|Year : 2019 | Volume
| Issue : 3 | Page : 162-166
Assessment of hepatitis B virus genotype D and interleukin-10, interferon gamma, and tumor necrosis factor-α in fulminant hepatic failure
Meher Rizvi1, Hiba Sami1, Mohd Azam1, Indu Shukla1, Haris M Khan1, MR Ajmal2
1 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of Medicine, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
|Date of Submission||04-Sep-2018|
|Date of Decision||29-Dec-2018|
|Date of Acceptance||20-Jan-2019|
|Date of Web Publication||13-Aug-2019|
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Genotypically, hepatitis B virus (HBV) is divided into 8 groups, A–H. The interaction between HBV replication and immune responses against HBV infection plays an important role in the pathogenesis of virus infection. Aims and Objectives: In an attempt to elucidate the role of immunomodulatory, pro-inflammatory, and anti-inflammatory mediators in the pathogenesis of HBV genotype D fulminant hepatitis (FH), we assessed the role of interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in such patients. Subjects and Methods: Two hundred and seventy-six cases of acute hepatitis among which 37 cases of fulminant hepatic failure were screened. A genotyping system based on polymerase chain reaction using type-specific primers was used in this study (Sami et al. 2013). TNF-α, IL-10 levels, and IFN-γ serum level were measured by ELISA. Results: Genotype D was detected in 10 cases (27.02%) out of 37 FH patients. Mean IL-10, IFN-γ, and TNF-α levels in FH patients were significantly higher than the healthy controls at 127.6 ± 73.45 pg/ml (P < 0.001), 10.6778 ± 5.23 pg/ml (P < 0.0001), and 62.9000 ± 21.67 pg/ml (P < 0.001), respectively. IFN-γ and IL-10 levels were much higher in cases of hepatitis B e-antigen (HBeAg) seronegative individuals, mean levels being 20.77 pg/ml and 36.53 pg/ml, respectively. Using multiple linear regression analysis, the presence of HBeAg was inversely associated with TNF-α. Conclusion: This study clearly highlights the excessive dysregulated response of the three cytokines, more so of IL-10 which appears pivotal in the pathogenesis of FH. Interventions leading to suppression of IL-10 levels may be beneficial in improving the patient outcome.
Keywords: Fulminant hepatitis, genotype D, Hepatitis B virus
|How to cite this article:|
Rizvi M, Sami H, Azam M, Shukla I, Khan HM, Ajmal M R. Assessment of hepatitis B virus genotype D and interleukin-10, interferon gamma, and tumor necrosis factor-α in fulminant hepatic failure. CHRISMED J Health Res 2019;6:162-6
|How to cite this URL:|
Rizvi M, Sami H, Azam M, Shukla I, Khan HM, Ajmal M R. Assessment of hepatitis B virus genotype D and interleukin-10, interferon gamma, and tumor necrosis factor-α in fulminant hepatic failure. CHRISMED J Health Res [serial online] 2019 [cited 2019 Aug 20];6:162-6. Available from: http://www.cjhr.org/text.asp?2019/6/3/162/264372
| Introduction|| |
Fulminant hepatitis (FH) is a clinical syndrome consisting of sudden and severe liver injury that results in hepatic encephalopathy and acute liver failure., The rate of mortality from FH remains very high, although intensive medical care and implementation of the latest therapies, including liver transplantation, are better than ever before. The pathogenesis leading to the development of FHB is still being vigorously explored. Although enhanced replication of the virus , and an exuberant immune response mounted by the host  are considered significant factors in the pathogenesis, several issues are yet to be understood.
Recently, many reports have discussed the influence of the genotypes on outcome of hepatitis B virus (HBV) infections. It was reported that the intracellular accumulation of HBV DNA may play a role in inducing liver damage. Genetic analysis of HBV has revealed six different genotypes, A to F, based on an intergroup divergence in nucleotide sequence of 8% or more., These genotypes vary in geographical prevalence and in clinical and serological outcomes. While it is well-known that patients with FH have elevated levels of various cytokines, genotype-specific cytokine studies have not been extensively conducted.
In an attempt to elucidate the role of immunomodulatory, pro-inflammatory, and anti-inflammatory mediators in the pathogenesis of HBV genotype D FH, we assessed the role of interferon gamma (IFN-γ), tumor necrosis factor- α (TNF-α), and interleukin-10 (IL-10) in such patients.
| Subjects and Methods|| |
The study was conducted over a period of 1½ years from January 2013 to November 2014 in our institute. During this period, 276 cases of acute hepatitis presented to our facility among which 37 cases of fulminant hepatic failure (FHF) were screened after taking informed consent. This study was conducted after obtaining permission from institutional ethics committee of J. N. Medical College and the procedures followed in the study were in accordance with institutional guidelines.
Patients with autoimmune hepatitis, alcoholic hepatitis, drug-induced hepatitis, human immunodeficiency virus coinfection, patients giving a history of recent infection, surgery, trauma within the preceding 2 months, renal insufficiency, or with other acute or chronic inflammatory diseases were excluded from this study. None of the participants had received any antiviral or immunosuppressive therapy before or during the course of this study.
Collection of specimen
For all serological assays, venous blood samples were obtained after taking an informed consent. After centrifugation, the serum was stored at −40°C until used for study.
Liver function tests (LFTs) such as serum amino alanine transaminase (ALT), serum aspartate aminotransferase (AST) and alkaline phosphatase, bilirubin (direct and indirect) total bilirubin, albumin, globulin, creatinine and international normalized ratio (INR) for prothrombin time (PT) were performed. Specific investigations such as ultrasonographic examination of liver, upper gastrointestinal (GI) endoscopy, and liver biopsy were performed wherever feasible.
All patients with FH were screened for hepatitis A virus (HAV), HBV, hepatitis C virus (HCV), hepatitis E virus (HEV), and HIV by commercially available ELISA kits: hepatitis B surface antigen (HBsAg), third-generation anti-HCV, fourth-generation anti-HIV (J. Mitra and Co. Pvt. Ltd., India), anti-HAV IgM, and anti-HEV IgM (DRG International, Inc., USA). HBc lgM antibodies were tested in HbsAg-positive samples using DRG Anti-Hepatitis B Core IgM Antigen ELISA kit (DRG International Inc., USA). Hepatitis B e antigen was tested in HbsAg-positive samples by DRG Hepatitis B e Antigen ELISA kit (DRG International Inc., USA).
The tests were performed according to the manufacturer's instructions.
Patients positive for HBV were enrolled for further study.
Fulminant hepatic failure /hepatic encephalopathy
The diagnosis of FHF/acute liver failure was made if encephalopathy developed within 8 weeks of the onset of symptoms of liver disease, in a patient with no prior known liver disease.,, The diagnosis and grading of complications of FHF were made on standard criteria., All variables included in determining the prognosis were those recorded at presentation. For complications, namely cerebral edema, renal failure, and GI bleeding, findings recorded within 48 h of hospitalization were included for analysis.
Thirty healthy age- and sex-matched blood donors with no clinical evidence of present or past involvement of liver disease, having normal LFTs, negative for all viral markers (HAV, HBV, HCV, HEV, HIV), and absence of jaundice were taken as controls for our study.
Genotyping of hepatitis B virus
HBV-positive cases were genotyped. DNA was extracted from 100 μL serum by phenol-chloroform extraction method. A 125 base pair sequence of the surface gene of HBV was amplified using a thermal cycler (Labnics, USA). A genotyping system based on polymerase chain reaction using type-specific primers was used in this study.
Interleukin-10, interferon gamma, and tumor necrosis factor-α assay
To measure IL-10 and TNF-α levels in serum of FHB-infected patients and in healthy controls, ELISA kits were procured from Orgenium, Finland while IFN-γ serum level was measured by ELISA kit from Diaclone, France. The ELISA kit protocols were followed as per the manufacturer's instructions. Absorbance was read at 450 nm in an automated ELISA plate reader (Thermo scientific). Standard curves of optical densities and corresponding concentrations of IL-10, IFN-γ, and TNF-α were plotted to determine their concentrations in serum samples. Results were expressed in picograms per milliliter (pg/ml). The detection limits of the IL-10 assay were 2 pg/ml while TNF-α and IFN-γ assay were 9 and 5 pg/ml.
Correlations between continuous variables were assessed by the Spearman rank test, corrected for ties, where a value of P > 0.25 (combined with P < 0.05) was considered significant. The nonparametric Mann–Whitney U-test was used to determine the significance of differences in continuous variables. The level of significance in all cases was set at a two-tailed P < 0.05. The cytokine values were presented as mean values with standard deviation.
| Results|| |
Genotype D was detected in 10 cases (27.02%) out of 37 FH patients. The demographic, clinical, serological, and biochemical characteristics of these patients are shown in [Table 1]. The mean age was 38.0 years (range, 27–69), and the ratio of male to female was 7:3. The majority (63.5%) of patients were in the third decades of life. The mean peak total bilirubin was 19.7, the mean peak alanine aminotransferase was 215 IU/ml, and the mean lowest prothrombin time was 1.4. It was observed that hyperbilirubinemia was five-fold higher in FHB cases than in the healthy control group while ALT and AST levels were four- to five-fold higher the controls.
|Table 1: Demographic profile, hepatitis B e antigen status, and biochemical profile of hepatitis B virus genotype D patients with fulminant hepatic failure and acute hepatitis|
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Serum cytokine levels
Mean IL-10, IFN-γ, and TNF-α levels in FH patients were significantly higher than the healthy controls at 127.6 ± 73.45 pg/ml (P < 0.001), 10.6778 ± 5.23 pg/ml (P < 0.0001), 62.9000 ± 21.67 pg/ml (P < 0.001), respectively. In the healthy controls, mean IL-10, IFN-γ, and TNF-α levels were much lower as seen in [Figure 1] and [Table 2], [Table 3].
|Figure 1: Cytokine levels in hepatitis B virus genotype D fulminant hepatitis cases in comparison to controls|
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|Table 2: Cytokine profile of patients with hepatitis B virus fulminant hepatitis genotype D|
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|Table 3: Multiple linear regression analysis to study the independent variable predicting amino alanine transaminase, aspartate aminotransferase, international normalized ratio, and serum bilirubin in fulminant hepatitis B virus cases|
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On analyzing the cytokines by multiple linear regression analysis in relation to transaminases, PT INR, and serum bilirubin, IL-10 was independently and strongly associated with all the four parameters. TNF-α was inversely associated with PT-INR and serum bilirubin. IFN-γ was independently associated with ALT and like TNF-α inversely associated with PT-INR and serum bilirubin as observed in [Table 3]. TNF-α alone was independently associated with age [Table 4].
|Table 4: Multiple linear regression analysis to study the independent variable predicting age|
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| Discussion|| |
As observed in a prior study from India  and in our previous study, the HBV genotype D was more prevalent than A among FHF (in communication). In East Asian countries where genotypes B and C are dominant, FH genotype C is associated with the more aggressive course of FH in comparison to genotypes A, B, and D. The FHF patients infected by genotype D were observed to have a shorter period of evolution than FHF patients infected by genotype A suggesting that genotype D has a fast progression course. To understand why genotype D had a faster progression course, we assessed the role that cytokines could play in the pathogenesis of FHB genotype D.
In this study, we assessed the role that cytokines could play in the pathogenesis of FHB and analyzed the serum concentrations of immunoregulatory cytokines (IFN-γ), pro-inflammatory cytokines (TNF-α), and anti-inflammatory cytokine (IL-10) in genotype D FHB cases.
Although levels of all three – IFN-γ, TNF-α, and IL-10 were significantly higher in FHB genotype D patients in comparison to the healthy controls, IL-10 was hugely elevated in comparison to the other two. This is especially relevant as IL-10 levels were very low in both acute hepatitis B (AHB) and chronic hepatitis B (CHB) (in communication).
FH is a life-threatening illness that results ultimately from the nearly complete destruction of the liver.
We found that at admission not only TNF-α but also IL-10 and IFN-γ were higher in persons with FH than in our prior studies on AHB and CHB, indicating that in addition to a massive pro-inflammatory reaction, a compensatory anti-inflammatory response contributes to the onset of FH.
Elevated levels of circulating IFN gamma, TNF-α, and IL-10 were associated with high mortality in patients with FH. IL-10 blood levels were directly related to the development of liver failure, as indicated by ALT, AST, and PT INR levels. IFN gamma was independently associated with higher ALT levels. On analyzing by multiple logistic regression, TNF-α and IFN gamma were inversely associated with PT-INR and serum bilirubin. These cytokines possibly could be used as additional prognostic markers of FH. In several studies of patients with sepsis and multiple organ dysfunction syndrome, persistently elevated levels of pro- and anti-inflammatory mediators were associated with increased mortality.,,, Thus, increased production of inflammatory cytokines in septic shock and FHF is associated with IL-10 hypersecretion, and both pro- and anti-inflammatory mediators may be influenced by each other.
Bone  inferred that an “immunologic dissonance” develops a pathophysiologic response that is out of balance and inappropriate for the person's biologic needs leading to multiple organ dysfunction syndrome.
This may well explain our results. Further studies will be required to elucidate how pro-inflammatory and anti-inflammatory cytokines can interact in the initiation and progression of the liver cell damage. In conclusion, IL-10 serum levels were high, even in the presence of elevated concentrations of TNF- α, in patients with FH, in contrast to levels of this cytokine in patients with AHB or CHB.
| Conclusion|| |
Attributing precise roles to any given cytokine in the context of HBV pathogenesis or clinical progression are complicated. However, this study clearly highlights the excessive dysregulated response of the three cytokines, more so of IL-10 which appears pivotal in the pathogenesis of FH. Interventions leading to suppression of IL-10 levels may be beneficial in improving the patient outcome. FHF continues to be a major challenge for the clinician because of its high mortality rate and undoubtedly requires a multidisciplinary approach. Innovative approaches involving immunomodulation of cytokines may be an effective approach in the management of FH patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]