The Angiopoietin-Tie-2 Axis in Children and Young Adults with Dengue Virus Infection in the Philippines

Abstract

Dengue virus (DENV) infection is associated with plasma leakage, which may progress to shock. The angiopoietin (Ang)-tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (Tie-2) axis regulates endothelial permeability. We examined the clinical utility of Ang-1, Ang-2, and the Ang-2-to-Ang-1 ratio for prediction of progression to severe DENV in a prospective cohort study of children and young adults (age 1 to <26 years) with DENV infection. Ang-1, Ang-2, Tie-2 were measured at presentation to an outpatient clinic in the Philippines from stored plasma by multiplex Luminex® assay. Patients were followed prospectively to document the clinical course (hospitalization, length of stay, intravenous fluid resuscitation, and transfer to a higher level facility). We included 244 patients (median age 9 years, 40% female). At presentation, 63 patients (26%) had uncomplicated dengue, 179 (73%) had dengue with warning signs, and 2 (0.82%) had severe dengue. One hundred eighty-one patients (74%) were hospitalized. Ang-1 levels were lower and Ang-2 higher in patients who required hospitalization. Ang-2-to-Ang-1 ratio >1 was associated with a relative risk of hospitalization of 1.20 (95% CI: 1.03-1.36, P = 0.016). A higher Ang-2-to-Ang-1 ratio was associated with longer length of hospital stay, higher frequency of transfer to a higher level facility, larger intravenous fluid requirement, hemoconcentration, and thrombocytopenia. Angiopoietin-2 was correlated with procalcitonin (Kendall's τ = 0.17, P = 0.00012), a marker of systemic inflammation, as well as soluble vascular cell adhesion molecule-1 (τ = 0.22, P <0.0001) and Endoglin (τ = 0.14, P = 0.0017), markers of endothelial activation. In conclusion, altered Ang-2-to-Ang-1 ratio can be detected early in the course of DENV infection and predicts clinically meaningful events (hospitalization, length of stay, and fluid resuscitation).

Figure 1.

Association of angiopoietins (Ang) and soluble tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 (Tie-2) with hospitalization for dengue virus infection. Angiopoietin-1 levels were lower (P = 0.013) (A) and Ang-2 levels were higher (P = 0.00026) (B) in patients who required hospitalization. (C) The Ang-2-to-Ang-1 ratio was statistically significantly higher in hospitalized patients (P = 0.00062). (D) Circulating levels of the soluble Tie-2 receptor (sTie-2) were elevated in patients who required hospitalization (P = 0.0048). (E) Receiver operator characteristic (ROC) curve analysis of Ang-2-to-Ang-1 ratio to discriminate between hospitalized and nonhospitalized patients (area under the ROC [AUROC] 0.64, 95% CI: 0.57–0.72, P = 0.00062). (F) The treating clinician’s decision to refer the patient for admission (AUROC 0.64, 95% CI: 0.58–0.71) was statistically improved with the added information provided by the Ang-2-to-Ang-1 ratio (AUROC 0.73, 95% CI 0.66–0.73, P = 0.0074). *P <0.05, **P <0.01, ***P <0.001.

Figure 2.

Association of angiopoietin (Ang)-2-to-Ang-1 ratio with DENV severity. (A) World Health Organization case classification indicated that Ang-2-to-Ang-1 ratio was statistically significantly higher in dengue with warning signs than uncomplicated dengue (P = 0.029). Two patients with severe dengue (Ang-2-to-Ang-1 ratio 0.18:0.50) are not shown on the graph. Among hospitalized patients, Ang-2-to-Ang-1 ratio >1 was predictive of longer length of stay (P = 0.0027) (B), frequency of transfer to a higher level facility (P = 0.019) (C), and larger volumes of intravenous fluids required to manage the plasma leak (P = 0.010) (D). (E) Hemoconcentration, a sign of plasma leakage, was associated with higher Ang-2-to-Ang-1 ratio (P <0.0001). (F) Thrombocytopenia was associated with higher Ang-2-to-Ang-1 ratio (P <0.0001). *P <0.05, **P <0.01, ***P <0.001.

Figure 3.

Correlation of angiopoietin (Ang)-1 and Ang-2 with platelet count, systemic inflammation, and endothelial activation. (A) Ang-1 was correlated with the platelet count (τ = 0.27, P <0.0001). (B) Ang-2 was correlated with procalcitonin, a marker of systemic inflammatory response to infection (τ = 0.17, P = 0.00012). (C and D) Ang-2 was correlated with markers of endothelial activation soluble vascular cell adhesion molecule (sVCAM-1) (τ = 0.22, P <0.0001) and endoglin (τ = 0.14, P = 0.0017). (E and F) Soluble tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 was also correlated with sVCAM-1 (τ = 0.15, P = 0.00036) and endoglin (τ = 0.27, P <0.0001).

Figure 4.

Normalization of angiopoietins (Ang) and soluble tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 (sTie-2) at convalescence (14–21 days after initial presentation). (A) Ang-1 levels were low at presentation and increased by a median of 4.7 ng/mL (interquartile range [IQR]: 1.1–11) after recovery from the acute illness (P <0.0001). (B) Ang-2 remained elevated at follow-up, increasing marginally by 0.35 ng/mL (IQR: –0.65 to 1.3) from baseline to last follow-up visit (P = 0.010). There were 91 patients (40%) whose Ang-2 decreased from baseline to follow-up and 136 patients (60%) whose Ang-2 increased from baseline to follow-up. (C) The Ang-2-to-Ang-1 ratio decreased by a median of 0.46 (IQR: 0.028–1.5, P <0.0001). (D) Soluble Tie-2 decreased by a median of 1.5 ng/mL (IQR: –2.9 to 6.4, P = 0.00099). *P <0.05, **P <0.01, ***P <0.001.