Quantitation of hepatitis C viraemia by a competitive reverse transcription-polymerase chain reaction system

Chronic hepatitis C infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. A quantitative assay to determine the level of hepatitis C (HCV) viraemia during treatment would be useful in determining the effect of antiviral agents. Such an assay has been developed with the principle of the method being the co-amplification of the viral genome isolated from the patient with an RNA competitor molecule (CM) using the competitive reverse transcription-polymerase chain reaction (RT-PCR). Known amounts of the CM compete for amplification with HCV RNA from the patient. To quantify each sample, 5 amplification reactions with titrated amounts of CM were performed. The CM can be distinguished from the normal HCV PCR product since it has been genetically altered to be a smaller molecule by the process of restriction digestion, ligation and reamplification. This quantitative method was used to monitor the viral load in 10 patients undergoing antiviral therapy with lymphoblastoid interferon. The level of HCV viraemia in these patients ranged from 10(9) to 10(12) genomes/ml serum. Declines in the level of viraemia were seen in 8 of the 10 patients after therapy. Since patients with low HCV viraemia levels are more likely to respond to interferon therapy in a sustained fashion, this method may also be employed to quantitate the level of viraemia in patients prior to interferon treatment, and may be an indicator of the dose and schedule of treatment. These results show that this quantitative method is useful in the monitoring of HCV viral load in patients.