Serum Proteome and Cytokine Analysis in a Longitudinal Cohort of Adults with Primary Dengue Infection Reveals Predictive Markers of DHF

Abstract

BackgroundInfections caused by dengue virus are a major cause of morbidity and mortality in tropical and subtropical regions of the world. Factors that control transition from mild forms of disease such as dengue fever (DF) to more life-threatening forms such as dengue hemorrhagic fever (DHF) are poorly understood. Consequently, there are no reliable methods currently available for early triage of DHF patients resulting in significant over-hospitalization.Methodology/Principal FindingsWe have systematically examined the proteome, cytokines and inflammatory markers in sera from 62 adult dengue patients (44 DF; 18 DHF) with primary DENV infection, at three different times of infection representing the early febrile, defervescence and convalescent stages. Using fluorescent bioplex assays, we measured 27 cytokines in these serum samples. Additionally, we used multiple mass spectrometry methods for iTRAQ-based comparative analysis of serum proteome as well as measurements of protein adducts- 3-nitrotyrosine and 3-chlorotyrosine as surrogate measures of free radical activity. Using multiple methods such as OPLS, MRMR and MSVM-RFE for multivariate feature selection and classification, we report molecular markers that allow prediction of primary DHF with sensitivity and specificity of >80%.Conclusions/SignificanceThis report constitutes a comprehensive analysis of molecular signatures of dengue disease progression and will help unravel mechanisms of dengue disease progression. Our analysis resulted in the identification of markers that may be useful for early prediction of DHF during the febrile phase. The combination of highly sensitive analytical methods and novel statistical approaches described here forms a robust platform for biomarker discovery.

Discovery and Characterization of Potential Prognostic Biomarkers for Dengue Hemorrhagic Fever

“…This discrepancy could be caused by a difference in methods (Western blot versus ELISA), or to sample size (HC = 6, DF = 6). Kumar and colleagues 17 observed elevated levels of Hx in DHF cases relative to DF cases over time, in contrast to our observation of lower Hx levels in DHF relative to DF (DPO = 0-5). This discrepancy may be caused by age differences between the samples analyzed or quantitation methodology (SELDI-TOF MS and ELISA versus isobaric tagging).…”

“…The Hx and Tf require further analysis to determine why our data differed from other researchers. 17,28,29 After further characterization of Vtn, Hx, and Tf, we may find that a combination of biomarkers has the greatest utility as a prognostic test for severe dengue as noted by other researchers. 7 Vitronectin is a multifunctional glycoprotein that circulates in the blood, usually as an inactive monomer, until it is recruited to regulate coagulation and platelet aggregation.…”

“…3 Previously identified markers of dengue severity include cytokines (i.e., tumor necrosis factor alpha [TNFα] and Interleukin 6 [IL-6]), vascular permeability proteins, clotting cascade regulators, and gene expression profiles. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] These severity markers are not, however, currently being used to guide patient management. 18,19 It is unclear why these severity markers are not being used, possibly because detection methods are expensive, slow, or require sophisticated equipment.…”

Dengue Patients with Early Hemorrhagic Manifestations Lose Coordinate Expression of the Anti-Inflammatory Cytokine IL-10 with the Inflammatory Cytokines IL-6 and IL-8

“…Indeed, several studies have presented many cytokines as predictive factors for dengue severity including IFN-γ, TNF-α, IL-6, IL-8, and IL-10. 17,24,25 However, the actual use of these potential markers to predict hemorrhagic manifestations (HM) of the disease is not clear due to the wide variation in study designs, timing, methods of sample collection/processing, and the precision in the identification of disease severity. Herein, we investigate cytokine expression profiles together with other parameters including viral load and dengue serotype, in addition to early expression of HM, during the initial 1-4 days of the onset of symptoms, in an attempt to define possible polarized responses in the earliest phases of the onset of disease and identify possible immune profiles related to more severe manifestations.…”

Biomarkers in differentiating clinical dengue cases: A prospective cohort study

“…However, VEGF is elevated in dengue with warning signs/ severe dengue [26], but care must be taken when comparing with studies conducted in children [27][28][29].…”