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Hepatitis C virus (HCV) RNA polymerase chain reaction (PCR) is widely used for diagnosis of HCV infection and evaluation of therapy. The sensitive hepatitis B virus (HBV) DNA PCR is often reserved for detection of quantities of HBV DNA that are insufficient for hybridization. Application of both PCR techniques is limited by their labor-intensity, potential for contamination, and substantial time required for analysis. To study HCV and HBV infections, occurring alone or in combination, we developed a combined one-step PCR method to detect HCV RNA and HBV DNA in a single serum specimen using oligoprimers from the HCV 5′ untranslated region and the HBV preS/S region. Specificity of the HBV and HCV PCR products was confirmed on the basis of their molecular sizes in positive samples, Southern blot hybridization, and negative controls. The sensitivities of the combined PCR were assessed using samples containing a wide range of defined amounts of HBV DNA and HCV RNA and were comparable with those obtained with conventional HBV DNA or HCV RNA PCR methods. The sensitivity of the combined method was further validated by the 100% concordance between results of its HBV and HCV components and those of conventional PCR methods in patients with HBV and/or HCV infections. The combined one-step HBV/HCV PCR is a sensitive, specific, rapid, and cost-effective method, especially suited for epidemiological screening and clinical diagnosis of HBV and HCV infections occurring alone or in combination.

Although detection of hepatitis C virus RNA with polymerase chain reaction has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmol/L MgCl2. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virus-infected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of hepatitis C virus (HCV) infection in fulminant hepatic failure is controversial. The frequency of serum HCV RNA positivity in previously reported patients with fulminant hepatic failure (FHF) of indeterminate cause ranged from 0 to 12% in the United States and Europe and from 43% to 59% in Asia. We assessed serum HCV RNA using polymerase chain reaction (PCR) and oligoprimers from the 5'UTR of the HCV genome in 26 consecutive patients with FHF. Another laboratory independently performed PCR on 21 of the serum samples using different oligoprimers from the 5'UTR and NS3 region of the HCV genome. Serum HCV RNA was detected in two of seven (28%) patients with hepatitis B, 9 of 15 (60%) with an indeterminate cause, and in none with hepatitis A (n = 2) or drug-induced hepatotoxicity (n = 2). HCV RNA PCR results were concordant between both laboratories in 17 of 21 (81%) of samples. In patients with an indeterminate cause, HCV RNA positivity was significantly associated with the transmission risk factor of low socioeconomic status and Hispanic ethnicity. Eighteen patients underwent liver transplantation (LT) and 15 (83%) survived. Among patients with FHF of indeterminate cause, recurrent or acquired HCV infection after transplantation occurred in three of five (60%) and one of four (25%) patients, respectively. Three of four (75%) patients with hepatitis C virus infection post-LT also developed histologic hepatitis. HCV appears to be the causative agent of a substantial number of cases of FHF classified as indeterminate in the Los Angeles area. Differences in patient populations or risk factors may explain the discordant incidences of HCV infection in FHF observed among different programs.