3), 50 mM KCl, Tween-20 0.01%, 0.2 mM deoxyribonucleotides, 2-4 pmol of each

primer, 2 mM MgCl2, and 0.5 units hot-start Taq DNA polymerase (RighTaq, Euroclone, Milan, Italy). Samples containing 10 ng of genomic DNA were subjected to 40 cycles of denaturation (at 95°C for 30 seconds), annealing (at 62°C for 30 seconds), and elongation (at 72°C for 30 seconds) using a Techne TC-412 thermal cycler. In a total volume of 20 μL, 10 μL of the amplicons were digested with 1 unit of the BstU-I restriction endonuclease (New England Biolabs, Hitchin, UK) at 60°C overnight. The digest fragments were 135, 82, and 25 bp for the C allele and 160 and 82 bp for the T allele variant. The fragments were resolved by electrophoresis on a 3.5% agarose gel after staining with ethidium bromide. As mentioned above, 144 out of 211 patients (68.2%) underwent a liver biopsy before starting therapy. Selleck PD-1/PD-L1 inhibitor Grade and stage were scored according to the Ishak system.17 All patients were treated with a combination therapy of PEG-IFN plus ribavirin. One hundred fifty-three patients (72.5%) received peginterferon alfa-2b (PegIntron, Schering-Plough, New Jersey, USA) at a dosage of 1.5 μg/kg/week, and 58 patients (27.5%) received peginterferon alfa-2a (Pegasys, Roche, Basel, Switzerland) at a dosage of 180 μg per week. In patients infected with HCV genotypes 1, 4, and 5, ribavirin (either Rebetol, Schering-Plough,

or Copegus, Roche) was administered according to body weight (1,000 mg/day for patients weighing <75 kg, 1,200 mg/day for patients weighing ≥75 kg); in the case of infection by genotypes 2 and 3, a single ribavirin PLX3397 supplier dose of 800 mg/day was used. The duration of therapy was 48 weeks for genotypes 1, 4, and 5 and 24 weeks for genotypes 2 and 3. Rapid viral response (RVR) was defined as an

undetectable serum HCV RNA (<50 IU/mL) level 4 weeks after starting therapy. Complete early viral selleck kinase inhibitor response (cEVR) was defined as an undetectable serum HCV RNA level 12 weeks after starting therapy. The end of treatment viral response (EOT) was defined as an undetectable serum HCV RNA level after completing the treatment schedule. Sustained viral response (SVR) was defined as an undetectable serum HCV RNA level at 24 weeks after stopping antiviral therapy. Patients who achieved EOT but reverted to a detectable HCV RNA level after stopping therapy were considered relapsers. Dropout was defined as discontinuation of antiviral therapy due to adverse effects. The stopping rule consisted of therapy discontinuation in HCV 1-, 4- and 5-infected patients who either failed to obtain a reduction in serum HCV RNA concentration of at least 2 log compared with baseline at week 12 or had a detectable serum HCV RNA level after 24 weeks of therapy.18-20 Patients who met stopping rule criteria for therapy discontinuation were defined as nonresponders.