CLINICAL-EVALUATION OF A SINGLE REACTION, DIAGNOSTIC POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF HEPATITIS-C VIRUS-RNA

Background/Aims: In the past few years the detection of HCV-RNA by pol ymerase chain reaction has become a well-established diagnostic tool f or patients with chronic hepatitis C. However, the lack of reproducibl e results between laboratories and the relatively high proportion of f alse-positive results, has indicated the need for a standardized and r eliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain rea ction assay based on a single, combined reverse transcription and ampl ification reaction and on the use of Uracil-N-glycosilase to prevent c arry-over contamination (Amplicor HCV, Roche Molecular Systems). Metho ds: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric syst em. Serum samples collected from 446 patients, including 181 with chro nic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in h emodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterm inate serology (RIBA indeterminate results), as well as from 121 contr ols were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. Results: In all cases a g ood concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99 .3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 disc ordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed pa tients, patients with autoimmune hepatitis and asymptomatic carriers o f anti-HCV, both assays produced results which were consistent with th e clinical diagnosis. In the former group, polymerase chain reaction w as able to identify the presence of active viral replication in some a ntibody negative samples. Conclusions: Taken together, these results i ndicate that the commercial, single-step polymerase chain reaction ass ay has the same clinical sensitivity and specificity as nested polymer ase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnosti c applications.