Correlation analysis.

<p>Plasma and sera samples from seronegative/healthy (SN/H, n = 45) and seropositive/chagasic (SP/C, n = 116) subjects were submitted to spectrophotometry analysis of various biomarkers of inflammation, oxidative stress, antioxidant status and cellular injury as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002364#s2" target="_blank">Materials and Methods</a>. Pearson's or Spearman's analysis was conducted to evaluate the strength of linear relationship between sera or plasma levels of biomarkers (among themselves) or with clinical disease category. Correlations coefficient (r) value of >0.8 was considered very strong and that of between 0.6–0.8 accepted as moderately strong <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002364#pntd.0002364-Chan1" target="_blank">[31]</a>. “-”indicates a significant correlation at p<0.01 was not present. ND: not determined.</p

Characterization of the subjects included in the study.

a<p>Subjects were screened for <i>T. cruzi</i>-specific antibodies by Wiener Chagatest-ELISA and Wiener Chagatest-HAI kits. Clinical exam included physical exam, electrocardiography and echocardiography.</p>b<p>Seronegative subjects from non-endemic areas were screened for <i>T. cruzi</i>-specific antibody response. Seronegative/cardiac patients exhibiting clinical symptoms were identified based upon blood levels of NT-proBNP to be >2000 pg/ml (normal <450 pg/ml).</p

SOD and GSH are indicators of compromised antioxidant status in chagasic subjects.

<p>Plasma <i>(</i><b><i>A, C, E</i></b><i>)</i> and sera <i>(</i><b><i>B, D, F</i></b><i>)</i> levels of activities of the superoxide dismutase (<b>A&B</b>) and glutathione peroxidase (<b>C&D</b>) were determined by spectrophotometry. The glutathione contents (<b>E&F</b>) were measured by GSSG-DTNB recycling assay.</p

Pair-wise correlation and modeling analysis.

<p>Pair-wise correlation analysis of (<b>A</b>) nitrite (µmol/mg protein) with LPO (µmol/ml) and (<b>B</b>) glutathione peroxidase (units/mg protein) with AOPP (µmol/ml) utilizing data from plasma analysis of seropositive/chagasic and seronegative/healthy subjects is shown. (<b>C&D</b>) MARS analysis was performed using 80% of the data for various biomarkers from seronegative/healthy and seropositive/chagasic subjects as training dataset (blue curve) and 20% of the remaining data as test dataset to assess the performance of the model (red curve). Shown in panel C is MARS analysis of plasma levels of the biomarkers that revealed model fits perfectly (AUC/ROC = 1) on the training data for LPO, nitrite and SOD (with AUC/ROC of 0.099955 with testing dataset). Shown in panel D is MARS analysis of sera levels of the biomarkers that revealed model fits perfectly (AUC/ROC = 1) on the training data for MPO, LPO, and nitrite (with AUC/ROC of 0.9589 with testing dataset). (<b>E&F</b>) Shown are pair-wise correlation analyses of MPO (E) and LPO (F) contents with clinical disease. Each dot represents an individual subject.</p

LPO and MDA are biomarkers of increased oxidative stress in chagasic subjects.

<p>The plasma <i>(</i><b><i>A&C</i></b><i>)</i> and sera <i>(</i><b><i>B&D</i></b><i>)</i> levels of lipid hydroperoxides (<b>A&B</b>) and malondialdehyde (<b>C&D</b>) were measured by spectrophotometry.</p

Inter-linked innate immune responses and oxidant/antioxidant status are major determinants of chronic Chagas disease.

<p><i>Trypanosoma cruzi</i> or danger-associated molecular patterns (DAMPs) released due to cellular injuries stimulate ROS (O₂^−•, H₂O₂, ^•OH) production by (1) NADPH oxidase–dependent oxidative burst in macrophages/monocytes and (2) mitochondrial membrane permeability transition and electron transport chain (ETC) inefficiency in non-phagocytes (e.g. cardiac myocytes). Likewise, <i>Tc</i> and DAMPs can activate phagocytes/neutrophils resulting in (3) iNOS-dependent ^•NO and (4) myeloperoxidase-dependent HOCl production. If these reactive species are not scavenged by (5) activation of antioxidants (e.g. superoxide dismutases (SOD), glutathione peroxidase (GPX) and glutathione (GSH), then highly stable free radicals, e.g., peroxynitrite (^•OONO), nitrosyl chloride and AOPPs are formed that (6) further inflict host cellular oxidative damage of lipids (e.g. LPO, MDA). The intracellular molecules (e.g., DNA, protein, lipids) that may be released from apoptotic or necrotic cells in oxidized form serve as DAMPs, sustaining the signaling activation of innate immune cells in chronically infected chagasic subjects.</p

Neutrophil (MPO) and macrophage (iNOS) activation indicate inflammatory state of seropositive/chagasic subjects.

<p>Plasma and sera samples were obtained from seronegative/healthy (SN/H), seropositive/chagasic (SP/C) and seronegative/cardiac (SN/C) subjects, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002364#s2" target="_blank">Materials and Methods</a>. Shown are the plasma <i>(</i><b><i>A,C,E</i></b><i>)</i> and sera <i>(</i><b><i>B,D,F</i></b><i>)</i> levels of MPO activity (<b>A&B</b>), AOPP contents (<b>C&D</b>) and nitrite levels (<b>E&F</b>). For all figures, data (mean of triplicate observations from each sample) are presented as box plot. The horizontal lines of the box (bottom to top) depict the lower quartile (Q1, cuts off lowest 25% of the data), median (Q2, middle value), and upper quartile (Q3, cuts off the highest 25% of the data). The lower and upper whiskers depict the smallest and largest non-outlier observations, respectively, and solid dots represent the outliers. The spacing between the different parts of the box indicates the degree of dispersion (spread). Standard deviation for triplicate observations for all samples was <12%. Significance is shown as *p<0.05, **p<0.05, and ***p<0.001.</p

Metabolic markers of tissue injury were not significantly altered in chagasic subjects.

<p>Shown are plasma levels of activities of the glutamate pyruvate transaminase (<b>A</b>) and creatine kinase (<b>B</b>), determined by spectrophotometry.</p

Characterization of the subjects included for screening of TcG1-,TcG2-, and TcG4-specific antibody responses in this study.

a<p>Subjects in Argentina were screened for <i>T. cruzi</i>-specific antibodies by Wiener Chagatest-ELISA and Wiener Chagatest-HAI kits. Clinical exam included physical exam, electrocardiography and echocardiography. Confirmation of leishmaniasis was obtained by parasitological test, Montenegro reaction, clinical demonstration, and two PCR approaches. One of the seropositive patient presenting acute infection was referred for chemotherapeutic treatment with Benznidazole.</p>b<p>Five of the seropositive/chagasic subjects from Argentina were positive for Leishmania infection, determined by two PCR approaches.</p>c<p>Sera samples from Chiapas Mexico were screened by epimastigote antigenic lysate-based ELISA, trypomastigote-based flow cytometry, and Chagas Stat-Pak immuno-chromatograpic test.</p>d<p>Seronegative subjects from non-endemic areas were screened for <i>T. cruzi</i>-specific antibody response using <i>T. cruzi</i> trypomastigote lysate as antigen source in ELISA assays. Seronegative/cardiomyopathy patients were identified based upon blood levels of NT-proBNP to be >2000 pg/ml (normal<450 pg/ml).</p><p>N/A: not available.</p

TcG1, TcG2 and TcG4 are recognized by antibody response in human subjects from Mexico.

<p>Sera samples obtained from volunteers living in the endemic areas of Chiapas Mexico were characterized as seropositive (+ve) and seronegative (−ve) by whole-parasite antigen based serology tests in the 1^st phase. The TcG1 (A), TcG2 (B) and TcG4 (C) specific antibody response was measured by ELISA, and data are presented as box plot (details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002018#pntd-0002018-g001" target="_blank">Fig. 1</a>).</p