Received: November 27, 2017
Accepted: March 21, 2018
Mediterr J Hematol Infect Dis 2018, 10(1): e2018023 DOI 10.4084/MJHID.2018.023
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Dengue infection is a rapidly spreading vector-borne disease and is
endemic in the Indian subcontinent. It has varied manifestations
ranging from subclinical infection to severe fatal shock syndrome. This
study aimed to estimate cytokine level in dengue patients and correlate
them with dengue severity.
Material and Methods
These 80 patients were further classified into nonsevere dengue (Group 1) and severe dengue (Group 2) by WHO classification of dengue 2009. Therefore, patients with and without warning signs were included in group 1, and those with severe manifestations like severe plasma leakage, severe bleeding, and severe organ impairment were included in group 2. There were 50 patients in group 1 and 30 in group 2. Blood samples were collected from both groups for complete hemogram, liver function test, renal function test and cytokine levels. Blood samples collected for estimation of cytokine levels were centrifuged to separate the serum and then stored at -80 degree Celsius. Serum levels of all three cytokines (INF-γ, IL-6, and TNF-α) were measured using ELISA kits. All admitted patients were monitored daily till discharge or death in the hospital. Both the groups were compared on the basis of serum cytokine levels along with clinical, biochemical and radiological parameters.
Statistical analysis. Data were recorded using a structured proforma and managed on an excel sheet. All qualitative variables were compared with Chi-square test or Fisher's exact test. Quantitative variables with a normal distribution were compared using Student's t-test for two groups and ANOVA test with post hoc Bonferroni correction for the three-group analysis. Quantitative variables not following a normal distribution such as cytokine levels were compared using non-parametric test (Kruskal-Wallis test). A p-value of less than 0.05 (<0.05) was considered as significant. The analysis was done between Group 1 (nonsevere dengue, n=50) and Group 2 (severe dengue, n=30).
|Figure 1. Frequency of symptoms in the two study groups.|
|Figure 2. Frequency of symptoms in the two study groups.|
|Table 1. Comparison of vital parameters among patients in the two groups.|
The most common clinical finding amongst all patients was positive tourniquet test (76%), followed by a rash (55.5%). The mean platelet count in group 2 (70364±50431/ul) was lower than in group 1 (93460±65000/uL), but this difference was not statistically significant. Mean AST levels in group 2 (451±633.2 IU) were significantly higher than in group 1 (96.5±157.4 IU, p=0.01). The mean serum ALT levels in group 2 (270.3±334.25 I.U.) were also higher than in group 1 (60.16±73.22 IU, p=0.01). Bleeding manifestations were more in group 2 (83.3%) as compared to group 1 (46%). The most common form of bleeding manifestation was skin petechiae, seen in 36% of group 1 patients and 63.3% of group 2 patients. Gastrointestinal bleeding (hematemesis, melena, or hematochezia) was present in 8% of group 1 and 56.6% of group 2. One patient had iliopsoas bleed and required surgical intervention (Figure 3).
|Figure 3. Distribution of various forms of bleeding manifestation among two groups.|
Tumor Necrosis Factor-α, Interleukin-6 and Interferon-γ levels were estimated in the serum of 80 patients from the sample collected on the day of the presentation. The median level of serum TNF-α in group 2 (62.5 pg/mL) was significantly higher than the median level in group 1 (20 pg/mL), (p=0.043). Similarly, the median level of serum IFN-γ in group 2 (10.25 pg/mL) was significantly higher than the median level in group 1 (8.5 pg/mL), (p=0.002). The median level of IL-6 was also higher in group 2 (29 pg/ml) as compared group 1 (14.2 pg/ml), but this result was not significant (p>0.05) (Table 2).
|Table 2. Comparison of cytokine levels between the two groups.|
Further, the median levels of both TNF-α and IFN-γ were calculated for each day of fever according to the day of fever on presentation and sample collection. The serum levels of TNF-α were significantly higher on day 2-3 of illness, followed by a fall on day 4-5 and a late upsurge in group 2 as compared to group 1. Similarly, IFN-γ levels were also plotted across the day of fever and significant increase was noticed on initial days of presentation followed by a fall and a second rise in the levels in group 2 (Figure 4, 5). The decline in the mid-phase is not expected but may be attributed to hemodilution due to intensive intravenous fluid therapy.
|Figure 4. Line diagram showing median levels of TNF-α plotted against days of fever.|
|Figure 5. Line diagram showing median levels of IFN-γ plotted against days of fever.|
Deranged liver function in dengue infection can be a result of the direct effect of the virus on hepatocytes or unregulated host immune response against the virus. Mahmuduzzaman and colleagues showed that both AST and ALT were significantly raised in patients with DHF as compared to those with dengue fever and increase in AST was much higher than the increase in ALT. Similarly, Pancharoen and coworkers also reported that levels of AST and ALT were significantly higher among patients with more severe disease. In present study too, the difference in both AST and ALT levels in the two groups were significant.
Studies in the recent past have highlighted the role of cytokines and other biomarkers in the pathogenesis of severe dengue and have studied the utility of these biomarkers as risk factors.[11,12] It has been clearly demonstrated that the inflammatory response associated with deregulated cytokine production perform critical roles in the development of severe dengue. The higher levels of cytokines like TNF- α, IL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7, and GM-CSF were associated with severe dengue fever in various studies.[14,15,16] In present study too, the levels of TNF-α and IFN-γ were significantly higher in severe dengue group (group 2). The elevated levels of cytokine in severe dengue make them good predictors of severity of dengue fever. Cytokine estimation at presentation can provide us a clue whether a patient is likely to develop severe manifestations of dengue or not. However, our study has certain limitations, like small sample size and the serotype of dengue virus was not studied. Further large prospective studies are warranted for better comprehension of the balance between circulating cytokines and their effect on the development of severe dengue.
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