Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes

Background: Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes. Methodology/Principal Findings: Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections. Conclusions/Significance: These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity. Author Summary: Epidemics of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and patient care. Developing a panel of biomarkers in acute-phase serum specimens for prognosis of severe dengue disease would be of enormous value for appropriate triaging of patients for management. Metabolomics offers great potential for identification of small molecule biomarkers (SMBs) for diagnosis and prognosis of dengue virus (DENV) infections. We identified metabolites that were associated with and differentiated DHF/DSS, DF and non-dengue (ND) febrile illness outcomes, primary and secondary virus infections, and infections with different DENV serotypes. These metabolites provide insights into metabolic pathways that play roles in DENV infection, replication, and pathogenesis. Some are associated with lipid metabolism and regulation of inflammatory processes controlled by signaling fatty acids and phospholipids, and others with endothelial cell homeostasis and vascular barrier function. Such metabolites and associated metabolic pathways are potentially biologically relevant in DENV pathogenesis. The diagnostic and prognostic efficacy of differentiating metabolites is currently being investigated. Our goal is to identify the most parsimonious SMB biosignature that, when combined with laboratory diagnostic results, eg., DENV NS1 or RNA detection, will provide the most efficient algorithm for dengue diagnosis and prognosis.