In this prospective cohort study, eligible consecutive hospitalized patients with ACLF were recruited from the Department of Infectious Diseases, Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (TCM), from November 2007 to July 2011. All recruited patients were examined by clinicians and were enrolled into the study according to the criteria of ACLF[17]. The inclusion criteria were: (1) age from 18 to 65 years; (2) the presence of hepatitis B surface antigen in the serum for at least 6 mo; (3) HBV DNA level > 10⁵ copies/mL; (4) alanine aminotransferase (ALT) level > 5 times the upper limit of normal; and (5) acute hepatic insult manifesting as jaundice (serum total bilirubin ≥ 171 μmol/L or a daily increase ≥ 17.1 μmol/L) and coagulopathy [international normalized ratio (INR) ≥ 1.5 or prothrombin activity < 40%], complicated within 4 wk by ascites and/or encephalopathy. The exclusion criteria were: (1) super-infection or co-infection with hepatitis A, C, D, E viruses, or human immunodeficiency virus; (2) coexistence of any other liver diseases, such as autoimmune hepatitis, alcoholic liver disease, drug hepatitis or Wilson’s disease; (3) hepatocellular carcinoma diagnosed by computed tomography; (4) coexistence of any other serious systemic or psychiatric diseases; (5) jaundice caused by obstructive or hemolytic diseases; (6) prolonged prothrombin time induced by blood system disease; and (7) a previous course of any antiviral, immunomodulator or cytotoxic/immunosuppressive therapy for chronic hepatitis or other illnesses within at least the preceding 12 mo.

The study protocol was in accordance with the Helsinki Declaration of 1975. The ethics committee of the Affiliated Hospital of Chengdu University of TCM approved the study. Written informed consent was obtained from each patient or their relatives before enrollment. Furthermore, the non-availability of artificial liver support therapy and liver transplantation facilities were also explained to the patients.

This was a prospective cohort study. All consecutive hospitalized patients spontaneously formed two cohorts (entecavir/lamivudine cohort), according to their preferences for antiviral therapy. Eligible subjects were given comprehensive internal medicine for 60 d (study period), and were followed up until 52 wk after enrollment (follow-up period) or death.

The sample size was calculated based on the data from previous studies[18,19], which suggested a survival rate in the lamivudine-treated group of 50% and a survival rate in the entecavir-treated group of approximately 65%. The match ratio was 1:1. The sample size in each group was 54, with a type I error (one-sided) of 5%, and a power of at least 80%[20]. On the assumption of a rate of 10% loss to follow-up, a target sample size of 119 was required.

All eligible patients were requested to accept antiviral treatment both in the study period and in the follow-up period. The entecavir cohort was given entecavir 0.5 mg/d (Baraclude, Bristol-Myers Squibb, China), while the lamivudine cohort was given lamivudine 100 mg/d (QO, GlaxoSmithKline, China). In addition, each patient was given standard comprehensive internal medicine during the study period. This routinely included absolute bed rest, barrier nursing, high calorie diet (35-40 cal/kg per day), lactulose, and intensive care monitoring, intravenous plasma, maintenance water, electrolyte and acid-base equilibrium monitoring, and prevention and treatment of complications. Patients also received albumin, terlipressin, and proton pump inhibitors if required. Enteral or parenteral nutrition was provided for patients whose caloric requirement was not fulfilled by mouth.

Patient clinical and laboratory data were collected prospectively. The latter included: (1) biochemical tests reflecting hepatocyte damage, for example serum ALT, total bilirubin (TBIL), albumin (ALB), and creatinine (CREA), all assayed using a colorimetric method (Automatic Analyzer 7170A, Hitachi, Japan); (2) INR for prothrombin time, performed following the manufacturer’s instructions (STA-evolution, STAGO, France); (3) serum HBV DNA, determined by a fluorescent quantifying polymerase chain reaction (PCR) method with a low limit of detection of 1000 copies/mL (Lightcycler-480, Roche, Switzerland); and (4) HBV markers, for example HBV antigens and antibodies, detected using commercially available enzyme immunoassays (Alisei Quality System, RADIM, Italy).

The severity of liver disease was assessed by the Child-Turcotte Pugh (CTP) score[21] and model for end-stage liver disease (MELD) score. The MELD score was calculated according to the following equation[22]: 3.78 × ln. [total bilirubin (mg/dL)] + 11.2 × ln INR+ 9.57 × ln [creatinine (mg/dL)] + 6.43 × (constant for liver disease etiology: 0 if cholestatic or alcoholic, 1 otherwise).

Patients were examined every 15 d in the first 60 d, followed by every 3 mo up to 52 wk. Clinical and laboratory data, adverse events, and compliance were monitored during the first 60 d of treatment, and both adverse events and compliance were monitored every 3 mo up to 52 wk.

The primary endpoint of this study was survival rate at day 60. Secondary endpoints were reduction in HBV DNA and ALT levels, and improvement in CTP and MELD scores at day 60, and survival rate at week 52. All patients were followed up and the outcome (recovery, bridging to artificial liver support therapy or liver transplantation, or death) of treatment was recorded. The date and cause of each death were documented.

The patients were questioned regarding adverse events. All adverse events, regardless of their possible association with antiviral drugs, were recorded.

Efficacy and safety analyses were performed according to intention-to-treat (ITT), and were conducted on the full analysis set (FAS). This set principally included the data from all patients receiving at least 1 dose of the study drugs. Partially missing data of the clinical evaluation were carried forward with the principle of the last visit carried forward. Deaths occurring in the study or follow-up period were not regarded as discontinued subjects.

Quantitative data were described by the mean ± SD. Independent-samples t test or Mann-Whitney U tests were performed to compare differences between quantitative data. A χ² test or Fisher’s exact test was performed to calculate differences between qualitative data. A survival curve was calculated by the Kaplan-Meier method and compared statistically using a log-rank test. Univariate and multivariate analyses were used to assess the associations between survival and independent variables, and multivariate Cox regression analysis with conditional stepwise forward was then used to calculate the relative risk ratios and 95%CI. All statistical tests were two-tailed, and a significance level (P) of 0.05 was used. The statistical tests were performed using the Statistical Package for the Social Sciences (SPSS version 17.0; SPSS Inc., Chicago, IL, United States).

Subject disposition during the study is shown in Figure 1. A total of 176 patients with SAE of CHB were assessed for eligibility, and 119 eligible patients were enrolled. One hundred and five patients completed the study, 57 in the entecavir group and 48 in the lamivudine group. Fourteen patients did not complete the study, but received treatment and had complete observations on at least one related record at a time point; the clinical data of these patients were analyzed on the basis of ITT. The FAS population included 119, with 65 in the entecavir group and 54 in the lamivudine group.

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Figure 1 Patient disposition during the study. PPS: Per protocol set; FAS: Full analysis set.

Baseline characteristics of all patients are shown in Table 1. There was no significant difference in age, gender, serum ALT, ALB, TBIL, PTA, CREA, Na, CTP and MELD score, HBV DNA level, HBeAg (±), or complications between the two groups before treatment (all P > 0.05).

Viral and host responses to nucleoside analog therapy in the two groups are presented in Table 2. Posttreatment HBV DNA levels in the entecavir and lamivudine group were significantly lower than pretreatment levels. Compared with patients in the lamivudine group, patients in the entecavir group had significantly lower HBV DNA levels at days 15, 30, 45, and 60 (all P < 0.001). Undetectable HBV DNA (lower than 1000 copies/mL) was observed in 33 of the 119 (27.7%) patients during treatment, and the entecavir group had a higher proportion of patients achieving undetectable viremia at days 45 and 60 (all P < 0.05). With regard to the ALT response between the two groups, there was a statistical difference at day 15 (P < 0.01), but no significant difference at days 30, 45, and 60 (all P > 0.05).

CTP and MELD scores in both groups of patients are presented in Table 3. Post-treatment CTP and MELD scores in the entecavir and lamivudine group were significantly lower than pretreatment scores. In addition, CTP scores at days 45 and 60 in the entecavir group were superior to those in the lamivudine group, with statistical significance (P < 0.05). Similarly, post-treatment MELD scores in the entecavir group were significantly lower than those in the lamivudine group (P < 0.05).

Of the 119 patients in FAS, 51 (78.5%) survived in the entecavir group and 35 (64.8%) survived in the lamivudine group at day 60 (P = 0.066 by log-rank test, Figure 2A). However, at the end of the follow-up period of 52 wk, 42 (64.6%) in the entecavir group and 26 (48.1%) in the lamivudine group survived (P = 0.038 by log-rank test, Figure 2B).

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Figure 2 Survival curve for the entecavir and lamivudine groups as determined by the Kaplan-Meier method. A: 60-d; B: 52-wk.

The causes of death were analyzed for 37 patients who had a complete and reliable death record. The major causes were hepatorenal syndrome (8/57, 14.0%), multiple organ failure (3/57, 5.3%), encephalopathy (2/57, 3.5%) and upper gastrointestinal bleeding (2/57, 3.5%) in the entecavir group; hepatorenal syndrome (10/48, 20.8%), multiple organ failure (5/48, 10.4%), encephalopathy (4/48, 8.3%), liver failure (2/48, 4.2%) and upper gastrointestinal bleeding (1/48, 2.1%) in the lamivudine group. There were no significant differences between the two groups (all P > 0.05).

On univariate analysis, gender (P = 0.020), HBeAg (±) (P = 0.048), undetectable HBV DNA at day 30 (P = 0.035), pretreatment CTP score (P = 0.026), and MELD score (P = 0.020) were found to be significantly associated with long-term survival. However, age, platelet count, serum ALT, HBV DNA level, and treatment with entecavir were not associated with fatal outcomes. In the forward Cox regression analysis, pretreatment MELD (B, 1.357; 95%CI: 2.138-7.062; P = 0.000), and undetectable HBV DNA at day 30 (B, 1.556; 95%Cl: 1.811-12.411; P = 0.002) were found to be unfavorable predictors of long-term survival.

None of the patients developed drug-induced severe lactic acidosis or other serious adverse events, and all patients tolerated the therapy without dose modification or early discontinuation.

Acute-on-chronic liver failure (ACLF) is a severe clinical syndrome. Spontaneous acute exacerbation of chronic hepatitis B is a leading cause of ACLF. Liver transplantation is the definitive therapy, but is limited by donor shortage and high costs. Thus, it is necessary to explore other effective therapies.

Lamivudine improved short-term prognosis in ACLF patients. However, continuous lamivudine therapy increased the risk of re-exacerbation after temporary relief, because of hepatitis B virus (HBV) variants with YMDD mutations. Entecavir is another oral anti-HBV compound with potent activity and low resistance. Theoretically, entecavir may be more suitable for the long-term treatment of ACLF due to severe reactivation of HBV.

The present study demonstrates that entecavir significantly reduced HBV DNA levels, decreased the Child-Turcotte-Pugh and model for end-stage liver disease (MELD) scores, and thereby improved the long-term survival rate in patients with spontaneous reactivation of hepatitis B presenting as ACLF. The pretreatment MELD score and virological response at 30 d affected patient survival.

The data from this study provide a rational basis for nucleoside analog treatment in clinical practice for patients with severe reactivation of HBV presenting as ACLF.

ACLF is a clinical syndrome where acute hepatic insult, manifesting as jaundice and coagulopathy, is complicated within 4 wk by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease.

The results in the study are interesting and may help clinicians to select anti-HBV therapy.