Introduction

Although dengue hemorrhagic fever (DHF) has a low rate of mortality, it is one of the diseases regarded as transmitted by arthropod vectors, which account for the highest percentage. DHF is endemic to more than 120 tropical and subtropical countries, thus rendering 55% of the global population susceptible to the risk of infection (World Health Organization, 2009). The disease has existed for nearly a century, yet its pathogenesis has yet to be fully understood, and the main determinant of its progression into severe cases has not been clearly defined. One such complication is dengue shock syndrome (DSS), which can lead to death. The incidence and effects of this form of the disease are difficult to predict through clinical examination.

Researchers have recently suggested that different cytokines play a role in the formation of severe DHF complications and the diagnosis of DHF shock. The fact that the cytokine system is very complex and that these molecules seem to be interesting indicators are relevant to disease severity and prognosis (Kleiner, Marcuzzi, Zanin, Monasta, & Zauli, 2013).

Materials and Methods

This prospective cohort study was conducted at Tien Giang General Hospital in Vietnam from December 2009 to November 2012. The participants selected were patients who were aged 18 months to 18 years, had a history of fever lasting less than 72 hours and were clinically suspected of having DHF. To determine a suitable sample size, this research used a formula that compares two ratios with a desired reliability of 95% [α = 0.05 Z (1-α/2) = 1.96, β = 10% Z (1-β) = 1.28]. The equation was adopted on the basis of Mustafa’s (2001). In the group with elevated concentrations of cytokines, the incidence rates of DHF with shock (P1) and non-shock DHF (P2) were 0.63 and 0.09, respectively [P = (P1 + P2)/2]. In line with this, the study needed at least 15 patients each under the shock and non-shock groups. According to (Hung et al., 2004), however, the incidence rate of shock accompanied DHF is 20.5%. Thus, the acceptable minimum sample size is N ≥ 75.

Venous blood samples were taken from each patient daily from the date of admission to date of discharge. Serologic tests for dengue virus (DENV) infection, namely, enzyme-linked immunosorbent assay (ELISA) for immunoglobulin M (IgM)/immunoglobulin G (IgG) (anti-DENV IgM/IgG ELISA) and the comparison of nonstructural protein-1 detection via reverse transcription-polymerase chain reaction and ELISA were conducted to confirm all cases of DHF. Cytokine concentration was detected using multiplex microbead immunoassay, which enabled the simultaneous detection of 10 cytokines: interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, interferon gamma-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Table 1 below shows the threshold of increase in cytokines obtained from the research of (Hung et al., 2004) and (Kleiner et al., 2013) on children.

Statistical analysis

Data were analyzed using Stata version 10.0. Descriptive statistics were used to express frequencies, and percentages were adopted to represent the characteristic variables of the sample. Quantitative variables with a normal distribution were compared by subjecting two patient groups (shock-accompanied DHF and non-shock DHF) to Student’s t-test and three patient groups to the analysis of variance. A p-value of less than 0.05 (<0.05) was considered significant.

Ethical considerations

This study was approved by the Scientific Research Committee of Tien Giang General Hospital. The collected data were used only for the purposes of this study. Participation was voluntary and anonymous, and the participants were asked to sign an informed consent form prior to enrollment in the research.

Results and Discussion

From December 2009 to November 2012, 234 patients were confirmed to be infected with DENV and were included in the study. Table 2 shows the characteristics of the participants.

Table 3 presents the results on differences in serum cytokine concentrations between the non-shock and shock-accompanied DHF groups, and Table 4 lists the average cytokine concentrations measured on the first day of hospitalization.

The findings regarding the relationship between cytokines and disease severity, DENV serotype and fever duration are displayed in Table 5, Table 6 and Table 7.

Sample characteristics

The rate of disease incidence among the patients in the non-shock DHF group (93.59%) was higher than that in the shock-accompanied DHF group (6.41%), which is also considerably lower than the percentage reported by (Mustafa, Elbishbishi, Agarwal, & Chaturvedi, 2001) for 58 cases. The author found an incidence rate of 69.1% in the non-shock DHF patients (levels I and II) and a rate of 30.9% among the patients suffering from shock-accompanied DHF (13 cases of level III, 13 cases of level IV). Most of the patients in the present study (76.5%) were re-infected at a rate of 23.5%. This result contrasts with that of Tang et al. (2011), whose survey on 353 hospitalized DENV patients in Southern China uncovered re-infection and infection rates of 39.9% and 60.1%, respectively. Among the patients in the current work, 41.45% and 50.85% were hospitalized upon contracting a fever on days 2 and 3, respectively. Only a few (7.69%) were hospitalized upon fever occurrence on day 1.

Rate of increase in cytokine concentrations in DHF patients

Of the subjects, 73.08% had increased IL-10, accounting for the highest percentage in the sample. The proportions of patients with increased IL-6 and IL-2 were 71.79% and 71.79%, respectively. The lowest proportion of patients with elevated IL-2 concentrations was 0.43%. This result is consistent with that derived by Rathakrishnan et al. (2012), who found lower concentrations of IL-12 in DHF patients than in healthy individuals.

The present study also found that the non-shock and shock-accompanied DHF patients all had comparable cytokine concentrations (p > 0.05) (Table 2). (Pacsa et al., 2000) research on 246 patients revealed that all cases of levels III and IV DHF exhibited increased cytokine concentrations; such elevation was found among 91% to 95% of patients with levels I and II DHF.

The relationship between average cytokine concentration and disease severity

The average concentrations of IL-2, IL-5, and IL-13, and especially IL-6, IL-10, and INF-γ, in the group with shock-accompanied DHF, were higher than those in the non-shock group, but the difference was statistically nonsignificant (p > 0.05, t-test). Conversely, the average concentrations of IL-1, IL-4, IL-12, and TNF-α in the shock-accompanied DHF group were lower than those in the non-shock group. Again, however, the difference between the groups was statistically nonsignificant (p > 0.05, t-test). (Bozza et al., 2008) discovered that concentrations of IL-1β, IFN-γ, IL-4, IL-6, IL-7, IL-13, and granulocyte-macrophage colony-stimulating factor are significantly higher among subjects with severe DHF than in those with mild DHF. The author also found that the concentration of macrophage inflammatory protein-1β was slightly higher in patients with mild DHF.

The relationship between average cytokines concentration and DENV serotype

The highest average concentrations of IL-2, IL-4, IL-13, and TNF-α were observed in the DENV-1–infected patients, followed by those infected with DENV-3 and DENV-2. The differences among these concentrations were statistically significant at 0.006, 0.023, 0.010, and 0.026, respectively. The average concentrations of IL-1, IL-5, IL-6, IL-10, IL-12, and INF-γ showed no statistically significant differences under various types of DENV infection (p > 0.05) (Table 5).

The relationship between average cytokines concentration and duration of fever

The highest average concentration of IL-12 was found in patients who developed a fever on day 1 of (5.60±13.04) disease. Then this concentration gradually decreased on day 2 (2.33±3.62) and day 3 (1.76±2.12). The difference among the concentrations was statistically significant (p = 0.003). The highest average concentrations of IL-1, IL-2, IL-4, IL-6, IL-13, and TNF-α also occurred in the patients who had a fever on day 1. Similarly, the concentrations slowly declined on days 2 and 3, but the difference was statistically nonsignificant (p>0.05). In contrast, the IL-10 and INF-γ concentrations tended to increase with the rising number of days with fever, with the lowest increase observed in the patients who developed this symptom on day 1. The concentrations increased on day 2 and were even higher in patients with have had a fever for three days, but the difference was statistically nonsignificant (p > 0.05). Chaturvedi, Agarwal, Elbishbishi, and Mustafa (2000) reported that TNF-α, IL-2, and IL-6 had the highest concentrations on day 2 and that IFN-γ concentrations occurred on day 2 and peaked on day 3. The author also stated that IL-10 and IL-5 levels increased on day 4, and IL-4 was elevated on day 6 after viral infection.

Conclusion

No difference in cytokine concentrations was found between the first day of admission and the day of shock complication occurrence in DHF patients. Increases in IL-10, IL-6, and IL-2 concentrations occurred among the majority of the subjects (>67%), and elevated IL-12 was exhibited by only 0.43% (the lowest) of the patients. The levels of IL-2, IL-4, IL-13, and TNF-α were highest in the DENV-1–infected group, followed by the DENV-3 and DENV-2 patients. The IL-12 concentrations decreased with the rising number of fever days.