Clinical Findings and Pro-Inflammatory Cytokines in Dengue Patients in Western India: A Facility-Based Study

BACKGROUND: Descriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: In 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-gamma, TNF-alpha, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version. Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-gamma were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-gamma and IL-8 were higher in early samples collected 2-5 days after onset than late samples collected 6-15 days after onset. IFN-gamma showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-gamma and IL-8 were positively associated with alanine transaminase levels. CONCLUSIONS/SIGNIFICANCE: Rash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-gamma and IL-8 levels had greater significance than absolute values in DHF pathogenesis

Laboratory findings in DF and DHF.

<p>Cases compared using chi-square test, <i>p</i>- value <0.05 considered significant.</p

Age-wise distribution of DF and DHF cases.

<p>Number of dengue cases plotted according to age – groups.</p

Cytokine levels (pg/ml) in early and late days of illness in DF and DHF patients.

<p>Scatter plot of levels of cytokine (pg/ml) in early (2–5) and late (6–15) days of illness. A) IFN-γ, B) TNF-α, C) IL-6 and D) IL-8. The two panels show DF and DHF cases. The mean levels are indicated with the red line. The cut-off (mean levels in healthy controls+2SD) for each cytokine is shown with a dashed line.</p

Demographics, Serotypes detected and disease categories.

<p>Demographics, Serotypes detected and disease categories.</p

Association of cytokine levels with clinical parameters of dengue patients.

<p>A. Association of levels of IFN-γ with ALT levels. B. Association of levels of IL-8 with ALT levels. C. Tukey box-whisker plot with median, range with the upper and lower quartiles and outliers of IL-8 levels in thrombocytopenic (platelet count <100,000) and non- thrombocytopenic (platelet count >100,000) dengue patients. A & B] Pearson correlation used for analysis, <i>p</i><0.05 considered significant. C] <i>p</i>-value calculated by ANOVA, <i>p</i><0.05 considered significant. Outliers showed as dots and those above the axis limit not shown in graph but included in analysis.</p

Levels of IFN-γ, TNF-α, IL-6 and IL-8 in DF and DHF patients.

<p>A. Mean with SEM levels (pg/ml) of cytokines. B. Number (%) of cases with increased (above cut-off) values of cytokines in DF/DHF. Analysis of variance was carried out for log transformed values of each cytokine and differences in groups analysed by using Tukey test.*<i>p</i>-value <0.05 when compared with controls. *<i>p</i>-value <0.05 when compared between DF and DHF. Comparison between number of cases for DF and DHF done using chi-square test. <i>p</i>-value <0.05 is shown.</p

Cytokine levels in primary and secondary DENV infections.

<p>Mean with SEM levels of four cytokines (pg/ml) in primary and secondary infections. Patients with IgM/IgG ≥1.78 had primary infections; IgM/IgG<1.78 had secondary infections. p-value calculated by analysis of variance of log transformed levels of cytokines. p<0.05 considered significant.</p

Clinical signs/symptoms in DF and DHF patients.

<p>Comparison between DF and DHF patients done by chi-square test, <i>p</i>-value <0.05 considered significant.</p