Inflammatory and cardiac biomarkers are differentially expressed in clinical stages of Chagas disease

Abstract

Background: Chagas disease has a long clinically silent period following Trypanosoma cruzi infection and before development of overt clinical pathology; detectable biomarkers of infection and pathogenesis are urgently needed. We tested 22 biomarkers known to be associated with cardiomyopathy to evaluate if a biomarker signature could successfully classify T. cruzi seropositive subjects into clinical Chagas disease stage groups.

Methods: This cross-sectional retrospective case-control study enrolled T. cruzi seropositive blood donors (BD) who were further characterized as having chronic Chagas cardiomyopathy (CC-BD) or not (nonCC-BD) and seronegative (SN) control donors; we also included clinically diagnosed Chagas cardiomyopathy patients (CC-P). All subjects underwent a health history questionnaire, medical examination, electro- and echocardiograms (ECG and Echo) and phlebotomy. Biomarkers were measured on blinded samples by luminex bead array and Ortho VITROS.

Results: A clear biomarker pattern was observed only in more severe cardiac disease; this pattern included significantly elevated levels of inflammatory cytokines IFN-γ, IL-6, IL-10 and TNF-α and soluble cardiovascular disease biomarkers CK-MB, troponin, myoglobin, VCAM and NTproBNP while there were lower levels of MPO, PAI-1, and MCP-1. The markers determined to be the most predictive of disease by ROC curve analysis were NTproBNP and T. cruzi PCR status.

Conclusions: Although many biomarkers demonstrated increased or decreased concentrations among the clinical forms of Chagas disease, NTproBNP and T. cruzi PCR were the only tests that would independently be of clinical value for disease staging, in concert with ECG, Echo and clinical assessments.

Keywords: Cardiomyopathy; Chemokines; Cytokines; Inflammation; Trypanosoma cruzi (T. cruzi).

Figure 1. Elevations in inflammatory cytokines in Chagas disease

There are higher inflammatory cytokines in chronic Chagas cardiomyopathy compared to the other groups of T. cruzi infected and seronegative individuals. IL-6 and IL-10 were significantly elevated compared to all groups (p<0.05). IFN-γ were significantly elevated compared to the seronegative donor group (p=0.004). TNF-α was elevated compared to T. cruzi seronegative donors (p=0.01). IL-1β concentrations were significantly elevated in T. cruzi infected groups compared to seronegative control donors (p<0.005).

Figure 2. Soluble cardiac biomarkers are differentially expressed in chronic Chagas cardiomyopathy disease stages

NTproBNP and Troponin were significantly elevated in CC-P and CC-BD compared to seronegative and/or Non-CC BD groups (p<0.005). CKMB was elevated in CC compared to other T. cruzi Ab+ and seronegative donor groups (p<0.05). Myoglobin and VCAM were elevated in CC-P compared to non-CC-BD and seronegative groups (p<0.005). There were reductions in other circulating biomarkers with disease severity; these markers included plasminogen activated inhibitor (PAI-1), myeloperoxidase (MPO) and monocyte chemotactic protein (MCP-1) (p<0.05).

Figure 3. Heatmap of group medians of all biomarkers and cardiac parameters

Each cell represents the median of the biomarker for the corresponding group. For each row, the medians are then scaled to mean 0 and standard deviation 1. The dendrogram is generated based on hierarchical clustering with complete linkage and 1-Pearson correlation as distance.

Figure 4. Receiver operator curve identifies potentially predictive biomarkers of Chagas cardiac pathology

The ROC curves show the prediction performance across all false positive rates (False Positives/False Positives + True Negatives) and true positive rates (True Positives/True Positives + False Negatives). AUCs (Area under curve) were calculated as prediction performance measures for each predictive model. We show 5 univariate biomarker models and the combined model which includes all 5 predictors. A random predictive model is shown for comparison.