Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

Abstract

SUMMARY:Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5Јnon-coding region (5ЈNCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5ЈNCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5ЈNCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5ЈNCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5ЈNCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV. (Lab Invest 2000, 80:851-856).

Characterization and evolution of NS5A quasispecies of hepatitis C virus genotype 1b in patients with different stages of liver disease

“…The rate of fixation of mutations observed in all our samples (0.77-7.65 Â 10 À3 s/nt/y) was similar to that previously described for this region Maekawa et al, 2000;Soguero et al, 2000;GimenezBarcons et al, 2001]. A mixed infection with two different HCV strains was detected in one patient (C19).…”

Dynamics of hepatitis C virus NS5A quasispecies during interferon and ribavirin therapy in responder and non-responder patients with genotype 1b chronic hepatitis C

“…Despite using different sets of primers and different reaction conditions, NS5A could not be amplified in three HCV RNA-positive samples (patient 5 at week 1 and patients 4 and 8 at week 2). The derived BTW-CONS amino acid sequence of these regions, obtained after alignment of all sequenced clones from every sample, was representative of the HCV-1b strains of our geographical area, as it matched that obtained upon alignment of over 300 isolates sequenced previously at our institution (Sáiz et al, 1998;Soguero et al, 2000;Giménez-Barcons et al, 2001;Puig-Basagoiti et al, 2001;Franco et al, 2003). The WTH-CONS sequence of each isolate remained invariable throughout the study, except in two patients (4 and 7).…”

Hepatitis C virus genotype testing in paraffin wax embedded liver biopsies for specimen identification

“…Soguero et al have used this technique as a research tool to carry out HCV genotyping in paraffin wax embedded pathology specimens. 10 Our present case report demonstrates the usefulness of this technique in a clinical setting. In Japan, there are many patients with chronic viral hepatitis, and liver biopsy will probably be performed more frequently in the future.…”