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

Date Published: February 25, 2016

Publisher: Public Library of Science

Author(s): Natalia V. Voge, Rushika Perera, Sebabrata Mahapatra, Lionel Gresh, Angel Balmaseda, María A. Loroño-Pino, Amber S. Hopf-Jannasch, John T. Belisle, Eva Harris, Carol D. Blair, Barry J. Beaty, Cameron P. Simmons.

Abstract: BackgroundEpidemic 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 FindingsExploratory 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/SignificanceThese 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.

Partial Text: Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) have emerged throughout the tropical and subtropical world with devastating consequences and are overwhelming public health capacity for diagnosis and patient care [1, 2]. Upon presentation early after disease onset, it is clinically impossible to differentiate dengue virus (DENV)-infected patients who will have an unremarkable DF disease episode from those who will progress to potentially fatal DHF/DSS [3–7]. Viral biomarkers that correlate with dengue severity include viremia titer and nonstructural protein 1 (NS1) concentration in the blood, secondary DENV infection, and infection with specific virus genotypes [8–11]. Host biomarkers associated with disease severity include multiple immune molecules, biochemical and physiological response indicators, and genetic polymorphisms [3, 4, 12–21]. Algorithms based upon clinical signs and laboratory test results have been proposed to predict dengue severity [22–28]. However, currently there are no standardized biomarkers or algorithms for prognosis of severe disease outcomes.

Our studies confirm that DENV infection perturbs the human metabolome [32]. Statistical analyses indicated that many metabolites and MFs identified by HILIC-LC-MS had statistically significant differences in abundance in pairwise comparisons of the DHF/DSS, DF, and ND diagnosis groups (Table 5 and S1 and S2 Tables). Cui et al. [32] demonstrated perturbation of many of the same metabolites in DF patients during the time-course of primary DENV infection. Metabolites that were perturbed in DHF/DSS and DF patients in both Nicaraguan and Mexican patients included lysoPCs (14:0, 16:0) and long-chain polyunsaturated fatty acids such as DHA, AA, and ALA. To determine if differentiating metabolites identified by HILIC-LC-MS could be identified using a different LC-MS platform and to more thoroughly explore the metabolome, a subset of serum samples were analyzed in the Purdue Metabolite Profiling Facility (PMPF) using reverse phase (RP)-LC-MS [37]. In confirmation, 54% (117/288) of differentiating metabolites detected by HILIC-LC-MS were also detected using a T3 column (Waters, Milford, MA) in RP-LC-MS. All of the 13 differentiating metabolites whose identities were confirmed by LC-MS/MS (Table 5) were detected by RP-LC-MS and differentiated the dengue diagnosis groups.



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