Association of hepatitis B e antigen and DNA viral load with severity of liver dysfunction and in-hospital outcomes in hepatitis B-related liver cirrhosis

Introduction

About 240 million people are infected with hepatitis B virus (HBV) worldwide (1). In patients with untreated chronic hepatitis B, the 5-year cumulative incidence of cirrhosis ranges from 8% to 20% (2). The 5-year mortality in patients with cirrhosis is 14–35% (3-7). Notably, 60% of cases with liver cirrhosis result from HBV infection in China (8).

Chronic HBV patients usually present either as positive or negative hepatitis B e antigen (HBeAg). Traditionally, the natural history of chronic HBV infection is usually divided into the immune tolerant phase, immune reactive HBeAg-positive phase, inactive HBV carrier state, HBeAg-negative chronic HBV infection phase, and HBsAg-negative phase (9-11). According to the updated guidelines, the natural history is defined as follows: HBeAg-positive chronic HBV infection, HBeAg-positive chronic hepatitis B, HBeAg-negative chronic HBV infection, HBeAg-negative chronic hepatitis B, and HBsAg-negative phase (2). HBV DNA viral load also plays an important role in the diagnosis and treatment of chronic HBV infection. As HBV continues to infect the liver, repeated inflammatory necrosis results in the regeneration and repair and activation of hepatic stellate cells. The abnormal deposition of extracellular matrix induces liver fibrosis and ultimately leads to liver cirrhosis (9,12). While the role of HBeAg status and HBV DNA viral levels for estimating the risk of individuals to progress to liver cirrhosis or hepatocellular carcinoma is well established in Asian and Caucasian cohorts (2), their clinical relevance as biomarkers in patients with HBV-related liver cirrhosis for complications and short-term mortality is unclear.

Herein, we conducted a retrospective monocentric study to evaluate the impact of HBeAg status and HBV DNA viral load on the clinical profiles and in-hospital mortality of patients with HBV-related liver cirrhosis.

Methods

Study design

All liver cirrhosis patients with chronic HBV infection who were consecutively admitted to our hospital between January 2012 and June 2014 were considered eligible for the study. All eligible patients were positive for HBsAg. The exclusion criteria were as follows: liver cancer or co-diagnosed with other malignant tumors; co-infection with hepatitis C virus or other chronic liver diseases; and a history of alcohol abuse. As our study endpoint was the in-hospital death, repeated admissions were included. The study protocol was approved by the Medical Ethical Committee of our hospital. The approval number was No. k[2015]39.

Laboratory tests

The following data were collected at the moment of the subjects’ admissions: age, sex, red blood cell count (RBC), hemoglobin (Hb), white blood cell count (WBC), platelet count (PLT), total bilirubin (TBIL), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), creatinine (Cr), calcium (Ca), sodium (Na), kalium (K), international normalized ratio (INR), acute upper gastrointestinal bleeding (AUGIB), HBsAg, hepatitis B surface antibody (HBsAb), HBeAg, hepatitis B e antibody (HBeAb), hepatitis B core antibody (HBcAb), and HBV DNA viral load. Child-Pugh and model for end-stage liver disease (MELD) scores were calculated according to the results of laboratory tests and grades of ascites and hepatic encephalopathy (HE) (13,14). In-hospital death was recorded.

The serum HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb expression and quantification of HBV DNA viral load were detected by the electrochemiluminescence immunoassay with relevant reagents (Huake Co. Ltd., Shanghai, China) at the Department of Laboratory Medicine of our hospital. Detectable HBV DNA viral load was defined as HBV DNA viral load was more than 200 IU/mL (1 IU/mL=5 copies/mL) (15).

Definition and diagnosis

Chronic HBV infection is defined as hepatitis B surface antigen (HBsAg) positivity for more than 6 months. The clinical diagnosis of liver cirrhosis is made on the basis of clinical presentations, medical imaging examination, and laboratory abnormalities (16,17). Ascites is classified as three grades: (I) mild ascites only detectable by ultrasound; (II) moderate ascites with symmetrical distension of the abdomen; and (III) tense ascites (18,19). Hepatic encephalopathy (HE) manifests as impaired disturbance of consciousness, abnormal behavior, or coma (20).

Statistical analysis

Statistical analysis was performed using the SPSS statistics 17.0.0 software. Continuous data were expressed as mean ± standard deviation or median (range), and were compared by the non-parametric tests. Categorical data were expressed as frequency (percentage), and were compared by the Chi-square tests. The correlation analyses were performed by the Spearman rank test. Logistic regression analyses were performed to check the independent risk factor for in-hospital death. A two-sided P<0.05 was considered to be statistically significant.

Results

A total of 428 HBsAg-positive patients with liver cirrhosis were enrolled in this study. Among them, 284 (284/428, 66.4%) patients were male, and the median age was 53.91 years (range, 25.62–86.93 years). The proportion of positive HBsAg, positive HBeAb, and detectable HBV DNA viral load was 11.9% (40/335), 50.7% (170/335), and 38.25% (109/285), respectively. None (0/428) had positive HBsAb. The median HBV DNA viral load in 109 patients with detectable HBV DNA viral load was 94,000 IU/mL (range, 220–48,000,000 IU/mL). The in-hospital mortality was 3.7% (16/428). The patient characteristics were shown in Table 1. The causes of death were as follows: gastrointestinal bleeding (n=5), sudden death and pulmonary embolism (n=1), and liver failure with multiple organ failure (n=10).

Comparison between HBeAg-negative and HBeAg-positive patients

Compared with HBeAg-negative patients, HBeAg-positive patients had significantly higher HBV DNA viral load, ALT, AST, ALP, GGT, and Child-Pugh score, higher proportions of moderate-large ascites and Child-Pugh class B-C, and lower ALB (Table 2). The in-hospital mortality was not significantly different between HBeAg-negative and HBeAg-positive patients (3.7% vs. 2.5%, P=0.695). After adjusting the Child-Pugh score, the HBeAg status remained not associated with in-hospital death in a multivariate logistic regression analysis (odds ratio =0.494; 95% CI, 0.057–4.274; P=0.522).

Comparison between patients with undetectable and detectable HBV DNA viral load

Compared with patients with undetectable HBV DNA viral load, patients with detectable HBV DNA viral load were significantly older and had significantly higher Hb, TBIL, ALT, AST, ALP, GGT, Cr, INR, Child-Pugh score, and MELD score, higher proportions of moderate-large ascites and Child-Pugh class B-C, lower ALB and Ca, and a lower proportion of AUGIB (Table 3). The in-hospital mortality was not significantly different between patients with undetectable and detectable HBV DNA viral load (2.3% vs. 6.4%, P=0.077). After adjusting the Child-Pugh score, detectable HBV DNA viral load remained not associated with in-hospital death in a multivariate logistic regression analysis (odds ratio =1.919; 95% CI, 0.486–7.578; P=0.353).

Comparison between patients with HBV DNA viral load <2,000 and >2,000 IU/Ml

Compared with patients with HBV DNA viral load <2,000 IU/mL, patients with HBV DNA viral load >2,000 IU/mL were significantly older and had significantly higher Hb, TBIL, ALT, AST, ALP, GGT, Cr, INR, Child-Pugh score, and MELD score, higher proportions of moderate-large ascites and Child-Pugh class B-C, lower ALB, Ca, and Na, and a lower proportion of AUGIB (Table 4). The in-hospital mortality was higher in patients with HBV DNA viral load >2,000 IU/mL than in those with HBV DNA viral load <2,000 IU/mL (7.7% vs. 2.1%, P=0.021). After adjusting the Child-Pugh score, HBV DNA viral load >2,000 IU/mL was not an independent risk factor for in-hospital death in a multivariate logistic regression analysis (odds ratio =2.154; 95% CI, 0.548–8.468; P=0.272).

Correlation analysis of HBV DNA viral load

In 109 patients with detectable HBV DNA, the HBV DNA viral load positively correlated with ALT, AST, ALP, BUN, INR, Child-Pugh score, and MELD score, and negatively correlated with RBC, Hb, ALB, and Ca (Table 5).

Discussion

Major findings of our study were as follows: (I) the in-hospital mortality of cirrhotic patients with HBV DNA viral load >2,000 IU/mL was significantly elevated, but this association was compromised after adjusting the Child-Pugh score; (II) positive HBeAg, detectable HBV DNA viral load, and HBV DNA viral load >2,000 IU/mL were all related to the degree of liver and renal dysfunction, as indicated by biomarkers, such as TBIL, ALT, AST, ALP, GGT, Cr, and Child-Pugh and MELD scores; and (III) there was a significant correlation of HBV DNA viral load with worsening liver function laboratory/features.

Clinical practice guidelines on the management of chronic hepatitis B published by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) indicated that age was an important predictive factor for the progression of liver cirrhosis in chronic HBV infection patients (2,21-24). Chinese clinical practice guideline also suggested that the risk of liver cirrhosis in HBV patients would be increased with age and high serum HBV DNA viral load, especially in patients with age above 40 years. Additional studies reported that liver injury was more significant in chronic HBV patients with age above 46 years (25-27). Consistent with these findings, our patients with HBV-related liver cirrhosis had a median age of 53.91 years (range, 25.62–86.93 years).

HBeAg status, liver function, serum HBV DNA viral load, and liver histology are important factors in determining the severity of liver diseases (2,21,23,24). HBeAg is a significant marker of viral infectivity and persistence, and plays an important role in the natural history of chronic HBV (28). Traditionally, HBeAg seroconversion is defined as negative HBeAg and positive HBeAb with an associated reduction in HBV viral replication and a lower infectivity in the natural history of infection. In our study, the prevalence of HBeAg-negative patients was 88.1% (295/335) and the prevalence of HBeAb-positive patients was 57.3% (169/295) in HBeAg-negative patients. The data suggested that most of HBV-related cirrhotic patients had experienced HBeAg seroconversion. Our study found that HBeAg-negative patients with cirrhosis had significantly better liver function than HBeAg-positive patients, indicating that lack of HBeAg seroconversion (and subsequent high viremia) posed a particular risk for cirrhotic patients. However, some studies reported that HBeAg-negative patients with or without cirrhosis still could have experienced a high viral replication and/or risk of viral hepatitis exacerbation (25,29,30). Similarly, HBeAg-negative HBV patients with elevated ALT and active histological changes could experience faster progression to liver cirrhosis than HBeAg-positive HBV patients (31). The potential reasons included: (I) a longer duration of infection in HBeAg-negative patients than in HBeAg-positive patients; and (II) core promoter mutations that might increase the replication efficacy of HBV (32-35).

Some studies had reported that long-term antiviral therapy could improve the survival of patients with HBV-related liver cirrhosis (36-38), but few studies explored the impact of HBeAg status and HBV DNA viral load on the severity of liver dysfunction and in-hospital outcomes in HBV-related liver cirrhosis patients. Our study revealed higher likelihood of adverse outcomes in cirrhotic patients with HBV DNA viral load, thereby suggesting that antiviral treatment is urgently required in such patients. This is consistent with the recommendations from EASL guideline regarding management of HBV (2,22). Unfortunately, information regarding use of antiviral therapy was unavailable in our study. Accordingly, future studies should explore whether the initiation of antiviral therapy could improve the outcomes of HBV-related liver cirrhosis patients with HBV DNA viral load >2,000 IU/mL.

A lower proportion of AUGIB and higher Hb levels were observed in patients with HBV DNA viral load >2,000 IU/mL. Similarly, HBV DNA viral load negatively correlated with RBC and Hb. These findings suggested that patients with higher HBV DNA viral load might suffer from less bleeding events but had worse liver function. Indeed, the major cause of hospital admission in patients with HBV DNA viral load >2,000 IU/mL might be liver dysfunction; by contrast, the major cause of hospital admission in patients with HBV DNA viral load <2,000 IU/mL might be acute gastrointestinal bleeding.

Our study had some limitations. First, not all HBV patients had data available for HBeAg, HBeAb, and HBV DNA viral load. Second, the diagnosis of cirrhosis was not confirmed by liver histology. Third, this was a single-center observational study and we did not collect the long-term follow-up data. Fourth, we are unable to dissect a potential role of antiviral therapy. Some of patients with undetectable HBV DNA viral load might have been on nucleoside/nucleotide analogues.

In conclusion, HBeAg and HBV DNA viral load are factors associated with the severity of liver dysfunction in HBV-related liver cirrhosis patients. More importantly, the in-hospital mortality was significantly higher in such patients with HBV DNA viral load >2,000 IU/mL, but it was not an independent risk factor for death, after adjusting the Child-Pugh score.

Acknowledgements

Funding: Liaoning Provincial Startup Foundation for PhD (No. 201501023) for Dr. Y Zhang.

Footnote

Conflicts of Interest: The authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/amj.2017.09.10). Xingshun Qi serves as an Editor-in-Chief of AME Medical Journal. Eric M. Yoshida serves as an unpaid Associate Editor-in-Chief of AME Medical Journal from Jun 2017 to Jun 2019. Nahum Mendez-Sanchez serves as an unpaid editorial board member of AME Medical Journal from Mar 2017 to Mar 2019. Fernando Gomes Romeiro serves as an unpaid editorial board member of AME Medical Journal from Apr 2017 to Apr 2019. Andrea Mancuso serves as an unpaid editorial board member of AME Medical Journal from Mar 2017 to Mar 2019. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study protocol was approved by the Medical Ethical Committee of our hospital. The approval number was No. k[2015]39. Informed consent was waived due to the retrospective nature of the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Liang TJ. Hepatitis B: the virus and disease. Hepatology 2009;49:S13-21. [Crossref] [PubMed]
2. European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370-98. [Crossref] [PubMed]
3. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45:507-39. [Crossref] [PubMed]
4. Tong S, Revill P. Overview of hepatitis B viral replication and genetic variability. J Hepatol 2016;64:S4-S16. [Crossref] [PubMed]
5. Sanchez-Tapias JM, Costa J, Mas A, et al. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterology 2002;123:1848-56. [Crossref] [PubMed]
6. Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol 2008;48:335-52. [Crossref] [PubMed]
7. Perz JF, Armstrong GL, Farrington LA, et al. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 2006;45:529-38. [Crossref] [PubMed]
8. Wang FS, Fan JG, Zhang Z, et al. The global burden of liver disease: the major impact of China. Hepatology 2014;60:2099-108. [Crossref] [PubMed]
9. Hadziyannis SJ, Papatheodoridis GV. Hepatitis B e antigen-negative chronic hepatitis B: natural history and treatment. Semin Liver Dis 2006;26:130-41. [Crossref] [PubMed]
10. Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49:652-7. [Crossref] [PubMed]
11. Brunetto MR, Oliveri F, Colombatto P, et al. Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers. Gastroenterology 2010;139:483-90. [Crossref] [PubMed]
12. McMahon BJ. The natural history of chronic hepatitis B virus infection. Semin Liver Dis 2004;24:17-21. [Crossref] [PubMed]
13. Child CG, Turcotte JG. Surgery and portal hypertension. Major Probl Clin Surg 1964;1:1-85. [PubMed]
14. Kamath PS, Kim WR. Advanced Liver Disease Study G. The model for end-stage liver disease (MELD). Hepatology 2007;45:797-805. [Crossref] [PubMed]
15. Liaw YF, Leung N, Kao JH, et al. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update. Hepatol Int 2008;2:263-83. [Crossref] [PubMed]
16. Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet 2014;383:1749-61. [Crossref] [PubMed]
17. Aube C, Bazeries P, Lebigot J, et al. Liver fibrosis, cirrhosis, and cirrhosis-related nodules: Imaging diagnosis and surveillance. Diagn Interv Imaging 2017;98:455-68. [Crossref] [PubMed]
18. Tsochatzis EA, Gerbes AL. Diagnosis and treatment of ascites. J Hepatol 2017;67:184-5. [Crossref] [PubMed]
19. Uojima H, Kinbara T, Hidaka H, et al. Close correlation between urinary sodium excretion and response to tolvaptan in liver cirrhosis patients with ascites. Hepatol Res 2017;47:E14-E21. [Crossref] [PubMed]
20. Vilstrup H, Amodio P, Bajaj J, et al. Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver. Hepatology 2014;60:715-35. [Crossref] [PubMed]
21. Terrault NA, Bzowej NH, Chang KM, et al. AASLD guidelines for treatment of chronic hepatitis B. Hepatology 2016;63:261-83. [Crossref] [PubMed]
22. European Association For The Study Of The L. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J Hepatol 2012;57:167-85. [Crossref] [PubMed]
23. Korean Association for the Study of the L. KASL Clinical Practice Guidelines. Management of chronic hepatitis B. Clin Mol Hepatol 2012;18:109-62. [Crossref] [PubMed]
24. Sarin SK, Kumar M, Lau GK, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int 2016;10:1-98. [Crossref] [PubMed]
25. Ormeci A, Aydin Y, Sumnu A, et al. Predictors of treatment requirement in HBeAg-negative chronic hepatitis B patients with persistently normal alanine aminotransferase and high serum HBV DNA levels. Int J Infect Dis 2016; [Crossref] [PubMed]
26. Yapali S, Talaat N, Lok AS. Management of hepatitis B: our practice and how it relates to the guidelines. Clin Gastroenterol Hepatol 2014;12:16-26. [Crossref] [PubMed]
27. Chen YC, Chu CM, Liaw YF. Age-specific prognosis following spontaneous hepatitis B e antigen seroconversion in chronic hepatitis B. Hepatology 2010;51:435-44. [Crossref] [PubMed]
28. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;64:51-68. [Crossref] [PubMed]
29. Funk ML, Rosenberg DM, Lok AS. World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants. J Viral Hepat 2002;9:52-61. [Crossref] [PubMed]
30. Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic hepatitis B. Hepatology 2001;34:617-24. [Crossref] [PubMed]
31. Alexopoulou A, Karayiannis P. HBeAg negative variants and their role in the natural history of chronic hepatitis B virus infection. World J Gastroenterol 2014;20:7644-52. [Crossref] [PubMed]
32. Tacke F, Gehrke C, Luedde T, et al. Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants. J Virol 2004;78:8524-35. [Crossref] [PubMed]
33. Chen CH, Lee CM, Lu SN, et al. Clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations affecting HBV e antigen expression in Taiwan. J Clin Microbiol 2005;43:6000-6. [Crossref] [PubMed]
34. Utama A, Siburian MD, Purwantomo S, et al. Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients. World J Gastroenterol 2011;17:708-16. [Crossref] [PubMed]
35. Huang Y, Deng H, Shan X, et al. Lower mutation frequency of BCP/precore regions in e antigen-negative chronic HBV-infected children instead of adults patients. PLoS One 2015;10:e0120733. [Crossref] [PubMed]
36. Perrillo R, Schiff E, Yoshida E, et al. Adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis B mutants. Hepatology 2000;32:129-34. [Crossref] [PubMed]
37. Xu Y, Zhang YG, Wang X, et al. Long-term antiviral efficacy of entecavir and liver histology improvement in Chinese patients with hepatitis B virus-related cirrhosis. World J Gastroenterol 2015;21:7869-76. [Crossref] [PubMed]
38. Goyal SK, Dixit VK, Shukla SK, et al. Prolonged use of tenofovir and entecavir in hepatitis B virus-related cirrhosis. Indian J Gastroenterol 2015;34:286-91. [Crossref] [PubMed]