Eligibility
Patients were recruited at Beijing Di Tan Hospital, Capital Medical University, People’s Liberation Army No. 302 Hospital, and Beijing You-An Hospital, Capital Medical University, from April 2013 to May 2015. All procedures related to this research were accorded morally with current laws as well as the creeds of the Declaration of Helsinki. The research was permitted by the Ethical Committee of Beijing You-An Hospital, Capital Medical University, Beijing Di Tan Hospital Capital Medical University, and People’s Liberation Army No. 302 Hospital (No.2 [2013]). All study participants gave their informed consent to participate in the study. The process of study selection and exclusion was shown in Fig. 1.
Patients selection
The inclusion criteria were the following: (1) aged 18 years or older; (2) previously diagnosed or undiagnosed HBV, HBsAg positive; and (3) all enrolled patients met the criteria for ACLF from the consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL) specified as follows: an acute hepatic insult manifesting as jaundice (serum bilirubin ≥ 5 mg/dl [≥ 85 μmol/l]) and coagulopathy (INR ≥ 1.5 or prothrombin activity ≤ 40%) complicated within 4 weeks by clinical ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease/cirrhosis [17, 18]. Diagnostic criteria for cirrhosis are made by history, physical examination, and previously available laboratory, fibrosis biomarkers (e.g., FIB-4 or FibroTest), endoscopic or radiologic investigations (ultrasound, CT abdomen or transient elastography [fibroscan]), or a previously liver biopsy history [19].
The exclusion criteria comprised the following: (1) uncontrolled bacterial infection or gastrointestinal hemorrhage before enrollment; (2) infection with hepatitis virus other than HBV, or human immunodeficiency virus; (3) autoimmune diseases, alcoholic liver disease, and drug-induced hepatitis; and (4) serious renal, cardiac, respiratory, neurologic diseases, or any detectable tumor.
The diagnostic criteria of complications included (1) gastrointestinal hemorrhage confirmed by endoscopy, (2) bacterial infection diagnosed by a positive culture result [20], (3) fungal infection diagnosed according to EORTC/MSG definition [21], (4) hepatorenal syndrome (HRS) diagnosed according to the International Ascites Club’s guidelines [22], (5) spontaneous bacterial peritonitis (SBP) diagnosed based on diagnostic paracentesis [23], (6) electrolyte disturbance defined as ≥ 1 electrolyte abnormalities of K+, Na+, and Cl−, (7) hypoalbuminemia diagnosed when albumin < 35 g/L, and (8) pleural effusion diagnosed by X rays or computerized tomography.
Study design
After investigators confirmed eligibility, patients were randomized (1:1 allocation ratio) to MP plus standard management (MP group) or standard treatments (control group) by computer-generated permutated block randomization (block size of four) stratified. Treatment was started when patients were enrolled and the day of enrollment was defined as day 1.
Patients were followed up at the baseline (0 days), 3 days, 7 days, 10 days, 14 days, 30 days, and then monthly until the 6th month. Collected information included laboratory tests, screening for complications. The alive status means significant improvement in clinical symptoms, bilirubin< 5 × upper limit of normal (5ULN), PTA > 30%, or INR < 1.5.
Treatment protocols
Standard treatments included antiviral drugs (lamivudine 100 mg/day, adefovir dipivoxil 10 mg/day, entecavir 0.5 mg/day, telbivudine 600 mg/day, or tenofovir disoproxil fumarate 300 mg/day based on individual’s condition before enrollment), nutritional support (1.5–2.0 g protein/kg/day and 35–40 kcal/kg/day), plasma exchange, and complications control such as anti-infection, administration of human serum albumin (10 g per day until serum albumin was 35 g/L), fresh frozen plasma (200 ml to 400 ml/day until the INR < 1.5), and vasoactive agents (terlipressin alone or in combination with noradrenaline to reverse septic shock). All treatments were performed based on the criteria of diagnostic and treatment guidelines for ACLF adopted by the Chinese Medical Association [24].
The MP group was given standard treatments combined with MP intravenously guttae for 7 days: 1.5 mg/kg/day, days 1–3; thereafter 1 mg/kg/day, days 4–5; and followed by 0.5 mg/kg/day, days 6–7.
Clinical and laboratory parameters
Laboratory tests measured PTA, prothrombin time (PT), INR, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, creatinine, blood urea nitrogen, cholesterol, cholinesterase, triglyceride, alpha-fatal protein (AFP), blood ammonia, hemoglobin, platelet, white blood cell, red blood cell, lymphocyte percentage, monocyte percentage, lymphocyte to monocyte ratio (LMR), and procalcitonin and neutrophil percentage with routine automated techniques. HBsAg and HBeAg were assayed using commercially available radioimmunoassay kits (Roche Diagnostics). Serum HBV-DNA was quantified using a cross linking chemical hybridization assay (Roche Diagnostics) and the detection threshold is 100 copies/ml. All hospitals use the same assay and standard operating procedure for the above indicators. Model for end-stage liver disease (MELD) score was calculated according to the original formula proposed by the Mayo Clinic [25].
Study outcomes
The primary endpoints were 6-month mortality and prognostic factors for 6-month survival. The secondary endpoints were adverse events and changes of laboratory indices during treatment.
Statistical analysis
Quantitative variables, expressed as means ± SD or medians (interquartile range), were compared using Wilcoxon, Kruskal–Wallis, or Student’s t tests, as appropriate. Qualitative variables, expressed as percentages, were compared using chi-square or Fisher’s exact tests. Ordinal enumeration variables were analyzed by rank sum tests. The changes of indices during follow up time were compared using variance analysis for repeated data. Kaplan-Meier survival curves were plotted and compared with log-rank tests. Significantly predictive factors of mortality in univariate models (P < 0.1) were included in multivariate Cox-regression models. ROC curve was used to identify the predictive value of indices for prognosis. All analyses were performed using SPSS (version 26.0, Chicago, IL, USA), and statistical significance was set up as a two-sided P value < 0.05. The formula was used to calculate the sample size: n1 = n2 = \( \frac{\left({q}_1^{-1}+{q}_1^{-1}\right){\left({Z}_{\alpha /2}+{Z}_{\beta}\right)}^2{S}^2}{\delta^2} \), where (α = 0.05, β = 0.10, 1-β = 0.90, σ = 8.8, S = 15.9). The required sample size was “70” for each group. Improvement of PTA by at least 30% is proposed to make a difference on the curative effect [26].
