Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction

Abstract

Background/Aims: The determination of HCV-RNA concentration in liver samples is likely to provide interesting insights for the study of disease progression and for evaluation of the efficacy of anti-viral therapy. Methods: a procedure was developed for the precise quantification of HCV-RNA in liver biopsies, based on the competitive reverse transcription-polymerase chain reaction technology. This competitive assay consists of the co-amplification of the target RNA with known amounts of a competitor RNA molecule containing the same sequence as the target plus an insertion in the middle, allowing resolution of the two amplification products by gel electrophoresis. Results: The amounts of HCV-genomic-RNA and β-actin mRNA (the latter being used as an internal standard to overcome the problem of reproducibility of quantitative RNA extraction) were evaluated in liver biopsies of 15 patients affected by hepatitis C virus-positive chronic liver disease at the time of diagnosis. All the patients underwent α-interferon therapy for 6 months and were subsequently followed for at least 1 further year after the end of treatment. Viral RNA concentration (which ranged from 2 to 2.7 × 10 5 HCV-RNA molecules per 10 16 β-actin molecules) directly correlated with the efficacy of treatment, indicating that low levels of viral replication in the liver are associated with a poor response to therapy. Conclusions: This study suggests that the determination of viral load in the liver is an important prognostic tool for the prediction of the efficacy of α-interferon therapy.