ELSEVIER Recurrent Hepatitis C Infection Following Orthotopic Liver Transplantation Is Predicted by Posttransplant Serum and Hepatic Allograft Viral Titers R. Cirocco, G.P. Fragulidis, D. Weppler, K. Zucker, M. Markou, X.A. Zervos, A. Viciana, V. Esquinazi, JR. Net-y, K.R. Reddy, E. Schiff, J. Miller, and A.G. Tzakis zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED 0 NE OF THE major causes of end-stage liver disease is hepatitis C related cirrhosis. Orthotopic liver trans- plantation (OLTx) restores liver function in these patients but there is recurrence of hepatitis C virus (HCV) disease in approximately 80% to 95% of these transplant recipients.’ The rate of recurrence in these patients varies over time. Factors that cause different rates of HCV progression are thought to be the amount of immunosuppressive therapy, HCV strain, and viral interference by poor antigen presen- tation. The recurrence of HCV is difficult to diagnose by histologic examination of liver biopsy sections. This evalu- ation is complicated in that two processes, HCV recurrence and/or rejection, may be occurring concomitantly. Thus, histologic diagnoses may not be reliably made as histologic features may overlap in the two conditions of rejection and recurrence. An assay that would enumerate the viral titer in serum and liver biopsies may be relevant in monitoring HCV progression in these transplant recipients. The aim of this study was to evaluate hepatitis C RNA levels in serum and liver allografts and to correlate them to recurrent HCV disease after OLTx. MATERIALS AND METHODS Posttransplant serum and liver tissue biopsies from liver transplant recipients (n = 23) with HCV were prospectivelyzp3 tested by a quantitative HCV polymerase chain reaction (PCR) test developed in our laboratory. Serum and liver biopsy material was secured simultaneously and stored at -70°C until RNA isolation. The liver biopsies were weighed and stored dry before freezing. RNA was isolated by the phenol isothiocyanate method of Chomczynaski and Sacchi.4 Briefly, 100 PL of serum, or biopsy tissue plus 100 PL of water, was added to 900 PL of RNA-ZOL B (Tel-Test Inc, Friendswood, Tex), vortexed for 15 seconds, and 100 PL of chloroform/isoamyl alcohol (ratio = 2411) was added and vortexed again. The mixture was centrifuged at 12,000 G for 15 minutes and the aqueous phase (0.5 mL) was mixed with 0.5 mL of isopropanol. The mixture then was vortexed and stored at -70°C for 30 minutes. The RNA pellet was isolated by centrifugation at 14,000 G and then stored at 4°C. This pellet was washed in 0.25 mL of 75% ethanol, the supernatant discarded, and the pellet dried in a vacuum. The resulting pellet was resuspended in 20 PL of RNase free water. Two microliters of this RNA were reverse transcribed (RT) into C-DNA with a RT-PCR kit (Roche Molecular, Branch- 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 29, 2839-2840 (1997) burg, NJ). The competitive PCR was carried out by adding a known amount of HCV competitor to the RT reaction tube. The resulting amplicons are separated by agarose gel electrophoresis. The gel is photographed and the negative is scanned by laser densitometry to ascertain the HCV titer for each specimen. Serum levels are measured in copies/ml and tissue in copies/mg. Recurrence of HCV was based retrospectively on clinical out- come in response to immunosuppressive regimen. Histologic ex- amination of liver biopsy specimens was done by a pathologist who was blinded to the HCV PCR assay evaluation and the HCV titers. All patients were part of an early postoperative alpha-interferon treatment protocol. RESULTS The patients were tested for HCV PCR levels in both the serum and biopsy and were divided into two groups: those diagnosed with recurrent HCV (n = 9) and those without recurrent disease (n = 14). These diagnoses were made retrospectively, based on the clinical outcome of the pa- tient. The overall follow-up for these patients was 9.2 months (range 5 to 15 months). Preoperative serum titers were evaluated by the branched DNA method (Chiron, Emeryville, Calif). The postoperative competitive RT- PCR results from these assays are shown in Table 1. All patients in the recurrent group had a clinical course con- sistent with recurrent HCV disease, and they showed an improvement in transaminase activity when immunosup- pression was decreased and a deterioration when immuno- suppression was increased. There was no statistically signif- icant difference in the preoperative serum RNA levels between the two groups (P = .71). There was a statistically higher level of serum and biopsy titers (viral burden) in the patients with recurrent HCV disease. Liver specimen RNA titers <lOOO copies/mg were not associated with recurrent histologic HCV disease. From the University of Miami, Department of Surgery, Division of Transplantation, Department of Medicine, Division of Hepa- tology, Miami, Florida. Address reprint requests to Georgios P. Fragulidis, MD, Uni- versity of Miami School of Medicine, Division of Transplantation, Department of Surgery (M840), PO Box 015809, Miami, FL 33136. 0041-l 345l97l$17.00 PII SOO41-1345(97)00699-4 2839