Cobas ampliprep taqman

We included adult (> 18 years) patients with HCV chronic infection that started IFN-free DAA therapy at Clinic Hospital, State University of Campinas (UNICAMP), Brazil, from December 2015 through July 2017. HCV genotypes 1, 2, and 3 were included. Chronic HCV infection was defined as the presence of HCV antibody (Abott AxSYM Anti-HCV 3.0; Abbott Laboratories, Wiesbaden, Germany) and detectable serum HCV RNA (Cobas Ampli Prep Taq Man; Roche Diagnostics Systems Inc., Almere, The Netherlands). Treatment-naive patients and those who previously failed to PEG-IFN and RBV or to PEG-IFN and RBV plus first generation PI were included. We excluded patients with HIV infection, post-liver transplant, and those who previously received SOF, DCV or SMV.

Blood samples were tested for Hepatitis B surface antigen (HBsAg), and antibodies against HBsAg (anti-HBsAg), Hepatitis B core antigen (anti-HBc),Hepatitis C (Anti-HCV) and HIV using Abbott ARCHITECT i2000SR Immunoassay System (Abbott Laboratories; Abbott Park, IL, USA) at the clinical laboratory of the First Hospital of Jilin University. All samples that were anti-HCV positive were confirmed by HCV-RNA test (COBAS AmpliPrep/TaqMan, Roche Diagnostics Ltd, Rotkreuz, Switzerland). Anti-HCV positive results were confirmed by recombinant immune blot assay (CHIRON RIBA HCV 3.0 SIA, Ortho Clinical Diagnostics, Johnson & Johnson, USA) in individuals who subsequently tested negative for HCV-RNA. HCV genotyping was performed by multicolor fluorescence polymerase chain reaction (PCR) using an HCV-RNA genotyping kit (BioAssay Science & Technology Co. Ltd., Beijing, China).

This protocol conformed to the ethics committee of Grenoble University Hospital (CHU-Grenoble) and the French Blood Service's (EFS-AuRA) Institutional Review Board and was declared under the number DC-2008-787 and DC-2011-1487. Written informed consent was obtained from all participants prior to their enrolment in this study. Blood samples were obtained from chronically HBV infected patients (HBV, n = 130) and healthy donors (HD, n = 85). Exclusion criteria included: infection with human immunodeficiency virus, co-infection with hepatitis C or D virus, other liver diseases, and current treatment with IFNα or immunosuppressive agents. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples using Ficoll-paque density gradient centrifugation according to the manufacturer's instructions (Eurobio). Plasma samples were collected and stored frozen. Serum HBsAg and viral load (HBV DNA) levels were quantified using the Abbott Architect i2000sr-QT assay (Abbott) and COBAS Ampliprep/Taqman (Roche), respectively. Liver biopsy samples were obtained from 29 HBV-infected patients and 33 non-viral infected patients. Liver tissue was reduced to cell suspensions by mechanical disruption. The clinical characteristics of the patients are summarized in Supplementary Table 1.