Quantitation of hepatitis C virus using an in-house real-time reverse transcriptase polymerase chain reaction in plasma samples

Even with the most advanced 3rd-generation assays, the serologic window period of hepatitis C virus (HCV) is approximately 74 days. HCV RNA detection would reduce the risk of transmission during this period. Furthermore, quantitation of HCV RNA is necessary for proper planning of treatment, monitoring disease progression, and assessing response to antiviral therapy. We have standardized an in-house HCV real-time reverse transcriptase polymerase chain reaction (RT-PCR) for screening and accurate quantitation and detection of HCV RNA in plasma samples. The in-house real-time assay was compared with a commercial assay using 100 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, HCV antibody, and HIV antibody. The lower limit of detection of this in-house HCV real-time RT-PCR as assessed against the World Health Organization (WHO) standard was 50 IU/mL. Interassay and intraassay coefficient of variation ranged from 1.3% to 6.4% and 0.0% to 2.3% respectively. Virus loads as estimated with this in-house HCV real-time assay correlated with the commercial artus HCV RG RT-PCR assay (r = 0.59, P < 0.0001). This assay could be used in screening and monitoring individuals on therapy, showing no genotype-dependent differences in detection.