer sequences were as follows; CXCL10: , TLR4: , GAPDH: . The 2(-Delta Delta C(T)) method was used to analyze relative gene expression data normalized to the housekeeping gene, GAPDH. Results are expressed as fold change relative to housekeeping gene in treatment versus vehicle-treated cultures.Total RNA was isolated from HBVEC and CD34p-values were determined using T-test, Mann-Whitney U-test, ANOVA or two-way ANOVA and Tukey’s post hoc comparisons where appropriate. Data is presented as mean ± standard error or median and interquartile range (IQR) unless otherwise stated. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 6.0 for Windows .Population distribution was determined using chi-squared test. Experiments were performed in triplicate and p < 0.0001 , WBC  and platelet   and malaria patients , < 0.0001 . There w 0.0001) .P. falciparum infection on circulating EPC populations, we measured frequency of EPC markers, both mature (CD45-CD34+VEGFR2+) and immature (CD34+VEGFR2+CD133+), in leukocyte fractions of whole blood from 42 randomly selected samples  using FACS analysis. Randomization was accomplished using a systematic sampling technique that selected every 12th subject for recruitment into the sub-study. The median frequencies of CD34+-HSPC and mature CD45-CD34+VEGFR2+-EPC were significantly decreased in malaria patients relative to non-malaria subjects; 0.5 (IQR 0.3–0.9) in non-malaria vs. 0.2 (0.1–0.3) in malaria patients, p = 0.0006 and 0.3 (IQR 0.1–0.7) in non-malaria vs. 0.1 (IQR 0.1–0.2) in malaria patients, p = 0.02 , malaria 26.9 μM (IQR 20.1–39.5), p < 0.0001, p < 0.0001, p < 0.0001, In 2012, Liu et al. reported significant increases in heme-induced HO-1 and CXCL10 in plasma in an experimental murine model of malaria and further confirmed their results in vitro . This stin vitro [in vitro and in vivo. FACS analysis was used to determine whether EPC exhibited altered expression of TLR4 in non-malaria versus malaria subjects. Median fluorescence intensity of TLR4 was assessed in 100,000 EPC events on a BD FACS Calibur. Statistical analysis of non-malaria versus malaria subsets indicated there was a significant increase in the median fluorescence intensity of TLR4 (CD284) expression on EPC in malaria patients (p = 0.04). These results confirmed previous reports indicating that pro-inflammatory factors, such as heme and CXCL10 are significantly associated with TLR4 expression in cells that modulate immunological responses [p < 0.0001, In 2012, Liu et al. reported significant increases in plasma heme induced HO-1 and CXCL10 in an experimental murine model of malaria and further confirmed their results in vitro . This stesponses . To dete+-HSPC in a concentration-dependent manner (p = 0.04 vs. vehicle) and the lethal dose for 50% (LD50) was obtained in both cell types with 60 μM heme, a physiologically relevant concentration in hemolytic diseases [50 doses and significant increases in apoptosis were observed (measured as percentage of TUNEL positive cells). Heme induced cell death via apoptosis in both HBVEC and CD34+-HSPC at 40 μM and 60 μM respectively  . Here wet manner . A signidiseases , 33. Thi+-HSPC in vitro using qRT-PCR. TLR4 mRNA was up-regulated in HBVEC and CD34+-HSPC when treated with heme at LD50 doses for 18 hr  complex that physically associates with TLR4 and induces signal transduction and TAK-242 is a TLR4 antagonist that suppresses ligand-dependent and -independent signaling. Induction of CXCL10 mRNA expression by heme was assessed using qRT-PCR in both HBVEC and CD34+-HSPC. Expression of CXCL10 mRNA increased 7-fold in HBVEC and 2-fold in CD34+-HSPC when exposed to 60 μM heme for 18 hr, data was analyzed using analysis of variance followed by Tukey’s multiple comparisons test  activation. In this study, individuals with malaria have significantly lower levels of EPC than observed in non-malaria. In addition, plasma levels of heme, heme oxygenase-1 (HO-1) and CXCL10 were significantly increased compared with non-malaria subjects.in vitro and in murine models of experimental cerebral malaria (ECM) [Plasmodium parasites scavenge erythrocytic hemoglobin. It has also been reported that free heme induces oxidative free radicals, leading to severe microvascular damage in sickle cell disease through induction of TLR4 [+ hematopoietic stem and progenitor cells, and these stem cell populations are depleted in individuals with severe malaria by an unknown mechanism [We have previously shown that free heme is a potent apoptotic factor as well as inducer of the pro-inflammatory chemokine CXCL10 in microvasculature (HBVEC) ia (ECM) , 30. Fre of TLR4 , 34, 35. of TLR4 , 36–40. echanism , 41. Hemechanism –44. DecrPlasmodium infection [+-HSPC. This confirmed in vivo observations of increased TLR4 expression in EPC populations in the presence of plasma heme and CXCL10 elevation. In addition, the TLR4 antagonist, TAK-242, dampened CXCL10 production in vitro in the presence of heme. Therefore, we propose a pathophysiological mechanism whereby heme mediates the TLR4 signaling pathway, resulting in overproduction of cytotoxic CXCL10. Thus depletion of EPC is a consequence of heme-induced CXCL10 production and TLR4-mediated apoptosis.The role of free heme in EPC depletion during severe malaria pathogenesis is poorly understood; therefore we explored the possibility that depletion of these endothelial cell precursors is mediated by TLR4 , 45, 46.nfection –49. In tTrypanasoma cruzi parasites are potent activators of TLR2 in both mice and humans and prolonged exposure to low doses of TLR4 activating LPS decreases the repopulating potential of murine hematopoietic stem and progenitor cells and increases inflammatory cytokine production [+ cells from individuals with myelodysplastic syndromes, express increased levels of TLR4 when compared to the constitutive expression of TLR4 in the absence of these hematological syndromes [P. falciparum infection, there is a subset of individuals that have polymorphisms in TLR4 and TLR9 that have been linked to severe disease in both CM and ECM [+-HSPC, are activated to increase expression of TLR4 in addition to excess CXCL10 production in the presence of increased heme, which mimics in vivo conditions in malaria. The expression of TLR4 by these populations makes them susceptible to the inflammatory effects of deleterious TLR4 activators TNFα and IFNγ, both of which are up-regulated in malaria [This study confirmed previous reports of the role of toll-like receptor activation in parasitic and inflammatory diseases. For example, glycosylphosphatidylinositol anchors from the protozoan parasite oduction , 50, 51.yndromes . It is a and ECM , 53, 54. malaria , 20, 55. malaria .in vitro conditions, though decreases in CXCL10 expression in CD34+-HSPC did not reach statistical significance. These findings indicate a resilience of CD34+-HSPC to receptor-mediated TLR4 signaling when compared to HBVEC. The high activation threshold in CD34+-HSPC may be cytoprotective and would explain why HBVEC had enhanced responses to heme, while CD34+-HSPC seemed to have dampened immunological response though susceptible to its apoptotic effects. Another possible explanation is that the high rate at which these cells undergo apoptosis in the presence of heme prevented detection of CXCL10 transcript in the presence or absence of TLR4 inhibitors.HBVEC showed significant reductions in expression of CXCL10 in the presence of the TLR4-blocking agent anti-CD14 Although this study assessed the role of heme in depleting EPC directly, another potential scenario is that mobilization of EPC from the bone marrow may be inhibited or inactivated in the presence of increased heme or other unknown cytotoxic factors , 58. The+-HSPC. Our results indicate that free heme is a major contributor to severe malaria pathophysiology by inducing apoptosis in HBVEC and CD34+-HSPC, which are vital BBB cellular components.In this study we have shown that heme-induced, TLR4-mediated CXCL10 expression contributes significantly to depletion of HBVEC and CD34One aspect of malaria pathogenicity, the role of polymorphisms in human CXCL10, HO-1 and TLR genes have been reported but were not assessed here , 60–62. Currently, there are few if any specific and sensitive biomarkers for prognosis of fatal malaria. Several host and parasite indicators of malaria infection have been identified as predictors of disease prognosis  including angiopoietins, elevated CXCL10 serum levels, CXCL10 polymorphisms and free heme , 63, 64.Heme induces production of apoptotic and inflammatory host factors, including CXCL10, and exacerbates malaria pathogenesis , 12. PreS1 Fig+-HSPC population was defined as CD45-CD34+ and the EPC population was defined as CD45-CD34+CD309+ from Forward Scatter/Side Scatter upon elimination of debris and RBC. Gating strategy included selection of CD45- events followed by gating for CD34+ or CD34+CD309+ double positive events.The CD34(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Background: The pathogenesis of malaria during pregnancy is not fully understood. A proinflammatory cytokine, macrophage migration inhibitory factor (MIF) is suggested as a factor involved in the pathogenesis of malaria during pregnancy.Methods: A cross-sectional study was conducted in Medani Hospital, Sudan to investigate MIF levels in placental malaria. Obstetrical and medical characteristics were gathered from each parturient woman using questionnaires. All women (151) were investigated for malaria using blood film and placental histology. MIF levels were measured using ELISA in paired maternal and cord blood samples.Results: There were noP. falciparum-positive blood films obtained from maternal peripheral blood, placenta or cord samples. Out of 151 placentae, four (2.6%), one (0.7%), 32 (21.2%) showed acute, chronic and past infection on histopathology examinations respectively, while the rest  of them showed no signs of infection.There was no significant difference in the median (interquartile) of maternal  and cord  MIF levels between women with a positive result for placental malaria infection (n=37) and women with a negative result for placental malaria infection (n=114). In regression models placental malaria was not associated with maternal MIF, hemoglobin or birth weight. MIF was not associated with hemoglobin or birth weight.Conclusion: There was no association between maternal and cord MIF levels, placental malaria, maternal hemoglobin and birth weight. Malaria during pregnancy can lead to adverse outcomes  e.g. anemia and low birth weight (LBW)4. Pregnant women in different regions of Sudan are susceptible to malaria, regardless of their age and parity5. Malaria is associated with adverse pregnancy outcomes such as anemia6 and LBW7, and it is the main cause of maternal mortality8.Malaria is a large public health problem in endemic tropical countries where there are over 30 million pregnancies at risk of malaria occur in Africa each yearPlasmodium falciparum–infected erythrocytes and accumulation of infected erythrocytes in placental intervillous spaces is responsible for the malaria-related pathologic changes in the placenta10. The exact mechanism by which malaria infection and placental inflammation result in fetal growth restriction and LBW is poorly understood. However, many chemokines and inflammatory cytokines are associated with malaria infection and malaria-related LBW11.The sequestration of12. MIF has been observed in the human endometrium, placental villi, cytotrophoblasts, and it has been implicated in implantation and other reproductive functions14. Several studies have demonstrated the role of MIF in modulating malaria severity and pathogenesis16. It has recently been reported that women with a positive result for placental malaria had significantly higher intervillous plasma MIF levels than women with a negative result for placental malaria17. There are few published data on MIF and placental malaria and none of them from Sudan. The current study was conducted in Medani Maternity Hospital, Central Sudan, to investigate MIF levels in women with placental malaria, and the effect – if any- on maternal hemoglobin and birth weight. This work is an addition to our previous research on malaria and its pathogenesis during pregnancy e.g. placental malaria infiltration19, hormones and cytokines21 complement, cytokines and malaria infections23.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine released from a variety of cells  and is implicated in the pathogenesis of sepsis, and inflammatory and autoimmune diseasesP. falciparum is the sole malaria parasite24.A cross-sectional study was conducted during the rainy and post-rainy season (September–November) 2013 in Medani Maternity Hospital, Central Sudan which is a referral tertiary hospital. Central Sudan is characterized by unstable malaria transmission andThe sample size of 151 women was calculated to have 80% power and to detect a difference of 5% at α=0.05 and 10% of women might not respond or have incomplete data.2. Newborns were weighed immediately following birth using a Salter scale and the sex of each newborn was recorded.After signing an informed consent form, information on history of obstetrics, medical history, antennal attendance characteristics, and bed net use was gathered from participants using questionnaires applied by a trained medical officer. Maternal weight and height were measured and body mass index was calculated and expressed as weight (kg)/height (m)5ml of blood  were taken and allowed to clot and centrifuged for 10 minutes at 3000 rpm and the serum was separated and stored at -20°C till the analyses.Maternal, placental, and cord blood films were prepared and stained using 10% Giemsa. If the slides were positive; the number of asexual parasites was counted per 200 leukocytes, assuming a leukocyte count of 8000 leukocytes/μl (for thick films) or per 1000 red blood cells (for thin films). Blood films were considered negative if no parasites were detected in 100 oil immersion fields of a thick blood film, which was double-checked in a blind manner by an expert microscopist. Maternal hemoglobin levels were measured by the HemoCue hemoglobinometer  and recorded.Placental histology. The method used for placental histology was mentioned previously20. In summary, a 3cm3 placental sample was obtained from the maternal surface at a location approximately halfway between the umbilical cord and the edge of the placenta. Each biopsy sample was immediately placed in 10% neutral buffered formalin. The buffer was used to prevent formation of formalin pigment, which has similar optical characteristics and polarized light activity as malaria pigment25. Placental biopsy samples were processed and were embedded in paraffin wax and 4mm thick slides were stained with hematoxylin-eosin and Giemsa. In these slides, placental malaria infection was characterized as follows26: uninfected (no parasites or pigment), acute (parasites in intervillous spaces), chronic , and previous .Maternal and cord serum levels were measured using a human MIF ELISA kit  by following the manufacturer’s protocol.Data were entered into a computer using SPSS for windows (version 16.0). MIF data were not normally distributed and were compared between groups using Mann-Whitney U test. Multivariate analyses were performed using binary models for placental malaria infection as the dependent variable and linear models with hemoglobin, birth weight, and MIF  levels as continuous dependent variables. Socio-demographic characteristics, education, antenatal care, residence, and placental malaria infections were the independent predictor of interest. Odds ratios (OR) and 95% confidence intervals (CI) were calculated and a P value of <0.05 was considered significant.The study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum, Sudan.P. falciparum-positive blood films obtained from maternal peripheral blood, placenta or cord samples. Out of 151 placentae, four (2.6%), one (0.7%), 32 (21.2%) showed acute, chronic and past infection on histopathology examinations respectively, while the rest  of them showed no signs of infection.The basic characteristics of the investigated women were shown inNone of the investigated factors were associated with placental malaria infection,There was no significant difference in the median (interquartile) MIF levels  between the maternal and cord samples.In linear regression placental malaria was not associated with maternal MIF, hemoglobin or birth weight. Likewise MIF levels were not associated with maternal hemoglobin or newborn birth weight .There was a significant association between maternal blood/placental and cord MIF ,et al., 2015 ‘Macrophage migration inhibitory factor and placental malaria infection in an area characterized by unstable malaria transmission in Central Sudan’Raw dataset for Eltayebhttps://www.gnu.org/software/pspp/.Basic characteristics of the women were gathered using questionnaires. Maternal and cord serum levels were measured using a human MIF ELISA kit. gestweeks=Gestational weeks; antenacare=had antinatal care ; usebednet=used bed nets ; useironfolic=used iron or folic acid ; Wt=weight ; Ht=height , Hb=haemoglobin ; birthwt=birthweight ; bloodgroup : 0=A, 1=B, 2=AB 3=O; MIFcord= MIF levels in the umbilical cord (ng/ml); MIFmother (ng/ml). Raw data file openable by PSPP, available atClick here for additional data file.Copyright: © 2015 Eltayeb R et al.2015Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).The main findings of the current study were; there was no significant difference in the MIF levels in women positive for placental malaria infection and women negative placental malaria infection negative. There was no association between MIF, hemoglobin and birth weight.et al. found no significant difference in the peripheral and cord MIF levels between women with placental malaria infections and women with placental malaria infections negative17. It is worth mentioning that in Singh’s later study the MIF levels in the intervillous space (which we did not measure) were significantly higher than the peripheral and cord levels and higher in women with placental malaria infection compared with women negative for placental malaria infection17. Furthermore, the observations of Singhet al. were based on microscopically-diagnosed placental malaria infection and in our cohort none of the women/placentae had microscopically detected malaria infections, except one that was diagnosed via histology. Yet high MIF was reported to be associated with adverse pregnancy outcome regardless of the presence of malaria infection17. Likewise, Chaisavaneeyakornet al.27 observed high levels of MIF in the intervillous blood, compared with that in both peripheral and cord plasma and that intervillous  MIF levels are associated with placental malaria among Kenyan women. Previous results obtained by Chaiyaroj and colleagues reported significantly higher MIF production by intervillous blood monocytes compared to peripheral ones and high MIF levels in placental plasma compared to paired peripheral plasma28. Increased secretion of MIF by syncytiotrophoblasts was observed previously using anin vitro system29. Generally MIF has been shown to play important roles during normal pregnancy30, as well as in preterm delivery31 and preeclampsia32 and therefore, intervillous MIF would be expected to be high.This goes with previous reports where Singh21. Furthermore, histologic studies have shown that malaria-infected placentae have high numbers of macrophages loaded with malarial pigment and these cells could have a critical role in the clearance of the malaria parasites25. Perhaps the high levels of MIF levels observed (by the later studies) in the placenta of women positive for malaria is induced by the malaria parasites that accumulated in the placenta, with MIF helping to retain macrophages in the placenta. Interestingly, it has been shown that MIF is effective in activating macrophages to clear/remove intracellular parasites e.g.Leishmania major33.We have previously shown that immunomodulatory hormones (cortisol), cytokines, monocytes and macrophages were implicated in the pathogenesis of malaria during pregnancy which affected pregnant women regardless of their age and parity7.As mentioned above, the malaria placental infections in the current study were past infections and this could explain the lack of significant difference in MIF levels. The other plausible explanation could be the submicroscopic/subpatent infections that we did not investigate in the current study. We have recently shown that in the same hospital, submicroscopic/subpatent infections that were detected by PCR rather than histology were significantly associated with low birth weightThe current study failed to show a significant association between maternal blood/placental and cord MIF levels, placental malaria, maternal hemoglobin or birth weight.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2015 Eltayeb R et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).F1000Research: Dataset 1. Raw dataset for Eltayebet al., 2015 ‘Macrophage migration inhibitory factor and placental malaria infection in an area characterized by unstable malaria transmission in Central Sudan’,10.5256/f1000research.7061.d10203934 I am in total favour of publishing reports of negative results, if the study is well conducted, as this one is. Malaria during pregnancy is an outstanding health issue that needs significant investigation. Biomarkers able to predict adverse outcomes are needed, and inflammation-related factors are a logical choice.This study on the correlation between MIF , placental infection and pregnancy outcomes  goes in this direction, and quite clearly shows that there is no correlation. The number of cases is possibly low, and no cases with blood malaria were found in the cohort. Although possibly preliminary, the study is nevertheless very clear and straight forward in its design, conduction and logical conclusions.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Proper study, well outlined and performed.Some minor critique regarding thickness of paraffin slides which should read 4 micromillimeter or with Greek letter μm, not "4mm". "strom" should read correctly "stroma". Finally, acronyms of authors in section "Authors contributions" are not clear to me with regard to the list of authors on title page. Please, amend, respectively.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Children under 5 represent 86% of annual malaria deaths in the world. Following increasing trends in international travel, cases of imported malaria are rising in North America. We describe the epidemiology of malaria diagnosed at a tertiary care pediatric center in the multicultural city of Toronto.Retrospective chart review of all laboratory confirmed malaria from birth to <18 years between July 1, 1997 and June 30, 2013. Epidemiological data, travel history, chemoprophylaxis history, as well as clinical presentation, diagnosis and treatment were extracted.Plasmodium falciparum malaria was identified in 76 (71%), Plasmodium vivax in 28 (26%). Median age of infected children was 6.7 years where 35% of children were born in Canada, 63% were recent or previous immigrants. Of those who resided in Canada, reason for travel included visiting friends or relatives (VFR) 95% and tourism or education (5%). Most common countries of infection were Ghana (22%), Nigeria (20%) and India (14%). Median parasitemia at presentation to our institution was 0.4% (IQR 0.1-2.3) with a maximum parasitemia of 31%. Nineteen (18%) met the WHO criteria for severe malaria due to hyperparasitemia, with 3 of these cases also meeting clinical criteria for severe malaria. One third of patients had a delay in treatment of 2 or more days. Ten percent of children had seen two or more primary health care professionals prior to admission. Prophylaxis was documented in 22 (21%), and out of those, 6 (27%) were appropriate for the region of travel and only 1 case was documented as adherent to their prescription. There were no cases of fatality.In total 107 children were diagnosed with malaria in the 16 year time period. Malaria continues to be a significant disease in returning travelers and immigrant or refugee populations. An increase in physician awareness is required. Appropriate pre-travel advice, insect protection measures, effective chemoprophylaxis is needed to reduce the incidence and improve the management of imported pediatric malaria. Plasmodium falciparum[Despite being largely preventable and treatable, malaria is estimated to kill 660 000 to 1 240 000 people per year with children under 5 representing 86% of annual malaria deaths in 2010 . Althouglciparum, 6, 7. Tlciparum.Children account for 15-20% of imported malaria cases and present distinctly from adults with malaria , 12. ImpDespite the growing threat of imported malaria, there have been few studies in North America, evaluating the epidemiology of imported malaria in children , 13. ThiThe objectives of the current study are twofold. First, describe the epidemiology of malaria diagnosed at SickKids over a 16 year period from 1997 to 2013. Second, identify populations at increased risk for malaria infection and at risk of severe malaria in Toronto. Understanding the epidemiology of imported malaria will support the design and implementation of targeted interventions for young travelers to endemic countries and others at risk both in Toronto and in similar large multi-cultural cities.This study is a retrospective review of all cases of laboratory-confirmed malaria from birth to <18 years, at SickKids between July 1, 1997 and June 30, 2013. Ethics was approved by the SickKids Institutional Review Board and access to clinical records were granted by Medical Records at SickKids, and laboratory records were granted by the Department of Microbiology. A positive malaria test result was defined as positive thin or thick smear or a positive rapid malaria test , BinaxNOW histidine-rich protein 2 test (T1) and BinaxNOW aldolase (T2) test were both conducted. Cases were identified through two methods: 1) SickKids microbiology records of positive thin or thick smears, and 2) ICD-9/10 codes from SickKids medical records. All positive thin or thick smears of malaria are sent to the Public Health Ontario Laboratories for confirmation. We included as cases only those which the Public Health Ontario Laboratory confirmed as positive by smear. There was a single case where the diagnosis of malaria was given to a patient (negative smears) due to positive rapid malaria test done at SickKids. This case had negative smears and negative rapid malaria test at PHL, and was not included in our study.After identifying cases, all medical charts were reviewed for demographic, history, laboratory, treatment and intervention data.Migration history extracted from the chart included 1) traveler/tourist from or to Canada, 2) a resident of Canada who was travelling abroad visiting friends or relatives 3) a recent immigrant to Canada 4) a recent refugee to Canada.A person whose travel originated from Canada, and whose purpose or the type of accommodation involved visiting friends and/or relatives, as documented in the chart was defined as visiting friends or relatives (VFR). An immigrant was defined as a person who was born outside of Canada, who has arrived to Canada for permanent residency and was not a refugee claimant. An immigrant on recent arrival was defined as an immigrant arriving to Canada at the visit considered in this study as being the point of exposure to malaria. An immigrant at a previous arrival was defined as a patient who arrived in Canada prior to the visit considered in this study as being the point of exposure to malaria. A refugee was defined as a person who had refugee status as documented by Interim Federal Health or on history taken by a healthcare worker.We defined ‘appropriate’ chemoprophylaxis as documentation that a patient was prescribed a anti-malarial prophylaxis regimen that was appropriate for planned travel itinerary. Severe malaria in this manuscript is defined based on WHO definition of severe malaria  which int-test was used to compare differences between two independent groups. Chi-squared test was used for categorical variables and Mann–Whitney U was conducted for continuous variables to compare differences between two independent groups where distribution was significantly different from normal. Results were considered significant when the probability of making a Type I error was <5% (p <0.05).Data was analyzed using SPSS . Statistical analysis of data was conducted using Kolmogorov-Smirnov Test for normality of distribution and Levene’s test was used to determine equality of variance. For variables with normal distribution student’s P. falciparum was identified in 76 (71%), P. vivax in 28 (26%), Plasmodium ovale in 2 (2%), and Plasmodium malariae in 1 (1%). The median age of infected children was 6.7 years (IQR 2.8 – 11.7) and 69 (64%) were boys  4–10) cases per year with no appreciable trend over the 16 years. P. falciparum infection were more likely to be born in Canada than those infected with P. vivax (p = 0.01).Out of the 99 (93%) of cases where country of birth was documented, 35 (35%) children were born in Canada. Sixty-two (63%) cases were recent or previous immigrants, 7 (7%) were documented as refugee claimants, 2 (2%) were recently internationally adopted children. Out of the 43 children that were documented to have had travel originating in Canada (Canadian born or previous immigrants), 41 (95%) were visiting friends or relatives (VFR) and 2 (5%) were traveling for tourism or studying abroad. Children with The most common locations of infection were West Africa (Ghana 24 (22%), Nigeria 21 (20%)), and South Asia (India 14 (13%), Pakistan 12 (11%))  were not prescribed any prophylaxis. Of those who were prescribed prophylaxis, 5 (23%) took chloroquine, 6 (27%) took mefloquine, one case took sulfadoxine and pyrimethamine, and one case took amodiaquine prior to arrival at the hospital. Appropriate prophylaxis for the region of travel was prescribed in 6 (27%) of cases, all but one of these cases recorded adherence to medication, however only in one case documented adherence to the medication was as directed. Documentation of past malarial history was available for 91 cases, of which 34 (37%) had reported previous episode of malaria.P. falciparum and those infected with P. vivax. There was no statistically significant difference in symptoms or signs between the groups. Notably, children infected with P. falciparum were more likely to have had more days of symptoms than those infected with P. vivax (p = 0.01).The most common symptoms described on history were fever (100%), vomiting 32%), headache (22%), and chills (20%). Fever was documented in the emergency room on presentation in 40% of cases, tachycardia in (20%), hepatosplenomegaly in 20%, hypotension in 5%, and CNS impairment  in 3%. Table 2%, headaP. falciparum infection compared to P. vivax infection (p = 0.03). One quarter (19) of children presenting with P. falciparum had severe malaria. All patient satisfying the WHO criteria for severe malaria had hyperparasitemia, and three cases had additional clinical features ). There were no significant differences in time away from Canada, time to presentation, delay in treatment, hospital stay between patients with non-severe and severe malaria.Median parasitemia was 0.4% (IQR 0.1 – 2.3), where the maximum parasitemia seen was 31%. Forty-six (43%) cases had parasitemia of = <0.1%. Of those with parasitemia >0.1%, the median parasitemia was 1% (IQR 0.4 – 5). There was a greater median parasitemia in children with P. falciparum, and 16 with P. vivax) between the years of 2006 to 2013. Binax T1 was positive for 22 cases (21 (95%) of which had P. falciparum) and Binax T2 was positive for 29 (16 (55%) of which were infected with P. falciparum). Binax T1 tests were negative for 2 children who were confirmed to have P. falciparum on blood smear. Similarly, Binax T2 tests were negative for 4 patients who were confirmed to have P. vivax. Binax T1 Sensitivity for P. falciparum 91.3%. Binax T2 sensitivity for P. falciparum 69.6%. Binax T1 is not sensitive for the detection of P. vivax whereas Binax T2 has a sensitivity of 75% for P. vivax. Our data is limited to patients who had positive microscopy for malaria, thus specificity cannot be calculated.Binax test results performed by the Ontario Public Health Laboratory were available for 42 patients  than those infected with P. vivax (p = 0.01). No child had severe anemia as defined by the WHO criteria for severe anemia . Mean platelet count for those presenting with P. falciparum was 116 × 103/μL, and for those presenting with P. vivax was 105 × 103/μL there were no statistically significant difference between groups.Children infected with P. falciparum were treated with single agent or combination of quinine (69%), atovaquone-proguanil (32%), sulfadoxine-pyrimethamine (16%), doxycycline (13%), clindamycin (22%), or single agent artesunate (5%). Intravenous quinine was used in the emergency room even for patients eventually discharged and treated subsequently with PO quinine as outpatients. Non-falciparum malaria infections were treated with single agent chloroquine (10%) or combination agents doxycycline and quinine (12%) and primaquine and chloroquine (46%) or primaquine and atovaquone-proguanil (32%). Sulfadoxine-pyrimethamine was predominantly used prior to the year 2001 and atovaquone-proguanil after 2001. Antimalarial treatment was refused in one case of an 8.5 year old boy exposed in Pakistan a year prior to presentation with 0.1% parasitemia of P. vivax. This child was followed up over the telephone after not presenting to the follow-up infectious disease clinic. Parents had preferred treatment with tincture of quinine despite a prescription of primaquine. Further telephone follow-up indicated the child was asymptomatic with no adverse events.Children infected with P. falciparum, were transferred to the pediatric intensive care unit. Of these five patients, length of stay ranged from 3–5 days. In total, nine patients received blood transfusions, 1 patient received exchange transfusion, 1 patient refused blood transfusion due to religious beliefs. There were no reported cases of mortality.Five patients (5%), all P. falciparum had longer stay compared to P. vivax (p = 0.02). Of those 9 patients who were treated as outpatients with P. falciparum, only one patient had percent parasitemia >0.1%. This patient was an 8 year old girl who had a parasitemia of 2%, arrived from Nigeria 2 days prior to presentation and was claiming refugee status. She was given IV quinine prior to discharge from the emergency department (ED), and follow-up with the Infectious Disease team at SickKids within 2 days.Table E. coli urinary tract infection). There were 10 cases that had a primary diagnosis other than malaria and had low malaria parasite load (<0.1%).Blood cultures were routinely collected for patients being admitted to hospital. Broad spectrum antibiotics was administered for 35 (33%) of patients, of which 3 (3%) eventually had a confirmed bacterial infection  public health agency of Canada <http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/>.Imported pediatric malaria remains an important cause of fever in returning travelers. Our study is consistent with other case reviews revealing that travelers visiting friends and relatives contribute the bulk of imported cases. Use of chemoprophylaxis was low and in only one case did we find that the appropriate drug was prescribed for the region of travel and that the patient reported proper adherence. This emphasizes that awareness amongst both travelers and primary care physicians play an important role in malaria prevention by providing pre-travel counseling and prescription of appropriate chemoprophylaxis along with non-pharmaceutical prevention of malaria . Physicians can refer to up to date chemoprophylaxis information from the Center for Disease Control Yellow Book “Health information for International Travel 2014”: <P. vivax, where 20% of patients were in Canada more than 2 months prior to presentation. Many patients had seen multiple primary care physicians . Primary care and ED physicians should be aware that vital signs and physical examination are often times nonspecific and that travel history may be remote.We also report the nonspecific signs and symptoms of malaria presentation of which fever is most often present in the history. However documentation of a fever in the ED was only present in 40% of our cases. Further, travel history was often remote particularly in those infected with The most common regions of acquisition of malaria reported in our study are consistent with those in the literature. In general, the top source country for newcomers in Canada are China, India, Pakistan. Source countries (between the year 2000 and 2013) with high risk of malaria are in order: Nigeria, Afghanistan, Cameroon, Democratic Republic of Congo, Guyana, Ghana, Rwanda, Uganda, and Honduras. In general, countries with English, such as Guyana, or French as a predominant language such may be choice countries for Canadians to travel or for tourism or education and as such may be overrepresented in our data set even though the risk of acquisition of malaria is by comparison low in these countries , 15.The BinaxNOW results reported in this study are consistent with reported sensitivities and specificities of this test in the literature , 19. ThiP. falciparum usually present within one month of travel, while those with P. ovale or P. vivax infections can present years later [According to the ‘Evidence-based clinical guidelines for immigrants and Refugees’ , there irs later , 27.The other major group in this study were the VFR children who are a group that should be targeted for pre-travel advice. These children VFRs are less likely to seek pre-travel health advice, take anti-malarial prophylaxis or take bite-prevention measures , 26, 28.It is important to engage the patient in pre-travel counselling as those that do take prophylaxis have a milder course, present with lower parasitemia rates, and if they adhere to the medication as prescribed rarely get malaria . ChemoprThe strengths of our study include the large number of years studied which spans changes in diagnosis techniques and treatment management. Furthermore the population of Toronto is a uniquely international population that represents modern trends in travel, tourism, and immigration. However the data that we include is from one institution and although the largest tertiary paediatric care hospital in the region with experienced infectious disease consultants, we do not present data from other institutions and outpatient clinics that would see returning travelers and vulnerable populations such as immigrants and refugees.Other implications from this study are that new immigrants/refugees are at high risk for malaria soon after their arrival to Canada. People new to many provinces in Canada, such as Ontario, do not have health care coverage for the first 90 days, in addition there have been heath care cuts to refugee claimant from certain countries , which tMalaria continues to be a significant disease in returning pediatric travelers and immigrant or refugee populations. Continued prevention with appropriate pre-travel advice and increase in physician awareness is required to reduce incidence and decrease delay to treatment.


Methadone maintenance treatment programs (MMTPs) are important public health intervention to control the human immunodeficiency virus (HIV) and the drug use problems. For expanding treatment coverage, publicly funded programs may be necessary for heroin users with low socio-economic status. We evaluated the difference of demographics, clinical features, and quality of life (QoL) of heroin users enrolled in publicly funded and self-paid MMTP and explored determinants influencing their attendance rate, respectively, for these two groups.A total of 234 heroin users enrolled in MMTP (129 in publicly funded and 105 in self-paid) between 2006 and 2008 self-reported the Taiwan version of the World Health Organization Quality of Life Instrument, Brief Version (WHOQOL-BREF) at baseline. Data regarding demographic and clinical features were collected during baseline interview. Methadone per 3-month attendance rates up to 18 months were conducted for each participant beginning from the index date.Self-paid group had a better QoL but lower treatment adherence than did the publicly funded group. Male and living alone were positive predictors on attendance rate for publicly funded group, and age of first heroin use and hepatitis C virus (HCV) seropositive were negative predictors. However, predictors on attendance rate for self-paid group were different from publicly funded group: HCV seropositive was a positive predictor and social QoL was a negative predictor.Findings of this study should be concerned with modifying original funding eligibility. Additional measures to explore what could impede treatment adherence are needed. Taiwan implemented a National Health Insurance (NHI) program from March 1995, offering a comprehensive, unified, and universal health insurance program to all citizens . HoweverAs for comparing the publicly funded and self-paid MMTP, we recommend using an important outcome index, quality of life (QoL). QoL refers to a subjective evaluation which is embedded in a cultural, social, and environmental context . There wIn addition to the QoL, adherence is another important issue for MMTP participants. Several studies from the West –21 foundUsing an 18-month follow-up data from one psychiatric center in south Taiwan, this observational study aimed to (1) examine the QoL difference between methadone maintenance patients presented for publicly funded and self-paid MMTP and (2) explore determinants influencing MMTP adherence using attendance rate, respectively, for these two groups.Diagnostic and statistical manual of mental disorders, fourth edition) criteria for opioid dependence; (3) having sufficient mental competence to understand and sign an informed consent; (4) residing near our treatment service site ; and (5) no other MMTP contraindication, such as severe liver cirrhosis, severe cognitive impairment, or behavioral disturbances. None of the participants had participated in any MMTP before the intake interview. At the intake interview, the participants were identified as publicly funded MMTP (n = 129) or self-paid MMTP (n = 105) groups based on whether they met the publicly funded eligibility criteria: (a) as a function of HIV status; (b) applying for deferred prosecution; and (c) had been incarcerated due to the Drug Act before and discharged during the recruited period. Methadone attendance of 18 months was conducted for each participant from the index date, and we divided the study period into six phases of 3 months duration each. The Hospital Ethics Committee of Jianan Psychiatric Center approved this study .A retrospective cohort study was conducted, and all recruited participants between March 2006 and July 2008 were diagnosed as opioid dependence by a board-certified psychiatrist from the Jianan Psychiatric Center research team. The inclusion criteria were as follows: (1) being more than 20 years old; (2) meeting the DSM-IV , which contains 28 items with 26 standard items from the original WHOQOL-BREF, and two Taiwanese national items . In addiIn addition to the WHOQOL-BREF, each MMTP patient completed a background information sheet including his or her birth date, gender, living status , educational years, age of first heroin use, heroin using years, employment status (fixed or not), and family drug using. After completing the WHOQOL-BREF and the background information sheet, each patient underwent a series of laboratory tests, including HIV, HBV, and HCV tests.χ2 tests (for frequency comparisons) and independent t tests (for mean comparisons). In addition, QoL scores of the two groups were separately compared with QoL scores of Taiwan population using one-sample t tests. The mean (±SD) QoL scores of Taiwan population are 14.06 ± 2.34 for the physical, 13.23 ± 2.15 for the psychological, 13.56 ± 2.29 for the social, and 12.72 ± 2.07 for the environment domains [Baseline characteristics and QoL scores between publicly funded and self-paid MMTP groups were compared using  domains .Generalized estimating equation (GEE) was used to examine the effects of several predictors on attendance rate. Because each participant had one to six attendance rates  from participating in the MMT program to 18 months later, using the GEE is adequate. All the analyses were done using SPSS 15.0 .t = 3.59, P < 0.001) and HIV carrier . The publicly funded group had significantly more heroin using years (9.25 ± 6.77 vs. 6.48 ± 3.86) and HIV carriers (n = 45 vs. 0) than the self-paid group. In addition, the self-paid MMTP participants had significantly higher QoL scores than the publicly funded MMTP participants in all QoL domains , living alone , age of first heroin use , and HCV carriers . As for the self-paid group, significant predictors were HCV carriers  and social QoL scores   Table .Table 3POur results are consistent with previous studies: heroin users had lower QoL scores as compared with the general population . This alWe found no demographic differences between heroin users under coverage of publicly funded and those of self-paid MMTP. However, QoL among publicly funded methadone patients was even worse. Although there was no demographic disparity between the publicly funded and self-paid MMTP, the publicly funded group had longer heroin abusing length and more HIV-seropositive patients than the self-paid group. This may explain the lower QoL  of the publicly funded group than that of the self-paid group. Previous studies indicate that people who have longer heroin use or who are HIV seropositive are more likely to have worse health status and QoL than their counterparts with shorter heroin use or HIV seronegative , 17.β = −0.054, 95 % CI = −0.124 to 0.016, P = 0.133). Gender difference of attendance pattern in our publicly funded group echo another recent finding [The most interesting finding is that predictors for MMTP attendance rate were different between publicly funded and self-paid groups. Our results of educational years and employment status as non-significant predictors are comparable to previous studies –34. Howe finding  that perβ = −0.068, P = 0.012). Therefore, we tentatively concluded that attendance pattern could be different between methadone patients contracted HCV and co-morbid HIV-HCV infection. However, certain baseline variables found to have an effect on the self-paid group used to predict attendance rate may have little effect on the publicly funded MMTP group. Further studies to examine the disparity between heroin users attending publicly funded and self-paid MMTP are urgently needed for policy makers.In addition to the demographic factors mentioned above, good social QoL and HCV seropositive were good predictors for worse and better methadone attendance, respectively, in our self-paid group. The negative influence of social QoL on the attendance rate could be explained by the misconception toward MMTP among their peer group, though we did not have solid evidences to justify this explanation, and future studies are warranted. Another explanation for the negative influence could be the good social interaction in the self-paid group. With good social interaction, the self-paid group tended to receive heroin from their friends and became less motivated to take methadone. On the other hand, the positive impact of HCV seropositive may be due to the prevalence and awareness of the self-paid patients. Methadone patients are found to have high prevalence but low awareness of HCV infection while attending MMTP , and theAlthough our results shed some light on the issues of QoL and treatment adherence for heroin users engaging in a MMTP, clinicians should interpret our results, including those in Tables The strength of this study was that newly admitted MMTP participants with better retention were our participants, while regular attendance could indicate better treatment outcome only under the circumstance of long enough treatment retention . Another advantage is that our study collected two kinds of important determinants (WHOQOL-BREF for measuring generic QoL and blood sample for confirming the chronic infectious disease) at baseline to predict attendance rate up to 18 months.However, this study has some limitations. First, our study involved only one site, limiting the generalization of its findings. Based on this limitation, readers need to know that our comparison results between MMTP patients and Taiwan general population were biased. Second, the study employed a cross-sectional design at the intake interview and the attendance rate may be affected by some potential important ongoing or time-dependent factors. Although previous Taiwan study reported no significant difference between HIV-positive and HIV-negative methadone patients , it shouTo the best of our knowledge, this is the first study comparing the QoL between publicly funded and self-paid heroin users and identifying their association of methadone attendance rate during 18 months follow-up. MMT is undoubtedly the key public health measures to control the HIV and the drug use problems. For treatment to be effective, regular attendance is necessary for reducing social costs in terms of drug-related legal and medical expenses. Previous study  has repo


Background. Unavailability of accurate, rapid, reliable, and cost-effective malaria diagnostic instruments constitutes major a challenge to malaria elimination. We validated alternative malaria diagnostic instruments and assessed their comparative cost-effectiveness. Method. Using a cross-sectional study design, 502 patients with malaria symptoms at selected health facilities in Ibadan between January and April 2014 were recruited consecutively. We examined malaria parasites using Cyscope®, QBC, and CareStart™ and results were compared to light microscopy (LM). Validity was determined by assessing sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Costs per hour of use for instruments and turnaround time were determined. Result. Sensitivity of the instruments was 76.0% (CareStart), 95.0% (Cyscope), and 98.1% (QBC). Specificity was 96.0% (CareStart), 87.3% (Cyscope), and 85.5% (QBC). PPV were 65.2%, 67.5%, and 84.7%, while NPV were 93.6%, 98.6%, and 99.4% for CareStart, Cyscope, and QBC with Kappa values of 0.75 (CI = 0.68–0.82) for CareStart, 0.72 (CI = 0.65–0.78) for Cyscope, and 0.71 (CI = 0.64–0.77) for QBC. Average cost per hour of use was the lowest ($2.04) with the Cyscope. Turnaround time was the fastest with Cyscope (5 minutes). Conclusion. Cyscope fluorescent microscope had the shortest turnaround time and is the most cost-effective of all the malaria diagnostic instruments evaluated. The world malaria report of 2014 showed that, as at 2013, millions of people at risk of malaria did not have access to interventions such as insecticide treated mosquito nets (ITNs), diagnostic testing, and artemisinin-based combination therapies (ACTs). As a result, 198 million cases and 584 000 deaths occur every year [Poor diagnosis hinders effective malaria control in many countries of SSA with associated economic impact. This is due to a combination of factors, including nonspecific clinical presentation of the disease, high prevalence of asymptomatic infection in some areas, lack of resources, insufficient access to trained health care providers and health facilities, and widespread practice of self-treatment for symptoms suggestive of malaria . One of Until recently, conventional Giemsa-stained light microscopy being the mainstay of malaria parasitological diagnosis in the absence of rapid tests is a major contributing factor to presumptive diagnosis. Though a valuable technique when performed correctly, it is unreliable and wasteful when poorly executed . ChallenThe introduction of newer and more rapid diagnostic techniques like Cyscope fluorescent microscopy (Cyscope), quantitative buffy coat fluorescent microscopy (QBC), and CareStart (a histidine-rich protein-2-based rapid diagnostic test (RDT) kit) was meant to overcome the challenges of using light microscopy. There is a need, however, to assess the performance and cost-effectiveness of these tools in order to guide further malaria management interventions in endemic and resource limited settings like Nigeria. This study was conducted to compare the diagnostic performance characteristics and cost-effectiveness of Cyscope , quantitative buffy coat , and CareStart  (a recently introduced RDT into the Nigeria market) for malaria parasite detection among clinically suspected malaria cases in a malaria-endemic environment.Malaria transmission has been intense, stable, and holoendemic in most parts of the country. Recent reports suggest some changes over time, such that, as at 2010, 85% of Nigerians lived in areas supporting mesoendemic transmission and 15% lived under conditions of hyper-holo-endemicity, with some areas within FCT and northeastern states supporting hypoendemicity . SeasonaWe carried out an evaluative cross-sectional study at the outpatient units of four health facilities in Ibadan, southwest Nigeria, between January 1 and April 30, 2014. The study sample size of 502 was calculated using a prevalence of 50% (due to nonavailability of published similar study as at the time of this study) with precision of 5% at 95% confidence interval and design effect of 1.5.We selected four health facilities comprising one tertiary health facility , one secondary health facility , and two primary health facilities  by simple random sampling in Ibadan southwest Nigeria as study sites. At the phlebotomy units of these facilities, patients suspected by clinicians to have malaria who presented with laboratory forms requesting malaria parasite investigation and consented to participate were recruited consecutively until the sample size of 502 was achieved. We defined a suspected case of malaria as a patient of any age for whom a clinician has made a decision to treat malaria with or without a history of fever or axillary body temperature ≥ 37.5°C. proforma register.Certified phlebotomists, following standard operating procedure, collected 1 mL of venous blood sample from each participant by venipuncture into an EDTA bottle. The specimen was tested for malaria parasites using the four diagnostic instruments, namely, light microscope, CareStart RDT, quantitative buffy coat, and Cyscope. The patients' biodata and the results of the performance of all the malaria diagnostic instruments were collected using a structured data collectionA WHO certified malaria microscopist examined the Giemsa-stained thick and thin films with a Zeiss light microscope  using the high power (40x) and the oil immersion (100x) objectives. Another WHO certified microscopist double checked the results. These microscopists, who hold bachelor degree in Medical Laboratory Science, were blinded to the outcome of other diagnostic instruments.μL of blood sample from each patient was applied to the preprepared slides of the fluorescent microscope, wet mounted with fluorescein dye, and viewed with the microscope. Presence (or absence) of malaria parasites was confirmed by viewing the fluorescent DNA of plasmodia under the microscope using ×40 objective.A trained and experienced medical laboratory scientist was engaged in the use of Cyscope malaria fluorescent microscope . Using the manufacturer's instruction, 10 A trained medical laboratory scientist with seven years of experience conducted QBC malaria test using QBC ParaLens Advance  according to the manufacturer's instruction. Plasmodium falciparum species) manufactured by Access Bio, Inc., New Jersey, USA, with Lot no. MO3B10 and expiry date July 2015. Manufacturer's instruction on standard operating procedures was followed.A trained medical laboratory scientist also tested individual specimens with CareStart, a histidine-rich protein-2 (HRP-2) RDT  as the time interval between receipt of the sample by the laboratory personnel who processed it and the time when results were generated following the standard operating procedure for each diagnostic instrument. The TaT of each procedure was monitored by trained research assistants using stopwatches and recorded in a structured data collection register.Cost-effectiveness analysis was calculated as the ratio of the resources used to the related effects, which is classified by comparison of the costs/input and consequences/outcome. The consensus paper on Guidelines on Health Economic Evaluation, Institut für Pharmaökonomische, Austria, was used for this analysis.The costs were separated into categories, namely, equipment, consumables cost, personnel, and miscellaneous items such as electricity.We made the following assumptions in costing the equipment: (i) that machine cost is per unit time of use, assuming uniform depreciation over time/lifespan of the equipment (fixed lifespan is 3 years for the entire machine), and (ii) that period of use was fixed at 8 hours per day.Machine cost per unit time of use = acquisition cost/total lifespan of equipment in real use days .Cost of reagents/consumables was calculated per session of use (8 hours/day).Personnel cost per unit time = standard monthly wage/hour, expressed in wage per hour. Basic staff qualification for CareStart usage is post-primary education while the staff qualification for other diagnostic tools is post-secondary education. Using the United States Office of Personnel Management, General Schedule Qualification Standards, 2014 (GS), given 8 hours/day and 21 working days, is as reflected in It was expected that each diagnostic tool produces a result (yield) for the malaria parasite test as the output or consequence of the use:Standard operating procedures (SOPs) were developed and validated for venipuncture and all laboratory procedures ensuring compliance with international practicing standard. Laboratory procedures were repeated by another experienced professional for each of the tools and a tie breaker observer was engaged where there were conflicting results. Data entered was double checked by the laboratory supervisor in charge to ensure consistency.Microsoft Excel (2008) and Epi Info version 7 were used for data entry, data cleaning, and analysis. We summarized data using proportions and means. Statistical analyses of the comparison of the diagnostic performance of the three methods of laboratory diagnosis of malaria parasites were carried out by assessing the sensitivity, specificity, and predictive values of all the instruments using light microscopy as a gold standard and inter-instrument agreement was analyzed using Kappa statistics at 5% level of significance. Plasmodium falciparum constituted 99% of malaria parasite species detected by LM. The sensitivity of the instruments compared with LM was 76% (CareStart), 95% (Cyscope), and 98.1% (QBC), while specificity was 96% (CareStart), 87.3% (Cyscope), and 85.5% (QBC). Positive predictive value was 84.7% for CareStart, 67.5% for Cyscope, and 65.2% for QBC, and negative predictive value was 93.6% (CareStart), 98.6% (Cyscope), and 99.4% (QBC)  .For inter-instrument agreement, all the diagnostic instruments tested, CareStart, Cyscope, and QBC, had good agreement with the light microscopy as shown by Kappa values of 0.71 (CI = 0.64–0.77), 0.72 (CI = 0.65–0.78), and 0.75 (CI = 0.68–0.82), respectively.The mean turnaround time for LM was 45.0 (standard deviation (SD): 1.9) minutes; for CareStart it was 17.4 (SD: 1.7) minutes; for Cyscope it was 4.5 (SD: 1.0) minutes; and for QBC fluorescent microscopy it was 8.5 (SD: 1.2) minutes . Per houThe cost-effectiveness ratio of LM was $10.77 per test; for CareStart it was $16.82 per test; for Cyscope fluorescent microscopy it was $2.04 per test; and for QBC fluorescent microscopy it was $5.89 per test . P. falciparum only in northeastern Tanzania and in Uganda however have reported sensitivities of 95.4% and 97.6% for Paracheck and Paracheck pf, respectively [This study validated three malaria parasites diagnostic test tools in comparison with light microscopy. Cyscope and QBC microscopes had sensitivity of 95% and above. This finding is in concordance with earlier studies in Nigeria and Uganda on sensitivity of diagnostic tools for malaria parasite detection which showed that QBC and Cyscope demonstrated higher sensitivity and CareStart lower sensitivity when compared with light microscopy , 12. Howectively , 14. CarThe absolute necessity for rational therapy in the face of ever increasing occurrence of drug resistance places increasing importance on the accuracy of malaria diagnosis . CurrentCyscope sensitivity complied with 95% WHO standard for sensitivity of rapid diagnostic test (RDT) . This fiWith respect to the operational characteristics of the tools, while light microscopy will produce about one result per hour, CareStart will produce three, and QBC and Cyscope will produce seven and twelve results per hour, respectively. Light microscopy is dependent on availability of electrical power for functionality. Only the Cyscope microscope has a standby battery as an alternative source of electric current.Light microscopy, QBC, CareStart, and Cyscope costs are $10.77, $5.89, $5.61, and $2.04, respectively, per hour of use. Considering the cost per hour of use of other tools, the cost of a test result from light microscopy is equivalent to cost of five test samples examined by Cyscope. In the same vein, costs of each of QBC and CareStart are equivalent to the cost of three test samples examined by Cyscope. Light microscopy, aside relatively cheap reagents, is the least cost-effective of all the instruments. falciparum malaria, the predominant Plasmodium species (94.8%) in Nigeria, which was corroborated in this study. Cyscope microscope for malaria detection is currently designed to detect only falciparum species. However, studies by Sousa-Figueiredo et al. from Uganda found that Cyscope detected all Plasmodium species but could not differentiate between them. Relatively high false positives were recorded in its usage from Uganda and Sudan which were attributed to small fluorescent bodies of unknown origin from the community/field mistaken as malaria parasites and submicroscopic parasitaemia among some pregnant women [ Plasmodium species. Although QBC has the best sensitivity and can detect all Plasmodium spp., the cost-effectiveness analysis and operational characteristics showed that its applicability may be more relevant in tertiary health care facilities.We found that Cyscope fluorescent microscope is a reliable diagnostic tool that is very sensitive and specific in diagnosingnt women , 19. FurAs with other diagnostic instruments, CareStart demonstrated best specificity and PPV while Cyscope showed better sensitivity and NPV. Cyscope microscope is a new technology that involves the use of lyophilized DNA probe in a fluorescein dye mounted on glass slide using wet preparation to detect malaria parasite in blood sample within 5 minutes, although it has likely chances of false positives especially in uncontrolled field setting . Its perThe Engineering and Physical Sciences Research Council, United Kingdom, holds that all new points of care diagnostics, including diagnostics for infectious diseases, should meet the ASSURED criteria, that is, Affordable by those at risk of infection, Sensitive with very few false negatives, Specific with very few false positives, User-friendly tests that are simple to perform and require minimal training, Rapid, to enable treatment at first visit, and Robust, for example, not requiring refrigerated storage, Equipment-free, and Delivered to those who need it. Based on these criteria and the need to expand malaria diagnostic services as part of a greater framework of health system strengthening within resource-limited settings , CyscopeThe findings of this study showed that CareStart, QBC fluorescent microscopy, and Cyscope fluorescent microscopy are valuable complements to light microscopy because they help expand the coverage of parasite-based diagnosis and minimize presumptive diagnosis. The cost of improved malaria diagnosis will inevitably increase, by investment in whether light microscopy or CareStart or both. However, such investment offers a more promising cost-effective strategy with the deployment of Cyscope fluorescent microscopy in appropriate settings.Cyscope fluorescent microscope had the least turnaround diagnostic time and the highest throughput and it is the most cost-effective of all the laboratory diagnostic instruments evaluated. We therefore strongly recommend Cyscope fluorescent microscope for malaria parasite detection especially in secondary and tertiary health care facilities where the request for malaria diagnosis is increasingly high.


Plasmodium falciparum invasion into the red blood cell (RBC) play an important role in clinical immunity to malaria. The protein family of parasite antigens known as P. falciparum reticulocyte binding protein-like homolog (PfRh) is required for RBC invasion. PfRh5 is the only member within the PfRh family that cannot be genetically deleted, suggesting it plays an essential role in parasite survival. This antigen forms a complex with the cysteine-rich P. falciparum Rh5 interacting protein (PfRipr), on the merozoite surface during RBC invasion. The PfRh5 ectodomain sequence and a C-terminal fragment of PfRipr were cloned and expressed in Escherichia coli and baculovirus-infected cells, respectively. Immunization of rabbits with these recombinant proteins induced antibodies able to inhibit growth of various P. falciparum strains. Antibody responses to these proteins were investigated in a treatment–re-infection study conducted in an endemic area of Papua New Guinea (PNG) to determine their contribution to naturally acquired immunity. Antibody titers to PfRh5 but not PfRipr showed strong association with protection against P. falciparum clinical episodes. When associations with time-to-first infection were analyzed, high antibody levels against PfRh5 were also found to be associated with protection from high-density infections but not from re-infection. Together these results indicate that PfRh5 is an important target of protective immunity and constitutes a promising vaccine candidate.Emerging evidence suggests that antibodies against merozoite proteins involved in  Plasmodium parasite is entirely responsible for malaria-associated pathology  surface followed by apical reorientation of the merozoite, tight junction formation and final entry into the erythrocyte  and the P. falciparurm reticulocyte binding protein-like homologs , which facilitates its expression on the merozoite’s surface for erythrocyte invasion  and when a child presented with symptoms at the local Mugil Health Centre (passive surveillance). Re-infection was detected by post-PCR ligase detection reaction–fluorescent microsphere assay (LDR-FMA) and light microscopy (LM) on Giemsa stained blood smears. Re-infections were categorized to (1) PCR-detectable, (2) LM-detectable, (3) LM-detectable re-infection with >500 parasites per μl, and (4) LM-detectable re-infection with >5000 parasites per μl. Clinical episode was defined as the presence of fever with ≥37.5∘C and >5000 parasites/μl. The study was approved by the Medical Research Advisory Committee (MRAC), PNG Ministry of Health, The Walter and Eliza Hall Institute Human Research Ethics Committee and the institutional review board of the Veteran’s Affairs Medical Center . Written consent was obtained from parents/guardians of all participants.Plasma samples were obtained from a prospective treatment–reinfection study of 206 children aged from 5 to 14 years conducted in Madang province of Papua New Guinea (PNG). Full details of this cohort have been previously described . BrieflyEscherichia coli expression and cloned into the pET-303 vector . The C-terminally HIS tagged expression product was refolded from the insoluble pellet material under standard conditions. PfRipr (AA604-1086) was cloned into pTri-Ex 2 vector for expression in baculovirus-infected Hi-5 cells . HIS-FLAG tagged PfRiPr protein was purified from the cell culture supernatant by incubation with M2-FLAG beads , followed by elution with FLAG peptide in NaCl-Tris buffer pH 8.0. The peptide was removed by dialysis prior to immunization. Purity and integrity of recombinant proteins were assessed in SDS–polyacrylamide gels . Rabbits were immunized twice with 100 μg PfRh5 in GLA-SE adjuvant , or three times with 200 μg PfRipr in Freund’s adjuvant. Total IgG was purified from immune sera as described  at a parasitemia of 0.5%. Parasitemia was counted after 48 h and specific growth inhibition calculated relative to parasites grown in non-immune IgG. Specific growth inhibition was calculated relative to parasites grown in non-immune IgG.Growth inhibition assays (GIA) using IgG isolated from immunized rabbits were performed as described . Briefly∘C. Empty sites were blocked with 5% skim milk for 1 h at 37∘C. After washing with 0.05% Tween-PBS, 100 μl of 1:2 serially diluted plasma samples were added to plates and incubated at 37∘C for 1 h. The plates were washed three times and incubated with a peroxidase-conjugated mouse anti-human antibody . Bound complexes were detected by reaction with tetramethy-benzidine  and H2O2. Absorbance was read at 450 nm. Plasma samples from malaria naïve anonymous Australian blood donors were included as negative controls. Antibody titers were calculated as the plasma dilution with an optical density (OD) value higher than that observed for negative controls at a 1/100 dilution.Microtiter plates were coated with recombinant PfRh5 (2 μg/ml) and PfRipr (0.5 μg/ml) in carbonate buffer pH 9.6 by overnight incubation at 4P. falciparum re-infection or clinical episodes. Cox proportional hazards modeling was used to calculate hazard ratios (HR) for time-to-first P. falciparum infection by PCR, LM, and time-to-first infection of >500 and >5000 parasites/μL. Poisson model was used to obtain incidence rate ratio (IRR) for the incidence of clinical malaria episodes throughout the study period. HR and IRR were adjusted for identified confounders. Differences in antibody titers between categorical variables were assessed using Wilcoxon rank sum test or Kruskal–Wallis tests.Statistical analysis was performed using STATA 9.2 . For analysis of associations with clinical outcomes, participants with no detectable parasitemia by PCR or LM during follow up were regarded as non-exposed and excluded from the analysis. Demographic and clinical variables were assessed as potential confounders. Kaplan–Meier method and log-rank test were used to explore associations between antibody titers and time to E. coli and baculovirus-infected insect cells, respectively. HIS-FLAG tagged proteins were purified with M2-FLAG beads. SDS-PAGE analysis of the purified recombinant proteins revealed highly pure protein preparations with predominant bands at 63 kDa corresponding to PfRh5 and 65 kDa corresponding to PfRipr .The PfRh5 ecotodomain sequence and the C-terminal PfRipr fragment from 3D7 were cloned and expressed in P. falciparum trophozoite cultures to determine their capacity to inhibit parasite growth. Figures 1B,C shows that both anti-PfRh5 and anti-PfRipr antibodies inhibit growth of P. falciparum 3D7, W2Mef, and FCR3 in a dose dependent manner. Incubation with 2 mg/ml of both antibodies resulted in ∼40% of growth inhibition of all parasite strains tested. Thus immunization with these novel recombinant proteins induces antibodies that prevent growth of multiple P. falciparum clones.To determine if the recombinant proteins were immunogenic, rabbits were immunized twice with 100 μg PfRh5 in GLA-SE adjuvant or three times with 200 μg PfRiPr in Freund’s adjuvant. Seroconversion of immunized rabbits was successfully confirmed by ELISA using PfRh5 and PfRipr recombinant proteins (data not shown). Total IgG was purified from immune sera 2 weeks after the last immunization and added to Figure 2 shows that anti-PfRh5 responses protected from high-density (≥5000 parasites/μL) parasitemia  but not against re-infection per se (assessed by PCR or LM). The association between antibody titers and the risk of acquiring new P. falciparum infections was also analyzed by Cox regression model. In agreement with previous studies with this cohort . However, a reduced risk of developing a high-density infection (>5000 parasites/μL) was observed for high anti-PfRh5 titers, even after adjustment for age and location of participants (Table 1).A preliminary screen conducted in the Madang Province of PNG suggested that antibodies to PfRh5 and to the EGF-like domain of PfRipr show an intermediate association with protection from clinical malaria . To furts cohort , age . A Poisson regression was used to test for associations between antibody levels and overall incidence of clinical disease. Increased anti-PfRh5 antibody responses were found to be strongly associated with reduced incidence of malaria clinical episode during the 6 month follow-up period . Moreover, anti-PfRh5 antibody titers were found to be significantly higher in individuals that did not experience clinical episodes compared to those that experienced 1, 2, or 3 malaria clinical episodes over the follow up period . Thus together these results indicate that naturally acquired anit-PfRh5 antibodies protect from parasitemia of high density and clinical illness.The association of anti-PfRh5 antibody responses with time-to-first clinical episode was also determined. Anti-PfRh5 antibody levels were found to be significantly associated with a protection from clinical malaria, as medium and high antibody responders showed 61.7 and 82.2% reduced risk of experiencing a clinical episode than low responders after the adjustment for confounders  appeared to be protected from parasitemia of high density (>5000 parasites/μl) but not from moderate-density parasitemia (>500 parasites/μl) or re-infection. Before adjustment for confounders, high responders showed a reduced risk to develop a high-density infection . However, this association did not reach statistical significance after adjustment for age and location of the study participants .The relationship between anti-PfRipr antibody titers and time to reinfection of different parasite densities was investigated in Kaplan–Meier survival curves and a log-rank test was used to determine the difference between high, medium and low responders. Table 2) as well as the overall incidence of malaria episodes throughout the 6 month follow-up period (Table 3) were similar amongst all responders, suggesting anti-PfRipr antibodies do not play an important role in clinical outcome.The association of anti-PfRipr antibody levels with risk of experiencing a malaria clinical episode was also determined. The risk of developing clinical episodes . To date, antibody responses to PfRipr have not been extensively investigated. Further work in other settings is required to determine whether this Plasmodium antigen is an important target of naturally acquired immunity to malaria.PfRh5 exists in a membrane-associated complex with the cysteine-rich protein PfRipr. We have previously found antibodies to e growth . Here imThere is an urgent need for a blood stage component in an anti-malarial vaccine to reduce parasite burdens responsible for morbidity and mortality in susceptible individuals. Development of a blood stage vaccine against malaria has been largely delayed mainly due to the high level of polymorphism in the few antigenic targets that were tested in clinical trials . Thus itConceived and designed the experiments: Chris Y. H. Chiu, Jennifer K. Thompson, Julie Healer, Alan F. Cowman, and Diana Silvia Hansen. Performed the experiments and analyzed the data: Chris Y. H. Chiu, Jennifer K. Thompson, Julie Healer, and Diana Silvia Hansen. Contributed essential reagents/materials/analysis tools: Lin Chen, Connie S. N. Li Wai Suen, Peter M. Siba, Lin Chen, Ivo Mueller, and Alan F. Cowman. Interpreted data and wrote the paper: Chris Y. H. Chiu, and Diana Silvia Hansen.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Mannose binding lectin, a plasma protein protects host from virus, bacteria, and parasites. Deficiency in MBL levels has been associated with susceptibility to various infectious diseases including P. falciparum malaria. Common MBL polymorphisms in promoter and coding regions are associated with decrease in plasma MBL levels or production of deformed MBL, respectively. In the present study, we hypothesized that MBL2 variants and plasma MBL levels could be associated with different clinical phenotypes of severe P. falciparum malaria.Methods: A hospital based study was conducted in eastern Odisha, India which is endemic to P. falciparum malaria. Common MBL-2 polymorphisms  were typed in 336 cases of severe malaria (SM)  and 131 un-complicated malaria patients (UM). Plasma MBL levels were quantified by ELISA.Results: Severe malaria patients displayed lower plasma levels of MBL compared to uncomplicated falciparum malaria. Furthermore, on categorization of severe malaria patients into various subtypes, plasma MBL levels were very low in MOD patients compared to other categories. Higher frequency of AB genotype and allele B was observed in MOD compared to UM . In addition, prevalence of YX genotype of MBL Y-221X polymorphism was also statistically more frequent in MOD case than UM (P = 0.009).Conclusions: The observations of the present study reveal that MBL-2 polymorphisms (codon 54 and Y-221X) and lower plasma MBL levels are associated with increased susceptibility to multi organ dysfunctions in P. falciparum malaria. Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. In a recent report, World Health Organization (WHO) estimates about198 million cases in 2013 and 1.2 billion subjects are at high riskworldwide , multi-organ dysfunction (MOD), and non-cerebral severe malaria (NCSM)  has been linked to susceptibility to severe malaria , renal failure (serum creatinine>3 mg/dl), and hepatic dysfunction . Patients with following criteria were excluded from current investigations: (i) Co-infection with other Plasmodium species, (ii) chronic disease like tuberculosis, chronic renal failure, cirrhosis of liver and autoimmune diseases like systemic lupus erythematosus and rheumatoid arthritis. Patients enrolled in the present study came from the coastal districts of the state having an average annual parasite index (API) of 6.67. 100 healthy controls were included from the same districts and selectively included so that both the groups are uniformly exposed during transmission of malaria. None of the controls reported history of clinical malaria in the last 5 years. They were essentially healthy and negative for demonstrable P. falciparum infection. Demographic characteristics of enrolled subjects are shown in Table In the present study, patients attending and/or admitted to Department of Medicine, SCB Medical College and Hospital, Cuttack, Odisha were included. Odisha is endemic for malaria and more than 85% cases are attributed to Genomic DNA was isolated from whole blood by Gen Elute Blood Genomic DNA Kit  according to manufacturer's instructions. MBL promoter polymorphisms (−550 H/Land −221 Y/X) and codon 54 polymorphism (A/B) were typed by double amplification refractory system (dARMS) and allele specific polymerase chain reaction (AS-PCR) respectively, as described earlier by us  kit (R&D Systems) according to manufacturer's instructions.http://www.tufts.edu/~mcourt01/Documents/Court%20lab%20-%20HW%20calculator.xls). Fisher's test was used for comparison of genotype, allele frequencies in different clinical categories of P. falciparum malaria. Odds ratios (ORs), 95% confidence intervals (95% CIs) were calculated by Graphpad prism 5.01. P value less than 0.01 was taken assignificant (Bonferroni correction for three SNPs 0.05/3 = 0.01). Mean plasma MBL levels in various clinical categories were compared by analysis of variance (ANOVA) followed by Turkey's multiple comparison post-test.Genotype and allele frequency were calculated by direct counting. Web based Microsoft Excel tools was employed to calculate Hardy-Weingberg equilibrium (P < 0.001) and uncomplicated malaria cases (P < 0.05). Furthermore, on analyses of the subtypes of SM, namely, CM, MOD, and NCSM, MOD patient showed least plasma MBL levels (107.1 ± 17.09 ng/ml) than CM (223.4 ± 27.66) and NCSM (268.5 ± 49.25). However, mean plasma MBL levels among different clinical categories of SM patients were not statistically significant. Mean plasma levels of MBL in HC was significantly higher compared to CM (P < 0.001), MOD (P < 0.001), and NCSM (P < 0.05).As shown in Figure 2 = 9.87, P = 0.001; MBL (H-550L): χ2 = 2.92, P = 0.08; MBL (Y-221X): χ2 = 9.98, P = 0.001].Among 100 healthy individuals, 15 were heterozygous and five were homozygous for codon 54 polymorphism. The prevalence of HL and LL genotypes of MBL (H-550L) polymorphism was found to be 25 and 69%, respectively. The distribution of YX and XX genotypes of MBL promoter (Y-221X) was 29 and 16% respectively in healthy individuals. Frequency of MBL polymorphisms  were comparable to our earlier report ; MOD (n = 20); NCSM (n = 10); UM (n = 12); and HC (n = 25)]. Association between MBL2 polymorphisms and plasma levels of MBL are shown in Figure P < 0.01) and BB (P < 0.01) genotypes . Furthermore, dominant inheritance model comparison reveled a significant association with susceptibility of MOD . Heterozygous (YX) for MBL promoter polymorphism (Y-221X) was more frequent in MOD cases than UM . Distribution of MBL2 (H-550L) genotypes were comparable among different clinical categories of P. falciparum malaria. Prevalence of MBL2 codon 54 heterozygous (AB), minor allele (B) and heterozygous for MBL2 (Y-221X) polymorphism (YX) was higher in severe malaria compared to UM, but these differences could not reach statistically significance levels after Bonferroni correction.Association of MBL2 polymorphism with mortality due to P. falciparum infection, distribution of codon 54, H-500L, Y-221X and combined genotypes was analyzed among subjects those died and survivors. No significant association of promoter, codon 54 polymorphism or combined genotypes was observed with malarial mortality (Data not shown).To find out association of P. falciparum malaria. As shown in Table P = 0.009, OR = 3.04, 95% CI = 1.25 to 7.37) and multi organ dysfunction  compared to uncomplicated malaria.As plasma MBL levels correlated with combined MBL2 genotypes, we analyzed distribution of MBL2 combined genotype in different clinical manifestations of P = 0.005, r = −0.46), serum urea  and alkaline phosphatase . These observations appear to be associational rather than a cause and effect relationship.We analyzed the possible correlation between plasma MBL levels and various clinical parameters and results are shown in Table P. falciparum malaria in an endemic region of Odisha, India. Our data suggest that low levels of MBL and MBL2 low producer genotypes are associated with multi organ dysfunction. Furthermore, in the current investigation we also confirm a correlation between MBL2 polymorphism with plasma levels of MBL.In the present case control study, we investigated the role of MBL in P. falciparum infection. MBL binds to infected erythrocytes  and Y-221X (YX) polymorphism had 2.34 and 2.06 fold higher chance of developing MOD. Furthermore, combined analysis of codon and promoter polymorphism revealed significant association of low producer genotype  with susceptibility to MODs (OR = 4.03). Earlier studies reported the role of MBL2 polymorphism in severe malaria. MBL2 codon 54 and 57 variants in Gabonese children, LXPA haplotype in Indian population  and promoter (H-550L) polymorphism deviated from HWE in line with our earlier observation  that modulates severity in AP was involved in design, performing experiments, analysis, interpretation, performed statistics, and writing the first draft of the manuscript. BD made a contribution in the design, data interpretation, work supervision and critically revising the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.