Combinations of Host Biomarkers Predict Mortality among Ugandan Children with Severe Malaria: A Retrospective Case-Control Study

Background: Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings: Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1-99.9) sensitivity and 88.8% (79.7-94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions: We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria

ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A(1), A(2), and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria

Infected O erythrocytes are phagocytosed more avidly by human macrophages.

<p><i>P. falciparum</i> (ITG and 3D7)-infected and uninfected A, B and O erythrocytes were incubated with A and O blood group macrophages for 90 minutes (ring-stage) or for 120 minutes (mature-stage). The phagocytic index was then calculated by determining the number of internalized parasites in ≥250 macrophages and the data were normalized to the average phagocytic index of infected A erythrocytes. (A) Data for ring-stage parasitized erythrocytes are presented as the combined results of two independent experiments. Data for each blood group (n = 3 donors/group) are shown as box-and-whiskers plot, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. There was a significant increase in the phagocytic uptake of ring-stage infected O erythrocytes compared to A and B infected erythrocytes (*p = 0.022, and **p = 0.007, respectively; Student's <i>t</i>-test for paired samples with Bonferroni correction for multiple comparisons). (B) Data for mature-stage parasitized erythrocytes represent the combined results of six independent experiments using multiple donors per group (A: n = 6; B: n = 3; O: n = 4) and are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. There was significantly enhanced phagocytic uptake of infected O erythrocytes observed compared to A and B infected erythrocytes (*p<0.01 and **p<0.05, respectively; Student's <i>t</i>-test with Bonferroni correction for multiple comparisons). (C) Macrophages were isolated from donors of blood groups A and O and phagocytosis assays were run in parallel. The blood group of the infected erythrocytes was found to influence phagocytic uptake, with infected O erythrocytes being preferentially phagocytosed (*p<0.01, two-way ANOVA), whereas the blood group of the macrophage had no effect on the uptake of infected A and O erythrocytes (p>0.05). Data represent three independent experiments normalized to the average phagocytic uptake of infected A erythrocytes by macrophages isolated from blood group A donors.</p

Blood group O (H antigen) status affects the phagocytic uptake of infected erythrocytes by human macrophages.

<p>Erythrocytes of blood group O and B were treated with B-zyme, thereby removing the terminal α-1,3-galactose from blood group B antigens. (A) Flow cytometric analysis with anti-B (clone 9621A8) and phycoerythrin (PE)-conjugated rat-anti-mouse kappa as secondary antibody. In the dot plot, the x and y axes represent FL1-derived fluorescence and PE-derived fluorescence, respectively, on logarithmic scales. Results show cleavage of the terminal galactose from B erythrocytes and enzymatic conversion from group B to erythrocytes which type as group O: (i) Native, untreated O erythrocytes, (ii) Native, untreated B erythrocytes, (iii) B-zyme-treated B erythrocytes. Group O cells mock-treated with B-zyme gave identical results to the group O untreated control (data not shown). (B) The phagocytic uptake was determined by counting the number of internalized infected erythrocytes in 250 individual macrophages and data was normalized to the average phagocytic index of infected untreated B erythrocytes. Data represent two independent experiments using the <i>P. falciparum</i> ITG clone. Each blood group is represented by four different donors. Bar graphs represent the mean±SEM. Significance was determined by Mann Whitney test with Bonferroni correction for multiple comparisons. There was an observed increase in the phagocytosis of untreated infected O erythrocytes when compared to untreated infected B erythrocytes (*p<0.05). The phagocytic index of the infected, B-zyme-treated B erythrocytes was significantly increased from phagocytic index of infected untreated B erythrocytes (**p<0.01). However, there was no significant difference in the uptake of treated vs. untreated infected O erythrocytes.</p

<i>P. falciparum</i> parasite invasion of A, B and O erythrocytes.

<p>Levels of parasite invasion and maturation during two cycles of growth of <i>P. falciparum</i> clone ITG in A, B and O erythrocytes. Data are presented as the combined results of 4 independent experiments using erythrocytes from healthy human volunteers with blood groups A (n = 7 donors), B (n = 4 donors) and O (n = 6 donors). Data are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. Statistical significance was determined by a one way ANOVA. Invasion is defined as the percentage of ring parasitemia, as measured 24 hours and 72 hours after inoculation. Maturation is defined as the percentage of trophozoite parasitemia, as measured 48 hours and 96 hours after inoculation. There were no significant differences observed in the invasion and maturation of <i>P. falciparum</i> parasites in A, B or O erythrocytes.</p

Hemichrome deposition and band 3 aggregation are increased in infected O erythrocytes.

<p>Hemichrome levels and band 3 aggregation were measured in uninfected, ring-stage infected and mature-stage infected A, B and O erythrocytes. (A) and (B) Hemichrome data represent the combined results of 6 independent experiments (means±SEM of ≥3 different donors per blood group) using <i>P falciparum</i> ITG and 3D7 clones and are expressed in nmol/ml of packed erythrocytes. Compared to infected A and B erythrocytes, hemichrome deposition was significantly increased in ring-stage infected (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002942#ppat-1002942-g007" target="_blank">Figure 7A</a>, *p = 0.005, **p = 0.038, respectively) and mature-stage infected O erythrocytes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002942#ppat-1002942-g007" target="_blank">Figure 7B</a>, *p = 0.013, **p = 0.024, respectively; Mann Whitney for two-tailed distribution). (C) Gel filtration chromatography effluents of Tween-20 membrane extracts of <i>P. falciparum</i>-infected A, B and O erythrocytes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002942#ppat-1002942-g007" target="_blank">Figure 7C</a>) were analyzed for aggregated band 3 protein as absorbance using the Bradford reagent at 595 nm and eosin-5-maleimide fluorescence indicating the location of band 3 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002942#ppat-1002942-g007" target="_blank">Figure 7C</a> insert). Increased high molecular weight aggregates of band 3 were observed in infected group O erythrocytes. The results presented are representative of 3 independent experiments.</p

Increasing phagocytosis of erythrocytes correlates with decreasing A antigen.

<p>A and O erythrocytes were typed using standard hemagglutinin techniques. Blood group A was further classified into A₁ or A₂ subgroups using <i>Dolichos biflorus</i> lectin. (A) Flow cytometric testing of A₁, A₂, O, and Bombay erythrocytes with anti-H (clone BRIC231) FITC conjugated antibody. FITC-derived fluorescence is displayed on the x axis on a logarithmic scale and the number of cells on the y axis. O (solid black line), A₂ (dotted black line), A₁ (dashed black line) and Bombay (filled grey). (B) A₁, A₂ and O erythrocytes infected with <i>P. falciparum</i> (ITG clone) were incubated with human monocyte-derived macrophages. The phagocytic index was calculated by determining the number of internalized parasitized erythrocytes within 250 macrophages. Data were normalized to the average phagocytic index of infected A₁ cells. Data represent 3 independent experiments and each blood group is represented by multiple donors, A₁ (n = 8/group), A₂ (n = 3/group), and O (n = 8/group). The box plots represent the median, interquartile and complete range. The phagocytosis of infected erythrocytes increased in an H antigen dose-dependent manner (r = 0.80, *p<0.0001; Spearman's correlation test).</p

Phosphatidylserine levels on <i>P. falciparum</i>-infected A, B and O erythrocytes.

<p>Annexin staining of A, B and O uninfected and <i>P. falciparum</i>-infected erythrocytes was evaluated. Freshly purified erythrocytes (n = 4 for each group) from healthy donors were left uninfected or were infected with ITG, 3D7 or E8B clones and stained by annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Data are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median level of percentage of annexin-V positive cells among total cell population. There were no significant differences observed between infected A, B, or O group erythrocytes.</p

Murine monocytes phagocytose infected O erythrocytes more efficiently <i>in vivo</i> than infected A or B erythrocytes.

<p>C57BL/6 mice were injected intraperitoneally with 50×10⁶ purified mature-stage <i>P. falciparum</i> parasites cultivated in human A, B or O erythrocytes. Three hours after injection, resident monocytes were collected, washed, and plated on glass coverslips. (A) The phagocytic index was calculated by counting the number of internalized parasites within 250 monocytes and the data were normalized to the average phagocytic index of infected A erythrocytes. Data represent three independent experiments using the <i>P. falciparum</i> strain ITG and each blood group is represented by at least 3 different donors, A (n = 5), B (n = 3) and O (n = 5). Bar graphs represent the mean±SEM. There was a significant increase in the phagocytic uptake of infected O erythrocytes compared to the phagocytic uptake of infected A or B erythrocytes (*p<0.05; Student's <i>t</i>-test with Bonferroni correction for multiple comparisons). (B) Photomicrographs show representative examples of increased uptake of mature-stage infected (i) O erythrocytes, versus mature-stage infected (ii) B erythrocytes. Images were acquired with an Olympus BX41 microscope and an Infinity capture camera at 1000× magnification.</p