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The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n ϭ 20) and extrahepatic (sera, n ϭ 32; PBMC, n ϭ 26 and fresh bone marrow cells, n ϭ 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5 Ј noncoding region, with or without a tag sequence, or in the nucleocapsid (CAP). Samples were selected to display different viral loads (10 5 -3 ϫ 10 7 genomic equivalent/ml or gram) and viral genotypes ( n ϭ 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, when either 5 Ј noncoding region primer pair was used, whereas both artifacts were dramatically reduced (

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