Fig 1.
Severe dengue disease occurs in both primary and secondary infections.
(A) Patients with primary and secondary dengue infections (N = 97) were stratified based on severity at the time of admission. DI–Dengue infection, DW- Dengue with Warning signs and SD–Severe Dengue. Patients with primary and secondary dengue infections with day of fever < 3 (B), <4 (C) or <5 (D) were stratified based on severity at the time of admission (N = 36, 45 and 64 respectively).
Table 1.
Clinical and laboratory data on the day of admission of patients with dengue illness.
Table 2.
Dengue serotyping.
Table 3.
Disease severity of enrolled patients at the time of admission (bleed 1) and repeat bleeds at around 48 h post-admission (bleed 2) with primary and secondary dengue infections.
Fig 2.
Dengue viremia does not correlate with disease severity.
(A) Dengue viral RNA levels in patients classified as primary and secondary dengue infections in bleed 1 (i) and bleed 2 (ii) with respect to the day of fever. An overlapping connecting line graph shows median values of the respective scatter plot. (B) Relationship between dengue plasma viremia and disease severity at the time of admission (i) and 48 h post admission (bleed 2) (ii) in patients with primary and secondary dengue infection. (C) Overall comparison of dengue viremia in all primary and secondary infections in bleed 1 (i) and bleed 2 (ii). Geometric mean value of the scatter plot is shown in all figures. Statistical significance was determined by Mann-Whitney test. ** P = 0.0074.
Fig 3.
Relationship between platelet count and primary/secondary infections.
(A) Platelet counts were measured at the time of admission, bleed 1 (i) or 48 h later, bleed 2 (ii) in patients with primary or secondary infections. (B) Platelet counts in bleed 1 (i) and bleed 2 (ii) of patients with primary or secondary infections categorized as per indicated disease severity. Median value is indicated. Statistical significance was determined by Mann-Whitney test. ** P< 0.005.
Table 4.
Quantitation of inflammatory mediators in dengue patients with different disease severities.
Fig 4.
Cytokine profile in dengue patients on indicated days of fever and disease severity.
Plasma levels of cytokines/chemokines/inflammatory mediators in dengue patients were measured by multiplex magnetic bead assays. (A) IFN-α (B) IFN-γ (C) IL-6, (D) IL-7, (E) IL-8, (F) IL-10 (G) MCP-1 (H) sCD40L, (I) TNF-α and (J) VEGF. Data are segregated into days of fever and disease severities within each group. Median value of cytokines in dengue illness (DI), dengue with warning signs (DW) and severe dengue (SD) is indicated by the respective color-coded bar. Dotted line represents the limit of detection. Statistical significance was determined by Mann-Whitney test.
Fig 5.
Cytokine profile in patients with primary and secondary infections Plasma cytokine/chemokine/inflammatory mediator levels in the plasma of primary and secondary dengue infection with indicated severities.
(A) IFN-α (B) IFN-γ (C) IL-7, (D) IL-12p70, (E) MCP-1, (F) sCD40L (G) IL-6 (H) IL-8, (I) IL-10 and (J).IP-10. Median values are indicated by the bar. Dotted line represents the limit of detection.
Table 5.
Correlation of viremia with secreted biochemical markers.
Fig 6.
Transient increase in intermediate monocytes at early time points in DENV infection and monocyte infection with DENV.
(A) Representative plots showing analytical multicolor flow cytometry of classical, non-classical and intermediate monocytes based on CD14 and CD16 expression ex vivo from PBMCs isolated from DENV-infected patients at indicated days of fever. (B) Cumulative frequencies of CD14+CD16+ intermediate monocytes as shown in scatter plot above. Unpaired, two-tailed t test was used for statistical analysis and P values were interpreted as * P<0.01 and ** P<0.001. (C) Ex vivo intracellular DENV staining was performed on total PBMCs stained with surface receptor antibodies for CD3, CD19, CD14, CD16 along with fixable viable dye to exclude dead cells. Representative histograms show intensity of DENV staining within each of the gated populations described. (D) Cumulative mean fluorescent intensities of DENV staining in the indicated cell populations (N = 31) is shown. Error bars represent Mean wth SD.
Fig 7.
Multivariate selection of features of dengue severity and recovery.
(A) The boxplots represent the distribution of variable importances after adjusting for the interactions among the variables. The minimal set of selected features (green) are the ones whose medians lie above the maximal possible median importance in the shadow data (see text for description). Eight parameters out of all available clinical and biochemical parameters were selected as important for prediction of the severity classes (SD, DW and DI). (B) Identifying markers of recovery from SD using the Boruta algorithm. All parameters from SD patients whose disease status improved clinically from first to second bleed were considered for analysis. Three delta parameters out of all the delta (absolute and relative changes across the first and second bleed, see text) were significantly important for prediction of recovery from SD and were consistent with findings in the univariate analysis.