A rapid method for the detection of hepatitis C virus RNA by in situ hybridization

The histologic detection of hepatitis C (Hep C) in paraffin-embedded tissue sections has been problematic. We describe the use of a rapid, i.e., less than 2 hours, in situ hybridization technique to detect Hep C by use of a manual capillary action system (MicroProbe Staining System) and a 24-base synthetic multibiotinylated oligonucleotide antisense probe from the prototype Hep C virus base sequence -16 to -13. This technique is rapid, i.e., it requires a 30-minute hybridization time and easily reproducible, and it uses a nonradioactive detection system, i.e., streptavidin-conjugated horseradish peroxidase. We tested formalin-fixed tissue from explanted human livers and found an overall sensitivity of 69%. Furthermore the technique is highly specific, with negative staining in normal liver tissue from four cases of partial hepatic resection for nontumoric diagnoses and in hepatic tissue obtained from cases of primary biliary cirrhosis, familial amyloidosis, and acute and chronic Budd-Chiari syndrome, for a total of seven confirmed Hep C-negative specimens. Staining with probes for Epstein-Barr virus NotI and EBER sequences was negative in Hep C-positive liver sections from both resected native livers at the time of transplantation and biopsy specimens from post-transplantation patients in whom Hep C has recurred. In those livers staining positively with probe against Hep C, the distribution was both patchy, i.e., staining only fairly well-preserved hepatocytes and not all hepatocytes, and intracytoplasmic, supporting the observations of previously published reports. Finally, the technique was employed on formalin-fixed liver biopsy specimens from four patients with recurrent Hep C; there was repeatedly positive staining in all cases. We think this technique could provide a clinically useful adjunct in the diagnosis of Hep C from formalin-fixed tissue from liver biopsy specimens and that it may be useful in the characterization of the pathophysiologic features of Hep C virus infection.