Direct detection of circulating hepatitis C virus RNA using probes from the 5' untranslated region.

Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. […] Abstract Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 983%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10' chimpanzee infectious doses5o/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards. (J. Clin. chronic HCV infection were included in this study. Diagnosis of HCV infection was based on exclu-sion of other etiologies for chronic liver disease and a positive HCV RNA PCR. One patient had evidence of concurrent chronic HCV and hepatitis B virus infection. 20 patients with acute and chronic liver diseases of other etiologies were studied as negative controls. In each control patient, the HCV RNA PCR was negative. The diagnoses in the control group included: acute hepatitis A (n = 2), chronic hepatitis B (n = 6), chronic hepatitis B and D (n = 1), chronic autoimmune hepatitis type 1 (n = 2), primary biliary cirrhosis (n = 1), Wilson's disease (n = 1), secondary biliary cirrhosis (n = 1), alcoholic hepatitis and/or cirrhosis (n = 4), cryptogenic cirrhosis (n = 1), and steatonecrosis secondary to obesity and hyperlipoproteinemia (n = 1). All serum samples were stored at before use.