Blood samples were centrifuged within 2 hours after venipuncture and the sera were frozen and stored until analysis. Antibodies against HCV were identified by third-generation enzyme-linked immunosorbent assay (Sanofi Diagnostics Pasteur Inc, Freiburg, Germany). Reactivity was confirmed by immunoblot analysis (Mikrogen, Munich, Germany). Patients whose sera were initially found repeatedly reactive in enzyme-linked immunosorbent assay but showed indeterminate results in immunoblot analysis were retested after 3 and 6 months. Hepatitis C virus RNA was detected by Roche Amplicor 2.0 (Roche Diagnostics, Mannheim, Germany) or by VERSANT HCV RNA qualitative assay (Bayer Diagnostics, Emeryville, Calif). Seventy-six individuals negative for HCV antibodies who still could have been in the incubation stage of acute HCV infection (lasting up to 26 weeks33) and 22 patients receiving immunosuppressive therapy were screened for the presence of HCV RNA by nucleic acid amplification techniques. Hepatitis C virus RNA was quantified by VERSANT 3.0 b-DNA Assay (Bayer Diagnostics). Typing of HCV isolates was performed as described in full details elsewhere.34,35 Serotyping was performed with Murex HCV Serotyping 1-6 Assay (Abbott Laboratories, Wiesbaden, Germany) according to the manufacturer's instructions. The primer sequences used for amplification of the HVR 1 of subtype 1b isolates were as follows: HVRO5 (nucleotides, nt 1290 to 1310, numbering according to Takamizawa et al36), 5′-TGGGATATGATGATGAACTGG (first polymerase chain reaction [PCR]); HVRO3 (nt 2007 to 2027), 5′-TCCGCA[C, T]GTCTT[A, G]GTGAACCC (reverse transcriptase and first PCR); HVRI5 (nt 1326 to 1346), 5′-CTAGTGGTGTCGCAG[C, T]T[A, G]CTC (second PCR); HVRI3 (nt 1782 to 1802), 5′-CGCGTAATGCCAGCAATA[A, T, G]GG (second PCR). Amplification of the HCV core region was performed as described previously.34,35 As area controls, 10 HCV isolates were obtained from patients within a radius of approximately 400 km from the hospital.37 Products of the second PCR were purified from the agarose gel (QIAquick PCR Purification Kit; QIAGEN, Hilden, Germany) and were subjected to direct sequencing in both directions (Dye Terminator DNA Sequencing Kit, Perkin-Elmer, Norwalk, Conn).