jury. Intrahepatic viral load appears to be significantly The balance between a direct cytopathic effect by hepincreased in patients with genotype 1b. IFN-a treatment atitis C virus (HCV) and immune-mediated injury redoes significantly lower intrahepatic HCV load. (HEPAmains unclear. This report aims to test the following TOLOGY 1996;23:676-687.) hypotheses: (1) that intrahepatic HCV load would correlate with the degree of liver injury; (2) that interferon alfa (IFN-a) would decrease intrahepatic HCV-RNA lev-
Persistent hepatitis C infection results in varying deels. Liver tissues (n Γ
56) were obtained from 47 patients grees of chronic liver injury. [5] Healthy carriers of the with chronic HCV (9 before and after IFN-a therapy). hepatitis C virus (HCV) have been described but seem Total RNA was isolated and quantitated for specific HCV uncommon. The pathogenesis of HCV-induced hepatic RNA by dot-blot polymerase chain reaction (DB-PCR) injury remains unclear and could be attributable to either using a standard curve created from synthetic HCV RNA direct cytopathic damage by HCV 8-10 or immune-mediof known titer to calculate actual RNA levels. A multivarated hepatic injury induced by HCV. [11][14] It is possible iate analysis was undertaken to determine the relationthat both may act simultaneously. ship of intrahepatic HCV-RNA levels with risk factors, One way to identify whether liver damage is attributlength of HCV exposure, and histological injury scores.
The confounding effect of HCV genotype was examined able to direct cytopathic damage is to examine whether by direct sequencing of the NS5b region. Liver HCV RNA the degree of viral load correlates with the degree of ranged from 10 2 to 3.1 1 10 7 molecules per microgram liver injury. Most studies addressing this question have total liver RNA. The multiple regression analysis showed measured the amount of HCV in serum. 10, Ideno effect of length of HCV exposure, risk factors, degree ally, HCV-RNA viral load should be estimated directly of bile duct damage, steatosis, or total Scheuer or Knofrom liver tissue itself, because serum levels of virus dell score on RNA levels. No significant confounding efmay be contributed to and influenced by extrahepatic fect of HCV genotype on the degree of liver injury was sources. Also, the presence of serum immune complexes observed. However, genotype 1b had a significantly may introduce further variables. Doubt has also been higher mean intrahepatic HCV-RNA load compared raised as to the validity of HCV-RNA quantitation in with the other genotypes detected. In the 9 patients who received IFN-a, treatment, 7 had no detectable HCV serum compared with plasma. 22 after treatment. This was associated with a significant
As well as the sample type, the method of HCV quandecrease in intrahepatic HCV-RNA levels (7.57 { 2.53 titation itself is important. A number of techniques 1 10 5 to 1.82 { 1.80 1 10 3 molecules per microgram total have been reported for quantitation of HCV by reverseliver RNA { SEM, n Γ
9, P Γ
.0005). These results do not transcription polymerase chain reaction (PCR) includdirectly support our hypothesis that increased intraheing competitive PCR, end-point titration, branched patic HCV load is associated with more severe liver in-DNA assay (bDNA) (Chiron Corp., Emeryville, CA), and internal standard RNA (Amplicor HCV Monitor quantitative assay, Roche Diagnostics Systems, Nut-Abbreviations: HCV, hepatitis C virus; PCR, polymerase chain reaction; ley, NJ). All of these have potential disadvantages, in-bDNA, branched DNA assay; IFN-a, interferon alfa; ALT, alanine transamicluding lack of resolving power of titer-based assays, nase; CH, chronic hepatitis; CIRR, cirrhosis; cDNA, complementary DNA; AlB, poor sensitivity of bDNA assays, and time-consuming aldolase B; AB6, outer sense primer; AB8, antisense primer; DB-PCR, dotpreparation of competitive RNA templates. Furtherblot polymerase chain reaction; EP-PCR, end-point titration polymerase chain reaction; N-PCR, nested polymerase chain reaction.
more, the reproducibility of these techniques has not From 1 The A. W. Morrow Gastroenterology and Liver Centre, and 2 Anatomi-been adequately documented.