Versant hcv genotype 2.0 assay lipa

FIB-4 was determined according to the equation of Sterling et al [16 (link)], FIB-4 score <1.45-3.25=F0-2 (none or moderate fibrosis) and FIB-4 score >3.25=F3-4 (advanced fibrosis or cirrhosis). CHC GT4 genotyping at screening was assessed by the VERSANT-HCV Genotype 2.0 Assay (LiPA) (Siemens, Germany). Also, genotyping for the IL-28B rs12979860 C/T polymorphism was performed using a polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay. HCV RNA was analyzed by quantitative PCR (qPCR), using a Roche Amplicor HCV monitor version 2.0 (Roche Molecular Systems, Inc., Branchburg, NJ) with lower detection limit <15 IU/mL. Hematological parameters were determined using a MICROS ABX autoanalyzer according to the manufacturer’s protocol.

All eligible patients underwent clinical examinations before treatment, after 12 weeks of the therapy period (EOT), and also 12 weeks after the therapy cessation. The patients underwent laboratory investigation including aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin content and total bilirubin, determined using reagent kits purchased from Human Diagnostics (Germany). The FIB-4 score was calculated using Sterling’s formula [12 ]. Interleukin-28B (IL-28B) genotype was estimated by use of polymerase-chain-reaction amplification (PCR), while HCV GT4 genotype was assessed by the VERSANT-HCV Genotype 2.0 Assay (LiPA) (Siemens, Germany). Quantitative polymerase chain reaction (qPCR) for HCV was assessed by Roche Amplicor HCV monitor version 2.0 (Roche Diagnostics, Branchburg, NJ), with lower detection limit < 15 IU/ml. However, hematological parameters were determined using an auto-analyzer (MICROS ABX) according to the manufacturer’s protocol.

HCV geno/subtype were determined pretreatment by sequencing a 329-base pair region within NS5B followed by basic local alignment search tool (BLAST) analysis. The results of the NS5B-based assay or, if missing, the results from the VERSANT HCV Genotype 2.0 Assay (LiPA) (Siemens Healthcare Diagnostics, Erlangen, Germany) or TRUGENE assay (Bayer HealthCare, Montville, NJ) were used.
HCV NS3/4A population sequencing using the conventional Sanger technique, as previously described, was performed pretreatment for all patients and postbaseline for patients treated with SMV/PegIFN/RBV who did not achieve SVR for any reason [7 (link)].
In vitro activity of SMV was assessed using genotype 1a or 1b replicons carrying site-directed mutants in a transient replicon assay, and cutoff values were used to differentiate between full susceptibility to SMV (≤2.0-fold reduction in SMV activity) and low-level versus high-level resistance (≥50-fold reduction in SMV activity) [6 (link), 11 (link)].
Resistance analyses considered 18 NS3 amino acid positions (36, 41, 43, 54, 55, 80, 107, 122, 132, 138, 155, 156, 158, 168, 169, 170, 174, and 175) associated with resistance to SMV or other HCV NS3/4A protease inhibitors, or that were considered to be of interest based on in vitro or in vivo observations in studies with SMV [7 (link)].

The HCV viral load was quantified using an Abbott RealTime HCV assay (Abbott Laboratories, Des Plaines, IL, USA; lower limit of detection 12 IU/mL). HCV genotype was assessed using a Versant HCV Genotype 2.0 Assay (LiPA) by Siemens (Siemens Healthcare Diagnostics, Tarry-town, NY, USA). Single-nucleotide polymorphisms (SNPs) of IL28B genomic region (rs12979860, rs8099917, rs12980275), and ITPA genomic region (rs1127354) were genotyped. The host genotypes were identified with iPLEX Gold (Sequenom, San Diego, CA, USA) at CapitalBio, a platform that could map SNPs.