Taqman assay

The baseline data including age, sex, biochemical investigations, virological tests, abdominal ultrasound, and major complications were collected at the time of admission and summarized in Table 1. Severity of the liver disease was assessed by Child–Turcotte–Pugh (CTP) and model for end-stage liver disease (MELD) scoring systems.
Serological tests for HBsAg and hepatitis B e antigen (HBeAg) were done by commercially available enzyme-linked immunoassays. The quantification of HBV DNA load was performed with the real-time polymerase chain reaction method (lower limit of detection 1000 copies/mL, Roche TaqMan assay).
Spontaneous bacterial peritonitis (SBP) and hepatorenal syndrome (HRS) was defined by International Ascites Club criteria.^{12 (link)} Hepatic encephalopathy (HE) with 4 grades (grade 1–4) was defined by the HE scoring algorithm (West Haven Criteria).^{13 (link)}

Clinical and laboratory information was collected at the time of admission. Information included age, sex, MELD score; major precipitating factor before the occurrence of HE during the stay in hospital, such as: infection, upper gastrointestinal bleeding, electrolyte disorder, PPI use, lactulose use, drugs to prevent hepatic coma, use of intestinal probiotic preparations and oral antibiotics; laboratory data including albumin, aminotransferase, r-GGT, total bilirubin, direct bilirubin, and creatinine levels, urea nitrogen, and the international normalized ratio (INR) of the prothrombin time, prothrombin activity and serological tests for hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAg), and anti-HBe which were measured by commercially available enzyme-linked immunoassays. HBV DNA estimation was done with the real-time polymerase chain reaction (PCR) method (lower limit of detection = 50 IU/mL, Roche Taqman assay). Of the 30 variables included in the univariate analysis, we included the variables with P < 0.1 which were clinically a common cause of HE into the Logistic regression. In the multivariate analysis, 14 variables were included in a stepwise regression.

Blood samples were taken at baseline, during treatment weeks 1, 2, 4, 5, 6, 8, 12, 24, 36 and 48 for BOC and 1, 2, 4, 8, 12, 24, 36, 48 for TEL, and at weeks 12, 24 and 48 post-treatment. HCV-RNA titres (Table 2) were measured at baseline, at treatment weeks 4, 8, 12, 16, 24, 28, 36, 40 and 48, and 12 and 24 weeks after treatment withdrawal. Analyses of the NS3 protease were performed on baseline and post-treatment samples from non-responders or patients with viral breakthrough/relapse. Patients 7, 14 and 21 had no bio-bank sample at treatment failure, thus blood samples taken later were analyzed.
HCV-RNA levels were measured with a real-time TaqMan assay (Roche Molecular systems) with a lower limit of detection of 15 IU/ml [14] (link) or an in-house real-time PCR method as described [15] (link). IL-28B genotyping was performed as described [16] (link).

We assayed the serum human immunodeficiency virus (HIV) antibody level in 15 patients. We also evaluated the frequencies of CD4⁺ and CD8⁺ T cells at admission and after amelioration of hepatitis in six patients. Six patients who provided written informed consent for evaluation of their CD4⁺ and CD8⁺ T-cells numbers were enrolled. We measured the plasma HIV RNA level using the Roche TaqMan assay (lower limit of detection, 20 copies/mL); we assayed CD4⁺ and CD8⁺ T-cells subsets by flow cytometry. Serum sIL-2R levels and CD4⁺ and CD8⁺ T-cell counts are routinely measured in our hospital when needed. These parameters served as measures of T-cell activation.